Enantiomeric Separation of Amphetamine, Methamphetamine

and Ring Substituted Amphetamines by Means of a

-Cyclodextrin-Chiral Stationary Phase
A. M. Rizzi 1. / R. Hirz 2 / S. C l a d r o w a - R u n g e 1 / H. Jonsson 1' 3

lnstitute of Analytical Chemistry, University of Vienna, W~ihringerstrage 38, 1090 Vienna, Austria
Department of Forensic Sciences, Federal Ministry of Interior, Liechtenwerderplatz, 1090 Vienna, Austria
Permanent adress: Institute of Chemistry, University of Uppsala, Sweden

Key Words
Column liquid chromatography
Chiral separation
Amphetamine
CYclodextrin

Summary
The enantioseparation of amphetamine, methamphetamine and various ring-substituted amphetamines by
Use of a chiral stationary phase carrying immobilized
native [~-cyclodextrin (13-CyD) selectors is reported.
The system is evaluated for resolving the specified
COmpounds directly without any derivatization and
after derivatization with phenyl isothiocyanate (PITC),
naphthyl isothiocyanate (NITC) and 6-aminoquinolylN-hydroxysuccinimidyl carbamate (AQC). This direct
enantioseparation is compared with the features of
~ndireet separation of diasteromeric derivatives after
reaction with the optically p u r e Marfey's reagent
employing a simple non-chiral alkyl-silica (RP-8)
COlumn. A selection of those methods best suited for
each single amphetamine is given.
Seventeen different samples of a m p h e t a m i n e ,
Confiscated by the Swedisch police, were analyzed with
respect to their enantiomeric composition. Within this
set of samples synthesized by the same method no
Significant deviation from a racemic ratio could be
Observed.

Introduction
The quantitative determination of the enantiomeric
COmposition of drugs submitted to governmental regulations and control has subjected to considerable
attention from the clinical as well as the forensic point
of view. Amphetamine, methamphetamine and various
related compounds obtained by ring-substitution, such
Chromatographia Vol. 39, No. 3/4, August 1994
0009-5893/94/08 0131-07

$ 3.00/0

as 3,4-methylenedioxyamphetamine and 3,4-methyenedioxymethamphetamine, act as central nervous system

stimulants and have become popular drugs of abuse [1].
The clinical and toxicological relevance of their enantiomeric composition result from the different activities
[2], toxicities [1] and different behavioura| effects [3] of
the enantiomers, where the S (+)-amphetamine is much
more potent in its stimulating action than the R(-)form. For the forensic scientist the information about
sample origin and method of clandestine preparation
which might be deduced from the enantiomeric composition [4] is of interest.
Enantiomeric separations of amphetamine and methamphetamine by various forms of HPLC have been
reported, (i) separation of the 3,5-dinitrobenzoyl derivatives by use of n-basic "Pirkle-type" stationary
phases such as the naphthyl urea phase [2, 5, 6] employing non-aqueous mobile phases, (ii) separation of the
diasteromeric 2,3,4,6-tetra-O-acetyl-13-D-glucopyranosyl isothiocyanate (GITC)- derivatives [4, 7] and the
diastereomeric FMOC-L-prolyI-derivatives [8] by use
of a simple non-chiral reversed phase type column. The
improved absorbance resulting from the fluorescencetagged labels allows the determination of drugs, even in
very low concentrations, in body fluids [9]. The use of
GC-MS for the determination of the enantiomeric
composition of some of the interesting ring-substituted
amphetamines from body fluids has been reported [10].
This paper deals with the enantioseparation of amphetamine, methamphetamine and its ring-substituted analogues listed in Figure 1, by use of a commercially
available chiral stationary phase (CSP) which carries
immobilized native 13-cyclodextrin selectors. Such
phases can be employed in a straightforward and
simple way using aqueous organic mixed mobile
phases. The resolutions achieved by this system for the
non-derivatized compounds are compared with those
obtained after derivatization of the target compounds
using phenyl isothiocyanate (PITC) and naphthyl
isothiocyanate (NITC) as well as the newly introduced
AccQ-Fluor TM reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). This direct enantio-

Original

9 1994 Friedr. Vicwcg & Sohn Vcrlagsgcsellschaft mbH

131

amphetamine

methamphetamine

I
4.-hydroxyamphetamine

4-methoxyamphetamine

3,4-methylenedioxymethamphetamine

3,4-methylenedioxyethamphetamine

oj

oj

2,5-0,me,hoxyamphetamine

2,5-dimethoxymethamphetamine

/.o

/o

0-~"

0.7
2, 5-dimethoxy--4-ethylamphetamine

I~r h . ~

..~o

I~rl

2,5-dimethoxy-4bromoamphetamine

/o

Figure 1
Chemical structures of the amphetanfines investigated

separation is compared with the features of indirect

separation of diastereomeric derivatives after reaction
with the optical pure Marfey's reagent employing a
simple nonchiral alkyl-silica (RP-8) column. For each
single amphetamine the various methods are discussed,
and best separation methods are indicated, taking into
account analysis time and effort for sample preparation. The established separation conditions were chosen for the chiral analysis of 17 different samples of
amphetamine confiscated by the Swedish police.

Experimental
Apparatus
A standard type liquid chromatographic system was
used consisting of a pump (L-6200, Intelligent Pump,
Merck-Hitachi, Tokyo, Japan), a syringe valve injector
(Rheodyne 7125, Cotati, USA) equipped with a 20 ~lloop and an UV-detector (L-4000, Merck-Hitachi)
connected to an integrator (D-2000, Chromatointegrator, Merck-Hitachi). Column temperature was controlled by use of a water jacket surrounding the column
and a thermostat (LTD 6, Grant, Herts, England).

Columns
For the direct ehiral separations a stainless-steel column (250 4.0 mm I.D.) prepacked with immobilized
132

13-cyclodextrin, d 5 t-tm ('Chiradex', E. Merck, Darnastadt, Germany) p was used. The separation of the
diastereomeric derivatives was performed on a stainless-steel column (125 x 4.0 mm I.D.) prepacked with
LiChropher 100 RP-8 dp 5 lam (E. Merck).

Chemicals and Standard Compounds

Standards of amphetamine and the amphetamine
related compounds listed in Figure 1 were obtained
from the U N I D O laboratories in Vienna. Amphetamine samples of different origin were obtained from
the Swedish police.
Methanol and acetonitrile were purchased in LiChrosolv grade, triethylamine (TEA) and acetic acid glacial
100 % in pro analysis grade, all from E. Merck. Water
was double destilled from a quartz apparatus and
processed through an Elgastat U H Q apparatus (Elga,
High Wycombe Bucks, UK). The eluents were premixed
and degassed in an ultrasonic bath before use.
Phenyl isothiocyanate (PITC), naphthyl isothiocyanate
(NITC) and Nc~-(2,4-dinitro-5-fluorophenyl)-L-alanin"
amide (Marfey's reagent) were purchased from Sigma
(Deisenhofen, Germany). 6-Aminoquinolyl-N-hydroXsuccinimidyl carbamate (AQC), commercially availa"
ble as AccQ-Fluor TM reagent, was obtained as a gift
from Millipore (Milford, MA, USA). Buffer salts for
the eluents (ammonium acetate) and the additives to
the derivatization solutions were of pro analysis grade,
all obtained from E. Merck.
Chromatographia Vol. 39, No. 3/4, August ] 994

Original

Chromatographic Conditions
Mobile phases consisted of aqueous buffer solutions
(0.1 M ammonium acetate, 0.1% TEA, pH adjusted
between 5.5 and 7.0, as specified) to which various
percentages of methanol were added.
Temperature was generally controlled within 0.1 ~
Measurements using the 13-CyD-column were carried
out at temperatures between 5 ~ and 40 ~ those
Using the RP-8 column at 20 ~
Flow rates between 0.3 and 0.5 ml/min were used with
the I~-CyD column, dependent on temperature and the
resultingcolumn back pressure. With the RP-8 column
the flow rate was 1 ml/min in all instances.
UV absorbance was monitored at wavelengths of
254 nm in all cases except for Marfey's derivatives
When a wavelength of 340 nm was chosen.
berivatizations were carried out according to Refs. [11]

(a)

ooo~
~

(b)

r
.s
O

L
time

(e)

(d)

~ ~

(e)

to [13] to obtain PITC-, N I T C - , A Q C - and Marfey's

derivatives, respectively.
uS~

Results and Discussion

(1}

Enantioseparation of Nonderivatized
Amphetamines
Using a I3-CyD-CSP it is possible to obtain at least
Partial e n a n t i o s e p a r a t i o n for most of the native amPhetamines listed in Figure 1. The enantioselectivity
Coefficients achieved under a p p r o p r i a t e c h r o m a t o graphic conditions are s u m m a r i z e d in T a b l e I, and
Chromatograms of resolved c o m p o u n d s are given in
Figure 2. Baseline separation is achieved for 4-hydroxyamphetamine, 3 , 4 - m e t h y l e n e d i o x y m e t h a m p h e t amine and 3 , 4 - m e t h y l e n e d i o x y e t h a m p h e t a m i n e only,
Where analysis times of about half an hour have to be
COnsidered. M e t h a m p h e t a m i n e , a drug of major impor-

time
Figure 2

Chromatograms of non-derivatized amphetamines: (a) methamphetamine, (b) 5-hydroxyamphetamine, (c) 4-methoxyamphetamine, (Sd) 3,4-methylenedioxymethamphetamine, (e) 2,5dimethoxyamphetamine.
Chromatographic conditions: 13-CyD-column, 250 x 4 man I.D.,
mobile phase: aqueous buffer (0.1 M ammonium acetate,
pH 7.0)/methanol; methanol content andd temperature as specified in Table I: flow rate: 0.5 ml/min. Time in minutes. Elution
order in the case of methamphetamine was S(+) before R(-).

Table !. Enantioseparation of nonderivatized amphetamines by the 13-CyD stationary phase. Enantio-

selectivity coefficients, c~, and capacity factors of the second eluted enantiomer, kS, at optimized eluent
and temperature conditions.
Chromatographic conditions: Elucnt-aqueous buffer (0.1 M ammonium acetate, pH 7) with specified
content of methanol; temperature as specified, flow rate 0.5 ml/min.
compounds

kS

amphetamine
methamphetamine
4-hydroxyamphetamine
4-methoxyamphetamine
3,4-methylenedioxymethanlphetanfine
3,4-1nethylenedioxyethamphetaminc
2,5-dimethoxyamphctanfine
2,5-dimethoxymethanlphctanainc
4-bromo-2,5-dimethoxyamphctamine
4-ethyl-2,5-dimethoxya,nphetamine

3.22
5.65
4.85
5.60
6.86
7.78
2.37
2.77
0.25
0.20

Qhromatographia Vol. 39, No. 3/4, August 1994

Original

0~
1.06
1.07
1.08
1.05
1.09

1.10
1.06

1.05
1.02
1.00

methanol % (v/v)
5
5
10
5
15
15
5
5
5
5

T [~
30
20
20
30
30
20
20
30
20
20

133

tance, is fairly well s e p a r a t e d (but not quite completely

to the baseline), 4 - m e t h o x y a m p h e t a m i n e , 2,5-dimetho x y a m p h e t a m i n e and 2 , 5 - d i m e t h o x y m e t h a m p h e t a m i n e
are s e p a r a t e d to an extent where the valley between the
e n a n t i o m e r i c p e a k s lies at about the half of the peak
height. The e n a n t i o m e r s of a m p h e t a m i n e , the most
frequently e n c o u n t e r e d drug, are insufficiently resolved. T h e rather rare a m p h e t a m i n e s 4-bromo-2,5d i m e t h o x y a m p h e t a m i n e and 4-ethyl-2,5-dimethoxyamp h e t a m i n e cannot be separated using this method.
With regard to mechanistic aspects, the d e p e n d e n c e of
the capacity factors reported in Table I, on size and
bulkiness of the analytes, deserves some attention.
W h e r e a s a m p h e t a m i n e , m e t h a m p h e t a m i n e and the
mono-substituted 4 - m e t h o x y a m p h e t a m i n e and 4-hyd r o x y a m p h e t a m i n e , as well as the bicyclic analogues
( 3 , 4 - m e t h y l e n e d i o x y m e t h a m p h e t a m i n e and 3,4-methy l e n e d i o x y e t h a m p h e t a m i n e ) seem to be able to penetrate the cavity of the selector, no full penetration
seems to be possible for the doubly substituted a m p h e t amines ( 2 , 5 - d i m e t h o x y a m p h e t a m i n e and 2,5-dimetho x y m e t h a m p h e t a m i n e ) and the triple substituted analogues 4 - b r o m o - 2 , 5 - d i m e t h o x y a m p h e t a m i n e and 4ethyl-2,5-dimethoxyamphetarnine. This hypothesis is
supported by the capacity factors found for the multisubstituted compounds: their k' values are lower than
those of m o n o s u b s t i t u t e d a m p h e t a m i n e s on the 13CyD-phase, whereas they are much larger when using
simple reversed-phase type c h r o m a t o g r a p h y .

(a)

Cb)

(c)

(d)

o.~[2

HNN~O Ot
Figure 3

Chemical structure of (a) PITC-, (b) NITC-, (c) AQC- and (d)
Marfey's dcrivatized amphctaminc.

cent 6-quinolyl-urea derivatives. The structures of

these derivatives are given in Figure 3. Derivatives of
this description are c h r o m a t o g r a p h e d on the [3-CyD"
CSP using m e t h a n o l concentrations of a b o u t 40 to
60 % (v/v). T h e corresponding capacity factors and
enantioselectivity data are given in T a b l e II.
Considering the P I T C derivatives first, it becomes
evident that a complete separation is readily achieved
for those a m p h e t a m i n e s carrying either none or only
one ring substituent and of the bicyclic analogueS.
Similar results were obtained for the N1TC derivatives
too, although in most cases the resolutions of the PITC
labeled c o m p o u n d s were superior. T h e m o r e bulky

Enantioseparation After Derivalizalion

Derivatization of a m p h e t a m i n e s with P I T C - and N I T C reagents yields stable thiourea derivatives, and with the
newly d e v e l o p e d A Q C - r e a g e n t (AccQ-Fluor), fluores-

Table 11, Separation of PITC-, N1TC- and ACQ-dcrivatized amphctamines by thc 13-CyD stationary
phase.
Chromatographic conditions: Eluent-aqueous buffer (0.1 M ammonium acetate, 0.1% TEA, pH 5.5)/
methanol. Content of methanol 60 % (v/v) for PITC- and NITC-derivatives and 50 % (v/v) for AQCderivatives.
NITC a

PITC a
k;
amphctaminc
methamphctamine
4-hydroxyamphctamine
4-methoxyamphetaminc
3,4-methylenedioxynletlaamphetamine
3,4-methylencdioxycthamphetamine
2,5-dimethoxyanlphctamine
2,5-dimethoxymethamphetaminc
4-bromo-2,5-dimethoxyamphetaminc
4-ethyl-2,5-dimethoxyarnphetamine

2.43
2.72
2.49
2.56
3.22
3.38

AQC b

ct

k~

Cc

1.34
1.30
1.32
1.36
1.21
1.21

2.01
2.19
1.90
1.88
1.77
1.96
9

1.17
1.10
1.11
1.13
1.21
1.20
,

k~

3.92
3.60
4.10
4.02
6.04c
5.48c
3.65d

1.30
1.29
1.33
1.34
1.00c
1.00c
1.21 d

1.61 d

1.07 d

2.04 d

l.l 0d

* k' values very low, peak of the compound was interfered by thc peak of thc dcrivatization reagents.
a
b
c
d
]34

temperature: 7 ~ flow rate: 0.4 mlhnin; content of methanol 60 % (v/v)

temperature: 20 ~ flow rate 0.5 ml/nlin; content of methanol 50 % (v/v)
content of methanol: 40 % (v/v)
content of methanol: 40 % (v/v) and temperature: 10 ~
Chromatographia Vol. 39, No. 3/4, August 1994

Original

dimethoxyamphetamines, partially excluded from penetrating the 13-CyD cavity in the non-derivatized form,
Were excluded even more in the derivatized state. They
Were eluted at the very beginning of the chromatogram
together with the residue of the derivatizing agents.
Consequently, these compounds cannot be enantioseparated as PITC or NITC derivatives at all.
Considering the AQC-derivatives, again an excellent
Separation is obtained for the small non- and monoSUbstituted amphetamines. With this type of derivatization, however, most of the bulky dimethoxyamphetamines can also be resolved. It is the only way shown in
this investigation to obtain direct resolution for 4bromo-2,5-dimethoxyamphetamine. On the other hand,
no resolution is achieved for the bicyclic derivatives,
3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethamphetamine. It is likely that the structure
of the complex formed between ~-CyD and these
analytes is quite different for the differently labeled
COmpounds. This has special significance for the separation of bicyclic as well as doubly substituted amphetamines.

Optimization of Enantioseparations
Optimization of enantioseparations is usually done by
Variation of two parameters-mobile phase composition
and temperature. With non-derivatized analytes the
effect of temperature is found to be small and therefore
not decisive, whereas the effects of organic modifier
COncentration can be considerable. Selection of low
modifier concentrations are in some cases necessary to
Obtain resolution.
With PITC-derivatized amphetamines the situation is
quite different. In this case the enantioselectivity
Coefficients can be increased significantly by decreasing
temperature (Figure 4). However, it is not affected
significantly by the methanol concentration in the
mobile phase, as documented in Table III. Analogous
dependencies can be observed for the NITC-derivatires. Capacity factors can be adjusted by reducing
nlethanol concentrations, independently of enantioselectivities which can be increased by reducing temperature. For the analysis of unknown amphetamine samPies, which is discussed later on, a mobile phase of 60 %
methanol and 40 % buffer and a temperature of 7 ~
Was chosen to combine rather low capacity factors with
high enantioselectivity. Under these conditions near
baseline separation is achieved in a quite short time.

10"C

20~

t6

S*C

=5

C3~

tb
c
..o
o

ti

II-

time

Figure 4
Chromatograms of PITC-amphetamine at different temperatures. Chromatographic conditions: 13-CyD-column,
250 4 mm I.D., mobile phase: aqueous buffer (0.1 M ammonium acetate, 0.1% TEA, pH 5.5)/methanol: 40/60 % (v/v); flow
rate: 0.5 ml/min.

Table II!. Influence of temperature and methanol concentration on the retention and enantioselectivity achieved for PITCamphelamine.
k~ denotes the capacity factors of the second eluted enantiomer.
PITC-amphetamine
temperature
[~

% (v/v) of methanol
70

30
20
10
5

0.47
0.65
2.11
2.65

5O

60
1.19
1.24

1.31

1.36
5.61

1.26
1.33

3.33

1.27

1.35

ty. Amphetamine and methamphetamine can be completely baseline separated using a column of 250 mm
length. This method is the only one separating the
enantiomers of 4-ethyl-2,5-dimethoxyamphetamine even
partially, but which might be of some use for differentiating between samples. The only compounds which
could not be separated at all were 4-methoxyamphetamine and 4-bromo-2,5-dimethoxyamphetamine. A few
examples using Marfey's derivatization are shown in
Figure 5.

Separation of Diastereomeric Derivatives

biastereomeric derivatives of the analytes were formed
by reaction with Marfey's reagent and were chromatographed on an octylsilica phase in a reversed phase
mode using between 50 and 60 % (v/v) methanol. The
COrresponding data are given in Table IV.
With this type of derivatization most of the amphetamines are separated, many of them with high selectiviChrornatographia Vol. 39, No. 3/4, August I994

Conclusion
With few exceptions, non-derivatized amphetamines as
well as their PITC-, NITC- and AQC-labeled derivatives were found to fulfil the structural requirements
for being enantioseparated by a native ~-CyD-CSP. In
many instances complete resolution could be achieved
Original

135

(a)

(b)

(c)

(iii) AQC-derivatization allows resolution of the small

as well as the doubly substituted amphetamines, and 4bromo-2,5-dimethoxy-amphetamine. (iv) Marfey's-derivatization to diastereomeric compounds is appropriate
for most of the compounds investigated. In particular,
it allows the partial separation of 4-ethyl-2,5-dimeth"
oxy-amphetamine. The special benefit of this method is
that it gives chiral resolution of most of the mentioned
compounds within rather short times using a standard
column only. An overview regarding the separability of
amphetamines utilizing different derivatization strategies is given in Table V. Direct enantioseparation by
the inclusion mechanism with !3-CyD and indirect
resolution via the formation of diasteromers are to
some extent complementary with respect to size and
bulkiness of the analytes. These results are expected to
be a more general conclusion, valid not only for the
group of amphetamines investigated.
The derivatization method discussed differ significantly
in time and effort required for sample preparation. ThiS
particular aspect favours the use of the AQC-derivati"
zation which is completed in a few seconds, does not
require time consuming evaporation of solvent compO"
nents and shows very little interference by peaks fro~
reaction components, especially when using fluoreS"
cence detection. This is the derivatization procedure of
choice for most of the amphetamines.
The final choice of the best suited method will depend
on the particular problem. For much testing e.g. routine
screening, base line separation is not really needed i~
order to give a crude information about whether
formulation is racemic or enantiomerically more or less
pure. In these instances the analysis of the nor~"
derivatized compounds offers distinct advantages, as
the total sample processing times is considerably
shorter, due to the absence of any derivatizatior~
procedures.
With the seventeen samples of amphetamine formula"
tions originating from clandestine preparations and
confiscated by the Swedish Police, enantiomeric resolu"
tion was carried out after derivatization with PITC"

r
9

r162

8
t~

0
l~

time
Figure 5

Chromatograms of diastereomeric Marfey's derivatives of (a)

3,4-methylenedioxymethamphetamine (55 % (v/v) methanol),
(b) 3,4-methylenedioxyethamphetamine (60 % (v/v) methanol)
and (c) 2,5-dimethoxymethamphetamine (55 % (v/v) methanol).
Chromatographic conditions: OS-column, 250 x 4 mm 1.D., mobile phase: aqueous buffer (0.1 M ammonium acetate, 0. I %
TEA, pH 5.5)/methanol, methanol content as specified; temperature: 20 ~ flow rate: 1.0 ml/min.
although in some cases the separation was not sufficient. However, except for the two triple substituted
compounds (4-bromo- and 4-ethyl-2,5-dimethoxyamphetamine), all amphetamines could be resolved by at
least one of these methods. 4-bromo-2,5-dimethoxyamphetamine could be only partially separated but for 4ethyl-2,5-dimethoxyamphetamine no sufficient separation was achieved. The most appropriate derivatization
for a compound of interest can be selected on the basis
of the data in the Tables I and II.
In summary: (i) chromatography of the underivatized
compounds results in baseline separation in the cases of
the 4-hydroxy-substituted and the bicyclic amphetamines only. (ii) PITC- and NITC-derivatization is
appropriate for all of the small amphetamine analogues. PITC derivatization is superior in most cases.

Table IV. Separation of diastereomeric Marfey's-dcrivatives on a non-chiral octylsilica (RP-8) column.

Chromatographic conditions: Eluent-aqueous buffer (0.1 M amnlonium acetate, 0.1% TEA, pH 5.5)/
methanol content as specified, temperature. 20 ~ Flow rate 1.0 ml/min.
compounds

methanol % (v/v)
55
k~

amphetamine
methamphetamine
4-hydroxyamphetamine
4-methoxyamphetamine
3,4-methylenedioxymethamphetamine
3,4-methylenedioxyethamphetamine
2,5-dimethoxyamphetamine
2,5-dimethoxymethamphetamine
4-bromo-2,5-dimethoxyamphetanline
4-ethyl-2,5-dimethoxyamphetamine

136

14.1
11.8
5.6
12.2
5.84
12.6
19.2
11.2
28.2

60
C~

k~

~x

4.01

1.49

3.82
10.7
14.4

1.00
1.04

1.08

1.08
1.31
1.00
1.23

1.58
1.08

1.18
1.00

1.17

Chromatographia Vol. 39, No. 3/4, August 1994

Original

TableV. Separability of amphetamines utilizing various derivatization strategies.

compounds

nonderivatized
insuff.
n.b.I.

amphetamine
methamphetamine
4-hydroxyamphetamine
4-methoxyamphetamine
3,4-methylenedioxymet hamphetamine
3,4-met hylenedioxyethamphetamine
2,5-dimethoxyamphetamine
2,5-dimethoxymethamphetamine
4-bromo-2,5-dimethoxyamphetamine
4-et hyl-2,5-dimet hoxyamphetaminc

n.b.I.

P1TC-

AQC-

,4-

n.b.l.
insuff.
insuff.

Marfey's
+

n.b.l.
+

+
+
+

n.b.I.
n.b.I.

+
+

n.b.1.

+
base line separation
n.b.I, separation not to baseline
insuff, separation insufficient
no separation

reagent under chromatographic conditions as specified

in Table II. Under these conditions the R(-)enantiorner elutes first. The enantiomeric ratio was evaluated
by the peak area ratios. All samples investigated
Showed a peak area relation (R)/(S) close to unity
(naean value 1.00 and standard deviation 0.02). This
naeans that all samples were racemic mixtures. Whether
the enantiomeric composition can provide useful pattern characterizing single samples with respect to their
Origin, method of synthesis and the cleaning procedures
has still to be evaluated. A wider study into the
COrrelation between the enantiomer ratio and the
Production technique has now been undertaken.

Acknowledgements
"l'he authors are grateful to Steve Aim for providing the
standard compounds; to E. Merck, Darmstadt, for
Providing the 13-CyD column, and to Millipore-Austria
for providing the AccQ-Fluor reagent.

References
[1] C.J. Schmidt, J. Pharmacol. Exp. Ther. 240, 1 (1987).
[2] L W. Wainel, 7". D. Doyle, J. Chromatogr. 259, 465 (1983).
[3] R. A. Glennon, R. Young, Pharmacol. Biochem. Behav.
20, 501 (1984).
[4] F. T. Noggle Jr., J. DeRuitet, C. R. Clark, J. Chromatogr.
Sci. 28, 529 (1990).
[5] T. D. Doyle, C. A. Brunner, D. G. Cunningham, Poster
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Received: Mar 11, 1994
Accepted: Apr 11, 1994

Qhromatographia Vol. 39, No. 3/4, August 1994

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