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ApplicationNOTE
CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRIC
AND TANDEM MASS SPECTROMETRIC ANALYSIS
AND IDENTIFICATION OF NON-STEROIDAL
ANTI-INFLAMMATORY DRUGS AND THEIR
METABOLITES IN HUMAN URINE *
Alison E. Ashcroft, Hilary J. Major, Waters Corporation, Tudor Road, Altrincham, Cheshire, WA14 5RZ, UK.
Ian D. Wilson, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.
Abstract
Capillary electrophoresis (CE) was coupled via a
triaxial sheath flow interface directly to the
negative ionization electrospray (ES) atmospheric
pressure source of a commercially available
tandem quadrupole mass spectrometer (MS). The
CE eluent was supplemented by a solvent make-up
flow and this, together with a low flow nebulizer
gas, produced a reliable and robust CE-ES-MS
interface.
Human urine extracts, after independent
administration of three non-steroidal antiinflammatory drugs, were analyzed and the major
metabolites identified using a combination of
molecular weight (i.e. (M-H)-) and fragmentation
data generated from both in-source collision
induced dissociation and low energy tandem MS
(MS-MS). The in-source collision induced
dissociation and MS-MS product ion scanning
spectra provided similar information.
* These data have appeared in
part in Anal. Proc., 32,
459, 1995.
MS-MS precursor ion scanning was used for the
ibuprofen-containing urine extract to screen
specifically for glucuronide metabolites, all of
which produced a common fragment ion at m/z
175 in negative ionization electrospray.
In conclusion, on-line CE-ES-MS (-MS) proved to be
a practical, viable technique for metabolite
monitoring and identification of the drugs
described, while minimizing sample clean-up.
ApplicationNOTE
Introduction
Capillary electrophoresis (CE) and related
techniques such as micellar electrophoresis have
found increasing use in the area of drug and
metabolite analysis in biological fluids1 due to the
low sample and solvent consumption and high
separation efficiencies. In parallel, CE coupled with
electrospray (ES) - mass spectrometry (MS) is
emerging as a viable on-line technique for a variety
of routine analyses2,3, and has been applied to
drug analysis4,5. In this paper, the high resolution
of CE combined with the sensitivity and specificity
of ES-MS and tandem ES-MS has been used to
produce a practical method for the detection and
identification of three non-steroidal antiinflammatory drugs and their metabolites in solid
phase extracts of human urine.
The drugs chosen were ibuprofen, flurbiprofen and
aspirin, (Schematic 1), all widely administered and
all of which produce highly polar metabolites
amenable to negative ionization ES. The aim of the
analyses was to produce both molecular mass
information and to generate structurally useful
fragment ions to aid the identification of the
metabolites. By studying known drugs the validity
of this approach can be estimated, and if
successful the methodology then applied to more
novel drug metabolism projects.
Experimental
All solvents were of HPLC grade purity, and all
reagents and chemicals of analytical grade purity.
All were used without further purification.
Drug Administration and Sample Preparation
Urine was obtained from a healthy male volunteer
for the period 0 to 2 hr post-administration of a
standard therapeutic dose of one of the chosen
drugs: ibuprofen (400 mg), flurbiprofen (100 mg),
or aspirin (600 mg), and then stored at -20°C until
required for extraction.
Solid phase extraction (SPE) of the urine was
performed using C18 bonded SPE cartridges
containing 500 mg of sorbent (Bond Elut, Varian,
supplied by Jones Chromatography Ltd., Hengoed,
UK). The cartridges were activated prior to
extraction by washing with methanol (5 mL)
followed by 0.1 M HCl (5 mL). Samples of urine (2
mL) were acidified to pH 2 with 0.1M HCl and
then applied to the activated cartridges. The
cartridges were then washed with 0.1M HCl (1 mL)
followed by elution with methanol (5 mL). The
collected methanolic eluates were reduced to
dryness using a stream of nitrogen, and stored at 20°C until required for analysis. For CE-MS the dry
residues were reconstituted in methanol (200 (µL).
Capillary Electrophoresis
CE was performed with an uncoated fused silica
capillary (75 µm i.d. x 375 µm o.d. x 100 cm
length) using a Beckman P/ACE System 2100
(Beckman Instruments Inc., Palo Alto, USA) with inline UV detection (200 nm) 19 cm from the
injection end of the capillary. New CE capillaries
were flushed with 1M NaOH, then water and
finally the buffer in use for the analyses. In-between
subsequent analyses the CE capillary was flushed
with buffer only. A pressure injection (5 or 10
seconds) representing approximately 30 nL was
used to introduce the sample into the capillary and
then a voltage (20 kV) applied to effect separation.
The CE buffer used was 20 mM aqueous
ammonium acetate taken to pH 9 with aqueous
ammonia solution.
Mass Spectrometry
The CE capillary was taken directly into
CO2H
CH 3
CH
the atmospheric pressure ES source via
the CE-ES triaxial sheath flow
probe/interface of a Micromass®
quadrupole mass spectrometer. The low
up flow of water: propan-2-ol (20:80,
v:v, 8 (µL/minute) which was introduced
coaxially through the probe thus ensuring
that mixing of the two mobile phases
CO2H
F
CH 2
Altrincham, Cheshire, UK) tandem
capillary necessitated the use of a make-
OCOCH 3
CH
Quattro II™ (Waters Corporation,
flow of liquid emerging from the CE
CO2H
CH 3
CH
CH 3
CH 3
C 15 H 13 FO2
244
C 9H 8 O 4
180
Flurbiprofen
Aspirin
(acetyl salicylic acid)
C 13 H 18 O2
206
Ibuprofen
Schematic 1. Chemical structures and formulae of ibuprofen, flurbiprofen and aspirin.
took place at the probe tip inside the
source and hence electrophoretic
Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30.
resolution was undisturbed.
ZEN CE57
100
Scan ES-
B 20.65
(a)
The probe tip was held at -4 kV and
C
nitrogen was used as both the drying gas
22.97
and nebulizing gas, the latter being
A
introduced through the triaxial probe.
20.23
%
The source temperature was maintained
D
at 70°C. Negative ionization was used
24.11
for all the analyses and mass accuracy
7.01
E
7.47
was ensured by calibration on a
31.10
24.42
14.24
separate introduction of a sugar mixture
0
using negative ionization ES analysis. All
analyses were acquired at unit resolution
ZEN CE57
100
200nm
AN AL OG
6.90
(b)
(50% valley definition).
3.54
3.95
Molecular mass information (i.e. (M-H)2.51
ions) was obtained using a low sampling
%
cone voltage (30 V). In order to generate
fragment ions in the source, this value
4.52
was increased to 50 V. For tandem mass
spectrometry (MS-MS) analyses, argon
gas was introduced into the collision cell,
which is situated between the first and
second quadrupole analyzers, and a
collision energy of 15 - 20 eV set.
0
T ime
10.00
20.00
30.00
40.00
Figure 1. (a) CE-MS total ion electropherogram for the separation of ibuprofen
metabolites isolated from human urine by solid phase extraction. The order of
elution of the peaks was: A. hydroxy ibuprofen glucuronide, 20.23 min.; B.
ibuprofen glucuronide, 20.65 min.; C. hydroxy ibuprofen, 22.97 min.; D.
ibuprofen, 24.11 min.; E. carboxylated ibuprofen glucuronide, 31.10 min.
(b) in-line CE-UV ( 200 nm) electropherogram.
ApplicationNOTE
Results and Discussion
Ibuprofen
Following oral administration to humans, ibuprofen is rapidly excreted6 as a mixture including the drug and
its hydroxy and carboxylic acid metabolites, together with the glucuronide conjugates of these three
species. Ibuprofen and its metabolites are efficiently extracted by SPE onto C18 bonded silica which
renders this a rapid, simple and convenient clean-up procedure from urine.
The CE-ES-MS analysis of the ibuprofen urine extract produced the UV and total ion mass spectrometric
electropherograms shown in Figure 1. The difference in retention times between the two traces results from
the fact that the UV detection is taking place approximately one-fifth of the way along the separation
column. A low sampling cone voltage (30V) had been set in the ionization source for this analysis to
optimize the production of the (M-H)- ions, and from these ions the molecular masses of the components
contained in this extract were established. The major components detected had molecular masses consistent
with those expected for ibuprofen (MW 206), its side-chain hydroxylated metabolite (MW 222), the
glucuronides of both these species (MWs 382 and 398 respectively), and the glucuronide of the side-chain
oxidized carboxylic acid metabolite (MW 412), (Schematic 2).
CH 3
CO2H
CH 3
CO2G
CH
CH
CH 2
CH 2
COH
CH
CH 3
CH 3
CH 3
CH 3
222
382
hydroxylated metabolite of
ibuprofen
glucuronide metabolite of
ibuprofen
CH 3
CO2G
CH 3
CO2H(G)
CH
CH
CH 2
CH 2
COH
CH
CH 3
CH 3
CH 3
CO2G(H)
398
412
hydroxylated glucuronide
metabolite of ibuprofen
carboxylated glucuronide
metabolite of ibuprofen
Schematic 2. Chemical structures of the major ibuprofen metabolites.
Figure 2 shows the electropherograms of
the (M-H)- ions of these components, in
comparison with the total ion
Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30.
a)
ZEN CE57
100
electropherogram. The CE-ES-MS peak
electropherogram is typically 12 seconds
for these compounds.
E
%
width measured from the
Scan ES411
31.10
7.63
21.89
0
b)
ZEN CE57
100
Scan ES205
24.11
D
%
1.63
4.57
7.63
8.50
12.43
16.00
24.99
20.65
0
ZEN CE57
c) 100
30.63
35.44
39.05
41.53 44.68
46.80
Scan ES221
22.97
C
%
23.33
24.47
0
d)
ZEN CE57
100
Scan ES381
20.65
B
%
0
e)
ZEN CE57
100
Scan ES397
20.29
A
%
2.82
0
f)
ZEN CE57
100
Scan ES-
20.65
22.97
20.23
%
24.11
7.01
24.42
14.24
31.10
0
T ime
10.00
20.00
30.00
40.00
Figure 2. CE-MS total ion (f) and selected ion electropherograms, (a) - (e), for the
separation of ibuprofen metabolites isolated from human urine by solid phase
extraction, showing the (M-H)- ions of ibuprofen, m/z 205; hydroxy ibuprofen, m/z
221; ibuprofen glucuronide, m/z 381; hydroxy ibuprofen glucuronide, m/z 397;
carboxylated ibuprofen glucuronide, m/z 411.
ApplicationNOTE
The analysis was repeated using a higher sampling cone voltage (50V) in order to achieve in-source
collision induced dissociation and generate structurally informative fragment ions for all of the components
in the urine extract. The low and high cone voltage spectra obtained for the glucuronide conjugates of
ibuprofen, hydroxylated ibuprofen, and carboxylated ibuprofen are compared in Figure 3. As these spectra
show, in-source collision induced dissociation generates structurally useful information by producing
diagnostic fragment ions. For example, the three glucuronide conjugates all exhibit a fragment ion at m/z
175 resulting from cleavage of the glucuronic acid portion of the molecules, and all three show a
complementary loss of 176 amu from their (M-H)- ions to form the (M-H)- ions of the corresponding
aglycones (i.e. m/z 205, 221, and 235) and/or decarboxylated aglycones (i.e. m/z 161, 177, and 191)
of ibuprofen, hydroxylated ibuprofen and carboxylated ibuprofen respectively. It can also be observed that
the intensity of the sodium adduct ions (M+Na-H)-, compared to the (M-H)- ions, is greater when a higher
sampling cone voltage is used, implying that the sodiated adduct is the more stable of the two. These results
indicate the potential of in-source collision induced dissociation on a single quadrupole mass spectrometer
to produce structurally informative fragment ions.
Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30.
a)
ZEN CE57
100
Scan ES-
411
%
433
220
0
b)
ZEN CE58
100
Scan ES-
113
175
433
%
73
65
191
193
257
411
0
c)
ZEN CE57
100
Scan ES-
397
%
398
0
d)
ZEN CE58
100
Scan ES-
113
175
397
193
%
0
e)
ZEN CE57
100
Scan ES-
381
%
382
0
f)
ZEN CE58
100
Scan ES-
113
175
381
193
%
205
0
m/z
50
100
150
200
250
300
350
400
450
500
Figure 3. The low and high sampling cone voltage spectra of ibuprofen glucuronide
(e and f), hydroxy ibuprofen glucuronide (c and d), and carboxylated ibuprofen
glucuronide (a and b).
For more specific fragmentation, and
hence structural information, MS-MS can
Ibuprofen disc 3, 20sec inj., 20mM amm. ac. pH 9, 20kV, D aughters, CE=15eV, Ar=3x10-3mbar.
a)
ZEN CE55
100
3: D aughters of 411ES-
175
be used with on-line CE-ES. Figure 4
shows the MS-MS product ion spectra
113
obtained from selectively and
independently transmitting the (M-H)- ions
%
of the same three glucuronide conjugates
through the first quadrupole analyzer into
193
the collision cell, and then monitoring the
fragment ions arising directly from these
using the second quadrupole analyzer.
411
235
157
0
b)
ZEN CE55
100
1: D aughters of 397ES-
175
193
113
Many similarities between the product ion
MS-MS spectra and the in-source collision
induced dissociation spectra are
apparent, in particular the characteristic
%
177
glucuronide ions described for Figure 3.
MS-MS data have the advantage of not
221
exhibiting solvent-related ions and
85
103
contain only those fragment ions which
arise directly from the selected precursor.
397
131
0
c)
ZEN CE55
100
2: D aughters of 381ES-
113
175
193
%
161
205
8589103
133 157
381
0
m/z
50
75
100
125
150
175
200
225
250
275
300
325
350
375
400
Figure 4. CE-MS-MS product ion spectra of the ibuprofen glucuronide conjugates:
(a) carboxylated ibuprofen glucuronide;
(b) hydroxy ibuprofen glucuronide;
(c) ibuprofen glucuronide.
ApplicationNOTE
a)
1, 10sec inj., 20mM amm. ac. pH 9, 20kV, Parents of m/z 175.
b) Ibuprofen disc(28.306)
Ibuprofen disc 1, 10sec inj., 20mM amm. ac. pH 9, 20kV, Parents of m/z 175.
ZEN CE47
100
Parents of 175ES-
18.16
Parents of 175ES411
ZEN CE47 808
100
T IC
A
C
%
18.54
413
209
333 341
B
c)
0
ZEN CE47 519 (18.190)
100
397
416 423 444 447
Parents of 175ES-
A
%
%
409
0
d)
ZEN CE47 528 (18.540)
100
Parents of 175ES-
381
C
28.31
B
%
449
384
439
0
T ime
5.00
10.00
15.00
20.00
25.00
30.00
451
0
m/z
200
220
240
260
280
300
320
340
360
380
400
420
440
Figure 5. CE-MS-MS ibuprofen metabolite precursor ion data from m/z 175:
(a) total ion electropherogram monitoring the precursor ions from m/z 175 in order to detect the
glucuronide conjugates, showing A. hydroxy ibuprofen glucuronide; B. ibuprofen glucuronide;
C. carboxylated ibuprofen glucuronide;
(b) The MS-MS precursor ion spectrum for carboxylated ibuprofen glucuronide;
(c) The MS-MS precursor ion spectrum for hydroxy ibuprofen glucuronide;
(d) The MS-MS precursor ion spectrum for ibuprofen glucuronide.
Ester glucuronide conjugates are very common metabolites of carboxylate-containing drugs and the ready
fragmentation of these metabolites to generate a common ion at m/z 175 can be exploited using MS-MS to
detect and identify selectively these conjugates in complex media such as urine extracts. This approach is
illustrated in Figure 5 which shows the electropherogram from the precursor ion scan of m/z 175. In this
case the second quadrupole analyzer was set to transmit only the m/z 175 ion while the first quadrupole
analyzer was scanned routinely, with the result that only those components which produce a fragment at
m/z 175 are monitored. The electropherogram shows three distinct components, whose precursor ion
spectra show specifically those ions from which the m/z 175 fragment arises directly. The data indicate that
the three components selectively monitored are indeed the three glucuronide conjugates discussed, and this
screening procedure provides a rapid and specific means of identifying a particular set or type of
compound in a complex extract.
Flurbiprofen
Flurbiprofen is another non-steroidal anti-
Flurbiprofen, disc 2, 20kV, 20mM amm ac pH 9, 5sec inj.
ZEN CE32
100
Scan ES-
17.56
inflammatory with some structural
17.34
similarities to ibuprofen. In humans, after
A
oral administration, it is also subject to
B
both “phase 1” metabolic hydroxylation
reactions and also “phase 2” reactions to
form glucuronide conjugates. After solid
phase extraction from human urine, online CE-ES-MS was used to separate and
identify the components in the extract.
Figure 6 shows the total ion mass
spectrometric electropherogram from the
CE-ES-MS analysis of the flurbiprofen
urine extract using a low sampling cone
voltage to optimize the (M-H)- ions. The
%
peaks corresponding to flurbiprofen (MW
244), flurbiprofen glucuronide (MW 420)
and the glucuronide of the hydroxylated
metabolite of flurbiprofen (MW 436)
have been identified on the
electropherogram, with retention times of
20.60, 17.56 and 17.34 minutes
respectively.
C
20.60
0
T ime
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Figure 6. CE-MS total ion electropherogram for the separation of flurbiprofen
metabolites isolated from human urine by solid phase extraction. The order of
elution of the peaks was: A. hydroxy flurbiprofen glucuronide, 17.34 min.; B.
flurbiprofen glucuronide, 17.56 min.; C. flurbiprofen, 20.60 min.
ApplicationNOTE
The analysis was also performed with a high
sampling cone voltage to induce in-source
fragmentation, and complementary pairs of spectra
for the two glucuronide conjugates are presented in
Figure 7. As the spectra indicate, the (M-H)- ions
are easily distinguished together with diagnostic
fragment ions at m/z 175 for the glucuronic acid
moieties, and their corresponding decarboxylated
aglycone ions. Although the two glucuronides were
barely resolved on the CE-MS electropherogram,
clean spectra without interfering ions from other
components have been obtained for both
components.
Flurbiprofen, disc 2, 20kV, 20mM amm ac pH 9, 5sec inj., CVF.
CE32 335 (17.345)
a) ZEN
100
Scan ES-
435
%
436
0
ZEN CE35 309 (16.000)
b) 100
Scan ES-
175
435
215
113
%
89
457
103
193
133
0
c)
ZEN CE32 338 (17.500)
100
Scan ES-
419
%
420
0
d)
ZEN CE35 307 (15.894)
113
100
Scan ES-
175
%
477
199
419
193
133
0
50
100
150
200
250
300
350
400
450
m/z
500
Figure 7. The low and high sampling cone voltage spectra of hydroxy flurbiprofen
glucuronide (a and b), and flurbiprofen glucuronide (c and d).
Aspirin
Aspirin (acetylsalicylic acid)7 is readily
Aspirin, cartridge 5, 20kV, 20mM amm ac pH 9, 5sec inj.
ZEN CE34
a) 100
metabolized in humans to salicylic acid
27.68
%
(2-hydroxybenzoic acid), which is then
mostly conjugated to glycine and
excreted in urine as salicylhippuric acid.
Scan ES-
28.45 29.90
22.20
0
ZEN CE34
b) 100
The glucuronide metabolites (both ester
Scan ES137
29.90
%
and ether) of salicylic acid are also
produced to a lesser extent, and in
addition minor metabolites include the
0
ZEN CE34
c) 100
hydroxylated metabolite gentisic acid, its
%
sulphate and glucuronic acid conjugate,
and gentisuric acid (the glycine
Scan ES194
28.40
28.19
0
ZEN CE34
d) 100
Scan ES187
27.68
conjugate).
%
The CE-ES-MS total ion electropherogram
from the analysis with in-line UV detection
0
ZEN CE34
e) 100
using a low sampling cone voltage to
Scan ES178
22.20
%
optimize the (M-H)- ions is illustrated in
Figure 8. The peaks were assigned as
0
ZEN CE34
f) 100
21.53
minutes), salicylhippuric acid (MW 195;
138; 29.90 minutes).
28.19
27.88
%
28.40 minutes), and salicylic acid (MW
Scan ES195
28.45
follows: hippuric acid (MW 179; 22.20
0
g)
ZEN CE34
100
Scan ES355
17.75
%
0
5.00
10.00
15.00
20.00
25.00
30.00
35.00
T ime
40.00
Figure 8. (a) CE-MS total ion and (b) - (g) selected ion electropherograms for the
separation of aspirin metabolites isolated from human urine by solid phase
extraction.
ApplicationNOTE
The analysis was repeated at a high sampling cone voltage and also using on-line MS-MS experiments in
order to generate structural information. For example, Figure 9 compares the low sampling cone voltage
spectrum for the component of retention time 28.40 minutes (Figure 8) with the MS-MS product ion spectrum
monitoring fragments of m/z 194. The assignment of salicylhippuric acid was made by observation of the
(M-H)- ion at m/z 194 in the former spectrum and the diagnostic fragment ions at m/z 150 and 93 in the
latter spectrum.
Aspirin, cartridge 5, 20kV, 20mM amm ac pH 9, 5sec inj.
a)
ZEN CE34 549 (28.399)
100
194
Scan ES-
%
195
0
b)
ZEN CE62 570 (29.476)
100
D aughters of 194ES-
150
93
%
194
0
m/z
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
Figure 9. (a) The low sampling cone voltage spectrum of salicylhippuric acid
compared with (b) its MS-MS product ion spectrum monitoring fragment ions from
m/z 194.
A Constant Neutral Loss (CNL) CE-ES-MSMS experiment monitoring those ions
Aspirin cartridge 5, 10sec inj., 20mM amm. ac. pH 9, 20kV, N eutral loss of 44.
a)
ZEN CE61 514 (26.583)
100
178
N eutral L oss 44ES-
which lose 44 amu was performed on this
urine extract to highlight specifically those
components containing carboxylate
functional groups by scanning both
quadrupole analyzers with an offset of 44
amu. Figure 10 shows two components of
%
the mixture monitored by this method:
salicylhippuric acid (MW 195) and
113
hippuric acid (MW 179), both glycine
conjugates. The method could constitute a
rapid screening technique for complex
extracts.
0
b)
ZEN CE61 670 (34.643)
100
N eutral L oss 44ES194
%
0
m/z
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
Figure 10. On-line CE-ES-MS-MS Constant Neutral Loss analysis monitoring ions
which lose 44 amu:
(a) hippuric acid (MW 179), and
(b) salicylhippuric acid (MW 195).
ApplicationNOTE
Summary
These studies show that CE coupled to negative ionization ES-MS can be applied to the detection and
identification of a range of non-steroidal anti-inflammatory drugs and their metabolites in urine, with
minimal sample clean-up necessary.
Molecular ion species (i.e. (M-H)-) ions were generated to confirm molecular weights, and in addition the
use of in-source collision induced dissociation and low energy tandem mass spectrometry facilitated
identification of the metabolites produced. Selective screening of the urine extracts for the presence of
ibuprofen glucuronide metabolites was achieved by on-line CE-ES-MS-MS monitoring the precursor ions of
the characteristic dehydrated glucuronic acid ion.
The system used was operated for several days without any maintenance, due in part to the fact that CE
requires only minimum amounts of dirty matrices to be injected. An environmental advantage of this
technique over LC-MS methods is that solvent consumption is minimized.
The results for each analysis were generated by injecting only 30 nL of the available, reconstituted urine
samples, which were collected following standard therapeutic doses of the drugs. The ability to detect
metabolites in this way could have a significant impact in the determination of such substances in biological
matrices for both drug metabolism studies and in areas such as forensic toxicology and the detection of
drugs of abuse.
References
1. D.K. Lloyd in “Biofluid and Tissue Analysis for Drugs, including Hypolipodaemics”, eds. E. Reid, H.M.
Hill, and I.D. Wilson, Royal Society of Chemistry, Cambridge, UK, p. 41 (1994).
2. J. Cai, J. Henion, J. Chromatogr., 703, 667 (1995).
3. M.W.F. Nielen, J. Chromatogr., 712, 269 (1995).
4. I.M. Johansson, R. Powelka, J.D. Henion, J. Chromatogr., 559, 515 (1991).
5. A.J. Tomlinson, L.M. Benson, K.L. Johnson, S. Naylor, J. Chromatogr., 621, 239 (1993).
6. S.S. Adams, R.G. Bough, E.E. Cliffe, B. Lessel, R.F.N. Mills, Toxicology and Applied Pharmacology, 15,
310 (1969).
7. K. Florey ed. in “Analytical Profiles of Drug Substances”, Academic Press, New York, p. 1 (1979).
ApplicationNOTE
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