JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom.

2001; 36: 12691280

Effect of eluent on the ionization efciency of avonoids by ion spray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization mass spectrometry
Jussi-Pekka Rauha,1 Heikki Vuorela1 and Risto Kostiainen2,3
1 2 3

Department of Pharmacy, Division of Pharmacognosy, PO Box 56, FIN-00014, University of Helsinki, Finland Department of Pharmacy, Division of Pharmaceutical Chemistry, FIN-00014, University of Helsinki, Finland Department of Pharmacy, Viikki Drug Discovery Technology Center (DDTC), University of Helsinki, Finland

Received 22 May 2001; Accepted 27 August 2001; Published online 11 October 2001

The effect of nine different eluent compositions on the ionization efciency of ve avonoids was studied using ion spray (IS), atmospheric pressure chemical ionization (APCI), and the novel atmospheric pressure photoionization (APPI), in positive and negative ion modes. The eluent composition had a great effect on the ionization efciency, and the optimal ionization conditions were achieved in positive ion IS and APCI using 0.4% formic acid (pH 2.3) as a buffer, and in negative ion IS and APCI using ammonium acetate buffer adjusted to pH 4.0. For APPI work, the eluent of choice appeared to be a mixture of organic solvent and 5 mM aqueous ammonium acetate. The limits of detection (LODs) were determined in scan mode for the analytes by liquid chromatography/mass spectrometry using IS, APCI and APPI interfaces. The results show that negative ion IS with an eluent system consisting of acidic ammonium acetate buffer provides the best conditions for detection of avonoids in mass spectrometry mode, their LODs being between 0.8 and 13 M for an injection volume of 20 l. Copyright 2001 John Wiley & Sons, Ltd.

KEYWORDS: avonoids; ion spray; APCI; APPI; buffer

INTRODUCTION
Flavonoids are a large group of polyphenolic natural products that are universally distributed in higher plants. The core structure of avonoids is a 2-phenylbenzopyranone, and the individual compounds differ from each other in the degree of unsaturation, pattern of hydroxylation, and type of linked sugar. Almost 6500 different avonoids are known.1 In addition to antioxidative activity, avonoids have multiple molecular targets, such as cyclo-oxygenase, oestrogen receptors and tumours.2 The best avonoid sources in the Western diet are onions, tea and red wine, and as dietary components they have, for example, been shown in epidemiological studies to prevent coronary heart disease.3 Since avonoids exist in complex natural matrices, selective and sensitive analytical methods are required. Several separation techniques, such as gas chromatography
Correspondence to: R. Kostiainen, Department of Pharmacy, Division of Pharmaceutical Chemistry, PO Box 56 (Viikinkaari 5E) FIN-00014, University of Helsinki, Finland. E-mail: risto.kostiainen@helsinki. Contract/grant sponsor: Graduate School in Pharmaceutical Research (Ministry of Education, Finland). Contract/grant sponsor: Tekes Technology Development Centre (Finland). Contract/grant sponsor: Finnish Ministry of Agriculture and Forestry.

(GC),4 thin-layer chromatography (TLC)5 and capillary electrophoresis (CE)6 have been applied. To date, however, the most widely used analytical method for avonoids is reversed-phase high-performance liquid chromatography (RP-HPLC) connected with UV/Vis or uorescence detection.7,8 However, the specicity of these techniques alone, except UV photodiode array detection, is not sufcient for unambiguous identication of various types of avonoid, and different kinds of mass spectrometry (MS) method have therefore been developed. Mass spectrometric detection combined with chromatography provides a wealth of structural information.9 Older LC/MS techniques such as moving belt (MB) and continuous-ow fast atom bombardment (CF-FAB), have been used in the analysis of avonoids,10,11 as well as GC/MS and electron impact (EI) MS.12 15 However, these methods have met a number of problems, which have been resolved by the use of atmospheric pressure ionization (API) systems. Among these techniques, recent interest has focused on electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and they have frequently become the method of choice for avonoids.16 18 A new ionization method for LC/MS, atmospheric pressure photoionization (APPI), has recently been introduced by Bruins and co-workers et al.19 Ionization is based on charge transfer to the analytes from dopant molecules (e.g. toluene)

DOI: 10.1002/jms.231

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that have been ionized using 10 eV photons produced by a vacuum-ultraviolet lamp. Photoionization is performed in a modied heated nebulizer (HN) APCI source. The use of APCI and ESI has been widely accepted for the analysis of avonoids.20 23 Owing to the soft ionization of APCI and ESI, the avonoid glycosides can be detected as protonated or deprotonated molecules together with diagnostic fragment ions. Since APCI and ESI offer easy operation and high sensitivity, they have replaced FAB techniques, which were used earlier for the analysis of avonoids.24,25 The effect of structure factors, such as the location of hydroxyl groups and sugar moieties, in relation to the mass spectra of avonoids has been studied using ESIMS26 and APCI-MS.27 The limit of detection for avonoids in full scan MS mode has been reported to vary from 0.1 to 40 ng per injection by ESI,22,28 which is reported to be more sensitive than APCI.29 However, when APCI is used in the single ion monitoring mode, a limit of quantication of as low as 10 pg per injection has been reached.30 When making a choice between different MS polarities and LC eluent conditions, the basic question is not only the detection sensitivity but also the structural information content of mass spectra or the intensity of the (de)protonated molecule signal when subsequent MS/MS is performed. Thus, in the literature on LC/MS applications, positive ion ESI has been preferred with very different aqueous phase solvent compositions: 1% formic acid,22 0.05% triuoroacetic acid (CF3 COOH)26 and 2 mM ammonium acetate.29 ESI in negative ion mode has been the choice with a 5% addition of formic acid in the eluent.20 For the same group of avonoids, 0.05% TFA as the aqueous phase21 in positive ion APCI, and 1% formic acid23 or 1% acetic acid27 in negative ion APCI have been used. The numerous applications of LC/ESI-MS and LC/APCI-MS indicate the suitability of these techniques for the analysis of avonoids. However, no detailed comparative study has been carried out on different API techniques and the effect of solvent on the ionization and detection sensitivity of avonoids. This work compares the applicability of current API techniques: IS, i.e. pneumatically assisted electrospray, and APCI, with a new ionization method, APPI, in the detection of avonoids. We present a comprehensive study on the effects of nine different HPLC mobile phase compositions on the ionization efciency of ve selected analytes.

and CF3 COOH from Fluka Chemie AG (Buchs, Switzerland) was protein-sequence analysis grade. Water was puried in a Milli-Q water purication system (Millipore, Molsheim, France).

Samples
The test mixture used for the solvent optimization studies contained (C)-catechin and isorhamnetin in a concentration of 0.2 mM, and vitexin, isoquercitrin and luteolin-30 ,7diglucoside in a concentration of 0.1 mM. For direct inlet studies with APPI, the former mixture was diluted to 1/5 with 40% of CH3 OHCH3 CN (3 : 4) and 60% of aqueous eluent (Table 1). For the determination of detection limits, samples were diluted to appropriate concentrations. The pKa values of the analytes were estimated using an ACD/ILab computer program (version 4.0, ACD Labs, Toronto, ON, Canada) in pKa prediction mode.

Selection of HPLC conditions

The chromatographic optimum for the mixing ratio of the organic solvents was selected using a chemometric LC method optimization program, Turbo Method Development (Perkin Elmer, Norwalk, CT, USA), according to the normal operating procedure presented by Rauha et al.31 The equipment used in optimizing the mixing ratio consisted of a Perkin Elmer Series 200 LC pump and autosampler (Norwalk, CT, USA), Perkin Elmer LC 235C diode array detector at the scanning wavelength of 195365 nm (Norwalk, CT, USA), and PE Nelson 600 series link (Norwalk, CT, USA). The system was controlled by means of a Digital Venturis 575 computer.

HPLC/MS instrumentation
LC/IS-MS and LC/APCI-MS conditions were optimized using chromatographic runs, and APPI-MS conditions were optimized using loop injections. The comparison of sensitivity between different methods was carried out in optimized conditions using HPLC/MS runs and determining the limits of detection for each analyte.
Table 1. Aqueous buffers used in solvent optimization studies. The organic component consisted of methanol and acetonitrile (3 : 4) Solvent pH 2.0 2.3 2.6 3.3 4.0 5.0 6.0 6.7 8.0 7.6 11.0 Preparation 0.05% CF3 COOH (v/v) 0.4% formic acid (v/v) 0.1% formic acid (v/v) pH adjusted with formic acid 10 mM ammonium acetatea 10 mM ammonium acetatea 10 mM ammonium acetatea 10 mM ammonium acetate 10 mM ammonium acetateb water 0.1% ammonium hydroxide (v/v)

EXPERIMENTAL Chemicals
(C)-Catechin (C-1251) was purchased from Sigma Chemical Co. (St Louis, USA), rutin from Merck (Darmstadt, Germany), and luteolin-30 ,7-di-O-glucoside (0085 S), vitexin (1232 S), isoquercitrin (1119 S) and isorhamnetin (0065) from Extrasynth se (Genay, France). The organic solvents used e [methanol (CH3 OH), acetonitrile (CH3 CN), tetrahydrofuran (THF) and toluene] were HPLC grade and obtained from Rathburn (Walkerburn, Scotland). Ammonium acetate, 25% ammonium hydroxide solution and dimethylsulfoxide (DMSO) from Merck (Darmstadt, Germany) were analytical grade; formic acid from Merck was Suprapur quality,

A B C D E F G H I J K
a b

pH adjusted with formic acid. pH adjusted with ammonium hydroxide.

Copyright 2001 John Wiley & Sons, Ltd.

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Separations were performed on a Prodigy ODS (3) column (100 4.6 mm I.D., Phenomenex, Torrance, CA, USA) with a particle size of 3 m and pore size of 10 nm. The chromatographic instrumentation consisted of Perkin Elmer Series 200 autosampler and two Series 200 Micro LC pumps (Norwalk, CT, USA). An efuent ow splitter (Acurate, LC Packings, Zurich, Switzerland) was used for the LC/IS-MS (1/100) and LC/APPI-MS (1/10) analyses. For loop injections (Rheodyne, Cotati, CA, USA), a constant ow of eluent was delivered with a Harvard Apparatus micro syringe pump (Harvard Apparatus, So. Natick, MA, USA). A PE Sciex API 300 LC/MS/MS triple quadrupole mass spectrometer (Sciex, Concord, ON, Canada), equipped with an IS, HN (APCI), and APPI interface, was used. The APPI interface (Machine Shop, University of Groeningen, Netherlands) has been described in detail by Bruins and co-workers.19 Nitrogen, produced with a Whatman 75-720 nitrogen generator (Whatman Inc., Haverhill, MA, USA), was used as curtain and auxiliary gas, and high-purity air (99.998%) was used as nebulizing gas. Mass spectra were acquired over the scan range of m/z 200700, using a step size of 0.2 Da and dwell time of 0.4 ms. MS and UV data were handled and the whole apparatus controlled by a Macintosh Power PC computer (Apple Computer Inc., Ireland). The instrumental conditions chosen are given in Table 2. LC/IS-MS and LC/APCI-MS gradient elution at a ow rate of 1 ml min 1 was performed as follows: 02 min 5% organic (isocratic), 212 min 595% organic (linear gradient), and 1215 min 95% organic (isocratic). The organic eluent consisted of CH3 OH and CH3 CN (3 : 4), whereas the whole series of different aqueous eluents was subjected to experimentation (AI, Table 1). For solvent selection for APPI, ve different solvents covering a wide pH range (C, E, H, J and K in Table 1) were tested. The constant solvent ow for loop injections consisted of 40% organic solvent (CH3 OH and CH3 CN 3 : 4) and 60%

aqueous component. The experiments were carried out using the operating conditions shown in Table 2. The samples prepared in the same solvents as the eluents were injected via a 50 l sample loop and analysed without chromatographic separation. Spectral data were collected from m/z 250 to 650 (step 0.100 Da and dwell time 0.5 ms), and the results were calculated as the sum of ve scans. High-purity nitrogen (99.998%) was used as lamp gas. An equimolar avonoid sample and the HPLC/MS gradient shown above were used to compare the three ionization techniques, as well as to determine the limits of detection for each optimized method. A signal-to-noise value of S/N D 3 was used as the boundary of acceptance.

RESULTS AND DISCUSSION Composition of the test solution and selection of the chromatographic method
The compounds in the test mixture represented three subgroups of avonoids: avonols (isorhamnetin and isoquercitrin), avones (vitexin and luteolin-30 ,7-diglucoside) and catechins (Fig. 1), and covered a wide range of polarity, thus making gradient elution a necessity. The calculated pKa values varied from about 6.6 to 9.5 (Fig. 1), and were in accordance with the reference values.32,33 The eluent compositions tested with IS, APCI and APPI are presented in Table 1. The compositions were selected for this study on the basis of their suitability for LC. The best isocratic separation was found with an CH3 OH:CH3 CN ratio of 3 : 4, and the organic phase was therefore similar in every eluent composition. The broad gradient of 5 to 95% organic solvent, which is not necessary in this particular case but which is normally required in phytochemical analyses, was used to increase the applicability of the results to the analysis of plant samples. The pH modiers satised the volatility requirements, and covered the pH range within

Table 2. Major operating parameters of the interfaces used in the solvent optimization studies ESI Parameter Sample introduction Injection volume (l) Flow rate to mass spectrometer (l min 1 ) Dopant ow rate (l min 1 ) Spray voltage (kV) Orice voltage (V) Needle current (A) Nebulizer temperature Nebulizing gas ow (l min 1 ) Nebulizing gas pressure (bar) Auxiliary gas ow (l min 1 ) Curtain gas ow (l min 1 ) Lamp current (mA) Lamp gas (l min 1 ) Pos HPLC 10 10 4.3 40 Neg HPLC 10 10 3.8 50 Pos HPLC 10 1000 APCI Neg HPLC 10 1000 Pos DI 50 200 25 1.3 30 300 5.6 2 1.6 0.75 1 APPI Neg DI 50 200 25 1.3 30 300 5.6 2 1.8 0.75 1

26 3.5 400 5.6 2.8 2.8

41 3.5 400 5.6 2.8 2.8

1.8

LC/APCI-MS in determination of detection limits: nebulizer temperature, 450 C. LC/APPI-MS in the determination of detection limits: sample introduction by HPLC; ow rate to mass spectrometer, 100 l min 1 ; dopant ow rate, 12.5 l min 1 ; nebulizer temperature, 350 C.

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OH 3 4 OH 7 5 O 2 3 OH OH OH O Isorhamnetin, MW=316 pKa =6.890.60 OH OH O

OCH3 OH

OH (+)Catechin, MW=290 pKa =9.500.10

OH OH Glu OH O OH O Glu O O OH Vitexin, MW=432 pKa =6.690.40 Glu O OH O Glu O OH O Isoquercitrin, MW=464 pKa =6.830.60 OH

OH

Luteolin-3,7-diglucoside, MW=610 pKa =6.640.40

Figure 1. Structures, nominal molecular weights and calculated pKa values of the avonoids used in the study.

the stability limits for the silica-based bonded stationary phase. The use of different aqueous eluents (AH) produced the same elution order and almost equal chromatographic resolutions, but the use of eluent I (pH 8.0) resulted in the co-elution of luteolin-30 ,7-diglucoside and vitexin.

Positive ion IS
The effects of eluent composition on the absolute abundance of the peak areas of the total ion chromatograms of the avonoids are presented in Fig. 2(a). The rst-order IS mass spectra show abundant [M C H]C ions, sodium adduct and some fragment ions for the partial identication of the avonoids under the operating conditions used in this work (Table 3). The composition of the eluents has no signicant effect on the relative abundance of the ions. It is worth noting that the spectra show only very weak ammonium adduct ions with eluents that contained ammonium acetate or ammonium hydroxide. Figure 2(a) shows that the best sensitivity was obtained with aqueous phase B, which included 0.4% formic acid (pH 2.3). A decrease in the concentration of formic acid (eluents BD) decreased the sensitivity for all the avonoids

studied. Since all avonoids are acids and their pKa values in water are between 6.6 and 9.5 (Fig. 1), they can be expected to be in a neutral form in the liquid phase, including at low pH values. It follows, therefore, that protonation of the avonoids occurs at the liquidgas interface of the droplet, i.e. in the incipient gas phase or via a proton transfer reaction in the gas phase, and the ionization efciency is dependent on the proton afnity (PA) of the avonoid. According to results for four avonoids presented by Alem n,34 the most a favoured site for protonation is the carbonyl oxygen, the PA of which is higher than that of ammonia. Since all the avonoids studied here are structurally close to those studied by Alem n, and include a conjugated carbonyl oxygen apart a from catechin, it can be concluded that their PAs are higher than or close to that of ammonia. The avonoids studied here are, therefore, fairly efciently protonated by all of the eluents. The PA of catechin (which lacks a carbonyl oxygen) is also high enough for efcient protonation, even though the absolute abundances measured for catechin were slightly lower those with the other avonoids. The sensitivity with eluent A (pH 2.0, adjusted with CF3 COOH) was signicantly lower than that with eluent

Copyright 2001 John Wiley & Sons, Ltd.

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(+)Catechin
25 20 15 50 40 30 20 10 A BCDE FGH I 0

Isorhamnetin
60 50 40 30

Vitexin
70 60 50 40 30 20 10 A BCDE FGH I 0

Isoquercitrin
30 25 20 15 10 5 A BCDE FGH I 0

Luteolin-3,7diglucoside

a
10 5 0

20 10 A BCDE FGH I 0

A BCDE FGH I

(+)Catechin
25 20 15 90 80 70 60 50 40 30 20 10 0 A BCDE FGH I

Isorhamnetin
7 6 5 4 3 2 1 0 A BCDE FGH I

Vitexin
7 6 5 4 3 2 1 0 A BCDE FGH I

Isoquercitrin
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 A BCDE FGH I

Luteolin-3,7diglucoside

10 5 0

A BCDE FGH I

(+)Catechin
35 30 25 140 120 100 80 60 40 20 0 C E H J K

Isorhamnetin
30 25 20 15 10 5 0 C E H J K

Vitexin
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 C E H J K

Isoquercitrin
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 C E H J K

Luteolin-3,7diglucoside

20 15 10 5 0

C E H J K

Figure 2. (a) Sum areas of ion chromatogram peaks of ions listed in Table 3 in the positive ion IS analyses using different eluent compositions. (b) Sum areas of ion chromatogram peaks of ions listed in Table 3 in the positive ion APCI analyses using different eluent compositions. (c) Intensities of protonated molecules in the positive ion APPI direct inlet analyses using different eluents (unit: 1 000 000 counts).

B (pH 2.3, adjusted with formic acid) even though the pH of eluent A was lower [Fig. 2(a)]. This may indicate that the counter ion [CF3 COO] has a stronger tendency to neutralize positive charges than the [HCOO] ion. The decreased sensitivity obtained with TFA has also been reported in earlier studies with a number of thiols,35 drugs36 and proteins.37 However, the decreased sensitivity with decreased concentration of formic acid (eluents BD) shows that the pH must be low enough for efcient ionization of avonoids in positive ion ion spray. The absolute abundances measured with eluents EI (pH 4.08.0), which had the same concentrations of ammonium acetate, were lower than those with B, and no significant differences were found for eluents EI. These results show that addition of ammonium ions decreases the sensitivity. Interestingly, the basic eluent I, which included ammonium acetate and ammonium hydroxide (pH 8), gave the same sensitivity as eluents EH. At pH 8, most of the avonoids are almost totally negatively charged in the liquid

phase, and a decreased sensitivity would be expected at pH 8. However, owing to the transfer of hydroxide ions from the eluent as a result of the high voltage at the tip of the sprayer and the evaporation of ammonia during the IS ionization process, it can be concluded that the pH in the droplets emitting gas-phase ions is signicantly lower than that of the eluent. Gatlin and Turecek,38 and later van Berkel and co-workers,39 reported that, in positive ion electrospray, acidity may be enhanced 103 104 -fold at the outer layer of the droplets. It follows, therefore, that the avonoids are neutralized in the small electrosprayed droplets even though they are negatively charged in the sample at pH 8. The protonation at pH 8 may also occur in the gas phase by proton transfer from ammonium ions, or by protons generated by solvent oxidation. This phenomenon, called wrong-way-round ionization, was also reported by Zhou and Cook in a recent paper.40 In conclusion, the use of a sufciently acidic eluent gives the best sensitivity in positive ion IS, although the use of ammonia makes the system robust and less dependent on

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Table 3. Mass spectrometric data of the avonoid spectra produced in optimized conditions; m/z (relative abundance). Buffers used in eluent: 0.4% formic acid [positive ion (PI) IS and APCI], 10 mM ammonium acetate adjusted to pH 4.0 [negative ion (NI) IS], 20 mM ammonium acetate adjusted to pH 4.0 (negative ion APCI), 5 mM ammonium acetate (APPI) Interface IS Ion mode PI PI PI PI PI NI NI NI NI NI PI PI PI PI PI NI NI NI NI NI PI PI PI PI PI NI NI NI NI NI Compound (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside (C)-catechin isorhamnetin vitexin isoquercitrin luteolin-30 ,7-diglucoside Ions (relative abundance) 581b (3), 344u (7), 313c (3), 291a (100), 273d (3) 339c (2), 317a (100), 302e (4) 455c (2), 433a (100), 415d (17), 397f (9), 343g (3), 313h (18), 283i (5) 519u (4), 487c (4), 465a (10), 303j (100) 633c (3), 611a (100), 449j (19), 287k (3) 617u (5), 579m (8), 327u (20), 311u (8), 289l (100), 245u (11) 631m (2), 315l (100), 300o (76) 431l (85), 341p (10), 311q (100), 283u (10) 463l (100), 301r (32), 300u (51) 609l (78), 447r (100), 285t (9) 332u (4), 291a (100), 273d (4) 317a (100), 302e (3) 433a (100), 415d (18), 343g (4), 313h (28), 295u (10), 283i (6) 465a (10), 303j (100) 611a (83), 449j (80), 287k (100) 335n (33), 289l (100), 245u (65) 361n (4), 315l (100), 300o (64) 431l (46), 341p (13), 311q (100), 283u (18) 463l (86), 301r (78), 300u (100) 655n (3), 609l (44), 493s (5), 447r (100), 285t (58) 291a (100) 317a (100) 433a (100), 415d (3), 313h (4) 465a (15), 303j (100) 611a (45), 449j (100), 287k (4) 289l (100), 245u (4) 315l (100), 300o (10) 431l (100), 341p (2), 311q (15) 463l (100), 301r (13), 300u (4) 609l (100), 447r (69), 285t (13)

APCI

APPI

[M C H]C . [2M C H]C . c [M C Na]C . d [M C H H2 O]C . e [M C H CH3 ]C . f [M C H 2H2 O]C . g [M C H C3 H6 O3 ]C . h [M C H C4 H8 O4 ]C . i [M C H C5 H10 O5 ]C . j [M C H Glu]C . k [M C H Glu Glu]C . l [M H] . m [2M H] . n [M H C HCOOH] . o [M H CH3 ] . p [M H C3 H6 O3 ] . q [M H C4 H8 O4 ] . r [M H Glu] . s [M H C HCOOH Glu] . t [M H Glu Glu] . u Not identied.
b

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(+)Catechin
70 60 50 100 80 60 40 20 C D E F G H I 0

Isorhamnetin
90 80 70 60 50 40 30 20 10 0

Vitexin
80 70 60 50 40 30 20 10 0

Isoquercitrin
60 50 40 30 20 10 C D E F G H I 0

Luteolin-3,7diglucoside

40 30 20 10 0

CD E F GH I

C D E F G H I

C D E F G H I

(+)Catechin
25 20 180 160 140 120 100 80 60 40 20 0

Isorhamnetin
18 16 14 12 10 8 6 4 2 0

Vitexin
8 7 6 5 4 3 2 1 0

Isoquercitrin
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 C D E F G H I

Luteolin-3,7diglucoside

15 10 5 0 C D E F G H I

C D E F GH I

C D E F G H I

C D E F G H I

(+)Catechin
70 60 50 160 140 120 100 80 60 40 20 0 C E H J K

Isorhamnetin
70 60 50 40 30 20 10 0 C E H J K

Vitexin
12 10 8 6 4 2 0 C E H J K

Isoquercitrin
1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 C E H J K

Luteolin-3,7diglucoside

40 30 20 10 0

C E H J

Figure 3. (a) Sum areas of ion chromatogram peaks of ions listed in Table 3 in the negative ion IS analyses using different eluent compositions. (b) Sum areas of ion chromatogram peaks of ions listed in Table 3 in the negative ion APCI analyses using different eluent compositions. (c) Intensities of deprotonated molecules in the negative ion APPI direct inlet analyses using different eluents (unit: 1 000 000 counts).

the pH. The chromatographic behaviour of the avonoids was also good when the most acidic solutions were used as aqueous eluents. Although CF3 COOH has no effect on the chromatographic behaviour of the avonoids compared with formic acid, the use of CF3 COOH should be avoided owing to its capacity for neutralizing positive charges.

Negative ion IS
The negative ion mass spectra of the avonoids (Table 3) show [M H] ion as the main peak, with the characteristic fragments [M H Glu] for O-glucosides and [M H 120] for vitexin. In addition to deprotonated molecules, the aglycons produced dimer [2M H] . Isorhamnetin produced the fragment [M H CH3 ] , the abundance of which increased along with the pH. The best sensitivity, especially for (C)-catechin, was obtained using eluent C, which included 0.1% of formic acid in the aqueous phase (pH 2.6) [Fig. 3(a)]. Furthermore, the sensitivity was better at pH 2.6 than at pH 3.3 (eluent

D). Since the pKa values of the avonoids are between 6.6 and 9.5, it can be expected that, in spite of the transfer of protons as a result of the high voltage at the tip of the sprayer and evaporation of formic acid from the droplets, the avonoids are in a neutral form in the small, electrosprayed droplets at pH 2.6. The obvious ionization mechanism at pH 2.6 is a proton transfer reaction between [HCOO] and avonoids at the liquidgas interface of the droplet or in the gas phase. The PAs for the phenoxide anion groups of the avonoids, owing to their electronegative chemical environment, will be lower than those presented for the o-OH-phenoxide anion (1392 kJ mol 1 )41 and for the m-OHphenoxide anion (1421 kJ mol 1 ).41 It follows that the PA of the [HCOO] ion (1415.4 kJ mol 1 )42 is higher than that for the deprotonated avonoids, and, therefore, a proton transfer reaction in the gas phase is thermodynamically possible. With increasing pH values (eluents EI, pH 48) the ion evaporation mechanism becomes more favoured. The results show, contrary to expectations, that the proton transfer

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reaction at a sufciently low enough pH produces more efciently deprotonated molecules than the ion evaporation mechanism at higher pH. The decreased sensitivity with eluents EI may be due to suppression by ammonium acetate or neutralization of negative charges by ammonium ions. This assumption was supported by the experiment in which the effect of ammonium acetate concentration was tested. The absolute abundances of isoquercitrin and isorhamnetin decreased to one half, and that of the other avonoids by 20%, when the buffer concentration was increased from 10 to 20 mM in eluent E. Experiments performed with 5 mM ammonium acetate buffer did not lead to any improvements in sensitivity. The absolute abundances were obtained by measuring the mass range m/z 200700, and then determining and summarizing the peak areas of the ion chromatograms derived from the avonoids. However, when the peak areas of the total ion chromatograms (TICs) are used, a better signal-to-noise ratio was achieved with eluent E than with eluent C, which otherwise gave the best sensitivity with extracted multiple ion chromatograms. This was because the chemical noise with eluent C was signicantly higher than that with eluent E. This demonstrates clearly that background ions play important role in the rst-step identication of unknown avonoids by LC/MS using full scan mode. In conclusion, eluent E is the most suitable for screening unknown avonoids by LC/MS.

Positive ion APCI

The positive ion APCI spectra of all the avonoids (Table 3) show protonated molecules and intense [M C H Glu]C fragment ions. The protonated molecules were base peaks for (C)-catechin, vitexin and isorhamnetin, whereas [M C H Glu]C ions were the base peaks of isoquercitrin and luteolin-30 ,7-diglucoside. The spectra did not show any adduct formation or notable dependence on the eluent used. The results show that the best sensitivity is achieved with aqueous eluents that included only formic acid (eluents BD). The use of 0.05% CF3 COOH (eluent A), instead of eluents BD, leads to decreased sensitivity [Fig. 2(b)]. In positive ion APCI, the components present in eluent A are H2 O, CF3 COOH, CH3 OH, and CH3 CN, for which the PAs are 691 kJ mol 1 , 712 kJ mol 1 , 754 kJ mol 1 and 779 kJ mol 1 , respectively.43 In this reactant gas mixture, protons are transferred to the weakest acid, i.e. to CH3 CN. It follows, therefore, that the addition of CF3 COOH does not signicantly change the composition of protonated gasphase reactant ions, and does not explain the decreased sensitivity with addition of CF3 COOH. However, the decreased sensitivity may be due to the formation of [CF3 COO] ions in positive ion APCI, and their capability to neutralize positive charges. This observation is in good agreement with the results obtained with positive ion IS. Notably better sensitivity was obtained for (C)catechin, isorhamnetin and isoquercitrin with eluents BD (pH 2.33.3), which did not include ammonium acetate, than with eluents EI (pH 4.08.0) that included ammonium acetate [Fig. 2(b)]. However, the sensitivity for vitexin and luteolin-30 ,7-diglucoside did not differ signicantly for

eluents BI. The addition of ammonium acetate changes the composition of the reactant ions (i.e. the eluent ions) in the gas phase, with the result that the initially formed protonated methanol, acetonitrile and water transfer their protons to the weakest acid, i.e. ammonia (PA D 854 kJ mol 1 ),43 and the role of ammonium ions in the ionization process increases. The PAs of catechin and isorhamnetin are obviously only slightly higher than that of ammonia, and the proton transfer reaction with ammonium ion is therefore less exothermic and obviously less efcient than that with the ions derived from the eluents BD, which did not include ammonia. However, the PAs of vitexin and luteolin-30 ,7-diglucoside are obviously higher than those of catechin and isorhamnetin, and the exothermicity of the proton transfer reaction is sufciently large with ammonium ions as well as with ions derived from eluents BD. The lower PA of catechin can be explained by the lack of carbonyl oxygen, which is thermodynamically the most favoured site for protonation (see above). In the case of isorhamnetin, the lower PA may be due to the possible formation of an internal hydrogen bond between the hydroxyl group at C3 and carbonyl oxygen (see Fig. 1). In conclusion, eluent B (containing 0.4% formic acid, and pH 2.3) is recommended for the avonoids in positive ion APCI. The results clearly show better sensitivity with aglycons than with glycosides, most likely due to the fact that the vaporizing temperature of glycosides is higher than that of aglycons. This was supported by the experiments with a nebulizer heated to different temperatures. The sensitivity was clearly increased by raising the temperature from 350 to 450 C, especially for glycosides. The increase in the probe temperature had no signicant effect on the fragmentation. As a result, 450 C was selected as the operating temperature in both APCI modes.

Negative ion APCI

The negative ion APCI spectra of all the avonoids (Table 3) show intense deprotonated molecules and some intense fragment ions. The characteristic fragment ions were [M H CH3 ] for isorhamnetin, as reported to be typical for methoxylated avonoids,44 and [M H 90] and [M H 120] for vitexin, typical for avonoid-Cglycosides.25,45 In general, the [M H] ions were base peaks for the analytes, except for luteolin-30 ,7-diglucoside, the base peak of which was [M H Glu] (m/z 447). Furthermore, formic acid adducts [M H C 46] and [M H C 46 Glu] were recorded with the eluents that included formic acid (CG). The relative abundances of the adduct ions increased with increasing formic acid concentration and polarity of the analyte. In negative ion APCI, the eluent composition has no signicant effect on the ionization efciency of avonoids [Fig. 3(b)]. The proton transfer reaction in negative ion APCI is possible if the PA of the reactant ion is higher than that of the deprotonated analyte. In negative ion APCI the weaker gas-phase bases transfer their protons to stronger bases, e.g. acetic acid transfers its protons to hydroxide ions. The weakest base among the eluent components tested is [HCOO] (PA D 1415.4 kJ mol 1 ).42 Since the PA of the

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Effects of eluent and API in LC/MS of avonoids

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deprotonated avonoids studied will be lower than that of [HCOO] (see section on Negative ion IS above), the proton transfer reaction is exothermic and efcient with all the eluents. The highest responses recorded using a TIC were obtained with eluent E (pH 4.0), although the responses recorded by selected ion chromatograms do not show any signicant differences between the eluents [Fig. 3(b)]. The addition of ammonium acetate to eluent E at concentrations of 550 mM had no signicant effect on the responses. Based on the chromatographic behaviour of the avonoids, 20 mM ammonium acetate adjusted to pH 4.0 with formic acid was found to be the most suitable buffer in negative ion APCI. The absolute abundances for the aglycones were also higher than those for the glycosides, which is in good agreement with the results obtained with positive ion APCI, and conrms that the vaporizing temperature has an effect on the sensitivity (as discussed above).

However, the ionization process is, so far, not very well understood.

Positive ion APPI

All the positive ion APPI spectra show an abundant protonated molecule and no radical cation. This indicates that the main ionization process is a proton transfer reaction between protonated eluent molecules and the avonoids with all the eluents tested (Table 3). There is no adduct formation, and the only fragmentation indicates a loss of glucose residues from glycosidic analytes. Fragmentation of the analytes in positive ion APPI was notably stronger than that in negative ion APPI (Fig. 4). In order to study the effect of dopant, all the measurements were made with and without toluene as a dopant. The sensitivity was 10100 times better with the dopant than without, verifying that the initial reaction in APPI is formation of a radical cation of the dopant. THF (ionization energy: 9.4 eV)43 was also tested as a dopant at a concentration of 10%. The results indicated that THF is suitable for LC/APPI-MS, but the sensitivity for avonoids with toluene was approximately 1.32 times better than that with THF. The effect of the solvent on the ionization efciency of avonoids in positive ion APPI was studied with ve different eluent compositions [Fig. 2(c)]. The results show that the best sensitivity was obtained with pure water (eluent J) as the aqueous eluent. The addition of ammonium acetate slightly decreased the sensitivity (eluent H), and the addition of formic acid (C and E) or ammonium hydroxide (K) signicantly so. The protonated CH3 CN is obviously the main reactant ion with eluent J, since the PA of CH3 CN (779.2 kJ mol 1 )43 is the highest of the components in eluent J and lower than that of the avonoids (see above). This means that proton transfer is exothermic and efcient, which explains the good sensitivity with eluent J. The effect of
3e+5

APPI
APPI is a novel ionization method in LC/MS recently introduced by Bruins and co-workers.19 The interface is based on the same conguration as APCI, but the discharge needle is replaced by a photoionization lamp. Dopant (toluene) is introduced into the vaporizer coaxially at a ow rate about one-tenth that of the efuent ow rate. The ionization energy of the dopant must be lower than the energy of the photons (10 eV) emitted by the photoionization lamp. The initial reaction in APPI is formation of a radical cation of the dopant, which may ionize the analyte through a charge exchange reaction if the recombination energy of the radical cation of the dopant (8.83 eV for toluene) is higher than that of the neutral analyte. Alternatively, the dopant radical cation initializes a process producing protonated eluent molecules, followed by a proton transfer reaction with the analyte.
6e+5

463 2e+5

303

4e+5

2e+5 301 0 200 300 400 500 600 700

1e+5 465 0 200

300

400

500

600

700

m/z
3e+5

m/z
1.5e+5

c
447

609

449

2e+5

1.0e+5 611

1e+5 285 0 200 300 400 500 600 700

5.0e+4 287 0 200 300 400 500 600 700

m/z
Figure 4. APPI-MS spectra of isoquercitrin detected in (a) negative ion and (b) positive ion mode, and spectra of luteolin-30 ,7-diglucoside detected in (c) negative ion and (d) positive ion mode.

m/z

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J.-P. Rauha, H. Vuorela and R. Kostiainen

ammonium acetate addition can be explained using the same arguments as for positive ion APCI. The decreased sensitivity with formic acid and ammonium hydroxide may be due to the formation of [HCOO] and [OH] gasphase ions, which may neutralize the positive charges. The absolute abundances in positive ion APPI are clearly lower for aglycones than for glycosides for the same reasons as discussed in the case of APCI (see above). Based on the LC behaviour of the avonoids, the most suitable eluent composition in LC/MS is eluent with 10 mM ammonium acetate as the buffer, although the highest responses were obtained with pure water.

Table 4. Limits of detection of avonoids (S/N D 3) using the respective ionization techniques. The results are shown in molar concentrations, in accordance with a sample injection volume of 20 l; XIC, band width 1 Da Limit of detection (M) Negative ion TIC IS (C)-Catechin Isorhamnetin Vitexin Isoquercitrin Luteolin-30 ,7-diglucoside APCI (C)-Catechin Isorhamnetin Vitexin Isoquercitrin Luteolin-30 ,7-diglucoside APPI (C)-Catechin Isorhamnetin Vitexin Isoquercitrin luteolin-30 ,7-diglucoside XIC Positive ion TIC XIC

Negative ion APPI

The negative ion APPI spectra of the avonoids (Table 3) show a deprotonated molecule, indicating that the main ionization mechanism is a gas-phase proton transfer reaction. The spectra were composed not only of deprotonated molecules and typical fragments, they also showed adducts of formic acid with eluents C and E, as was the case in negative ion APCI. Furthermore, a very small amount of [2M H] ions of aglycones was detected in negative ion APPI, as in negative ion IS. In negative ion APPI, the highest responses for the aglycones were obtained with pure water (eluent J) [Fig. 3(c)]. The addition of ammonium acetate decreased the sensitivity; however, for glycosides, signicantly higher responses were obtained with ammonium hydroxide (eluent K) than without. This may indicate the deprotonation of hydroxide groups in the glycoside moiety by hydroxide ions under basic conditions. However, with basic eluents the avonoids are ionized and the retention on the reversed-phase C18 column is poor, leading to signicantly decreased resolution in LC. The decreased sensitivity under acidic conditions may be due to neutralization of the negative charges by hydronium ions. Additional LC/APPI-MS runs were carried out in both ion modes using 5, 10 and 20 mM ammonium acetate buffers, and an increase in sensitivity occurred as the buffer concentration decreased. Therefore, it was concluded that, in order to make a compromise between chromatographic behaviour and sensitivity, aqueous ammonium acetate at a concentration of 5 mM (pH 6.7) represented the most suitable eluent system.

12 3.2 0.75 0.85 1.7 13 3.0 2.8 5.5 7.0 11 4.4 3.7 6.0 4.4

4.5 1.1 0.18 0.10 0.32 4.6 2.2 0.50 3.6 4.1 1.1 0.42 0.33 5.5 5.5

46 16 6.5 7.5 2.7 36 5.0 11 12 60 55 15 7.5 17 32

2.9 0.55 0.39 2.4 0.65 1.2 0.17 1.5 2.8 1.7 3.6 0.75 0.65 4.0 4.5

Comparison of the methods under optimized conditions

The IS, APCI and APPI LC/MS methods were compared with respect to detection sensitivity (S/N D 3) and their applicability for the analysis of avonoids with optimized eluent compositions. Extracted ion chromatograms produced with all the API techniques studied are presented in Fig. 5 and the spectra in Table 3. The limits of detection were determined from TICs and extracted ion chromatograms (XICs) (Table 4). As the selected conditions represent a compromise between several factors, such as chromatographic behaviour, individual properties of the analytes, and instrumental settings, broad generalizations must be avoided. XIC data show that there are no signicant differences in ionization efciencies between the positive and negative ion modes.

However, the detection limits by TIC were about two to nine times lower in negative ion modes than in positive ion modes, irrespective of the ionization technique tested (Table 4). Compared with the TIC data, the limits of detection determined from the XICs are 036 times lower, indicating the large variability of chemical noise with regard to different techniques, polarities, and gradient elution conditions. The respective ndings show that the chemical background noise in positive ion mode is stronger than that in negative ion mode. The limits of detection determined from the TICs and XICs in positive ion mode do not differ essentially between the techniques. The only exception is luteolin-30 ,7diglycoside, the detection limit of which is signicantly better using IS than with the other techniques. In negative ion mode, the TIC and XIC limits of aglycones, (C)-catechin and isorhamnetin, do not differ markedly between the techniques, but the detection limits of glycosides are clearly lower using IS than APCI or APPI. This phenomenon may be due to the thermal energy used with these two techniques. The results cannot be generalized because none of the optimized conditions can be ideal for all the analytes. For example, the values for glycosidic avonoids are lower with IS than APCI and APPI, but strongly enhanced limits might be achieved if a polymer-based stationary phase and a basic buffer in negative ion mode are used in APPI analyses, thus providing effective ionization of the glycosides. In our case, IS and APPI were performed with an efuent ow splitter that makes their virtual limits in-detector notably lower than those of APCI. This is an advantage, for example,

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1.4e+7 1.2e+7

8.0e+6 5

Intensity (cps)

Intensity (cps)

1.0e+7 8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0 0 2 4 6 8 10 12 14 1 2 3 4

6.0e+6 4.0e+6 2.0e+6 0.0 0 2 4 6 8 10 12 14

Time (min)
3.0e+7 2.5e+7

Time (min)
1.2e+7

c
Intensity (cps)

Intensity (cps)

1.0e+7 8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0

2.0e+7 1.5e+7 1.0e+7 5.0e+6 0.0 0 2 4 6 8 10 12 14

10

12

14

Time (min)

Time (min)

1.4e+7

e
Intensity (cps)

8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0

Intensity (cps)

1.2e+7 1.0e+7 8.0e+6 6.0e+6 4.0e+6 2.0e+6 0.0 0 2 4 6 8 10 12 14

10

12

14

Time (min)

Time (min)

Figure 5. Sum overlays of XICs of ions listed in Table 3 by (a) negative ion IS, (b) positive ion IS, (c) negative ion APCI, (d) positive ion APCI, (e) negative ion APPI, (f) positive ion APPI. LC/MS elution order in each chromatogram: 1, (C)-catechin; 2, luteolin-30 ,7-diglucoside; 3, vitexin; 4, isoquercitrin; 5, isorhamnetin.

when using microbore columns with lower eluent ow rates, thus avoiding efuent splitting. However, the results show that negative ion IS with an eluent system containing acidic ammonium acetate buffer provides the best conditions for detection of avonoids in MS mode.

higher limits of detection. The limits of detection are at about the same level with all the techniques, but, compared with the others, a better detection is achieved using IS in negative ion mode, where the sensitivity is strongly affected by gas-phase reactions.

Acknowledgements CONCLUSIONS
We have demonstrated that the eluent composition has a signicant effect on the ionization efciency of avonoids using all of the LC/MS techniques tested. The effect is greater with APPI than with IS or APCI, but the differences in ionization sensitivity even out when optimized eluent compositions are utilized. In these optimized eluent conditions, there is no marked difference in absolute ionization efciency between the positive and negative ion modes. However, the background noise is stronger in positive ion mode than in negative ion mode, leading to
The study was supported by the Graduate School in Pharmaceutical Research (Ministry of Education, Finland), Tekes Technology Development Centre (Finland), and the Finnish Ministry of Agriculture and Forestry.

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J. Mass Spectrom. 2001; 36: 12691280