(S)-(-)-N-(Pentafluorobenzylcarbamoyl)prolyl chloride: A chiral derivatisation reagent designed for gas chromatography/negative ion chemical ionisation mass spectrometry of amino compounds

Article abstract of DOI:10.1002/rcm.6146

RATIONALE The synthesis of a novel chiral derivatisation reagent, (S)-(-)-N-(pentafluorobenzylcarbamoyl)prolyl chloride is described which is preferably useful for negative-ion chemical ionisation mass spectrometry. METHODS Preparation of the reagent followed a general strategy used to prepare enantioselective reagents based on the N-substitution of L-proline. Pentafluorobenzyl chloroformate smoothly reacted with L-proline to give the desired derivatisation reagent after conversion into the acyl chloride. The product was sufficiently pure to be used in the following steps without any additional purification. RESULTS The reagent was tested against selected chiral and non-chiral analytical targets. Chromatographic enantioseparation was at least equal to the commonly used (S)-(-)-N-(heptafluorobutyryl)prolyl derivatives. The derivatives exhibit excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high ion current for the targeted m/z during analysis. With electron ionisation, the fragmentation that occurs is mainly directed by the introduced group. Enantioseparation with gas chromatography/negative-ion chemical ionisation mass spectrometry of the derivatives was demonstrated for the enantiomers of amphetamine, α-aminocaprylic acid methyl ester and threo-methylphenidate. CONCLUSIONS The new derivatisation reagent shows highly improved mass spectral properties for negative-ion chemical ionisation mass spectrometry and is thus suitable for sensitive chiral detection of amino compounds. The reagent extends the applicability of dissociative resonance electron capture using pentafluorobenzyl derivatives to chiral analysis. Copyright

Full text of DOI:10.1002/rcm.6146

Research Article
Received: 11 November 2011
Revised: 22 December 2011
Accepted: 23 December 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 592–598
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6146
(S)-(À)-N-(Pentafluorobenzylcarbamoyl)prolyl chloride: a chiral
derivatisation reagent designed for gas chromatography/negative
ion chemical ionisation mass spectrometry of amino compounds
*
Hans J. Leis and Werner Windischhofer
University Children’s Hospital, Research Unit of Osteology and Analytical Mass Spectrometry, Auenbruggerplatz 34/II,
A-8036 Graz, Austria
RATIONALE: The synthesis of a novel chiral derivatisation reagent, (S)-(À)-N-(pentafluorobenzylcarbamoyl)prolyl
chloride is described which is preferably useful for negative-ion chemical ionisation mass spectrometry.
METHODS: Preparation of the reagent followed a general strategy used to prepare enantioselective reagents based on
the N-substitution of L-proline. Pentafluorobenzyl chloroformate smoothly reacted with L-proline to give the desired
derivatisation reagent after conversion into the acyl chloride. The product was sufficiently pure to be used in the following
steps without any additional purification.
RESULTS: The reagent was tested against selected chiral and non-chiral analytical targets. Chromatographic enantioseparation
was at least equal to the commonly used (S)-(À)-N-(heptafluorobutyryl)prolyl derivatives. The derivatives exhibit
excellent mass spectral properties under negative ion chemical ionisation, i.e. reduced fragmentation and thus high
ion current for the targeted m/z during analysis. With electron ionisation, the fragmentation that occurs is mainly
directed by the introduced group. Enantioseparation with gas chromatography/negative-ion chemical ionisation mass
spectrometry of the derivatives was demonstrated for the enantiomers of amphetamine, a-aminocaprylic acid methyl
ester and threo-methylphenidate.
CONCLUSIONS: The new derivatisation reagent shows highly improved mass spectral properties for negative-ion
chemical ionisation mass spectrometry and is thus suitable for sensitive chiral detection of amino compounds. The
reagent extends the applicability of dissociative resonance electron capture using pentafluorobenzyl derivatives to chiral
analysis. Copyright © 2012 John Wiley & Sons, Ltd.
Chiral separation has become more and more important in
biochemical and pharmaceutical analysis. Drugs are often
administered as a racemic mixture, but in many cases only
one enantiomer displays biological activity or at least enhanced
activity.[1,2] Thus, for tracing the bioavailability of racemic
drugs and establishing pharmacokinetic profiles, chromato-
graphic separation of enantiomers is a crucial task. Many
reagents are available for chiral derivatisation to yield deriva-
tives amenable to gas chromatography (GC) analysis. With
chiral derivatisation, enantiomers can be resolved on non-chiral
columns. Among the reagents used, perfluoroacylated L-prolyl
chlorides are widely used.[3,4] These derivatives are suitable for
a variety of GC detectors, including electron capture and mass
spectrometry.[5,6] In the latter case, however, there are some
inconveniences encountered. The complex structural features
of the group introduced during derivatisation yields the major
fragment ions observed in the mass spectra, especially under
electron ionisation (EI) conditions, whereas target-specific
fragmentation is poor. On the other hand, using the highly
fluorinated derivatives, negative ion chemical ionisation (NICI)
can also be applied.[7] Basically, resonance electron capture
(REC) and dissociative REC are the mechanisms involved,
where REC produces a molecular radical anion in its
excited state that must be stabilised to prevent electron
autodetachment, whereas dissociative REC results in non-
radical anions with a distribution of internal energies, depend-
ing largely on the leaving group and its ability to absorb excess
internal energy, which increases with its size.[8–11] The REC
process also adds another dimension of selectivity to the
analytical process as it strongly depends on the electron affinity
of the target molecule, and thus on its electronegativity,
rendering it as electrophoric. Unfortunately, many analytical
targets possess only low electron affinity, and hence chemical
derivatisation is usually employed to enhance NICI response.
There are many reagents available, the most widely used com-
pounds are perfluoroacyl[12–15] and pentafluorobenzyl[16–18]
(PFB) derivatives. For perfluoroacylates REC response is
usually high, but the molecular radical anion exhibits low
abundance in many cases. Instead of this, sequential loss of
hydrogen fluoride (HF) from the molecular anion is observed,
sometimes displaying a complex fragmentation pattern in the
NICI mass spectra and hence reducing the ion current of the
’diagnostic’ fragment chosen for quantification.[19,20] This was
also found for the chiral analysis of amino compounds using
(S)-(À)-N-(heptafluorobutyryl)prolyl derivatives.[21] Thus, deri-
vatives yielding low fragmentation under NICI with ions in
the higher mass range are ideally suited for enhancement of
sensitivity. This is met by PFB derivatives of carboxylic acids
* Correspondence to: H. J. Leis, University Children’s Hospital,
Research Unit of Osteology and Analytical Mass Spectrom-
etry, Auenbruggerplatz 34/II, A-8036 Graz, Austria.
E-mail: hans.leis@medunigraz.at
Rapid Commun. Mass Spectrom. 2012, 26, 592–598
Copyright © 2012 John Wiley & Sons, Ltd.

Chiral derivatisation reagent for GC/NICI-MS
and phenols, where both, the carboxylate or phenolate anion
and PFB radical formed by dissociative REC are stabilised
by resonance and hence produce virtually only these ions
without any further fragmentation in many cases.[22] For a
long time these extremely sensitive derivatives were
only available for carboxylates and phenolates, using PFB
bromide as a reagent, but recently two reagents have been
described for the ultrasensitive non-chiral analysis of amino
compounds bearing the PFB ester moiety.[23–26] It was thus
the aim of this study to develop a new chiral reagent
that produces derivatives of amino compounds with similar
NICI response relying on dissociative REC of PFB ester
functionalities together with adequate chromatographic
separation of enantiomers on the GC, using amphetamine,
methylphenidate, nortriptyline paroxetine and a-aminocaprylic
acid as model compounds.
Synthesis of (S)-(À)-N-(pentafluorobenzylcarbamoyl)prolyl
chloride (PCPCl)
(S)-(À)-N-(pentafluorobenzylcarbamoyl)proline (0.134 mmol)
was dissolved in dichloromethane (1 mL), cooled on an ice bath
and thionyl chloride (200 mL) was slowly added. The solution
was left at room temperature for 20 min and then the solvent
and reagent were removed under a stream of nitrogen. The resi-
due was redissolved in 10 mL of dichloromethane to give the
working solution of (S)-(À)-N-(pentafluorobenzylcarbamoyl)
prolyl chloride (PCPCl) at a concentration of 13.4 mM. The
reagent was prepared freshly each day. Reaction yield was
estimated to 97.2%. Additionally, purification of the reagent
can be achieved as follows: The oily residue was extracted with
hexane/dichloromethane (9:1, v/v) and the extract filtered
over a short column of silica gel (Merck Kieselgel G60). After
washing with the same solvent, the solution was dried under
nitrogen and the purified residue redissolved in the appropri-
ate amount of dichloromethane.
EXPERIMENTAL
Derivatisation with PCPCl
Racemic (Æ)-a-aminocaprylic acid was converted into the
methyl ester prior to PCPCl treatment with ethereal diazo-
methane. Aqueous solutions of racemic (Æ)-a-aminocaprylic
acid methyl ester, racemic (Æ)-amphetamine sulphate,
racemic (Æ)-threo-methylphenidate.HCl, nortriptyline.HCl
and 4R,3S-paroxetine.HCl (1 mg each) were made alkaline
with 3 M NaOH and extracted with n-hexane. An aliquot of
the hexane phase (100 mL) was treated with 200 mL of reagent
solution for 30 min at room temperature. Solvent was
removed under nitrogen and the residue reconstituted in
100 mL ethyl acetate. A volumes of 1 mL was subjected to
analysis by GC/EI-MS and GC/NICI-MS. Alternatively, an
optimised procedure can be used as exemplarily shown for
amphetamine and methylphenidate: 1 mL of carbonate buffer
(pH 9.0) was added to the sample followed by addition of
1 mL dichloromethane or hexane (this could also be the
organic phase after extraction from alkalinised aqueous
samples). Then 50 mL of PCPCl (3.4 mM in dichloromethane)
were added and the mixture shaken on a rotary shaker
for 15 min. The organic phase was collected and brought
to dryness under a stream of nitrogen. The residue was
reconstituted in 100 mL of ethyl acetate and analysed by
GC/NICI-MS.
Chemicals and reagents
Pentafluorobenzyl alcohol, N,N-dimethylaniline and phosgene
were supplied by Aldrich (Vienna, Austria). Silylation grade
pyridine was obtained through Pierce (Rockford, IL, USA). Race-
mic (Æ)-threo-methylphenidate hydrochloride was from Medice
(Iserlohn, Germany). Enantiomeric pure 4R,3S-paroxetine
((À)-trans-4R-(4’-fluorophenyl)-3S-((3’,4’-methylenedioxy-
phenoxy)methyl)piperidine) was from Gedeon Richter
(Budapest, Hungary). (R)-(+)-a-Methylbenzylamine(PEA)chira-
Select of certified enantiomeric composition (R:S ≥99.5:0.5) was
purchasedfromFluka(Buchs,Switzerland).Allothersubstances,
solvents and reagents of analytical grade were from Merck
(Darmstadt, Germany).
Synthesis of pentafluorobenzyl chloroformate
Pentafluorobenzyl chloroformate (PFBCLF) was prepared as
described elsewhere.[27,28] Briefly, pentafluorobenzyl alcohol
and N,N-dimethyl aniline in ether were added to a cooled
(0 ꢀC) solution of phosgene in toluene. After stirring overnight
the solution was concentrated under a well-ventilated hood.
The crude product was distilled to yield the pure compound
as a colorless liquid.
Gas chromatography/mass spectrometry
A Finnigan TRACE 8000 gas chromatograph coupled to a
Synthesis of (S)-(À)-N-(pentafluorobenzylcarbamoyl)-proline
Finnigan TRACE quadrupole mass spectrometer and
a
L-Proline (10 mmol) was dissolved in 5 mL of NaOH (2 M) on
an ice bath and 1 mL of pyridine was added. To the stirred
solution, PFBCLF (10 mmol) and NaOH (2 M, 5 mL) were
added alternatingly in 5 portions. After addition, the reaction
mixture was stirred for 30 min at room temperature. The mix-
ture was acidified with 50% HCl and the separated oil extracted
with diethyl ether. The organic phase was washed with water
and dried over sodium sulphate. After removal of the solvent
the product was obtained as a colorless oil in 80.8% yield.
Identity and purity were checked by GC/EI-MS after
Finnigan ISQ GC/MS system were used (Thermo Scientific,
Vienna). The gas chromatograph was fitted with a BPX5
fused-silica capillary column (15 m  0.25 mm i.d., SGE). The
ion source temperature was 150ꢀC (TRACE) and 200ꢀC (ISQ).
The injector was operated in the splitless mode at 280 ꢀC.
Helium was used as a carrier gas at a constantꢀflow rate of
1.5 mL/min. Initial column temperature was 60 C for 1 min,
followed by an increase of 40ꢀC/min to 315ꢀC and an iso-
thermal hold of 10 min. The mass spectrometer transfer line
ꢀ
was kept at 320 C. NICI was performed with methane as a
conversion into the methyl ester with ethereal diazomethane
moderating gas at an electron energy of 70 eV and an emission
current of 0.250 A (TRACE) or 0.050 A (ISQ). Methane flow
was adjusted to 1.5 mL/min. EI was performed at an electron
+
solution (EI-MS: m/z 353 (M● ; 1.6); m/z 294 (100); m/z 250 (65);
m/z 181 (40)).
Rapid Commun. Mass Spectrom. 2012, 26, 592–598
Copyright © 2012 John Wiley & Sons, Ltd.
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H. J. Leis and W. Windischhofer
energy of 70 eVand an emission current of 0.250 A (TRACE) or
0.050 A (ISQ). Resolution factors (Rs) and separation factors (a)
were calculated under these conditions.
in Fig. 2. The following fragment ions can be assigned to the
+
PCP moiety: m/z 181 (C7H2F●5+), m/z 250 (C11H9F5NO●
,
presumably resulting from m/z 294 by neutral loss of CO2),
m/z 294 (C12H9F5NO●2+) and m/z 322 (C13H9F5NO●3+). For the
amphetamine derivative, the only fragment ion containing at
least the chiral carbon and the amino nitrogen of the original
target with considerable intensity can be found at m/z 365
(C15H14F5N2O●3+). For PCP-threo-methylphenidate, m/z 405 is
formed by a-cleavage to the piperidinyl nitrogen. Similar EI
mass spectra have been obtained for PCP derivatives of all
compounds tested. Molecular ions are absent or weak, as are
fragment ions at higher m/z values. Thus, the majority of
fragmentation of PCP derivatives under EI conditions is rather
group-specific for the introduced moiety, but less for the
original target structure. Although of possible analytical use,
it would be desirable to detect more specifically the target
structure and reduce fragmentation in order to carry a high
proportion of the total ion current in only a few fragment ions
for the sake of low detection limits.
RESULTS AND DISCUSSION
Preparation of the PCPCl reagent
The procedure for the preparation of PCPCl followed a general
strategy used to prepare enantioselective reagents based on the
N-substitution of L-proline.[29–31] PFBCLF smoothly reacted
with L-proline to give the desired starting compound. The
product was sufficiently pure to be used in the following steps
without any additional purification. Only one single peak
appeared in the chromatogram of the PCP methyl ester. For
derivatisation reactions, PCPCl can be prepared freshly by
the simple procedure described. We have used the general
procedures described herein to prepare the PCP derivatives of
various compounds. Quantitative conversion occurred with
amphetamine and methylphenidate under the conditions of
extractive acylation, as judged by subsequent treatment with
heptafluorobutyric anhydride and analysis of the relative
derivative amounts. Direct transfer of extraction solvent
from aqueous matrix to the carbonate buffer without any
condensation steps prevents loss of volatile samples. PCPCl
can thus be regarded useful for the conversion of primary and
secondary amines, as well as of a-amino acids, and can be
applied to the same range of applications as the widely used
heptafluorobutyryl-substituted derivative. The structures of
the reagent and the target compounds tested are shown in Fig. 1.
NICI mass spectrometry of PCP derivatives
NICI as a soft ionisation technique can be expected to show
reduced fragmentation and more high-mass ions as compared
to EI. This is, however, only true to a certain extent when
perfluoroacyl-prolyl derivatives are measured. Although deliv-
ering fragment ions in the higher m/z range up to the molecular
ion, fragmentation is quite intense and results predominantly
from the sequential loss of HF from the molecular anion.[21]
We have observed this fragmentation pattern also after non-
chiral dervatisation with heptafluorobutyric anhydride,[19,20]
thus suggesting the fluoroacyl part is mainly responsible for
directing this pathway. On the other hand, fragmentation
under NICI conditions can be drastically reduced to one or
two fragment ions with striking intensity in certain cases:
Pentafluorobenzyl derivatives of carboxylic acids undergo
dissociative resonance electron capture when subjected to NICI
that leaves the carboxylate anion with high stability and high
EI mass spectrometry of PCP derivatives
As expected, the EI mass spectra of PCP derivatives of amino
compounds are largely dominated by fragment ions arising
from the PCP substructure. There is no difference in the spectra
of the enantiomers. As typical examples, the EI mass spectra of
PCP-threo-methylphenidate and PCP-amphetamine are shown
F
H
N
F
F
F
O
NH2
F
O
NH2
O
O
O
N
O
NH2
F
Cl
1
2a
2b
3
4
H
H
O
O
N
N
OH
OH
H3COOC
COOCH3
NH2
NH2
H
H
5a
5b
6a
6b
Figure 1. Structural formulae of PCPCl reagent (1), R-(À)-amphetamine (2a), S-(+)-amphetamine (2b),
4R,3S-paroxetine (3), nortriptyline (4), S-(+)-a-aminocaprylic acid (5a), R-(À)-a-aminocaprylic acid (5b),
2R,2’R-threo-methylphenidate (6a) and 2S,2’S-threo-methylphenidate (6b).
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Chiral derivatisation reagent for GC/NICI-MS
F
F
F
F
F
m/z 181
181
100
A
O
80
O
118
N
250
294
m/z 322
70 91
60
40
20
0
O
365
322
m/z 294
NH
161
339
m/z 365
50
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z
F
F
F
F
181
100
80
60
40
20
0
B
O
F
250
294
O
O
N
m/z 405
405
H
N
H COOC
H
84
142
322
264
50
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z
Figure 2. EI mass spectra of PCP-amphetamine (A) and PCP-threo-methylpheni-
date (B).
abundance.[8,22] We have observed this unique feature also with
Gas chromatography of PCP derivatives
pentafluorocarbamates and pentafluorocarbonates[32,33] and,
more recently, after derivatisation of amino compounds with the
new reagents o-(pentafluorobenzyloxycarbonyl)benzoyl chloride
and o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl
chloride.[23–26] It could thus be expected that PCP derivatives
would also underly this fragmentation mechanism and
yield similar results. The NICI mass spectra of the tested
target compounds after PCP derivative formation are given in
Fig. 3.
As expected, fragmentation is largely reduced. The resonance-
stabilised carboxylate anion is the only fragment ion observed
in the spectra of (achiral) PCP-nortriptyline and enantiopure
PCP-4R,3S-paroxetine. The PCP derivatives of amphetamine,
threo-methylphenidate and a-aminocaprylate, however, exhibit
additional neutral loss (CO2) from the carboxylate anion to show
a second fragment ion. The intensity ratio of these two fragment
ions is likely to reflect differences in the stabilisation of the
carboxylate anion and hence internal energy distribution.
In general, the NICI spectra of the two enantiomers are
identical, differing only in the intensity ratio between the
two fragment ions, as can seen in the NICI mass spectra of
PCP-threo-methylphenidate enantiomers in Fig. 4. As this
enantiospecific difference in neutral loss reaction is consider-
able, it also effects the relative peak areas of the enantiomers
in the chromatogram given in Fig. 5(B), showing the trace
for m/z 373. If, on the other hand m/z 329 was monitored,
the relative intensities were exactly opposite, and equal
distribution was seen monitoring the total ion current (results
not shown) The same behaviour was also observed to a
minor extent for the enantiomers of amphetamine and
a-aminocaprylate.
During GC analysis, PCP derivatives displayed similar
enantioseparation like the widely used heptafluorobutyrylprolyl
(HFP) derivatives. As shown in Fig. 5, the PCP derivatives of
R-(À)-amphetamine and S-(+)-amphetamine are baseline
resolved, even though a rapid heating rate of the GC column
(40 ꢀC/min) was employed. The early eluting enantiomer from
the GC is R-(À)-amphetamine, as judged by the analysis of the
pure enantiomer. An even better separation is achieved with
threo-methylphenidate. Unfortunately, no pure enantiomers of
this compound were available to our laboratory to assign the
elution order on the GC column. A comparison of the calculated
peak resolution factors and peak separation factors of HPC and
PCP derivatives is shown in Table 1. Enantioresolution is
obviously dependent on the chiral target and the chiral reagent
must be selected accordingly. Nevertheless, as seen from the
data in Table 1, PCP derivatives provide sufficient resolution
for chiral analysis of the compounds.
In general, retention times of PCP derivatives are considerably
longer as compared to HFP derivatives due to the bulky
pentafluorobenzylcarbamoyl group. This makes this new
reagent particularly interesting for the chiral analysis of low
molecular weight amino compounds. We have detected
amphetamine and methylphenidate enantiomers down to
50 pg (corresponding to 1.8 pg injected) with a signal-to-noise
ratio of 100:1. This demonstrates high sensitivity of the deriva-
tive with NICI detection in the femtomole range.
Enantiopurity of the PCP reagent
When enantiomeric R-(À)-amphetamine and S-(+)-amphetamine
were analysed independently as PCP derivatives, only one
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H. J. Leis and W. Windischhofer
Figure 3. NICI mass spectra of PCP-amphetamine (A), PCP-threo-methylphenidate (B), PCP-nortriptyline (C), PCP-paroxetine (D),
and PCP- a-aminocaprylic acid (E).
373
expectedly showed only one single GC peak as well.
100
Enantiopurity of the reagent was finally checked with PEA
80
chiraselect of certified enantiomeric composition. No change
in the enantiomeric ratio was observed after PCPCl
derivatisation.
60
40
20
329
350
0
50
100
150
200
250
300
m/z
400
450
500
373
100
CONCLUSIONS
80
60
40
20
0
In this study we report the synthesis of a new chiral reagent,
PCPCl. The compound is reactive towards primary and
secondary amines and displays ideal characteristics for
analysis by GC/NICI-MS. Enantioseparation is comparable
to the established heptafluorobutyryl-substituted prolyl
derivatives. The unique NICI mass spectra obtained allow
specific detection. The reagent can be prepared freshly on
demand by a simple procedure, as the more demanding
PFB-chloroformate is commercially available. The PCP
derivatives are sufficiently stable, as repeated injections of
one prepared sample on several consecutive days did not
show any loss of signal intensity.
329
50
100
150
200
250
300
350
400
450
500
m/z
Figure 4. NICI mass spectra of PCP-threo-methylphenidate enan-
tiomers (first eluting: upper trace, second eluting: lower trace).
single corresponding GC peak was detected. The same was
observed with 4R,3S-paroxetine. This suggests a high degree
of enantiopurity. Nortriptyline,
a
non-chiral molecule,
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Chiral derivatisation reagent for GC/NICI-MS
6,58
100
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6,64
A
m/z 275
80
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R-(-)-amphetamine
60
S-(+)-amphetamine
40
20
0
5,0 5,2 5,4 5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0
Time (min)
7,98
100
80
60
40
20
0
B
m/z 373
(2R,2´R)-threo-methylphenidate and
(2S,2´S)-threo-methylphenidate
7,76
6,5 7,0 7,5 8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5
Time (min)
6,53
6,50
100
80
60
40
20
0
m/z 269
C
R-alpha-aminocaprylate methyl ester and
S-alpha-aminocaprylate methyl ester
5,0
5,5
6,0
6,5
7,0
7,5
8,0
8,5
9,0
Time (min)
Figure 5. Chromatograms obtained after analysis of racemic
amphetamine (A), racemic threo-methylphenidate (B), and
racemic a-aminocaprylic acid methyl ester (C). The compounds
were converted into the PCP derivatives and analysed by
GC/NICI-MS.
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Table 1. Peak resolution factors (Rs) and separation factors
(a) of HPC- and PCP derivatives during enantioselective
analysis by GC/NICI-MS
HPC
derivatives
PCP
derivatives
Rs
a
Rs
a
amphetamine
2.381
10.303
2.857
1.011
1.030
1,013
3.030
5.769
1.250
1.008
1.021
1.005
methylphenidate
a-aminocaprylic
acid methyl ester
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