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J. Eric Brewer A Study of Polyethoxylated Alkylphenols by Packed Column Supercritical
ebrewer@j-chrom-sci.com Fluid Chromatography
Editorial Assistant B.J. Hoffman and L.T. Taylor ..........................................................................................................61
Kevin Bailey
An Isocratic Liquid Chromatographic Method with Diode-Array Detection for
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Dean Rood Roger K. Gilpin the Simultaneous Determination of -Tocopherol, Retinol, and
Brian A. Bidlingmeyer Five Carotenoids in Human Serum
S. Gueghuen, B. Herbeth, G. Siest, and P. Leroy ............................................................................69
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Director of Marketing Services M.A. Hable, J.B. Sutphin, C.G. Oliver, R.M. McKenzie, E.F. Gordon, and R.W. Bishop..................77
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Production Displacement Study on a Vancomycin-Based Stationary Phase Using
Roberta Knight, Manager N-Acetyl-D-Alanine as a Competing Agent
Dana Neiman I. Slama, C. Ravelet, A. Villet, A. Ravel, C. Grosset, and E. Peyrin..................................................83
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Stephanie Graffuis-Cain, WebMaster Furosine Determination Using Ion-Pair Reversed-Phase High-Performance
Pamela Kintzel Liquid Chromatography
Editorial Advisory Board M.A. Serrano, G. Castillo, M.M. Muñoz, and A. Hernández ..........................................................87
Lars Blomberg R. Kaliszan
Phyllis R. Brown J.J. Kirkland
The Use of Nonendcapped C18 Columns in the Cleanup of Clenbuterol and a
Kenneth A. Cohen S.F.Y. Li New Adrenergic Agonist from Bovine Liver by Gas Chromatography–Tandem
Tibor Cserháti C.E. Lin
Neil D. Danielson C.H. Lochmüller
Mass Spectrometry Analysis
William A. Dark Fernando M. Lanças M. Fiori, C. Cartoni, B. Bocca, and G. Brambilla ...........................................................................92
Gerald D. Dupré David C. Locke
R. Gilpin Fred Rabel Simultaneous High-Performance Liquid Chromatographic Determination of
G. Guiochon M.L. Riekkola
Jaroslav Janák R.P.W. Scott Paracetamol, Phenylephrine HCl, and Chlorpheniramine Maleate in
Kiyokatsu Jinno A.M. Siouffi Pharmaceutical Dosage Forms
S. Bart Jones Larry T. Taylor
Donald E. Willis
H. S˛enyuva and T. Özden..............................................................................................................97

Evaluation of Select Variables in the Ion Chromatographic Determination of

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Journal of Chromatographic Science, Vol. 40, February 2002
A Study of Polyethoxylated Alkylphenols by Packed
Column Supercritical Fluid Chromatography
Brian J. Hoffman and Larry T. Taylor
Virginia Tech, Department of Chemistry, Blacksburg, VA 24061-0212
Abstract
Alkylphenol polyethoxylates (APEs) are a widely used group of
nonionic surfactants in commercial production. Characterization of
the composition of APE mixtures can be exploited for the
determination of their most effective uses. In this study sample
mixtures contain nonylphenol polyethoxylates and octylphenol
polyethoxylates. The separation of individual alkylphenols by
ethoxylate units is performed by supercritical fluid chromatography
(SFC)-UV as well as normal-phase high-performance liquid
chromatographic (HPLC)-UV employing packed columns. The
stationary phase and column length are varied in the SFC setup to
produce the most favorable separation conditions. Additionally,
combinations of packed columns of different stationary phases are
tested. The combination of a diol and a cyano column is found to
produce optimal results. An advantage of using packed columns
instead of capillary columns is the ability to inject large amounts of
sample and thus collect eluted fractions. In this regard, fractions
from SFC runs are collected and analyzed by flow injection
analysis–electrospray ionization–mass spectroscopy in order to
positively identify the composition of the fractions. In comparing
the separation of APE mixtures by SFC and HPLC, it is found that
SFC provides shorter retention times with similar resolution. In
addition, less solvent waste is produced using SFC.
Introduction
Alkylphenol polyethoxylates (APEs) are referred to as nonionic
surfactants. Since the mid 1940s, APEs have been used commer-
cially for their surfactant ability. The term surfactant includes
surface-active compounds characterized by their ability to con-
centrate at surfaces and form micelles in solution (1). They have
been used in a wide variety of applications including industrial
process aids, dispensing agents in paper and pulp production,
emulsifying agents in latex paints and pesticide formulations,
flotation agents, industrial cleaners (metal surfaces, textile pro-
cessing, and food industry), and household cleaners (1). These
compounds are commercially available as oligomeric mixtures
with varying ethoxylate chain lengths as well as varying alkyl
sizes. Certain APEs have been determined to be estrogenic in fish,
birds, and mammals (2).
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
APEs contain two main molecular regions: the polyethoxylate
(POE) chain (EO) is polar and thus hydrophilic and the alkyl-
phenol is the hydrophobic area. The hydrophilic nature of the EO
is attributed to the hydration of the ether-linked oxygen atoms
(3). A technical synthesis of APEs start with phenol, which is alky-
lated by trimethylpentane and thus produces octylphenol (OP), or
by nonene isomers, which forms nonylphenol (NP) in an acid-cat-
alyzed process. Ethoxylation is performed by using KOH–ethanol
as a catalyst with a known ratio of ethylene oxide to the
alkylphenol (1). The reaction results in an oligomeric mixture of
the alkylphenol containing an EO chain of varying lengths.
The separation and identification of the components of an APE
mixture can be useful for the determination of their most effec-
tive applications. Several different types of chromatography have
been studied previously in efforts to achieve better separation
conditions. Gas chromatography (GC) coupled with flame ioniza-
tion detection as well as mass spectrometry (MS) has been used in
the analysis of APEs (4). Isomers of each oligomer tend to be sep-
arated into clusters by GC. Usually, it is necessary to derivatize
samples containing APEs for analysis by GC, because the com-
pounds are not very volatile. GC poorly separates higher molec-
ular-weight oligomers because of their lower volatility.
High-performance liquid chromatography (HPLC) has been
used to separate APEs of higher mass oligomers. Both reversed-
phase (3) and normal-phase (5–7) chromatographic separations
have been performed on solutions containing APEs. Each
oligomer is separated by an ethoxylate unit, and isomers of each
oligomer tend to coelute. Recently, Gundersen used a graphitic
carbon column in research to separate isomers of individual
ethoxylated alkylphenols by HPLC (8). Ferguson et al. used
reversed-phase HPLC–electrospray ionization (ESI)–MS to ana-
lyze APEs and their metabolites in aquatic environments (9).
Normal-phase HPLC–ESI–MS was used by Shang et al. to quanti-
tate NPEOs in marine sediment (10).
In addition to traditional forms of chromatography, supercrit-
ical fluid chromatography (SFC) has been employed for APE sep-
aration. SFC has advantages over both HPLC and GC. SFC can
operate at lower temperatures than GC, allowing samples that are
thermally labile to be analyzed. Supercritical fluids have densities
similar to liquids and diffusivities similar to gases. These qualities
allow large molecular-weight molecules that are not volatile to be
61

separated by SFC similar to HPLC but with shorter retention
times because of the physical properties of supercritical fluids.
This reduces solvent waste and decreases the total analysis time.
Capillary-column SFC using flame ionization detection (11,12)
has been used to separate both NPEO and OPEO. Because a
sample is generally destroyed by this method, it is not possible to
directly determine analyte identity. Peak identity can be surmised
by comparing retention times of samples with other APE mix-
tures that contain a large fraction of a known single oligomer. A
disadvantage associated with capillary columns is the inability to
inject large sample volumes, which precludes semipreparative
fraction collection.
In addition, OPEO mixtures have been separated on packed-
column SFC using reversed-phase (13,14) and normal-phase
(15,16) packing material. Both Takeuchi and Saito and Giorgettie
et al. used C18 packed columns to separate OPEO samples by
SFC. Takeuchi and Saito found that a microcolumn (1.0 × 500
mm) had the best separation performance, but a semimicro-
column (1.7 × 250 mm) produced the best results. A conventional
column (6.0 × 250 mm) was used in their research for preparative
purposes. Packed-column SFC allows larger amounts of sample
to be injected into the system for the semipreparative collection
of analyte fractions. Giorgettie et al. studied mixed mobile phases
using the addition of a modifier in order to make their mobile
phase more polar. They used pressure programming and a modi-
fier additon to produce optimum separations. Highly efficient
separations were produced under constant modifier concentra-
tion and pressure programming.
The object of this study was to compare the ability of normal-
phase packed columns to separate APEs on an SFC system.
Individual packed columns as well as stacked packed columns of
different stationary phases were used in the SFC experiments.
Additional goals of this study were to identify the components
that gave rise to the chromatographic peaks in hopes of pro-
ducing individual ethoxylated alkylphenol standards. Fractions
that contain a single ethoxylate compound could later be used as
standards for quantitating APEs in a variety of applications. A
comparison of the ability of SFC and HPLC to separate APEs
using normal-phase packed columns was also studied.
Experimental
Packed-column SFC
A Berger (Newark, DE) SFC system was used in the SFC anal-
ysis. A Berger autosampler with a 10-µL injection loop was used
for conventional sample analysis, and a 75-µL injection loop was
used for the injection of semipreparative samples. SFC-grade
carbon dioxide (Air Products and Chemicals, Inc., Allentown, PA)
was used with methanol (Burdick & Jackson, Muskegon, MI) as a
modifier. The mobile phase flow rate was 2.0 mL/min. The oven
temperature was set at 60°C, and the outlet pressure was kept at
120 atm. Absorbance was read at 225 nm by a diode-array
detector. The detection wavelength was determined by finding the
maximum absorbance of an individual APE sample by obtaining
its UV–vis spectrum. Supelcosil LC-Diol, Supelcosil LC-CN
(Supelco, Bellefonte, PA), and Spherisorb NH2 (Waters, Millford,
62
Journal of Chromatographic Science, Vol. 40, February 2002
MA) columns were used for the chromatographic separation of
the APE mixtures. All columns measured 4.6 × 250 mm with a
5-µm particle size. A diol bonded silica guard column was used.
Normal-phase HPLC
For HPLC analysis, a Hewlett-Packard (Little Falls, DE) 1050
Series HPLC system was used with a variable wavelength detector
(reading 225 nm) and an inline vacuum degasser. Injections were
made manually with a Rheodyne (Rohnert Park, CA) injector
equipped with a 20-µL injection loop. Data were collected and
chromatograms were processed by MassLynx software (Fisions
Instruments, Altricham, U.K.). A Supelcosil LC-Diol column (4.6
× 250 mm, 5 µm) was used for the chromatographic separation of
the APE mixtures.
Flow injection analysis–MS
A Fisions Instruments VG Platform MS was used for the mass
analysis of collected sample fractions. All samples were analyzed
under positive ESI. A syringe pump (Harvard Apparatus, South
Natick, MA) supplied an 80:20 methanol–water mobile phase to
the probe. Samples were injected by a Rheodyne injector
equipped with a 20-µL injection loop. Nitrogen was used as both
the drying and sheath gas. Data were collected and analyzed by
MassLynx software.
Alkylphenol samples
POE-(4)-NP (ChemService, West Chester, PA) and Triton N-101
(Sigma-Aldrich, Milwaukee, WI) were used as NPEO mixtures.
POE-(5)-tert-OP (ChemService) was used as an OPEO mixture.
All of the samples that were analyzed by SFC were dissolved in
methanol, and samples analyzed by normal-phase HPLC were
dissolved in hexane. The Triton N-101 sample that was used for
HPLC was dissolved in 9:1 hexane–acetone in order to increase
solubility. HPLC samples were prepared at approximately 1.0
mg/mL, and SFC samples were prepared at approximately 2.0-
mg/mL concentrations.
Semipreparative SFC
A tee was placed inline between the column and diode-array
detector of the SFC system, splitting effluent approximately 75%
to the collection and 25% to the detector. Eluent was diverted
using a portion of fused-silica capillary tubing. Fractions were
collected in preweighed 16-mL collection vials. Absorbance was
monitored, and fractions were collected manually between min-
imum absorbance values. POE-(4)-NP and POE-(5)-tert-OP were
separated in this fashion. Fractions were evaporated by nitrogen
blow-down on a hot plate. The remaining residue was weighed.
The fractions were then diluted to 10.0 mL with methanol.
Fractions were analyzed by SFC-UV followed by flow injection
analysis (FIA)–ESI–MS for purity.
FIA–ESI–MS method
SFC-collected fractions were evaporated by nitrogen blow-
down and weighed. Collected fractions were then dissolved in
methanol. Optimal MS settings were found by injecting each frac-
tion and tuning the instrument. Fractions were then reinjected,
and mass-spectral data were recorded and analyzed. The source
temperature was set at 100°C. ESI nebulizing gas flow was set at
Journal of Chromatographic Science, Vol. 40, February 2002
20 L/h, and the drying gas flow was 300 L/h. Samples were
recorded in full-scan mode from m/z 200 to 700. The cone voltage
ranged from 52 to 75 V, and the high voltage lens and ESI capil-
lary voltage were kept at 0.88 and 3.46 kV, respectively.
HPLC method
Hexane and isopropanol were used as the mobile phase. A linear
gradient was used starting with 100% hexane and then changing
to 70:30 hexane–isopropanol over 30 min. From t = 30 to 35 min,
the mobile phase was returned to 100% hexane and held for
5 min in order to equilibrate. POE-(4)-NP and POE-(5)-tert-OP
were separated in this fashion.
Results and Discussion
APEs are complex mixtures that provide moderate challenges
for chromatographic techniques. Our research studied how the
total column length, stationary phase, and column stacking order
of different stationary phases affect the SFC separation of ethoxy-
late units in APE mixtures. Our goal was to find a setup that pro-
duced the best separation. In order to accomplish this we kept all
system parameters constant throughout the study other than
column setup and modifier gradient. All of the columns used
were uniform in size (4.6 × 250 mm, 5 µm) in order to allow us to
verify the effect of column length and packing material. POE-(4)-
NP was used in all of the diol column studies because of its short
elution time.
POE-(4)-NP was separated on a combination of one-, two-, and
three-packed diol columns connected in series to study the effect
of column length (Figure 1). A single diol column poorly sepa-
rated the sample. Baseline separation was not achieved with a
Figure 1. Packed-column supercritical fluid chromatograms using stacked diol
columns: (A) one Supelcosil LC-Diol column, (B) two Supelcosil LC-Diol
columns, and (C) three Supelcosil LC-Diol columns. The sample used in each
chromatogram was POE-(4)-NP (2.0 mg/mL). A linear modifier gradient was
used by the following program: 10.0% methanol was increased to 26.0% at a
rate of 0.6%/min with a 2.0-min hold and then returned to 10.0% in 4.0 min
followed by a 2.0-min hold.
single column. SFC separation on two diol columns increased
separation, but early eluting peaks were not baseline separated.
Using two diol columns, SFC separation was comparable with
normal-phase HPLC using one diol column. For comparison,
POE-(4)-NP, POE-(5)-tert-OP, and Triton N-101 were separated by
SFC on two diol columns and HPLC on one diol column (Figures
2–4). The retention time of the chromatographic peaks for SFC
separation using two diol columns was considerably lower than
normal-phase HPLC separation using one diol column (Tables I
and II shows data for the NPEO sample and Table III shows data
for the OPEO sample). The addition of a third diol column to the
SFC system generated a better separation, but later-eluting peaks
began to broaden.
The effect of the stationary phase on separation was sequen-
tially tested using a single diol, amino, and cyano column (Figure
5). The retention of oligomers with longer ethoxylated units
varied with each stationary phase tested. The diol column had the
least retention, the amino column had intermediate retention,
and the cyano column had the greatest retention. It was not pos-
sible to elute all of the compounds off the cyano column using the
corresponding gradient. In general, a larger methanol modifier
concentration was needed to elute longer ethoxylate-chain com-
Figure 2. Chromatograms of POE-(4)-NP using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
63

pounds. Because of this, we can conclude that APEs with a longer
ethoxylate chain are more polar than those with shorter chains.
Following this reasoning, the cyano column must be the most
polar stationary phase because it retained the more polar compo-
nents longer, and the diol column is the least polar.
Columns with different stationary phases were coupled in
series to test how the arrangement would affect the retention of
an APE sample. Two column arrangements were tested. The first
consisted of one diol column followed by one cyano column. The
second setup contained three columns, a diol column, a cyano
column, and an amino column in series (Figure 6). A steeper gra-
dient was needed than previously used in order to elute all of the
compounds because of the presence of the cyano column (as pre-
viously mentioned). The modifier gradient that was used is
described in Figure 6.
One of our goals in this study was to achieve separation that
would allow us to easily collect individual oligomers for use as
standards. The combined diol–cyano setup rendered shorter
retention times than the combined diol–cyano–amino setup;
therefore, this arrangement was used for preparative fraction col-
lection. In the chromatograms of stacked columns using different
stationary phases, peak splitting was observed for later-eluting
Figure 3. Chromatograms of POE-(5)-tert-OP using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
64
Journal of Chromatographic Science, Vol. 40, February 2002
peaks. POE-(4)-NP and POE-(5)-tert-OP were separated, and five
fractions of each sample were collected. A large volume (75 µL) of
concentrated sample was injected six to eight times in the collec-
tion process. Isolated fractions were reanalyzed both by SFC for
purity (Figures 7 and 8) and FIA–ESI–MS for identification. The
concentrations used for the semipreparative work caused the
chromatographic peaks to significantly broaden and in some
cases combine. Because of this phenomenon we were not able to
collect individual fractions of the two initial oligomers of POE-
(4)-NP and fractions of the three initial oligomers of POE-(5)-tert-
OP as evidenced by the SFC-UV of the early fractions.
FIA–MS was used to identify the components in each fraction.
ESI–MS was chosen because it is amenable to high-molecular-
weight analytes and works well with liquid mobile phases.
Samples were dissolved in methanol (a compatible solvent for
ESI–MS), which made ESI–MS a desirable tool for fraction iden-
tification. It was possible to produce sodium-adducted molecular
ions rather easily. In order to create an optimum response, the
fractions were first injected and the cone voltage varied in order
to produce the greatest response for each individual analyte. After
MS tuning conditions were perfected, the fractions were rein-
jected into the instrument. A spectrum was created between
Figure 4. Chromatograms of Triton N-101 using (A) normal-phase HPLC-UV
with one Supelcosil LC-Diol column and (B) SFC-UV with two Supelcosil LC-
Diol columns. The peak annotations represent the number of ethoxylate units.
Journal of Chromatographic Science, Vol. 40, February 2002

m/z 200 and 700 by averaging scans of the injected sample.
Figures 9 and 10 show the average mass spectrum of each fraction.
The spectra confirm that each chromatographic peak varied by
one ethoxylated unit (a separation of m/z 44 represents an ethoxy-
late unit). It was possible to identify NP3EO through NP7EO in
basically pure collected fractions of POE-(4)-NP and OP5EO
through OP8EO in fractions collected from POE-(5)-tert-OP.
Major ion peaks consisted of Na+ adduct ions, and minor peaks
were produced by K+ adduct ions under positive electrospray con-
ditions. Trace levels of sodium and potassium must be present in
the mobile phase that was used for FIA–ESI–MS because elec-
trolyte was not added to the solutions. According to Okada’s
research (17), APEs have an affinity for alkali metals and have a
flexible structure that allows them to form complexes with alkali
metals. This explains the ion pairing seen in the mass spectra.
Crescenzi et al. performed an experiment to see if the detector
response would decrease because of the complexation of
oligomers competing for the limited metal pool available. When
equivalent amounts of ethoxylated compounds were analyzed by
ESI–MS, it was found that the detector response increased expo-
nentially from 1 to 6 EO units and then leveled off at 8 EO units
(the scope of the study) (18). A decrease in signal was most notice-
able for lower ethoxylated oligomers. This can be explained by
noting that ethoxylated compounds can form increasingly stable
complexes with alkali metal ions as the EO unit number increases
(17).

Table I. Chromatographic Peak Retention Times of Table III. Chromatographic Peak Retention Times of
POE-(4)-NP (NPEO) Separated by SFC Using Two POE-(5)-tert-OP (OPEOs) Separated by SFC Using Two
Supelcosil LC-Diol Columns and HPLC Using One Supelcosil LC-Diol Columns and HPLC Using One
Supelcosil LC-Diol Column Supelcosil LC-Diol Column

EO unit SFC RT* HPLC RT EO unit SFC RT* HPLC RT

2 7.18 9.14 2 6.79 9.28

3 7.86 9.93 3 7.48 10.06
4 8.64 10.66 4 8.20 10.92
5 9.68 11.82 5 9.05 12.08
6 10.61 13.08 6 10.00 13.39
7 11.56 14.46 7 10.96 14.75
8 12.49 15.83 8 11.91 16.10
9 13.43 17.28 9 12.88 17.51
10 14.37 18.74 10 13.86 18.96
11 15.16 11 14.80
12 15.76
* RT, retention time.
* RT, retention time.

Table II. Chromatographic Peak Retention Times of Triton

N-101 (NPEOs) Separated by SFC Using Two Supelcosil
LC-Diol Columns and HPLC Using One Supelcosil
LC-Diol Column

EO unit SFC RT* HPLC RT

2 7.29 9.88
3 8.02 10.68
4 8.83 11.84
5 9.75 13.08
6 10.66 14.33
7 11.57 15.55
8 12.48 16.80
9 13.36 18.06
10 14.20 19.39
11 15.03 20.68
12 15.84 22.29
13 16.61 24.11
14 17.37 Figure 5. Packed-column supercritical fluid chromatograms using single
15 18.10 columns of different polar packing material: (A) Supelcosil LC-Diol column,
16 18.81 (B) Spherisorb NH2 column, and (C) Supelcosil LC-PCN column. The sample
17 19.58 used in each chromatogram was POE-(4)-NP (2.0 mg/mL). A linear modifier
18 20.10 gradient was used by the following program: 10.0% methanol was increased
to 26.0% at a rate of 0.6%/min with a 2.0-min hold and then returned to 10.0%
* RT, retention time. in 4.0 min followed by a 2.0-min hold.

65

It was important to perform chromatographic separations with
absorbance detection on the fractions as well as MS analysis, thus
allowing us to positively identify sample components because MS
could not detect all of the compounds present. The first fraction
of both POE-(4)-NP and POE-(5)-tert-OP contained more than
one compound (as seen in their SFC-UV chromatograms). The
sodium ion affinity of the smaller ethoxylate chain compounds is
lower than the larger chain oligomers, and because of this they
Figure 6. Packed-column supercritical fluid chromatograms using stacked
columns of different polar stationary phases: (A) one Supelcosil LC-Diol
column and one Supelcosil LC-PCN column and (B) one Supelcosil LC-Diol
column, one Supelcosil LC-PCN column, and one Spherisorb NH2 column.
The sample used in each chromatogram was POE-(4)-NP (2.0 mg/mL). Multiple
linear modifier gradients were used by the following program: 10.0% methanol
was increased to 13.2% by 0.5%/min and then continued to 14.4% at
0.7%/min, 16.6% at 0.8%/min, 20.0% at 1.0%/min, 40.0% at 8.0%/min (held
for 5.0 min), and then returned to 10.0% at 15.0%/min.
Figure 7. Supercritical fluid chromatograms of collected POE-(4)-NP fractions.
Separation was conducted on one Supercosil LC-Diol column and one
Supelcosil LC-PCN column in series (the system settings were the same as
Figure 3).
66
Journal of Chromatographic Science, Vol. 40, February 2002
were not detectable in the mass spectra.
APEs can be categorized by their average ethoxylate unit value.
According to Wang and Fingas (3), all of the oligomers have
almost identical molar absorptivity, which allows integrated chro-
matographic peak areas to be used directly to determine the mole
fraction of each oligomer. POE-(4)-NP contained NP predomi-
nantly with short ethoxylate chains. NP2EO through NP11EO
were observed in its SFC-UV separation. An average ethoxylate
Figure 8. Supercritical fluid chromatograms of collected POE-(5)-tert-OP frac-
tions. Separation was conducted on one Supelcosil LC-Diol column and one
Supelcosil LC-PCN column in series (the system settings were the same as
Figure 3).
Figure 9. Positive-ion FIA–ESI–MS of POE-(4)-NP fractions operated in full-scan
mode. Ions were in the form of (M+Na)+ and each were separated by m/z 44
(the mass of one ethoxyl unit): (A) fraction 1, cone voltage of 59 V; (B) fraction
2, cone voltage of 53 V; (C) fraction 3, cone voltage of 62 V; (D) fraction 4, cone
voltage of 65 V; and (E) fraction 5, cone voltage of 67 V. Each spectrum was
averaged over the sample injection peak.
Journal of Chromatographic Science, Vol. 40, February 2002
unit value of 4.20 was calculated from peak areas. POE-(5)-tert-
OP had a similar distribution as POE-(4)-NP. Its average ethoxy-
late unit value was calculated as 4.48, and it contained OP2EO
through OP12EO in its SFC-UV separation. Triton N-101 con-
tained a greater range of NPEOs. Its calculated average ethoxylate
unit was 9.97. NP2EO through NP18EO were observed in its SFC-
UV chromatogram. Higher EO peaks were detected in SFC sepa-
rations, which were not detected by HPLC analysis. Wang and
Fingas produced similar average EO unit values from their capil-
lary SFC data. Their analysis of Igepal CO430 (trade name for
POE-(4)-NP), Triton X-45 (trade name for POE-(5)-tert-OP), and
Triton N-101 produced average EO values of 4.14, 4.50, and 9.52,
respectively (11,12). We used the chromatographic data from the
SFC-UV separations on two diol columns to calculate our average
EO values.
Conclusion
Normal-phase packed-column SFC produced a similar separa-
tion of APE mixtures compared with normal-phase HPLC.
Column length, stationary phase, and column combinations with
different stationary phases all affected the separation of the APE
mixtures tested. Longer column lengths increased the separation
of oligomers. More-polar stationary phases retained oligomers
with larger ethoxylate units for a longer time. A combination of
columns with different stationary phases produced separations
combining both the effects of longer columns and the separation
ability of each stationary phase. Retention times for SFC separa-
Figure 10. Positive-ion FIA–ESI–MS of POE-(5)-tert-OP fractions operated in
full-scan mode. Ions were in the form of (M+Na)+ and each were separated by
m/z 44 (the mass of one ethoxyl unit): (A) fraction 1, cone voltage of 63 V; (B)
fraction 2, cone voltage of 68 V; (C) fraction 3, cone voltage of 65 V; (D) frac-
tion 4, cone voltage of 75 V; and (E) fraction 5, cone voltage of 75 V. Each spec-
trum was averaged over the flow injection peak.
tions were notably shorter than normal-phase HPLC. One of
SFC’s advantages is its ability to use longer combined column
lengths without elevated back pressure, which occurs in HPLC.
Combining multiple columns with different stationary phases
seemed to provide the best separation.
An advantage of using packed columns over the use of capillary
columns is the ability to inject larger amounts of sample and col-
lect eluted fractions. It is possible to isolate and identify individual
APEs. Additionally, it is possible to identify the remaining chro-
matographic peaks because of each peak differing by one ethoxy-
late unit. Our study demonstrated the importance of using both
absorbance detection as well as MS. MS alone did not show all the
components of our initial fractions because of the decreased
detector response.
Less solvent waste was produced using SFC compared with
HPLC. Each SFC separation that used cyano packing as part of its
column arrangement used 6.7 mL of methanol. The remaining
SFC setups (the studies of column length and stationary phase)
used 11.8 mL of methanol. All separations performed by normal-
phase HPLC used 34.75 mL of hexane and 5.25 mL isopropanol
for a combined volume of 40 mL. The HPLC system used almost
600% more solvent than the SFC system using a cyano stationary
phase and over 330% more than the other SFC setups studied
(this is not including the volume of solvent needed to initially
equilibrate the systems). The reduction of solvent waste is an
important step of reducing pollution.
Because of the fact that APEs are used as industrial cleaners and
other processing aids, they enter wastewater and end up in
sewage treatment plants. Some APE waste is transferred into the
environment and metabolized into lower ethoxylated alkylphe-
nols, which are considered endocrine disrupters (2). APEs have
been found in fish, river sediment, and other environmental sam-
ples through analytical techniques (1,4,9,10,18–22). The results
of our study could lead to the further use of the method developed
for applications in the analysis of environmental samples.
Additionally, our method could be altered for use in a future
large-scale separation and collection of individual ethoxylated
alkylphenols. Access to standards of individual ethoxylated
alkylphenols is important for their quantitative analysis.
Acknowledgments
We would like to acknowledge Dr. Clifford P. Rice (USDA
ARS/NRI/EQL, Beltsville, MD) for APE information and Air
Products and Chemicals, Inc. for supplying SFC-grade carbon
dioxide.
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Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
An Isocratic Liquid Chromatographic Method with
Diode-Array Detection for the Simultaneous
Determination of α-Tocopherol, Retinol, and
Five Carotenoids in Human Serum
Sonia Gueguen1, Bernard Herbeth1, Gérard Siest1, and Pierre Leroy2
1Inserm U525, Centre de Médecine Préventive, 2 rue du Doyen Jacques Parisot, 54500 Vandoeuvre-lès-Nancy, France and 2Thiols and
Cellular Functions, Faculté de Pharmacie, Université Henri Poincaré Nancy 1, 30 rue Lionnois, 54000 Nancy, France
Abstract
An isocratic high-performance liquid chromatography (HPLC)
method for the simultaneous determination of α-tocopherol,
retinol, and five carotenoids (lutein–zeaxanthin, β-cryptoxanthin,
lycopene, and α- and β-carotene) in human serum is described.
Serum samples are deproteinized with ethanol and extracted once
with n-hexane. Resulting extracts are injected onto a C18 reversed-
phase column eluted with methanol–acetonitrile–tetrahydrofuran
(75:20:5, v/v/v), and full elution of all the analytes is realized
isocratically within 20 min. The detection is operated using three
channels of a diode-array spectrophotometer at 290, 325, and
450 nm for tocopherol, retinol, and the carotenoids, respectively.
An internal standard is used for each channel, which improves
precision. The choice of internal standards is discussed, as well as
the extraction protocol and the need for adding an antioxidant
during the extraction and chromatographic steps. The analytical
recoveries for liposoluble vitamins and carotenoids are more than
85%. Intra-assay relative standard deviation (RSD) values (n = 20)
for measured concentrations in serum range from 3.3% (retinol)
to 9.5% (lycopene), and interassay RSDs (n = 5) range from 3.8%
α-tocopherol) to 13.7% (ββ-cryptoxanthin). The present method is
(α
used to quantitate the cited vitamins in healthy subjects (n = 168)
from ages 9 to 55 years old.
Introduction
Retinol (vitamin A) and α-tocopherol (vitamin E) are nonen-
zymatic antioxidants (1). Vitamin A acts as a direct “scavenger”
of reactive oxygen species (ROS) and is also thought to inhibit
free radical synthesis via increasing the activity of detoxifying
systems (2).
* Author to whom correspondence should be addressed: email pierre.leroy@pharma.uhp-nancy.fr.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Vitamin E protects unsaturated fatty acids located in both cell
and organelle membranes against endo- and exogenous free rad-
icals and ROS, which are involved in the initiation and extent of
membrane damages caused by nonenzymatic lipid peroxidation
(3,4). Carotenoids act as ROS and free radical scavengers (5),
stimulants of immune response (6), and anticarcinogenic agents
(7). Because of their wide variety of functions and biological
roles, clinical interest in the evaluation of retinol, α-tocopherol,
and carotenoids has increased in recent years owing to their role
as antioxidants, which may be important in reducing the risk of
numerous diseases including cancer (8–11), coronary heart dis-
ease (12,13), and diabetes mellitus (14–18).
Thus, rapid, simple, sensitive, and selective methods for the
simultaneous determination of these antioxidants in biological
fluids are needed. As a matter of fact, the measurement of an
individual class of antioxidants such as thiols (19), hydrophilic,
or liposoluble vitamins provides more information for the mech-
anistic evaluation of a clinical disease linked to oxidative stress
than a total antioxidant status assay (20).
Numerous spectroscopic and separative methods have already
been reported for the assay of retinol, α-tocopherol, and
carotenoids in plasma or serum, and among them high-perfor-
mance liquid chromatography (HPLC) is one of the most pow-
erful analytical tools for this purpose (21–25).
Both normal-phase (26–28) and reversed-phase (29–35) HPLC
conditions have been widely used. However, many of these
methods include gradient elution (36–39), flow rate (34,36),
wavelength time-programmation (36,40), a switching device
between coupled columns (41,42), and the use of two different
detectors in series (43,44). All of these approaches are time-con-
suming because of their long-equilibration period between each
run and troublesome because of the hyphenated systems needed.
Indeed, the main difficulty for the simultaneous determination
of liposoluble vitamins and carotenoids results from their dif-
ferent spectral characteristics (absorption maxima vary in the
69
 Journal of Chromatographic Science, Vol. 40, February 2002

range of 292 to 450 nm). This problem has been solved by using (Millipore Milford, MA). Tert-butylated hydroxytoluene (BHT)
multichannel UV–vis spectrophotometric detectors (31,37,40, was purchased from Sigma-Aldrich (St. Quentin Fallavier,
45–47). More recently, a technique combining both isocratic elu- France).
tion in reversed-phase mode and diode-array detection was All-trans retinol (henceforth simply referred to as retinol),
reported, providing selectivity between the three classes of retinol acetate, α-tocopherol, α-tocopherol acetate, and
liposoluble vitamins and thus a convenient way for their simul- β-carotene standards were obtained from Fluka (Buchs,
taneous measurements (32). Switzerland). Zeaxanthin and β-cryptoxanthin were a generous
For all these methods, the preanalytical treatments, especially gift from Hoffman-Laroche (Basle, Switzerland). Lycopene and
the extraction procedure relying upon either liquid–liquid echinenone were purchased from CaroteNature (Lupsingen,
(26–28,30–35,39,43,47,48) or solid–liquid (38,49,50) partition, Switzerland). Stock solutions of retinol, α-tocopherol, and their
are critical steps to obtain reliable data. corresponding internal standards (acetate form) were prepared
This study deals with some improvements of a previously in ethanol (EtOH) added with 0.01% (w/v) BHT. Carotenoids
reported method (32); the full validation of the optimized assay; were prepared in tetrahydrofuran (THF) added with 0.01% BHT.
and its use to quantitate retinol, α-tocopherol, lutein–zeaxan- Stock solutions were protected from light in ambered glass bot-
thin, β-cryptoxanthin, lycopene, and α- and β-carotene in tles, titrated by spectrophotometry using their specific
healthy subjects. absorbance (Table I), and stored under nitrogen at –80°C for up
to 2 mo. The concentrations of stock solutions were 0.25–0.5
mg/mL for retinol and retinol acetate, 3–4 mg/mL for α-toco-
pherol and α-tocopherol acetate, and 0.1–0.2 mg/mL for
Experimental carotenoids.
Daily working solutions for calibration curves were prepared
Chemicals, reagents, and standards by diluting stock solutions in EtOH containing 0.01% BHT. The
All solvents and reagents used were of analytical- or HPLC- ranges of tested concentrations are indicated in Table II. An
grade. Ultrapure water was prepared using a Milli-Q system internal standard mixture containing retinol acetate, α-toco-
pherol acetate, and echinenone was also prepared
Table I. Characteristics of Standards Used daily following a similar procedure (combining
100 µL of each stock solution of internal standard
Maximum and diluting the volume to 20 mL with
Molecular weight wavelength EtOH–0.01% BHT). All the operations were per-
Compounds (g/mol) (nm) A1%1 cm* ε (mol–1/L/cm–1) formed by handling solutions in darkness and ice.
The standards of β-carotene and zeaxanthin
Retinol 286.5 325 1835 (32,61) 52573 were used to quantitate α-carotene and both
Retinol acetate 328.5 326 1550 (32,61) 50912 lutein and zeaxanthin, respectively.
α-Tocopherol 430.7 292 75.8 (45) 3265
α-Tocopherol acetate 472.8 290 40 (32) 1891
Echinenone 550.9 458 2244 123622
Blood collection and storage conditions
(Hoffmann-Laroche data source) Blood was collected at the antecubital vein of
Lutein–zeaxanthin 568.9 452 2765/2416 (45) 157301/137446 168 healthy control subjects from ages 9 to 55
β-Cryptoxanthin 552.9 452 2486 (45) 137451 years old (informed consent was obtained, and
Lycopene 536.9 472 3450 (32,61) 185231 the research protocol was in agreement with the
β-Carotene 536.9 450 2620 (35) 140667 Helsinki Declaration) in a reclined position in dry
* In EtOH as the solvent. Data references appear in the parentheses.
tubes (Vacutainer Tube, Becton Dickinson,
Grenoble, France). Blood samples were cen-

Table II. Equations of Calibration Curves and Values of LODs and LOQs*
Equations of calibration curves
Concentration Slope† Intercept Correlation LOD LOQ
range (µmol/L) (SD‡, n = 5) (SD, n = 5) coefficient† (µmol/L) (µmol/L)

Retinol 0.45–7.50 0.16 (0.015) 0.021 (0.016) 0.998 0.45 0.66

α-Tocopherol 4.80–80.0 0.01 (0.000) 0.027 (0.008) 0.996 2.64 5.36
Lutein–zeaxanthin 0.10–1.90 0.35 (0.034) 0.024 (0.006) 0.997 0.06 0.11
β-Cryptoxanthin 0.09–1.50 0.34 (0.031) 0.022 (0.019) 0.996 0.03 0.09
Lycopene 0.12–1.90 0.24 (0.018) 0.018 (0.020) 0.997 0.03 0.08
β-Carotene 0.13–2.00 0.35 (0.019) 0.014 (0.006) 0.997 0.03 0.06

* Each calibration curve included six points, and each point was assayed in five replicates.
† Calculated by internal standardization: (standard peak area/internal standard peak area)/standard concentration.
‡ SD, standard deviation.

70
Journal of Chromatographic Science, Vol. 40, February 2002
trifuged (1500 g for 15 min at 4°C) within 2 h after collection,
and resulting serum samples were frozen in liquid nitrogen until
HPLC analysis.
Serum sample treatment
All the handling operations were carried out in darkness. The
serum samples were rapidly thawed at room temperature,
homogenized, and 200 µL was transferred into a borosilicate
glass tube kept on ice and 300 µL of the internal standard mix-
ture added. After mixing with a vortex for 20 s, proteins were pre-
cipitated by adding 200 µL of EtOH–0.01% BHT, and the volume
was diluted to 1 mL with ultrapure water. After mixing with an
orbital shaker at 2500 rpm for 1 min, 2 mL of n-hexane–0.01%
BHT was added. The samples were shaken for 1 min and cen-
trifuged at 2700 g for 20 min at 4°C.
The organic layer was carefully transferred into a glass tube
and evaporated to dryness under a stream of nitrogen at room
temperature. The dried residue was redissolved in 25 µL of
THF–0.01% BHT and vortexed for 30 s. A 75-µL amount of
mobile phase was added, and the resulting mixture was vortexed
for another 30 s. Samples were then transferred to 200-µL insert
vials and placed into the HPLC autosampler.
HPLC system and operating conditions
The HPLC system consisted of an isocratic solvent delivery
pump (Model Kontron Instruments 422), an autosampler
equipped with a 20-µL injection loop, a cooling sample tray and
a column oven (Model AS-300, ThermoQuest, Les Ulis, France),
a UV–vis diode-array detector (Model Gold LC-168, Beckman
Coulter, Fullerton, CA), and data-processing software (Gold New,
Beckman).
A guard column (8- × 3-mm i.d.) packed with Nucleosil C18
(5 µm) (Macherey Nagel, Duren, Germany) and an analytical
column (250- × 3-mm i.d.) packed with Nucleosil 100 C18 (5 µm)
(Macherey Nagel) were eluted with a mobile phase consisting of
a mixture of methanol–acetonitrile–tetrahydrofuran (75:20:5,
v/v/v) containing 0.01% (w/v) BHT. The mobile phase was filtered
through a 0.45-µm Nylon membrane and was used at a column
temperature of 35°C and a flow rate of 0.6 mL/min. Three chan-
nels corresponding with different wavelength values were used
to acquire data for the selective monitoring of α-tocopherol (290
nm), retinol (325 nm), and carotenoids (450 nm) and their
respective internal standard. During analysis, the tray compart-
ment containing sample vials was cooled at 5°C. After each
working period (approximately 50 samples), it was necessary to
rinse the column with methanol at a flow rate of 0.6 mL/min for
20 min to eliminate highly hydrophobic compounds and prevent
the loss of column efficiency.
Calculation
The vitamin concentrations were determined from a standard
curve of the peak-area ratio of the analyte–internal standard
plotted against the concentration of analyte (expressed in micro-
moles per liter). A linear least-square regression analysis was
performed for each analyte, and the standard curve was repeated
if the correlation coefficient was below 0.990.
The detection limit (LOD) and the quantitation limit (LOQ)
were expressed, respectively, as:
LOD = (a0 + 3sa0) / a1 Eq. 1
and
LOQ = (a0 +10sa0) / a1 Eq. 2
where a1 is the slope, a0 the intercept, and sa0 the standard devi-
ation of the intercept (51).
Quality control
A human serum pool made with 1 mL of fresh serum from 100
healthy subjects and stored at –80°C was used for the routine
quality control. Aliquots were extracted and analyzed according
to the same procedure that was described previously. Evaluation
of the method performance was assessed by comparing the
results of the quality control with the means and relative stan-
dard deviations (RSDs) calculated using results from several pre-
liminary runs (n = 20 per day for five days).
Results and Discussion
Optimization of sample treatment and HPLC technique
The basic method used in this study has been described by
Talwar et al. (32). Some modifications relating to the internal
standards, the sample preparation procedure, and the use of an
antioxidant during both the extraction and chromatography pro-
cesses have been made. We chose this method because it allows
in a fast and easy way the simultaneous separation of the two
classes of lipophilic vitamins (namely retinol, α-tocopherol, and
carotenoids). Our main objective was to measure simultaneously
lipophilic vitamins and carotenoids, which are the most abun-
dant in human serum. Thus, the separation of the isomers of
retinol, α-tocopherol, and carotenoids did not appear relevant
for our present epidemiological studies.
In most methods, the use of an antioxidant during sample
treatment was demonstrated to be necessary to prevent a signif-
icant loss in carotenoid contents, especially lycopene and
β-carotene (32,37,39,40,47). Thus, we initially added 0.01%
ascorbic acid to the organic solvents used for the standards
preparation (EtOH and THF) and to the mobile phase, as indi-
cated by Talwar et al. (32). After analyzing the same sample
several times, we observed a decrease of the carotenoid concen-
trations, indicating degradation as a function of time. We tested
another antioxidant (BHT) that is widely used in other methods
(37,39,47) and added it to the mobile phase and all the solvents
(EtOH, THF, and hexane) used for the standard and sample
preparation. Indeed, hexane containing BHT efficiently pro-
tected the carotenoids from degradation during the evaporation
of the extractive organic layers, and the addition of BHT to the
mobile phase also prevented any loss of these analytes and prob-
ably helped increase the longevity of the column by neutralizing
peroxides present in THF. Moreover, we observed that decreasing
the evaporation temperature from 40°C to room temperature
significantly increased carotenoid recoveries, as already noted by
different authors (39,43).
Other parameters have to be optimized in order to provide the
best conditions for the extraction of liposoluble vitamins and
71

carotenoids. The addition of ultrapure water to the deproteinized
serum with EtOH has been noted to improve the recoveries of
carotenoids and liposoluble vitamins (37,52). We tested several
EtOH–water proportions in the 1:4 to 1:1 range (v/v) in order to
obtain the highest recoveries, and we selected the 1:1 (v/v) pro-
portion. Single and double extraction steps with n-hexane (an
increase of the shaking period) were tested, but no significant
improvement of recoveries was observed.
The method previously described (32) used two internal stan-
dards: retinol acetate as an internal standard for retinol, and
α-tocopherol acetate as an internal standard for both α-toco-
pherol and carotenoid. We used a third internal standard (echi-
A throughput of samples (approximately 30 per day).
Figure 1. Typical chromatograms corresponding with a mixture of retinol,
α-tocopherol, and carotenoid standards: (A) channel 1, diode-array detection
at 290 nm for α-tocopherol and α-tocopherol acetate; (B) channel 2, diode-
array detection at 325 nm for retinol and retinol acetate; and (C) channel 3,
diode-array detection at 450 nm for carotenoids and echinenone. The peak
numbers are as follows: (1) 26 µmol/L α-tocopherol, (2) α-tocopherol acetate
(the internal standard), (3) 2.43 µmol/L retinol, (4) retinol acetate (internal stan-
dard), (5) 0.62 µmol/L lutein–zeaxanthin, (6) 0.49 µmol/L β-cryptoxanthin, (7)
echinenone (internal standard), (8) 0.62 µmol/L lycopene, (9) α-carotene, and
(10) 0.65 µmol/L β-carotene.
72
Journal of Chromatographic Science, Vol. 40, February 2002
nenone) for the quantitation of the carotenoids. Echinenone is a
synthetic carotenoid and has a structure and chemical properties
very similar to the naturally occurring carotenoids in serum.
Thus, the use of echinenone is preferable to the use of retinol
acetate and α-tocopherol acetate or tocol currently used in other
methods (34,43,47), because it is detected at the same wave-
length as the other carotenoids and is light- and temperature-
sensitive as other carotenoids. Thus, the use of three internal
standards allows for a better quality control and helps to correct
analytical variations occurring for each liposoluble vitamin and
carotenoid during the extraction and chromatography pro-
cesses.
Because no loss of analytes was observed in serum extracts
kept in darkness for at least 24 h at 5°C, as already reported (39),
the automation of the technique was possible with a high
Several methods have been developed to measure the main
Figure 2. Typical chromatograms corresponding with an extract of a human
serum sample: (A) channel 1, diode-array detection at 290 nm for α-tocopherol
and α-tocopherol acetate; (B) channel 2, diode-array detection at 325 nm for
retinol and retinol acetate; and (C) channel 3, diode-array detection at 450 nm
for carotenoids and echinenone. Peak numbers are the same as Figure 1.
Journal of Chromatographic Science, Vol. 40, February 2002

carotenoids present in serum in one run simultaneously with The LOD and LOQ values agree with previous data in the liter-
α-tocopherol and retinol (30–34,37,47). Most carotenoids are ature (32). In order to calculate recoveries, a pooled serum was
detected at 450 or 473 nm, but α-tocopherol and retinol can only spiked with 20 µL of combined standards to provide the added
be detected at 290 and 325 nm, respectively. Most of the previ- concentrations of 0.7 µmol/L retinol, 8.7 µmol/L α-tocopherol,
ously mentioned methods therefore require the use of several and 0.15 to 0.2 µmol/L carotenoids. The spiked serum samples
detectors in series (43,44) and a multiwavelength detector either (n = 5) were then extracted using a single extraction step with
with simultaneous monitoring at different wavelengths n-hexane. Recoveries found were 99.6% ± 11.1% for retinol,
(31,36,37,40,53) or a change in the detection wavelength during 91.2% ± 2.0% for retinol acetate, 109.4% ± 13.4% for α-toco-
the run (30,32,44,47). The need for simultaneous detection at pherol, 101.2% ± 3.0% for α-tocopherol acetate, 112.6% ±
different wavelengths is illustrated by the retinol and 22.2% for lutein–zeaxanthin, 104.3% ± 9.1% for β-crypto-
lutein–zeaxanthin that elute within a 0.3-min interval and have xanthin, 109.4% ± 31.0% for lycopene, 85.1% ± 8.5% for
to be detected at 325 and 450 nm, respectively. Typical chro- β-carotene, and 95.6% ± 9.5% for echinenone. The different
matograms of a standard mixture and an extracted human behaviors of carotenoids with regard to extraction using
serum are shown in Figures 1 and 2. The chromatograms n-hexane has already been reported by Barua et al. (48). The cal-
revealed elution and baseline resolution between all the analytes culated recoveries in this study are satisfactory and comparable
of interest except for lutein and zeaxanthin, which are not sepa- with previously reported values (30,32,33).
rated by this method. The internal standard echinenone was In order to check the precision of the method, a human serum
eluted between β-cryptoxanthin and lycopene and thus did not pool was analyzed 20 times during the same day to assess the
interfere with the other carotenoids analyzed. Several additional repeatability. This operation was repeated 5 times over a period
carotenoids not identified as of yet appeared between the peak of of one month to evaluate the interassay precision. The intra- and
lutein–zeaxanthin at 4 min and β-cryptoxanthin at 8 min. Before interassay variations calculated for each vitamin are shown in
validation of the HPLC method, we have realized
a selectivity study, and BHT has been analyzed Table III. Precision of the HPLC Assay of Liposoluble Vitamins and
with other analytes to see any potential chro- Carotenoids in Serum
matographic interference. BHT elutes with a
Within run Between run
short retention time (within 3 min) and is only
detectable at 290 nm, thus no interference with Analyte Concentration* (µmol/L) %RSD Concentration† (µmol/L) %RSD
vitamins was observed.
Retinol 1.90 (0.06) 3.3 2.1 (0.09) 4.4
α-Tocopherol 34.9 (1.31) 3.8 29.3 (1.1) 3.8
Assay validation and quality control of the
Lutein–zeaxanthin 0.65 (0.02) 3.8 0.51 (0.02) 4.5
HPLC method β-Cryptoxanthin 0.13 (0.01) 7.8 0.10 (0.01) 13.7
The quantitation was achieved using the Lycopene 0.53 (0.05) 9.5 0.28 (0.04) 12.5
internal standardization mode. Data concerning α-Carotene 0.18 (0.02) 8.8 0.14 (0.02) 12.1
linearity (the linearity range for each liposoluble β-Carotene 0.57 (0.04) 6.7 0.52 (0.05) 9.1
vitamin was selected according to its physiolog-
* Mean (standard deviation), n = 20.
ical values), LOD, and LOQ are indicated in Table † Mean (standard deviation), n = 5.

II (and precision in Table III).

Table IV. Concentrations of Retinol, α-Tocopherol, and Carotenoids in Millimoles per Liter Measured in the Serum of 168
Healthy Subjects from Ages 9 to 55 Years Old and a Comparison with Other Studies
Present study*
Men Women Talwar Steghens Olmedilla Sowell
Compound 9–20 years old 21–55 years old 9–20 years old 21–55 years old et al.*,† (32) et al.*,‡ (37) et al.*,§ (54) et al.**,†† (31)

Retinol 1.37 (0.36) 2.18 (0.43) 1.36 (0.31) 1.86 (0.53) 2.00 (0.60) 1.84 (0.80) 1.71 (0.39) 1.91 (1.05–2.97)
α-Tocopherol 20.6 (4.08) 29.7 (8.16) 23.6 (10.9) 26.6 (6.38) 29.6 (7.60) 33.0 (6.67) 32.7 (7.40) 25.7 (13.9–47.0)
Lutein 0.42 (0.12)‡‡ 0.43 (0.24)‡‡ 0.49 (0.23)‡‡ 0.52 (0.25)‡‡ 0.26 (0.11)‡‡ 0.71 (0.30)‡‡ 0.24 (0.21)‡‡ 0.36 (0.14–0.74)‡‡
Zeaxanthin –‡‡ –‡‡ –‡‡ –‡‡ –‡‡ 0.09 (0.05) 0.07 (0.04) –‡‡
β-Cryptoxanthin 0.13 (0.08) 0.13 (0.11) 0.19 (0.14) 0.17 (0.12) 0.55 (0.11) 0.35 (0.27) 0.60 (0.47) 0.22 (0.05–0.52)
Lycopene 0.33 (0.16) 0.28 (0.16) 0.31 (0.16) 0.32 (0.22) 0.37 (0.18) 0.56 (0.43) 0.42 (0.24) 0.40 (0.11–0.80)
α-Carotene 0.08 (0.06) 0.10 (0.13) 0.13 (0.11) 0.14 (0.14) 0.07 (0.04) 0.36 (0.26) 0.07 (0.05) 0.08 (0.02–0.22)
β-Carotene 0.49 (0.43) 0.42 (0.29) 0.60 (0.37) 0.64 (0.72) 0.38 (0.20) 0.81 (0.45) 0.37 (0.23) 0.34 (0.07–0.88)

* Means (standard deviation).

† Concentrations in serum for men and women ranging from ages 19 to 62 years old, n = 111.
‡ Concentrations in serum for women ranging from ages 35 to 50 years old, n = 96.
§ Concentrations in serum for women ranging from ages 25 to 59 years old, n = 54.

** Concentrations in serum for men and women ranging from ages 4 to 93 years old, n = 3480.
†† Means (concentration range).
‡‡ Sum of lutein and zeaxanthin (peaks not separated).

73

Table III. The RSDs ranged from 3.3% (retinol) to 9.5%
(lycopene) for intra-assay precision and 3.8% (α-tocopherol) to
13.7% (β-cryptoxanthin) for interassay precision. The RSD
values obtained for some carotenoids were comparable with
those reported for most of the other assays (16,31,32). However,
the RSDs obtained for retinol and α-tocopherol were lower than
those reported in these methods. This serum pool was then used
for routine quality control.
Assay of liposoluble vitamins and carotenoids in a healthy
population
The validated method was applied to the measurement of
retinol, α-tocopherol, and five carotenoids in the serum of 168
healthy Caucasian subjects (Table IV). In comparison with previ-
ously published studies, including more than 25 subjects
(31,32,37,54), the value ranges were comparable for most of the
liposoluble vitamins and carotenoids measured except for
lutein–zeaxanthin, which was higher than the value found by
Talwar et al. (32) but similar to other studies (31,54). Similar
results were demonstrated in a previous study by De Leehneer et
al. (55). This fact can probably be explained by differences
between the populations involved in the different studies. We can
also notice that lutein–zeaxanthin and lycopene were in the
serum in significant quantities, thus β-carotene could not be
measured alone as a representative marker of the serum
carotenoids. As a matter of fact, the carotenoids exhibited dif-
ferent distributions between subjects, tissues (56,57), and food
(58). Moreover, their antioxidant capacities and functions may
differ at the cellular level (59). More recently, an HPLC system
coupling two different C18 columns has been reported for the
separation of 13 carotenoids in plasma (60), but the overall run
time for one sample reached 50 min, which limits the
throughput, and thus no important additional epidemiological
information was given.
Conclusion
The reported HPLC method devoted to the assay of liposoluble
vitamins and carotenoids in serum permits the separation of the
main carotenoids (lutein–zeaxanthin, β-cryptoxanthin, lyco-
pene, α- and β-carotene, retinol, and α-tocopherol) within 20
min, which allows a high throughput of samples. The method
was run for several months in the routine laboratory and has
clearly proven its reliability. Because of its specificity and sensi-
tivity for a great number of liposoluble vitamins corresponding
with important serum antioxidant biomarkers, this method has
an evident interest for nutritional and epidemiological studies
and is now applied to various pathological groups such as alco-
holic and Type I diabetic patients.
Acknowledgments
This project was supported in part by a grant from the
Association de la Recherche sur le Cholesterol (ARCOL). The
74
Journal of Chromatographic Science, Vol. 40, February 2002
authors gratefully acknowledge the technical staff of the Center
of Preventive Medicine for their kind participation. The authors
thank the Société Francophone des Biofacteurs et Vitamines for
giving them the opportunity to participate in an interlaboratory
quality control.
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Journal of Chromatographic Science, Vol. 40, February 2002
A Procedure for Sampling and Analysis of Air for
Energetics and Related Compounds
Michael A. Hable, Joseph B. Sutphin, Curtis G. Oliver, Robert M. McKenzie, Eleonor F. Gordon, and Richard W. Bishop
The U.S. Army Center for Health Promotion and Preventive Medicine, 5158 Blackhawk Road, Aberdeen Proving Ground, MD 21010-5403
Abstract
A procedure for the sampling and analysis of energetics and related
compounds in the atmosphere is described. The basic procedure
consists of the collection of air samples using sampling cartridges
containing XAD-2 resin, extraction of the resin with isoamyl acetate,
and an analysis of the extract using gas chromatography with
electron capture detection. Modifications and additions to this
procedure are discussed, such as the use of a prefilter before the
resin sampler to collect particulates and the use of a mass selective
detector to analyze for some propellant compounds of interest or
for quantitative confirmation purposes. Two differing sizes of
samplers are evaluated according to the air volumes required for
collection. The procedure is tested through the analysis of spiked
resin samples, which had air pulled through them for periods of
time corresponding with the required sampling volumes. This
procedure has application toward the measurement of energetic
residues in atmospheres resulting from weapons testing and
operations during training exercises involving munitions.
Introduction
The quantitative measurement of the residual amounts of ener-
getics and related compounds in the environment has been rou-
tinely performed for over three decades. There are numerous
methods used to analyze soils and waters for nitroaromatics,
nitramines, and other compounds related to U.S. munitions
(1–6). The U.S. Army has used many of these methods in the
course of environmental monitoring to protect the health and
safety of soldiers and the general population. It has also relied on
these methods to measure soil and water contamination from
explosives during environmental cleanup operations. The proce-
dures generally involve gas chromatographic (GC) and high-per-
formance liquid chromatographic (HPLC) analyses, but there are
also thin-layer chromatography and immunoassay methods that
are useful as field screening tests (7–8).
Additionally, there are methods used to monitor selected com-
pounds such as trinitrotoluene (TNT) and dinitrotoluenes in
workplace atmospheres (9–10). This monitoring is used to ensure
that munitions workers are not exposed to harmful levels of these
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
compounds. However, there has been little done toward environ-
mental air monitoring for energetics other than the specific case
of stack emissions produced during weapons destruction by
incineration. The primary impetus for stack monitoring has been
to determine destruction efficiencies associated with the pro-
cesses used to burn the munitions feedstocks. The measurement
of energetic and related compounds in the general atmosphere
from a health-risk standpoint has become an issue only in the last
few years.
The U.S. Army has recognized the need to perform air moni-
toring for energetics, partially because of public concern about
air-quality issues in areas near U.S. military reservations. There
are operations during weapons testing and training that are
potentially capable of putting measurable quantities of energetics
and related compounds into the atmosphere. As a result, the
Army Center for Health Promotion and Preventive Medicine
(USACHPPM) has determined the need to modify current air-
sampling methodologies and analytical techniques to provide
monitoring efforts for a suite of explosives compounds, including
those commonly analyzed for by soil and water methods. The list
of compounds of concern includes the nitroaromatics (such as
TNT, tetryl, and their precursors and breakdown products) and
nitramines (such as hexahydro-1,3,5-trinitro-1,3,5-triazine
(RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine
(HMX)). There are also other propellant compounds of occasional
concern, including nitroglycerin, dibutyl- and dioctyl-phthalates,
diphenylamine, and pentaerythritol tetranitrate (PETN).
There are U.S. Environmental Protection Agency (EPA) air-
sampling procedures that employ sampling devices containing
XAD-2 resin to trap polynuclear aromatic hydrocarbons from
ambient air and semivolatile organic hazardous compounds in
stack emissions (11,12). USACHPPM has successfully used modi-
fications of several types pertaining to the XAD-2 sampling trains
for the collection of stack emissions for energetic residues. We
decided therefore to investigate the use of glass cartridges packed
with XAD-2 resin for general atmospheric sampling for the ener-
getics and propellant compounds. Preliminary tests were con-
ducted using PS-1 cartridges manufactured for EPA Air Toxics
Method TO-13 for polynuclear aromatic hydrocarbons, and a field
study was successfully performed using these cartridges.
Recently, newly designed cartridges have been employed. These
77

cartridges are somewhat more robust during shipping and han-
dling than the original types and are compatible with the sam-
pling requirements of the U.S. Army during weapons testing.
These cartridges are of two types: the first being a modification of
the original PS-1 design used for high-volume sampling (Figure
1A) and the second a smaller two-section cartridge designed for
shorter test intervals (Figure 1B).
The analytical approach has generally been to use a modifica-
tion of existing USACHPPM GC procedures for the nitro-con-
taining compounds. These procedures use electron-capture
detection and have been used for many years in our laboratories
to reliably quantitate these compounds from a variety of matrices
(3,4,10,13). GC using a mass selective detector (MSD) was chosen
as the most expedient means of analyzing for the phthalate esters
and diphenylamine, because all can be done in a single GC run.
The XAD-2 resin was solvent desorbed with isoamyl acetate in
order to place the analytes into solution prior to analysis. Isoamyl
acetate has proven to be an excellent solvent for the compounds
of interest. It also provides superior response and reproducibility
with the electron-capture detector compared with other solvents
tried (such as acetonitrile). Finally, it helps to minimize chro-
matographic problems that can arise with moisture-laden sam-
ples because it is not water miscible (and thus does not retain the
water).
The sampling cartridges, the chromatographic and analytical
procedures used to analyze for the compounds of concern, and
the test results from the spike studies conducted with the sam-
plers will be described.
Experimental
Air-sampling cartridges
The initial testing was done using PS-1 sampling cartridges
packed with 55 g of XAD-2 resin. Subsequent tests with these
A B
Figure 1. Cartridge designs for XAD-2 samplers used with energetics sampling
in air: (A) 50-g cartridge with a modified PS-1 design used for high-volume
sampling and (B) 10-g two-section XAD-2 cartridge used for shorter time
sampling.
78
Journal of Chromatographic Science, Vol. 40, February 2002
samplers used 50 g rather than 55 g of the XAD-2, primarily as a
matter of convenience. These cartridges are designed such that
the glass cartridge contains a metal screen at the bottom to retain
the resin during sampling, and the resin is sandwiched above the
screen between two sections of glass wool. Design specifications
for these cartridges vary, but the basic size of the inside sampling
bed is 4 × 2.25 inches. The actual sampler and its calibration and
use has been described elsewhere (11).
The modified sampling cartridges were manufactured by Ace
Glass Inc. (Vineland, NJ). The larger size being tested still
employed 50 g of XAD-2 resin, but the resin was retained by a
metal screen/metal mesh combination at both ends of the car-
tridge, with some glass wool at the outlet end only. The glass car-
tridge body and contents were held together using Teflon end
fittings. The smaller size cartridge used two 10-g sections of XAD-
2 resin separated by glass wool. It also contained metal screens
and mesh at both ends and glass wool between the screen/mesh
and resin at the outlet end of the cartridge. The inner diameter of
the glass body was smaller, but the cartridge was similar to the
large one in its use of metal screens and Teflon end fittings (as
shown in Figure 1B). The second section of the smaller cartridge
was used as a back-up to measure breakthrough. If more than
20% of the total of an analyte was found in this section, then the
cartridge was considered to have been oversampled. Both types of
cartridges were compatible with the sampling devices used with
the original PS-1 cartridges and were used in the same fashion.
The XAD-2 resin used for packing the cartridges was a
styrene–divinylbenzene porous polymer. It was purchased from
Restek Corporation (Bellefonte, PA) under the name “Ultra Clean
XAD-2 Resin”. It was found to be sufficiently clean because it did
not require further purification for application toward energetic
sampling. It was noted, however, that its appearance varied
between different lots of the material. This did not seem to affect
the resin’s adsorbent properties, but it had other effects (as will be
described).
Recovery tests
The ability of the XAD-2 cartridges to retain the compounds of
concern while large volumes of air were passed through them was
tested. Solutions containing known amounts of the analytes in
acetonitrile were spiked into the front part of the resin within a
cartridge (in the case of a two-section cartridge the front section
was spiked). The cartridges were placed in a PS-1 sampling appa-
ratus, and clean ambient air was pulled through in the same way
as it is generally done with actual sampling in the field. The car-
tridges were then returned to the laboratory for the analysis and
evaluation of analyte retention.
Analytical procedures
The XAD-2 from sampled cartridges was transferred to 250-mL
glass bottles with Teflon-lined caps. The two section cartridges
used one bottle per section. Isoamyl acetate (Aldrich, Milwaukee,
WI) was added to the containers to desorb the analytes of interest
from the resin. The nominal amounts used were 100 mL for the
large size samples and 25 mL for the small size samples. Usually,
these amounts were sufficient to cover all the resin in a jar, but
occasionally they had to be increased to 125 mL and 40 mL,
Journal of Chromatographic Science, Vol. 40, February 2002
respectively, as a result of the excessive swelling of the resin when
placed in the solvent. The reason for this was not known but
appeared to vary with the lot of resin used to pack the cartridge.
The jars or vials were agitated for 2 h on a platform-type shaker in
order to ensure adequate resin–solvent contact, then allowed to
sit for at least 18 h in a refrigerator at 5°C. A portion of the solvent
Figure 2. Chromatogram for energetics analysis on a 7-m, 0.53-mm-i.d., 1.0-
µm film DB-1 column with electron-capture detection: (1) nitrobenzene, RT =
1.57; (2) 2-nitrotoluene, RT = 2.03; (3) 3-nitrotoluene, RT = 2.29; (4) 4-nitro-
toluene, RT = 2.41; (5) nitroglycerin, RT = 3.26; (6) 1,3-dinitrobenzene, RT =
3.97; (7) 2,6-dinitrotoluene, RT = 4.13; (8) 2,4-dinitrotoluene, RT = 4.84; (9)
3,4-dinitrotoluene, RT = 5.37; (10) 1,3,5-trinitrobenzene, RT = 6.32; (11)
2,4,6-TNT, RT = 6.88; (12) RDX, RT = 8.62; (13) 4-amino-2,6-dinitrotoluene,
RT = 11.04; (14) 2-amino-4,6-dinitrotoluene, RT = 12.14; (15) tetryl, RT =
13.80; and (16) HMX, RT = 19.56.
extract was subsequently placed in an autosampler vial prior to
analysis for energetics and propellant compounds.
Energetic stock standards were purchased as 1.0-mg/mL solu-
tions. All of them were available from AccuStandard Inc. (New
Haven, CT) except nitroglycerin, which was purchased from
Cerilliant (Austin, TX). The diphenylamine and phthalate esters
were available as neat materials from Aldrich. Working standards
in isoamyl acetate were prepared from the stock standards (or
neat materials). The energetic and nitroglycerin standards ranged
from 0.01 to 2.0 µg/mL (0.02 to 4.0 µg/mL for HMX), and the
standards for MSD analysis were from 0.5 to 5.0 µg/mL.
The chromatographic analyses for the energetics and nitroglyc-
erin were conducted using Agilent Technologies (Wilmington,
DE) Model 6890 GCs equipped with electron-capture detectors.
Chromatographic runs (shown in Figure 2) were typically made
using a DB-1 column (J&W Scientific, Folsom, CA) (0.53-mm i.d.,
1.0-µm film thickness) cut to 7 m in length. The GC oven was
temperature programmed from 80°C at 15°C/min to 140°C, then
to 170°C at 3°C/min, and finally to 200°C at 5°C/min and held for
3.0 min. The helium carrier gas was programmed from 2.0 psig
(held for 13.0 min) to 4.0 psig at a rate of 150 psig/min and held.
The dilution gas was nitrogen at 30 mL/min. The injection-port
temperature was set at 225°C, and the injection-port liner was a
Silcosleeve (Restek) with a Silcosteel seal used in splitless mode.
The Ni-63 electron capture detector temperature was 250°C. Data
processing was done using Turbochrom (PE Nelson, Shelton,
CT). An Agilent Model 7673A autosampler was used to make the
injections (the injection volume was 1.0 µL).
Analyses for the phthalate esters and diphenylamine were done
using an Agilent Technologies 5792 MSD interfaced with an
Agilent 5890 GC. The analytical column was an RTX-5ms column
(Restek) (0.25-mm i.d., 0.25-µm film thickness) that was 30 m in
length. The GC oven was temperature programmed from 80°C at
30°/min to 260°C and then held for 6.0 min. The helium carrier
gas was set to a constant pressure of 14 psi. The injection-port
temperature was set at 275°C, and the injection-port liner was a
Silcosleeve with a Silcosteel seal used in splitless mode. The
GC–MSD interface temperature was 260°C. An Agilent Model
7673 autosampler was used to make the injections (the injection
volume was 3.0 µL). The detector was scanned from m/z 45 to 300
after a 5-min solvent delay. Data processing was done using
Agilent ChemStation software. Figure 3 shows a typical chro-
matogram of the three analytes.
Results and Discussion
Sampling
All recovery tests were done using spiked cartridges. We recog-
nize that the ideal way to evaluate the cartridges would have been
to sample atmospheres containing known concentrations of the
target analytes, but unfortunately this was not an option. It would
be difficult (if not impossible) to generate stable atmospheres of a
known vapor concentration for many of the compounds. The sit-
uation was further complicated by the requirement to sample
very large air volumes (a small test chamber would be inadequate
for such testing). Fortunately, the ability of XAD-2 to trap ener-
79
 Journal of Chromatographic Science, Vol. 40, February 2002

getics has been demonstrated by actual field sampling. XAD-2 tridges are presented in Table I. These cartridges had 2.6 to 2.7 m3
resin has been successfully used by USACHPPM over the last of air pulled through them. Similar tests on the larger cartridges
dozen years to sample for some of the target compounds of this (but with 130 m3 of air) are shown in Table II.
study. It has also been used for gas sampling during the testing of
proprietary methodology used for the destruction of chemical
Table I. Tests Using a Small Cartridge Containing Two
munitions. The multisection sampling tubes used in both
10-g XAD-2 Resin Sections*
instances were of a different design than the cartridges described
in this study, but the resin was the same. Recent field-sampling %Relative
events using the cartridges described in this study have also Compound %Recovery standard deviation
shown that the resin is effective in trapping the energetics with
minimal breakthrough. We are confident that these spiking tests 2,6-Dinitrotoluene 95 18.8
provide an adequate further indication of the utility of these car- 2,4-Dinitrotoluene 93 18.1
tridges for energetic trapping and retention. 3,4-Dinitrotoluene† 100 21.9
The recovery results for seven replicate tests on the smaller car- 2,4,6-TNT 101 14.5
RDX 125 17.3
HMX 118 14.3
2-Nitrotoluene 77 16.9
3-Nitrotoluene 94 5.0
4-Nitrotoluene 95 19.3
Nitrobenzene 85 15.6
1,3-Dinitrobenzene 93 17.7
1,3,5-Trinitrobenzene 94 16.5
4-Amino-2,6-dinitrotoluene 102 15.5
2-Amino-4,6-dinitrotoluene 109 14.3
Tetryl 100 18.3
Nitroglycerin 125 17.8
Diphenylamine 91 8.3
Di-n-butylphthalate 113 8.4
Dioctylphthalate 109 8.2

* 2.6–2.7 m3 volume sampled. Seven spikes at 15 µg (energetics) or 75 µg (propellants).

† Surrogate compound. Four replicates were tested.

Table II. Tests Using a Modified PS-1 Cartridge

Containing One 50-g XAD-2 Resin Section*

%Relative
Compound %Recovery standard deviation

2,6-Dinitrotoluene 88 8.1
2,4-Dinitrotoluene 89 7.2
3,4-Dinitrotoluene† 87 6.2
2,4,6-TNT 91 6.9
RDX 101 5.2
HMX 107 17.7
2-Nitrotoluene 99 13.9
3-Nitrotoluene 103 18.8
4-Nitrotoluene 114 17.2
Nitrobenzene 89 9.1
1,3-Dinitrobenzene 87 7.4
1,3,5-Trinitrobenzene 85 7.9
4-Amino-2,6-dinitrotoluene 93 6.6
2-Amino-4,6-dinitrotoluene 103 5.1
Tetryl 96 11.2
Nitroglycerin 104 16.0
Diphenylamine 88 3.9
Di-n-butylphthalate 100 3.5
Figure 3. Total ion chromatogram for propellant analysis of 5.0 µg/mL Dioctylphthalate 106 3.3
diphenylamine and phthalate esters on a 30-m, 0.25-mm-i.d., 0.25-µm film
RTX-5MS column with mass selective detection: (1) diphenylamine, RT = * 130 m3 volume sampled. Seven spikes at 100 µg (energetics) or 500 µg (propellants).
† Surrogate compound. Six spikes were done for this compound.
5.25; (2) di-n-butylphthalate, RT = 6.35; and (3) dioctylphthalate, RT = 11.55.

80
Journal of Chromatographic Science, Vol. 40, February 2002
The recoveries were acceptable and no breakthrough was
observed in any of the tests of the small cartridges. The effective-
ness of desorbing the resin via shaking was tested by multiple
extractions of spiked resins. One hour appeared to be sufficient to
recover all the analytes, but two hours (followed by standing
overnight) is recommended to ensure for full recovery. The
shakeout procedure was much simpler than the Sohxlet proce-
dure used for desorbing XAD-2 with methylene chloride as man-
Figure 4. Chromatogram for energetics verification analysis on a 9-m, 0.53-
mm i.d., 1.0-µm film DB-210 column with electron capture detection: (1)
nitrobenzene, RT = 1.58; (2) 2-nitrotoluene, RT = 1.86; (3) 3-nitrotoluene, RT
= 2.12; (4) 4-nitrotoluene, RT = 2.25; (5) 2,6-dinitrotoluene, RT = 4.17; (6)
nitroglycerin, RT = 4.31; (7) 1,3-dinitrobenzene, RT = 4.39; (8) 2,4-dinitro-
toluene, RT = 4.89; (9) 3,4-dinitrotoluene, RT = 5.66; (10) 2,4,6-TNT, RT =
6.53; (11) 1,3,5-trinitrobenzene, RT = 6.75; (12) 4-amino-2,6-dinitrotoluene,
RT = 7.21; (13) 2-amino-4,6-dinitrotoluene, RT = 7.65; (14) RDX, RT = 7.86;
(15) tetryl, RT = 8.68; and (16) HMX, RT = 12.67.
dated in the EPA organics procedures (11,12). A Sohxlet extrac-
tion using isoamyl acetate would be difficult to conduct because
of the high boiling temperature of the solvent. Extraction using
methylene chloride is not recommended because it is a poor sol-
vent for the nitramine compounds.
Analysis
The chromatographic procedures used to analyze the resin
extracts were not complex for most of the analytes, but several
potentially required some adjustment of analytical conditions.
The nonpolar (DB-1) primary column we used for energetic anal-
ysis was capable of separating all of the analytes of interest except
PETN in a relatively short time. Most of the compounds were not
subject to interferences when used to analyze XAD-2 extracts
from ambient air samples. However, there may be occasional
background interferences with the peaks for the nitrotoluene iso-
mers or nitroglycerin depending on the lot of resin, isoamyl
acetate used, or both. When necessary, a column containing a dif-
ferent liquid phase was used to quantitate compounds that could
not be determined with the primary column. Secondary column
analysis was also routinely done in order to verify positive detec-
tions on the primary column. A polar DB-210 column (J&W
Scientific) was useful for this purpose. The temperature program
was from 80°C to 240°C, and the carrier gas (H2) was pro-
grammed from 1.5 to 9 psig. Chromatographic conditions can be
varied, but a short column is recommended if HMX verification is
required (shown in Figure 4). HMX is very reactive; a fast flow rate
and temperature program is required to get it through the polar
column before peak degradation begins to occur.
One important factor that must be considered when per-
forming GC analyses for energetic compounds is the use of a
clean, properly silanized injection-port liner. Commercially pre-
pared liners such as Silcosleeve are recommended. Peaks for the
more reactive compounds (especially HMX and the aminodinitro-
toluene isomers) will show distorted peak shapes or disappear
entirely if the liner is dirty or not silanized. On-column injections
are not recommended with this analysis because reproducibility
is not as good as with the splitless injections and column life may
be shortened.
A 30-m RTX-5ms column is recommended for the propellant
compound analysis on the GC–MSD, but a shorter column (10 m)
can be used. The only consideration with a shorter column is the
separation of the diphenylamine from the isoamyl acetate solvent
(there is not much separation between the two). If any of the later-
eluting energetic compounds (trinitrobenzene and subsequent)
(Figure 2 shows the elution order for energetics on the DB-1 and
RTX-5ms columns) are present in the samples, they may be
detected during the propellant compound scan if they are present
in high enough concentrations. The earlier compounds elute
with the solvent front and HMX is not seen. HMX possibly breaks
down either when it contacts the metal parts of the detector or is
so slowly eluted from the column that its peak flattens out com-
pletely (or both).
The reporting limit for the energetics and nitroglycerin based
on the lowest injected standard is 0.4 µg for each compound (0.8
µg for HMX) per cartridge for the small cartridge if 40 mL of the
desorbing solvent is used. It is 1.0 µg for each compound (2.0 µg
for HMX) for the larger cartridge desorbed with 100 mL. Similarly,
81

the reporting limits for the propellant compounds are 20 µg and
50 µg, respectively, based on their lowest injected standard.
The propellant compound PETN was not included during the
conduct of these tests because it was difficult to separate chro-
matographically from RDX. The two compounds coelute on both
the primary and secondary GC columns that were used during
the test analyses. Because RDX is a major component of many
U.S. high explosives, it was considered the more important com-
pound to evaluate during these cartridge studies. A subsequent
check using other chromatographic columns has shown that a
DB-1301 column (J&W Scientific) can separate the two com-
pounds. This column can be used when both RDX and PETN are
potentially present in a sample. PETN is structurally related to
nitroglycerin and thus is probably similarly collected and retained
by the XAD-2 resin. PETN spiked onto XAD-2 and desorbed with
isoamyl acetate showed good extraction efficiency, but no tests
have been conducted using spike cartridges with air pulled
through them.
The XAD-2 resin cartridge was designed to collect the analytes
of interest in the vapor state, but probably serves as a particulate
trap, also. A prefilter can be placed within the cartridge (or in a
separate housing before the cartridge) if differentiation between
the two physical forms is desired. A filter sampled separately from
the resin should be placed in desorbing solvent soon after collec-
tion, because many of the compounds of interest will evaporate or
sublimate from a filter. A filter included within a cartridge would
just transfer evaporating compounds to the adjoining resin. This
is the basis of the design of small sampling tubes containing
Tenax resin used in industrial hygiene applications involving air
sampling for TNT and other substances (9,13).
Conclusion
A sampling and analytical procedure has been devised and vali-
dated for the measurement of energetics and related compounds
in the atmosphere. The sampling cartridge is a successful modifi-
cation of other resin-containing devices used for the collection of
semivolatile organic compounds in air. GC with electron-capture
detection provides a very sensitive and reliable technique for the
quantitation of nitro-compounds collected on the sampling
media. Other compounds that may be of interest (such as the pro-
pellant components described in this study) can easily be quanti-
tated using GC–MSD.
82
Journal of Chromatographic Science, Vol. 40, February 2002
References
1. U.S. Environmental Protection Agency. Test Methods for Evaluating
Solid Waste, Physical/Chemical Methods, SW-846 Update III. Office
of Solid Waste, Washington, D.C., 1997.
2. M.E. Walsh and T. Ranney. Determination of nitroaromatic,
nitramine, and nitrate ester explosives in water using solid-phase
extraction and gas chromatography-electron capture detection:
comparison with high-performance liquid chromatography.
J. Chromatogr. Sci. 36: 406–16 (1998).
3. M. Hable, C. Stern, C. Asowata, and K. Williams. The determination
of nitroaromatics and nitramines in ground and drinking water by
wide-bore capillary gas chromatography. J. Chromatogr. Sci. 29:
131–35 (1991).
4. F. Belkin, R.W. Bishop, and M.V. Sheely. Analysis of explosives in
water by capillary gas chromatography. J. Chromatogr. Sci. 24:
532–34 (1985).
5. M.E. Walsh and T. Ranney. Determination of Nitroaromatic,
Nitramine, and Nitrate Ester Explosives in Soils Using GC-ECD.
CRREL Special Report 99-12. U.S. Army Cold Regions Research and
Engineering Laboratory, Hanover, NH, 1999.
6. T.F. Jenkins, M.E. Walsh, P.W. Schumacher, P.H. Miyares, C.F. Bauer,
and C.L. Grant. Liquid chromatographic method for the determina-
tion of extractable nitroaromatic and nitramine residues in soil.
J. AOAC 72: 890–99 (1989).
7. P.G. Thorne and K.F. Myers. Evaluation of Commercial Enzyme
Immunoassays for the Field Screening of TNT and RDX in Water.
CRREL Special Report 97-32. U.S. Army Cold Regions Research and
Engineering Laboratory, Hanover, NH, 1997.
8. S. Nam. On-Site Analysis of Explosives in Soil. Evaluation of Thin-
Layer Chromatography for Confirmation of Analyte Identity. CRREL
Special Report 97-21. U.S. Army Cold Regions Research and
Engineering Laboratory, Hanover, NH, 1997.
9. OSHA Sampling and Analytical Methods, ORG 044, 2,4-
Dintitrotoluene (DNT) and 2,4,6-Trinitrotoluene (TNT). U.S. Dept. of
Labor. Salt Lake City, UT, 1983, http://www.oshaslc.gov/dts/sltc/
methods/organic/org044/org044.html.
10. R.W. Bishop, J.L. Kennedy, G.E. Podolak, and J.L. Ryea, Jr. A field
evaluation of air sampling methods for TNT and RDX. Am. Ind. Hyg.
J. 49(12): 635–38 (1988).
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Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
Displacement Study on a Vancomycin-Based Stationary
Phase Using N-acetyl-D-Alanine as a Competing Agent
Ines Slama, Corinne Ravelet, Annick Villet, Anne Ravel, Catherine Grosset, and Eric Peyrin*
Laboratoire de Chimie Analytique, UFR de Pharmacie de Grenoble, UJF, Domaine de la Merci, 38700 La Tronche, France
Abstract
The analysis of the binding data of D,L-dansyl amino acids on a
vancomycin stationary phase is investigated in relation to the
addition of N-acetyl-D-alanine in the mobile phase. This eluent
additive acts as a specific competing agent for the aglycone pocket
of the immobilized chiral selector. A model taking into account both
stereoselective and nonstereoselective interactions between the
solutes and the stationary phase is used to fit the experimental data.
From the results, the theoretical approach is considered to be
adequate to describe the competing agent dependence on solute
retention. To the best of our knowledge, this report constitutes the
first example of a displacement study on a macrocyclic antibiotic
stationary phase. This work shows that dansyl amino acids bind to
the active aglycone pocket of the selector and that this interaction is
enantioselective. The results also demonstrate that additional
enantioselective sites at the vancomycin surface are involved in the
chiral discrimination of these solutes.
Introduction
Various experimental approaches have been proposed to ana-
lyze the mechanisms of enantioseparation on chiral stationary
phases (CSPs). For example, some studies have examined the
temperature effects on retention and enantioselectivity (1–4). The
changes in enthalpy and entropy associated with the transfer of
the solute can be extracted from linear van’t Hoff plots and ana-
lyzed in order to obtain information about the driving forces
implied in the association process. Another approach for studying
the interactions between an enantiomer and a selector involves
the variation of the mobile phase composition (5–10). Such an
investigation has been carried out for several solute–CSP associa-
tions by varying the proportion of the organic modifier (6), the pH
(7,8), or the ionic strength (9,10) of the mobile phase.
The competitive (or displacement) approaches constitute
another powerful tool to examine the retention behavior of enan-
tiomers in the chiral selective environment. Classically, it may be
carried out by injecting one compound as the solute while a fixed
* Author to whom correspondence should be addressed: email eric.peyrin@ujf-grenoble.fr.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
concentration of a possible competing agent is passed through
the column in the mobile phase. Several examples of such studies
have been reported with immobilized proteins. Wainer et al.
(11–13) have studied thoroughly the competitive displacement of
various drugs from a human serum albumin (HSA) stationary
phase by different competing agents. Hage et al. (14–16) have also
studied the effects of additives (such as digitoxin, acetyldigitoxin,
or chiral compounds) on the solute retention for immobilized
HSA. More recently, this approach has been applied with success
to the study of enantiomer binding to the cellobiohydrolase sta-
tionary phase, using cellulose as a competing compound (17).
Also, the investigation of the retention and the enantioselectivity
of a new CSP (immobilized fatty-acid-binding protein) has been
carried out for a large number of chiral compounds through dis-
placement studies (18).
In this study, the displacement concept was applied specifically
to the investigation of the enantioselective and nonselective
binding of test solutes (D,L-dansyl amino acids) on a vancomycin
stationary phase. In order to obtain information about the role of
the vancomycin aglycone pocket in the enantioselectivity process,
the solute retention factor was plotted against the concentration
of the eluent N-acetyl-D-alanine (Ala). N-acetyl-D-Ala was used as
a competing agent because it is able to bind specifically to this site
(19). Using a general model describing the competing agent con-
centration dependence on the solute binding, the retention
parameters as well as the association constant between N-acetyl-
D-Ala and vancomycin were determined. The results will be dis-
cussed in relation to the variation of enantioselectivity in order to
provide a precise picture of the enantiomer–selector association
process.
For high-performance affinity chromatography in the zonal
elution mode, the total retention factor (k) is a direct measure of
the solute interactions within the column. The parameter k is
related to the number of binding sites of the analyte to the sta-
tionary phase and the equilibrium constants for the solute at
these sites (13):
Eq. 1
where βi is the equilibrium constants at the individual sites (i), ji
83

the fraction of each type of site, m the total moles of binding sites,
Vm the void volume, and ki the respective contributions to the
total retention factor.
The binding of a chiral compound to a CSP can involve two
kinds of sites at the surface of the selector: one class of nonselec-
tive binding sites with lower affinity and another class of enan-
tioselective sites with higher affinity (17). Thus, k can be
simplified as follows:
Eq. 2
where the two terms are the sums of the retention factors corre-
sponding with selective (s) and nonselective (ns) interactions,
respectively. For a pair of enantiomers, it is expected that the kns
terms are identical while the ks contributions differ in relation to
the stereoselectivity.
Williams et al. (18,19) have previously shown by nuclear mag-
netic resonance and modeling studies that a ligand such as
N-acetyl-D-Ala is specifically bound in a 1:1 stoichiometry to the
pocket of the aglycone of vancomycin (the structure of van-
comycin is shown in Figure 1) via hydrophobic interactions and
hydrogen bonds. This is explained by the fact that this antibiotic
acts on bacteria by binding to cell wall mucopeptide precursors
terminating in D-Ala. If N-acetyl-D-Ala is used as a mobile phase
additive, it is expected that it will interfere on the retention of
solutes interacting with the specific aglycone pocket. Thus,
assuming that the pocket of the vancomycin aglycone constitutes
one of the enantioselective sites of vancomycin for dansyl amino
acid, equation 2 can be modified as follows:
Eq. 3
where kls is the part of the retention factor implying the solute
enantioselective binding to the aglycone pocket, kns the part of
Figure 1. Vancomycin structure with the aglycone binding pocket (indicated
by the arrow) (19).
84
Journal of Chromatographic Science, Vol. 40, February 2002
the retention factor involved in the nonspecific binding, and ks
the part of the retention factor corresponding with enantioselec-
tive interaction unaffected by the competing agent. K is the asso-
ciation constant between N-acetyl-D-Ala and vancomycin and c
the N-acetyl-D-Ala (competing agent) concentration. This simpli-
fied model allows for a simple estimate of the role of the aglycone
pocket on the enantioseparation of dansyl amino acids on immo-
bilized vancomycin. Also, it constitutes a valuable tool to explore
the exact contributions of enantioselective interactions in the
overall retention process of these solutes.
Experimental
Apparatus
The HPLC system consisted of an LC 10AT Shimadzu pump
(Touzart et Matignon, Courtaboeuf, France), a Rheodyne Model
7125 injection valve (Interchim, Montluçon, France) fitted with a
20-µL sample loop, and a Shimadzu SPD-10A UV–vis detector. An
Astec 150- × 4.6-mm Chirobiotic V HPLC column (packed with a
stationary phase produced by the chemical bonding of the macro-
cyclic glycopeptide vancomycin to a 5-µm silica gel) was used
with controlled temperature in an Igloocil oven (Interchim). The
mobile phase flow rate was 0.8 mL/min.
Reagents and operating conditions
D,L-amino acids were obtained from Sigma Aldrich (Saint-
Quentin, France). Methanol (HPLC grade), trisodium citrate, and
citric acid were supplied by Prolabo (Paris, France). Water was
obtained from an Elgastat option water purification system (Odil,
Talant, France) fitted with a reverse osmosis cartridge. The
column temperature was maintained at 25°C for all the experi-
ments. The mobile phase consisted in citrate buffer (pH
7.0)–methanol (90:10, v/v). The variation range of the N-acetyl-D-
Ala concentration was 0 to 20mM. In order to examine the con-
centration dependencies of the solute retention corresponding
with the binding capacity of the immobilized vancomycin, reten-
tion measurements were related to varying amounts of injected
solute. Solute samples were prepared at different concentrations
in the mobile phase from 0.125 to 10 µg/mL. The retention factor
versus the sample amount plots exhibited a plateau at a sample
concentration lower than 0.625 µg/mL, followed by a small
decrease at higher solute concentrations. Thus, 20 µL of each
solute at a concentration of 0.250 µg/mL was injected in triplicate
(i.e., in linear conditions) (20).
Results and Discussion
The retention factor values for D,L-dansyl amino acids on
immobilized vancomycin were determined in relation to the con-
centration of N-acetyl-D-Ala in the mobile phase (0 to 20mM). The
coefficients of variation for the k values were < 0.5%, indicating a
high reproducibility and a good stability for the chromatographic
system. The k values were plotted against c for all the compounds.
Figure 2 shows the k versus c plots of D,L-dansyl amino acids. In
Journal of Chromatographic Science, Vol. 40, February 2002

all cases, the retention factors decreased when the competing
agent concentration increased. Equation 3 was fitted to the exper-
imental data using a nonlinear regression procedure. The values
of the various parameters of equation 3 are shown in Table I. The
nonlinear regression coefficients (R) were higher than 0.987.
Thus, it appears clearly that the behavior of the solutes was well-
described by the model taking into account the competition
between N-acetyl-D-Ala and dansyl amino acids at the aglycone
pocket of vancomycin. For all the dansyl amino acid enantiomers,
the association constant between the competing agent and van-
comycin varied between 820 and 1300 M–1. This demonstrated
that all the solutes studied (D- and L-enantiomers) interacted with
the aglycone pocket. The K average value was similar to the asso-
ciation constant value reported for the N-acetyl-D-Ala binding to
the macrocycle binding pocket (i.e., 1300 M–1 at 23°C) (18). From
the magnitude of the retention factor kls, approximately 45–50%
of the total binding observed for these compounds was dependent
on the association with the aglycone pocket. The L-enantiomers
exhibited a value of approximately 45%, and the D-enantiomers
were bound at approximately 50% to this active site. This demon-
strates that the aglycone pocket is implied in the chiral discrimi-
nation. Moreover, the apparent enantioselectivity (kD /kL )
decreased when c increased (as shown in Figure 3). However,
when the aglycone site was saturated by N-acetyl-D-Ala at a high
displacer concentration (as shown in Figure 1, in which a roughly
constant value of the solute retention factor is observed for the
concentration range between 10 and 20mM), a substantial enan-
tioselectivity remained for all the enantiomeric pairs. This
suggests that other enantioselective sites are involved in
the chiral discrimination of dansyl amino acids. The enantiose-
lectivity values from the solute binding to the aglycone pocket
(αp= klsD / klsL) as well as the “residual” enantioselectivity values
(αr =knsD+ksD/knsL+ksL) were calculated (Table II). Such an obser-
vation is unusual in comparison with the results of other dis-
placement studies carried out on protein CSPs. Using
immobilized protein, only one enantioselective site is generally
involved in the chiral discrimination process (11–17). This orig-
inal behavior, observed for the chiral recognition on a van-
comycin stationary phase, can be explained by the fact that this
type of macrocycle contains several accessible chiral interaction
sites. Moreover, Berthod et al. (21) have previously shown that
carbohydrate moieties of the teicoplanin stationary phase offer
enantioselective binding sites for various enantiomers. Thus, it is
quite possible for dansyl amino acid enantiomers to interact with
some chiral environments of the selector in addition to the active
aglycone site. For the four pairs of dansyl amino acid enan-
tiomers, a significant difference was observed for the αp values
(from 1.26 to 1.48). This demonstrates that the aglycone pocket is

Figure 2. Plot of k versus c for D,L-dansyl amino acids: dansyl valines D (+) and
▲ ), dansyl serines D (●) and L (●
L (▲ ●), dansyl leucines D (■) and L (■ ■ ), and Figure 3. Plot of the apparent enantioselectivity (αapp) versus c for all enan-
dansyl phenylalanines D (◆) and L (◆ ◆). tiomeric pairs: D,L-dansyl valine (◆); D,L-dansyl serine (+); D,L-dansyl leucine
(■); and D,L-dansyl phenylalanine (●).

Table I. Determination of the Model Parameters by

Fitting Equation 3 to the D,L-Amino Acid Enantiomer Table II. Determination of the Enantioselectivity Values
Retention Factors on Immobilized Vancomycin for All the Enantiomer Pairs

k ns + k s k ls K (M –1) α app* α p† α r‡
(kD/kL) (klsD/klsL) (knsD + ksD/knsL + ksL)
D-dansyl valine 1.51 1.58 820
L-dansyl valine 1.32 1.07 1118 Dansyl valine 1.26 1.47 1.14
D-dansyl serine 0.99 1.01 882 Dansyl serine 1.29 1.48 1.09
L-dansyl serine 0.91 0.68 1249 Dansyl leucine 1.18 1.26 1.11
D-dansyl leucine 1.49 1.62 918 Dansyl phenylalanine 1.18 1.28 1.08
L-dansyl leucine 1.45 1.18 1144
* The apparent enantioselectivity.
D-dansyl phenylalanine 2.49 2.58 1080 † The enantioselectivity resulting from the compound binding to the aglycone pocket.
L-dansyl phenylalanine 2.29 2.00 1305 ‡ The residual enantioselectivity.

85

responsible for different enantioselective interactions in relation
to the structure of the compounds. Two solute groups can be dis-
tinguished: (a) D,L-dansyl leucine and dansyl phenylalanine with
a lower αp and (b) D,L-dansyl valine and dansyl serine with a
higher αp (Table II). Such a behavior can be explained by a steric
hindrance phenomenon. The bulky dansyl amino acid (the first
solute group mentioned) could limit access to the aglycone
binding pocket, and thus the chiral recognition would be
reduced. This is confirmed by the fact that D,L-dansyl tryptophan
enantiomers (the most bulky of the dansyl amino acids) were not
separated on this CSP (data not shown). Also, this result agrees
well with the findings of the enantioseparation of amino acid
derivatives on a teicoplanin stationary phase (21,22).
Conclusion
This work demonstrates the interest to use N-acetyl-D-Ala as a
competing agent for the aglycone pocket of the macrocyclic
antibiotic stationary phase. From the results, it is shown that the
dansyl amino acids interact substantially with this active site
(nearly 50% of the k value) of the vancomycin stationary phase.
Furthermore, it is demonstrated that the aglycone pocket is an
effective enantioselective site, but another class of sites is also
involved in the chiral discrimination. This type of displacement
study could be applied similarly to the investigation of the relative
contribution of enantioselective and nonselective interactions for
the solute binding on other types of commercially available
macrocycles (such as teicoplanin or ristocetin A) that contain a
similar aglycone pocket.
References
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Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
Influence of Hydrolysis, Purification, and Calibration
Method on Furosine Determination Using
Ion-Pair Reversed-Phase High-Performance
Liquid Chromatography
M.A. Serrano, G. Castillo, M.M. Muñoz, and A. Hernández*
Departamento de Nutrición y Bromatología, Campus Universitario, Universidad de Alcalá, 28871-Alcalá de Henares (Madrid), Spain
Abstract
The influence of HCl concentration (6M, 8M, and 10M) and the
ratio of sample protein to acid (1 or 5 mg of protein per mL of
acid) on furosine formation during sample hydrolysis is studied.
The conditions that maximize furosine formation are 10M HCl in
the ratio of 1 mg of protein to 1 mL of acid. Purification of the
hydrolysate by solid-phase extraction is also considered by
examining the effect of hydrolysate volume and volume of 3M HCl
used to elute the furosine. Furosine quantitation is carried out
using the standard additions and external standard methods. The
results indicate that there is no interference by the sample matrix
and that external calibration is adequate.
Introduction
Furosine (ε-N-(2-furoylmethyl)-L-lysine) is an amino acid
formed during the acid hydrolysis of such Amadori products as
fructoselysine, lactuloselysine, and maltuloselysine, which are
generated in the early stages of the Maillard reaction during the
heat processing of foods (1). For that reason, estimates of the
extent of protein damage caused by heating in the first stages
of that reaction are often based on determinations of the
amount of furosine that forms during the acid hydrolysis of
foods.
Furosine determinations may be carried out by ion-exchange
chromatography (IEC) (2–4), gas chromatography (5), and
ion-pair reversed-phase high-performance liquid chromatog-
raphy (RP-HPLC) (6–12). Recently, capillary electrophoresis
(CE) methods of furosine determination have also been devel-
oped (13–15).
The first step in furosine analysis is hydrolysis of the sample
proteins. Although optimization of the hydrolysis step has
* Author to whom correspondence should be addressed: email amelia.hernandez@uah.es.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
been considered in previous research (2,3,8–12,16), Resmini et
al. (8) pointed out that more research on the effect of acid
hydrolysis on furosine formation was needed. Additional issues
associated with the assay include whether hydrolysate cleanup
by solid-phase extraction improves assay performance and
what calibration strategy produces optimum performance.
As a consequence of the foregoing, the object of this study
was to examine the influence of HCl concentration and ratio on
furosine formation and to establish the most suitable condi-
tions for hydrolysate purification and furosine quantitation
based on the chromatographic conditions developed by Del-
gado et al. (9).
Experimental
Chemicals and reagents
The furosine standard with a purity of approximately 70%
was obtained from Neosystem (Strasbourg, France). HPLC-
grade acetonitrile was from Scharlau (Barcelona, Spain). The
other analytical reagent-grade chemicals were from Merck
(Darmstadt, Germany). Water was quartz-distilled and deion-
ized using the Milli-Q system (Millipore, Bedford, MA).
Equipment
The HPLC apparatus consisted of a Model 110B pump and a
Model 210A injector from Beckman (Berkeley, CA) equipped
with a 20-µL loop and a KNK-029.757 UV–vis detector (Konik
Instruments S.A., Barcelona, Spain). Peak areas were deter-
mined with the aid of an SP-4290 recorder–integrator (Spectra-
Physics, San Jose, CA).
Samples
The trials were performed using two samples (A and B) of
a powdered enteral formula and two samples (A and B) of
87

powdered whole milk. Caseinate was the protein source for the
enteral formula, and the protein content was 20%. The protein
content of the powdered whole milk was 25%.
Sample hydrolysis
In order to determine the influence of HCl concentration
and ratio on furosine formation during hydrolysis, three HCl
concentrations (i.e., 6M, 8M, and 10M) and two ratios of sample
protein to acid volume (i.e., 1 and 5 mg of protein per mL of
acid) were tested. A quantity of sample accurately weighed was
poured into a 250-mL Pyrex screw-cap flask, and an appropriate
quantity of acid was added. Hydrolysis was carried out in a
nitrogen atmosphere at 110ºC for 24 h. After the hydrolysate
had cooled to room temperature, it was filtered through No. 52
Whatman paper (Whatman, Maidstone, U.K.), and the screw-
cap flask was washed out with Milli-Q water. All the liquids were
collected in a volumetric flask that was then topped off with
Milli-Q water.
For the sample protein-to-acid ratio trials using 1 mg of
protein per mL of acid, approximately 0.20 g of the powdered
milk or 0.25 g of the enteral formula was weighed out and
hydrolyzed with 50 mL of acid, and the volume was diluted to
100 mL. For the sample protein-to-acid ratio trials using
5 mg of protein per mL of acid, approximately 0.40 g of the
powdered milk or 0.50 g of the enteral formula was weighed
and hydrolyzed with 20 mL of acid, and the volume was diluted
to 50 mL.
Hydrolysate purification
The different sample hydrolysates were purified using a
Sep-Pak C18 cartridge (WAT020515, Waters, Milford, MA). The
cartridges were prewetted with 5 mL of methanol and 10 mL
of Milli-Q water before use.
Different trials were performed to ascertain the optimal con-
ditions for purification. Aliquots of 0.5 and 1 mL of filtered
hydrolysate of an enteral formula were gradually loaded onto
the cartridge, and the displaced liquid was collected in an evap-
oration flask, being careful not to allow air to enter the car-
tridge. Elution of the furosine on the cartridge was then carried
out using 3 or 5 mL of 3M HCl, the eluate being collected in
the same flask. The solution thus obtained was evaporated to
dryness in a rotary evaporator at 30ºC. The dry residue was
reconstituted with acetonitrile–Milli-Q water–formic acid
(20:79.8:0.2). Other aliquots of the same filtered hydrolysate
were injected onto the chromatograph without undergoing
purification.
Other parallel trials were performed using 0.5 mL filtered
hydrolysate of the enteral formula and the powdered whole
milk in the same conditions to corroborate the effect of col-
lecting or discarding the displaced liquid when running the
hydrolysate through the cartridge.
Quantitative analysis
Quantitation was performed using the external standard
method. A stock solution with approximately 140 µg/mL of
pure furosine standard was prepared by dissolving the total
amount of a commercial vial in 0.1M HCl. This stock solution
was stored under refrigeration at 4ºC.
88
Journal of Chromatographic Science, Vol. 40, February 2002
An appropriate aliquot of the stock solution was evaporated
to dryness in a rotary evaporator, and the dry residue was
reconstituted with an appropriate volume of acetonitrile–Milli-
Q water–formic acid (20:79.8:0.2). Using that same solvent,
eight standard dilutions ranging in concentration from 1 to 8
µg/mL were prepared from the solution that was obtained. A
calibration curve was obtained by plotting the peak areas versus
the micrograms per milliliter of furosine injected.
Calibration by the standard additions method was also tested.
For this purpose, five solutions of each sample (powdered
enteral formula and powdered milk) were prepared by taking a
uniform quantity of hydrolysate and increasing quantities of
furosine standard (ranging from 1 to 5 µg/mL). Thus, two
standard additions curves were obtained.
All standard solutions and samples were injected twice.
Chromatographic conditions
Furosine was determined by ion-pair RP-HPLC according to
the method of Delgado et al. (9). Separations were carried
out on a Spherisorb ODS2 5-µm column (250- × 4.6-mm i.d.)
(Phenomenex, Torrance, CA) thermostatted at 30ºC. The
mobile phase was 5mM sodium heptane sulfonate with 20%
acetonitrile and 0.2% formic acid at a flow rate of 1.2 mL/min.
Detection was carried out at 280 nm.
Results and Discussion
The chromatographic method of Delgado et al. (9) was
selected because it was an isocratic method in which furosine
was eluted after a short retention time, thereby reducing
the analysis time. In view of the discrepancies concerning
hydrolysate purification contained in the literature, the first
question addressed was whether or not a purification stage
was necessary and what the optimum purification conditions
were.
Figure 1 presents the chromatograms for an unpurified and
purified hydrolysate of an enteral formula. The furosine eluted
after a retention time of 10 min. On the chromatogram for the
unpurified hydrolysate, a series of small spikes can be observed
along the entire baseline, with two spikes being located quite
close to the furosine peak. The chromatogram for the purified
hydrolysate presented a more stable baseline and a smaller
number of peaks, which translates into better separation and
integration. Furthermore, the lifetime of the chromatographic
column was extended by purification of the hydrolysates.
Two trials were run to test the purification conditions. The
first trial was carried out on a hydrolyzed enteral formula.
Two different volumes of hydrolysate (0.5 and 1 mL) were puri-
fied, with the displaced liquid collected in both cases. In addi-
tion, two different volumes (3 and 5 mL) of 3M HCl for furosine
elution were tested. Other aliquots of hydrolysate were injected
without purification of any kind. Four replications were per-
formed for each set of conditions.
The results are presented in Table I. There was no significant
difference in the furosine values obtained under either set of
conditions when 0.5 or 1 mL of the hydrolysate was run
Journal of Chromatographic Science, Vol. 40, February 2002

through the cartridge. Thus, the step of

solid-phase extraction can be used not
only for purification of the hydrolysate
but also to increase the amount of furo-
sine in the dry residue obtained, provided
that the displaced liquid is also collected.
This advantage may be quite useful when
analyzing samples containing smaller
quantities of furosine, such as pasteur-
ized milk.
In addition, the amount of furosine
obtained on elution with 3 mL of HCl was
9–12% lower than the amount obtained in
the unpurified hydrolysate injected
directly onto the column. However, elu-
tion carried out using 5 mL of HCl yielded
the same amount of furosine as in the
unpurified hydrolysates. Thus, collecting
the displaced liquid when running the
hydrolysate through the cartridge and
eluting with 5 mL of 3M HCl implies a
furosine recovery of 100% and makes it
unnecessary to use a correction factor.
Another trial was carried out to con-
Figure 1. Chromatograms for an (A) unpurified and (B) purified hydrolysate of an enteral formula.
firm the previously mentioned results and
to try to elucidate the effect of discarding
or collecting the displaced liquid when
Table I. Effect of Hydrolysate Volume Purified and 3M HCl Volume Used as the
running the sample through the car- Elution Solvent on the Furosine* Determination in a Powdered Enteral Formula†
tridge. In this trial samples of both an
enteral formula and powdered milk were Volume of hydrolysate run through the Sep-Pak cartridge
used. Two hydrolysates were obtained
from each sample. Parallel trials were per- Enteral formula A 0.5 mL 1 mL
formed using 0.5 mL of each hydrolysate,
Unpurified hydrolysate 66.21 ± 2.48 66.70 ± 0.71
discarding or collecting the displaced Purified hydrolysate
liquid from the cartridge, and eluting the Elution with 3 mL 3M HCl 58.22 ± 0.78 60.73 ± 0.95
furosine with 3 or 5 mL of 3M HCl. Table Elution with 5 mL 3M HCl 66.29 ± 0.71 66.68 ± 0.01
II summarizes the results, which shows
that the furosine values in all cases were * Milligrams per 100 g of product. Values are the means of four replications ± standard deviation.
† Hydrolysate prepared using 6M HCl at a ratio of 5 mg protein to 1 mL HCl.
higher when the displaced liquid was not
discarded than when it was discarded,
though the difference was small (approx-
imately 1% to 3%) and only statistically Table II. Effect of Collecting or Discarding the Liquid Displaced from the
significant for the enteral formula. The Cartridge by Hydrolysate and the 3M HCl Volume Used as the Elution
Solvent on the Furosine* Determination in a Powdered Milk† and a Powdered
values were also higher when the furo-
Enteral Formula†
sine was eluted using 5 mL of acid instead
of 3 mL, but in this case the difference Displaced hydrolysate
was higher (approximately 9%) and sta-
tistically significant for both samples. Use Sample Discarded Collected
of a larger volume of HCl for elution in
Powdered milk B
laboratory tests confirmed that the same Elution with 3 mL 3M HCl 120.83 ± 6.00 124.05 ± 2.34
results were obtained. Elution with 5 mL 3M HCl 131.94 ± 2.87 132.86 ± 2.55
It can therefore be concluded that, in
the conditions of the experiment, the Powdered enteral formula B
effect of using a larger volume of HCl to Elution with 3 mL 3M HCl 128.25 ± 4.56 133.12 ± 4.41
elute the furosine was greater than col- Elution with 5 mL 3M HCl 139.49 ± 4.99 143.53 ± 6.91
lecting or discarding the displaced liquid.
* Milligrams per 100 g of product. Values are the means of four replications ± standard deviation.
Nevertheless, if a larger volume of † Hydrolysate prepared using 10M HCl at a ratio of 1 mg of protein to 1 mL HCl.

hydrolysate were to undergo purification,

89
 Journal of Chromatographic Science, Vol. 40, February 2002

the error produced by discarding the displaced liquid could
become considerable. The rest of the trials were performed in
the optimum conditions described previously.
Conflicting information has been reported in terms of the
most appropriate calibration method to use with both the use
of the external standards (8,17) and standard additions (9,12)
mentioned. Because calibration by means of the standard addi-
tions method makes analysis both longer and more compli-
cated because a separate calibration curve is required for every
sample with a different composition, the calibration condi-
tions were studied. Two calibration curves (one for the pow-
dered milk and another for the enteral formula) were obtained
by the standard additions method by adding increasing quan-
tities from 1 to 5 µg/mL of the furosine standard to the corre-
sponding previously hydrolyzed samples. Two other external
standard calibration curves were also obtained.
Table III summarizes the results of the calibration study.
The slopes of the two calibration curves, the external stan-
dard calibration curve, and the standard additions calibration
curve were quite similar. A statistical comparison of the slopes
of the two curves found no statistically significant differences
between them (P < 0.05). Therefore, it can be concluded that
there were no effects attributable to the hydrolysate matrix of
either of the samples tested, even though they differed con-
siderably in composition.
The furosine concentration in the samples was also calcu-
lated using the two calibration curves, and the furosine
recovery (%) was calculated by the ratio between furosine
determined with standard additions and external standard
curve equations (Table III). The recovery was 100%, which
again confirmed that there were no differences attributable to
the sample matrix.
One of the drawbacks to the standard additions method is
that sample concentration is calculated by extrapolation rather
than by interpolation while it is in the external standard cali-
bration method. For this reason, the accuracy of the external
standard method was also evaluated by calculating the recovery
of furosine as the known (increasing quantities of the standard
were added to the samples). The recoveries are shown in Table
IV. Mean recovery values for the two samples were nearly 100%,
with coefficients of variation lower than 0.2%. Thus, calcula-
tion error resulting from extrapolation would appear to be
minimal, and calibration by the external standard method is
adequate.

Table III. Calibration Curve Equations and Furosine Recovery for a Powdered Enteral Formula and a Powdered Milk

External standard calibration Standard additions calibration

Furosine injected Furosine injected Recovery
Sample Curve equation (µg/mL) Curve equation (µg/mL) (%)

Enteral formula A y = 71526.1x – 2547.1 3.39 ± 0.01 y = 70428.9x + 239409 3.40 100.2
s.e.* = 1454 s.e. = 941
r 2† = 0.9999 r 2 = 0.9998

Powdered milk A y = 71481.4x – 2100.8 2.10 ± 0.01 y = 70304.2x + 147529 2.09 99.7
s.e. = 2157 s.e. = 1210
r 2 = 0.9999 r 2 = 0.9999

* s.e., standard error of estimation.

† Determination coefficient.

Table IV. Percentage Furosine Recovery for a Powdered Table V. Effect of HCl Concentration and Ratio During
Enteral Formula and a Powdered Milk Hydrolysis on Furosine* Formation in a Powdered
Enteral Formula and a Powdered Milk
Furosine (µg/mL)
Recovery
Hydrolysis conditions Enteral formula A† Powdered milk A†
Sample Initial Added Recovered (%)
1 mg protein to 1 mL acid
Enteral formula A 3.39 0.95 4.30 99.08
10M HCl 546.22 ± 4.39 (100) 322.95 ± 0.95 (100)
1.42 4.78 99.38
8M HCl 460.45 ± 2.41 (84.3) 289.37 ± 4.55 (89.6)
1.89 5.25 99.43
6M HCl 332.66 ± 1.24 (60.9) 195.63 ± 0.43 (60.6)
2.37 5.72 99.31
Mean value 99.30 ± 0.15 5 mg protein to 1 mL acid
10M HCl 493.59 ± 4.40 (100) 306.39 ± 0.98 (100)
Powdered milk A 2.10 0.95 3.02 99.02 8M HCl 428.14 ± 5.56 (86.7) 268.89 ± 5.53 (87.8)
1.89 3.94 98.75 6M HCl 320.26 ± 2.56 (64.9) 161.66 ± 1.47 (52.8)
2.84 4.89 98.99 * Milligrams per 100 g protein. Values are the means of three replications ±
3.79 5.84 99.15 standard deviation.
† Difference in percentage furosine formation with respect to the values obtained
Mean value 98.98 ± 0.17 using 10M HCl in brackets.

90
Journal of Chromatographic Science, Vol. 40, February 2002
The effect of different hydrolysis conditions on the amount
of furosine formed was tested under optimal purification con-
ditions using external standard calibration. Three acid con-
centrations (6M, 8M, and 10M) and two quantities of protein
per mL of acid (1 mg of dilute hydrolysis and 5 mg of concen-
trated hydrolysis) were tested. Samples of the enteral formula
and the powdered milk were used, and the results were
expressed as milligrams of furosine per 100 g of protein in the
sample. The results of these trials appear in Table V.
The amount of furosine formed increased with increasing
acid concentration in both the dilute and concentrated hydrol-
ysis of both samples. Furosine formation was thus highest for
the 10M HCl. The difference in the level of furosine formation
was greater between the 6M and 8M acid concentrations than
between the 8M and 10M acid concentrations for both the
milk and the enteral formula.
The protein-to-acid ratio during hydrolysis also affects furo-
sine formation. Furosine formation was in all cases higher for
the ratio of 1 mg of protein to 1 mL of acid (in other words, for
dilute hydrolysis). Nevertheless, the influence of HCl concen-
tration was much greater than that of the sample protein-to-
acid ratio during hydrolysis. The dilute ratio has customarily
been recommended for the analysis of total amino acids (16).
The increase in furosine formation according to HCl acid
concentration is in agreement with the results obtained for dif-
ferent foods by other researchers (2,3,11,12,17).
No information concerning the influence of the protein-to-
acid ratio on furosine formation was found in the literature.
Based on the experimental results, it can be concluded that
optimum hydrolysis conditions to maximize furosine forma-
tion are 10M HCl in the ratio of 1 mg of protein to 1 mL of acid.
Because furosine is formed from the Amadori products during
the hydrolysis of foodstuffs, its concentration was used to eval-
uate thermal damage sustained by foods during processing, and
it will therefore in all cases be appropriate to try to maximize
furosine formation to ensure the maximum correctness of the
evaluation. This is even more important in samples that
undergo milder heat processing.
In liquid foods the concentration of the HCl added to the
samples should be higher, so that the final acid concentration
during hydrolysis will be 10M. Although the concentration of
commercial HCl acid concentrate is approximately 11.9M, con-
centrations higher than 10M were not tested in order to ensure
comparability of the results for the solid and liquid samples.
Acknowledgments
This study was supported by a grant awarded by the
Comisión Interministerial de Ciencia y Tecnología (Project
ALI97-0606-C02-02) of the Spanish Ministry of Education and
Science.
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1. H. Erbersdobler and H. Zucker. Untersuchungen zum gehalt an
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3. I. Molnár-Perl, M. Pintér-Szakács, R. Wittmann, M. Reutter, and
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4. J. Hartkopf and H. Erbersdobler. Stability of furosine during ion-
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7. G.H. Chiang. A simple and rapid high-performance liquid chro-
matographic procedure for determination of furosine, lysine-
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of furosine in milk and dairy products by a direct HPLC method.
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9. T. Delgado, N. Corzo, G. Santa-Maria, M. L. Jimeno, and
A. Olano. Determination of furosine in milk samples by ion-pair
reversed phase liquid chromatography. Chromatographia 33:
374–76 (1992).
10. T. Maroni and P. Lazzari. Determinazione di furosina in HPLC con
metodo isocratico. Ind. Aliment. 33: 964–66 (1994).
11. T. Henle, G. Zehetner, and H. Klostermeyer. Fast and sensitive
determination of furosine. Z. Lebensm. Unters. Forsch. 200:
235–37 (1995).
12. E. Guerra-Hernández and N. Corzo. Furosine determination in
baby cereals by ion-pair reversed-phase liquid chromatography.
Cereal Chem. 73: 729–31 (1996).
13. A. Tirelli and L. Pellegrino. Determination of furosine in dairy
products by capillary zone electrophoresis. A comparison with the
HPLC method. Ital. J. Food Sci. 4: 379–85 (1995).
14. L. Del Giovine and A. Bocca. Elettroforesi capillare applicata
all’analisi della furosina nel latte. Riv. Sci. Aliment. 3: 247–52 (1996).
15. A. Tirelli. Improved method for the determination of furosine in food
by capillary electrophoresis. J. Food Prot. 61: 1400–1404 (1998).
16. J.W. Finley. Digestibility and Amino Acid Availability in Cereals
and Oilseeds. J.W. Finley and D. Hopkins, Eds. Am. Assoc. Cereal
Chem., St Paul, MN, 1985, pp. 15–30.
17. F. Evangelisti, C. Calcagno, S. Nardi, and P. Zunin. Deterioration
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Manuscript accepted November 26, 2001.
91

The Use of Nonendcapped C18 Columns in the Cleanup
of Clenbuterol and a New Adrenergic Agonist from
Bovine Liver by Gas Chromatography–Tandem
Mass Spectrometry Analysis
Maurizio Fiori, Claudia Cartoni, Beatrice Bocca, and Gianfranco Brambilla*
Istituto Superiore di Sanità—Laboratorio di Medicina Veterinaria, viale Regina Elena 299, I-00161 Rome, Italy
Abstract
More specific official methodology is needed to survey the illegal
use of clenbuterol in animal production plus the synthesis of new
compounds that currently elude routine analytical methods. The
identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-
dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide
(known as compound A) in animal feed has prompted studies to
verify if the existing cleanup procedures developed for clenbuterol
are really effective. This study considers the ion-exchange
mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX),
mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE)
solid-phase extraction (SPE) columns. Results indicate that
compound A (by contrast with clenbuterol) is not efficiently
retained on the CN, SCX, and MPH SPE columns (recovery
< 10%). This finding thus leads to the development of a gas
chromatography–tandem mass spectrometry procedure based on
C18NE SPE that is able to purify both agonists from bovine livers
spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for
clenbuterol and 92% for compound A.
Introduction
The development of a multiresidue analytical strategy to
survey the abuse of adrenergic agonist drugs used as growth
promoters in animal production is mainly based on the selec-
tivity of the cleanup procedures and the specificity of the detec-
tion systems (1,2). Such requirements have been fundamental
steps for the analytical toxicology investigations related to
human poisonings described after the ingestion of clenbuterol-
contaminated bovine liver and meat (3,4). Most of the analytical
procedures reported as effective include an ion-exchange step in
* Author to whom correspondence should be addressed.
92 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of Chromatographic Science, Vol. 40, February 2002
the multiresidue cleanup of beta agonists. This procedure
involves the interaction between the secondary amino group
shared among all beta agonists and the cyanopropyl (CN) (5)
and sulfonic cation exchange (SCX) (6,7) functional groups of
solid-phase extraction (SPE) columns. The recent characteri-
zation of a new clenbuterol-like drug, N1-(2-(4-amino-3,5-
dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (8)
(known as compound A), with an amide substitution of the
nitrogen atom on the alkylic chain (Figure 1) prompted our
group to verify if the cleanup procedures most in use were
really selective for such a molecule and explore any possible
alternatives. The aim was to limit as much as possible false
negative results that could compromise the reliability of the
results of forensic investigations, thus exposing consumers to
the previously mentioned toxicological risk.
Experimental
Equipment
Cleanup procedures were performed on a Supelco (Milan,
Italy) vacuum manifold device. A high-performance liquid chro-
matography (HPLC) System Gold, a Model 126 pump, a Model
168 diode-array detector (DAD), and a Model 501 autosampler
(Beckmann Analytical, S. Ramon, CA) were used to assess the
performance of standards on different SPE columns. Chro-
matographic conditions consisted of a reversed-phase (RP) C18
Lichrosphere Select B column (250 × 4 mm, 5 µm) (Merck,
Darmstadt, Germany), a mobile phase of 0.01M sodium acetate
(pH 3.0) (A) and acetonitrile (B), and a linear gradient from 10%
to 100% B in 20 min. The flow rate was 1.0 mL/min, the DAD
was set at 245 and 305 nm, the bandwidth was 4 nm, and the
spectra recorded in the range of 220 to 350 nm.
An analytical performance on spiked livers at residue levels
Journal of Chromatographic Science, Vol. 40, February 2002

of 0.5, 1.0, and 2.0 ng/g were carried out on a GCQ ion trap
detector (ThermoQuest Italia, Milan, Italy) with a CP-SIL 8CB-
MS FS 30X.25(.25) capillary column (Chrompack Italia, Milan,
Italy). The injector temperature was set at 250°C and was in
splitless mode. The constant velocity of the carrier gas (He) was
40 cm/s. The oven temperature program raised from 70°C to
230°C in 11 min (20°C/min), then raised to 280°C in 10 min
(5°C/min), and was held for 5 min at 280°C. The GCQ acquisi-
tion was in electron-impact mode (70 eV), the multiplier was
set at 1300 V, and the resolution 0.5 amu. For clenbuterol the
precursor ion was m/z 262; the width was 4; the excitation volts
1.1; and the product ions were m/z 188–192, 225–229, and
262–264. For compound A the precursor ion was m/z 262; the
width was 4; the excitation volts 1.2; and the product ions
were m/z 73–77, 188–192, and 262–264.
Liver extraction was performed by an ultraturrax apparatus
and a rotary evaporator (Buchii, Zurich, Switzerland).
Evaporation under a nitrogen stream and trimethylsilyl
derivatization of the extracts were carried out on a Heat Block
(Pierce Italia, Milan, Italy).
Materials
Clenbuterol HCl (Sigma, Milan, Italy) and compound A
(courtesy of Prof. G. Boatto, Dept. of Pharmacology University
of Sassari, Sassari, Italy) were used as pure standards. Meth-
anol, acetonitrile, and all other reagents and solvents were of
analytical grade. Bakerbond CN propyl SPE (100 mg) (CN), aro-
matic sulfonic acid (100 mg) (SCX), light-load (500 mg) C18
nonendcapped (C18NE) (J.T. Baker Italia, Milan, Italy), Bond
Elut certify columns (200 mg) (mixed phase, MPH) (Varian
Italia, Milan, Italy), and Extrelut 20 columns (Merck) were
also used.
Figure 1. Structures of compound A and clenbuterol.
A standards phosphate buffer (PBS) (0.1M, pH 6.0) and 100%
and 20% methanol stock solutions were prepared at 1 mg/mL
and stored at +4°C. Working solutions were freshly obtained by
dilutions in the appropriate solvents in the range of 100.0 to
0.1 µg/mL.
Samples
Seven different incurred beef livers (previously tested as
negative for beta agonists) were spiked at levels of 0.5, 1.0,
and 2.0 ng/g each by the addition of 25, 50, and 100 µL of the
0.1-µg/mL PBS working solutions, respectively. Such spiking
levels have been chosen according to the pharmacokinetics
data of clenbuterol in calves (9). Analyses were repeated on
three different sessions, and recoveries and reproducibility
were calculated according to Gowick et al. (10).
Analytical procedure
Different protocols were followed depending on the SPE
sorbent tested. From fractions of 1 mL from the applications,
washing and elution steps were collected separately, brought to
dryness, and resuspended in a 200-µL HPLC mobile phase to
assess recovery. For each SPE procedure considered, repro-
ducibility was assessed on 12 replicates on two different days.
The CN procedure was done according to Musch and Massart
(5). Columns were conditioned with 2 mL of MeOH followed by
1 mL H2O, not allowing the column to dry. Then, 1 mL of
20% of a 100-µg/mL MeOH standard working solution was
applied at a flow rate of 0.5 mL/min. The column was washed
with 3 mL of MeOH and eluted by another 3 mL of MeOH,
which had 1% triethylamine (TEA) as the counter ion.
The SCX procedure was as follows. Columns were condi-
tioned with 2 mL MeOH and 1 mL 0.1M acetic acid. Then, 1 mL
of the standard working solution (20% MeOH) was applicated
at a flow rate of 0.5 mL/min. After washing with 3 mL MeOH,
elution was performed with 3 mL MeOH (1% TEA).
The MPH columns were performed as follows. According to
the procedure described by Montrade et al. (6), columns were
conditioned with 2 mL MeOH and 0.1M PBS (pH 6.0). Appli-
cation of the standard working solution (100 µg/mL) in PBS
(0.1M, pH 6.0) was performed at a flow rate of 0.5 mL/min.
Rinsing with 1 mL 1.0M acetic acid, washing with 3 mL MeOH,

Table I. Recovery Expressed as the Percentage of Compound A and Clenbuterol* on CN, SCX, MPH, and C18NE SPE
Columns†

CN SCX MPH C18NE

Fraction Compound A Clenbuterol Compound A Clenbuterol Compound A Clenbuterol Compound A Clenbuterol

Application 22 ± 3 n.d.‡ 78 ± 4 n.d. n.d. n.d. n.d. n.d.

First washing 64 ± 5 2±2 22 ± 3 n.d. 56 ± 81 2±1 n.d. n.d.
Second washing 13 ± 3 n.d. n.d. n.d. 27 ± 5 n.d. n.d. n.d.
Third washing 3±4 n.d. n.d. n.d. 12 ± 3 n.d. 18 ± 5 n.d.
First elution n.d. 71 ± 4 n.d. 82 ± 3 n.d. 78 ± 4 29 ± 6 66 ± 3
Second elution n.d. 28 ± 3 n.d. 24 ± 1 n.d. 21 ± 2 49 ± 3 29 ± 2
Third elution n.d. n.d. n.d. n.d. n.d. n.d. 4±2 1±2

* Mean ± standard deviation, n = 12.

† 100 µg of each compound loaded.
‡ n.d., not determined.

93
 Journal of Chromatographic Science, Vol. 40, February 2002

and eluting by 3 mL MeOH (1% TEA) was then performed.
C18NE columns were conditioned with 3 mL MeOH and
1 mL H2O. Application of the working standard solution (20%
MeOH) involved washing with 3 mL of 50% MeOH and elution
with 4 mL MeOH (1% TEA).
Blank and spiked liver samples
From each liver 5 g was sampled with the addition of 200 µL
of the appropriate internal standard. A 0.1-µg/mL working
solution in PBS (0.1M, pH 6.0) (clenbuterol for compound A
analysis and vice-versa) corresponded with 4 ng/g. The liver
samples were minced in 50-mL polypropylene tubes by ultra-
turrax in 20 mL HCl (0.5 N). After sonication (RT = 15 min),
the samples were allowed to hydrolyze overnight at room tem-
perature under shaking. Supernatants were recovered by cen-
trifugation (3000 g, 30 min, and 4°C), thus removing the upper
solid lipid layer. After the addition of 200 µL NaOH (10 N)
under vortexing, the liquid was adsorbed on Extrelut 20

Table II. Recovery Study* by GC–MS–MS from 20 Livers Spiked at Residue Levels of 0.5, 1.0, and 2.0 ng/g

Retention Ions Spiked level Mean

Drug time (m/z) (ppb) N recovery CVR CC alpha CC beta

Clenbuterol 14.37 262 0.5 21 93.9 4.2 0.55 0.58

225 1.0 21 93.0 1.6
188 2.0 21 94.1 1.8

Compound A 20.47 262 0.5 21 93.2 3.1 0.53 0.56

188 1.0 21 93.4 2.4
73 2.0 21 91.4 2.5

* Mean ± standard deviation.

Figure 2. Elution profile of clenbuterol (RT = 10.73) and compound A (RT = 15.69) from RP-HPLC–DAD analysis with a linear gradient of 10% to 100%
acetonitrile in 20 min.

94
Journal of Chromatographic Science, Vol. 40, February 2002
columns (1), and the elution was performed by a 60-mL mix-
ture of n-hexane–dimethyl chloride (8:2, v/v). Organic phase
collected in a 100-mL glass round bottom flask was evapo-
rated to dryness. Residue resuspended in 1 mL 20% MeOH
was loaded on C18 SPE columns, according to a 2-mL washing
(50% MeOH) and 4-mL MeOH (1% TEA) elution. The eluate
was evaporated under a nitrogen stream on the heater block
and derivatized with 20 µL N,O-bis(trimethylsilyl)trifluoro-
acetamide (1% trimethylchlorosilane, 60 min, 60°C) (1). We
injected 2 µL in the gas chromatography (GC)–tandem mass
spectrometry (MS–MS) system. Calibration curves for each
analytical session (in the range of 0.20 to 4.00 ng injected) were
built for each analyte.
Calculations
For HPLC analysis, calibration curves were built by plotting
the peak area of the analyte versus its nominal concentration.
For the GC–MS–MS analysis validation study, we considered
the sum of the area of the precursor ion and two product ions
both for the analyte and the internal standard (clenbuterol for
compound A analysis and vice-versa).The peak-area ratio (ana-
lyte–internal standard) was plotted against the nominal con-
centration of the analyte. The calculation of the decision limits
for CC alpha (the smallest content of the analyte in liver that
may be confirmed with 95% probability) and CC beta (the
smallest content of the analyte from which sample is truly
violative with a confidence limit of 99%) on livers spiked at
residue levels was performed according to the statistical
approach of Gowick et al. (10).
Results and Discussion
SPE
The elution profile expressed as the recovery rate of clen-
buterol and compound A from different SPE columns is
A B
Figure 3. GC–MS–MS analysis of a blank and spiked liver for compound A: (A) clenbuterol used as the internal standard spiked at 4.0 ng/g, (B) the blank liver
extract for compound A, and (C) a liver extract spiked with compound A at 1.0 ng/g.
reported in Table I. The coefficient of regression for the cali-
bration curves were r = 0.9976 for clenbuterol and r = 0.9973
for compound A.
Method validation
The results of the recovery study by GC–MS–MS on livers
spiked at the residue levels are reported in Table II with method
performances. Regression curves over three analytical sessions
were r = 0.9985 for compound A and r = 0.9987 for clen-
buterol. It is worth noting that the European Commission
suggested the acquisition of one parent ion and two product
ions for the unambiguous GC–MS–MS identification of the
forbidden substances. For this purpose, the chromatograms of
a blank liver extract for compound A and a liver extract spiked
at 1.0 ng/g (clenbuterol as the internal standard added at 4.0
ng/g) are shown in Figure 2, in which the traces reported on
the figure from the top to the bottom refer to the total ion cur-
rent, the precursor ion, the first product ion, the second
product ion, and the sum of the ions (precursor + product
ions), respectively.
Discussion
A comparison of the results of the recovery study on SCX and
CN columns for clenbuterol and compound A indicates the
former bases’ binding mainly on the ion-exchange interaction
of the secondary amino group. The lack of such a function in
compound A greatly affects the recovery, thus demonstrating
that the primary amino group shared among both compounds
is weakly active charged (its pKa is lowered) and sterically hin-
dered by the two chlorinated atoms (Figure 1). The behavior of
compound A (more retained on propyl CN columns during the
application) can be mainly addressed to hydrophobic interac-
tions (Table I). Such hydrophobic interactions that are present
in MPH columns as C8 alkylic chains are not sufficient to retain
compound A during the methanolic washing, which is a basic
step to improve the selectivity of such “cleanup” procedures.
These considerations suggest the use of stronger hydro-
C
95

phobic interactions, coupled with the presence of an ion-
exchange mechanism for clenbuterol (represented by the free
silanols groups in the case of C18NE columns). In order to
allow the electrostatic interactions of clenbuterol, the flow
rate in the application was reduced to 0.5 mL/min. The infor-
mation derived from the RP-HPLC analysis of such drugs
showed that compound A was eluted only in the presence of
100% acetonitrile (Figure 3). This behavior has been con-
served on C18NE SPE columns that allow up to 2 mL 50%
MeOH washing without any appreciable loss of compound A.
On this basis, an appropriate SPE procedure was developed
on liver extracts. In order to limit as much as possible the
presence of interfering substances that could act as counter
ions, a preliminary extraction step on Extrelut columns at
alkaline pH was carried out, which proved to be effective to
concentrate clenbuterol and anilino-like compounds in the
organic phase. The limited losses in the recoveries reported in
Table II referred to the overall procedure and can be reasonably
addressed to the sample handling.
Conclusion
The results encourage the use of C18NE SPE columns,
which are able to work at the same time with RP and ion-
exchange mechanisms and give satisfactory recoveries for two
compounds with quite different chromatographic behavior.
The pharmacokinetics evidence that clenbuterol could persist
in bovine liver at residue levels above 1 ng/g for at least 400 h
from the withdrawal of a growth promoting treatment (9) sug-
gests that this approach could represent a realistic tool for
the survey of the illegal use of such a drug and its related sub-
stances in animal productions, with an irrelevant probability of
having false positive (CC alpha) or false negative (CC beta)
results.
Acknowledgments
We would like to thank Prof. Gianpiero Boatto (Department
of Pharmacology, University of Sassari) for the characterization
96
Journal of Chromatographic Science, Vol. 40, February 2002
of compound A and Mr. Giovanni Bartolini (Istituto Superiore
di Sanità) for the technical help. This work was supported by
Ministero della Sanità, Project No. 98/JG.
References
1. The Use of Immunoaffinity Columns in Multi-Residue and Con-
firmation Analysis of Beta Agonists in Biological Samples. L.A. van
Ginkel, H.J. van Rossum, and R.W. Stephany, Eds. Commission of
the European Communities, Bilthoven, The Netherlands, April
22–26, 1991.
2. A. Polettini, M.C. Ricossa, A. Groppi, and M. Montagna. Deter-
mination of clenbuterol in urine as its cyclic boronated derivative
by gas chromatography-mass spectrometry. J. Chromatogr. B 564:
529–35 (1991).
3. G. Brambilla, T. Cenci, F. Franconi, R. Galarini, A. Macrì, F. Ron-
doni, M. Strozzi, and A. Loizzo. Clinical and pharmacological
profile in a clenbuterol epidemic poisoning of contaminated beef
meat in Italy. Toxicol. Lett. 114: 47–53 (2000).
4. Centro Nacional de Epidemiología. Intoxicación alimentaria rela-
cionada con Clenbuterol. España 1993–1994. Bol. Epidem.
Microbiol. 1(12): 229–31 (1993).
5. G. Musch and D.L. Massart. Isolation of basic drugs from plasma
using solid-phase extraction with a cyanopropyl-bonded phase.
J. Chromatogr. 432: 209–22 (1988).
6. M.P. Montrade, B. Le Bizec, F. Monteau, B. Siliart, and F. Andrè.
Multi-residue analysis of beta agonist drugs in urine of meat pro-
ducing animals by gas chromatography-mass spectrometry. Anal.
Chim. Acta 275: 253–68 (1993).
7. B.F. Spisso, C.C. Lopez, M.A.S. Marques, and F.R.A. Neto. Deter-
mination of beta 2 agonists in bovine urine: comparison of two
extraction/clean up procedures for high-resolution gas chro-
matography-mass spectrometry analysis. J. Anal. Toxicol. 24:
146–52 (2000).
8. B. Neri, R. Cozzani, M. Di Pietrogiacomo, G. Brambilla, M. Fiori,
C. Testa, and G. Boatto. HRGC-MS EI and CI for the identification
and characterisation of a new clenbuterol-like substance. Adv.
Mass Spectrom. 15: 587–88 (2001).
9. M.J. Sauer, R.J.H. Pickett, S. Limer, and S.N. Dixon. Distribution
and elimination of clenbuterol in tissues and fluids of calves fol-
lowing prolonged oral administration at growth-promoting dose.
J. Vet. Pharmacol. Therap. 18: 81–86 (1995).
10. P. Gowik, B. Julicher, and S. Uhlig. Multi-residue method for
non-steroidal anti-inflammatory drugs in plasma using high-per-
formance liquid chromatography-photodiode-array detection.
Method description and comprehensive in-house validation.
J. Chromatogr. B Biomed. Sci. Appl. 716(1-2): 221–32 (1998).
Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
Simultaneous High-Performance Liquid
Chromatographic Determination of Paracetamol,
Phenylephrine HCl, and Chlorpheniramine Maleate in
Pharmaceutical Dosage Forms
S
Hamide çenyuva* and Tuncel Özden
Instrumental Analysis Centre, Scientific and Technical Research Council of Turkey, 06530, Ankara, Turkey
Abstract
A rapid, precise, and specific high-performance liquid
chromatographic method is described for the simultaneous
determination of paracetamol, phenylephrine HCl, and
chlorpheniramine maleate in combined pharmaceutical dosage
forms. The method involves the use of a µBondapak CN RP
analytical column (125 Å, 10 µm, 3.9 × 150 mm) at 22°C as the
stationary phase with the mixture of acetonitrile and phosphate
buffer (pH 6.22, 78:22) as the mobile phase. Derivatization of the
drugs is not required. The method is applied to commercial
pediatric cough–cold syrups, tablets, and capsules marketed in
Turkey. The relative standard deviation for 10 replicate
measurements of each drug in the medicaments is always less
than 2%.
Introduction
Combinations of decongestant, antihistaminic, and analgesic
preparations are widely used for cough and cold treatment.
Several methods have been described for the quantitative deter-
mination of these drugs. High-performance liquid chromatog-
raphy (HPLC) methods have been investigated by many workers.
Most of them were based on ion-pair formation, and the detection
methods were typically based on measuring the UV absorbance of
the analytes (1–9).
The methods given in the literature were applied to these active
ingredients, but the methods could only determine two compo-
nents simultaneously. In the given methods, paracetamol and
phenylephrine HCl give peaks with the same retention times.
Other analytical techniques such as derivative spectrophotometry
(2), high-performance thin-layer chromatography (10), liquid
chromatography (LC)–mass spectrometry (11), gas–liquid chro-
* Author to whom correspondence should be addressed: hsenyuva@tubitak.gov.tr.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
matography (12), and spectrofluorometric (13) methods have
also been reported, but none of the methods are applicable for the
simultaneous determination of three components with a large
excess of paracetamol content.
In USP 24, the determination of these components has also
been performed with HPLC, but all of them were determined sep-
arately and the method does not involve simultaneous determi-
nation (14).
In this study, we propose to employ UV detection to determine
active ingredients in cough–cold syrups, tablets, and capsules
after HPLC separation.
The advantages of the proposed method are that the method
works well for all cold drugs with UV absorptivity, the detector
response for all drugs are similar, they are easily applicable in
large excess amounts of paracetamol drugs without any fitting
into one another, and they have a very short analysis time of
approximately 4 min.
Experimental
Apparatus
The system consisted of a Hewlett Packard (Waldborn,
Germany) Series 1100 LC including an HP UV–vis detector,
vacuum degasser, gradient pump module, auto injector with a
variable injection valve, and column compartment oven. A
µBondapak CN RP analytical column from Waters (Milford, MA)
(125 Å, 10 µm, 3.9 × 150 mm) was used. Instrumental settings
were a flow rate of 1.5 mL/min, a column temperature at 22°C,
and a detector wavelength of 265 nm.
Materials and reagents
All the drugs were of USP quality. Methanol and acetonitrile
were obtained from J.T. Baker (Griesheim, Germany) in HPLC
gradient grade. Orthophosphoric acid and triethylamine were
obtained from Merck Inc. (Darmstadt, Germany). The water used
97

was distilled and deionized by using a Millipore (Vienna, Austria)
Milli-Q ultrapure system. Other chemicals were of analytical or
HPLC grade.
Mobile phase
The mobile phase consisted of an aqueous solution of phos-
phate buffer (pH 6.22) and acetonitrile (22:78, v/v). The phosphate
buffer was prepared by dissolving 1.36 mL orthophosphoric acid
in 1 L water. Triethylamine was added to the phosphate buffer
solution in order to adjust the pH to 6.22. Acetonitrile and water
were previously filtered under vacuum through 0.45-µm nylon
filters before injection into the HPLC apparatus.
Standard stock solutions
Standard solutions were prepared by dissolving the drugs in
methanol and diluting them to the desired concentrations.
Paracetamol
A 320-mg sample of paracetamol was accurately weighed and
dissolved with methanol up to volume in a 10-mL volumetric
flask. A 31.2-µL volume of this solution was again diluted with
methanol to volume in a 10-mL volumetric flask.
Figure 1. A typical HPLC chromatogram of the standards: paracetamol (1.13 min), phenylephrine HCl
(2.13 min), and chlorpheniramine maleate (3.44 min).
Figure 2. A typical HPLC chromatogram of pediatric cough–cold syrup: paracetamol (1.13 min),
phenylephrine HCl (2.13 min), and chlorpheniramine maleate (3.44 min).
98
Journal of Chromatographic Science, Vol. 40, February 2002
Phenylephrine HCl
A 10-mg sample of phenylephrine HCl was accurately weighed
and dissolved with methanol up to volume in a 10-mL volumetric
flask. A 1000-µL volume of this solution was again diluted with
methanol to volume in a 10-mL volumetric flask.
Chlorpheniramine maleate
A 10-mg sample of chlorpheniramine maleate was accurately
weighed and dissolved with methanol up to volume in a 50-mL
volumetric flask.
Standard mixture solution
A standard mixture solution was prepared from these stock
solutions by mixing 2000 µL of a paracetamol standard solution,
152 µL of a phenylephrine HCl standard solution, 14.8 µL of a
chlorpheniramine maleate standard solution, and 1232 µL
methanol.
Sample preparations
Syrup samples
The syrup solution was homogenized by shaking and diluted
with methanol to give a final concentration of 40 to 120 µg for
paracetamol, 1 to 4 µg for phenylephrine HCl, and 0.3 to 1 µg for
chlorpheniramine maleate in 1 mL.
Tablet and capsules
Twenty tablets or capsule contents were
weighed, their mean weight determined, and they
were finely powdered. An equivalent weight of the
tablet or capsule content was transferred into a
10-mL volumetric flask containing 6 mL
methanol, ultrasonicated for 20 min, and diluted
to 10 mL with methanol. The solution was filtered
through a 0.45-µm nylon filter.
This solution was again diluted with methanol
to give a final concentration mentioned in the
syrup samples.
Results and Discussion
Calibration and linearity
An external standard method was used for quan-
titative determinations. Triplicate 1-, 3-, 6-, 8-, 9-,
10-, and 15-µL injections were made for the stan-
dard mixture solution. The retention times of the
standards were 1.13 min for paracetamol, 2.13
min for phenylephrine HCl, and 3.44 min for
chlorpheniramine maleate. A typical HPLC chro-
matogram of the standard mixture is shown in
Figure 1. The calibration graphs were obtained by
plotting the peak area against the concentration of
the drugs. In the simultaneous determination, the
calibration graphs were found to be linear in the
mentioned concentrations (the correlation coeffi-
cients are shown in Table I).
Precision (reproducibility)
The precision of the method was studied by
Journal of Chromatographic Science, Vol. 40, February 2002

determining the concentrations of each drug in a syrup, capsule, Recovery tests

and tablet ten times. The results of the precision study (shown in
Table I) indicate that the method is reliable (relative standard
deviation percentage < 2).
Table I. Results of the Precision and Linearity Study*
Recovery tests were performed by adding a known amount of
each drug to a cough–cold syrup and tablet where it was known
to be absent.
The mean results of five analyses ranged from 97.10 to 99.53
(Table II), and these can be considered to be good recoveries.

Precision Coefficient of Linearity Determination of the limit of detection and quantitation

Ingredients (%RSD†) correlations (µg/mL) The limit of detection (LOD) was defined as the concentration
of phenylephrine HCl and chlorpheniramine maleate (calculated
Paracetamol 0.13 0.9999 25–120 as 0.0325 µg/mL and 0.0279 µg/mL, respectively) that produce
Phenylephrine HCl 1.95 0.9999 0.3–10 analytical signals equal to thrice the deviation of the background
Chlorpheniramine maleate 1.36 0.9999 0.2–3 signals. The limit of quantitation (LOQ) was the lowest levels of
* n = 10. phenylephrine HCl and chlorpheniramine maleate (determined
† %RSD, relative standard deviation percentage.
to be 0.251 µg/mL and 0.184 µg/mL, respectively) in the simulta-
neous quantitative assay. The relative standard deviation per-
centage results of the LOQ studies were 1.29 for phenylephrine
Table II. Results of the Recovery Tests for the Drugs HCl and 2.51 for chlorpheniramine maleate (n = 10).
Under Study by the Proposed Method*
Selectivity
Amount Selectivity was assessed by a quality control of the chro-
Matrix Ingredient added % Recovery†
matograms obtained from samples and placebo. Possible interfer-
Syrup (mg/mL) Paracetamol 30.0 99.73 (0.18)
ences resulting from substances present in the medicaments
Phenylephrine HCl 1.0 98.40 (1.55) were not observed.
Chlorpheniramine maleate 0.2 98.00 (4.55)
Determination of active ingredients in pharmaceutical
dosage forms
Tablet (mg) Paracetamol 325.0 99.99 (0.04)
Phenylephrine HCl 5.0 100.56 (1.56) The contents of three drugs in ten different pediatric
Chlorpheniramine maleate 2.0 99.60 (0.65) cough–cold syrups, capsules (Coldeks), and tablets for each brand
(Dristan and Deflu) were determined by the proposed method,
* n = 5.
† Relative standard deviation shown in parentheses.
and the results are presented in Table III.
The chromatogram of a pediatric cough–cold syrup is shown in
Figure 2.
Table III. Assay Results of Active Ingredients in
Commercial Samples*

Label Found %Label

Conclusion
Samples Ingredient value (mg) (mg) claim
The concentration of phenylephrine HCl, chlorpheniramine
Syrup (mg/mL) Paracetamol 32 32.100 100.30 maleate, and a large excess of paracetamol in pharmaceutical
Phenylephrine HCl 1 1.000 100.00 samples can be satisfactorily determined using HPLC with UV
Chlorpheniramine detector. This study has shown that UV detection is a sensitive,
maleate 0.2 0.200 100.00 reliable, reproducible, and accurate method for the determina-
tion of the active ingredients in pediatric cough–cold syrups, cap-
Dristan Paracetamol 325 325.010 100.00 sules, and tablets.
Phenylephrine HCl 5 5.037 100.74 The method is straightforward and simpler than the commonly
Chlorpheniramine
used HPLC methods involving ion pairing or derivatization. As
maleate 2 2.019 100.99
can be seen in the figures, 3.5 min is enough for all of the active
Deflu Paracetamol 300 300.051 100.02 ingredients to be released.
Phenylephrine HCl 5 5.019 100.38 This method has been found suitable for the routine analysis of
Chlorpheniramine the pharmaceutical dosage forms in quality control and R&D lab-
maleate 2 2.010 100.50 oratories for products of similar type and composition.

Coldeks Paracetamol 325 325.015 100.01

Phenylephrine HCl
Chlorpheniramine
maleate
* Average of 10 analyses.
5 5.012 100.24
References
1 1.006 100.60
1. O.W. Lau and C.S. Mok. High-performance liquid chromatographic
determination of active ingredients in cough–cold syrups with indi-
rect conductimetric detection. J. Chromatogr. A 693: 45–54 (1995).

99

2. N. Erk and M. Kartal. Simultaneous high performance liquid chro-
matographic and derivative ratio spectra spectrometry determination
of chlorpheniramine maleate and phenylephrine hydrochloride.
Il Farmaco. 53: 617–22 (1998).
3. R.N. Raju, P. Srilakshimi, and U.M. Krishina. Simultaneous determi-
nation of chlorpheniramine maleate, ephedrine hydrochloride and
codeine phosphate in cough syrups by reverse-phase high-perfor-
mance liquid chromatography. Indian Drugs 29: 408–11 (1992).
4. G. Liang, H. Qian, L. Zhu, Y. Wang, and L. Sun. Simultaneous deter-
mination of paracetamol and chlorpheniramine by RP-HPLC.
Zhongguo Yaoxue Zazhi. 29: 46–48 (1994).
5. I.L. Honigberg, J.T. Stewart, and A.P. Smith. Liquid chromatography
in pharmaceutical analysis: determination of cough–cold mixtures.
J. Pharm. Sci. 63: 766–69 (1974).
6. J.O. De Beer, C.V. Vandenbroucke, and D.L. Massart. Experimental
design for the rapid selection of separation conditions for methyl and
propyl parahydroxybenzoate, phenylephrine hydrochloride and
chlorpheniramine maleate by ion-pair liquid chromatography.
J. Pharm. Biomed. Anal. 12: 1379–96 (1994).
7. B.R. Thomas, X.G. Fang, P. Shen, and S. Ghodbane. Mixed ion pair
liquid chromatography method for the simultaneous assay of
ascorbic acid, caffeine, chlorpheniramine maleate, dextromethor-
phan HBr monohydrate and paracetamol in Frenadol sachets.
J. Pharm. Biomed. Anal. 12: 85–90 (1990).
8. A.I. Gasco-Lopez, R. Izquierdo-Hornillos, and A. Jiminez.
Development and validation of high performance liquid chromatog-
100
Journal of Chromatographic Science, Vol. 40, February 2002
raphy method for the determination of cold relief ingredients in
chewing gum. J. Chromatogr. A 775: 179–85 (1997).
9. G. Indrayanto, A. Sunarto, and Y. Adriani. Simultaneous determina-
tion of phenylpropanolamine hydrochloride, caffeine, paracetamol,
glycerylguaiacolate and chlorpheniramine maleate in Silabat tablet
using HPLC with diode array detection. J. Pharm. Biomed. Anal. 13:
1555–59 (1995).
10. M. El-Sadek, A. El-Shanawany, A. Aboul-Khier, and G. Ruecker.
Determination of the components of analgesic mixtures using high-
performance thin layer chromatography. Analyst 115: 1181–84
(1990).
11. C. Celma, J.A. Allue, J. Prunonosa, C. Peraire, and R. Obach.
Simultaneous determination of paracetamol and chlorpheniramine
maleate in human plasma by liquid chromatography–tandem mass
spectrometry. J. Chromatogr. A 870: 77–86 (2000).
12. O.W. Lau and Y.M. Cheung. Simultaneous determination of some
active ingredients in cough–cold syrups by gas–liquid chromatog-
raphy. Analyst 115: 1349–53 (1990).
13. J.A. Arancibia, A.J. Nepote, G.M. Escandar, and A.C. Olivieri.
Spectrofluorimetric determination of phenylephrine in the presence
of large excess of paracetamol. Anal. Chim. Acta. 419: 159–68
(2000).
14. The United States Pharmacopoeia 24. The National Formulary 19,
U.S. Pharmacopeial Converntion Inc., Rockville, MD, 2000.
Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
Evaluation of Select Variables in the Ion
Chromatographic Determination of F –, Cl –, Br –,
NO3–, SO4–2, and PO4–3 in Serum Samples
Z. Benzo1,*, A. Escalona3, J. Salas1, C. Gómez1, M. Quintal1, E. Marcano1, F. Ruiz1, A. Garaboto1, and F. Bartoli2
1Centro de Química and 2Dpto. de Biologia estructural, Instituto Venezolano de Investigaciones Científicas, IVIC, Apdo. Postal 21827,
Caracas 1020-A, Venezuela and 3Centro de Química Analítica, Facultad de Ciencias, Universidad Central de Venezuela, Apdo. Postal 47102,
Caracas 1041-A, Venezuela
Abstract
A full experimental design at two levels is applied for the estimation
of the significance of select factors that may influence the ion
chromatography (IC) determination of F–, Cl–, Br–, NO3–, SO4–2, and
PO4–3 in serum samples. The factors studied are various sample
deproteinization procedures, eluent composition, and flow rates.
Deproteinization using either acetonitrile–NaOH or ultrafiltration
can be used in order to obtain a significant protein removal before
IC analysis; however, the former is recommended because it is less
time-consuming and cheaper. Better resolution is obtained when
a sodium hydroxide solution is used as the eluent. There is no
influence of the sample’s deproteinization procedures on the
chromatographic resolution.
Introduction
The ability to determine the concentration of physiologically
and pathologically related anions with a single sample treatment
and technique is of great importance. Ion chromatography has
become one of the most powerful tools for the quantitative anal-
ysis of anions in a wide variety of matrices. One of the problems
associated with serum anion determination by this technique is
the lack of similar sensitivity for all the anions to be determined
simultaneously. Of all the inorganic anions present in human
serum, chloride, bicarbonate, and phosphate are the most rou-
tinely determined. However, the demand for sulfate, bromide,
nitrate, and fluoride serum analysis is increasing because of the
clinical information derived from the levels in serum. The impor-
tance in human physiology of the inorganic anions sulfate (1),
bromide (2–4), nitrate (5,6), and chloride (7) have been described.
The biochemical analysis of metabolites in whole blood and
serum requires initial deproteinization to remove hemoglobin
and other proteins that may interfere with the assays. Different
* Author to whom correspondence should be addressed: email zbenzo@ivic.ve.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
agents have been used. One of them is perchloric acid (8,9); how-
ever, it has the disadvantage that percholorate inhibits some
enzyme reactions considerably and it has to be removed by pre-
cipitation with potassium hydroxide. A simple deproteinization
procedure has been reported by Khan et al. (10) involving the use
of sulfosalycylic acid (SSA). However, the SSA agent is often con-
taminated with sulfate. Tricholoroacetic acid has been reported
(11) as a time-consuming procedure and interferes with the
anion elution profile. The precipitation method based on acetoni-
trile (ACN) (12) has not been successful because proteins are not
quantitatively precipitated by this method, which limits the life-
time of the separator column to a maximum of two months.
Centrifugation or ultracentrifugation has been reported (11) as
an efficient method for the deproteinization of serum samples.
A number of studies have reported the determination of some
inorganic anions in serum by ion chromatography. These have
included the simultaneous determination of inorganic phos-
phate, bromide, nitrate, and sulfate in human serum (1) and the
determination of thiocyanate (13), bromide (14), and sulfate (15).
In measuring systems in which the signal is linearly related to the
component of interest, matrix components, and instrumental
parameters, factorial analysis is a convenient tool to apply.
Factorial approaches to experimental designs contrast with sim-
plex approaches in that several experiments can be performed
simultaneously and are used to calculate the main effects and the
interaction effects of several factors. Full factorial designs at two
levels of variation for the input factors are often used in analytical
chemistry (16,17).
This research deals with the optimization of a method for the
simultaneous analysis of chloride, fluoride, nitrate, bromide, sul-
fate, and phosphate in human serum by isocratic ion chromatog-
raphy. A factorial design at two levels was applied in order to
estimate the magnitude of the main effects and various two-factor
interactions under the experimental conditions of interest. It fur-
ther allowed for a better interpretation of the results obtained.
The evaluation of parameter significance is a very important step
in the optimization procedure. The selection criterion for
choosing the factors involved in this design was dictated by
101
 Journal of Chromatographic Science, Vol. 40, February 2002

variables that may influence the resolution such as deproteiniza-
tion treatment, eluent composition, and flow rate.
Experimental
Apparatus and reagents
The ion chromatographic equipment used was a Dionex
(Sunnyvale, CA) DX 500 system with a 100-µL injection loop, an
Ion Pac AS11 analytical column (4 × 250 mm), and an Ion Pac
AG11 Guard column (4 × 50 mm) (Dionex). The column temper-
ature was 25°C. The apparatus was equipped with an ion fiber
suppressor ASRS-11-4 mm (Dionex Anion self regenerating) that
was continuously regenerated by water and a conductimetric
detector (Dionex) PeakNet Chromatography Workstation.
All chemicals were of analytical-reagent grade. Sodium
hydroxide was obtained from Merck (Darmstadt, Germany), and
Na2CO3, NaHCO3, NaCl, NaNO3, NaF, Na2SO4, Na3PO4, and KBr
(> 99.9% pure) were from Aldrich (St. Louis, MO). Milli-Q deion-
ized water was used throughout (Milli-Q water purification
system, Millipore, MA) as well as HPLC-grade ACN (EM Science,
NJ). Ultrafree-Cl low binding cellulose 10,000 nominal molecular
weight limit filters were used (Millipore) as well as 0.45- and 0.47-
µm nylon filters (Alltech, Deerfield, IL).
A protein assay was performed with the Coomassie Plus Protein
assay reagent kit (Pierce, Rockford, IL).
Proteins that are present in high concentration must be removed
before the sample is injected, because they negatively affect the
separation efficiency of the ion-exchange columns.
The principal objective of this study was to find an efficient, fast,
and reliable deproteinization method that yields a solution suit-
able for subsequent ion chromatographic analysis. The three
deproteinization methods described in the Experimental section
were applied, and the resulting percentages of protein removal
were 21.5%, 93.3%, and 98.6% for the ACN, ACN–NaOH, and
ultracentrifugation treatments, respectively.
After the efficiency of these methods was verified, it was decided
to choose the ACN–NaOH mixture and ultrafiltration depro-
teinization procedures as variables to be considered in the exper-
imental design in order to observe their influence on the
chromatographic resolution.
Optimization
In order to obtain proper information on the significance of the
factors mentioned, a full 24 factorial design at two levels was
applied. This type of design involved sixteen experiments. Four
factors were examined in this design: a sodium hydroxide solu-
tion (X1), a sodium carbonate–sodium bicarbonate mixture (X2),
Table I. Experimental Variables Considered in the
Application of the 24 Full Factorial

Serum samples Natural Coded

A pooled serum sample was obtained from the Medical Center variable variable Level (+1) Level (–1)
at our research institute. Each deproteinization method was per-
formed on an aliquot of this pooled sample. NaOH X1 12mM 6mM
Na2CO3–NaHCO3 X2 3mM,2.4mM 0
Sample treatment Flow rate X3 1.5 mL/min 1.0 mL/min
A preliminary study was carried out in order to assess the effi- Deproteinization
ciency of protein removal by different methods. For this reason, procedure X4 ultrafiltration NaOH–ACN
three deproteinization treatments that have been reported in the
literature were tested: ACN (18,19), ACN–NaOH (20), and ultrafil-
tration (19). Protein quantitation was carried out on an aliquot of Table II. 24 Experimental Design*
the same serum sample after each deproteinization treatment,
measuring the absorbance at 595 nm. Resolution
The ACN treatment consisted of mixing 0.2 mL of serum Experiment X1 X2 X3 X4 criterion
sample with an equal volume of ACN, then centrifuging at 2000
× g for 5 min. 1 –1 –1 –1 –1 11.36
For the ACN–NaOH treatment, 500 µL of a serum sample, 2 1 –1 –1 –1 10.62
50 µL of NaOH (2M), and 150 µL of deionized water were added 3 –1 1 –1 –1 12.30
and shaken for a few seconds. Then, 1 mL of ACN was added and 4 1 1 –1 –1 7.41
vortex mixed for 10 s. The resulting mixture was centrifuged for 5 –1 –1 1 –1 11.03
6 1 –1 1 –1 10.99
5 min at 755 × g. Finally, 1 mL of the supernatant solution was
7 –1 1 1 –1 11.50
diluted with 5 mL of deionized water and injected into the chro- 8 1 1 1 –1 7.14
matograph. 9 –1 –1 –1 1 10.04
For the ultrafiltration, a 1:10 diluted serum sample was filtered 10 1 –1 –1 1 10.84
for 30 min and injected. 11 –1 1 –1 1 12.69
12 1 1 –1 1 7.56
13 –1 –1 1 1 10.52
Results and Discussion 14 1 –1 1 1 11.07
15 –1 1 1 1 11.32
Protein removal 16 1 1 1 1 9.26
The sample preparation for ion chromatographic analysis of * Resolution taken as the response.
serum is much more elaborate than for other physiological fluids.

102
Journal of Chromatographic Science, Vol. 40, February 2002

the flow rate (X3), and a deproteinization procedure (X4) (Table I). student t-test with a 0.05 significance level. Table III shows these
Two levels were chosen associated with the –1 and +1 levels of the results. The variables of X1 at the –1 level (6mM), X2 at the –1
corresponding coded variables. These were selected on the basis level, and the interaction of X1 and X2 resulted as being significant
of the literature review on this topic, and the deproteinization at the 95% level. The influence of X1 revealed that a better reso-
procedures were selected according to the results obtained and lution was obtained when this sodium hydroxide concentration
described in the previous section. was used. The use of the Na2CO3–NaHCO3 mixture did not influ-
The selected experimental response was a criteria of peak reso- ence the resolution, and this agreed with the result of the signifi-
lution (Rs) and the peak area. This criterion of resolution con- cance test. The X1–X2 interaction revealed that an improvement
sisted in the addition of the resolution values of all consecutive on the resolution was obtained when both mixtures were used.
peaks plus the number of peaks that appears in the chro-
matogram (21). Table II shows the results. Identification
The significance was evaluated through the application of the Figure 1 shows the chromatograms of an aqueous solution run
at an eluent composition of 6 and 12mM NaOH (Figures 1A and
Table III. Calculation of the Effects 1B) and a serum sample using isocratic conditions (Figures 1C
and 1D). Peak identification was based on retention times.
Estimated Standard Experimental Probability As it can be seen from these chromatograms, in general, the
Effect value deviation t-value level aqueous standard and the serum samples exhibited a well-defined
resolution and symmetrical peaks (not broadened) in less than 16
b0 10.42 0.1613 64.58 0.0000
min. Shorter retention times, mainly for the monovalent and
b1 –1.0574 0.1613 –6.55 0.0012*
b2 –0.4545 0.1538 –2.95 0.0317*
divalent anions, were observed when changing the concentration
b3 –0.0619 0.1613 –0.10 0.7039 of the eluent mixture (12mM). Furthermore, higher signals were
b4 0.1256 0.1613 0.77 0.4714 obtained at this condition. Observed in the chromatogram of the
b12 –1.0655 0.1538 –6.92 0.0012* serum sample was the appearance of a second peak (probably
b13 0.2638 0.1945 1.35 0.2331 acetate), which was coeluted with fluoride.
b14 0.1955 0.1613 1.21 0.2797 Figures 1C and 2 show the effect on the resolution from the
b23 –0.0945 0.1538 –0.61 0.5659 deproteinization treatments. It can be observed that there was not
b24 0.2514 0.1538 1.63 0.1630 a significant change between the results obtained. Therefore,
b34 0.0621 0.1613 0.38 0.7158 either treatment (deproteinization by using ACN–NaOH or by
* Significant effect.
ultrafiltration) could be used. This result was consistent with that
obtained in the experimental design in which the deproteiniza-

Figure 1. Chromatograms of an aqueous solution run at two different eluent compositions (A and B) and a serum sample using two different isocratic conditions (C and
D): (A) an aqueous standard of 6mM NaOH eluent and 1-mL/min flow rate; (B) an aqueous standard of 12mM NaOH eluent and 1-mL/min flow rate; (C) a serum
sample of 6mM NaOH eluent, ACN–NaOH deproteinization, and 1-mL/min flow rate; and (D) a serum sample of 12mM NaOH eluent, ACN–NaOH deproteiniza-
tion, and 1-mL/min flow rate.

103

tion treatment did not have any statistical significance. However, 5 min compared with the 30 min that it takes for the ultrafiltra-
we recommend the ACN–NaOH mixture because it is less time- tion procedure).
consuming (the sample can be deproteinized in approximately
Figure 2. Chromatogram of a serum sample with ultrafiltration: 6mM NaOH eluent and 1-mL/min flow taking into account the peak area as a measure-
rate.
Figure 3. Chromatogram of a serum sample with ACN–NaOH: 6mM NaOH eluent and 1.5-mL/min with it being able to analyze the inorganic anions
flow rate.
Figure 4. Chromatogram of a serum sample with ultrafiltration: 6mM NaOH–Na2CO3–NaHCO3
eluent and 1-mL/min flow rate.
104
Journal of Chromatographic Science, Vol. 40, February 2002
Even when the flow rate was not significant, according to the
results from the significance test a flow rate of 1.5
mL/min (+1 level, X3) is more convenient if anal-
ysis time is considered important (as shown in
Figures 1C and 3).
The significance of the b12 interaction was veri-
fied by the use of the interaction diagrams (22).
The b12 interaction at the –1 level revealed that the
resolution was improved when the NaOH (6mM)
and Na2CO3–NaHCO3 mixtures were used. The
chromatogram resulting from this experimental
condition is shown in Figure 4. However, this con-
dition is not recommended when fluoride has to
be determined, because its peak appeared within
the water dip.
In order to further evaluate the efficiency of the
deproteinization procedures, an evaluation of the
signal sensitivity was carried out for each anion by
ment of the response in the experimental design.
The result of this study (not shown) led to the con-
clusion that the treatment procedure does not
have any significant effect on the signal sensitivity.
Because of the complexity of the serum matrix,
the column efficiency was checked in order to see
if any modification (probably occasioned by the
matrix) could have affected it. For this reason, a
control solution containing all the anions consid-
ered in this study was passed through the column
before and after running the serum samples for
their analysis (shown in Figure 5). In order to
evaluate column efficiency, the theoretical plates
were calculated for each anion before and after the
set of experiments involved in this research, and
no significant change was observed. No change in
the signal sensitivity and resolution was obtained.
Therefore, it can be said that the serum matrix did
not alter the column’s original characteristics
in this matrix under the conditions developed in
this work.
Conclusion
It has been shown in this work that the simulta-
neous determination of six important physiolog-
ical anions in human serum is possible using ion
chromatography under isocratic conditions
without altering the column’s original conditions
and thus its efficiency. This is an important aspect,
taking into account the complexity of the serum
matrix. Scarce information has been given about
column integrity after this type of analysis.
The application of the experimental design to
Journal of Chromatographic Science, Vol. 40, February 2002

this particular application for evaluating the significance of input Acknowledgments

parameters for analytical determinations allows for the obtaining
of important information on the variables that most influence the
determination of the anions in this matrix and on the interactions
between chemical and instrumental parameters.
There was no influence of the sample’s deproteinization proce-
dures on the chromatographic resolution.
Table IV. Data for the Anions in Figure 5A
The authors gratefully acknowledge the valuable support of the
program BID-CONICIT through the project QF-10 and Prof. Luis
Gómez of the chromatography laboratory at the Analytical
Chemistry Center (UCV) for his valuable assistance.
This research is dedicated to our late and dearest colleague
Santos Melendez who could never fulfill his dream of working
with us in this project.

Anion Retention time (min) Area

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Fluoride 1.82 71263
Chloride 2.30 674127
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Nitrite 2.52 42713
simultaneous determination of inorganic phosphate, bromide, nitrate
Bromide 3.48 91697 and sulphate in human serum. Clin. Chim. Acta. 132: 63–71 (1983).
Nitrate 3.60 294013 2. Z. Khalkhali and B. Parsa. “Measurement by Non-Destructive
Sulfate 4.48 1065847 Neutron Activation Analysis of Bromine Concentration in the
Secretions of Nursing Mothers. In Nuclear Activation Techniques in
the Life Sciences. International Atomic Energy Agency, Vienna,
Austria, 1972, pp. 461–66.
Table V. Data for the Anions in Figure 5B 3. L.-O. Plantin. In Nuclear Activation Techniques in the Life Sciences.
International Atomic Energy Agency, Vienna, Austria, 1972, pp.
Anion Retention time (min) Area 461–66.
4. D.L. Trump and M.C. Hochberg. Bromide intoxication. Johns
Fluoride 1.82 71282 Hopkins Med. J. 138: 119–23 (1976).
Chloride 2.27 666512 5. M. Ferrant. Methemoglobinemia. J. Pedriatr. 29: 585–92 (1946).
Nitrite 2.48 37662 6. M. Cornblath, A.F. Hartmann. Methemoglob-inemia in young
Bromide 3.43 107169 infants. J. Pedriatr. 33: 421–25 (1948).
7. K. Arai, F. Kusu, N. Noguchi, K. Takamura, and H. Osawa. Selective
Nitrate 3.55 284737
determination of chloride and bromide ions in serum by cyclic volat-
Sulfate 4.25 1074038 mmetry. Anal. Biochem. 210: 109–13 (1996).
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13. Y. Michigami, T. Takahashi, F. He, Y. Yamamoto,
and K. Ueda. Determination of thiocyanate in
human serum by ion chromatography. Analyst 113:
389–92 (1988).
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chromatographic determination of bromide in
serum. Clin. Chem. 30: 781–83 (1984).
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by controlled-flow anion chromatography.
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Investigation by experimental design and regression models of the
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Manuscript accepted September 5, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
Temperature Effect on Peak Width and Column
Efficiency in Subcritical Water Chromatography
Yu Yang1,*, Lori J. Lamm1, Ping He1, and Toru Kondo2
1Department of Chemistry, East Carolina University, Greenville, NC 27858 and 2Fuji Silysia Chemical Ltd., Kasugai-Shi, Aichi-Ken,
487-0013, Japan
Abstract
Subcritical water has been recently employed as the mobile phase
to eliminate the use of organic solvents in reversed-phase liquid
chromatography. Although the influence of temperature on
retention in subcritical water chromatography has been reported,
the temperature effect on peak width and column efficiency has not
yet been quantitatively studied. In this work, several polar and
chlorinated compounds are separated using pure subcritical water
on Zorbax RX-C8, PRP-1 (polystyrene–divinylbenzene), Hypersil
ODS, and ZirChrom-polybutadiene columns. Isothermal separations
are performed at temperatures ranging from 60°C to 160°C. The
retention time and peak width of analytes are reduced with
increasing temperature. However, the column efficiency is either
improved or almost unchanged with the increasing temperature in
the low-temperature range (lower than the 100°C to 120°C range),
but it is decreased when temperature is further raised in the high-
temperature range (higher than the 100°C to 120°C range).
Therefore, a maximum in column efficiency is obtained at
temperatures within the 100°C to 120°C range in most cases.
Introduction
Reversed-phase liquid chromatography (RPLC) is a very
popular separation and analysis technique used today.
Unfortunately, organic solvents are required to achieve separa-
tion in RPLC. An enormous amount of these organic solvents is
consumed every day worldwide. These organic solvents are
expensive in terms of both purchasing and waste disposal. In
addition, they are also potentially harmful to the laboratory envi-
ronment and the operator. Therefore, searching for nontoxic sol-
vents as the mobile phase for RPLC is of great interest.
Ambient water is too polar to serve as an eluent for reversed-
phase separation. Fortunately, the polarity of water decreases
with increasing temperature. Therefore, the solubility of organic
compounds is dramatically increased in water at elevated tem-
* Author to whom correspondence should be addressed: email yangy@mail.ecu.edu.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
peratures (1–4). For example, the solubility of some pesticides
and polycyclic aromatic hydrocarbons is increased several orders
of magnitude by raising the water temperature from ambient to
200°C (1–3). Thus, liquid chromatographic (LC) separations can
be achieved by using high-temperature (subcritical) water
(5–11). With two additional components (an oven and a back-
pressure regulator or restrictor), a conventional high-perfor-
mance liquid chromatographic (HPLC)–UV system can be easily
modified to a subcritical water separation system. The oven is
used to provide the temperature for subcritical water separation,
and the backpressure regulator prevents water from boiling
when working with temperatures higher than 100°C. The UV
detector is placed outside the oven. Depending on the separation
temperature and the flow rate of water used, the temperature of
the water eluent in the UV flow cell varies, but it is lower than the
oven temperature. If a backpressure regulator or a short packed
LC column is used to provide the backpressure, they are nor-
mally connected to the outlet of the UV flow cell. Thus, the flow
cell is under pressure and may be damaged.
Most reports on subcritical water chromatography mainly
focus on testing the feasibility of using subcritical water as the
mobile phase for reversed-phase separation (5–11). Even though
the effect of water temperature on the retention is mentioned in
some of these reports (5–11), a quantitative study of the temper-
ature effect on peak width and column efficiency in subcritical
water separation has not yet been reported. It should be pointed
out that the effects of temperature on retention, viscosity, diffu-
sivity, and the number of plates have been well-investigated in
conventional HPLC (12–17). However, the temperature range
was generally much narrower and normally went up to 80°C. In
addition, organic solvents were involved in the mobile phases of
these studies (12–17).
In this work, pure water at elevated temperatures and pres-
sures was used as the eluent to separate several polar analytes
and chlorophenols on four commercial columns, which
included the Zorbax RX-C8, polymeric PRP-1, Hypersil ODS, and
ZirChrom-PBD columns. Separations were performed at tem-
peratures ranging from 60°C to 160°C in an isothermal manner.
The peak width was monitored and the number of theoretical
plates was calculated to evaluate the temperature effect on
column efficiency.
107

Experimental
Separation columns
A polystyrene–divinylbenzene column (PRP-1, 250- × 4.1-mm
i.d.) was purchased from Hamilton Company (Reno, NV). A
Zorbax RX-C8 column (250- × 4.6-mm i.d.) was obtained from
DuPont (Wilmington, DE). A Hypersil ODS column (100- × 4.6-
mm i.d.) (Keystone, Bellefonte, PA) was used to separate a phenol
mixture. Because the recently developed zirconia-based columns
have shown excellent thermal stability and column efficiency
(18–20), a ZirChrom-polybutadiene (PBD) column (100- × 2.1-
mm i.d.) (ZirChrom Separation Inc., Anoka, MN) was also
employed in this study. The particle size was 3 µm for the
ZirChrom-PBD column and 5 µm for the other three columns.
Reagents
All analytes used in this study were obtained from Sigma (St.
Louis, MO). The stock solutions of the solutes were prepared in
methanol (HPLC grade) (Fisher Scientific, Fair Lawn, NJ). The
deionized water (18 MΩ) was prepared in our laboratory using a
Sybron/Barnstead (Boston, MA) system. All mobile phases were
purged using helium gas prior to each use.
Subcritical water separation
A homemade subcritical water chromatography–UV system
was employed in this work. A Hewlett-Packard (Avondale, PA)
gradient pump Series 1050 was used to deliver the mobile phase.
The flow rate was 0.2 mL/min for the ZirChrom-PBD column and
1 mL/min for the other three columns. The outlet of the pump
was connected to a Valco injector fitted with a 2-µL sample loop
(purchased from Keystone Scientific). The injector was located
just outside a Fisher Isotemp oven. A piece of stainless steel
tubing (100-cm × 0.005-inch i.d.) (Keystone) was connected
between the injector and the separation column as a preheating
coil. Both the preheating coil and the separation column were
placed inside the oven. The preheating coil acted like a high-tem-
perature water reservoir to ensure that the water eluent reached
the desired temperature before entering the separation column.
Because water will be vaporized at 1 atm and temperatures at
100°C or higher, backpressure must be applied to the outlet of the
column in order to keep water in the liquid state. There are sev-
eral reasons for avoiding steam in subcritical water chromatog-
raphy–UV systems. The water mobile phase may stay in liquid
near the column inlet, thus causing steam to form inside the sep-
aration column near the outlet end if there is not enough back-
pressure applied. Thus, the mobile phase exists as two separate
phases (liquid water and steam) in the separation column. In
addition, the UV signal strongly fluctuates if steam exists in the
system. This means that the UV detector is not stable when steam
passes through the flow cell. In this study, a capillary restrictor
(7-cm × 75-µm) (Polymicro Technologies Inc., Phoenix, AZ) was
placed outside the oven and between the separation column and
the UV flow cell in order to ensure that the water inside the sepa-
ration column stayed in the liquid state at higher temperatures.
Connection unions (1/16 inch to 1/16 inch) (Supelco, Bellefonte,
PA) were used to connect the restrictor. By 1/16-inch stainless steel
tubings, the inlet of union 1 and the outlet of union 2 were con-
nected to the column and UV detector, respectively. The fused-
108
Journal of Chromatographic Science, Vol. 40, February 2002
silica capillary restrictor was connected with both unions using
graphite ferrules (Alltech, Deerfield, IL). We evaluated the influ-
ence of the restrictor dimension on the retention time using
restrictors having 7 to 30 cm in length and 51 to 103 µm in inner
diameter. However, there was no significant effect of the restrictor
dimension on the retention time. A restrictor with a length of
7 cm and a 75-µm inner diameter was chosen for all of the exper-
iments reported in this work. An LDC variable wavelength
detector (spectro Monitor 3200, Riviera Beach, FL) was used in
this separation system. The UV detector was set at a wavelength of
254 nm for the entire work.
After purging the deionized water with helium, the water was
continuously pumped through the separation column at either
0.2 or 1 mL/min, depending on the columns used. Then, the
oven was turned on and set to a desired temperature. In order to
ensure that separations were carried out at the set temperature,
the first injection was not made until approximately 20 min after
the oven temperature was reached. This allowed the stationary
phase in the packed column and the mobile phase to equilibrate
to the desired temperature. It should be noted that the tempera-
ture of the stationary phase and the mobile phase inside the
column lagged behind the oven temperature by approximately 5
to 20 min, depending on the temperature employed. A Hewlett
Packard 3396 Series II integrator was used as the data-recording
device. The peak width monitored in this work was at half-
height, and the number of theoretical plates (N) was computed
using the following equation:
N = 2π(tRH/A) 2 Eq. 1
where H and A are the peak height and area, respectively.
Results and Discussion
Zorbax RX-C8 column
The Zorbax RX-C8 column was first used to study the temper-
ature effect on the peak width and column efficiency. The test
solutes in this study included pyridine, benzamide, catechol, and
guaiacol. The temperature used for the separation of these ana-
lytes ranged from 60°C to 100°C because this column was
proven to be thermally stable at temperatures up to 100°C for
several thousand column volumes (18). In case of coelution, the
analytes were injected individually. It is known that the retention
time is decreased with increasing temperature. The same trend
was observed in this study with all four solutes tested. For
example, pyridine was not eluted until approximately 44 min at
60°C (as shown in Figure 1A) (t0 = ~2.4 min). However, the same
analyte was eluted within approximately 16 min at 100°C. It
should be noted that the decrease in retention with increasing
temperature was in an almost linear fashion (Figure 1A). Figure
1B demonstrates the temperature effect on the peak width for
the test analytes. Because the viscosity of water decreased dra-
matically when the temperature was raised (as shown in Table I)
(21,22), the diffusivity was greatly enhanced. Thus, narrower
Journal of Chromatographic Science, Vol. 40, February 2002

peaks were obtained at elevated temperatures. Similar to the
retention time, the reduction in the peak width with increasing
temperature was not dramatic.
The influence of temperature on column efficiency is demon-
strated in Figure 1C. Based on the type of curves in Figure 1C,
the solutes can be divided into two groups. The first group
includes benzamide and pyridine. The peak efficiency of these
two solutes was significantly improved with increasing tempera-
Table I. Temperature Effect on the Viscocity of Water*
Viscocity (cP)
Temperature (°C) At 50 bar At 100 bar
ture. The number of theoretical plates obtained at 100°C was
53–84% higher than that at 60°C for benzamide and pyridine.
This uptrend temperature effect on efficiency was achieved
because the reduction in retention was slower than the reduc-
tion in peak width when the temperature was raised. This phe-
nomenon can be seen from Figures 1A and 1B. When the
temperature was increased from 60°C to 100°C, the reduction in
retention time and peak width for benzamide was 49% and 62%,
respectively. However, the plate number of catechol and guaiacol
was almost unchanged when the temperature was raised from
60°C to 100°C. This means that both the retention time and peak
width of catechol and guaiacol were decreased with similar rates
when the temperature was increased.

25 0.8898 0.8889 PRP-1 column

50 0.5479 0.5487 Because the polymeric PRP-1 column is thermally stable at
100 0.2836 0.2849 temperatures up to 160°C based on our previous work (18), the
150 0.1832 0.1844 temperature range was expanded to 160°C to evaluate the tem-
200 0.1345 0.1357 perature effect on the column efficiency with a greater tempera-
250 0.1061 0.1075
* Obtained from references 21 and 22.

Figure 1. Temperature effect on (A) the retention time, (B) peak width, and Figure 2. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates for separation on the Zorbax RX column. (C) number of theoretical plates for separation on the PRP-1 column.

109
 Journal of Chromatographic Science, Vol. 40, February 2002

ture range. Therefore, the temperature effect on the peak effi- 140°C. Because the inner diameter of the ZirChrom-PBD
ciency of the same or similar solutes (resorcinol, catechol, ben- column was 2.1 mm, a flow rate of 0.2 mL/min was used for this
zamide, and pyridine) was also investigated by using the PRP-1 column. Similar to separations on the Hypersil ODS column, the
column. The separation temperatures ranged from 60°C to column efficiency was either increased or unchanged when the
160°C at an interval of 20°C. The analytes were injected individ- temperature was raised from 60°C to 100°C (as shown in Figure
ually in case of coelution at higher temperatures. The retention 4C), but the plate number (equivalent to a 25-cm column) was
time and peak width were also significantly decreased with decreased when the temperature was further increased from
increasing temperature as shown in Figure 2 (t0 = ~2.0 min). The 100°C to 140°C. However, the decrease in efficiency was less sig-
reduction in retention time and peak width was up to 80% for nificant for this zirconia-based column compared with that for
these analytes by raising the separation temperature from 60°C the Hypersil column. As can be seen from Figure 4C, increasing
to 160°C. This means that the analysis was 7–9 times faster at the temperature from 60°C to 140°C resulted in either no
160°C than that at 60°C. decrease or a very little decrease (approximately 15%) in effi-
The effect of temperature on peak efficiency is depicted in ciency with the ZirChrom-PBD column but a typical 40%
Figure 2C. Similar to the separation on the Zorbax column, decrease with the Hypersil ODS column. This means that the
uptrend curves of temperature versus column efficiency were
obtained in the temperature range of 60°C to 120°C. However, the
number of theoretical plates was decreased when the temperature
was further raised to 160°C. Thus, the peak efficiency reached a
maximum at temperatures in the 100°C to 120°C range. This
maximum of peak efficiency shows that the reduction in reten-
tion time was smaller than the reduction in peak width at the low-
temperature range, and the decrease in retention time was
greater than the decrease in peak width at the high-temperature
range. This phenomenon can be clearly seen from Figures 2A and
2B. For example, the retention time of resorcinol was reduced
35% while its peak width experienced a 48% reduction when tem-
perature was raised from 60°C to 80°C (low-temperature range).
However, the opposite phenomenon was observed at the higher
temperature range. When the temperature was increased from
140°C to 160°C, the decrease in retention time and peak width for
catechol was 20% and 8%, respectively.

Hypersil ODS column

In order to further explore the effect of temperature on
column efficiency, a mixture of phenol, 2-chlorophenol, and 2,3-
dichlorophenol was separated using a Hypersil ODS column.
Even though the thermal stability of this column is poorer than
the PRP-1 column, we still used a temperature range of 60°C to
140°C. Because the column was exposed to high temperatures
only for several hours (the most) in this study, the thermal sta-
bility did not get significantly worse within this short period of
time based on our previous study (18). Again, both the retention
time (t0 = ~1.0 min) and peak width were decreased with
increasing temperature as shown in Figures 3A and 3B. The
number of theoretical plates (equivalent to a 25-cm column) was
slightly increased for chlorophenols but stayed almost
unchanged for phenol when the temperature was increased from
60°C to 100°C. Further raising the temperature from 100°C to
140°C caused a significant decrease in column efficiency (as
demonstrated in Figure 3C). This was in agreement with the
results obtained by using the PRP-1 column even though dif-
ferent analytes were used.

ZirChrom-PBD column
Based on references 19 and 20, the ZirChrom-PBD column
was stable at temperatures up to the range of 150°C to 200°C.
Therefore, the phenol mixture was also separated on the
ZirChrom-PBD column at temperatures ranging from 60°C to
Figure 3. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates (equivalent to a 25-cm column) for separation
on the Hypersil ODS column.

110
Journal of Chromatographic Science, Vol. 40, February 2002
zirconia-based column is more suitable for separations at higher
temperatures. Another benefit associated with the ZirChrom-
PBD column is that the analysis time required by this column
was much shorter than that required by the Hypersil column. As
shown in Figures 3 and 4, separation on the ZirChrom-PBD
column was approximately four times faster than that on the
Hypersil column, but the column efficiency of the ZirChrom-
PBD under the fast analysis conditions was still competitive
compared with that obtained by the Hypersil column.
Mechanism for the temperature effect on column efficiency in
subcritical water chromatography
To the best of our knowledge, this is the first report that quan-
titatively describes the effect of water temperature on column
Figure 4. Temperature effect on (A) the retention time, (B) peak width, and
(C) number of theoretical plates (equivalent to a 25-cm column) for separation
on the ZirChrom-PBD column.
efficiency over a wide temperature range in subcritical water
chromatography. In many cases, a maximum in column effi-
ciency in subcritical water chromatography was obtained when
the water temperature was varied in this work. We believe that
this maximal efficiency was caused by two factors that are asso-
ciated with water temperature. The first one is the mass transfer
that improves the efficiency, and the second is the longitudinal
diffusion that worsens the column efficiency.
It is well-known that the diffusivity or diffusion coefficient of
the mobile phase (Dm) is directly proportional to the absolute
temperature and inversely proportional to the viscosity of the
mobile phase. Because the viscosity of water is decreased with
increasing temperature as demonstrated in reference 22 (Table I),
the diffusivity (mass transfer) of water is dramatically increased
and the mass transfer resistance (the Cm term in the van Deemter
equation) is greatly decreased at elevated temperatures. Thus,
narrower bands and higher column efficiency should be expected
with increasing temperature. This is why the number of theoret-
ical plates was generally increased when the temperature was
raised from 60°C to the 100°C to 120°C range (as illustrated in
Figures 1–4). Therefore, we believe that mass transfer may domi-
nate the subcritical water separation process at the lower temper-
ature range (lower than the 100°C to 120°C range).
By increasing the temperature from 100°C to 120°C, the diffu-
sivity is further increased and even better mass transfer results.
However, the better mass transfer also causes a greater axial
molecular diffusion (longitudinal diffusion, the B term in the van
Deemter equation), which makes the column efficiency become
poorer. Therefore, the higher the temperature, the greater the
longitudinal diffusion (B is directly proportional to Dm in the van
Deemter equation) and the lower the column efficiency. This is
the reason why the number of plates was decreased when the
temperature was raised from the 100°C to 120°C range to the
140°C to 160°C range (Figures 2–4). Therefore, longitudinal dif-
fusion may be the dominating factor that controls the subcritical
water separation at the higher temperature range. Carr et al.
(20) reported that the column efficiency was decreased for sepa-
rations using organic solvent–water mixtures at temperatures of
150°C and 200°C, although the authors indicated that this might
be caused by the interaction of molecules with the column walls
at higher temperatures (20).
Because increasing the separation temperature causes lower
mass transfer resistance (the C term in the van Deemter equa-
tion decreases) but also greater longitudinal diffusion (the B
term in the van Deemter equation increases), a maximal column
efficiency may be observed. However, if the decrease in the C
term and increase in the B term are similar in the lower temper-
ature range, then they compensate each other. Thus, the effi-
ciency will stay unchanged when the temperature is increased
from low temperature to the 100°C to 120°C range. This is evi-
denced in Figures 1, 3, and 4. However, at a higher temperature
range the increase in the B term always exceeds the decrease in
the C term. Therefore, the column efficiency was always
decreased when the temperature was further raised. This may
explain why the number of plates was always decreasing with all
of the columns and solutes tested when the temperature was
increased from the 100°C to 120°C range to the 140°C to 160°C
range (as shown in Figures 2–4).
111

Conclusion
The elution of several polar analytes has been achieved by
using pure water and four different types of commercially avail-
able reversed-phase columns at elevated temperatures under
moderate pressure to keep the water in the liquid state. A fused-
silica capillary restrictor was connected between the separation
column and the UV flow cell to provide the backpressure needed
to avoid water from boiling at higher temperatures. For all of the
analytes studied and columns used, the peak width was
decreased with increasing water temperature. For example, the
peak width of benzamide obtained on the PRP-1 column was
reduced by as much as 93% by raising the temperature from
60°C to 160°C. However, the column efficiency was either
improved or remained unchanged initially but then decreased
with increasing temperature. Thus, a maximum in efficiency was
observed at temperatures in the 100°C to 120°C range in most
cases.
Acknowledgments
This research was supported by an award from Research
Corporation (CC4607). The authors would also like to thank
ZirChrom Separations Inc. for providing the ZirChrom-PBD
column. Toru Kondo acknowledges Fuji Silysia Chemical Ltd.
and the Department of Chemistry at East Carolina University for
providing the opportunity to conduct this research.
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eluent: a neglected tool in high-performance liquid chromatog-
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Manuscript accepted December 7, 2001.
Journal of Chromatographic Science, Vol. 40, February 2002
A High-Pressure Liquid Chromatographic–Tandem
Mass Spectrometric Method for the Determination of
Ethambutol in Human Plasma, Bronchoalveolar Lavage
Fluid, and Alveolar Cells
John E. Conte, Jr.1,2,3,*, Emil Lin4, Yeping Zhao4, and Elisabeth Zurlinden1
1Department of Epidemiology and Biostatistics, Infectious Diseases Research Laboratory, 2Department of Medicine, 3Department of
Microbiology and Immunology, and 4Department of Biopharmaceutical Sciences, University of California, San Francisco, 350 Parnassus
Avenue, Suite 507, San Francisco, CA 94117
Abstract
A technique is presented for the specific and sensitive determination
of ethambutol concentrations in plasma, bronchoalveolar lavage
(BAL), and alveolar cells (AC) using a high-pressure liquid
chromatographic (HPLC)–tandem mass spectrometric (MS–MS)
method. The preparation of samples requires a deproteinization step
with acetonitrile. The retention times for ethambutol, neostigmine
bromide, and propranolol are 2.0, 1.4, and 1.1 min, respectively, with
a total run time of 2.8 min. The detection limits for ethambutol are
0.05 µg/mL for plasma and 0.005 µg/mL for the BAL supernatants and
AC suspensions. The assay has excellent performance characteristics
and has been used to support a study of the intrapulmonary
pharmacokinetics of ethambutol in human subjects.
Introduction
Ethambutol has a primary role in the treatment of tuberculosis
and is recommended with isoniazid, rifampin, and pyrazinamide
as initial therapy (1). Ethambutol is rapidly absorbed and has a
bioavailability of 7% after oral administration (2,3). Under fasting
conditions, the maximum concentration (mean ± standard devi-
ation, SD) of the drug in serum is 4.5 ± 1.0 µg/mL and the time
to maximum concentration is 2.5 ± 0.9 h (2). The minimum
inhibitory concentration of ethambutol for M. tuberculosis
ranges from 0.5 to 2 µg/mL in broth media (4). A microbiological
assay (detection limit of 0.4 µg/mL) using M. smegmatis as the
test organism for determining ethambutol in serum has been
reported (5). A gas chromatographic (GC)–mass spectrometric
(MS) method has been used for the determination of ethambutol
in tablets (6). A GC–liquid chromatographic (LC) assay with
improved performance characteristics (detection limit of 0.1
µg/mL in plasma) has been used to study the pharmacokinetics of
* Author to whom correspondence should be addressed.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
ethambutol in humans (3,7–9) and rabbits (10), and a high-pres-
sure liquid chromatographic (HPLC) method for the determina-
tion of ethambutol in plasma (detection limit of 10 ng/mL) and
urine (detection limit of 10 µg/mL) has been described (11).
We report the use of a sensitive HPLC–tandem mass spectro-
metric (MS–MS) technique to measure ethambutol in human
plasma, bronchoalveolar fluid (BAL) (detection limit of 0.05
µg/mL), alveolar cells (AC) (detection limit of 0.005 µg/mL), and
plasma (detection limit of 0.05 µg/mL). Compared with other
methods, the technique has the advantages of increased sensi-
tivity and a capability to analyze small sample volumes. The speci-
ficity of HPLC–MS–MS detection greatly minimizes the risk of
interference from other substances. This is especially important
when analyzing specimens from patients such as those with AIDS
who are taking numerous concomitant medications. It currently
is being used to support a phase-one study of the intrapulmonary
pharmacokinetics of ethionamide in normal subjects and sub-
jects with AIDS.
Experimental
Chemicals
All solvents and chemicals were HPLC grade except ammo-
nium acetate, which was certified. A 1.0-mg/mL solution of
ethambutol HCl (Lederle Laboratories, Wayne, NJ) was made in
50% methanol and stored refrigerated. This solution was further
diluted to produce working stock solutions of 0.1, 1.0, and 10
µg/mL of ethambutol. Stock solutions of 1.0 mg/mL neostigmine
bromide (Aldrich Chemical Co., Milwaukee, WI) and propranolol
(USP Reference, Rockville, MD) were prepared in 50% methanol.
Neostigmine bromide and propranolol were then diluted to a con-
centration of 0.050 µg/mL in acetonitrile and used as the internal
standard for plasma, and propranolol was diluted to 0.300 µg/mL
and used as the internal standard for BAL and AC.
113

Instrumental
Chromatography
The mobile phase (containing 80% acetonitrile, 4mM ammo-
nium acetate, and 0.10% trifluoroacetic acid) was run through a
hypersil silica column (50- × 4.6-mm i.d., 5-µm particle size) at a
Figure 1. Daughter ion spectra and chemical structures of ethambutol using the
Sciex APCI mode.
Figure 2. Daughter ion spectra and chemical structures of neostigmine (the
internal standard) using the Sciex APCI mode.
114
Journal of Chromatographic Science, Vol. 40, February 2002
flow rate of 0.8 mL/min using a Shimadzu (Columbia, MD) LC-10
AD pump. Extracts from samples were injected onto the system
with a Waters (Milford, MA) Intelligent Sample Processor 717
Plus. The retention times for ethambutol, neostigmine, and pro-
pranolol were 2.0, 1.4, and 1.1 min, respectively, with a total run
time of 2.8 min.
MS
We used two different MS systems during the development and
validation of this assay to explore different types of MS equipment.
Neostigmine bromide was the internal standard used for the
plasma and BAL that were assayed on the PE Sciex API III
(PerkinElmer, Foster City, CA), whereas propranolol was used as
the internal standard for the assays in plasma, BAL, and ACs per-
formed on the Micromass (Manchester, U.K.) Quattro LC. Peak
detection and area determinations for some plasma and BAL were
Figure 3. Daughter ion spectra and chemical structures of ethambutol using the
Micromass Quattro LC electrospray mode.
Figure 4. Daughter ion spectra and chemical structures of propranolol (the
internal standard) using the Micromass Quattro LC electrospray mode.
Journal of Chromatographic Science, Vol. 40, February 2002
carried out with a PE Sciex API III.
The MS used the following settings and conditions. The mul-
tiple reaction monitor scanning mode was set at m/z 205–116 for
ethambutol and m/z 209–71 for neostigmine (Figures 1 and 2).
Atmospheric pressure chemical ionization (APCI)–positive ion-
ization was used. The sample inlet used a heated nebulizer at
450°C. The discharge current was +3 µA. The gas curtain flow was
1.2 L/min (N2 = 99.999%). The nebulizer pressure was 551.4 kPa.
The collision gas consisted of a 9.99% nitrogen–90.01% argon
mixture (set at 250 × 1012 molecules/cm2). Peak detection for the
ACs and some plasma and BAL specimens was carried out on a
Micromass Quattro LC. For these specimens the reaction channel
was m/z 205.35–116.10 for ethambutol and m/z 260.18–115.95
A 0.010, 0.020, 0.040, 0.080, 0.160, 0.320, and 0.640 µg/mL of
B trile containing 0.050 µg/mL neostigmine or propranolol as the
Figure 5. Chromatograms of blank plasma: (A) the internal standard and
(B) ethambutol.
A supernatant standards and samples were prepared by adding 0.5
B the internal standard (0.300 µg/mL propranolol) was added to 250
Figure 6. Chromatograms of a study subject’s plasma obtained 4 h after the fifth
dose of 15 mg/kg ethambutol administered once a day: (A) the internal standard
and (B) ethambutol. The ethambutol concentration was 0.734 µg/mL.
for propranolol (Figures 3 and 4). Electrospray–positive ioniza-
tion with a flow rate of 0.2 mL (5-to-1 split ratio of 1.0 mL/min)
to the Micromass system was used. The sample inlet used a heated
nebulizer. The sample cone was set to 25 V for ethambutol and
35 V for propranolol. The energy collision was set to 15.0 eV for
both ethambutol and propranolol. A Macintosh Quadra 800 com-
puter (Apple Computers, Cupertino, CA) was used for peak inte-
gration and analysis.
Sample preparation
Standard curves
Plasma standard curves were prepared by adding appropriate
volumes of ethambutol working stock solutions into 0.2 mL of
blank plasma to yield the concentrations of 0.05, 0.10, 0.20, 0.40,
0.80, 1.2, 1.6, and 2.4 µg/mL of ethambutol. The standards for
BAL supernatants were spiked to yield concentrations of 0.005,
ethambutol. The AC suspension standards were spiked to yield
concentrations of 0.005, 0.010, 0.020, 0.040, 0.100, 0.400, 0.800,
1.600, and 2.000 µg/mL ethambutol. Standard curves were con-
structed by plotting a 1/y weighted least-squares linear regression
of ethambutol to the internal standard peak-area ratios versus the
spiked concentration of ethambutol.
Preparation of plasma standards and samples
In order to ensure consistency of recovery, 200 µL of acetoni-
internal standard was added to 0.2 mL plasma standards and sam-
ples. After vortexing, an additional 0.2 mL of the internal standard
solution was added. After vortexing and then centrifuging for
5 min at 1800 × g, the solvent phase was transferred to a 400-µL
microfuge tube, and 2.0 µL were injected onto the HPLC system.
Preparation of BAL supernatants and AC pellet standards
and samples
A cell count and differential was performed on the BAL lavage
fluid, then a 30-mL aliquot was centrifuged at 400 × g for 5 min
and the supernatant immediately separated from the cells. BAL
mL of the internal standard solution (0.015 µg/mL neostigmine
or 0.150 µg/mL propranolol) to 0.25 mL of the sample, vortexing,
and then centrifuging for 5 min at 1800 × g. The solvent phase
was transferred to a 400-µL microfuge tube, and 2.0 µL were
injected onto the HPLC system.
ACs were resuspended volumetrically in deionized water and
sonicated for 2 min on a Fisher 550 dismembrator (Fisher
Scientific, Santa Clara, CA) to lyse the cells. A 250-µL volume of
µL of an AC cell suspension and vortexed. A 250-µL volume of ace-
tonitrile was added and mixed by vortexing. Following centrifu-
gation for 5 min at 1800 × g, 2 µL of the solvent phase was
injected onto the HPLC system.
Preparation of controls for method validation
Two sets of stock solutions were prepared; one was used for
spiking standards and the other for spiking controls. Measured
amounts of plasma were spiked at 0.15, 0.4, 0.8, and 1.4 µg/mL;
aliquoted; and frozen at –70°C for stability studies. Aliquots were
115
 Journal of Chromatographic Science, Vol. 40, February 2002

analyzed in duplicate weekly over a period of six weeks. In order
to assess interday reproducibility, standard curves with controls
spiked at concentrations of 0.1, 0.3, 1.2, and 2.4 µg/mL were ana-
lyzed on five different days. Intraday reproducibility was assessed
by analyzing six preparations of each of the four concentrations
on the same day. The validation for BAL supernatants was carried
out in the same time frames as for plasma, with controls spiked at
concentrations of 0.015, 0.04, 0.16, and 0.24 µg/mL. The valida-
tion for ACs was performed at concentrations of 0.010, 0.40, and
1.60 µg/mL.
tration) (13), and percentage accuracy (14) were calculated. The
detection limit was defined as the lowest point of the standard
curve. Drug concentrations in epithelial lining fluid (ELF) were
calculated using the urea diffusion method, and AC concentra-
tions were calculated using cell counts in alveolar fluid as we have
previously reported (15–17).
Results and Discussion

Statistics Linearity, assay precision, recovery, and accuracy assessments

The statistical analysis was performed using the PROPHET HPLC–MS–MS chromatograms of ethambutol and the internal
Computer Resource (12). Linearity (r2), precision (coefficient of standard in plasma, BAL supernatant, and AC suspension are
variation, CV), recovery (relation of test result to the true concen- shown in Figures 5–10. The detection limits for ethambutol were
0.05 µg/mL for plasma and 0.005 µg/mL for the BAL supernatants

A
A

B B

Figure 7. Chromatograms of blank BAL supernatant: (A) the internal standard Figure 9. Chromatograms of blank AC suspension: (A) the internal standard and
and (B) ethambutol. (B) ethambutol.

A A

B B

Figure 8. Chromatograms of a study subject’s BAL supernatant obtained 4 h Figure 10. Chromatograms of a study subject’s AC suspension obtained 4 h
after the fifth dose of 15 mg/kg ethambutol administered once a day: (A) the after the fifth dose of 15 mg/kg ethambutol administered once a day: (A) the
internal standard and (B) ethambutol. The ethambutol concentration was 0.053 internal standard and (B) ethambutol. The ethambutol concentration was 0.316
µg/mL. µg/mL.

116
Journal of Chromatographic Science, Vol. 40, February 2002

and AC suspensions. The detection limit referred to the lowest 12.67% ± 4.59% (ranging from 6.0% to 20.0%), respectively
point of the standard curve and was at least five times the noise (Tables I–III).
level. The mean ± SD of the r2 from 24 standard curves (8 in The mean (± SD) recoveries and the ranges of the assays for
plasma, 8 in BAL, and 8 in ACs) was 0.9941 ± 0.0060. Results for intraday and interday determinations together in plasma, BAL
the assay precision, recovery, and accuracy assessments in the supernatants, and ACs were 105.91% ± 7.73% (ranging from
plasma, BAL, and AC suspensions are summarized in Tables I–III. 93.3% to 119.0%), 95.94% ± 10.43% (ranging from 80.0% to
106.88%), and 105.48% ± 3.60% (ranging from 100.00% to
CV 110.00%), respectively (Tables I–III). The accuracy ranges for all
The mean (± SD) CVs and the ranges of the assay for intraday of the determinations in plasma, BAL supernatants, and ACs were
and interday determinations together for plasma, BAL super- –6.67% to 19.0%, –20.0% to 6.88%, and 0.0% to 10.0%, respec-
natants, and ACs were 7.81% ± 2.02% (ranging from 3.9% to tively (Tables I–III).
10.14%), 6.46% ± 3.69% (ranging from 1.42% to 11.42%), and
Stability
Table I. Assay Precision, Recovery, and Accuracy for The results of repeated determinations of ethambutol in spiked
Ethambutol Determination in Plasma plasma, BAL supernatants, and ACs stored at –70°C revealed no
significant degradation of the drug. These determinations were
Measured performed over a period of 4 mo for plasma, 7 weeks for BAL
Spiked concentration
concentration (mean ± SD) Recovery* Accuracy†
Table III. Assay Precision, Recovery, and Accuracy for
(µg/mL) (µg/mL) CV (%) (%) (%)
Ethambutol Determination in Alveolar Cells
Intraday‡ (n = 6)
Measured
2.4 2.24 ± 0.227 10.1 93.33 –6.67
Spiked concentration
1.2 1.30 ± 0.111 8.6 108.33 8.33
concentration (mean ± SD) Recovery* Accuracy†
0.3 0.32 ± 0.012 6.1 106.67 6.67
(µg/mL) (µg/mL) CV (%) (%) (%)
0.1 0.12 ± 0.011 9.2 119.00 19.00
Intraday‡ (n = 6)
Interday§ (n = 10)
1.600 1.643 ± 0.099 6.0 102.69 2.69
2.4 2.35 ± 0.168 7.2 97.92 –2.08
0.400 0.423 ± 0.053 12.6 105.75 5.75
1.2 1.26 ± 0.114 9.1 105.00 5.00
0.010 0.010 ± 0.001 14.5 100.00 0.00
0.3 0.33 ± 0.013 3.9 110.00 10.00
0.1 0.107 ± 0.009 8.3 107.00 7.00
Interday§ (n = 10)
* Measured/spiked × 100%. 1.600 1.707 ± 0.207 12.1 106.69 6.69
† (Measured – spiked)/spiked × 100%.
0.400 0.431 ± 0.047 10.8 107.75 7.75
‡ Six separately spiked samples at each of four concentrations.
§ Plasma spiked at four concentrations and analyzed in duplicate on five different days. 0.010 0.011 ± 0.002 20.0 110.00 10.00

* Measured/spiked × 100%.
† (Measure – spiked)/spiked × 100%.
‡ Six separately spiked samples at each of three concentrations.
Table II. Assay Precision, Recovery, and Accuracy for § Plasma spiked at three concentrations and analyzed in duplicate on five different days.

Ethambutol Determination in BAL Supernatant

Measured
Spiked concentration
Table IV. Ethambutol Concentrations* in Plasma, ELF, and
concentration (mean ± SD) Recovery* Accuracy†
AC in Five Adult Volunteer Subjects
(µg/mL) (µg/mL) CV (%) (%) (%)
Subject Subject Subject Subject Subject
Sample #1 #2 #3 #4 #5
Intraday‡ (n = 6)
0.240 0.248 ± 0.004 1.4 103.33 3.33
Plasma
0.160 0.171 ± 0.004 2.2 106.88 6.88
(2 h after fifth dose†) 3.41 1.79 1.15 1.75 0.86
0.040 0.040 ± 0.002 6.2 100.00 0.00
Plasma
0.015 0.012 ± 0.001 4.4 80.00 –20.0
(4 h after fifth dose) 4.99 1.15 2.11 1.90 2.39
ELF‡
Interday§ (n = 12)
(4 h after fifth dose) 3.61 1.14 3.05 1.80 2.51
0.240 0.238 ± 0.023 9.9 99.17 –0.83
AC§
0.160 0.165 ± 0.011 6.4 103.13 3.13
(4 h after fifth dose) 64.82 18.92 59.98 108.9 35.42
0.040 0.038 ± 0.004 11.4 95.00 –5.0
0.015 0.012 ± 0.001 9.8 80.00 –20.0 * All concentrations are given in micrograms per milliliter.
† A single oral daily dose of 15 mg/kg was given for 5 days.

* Measured/spiked × 100%. ‡ The amount of ELF collected in the BAL fluid was calculated from the urea
† (Measured – spiked)/spiked × 100%. concentration in BAL and serum, as previously reported (15–17).
‡ Six separately spiked samples at each of four concentrations. § The concentration of ethambutol in ACs is given as micrograms per milliliter of
§ Plasma spiked at four concentrations and analyzed in duplicate on six different days. cell volume and was calculated as previously reported (15–17).

117

supernatant, and 9 mo for ACs (data not shown). The mean (± SD)
CV of the stability studies at four concentrations in plasma and
BAL supernatant were 8.38% and 7.36%, respectively. Repeat
analyses of BAL pellets from four study subjects resulted in a
mean (± SD) CV of 0.10%.
Patient data
The concentrations of ethambutol in plasma, BAL supernatant,
and ACs in five of forty subjects who participated in an NIH-sup-
ported study of the intrapulmonary pharmacokinetics of ethamb-
utol are summarized in Table IV. Bronchoscopy and BAL were
performed, and blood was drawn at 4 h following the last dose of a
5-day course of 15 mg/kg ethambutol. Blood samples were also
obtained 2 h after the last dose. From this preliminary analysis, it
can be seen that ethambutol concentrations in plasma ranged from
0.86 to 3.41 µg/mL at 2 h and 1.15 to 4.99 µg/mL at 4 h after the last
dose was administered. The concentrations in ELF ranged from
1.14 to 3.61 µg/mL and in AC ranged from 18.92 to 108.9 µg/mL.
Conclusion
We have developed a sensitive HPLC–MS–MS assay that pro-
vides specific, rapid, and reliable determinations for ethambutol
in small volumes of plasma, BAL, and AC. The preparation of
plasma, BAL supernatant, and AC samples requires a depro-
teinization step. The stability data indicated that no significant
drug degradation occurred in plasma, BAL supernatant, or ACs
stored at –70°C over a period of 4 mo, 6 weeks, and 9 mo, respec-
tively. The linearity of the standard curve in the range described
was excellent. Assay precision was high for plasma, BAL, and ACs.
The performance characteristics of this assay make the method
suitable for clinical and pharmacological studies, particularly
those that are designed to quantitate the intrapulmonary concen-
tration of drugs.
This method is currently being used to support phase-one
studies of the pulmonary pharmacokinetics of ethambutol in
patients with tuberculosis and normal volunteers. In this prelim-
inary analysis, ethambutol concentrations in plasma and ELF
appear to be similar (i.e., the drug diffuses passively from plasma
into ELF). Ethambutol concentrations are considerably greater
in the AC than in plasma or ELF, indicating that ethambutol is
concentrated in ACs. This finding may be of importance in the
treatment of tuberculosis, which is an intracellular infection. A
complete analysis of this pharmacokinetic study will be published
elsewhere.
Acknowledgments
This work was carried out with funds provided by NIH Grant
#AI36054 and NIH Grant #5 MO1 RR-00079 (General Clinical
Research Center) at the University of California, San Francisco.
118
Journal of Chromatographic Science, Vol. 40, February 2002
The authors would like to thank Ganfeng Wong for assay devel-
opment, Margareta Andersson for performing the assays, and Eve
Benton for manuscript preparation.
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Manuscript accepted December 7, 2001.