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Determination of Peptidoaminobenzophenone
(2-o-chlorobenzoyl-4-chloro-N-methyl-N’glycylglycinanilide) and its Metabolites in
Human Plasma by Glass Capillary Gas
Chromatography Negative Ion Chemical
Ionization Mass Spectrometry-1
Shin’ichiro Hashimoto, Eiichi Sakurai, Minoru Mizobuchi and Shir6 Takahashit
Department of Drug Metabolism, Shionogi Research Laboratories, Shionogi & Co. Ltd, Fukushima-ku, Osaka 553,
Japan
Ken-ichi Yamamoto
Department of Pharmacology, Shionogi Research Laboratories, Shionogi & Co. Ltd, Fukushima-ku, Osaka 553, Japan
Takashi Momose
Department of Anesthegiology, National Nagoya Hospital, Naka-ku, Nagoya 460,Japan
A highly sensitive and selective glass capillary gas chromatographic negative ion chemical ionization mass
spectrometric assay was developed to measure peptidoaminobenzophenone(2-o-chlorobenzoyl-4-chloro-Nmethyl-N’-glycylglycinanilide)and its metabolites; chlorodiazepam, chlorodesmethyldiazepam and lorazepam
in human plasma. As peptidoaminobenzophenone underwent pyrolysis during gas chromatography, it was
converted to a thermally stable aminoquinolone. Internal standards for these compounds were the respective
deuterium labelled compounds. Calibration curves were prepared for the range of 1-100 ng rn1-l. Interference
by endogenous substances was negligible in the isobutane negative ion chemical ionization mode in contrast
to the electron impact or positive ion chemical ionization mode. This method was used to determine the plasma
levels in humans following oral administration of two 5 mg doses.
INTRODUCTION
Peptidoaminobenzophenone (l), developed in our
laboratory as a minor tranquilizer, is a prodrug of 1,4benzodiazepine;’ it gives chlorodiazepam (2) in plasma
as the major metabolite. The low dosage used and its
extensive metabolism necessitates a highly sensitive and
selective analytical method to measure the amount of
1 and its metabolites in human plasma.
Several analytical methods are available for quantifying benzodiazepines such as pulse polarography,*
flu~r om e t r y, ~radioimmunoassay;
and thin-layer
chromatography (TLC),5 but the most widely used one
has been electron capture detection (ECD) gas
chromatography6 along with several examples using gas
chromatography mass spectrometry (GC/MS).’” Compound 1 and its metabolites in dog and human plasma
were quantified with ECD gas chromatography by Agoh
et a1.8
The sensitivity of gas chromatography electron
impact (EI) mass spectrometry is not necessarily high
compared with ECD gas chromatography, but the negative ion (NI) chemical ionization (CI) mode affords the
highest sensitivity for the quantification of ~ l o n a z e p a m . ~
The present work describes a highly sensitive and
selective glass capillary gas chromatographic NICI mass
spectrometric analysis of compound 1 and three of its
metabolites in human plasma. The metabolites detert Author to whom correspondence should be addressed.
mined in this study were chlorodiazepam (2), chlorodesmethyldiazepam (3) and lorazepam (4) as shown in
Scheme 1.
CHI
c,%
CH2NHCOCH2NHr
CI
(HP)
I
2
3
4
Scheme 1. Postulated metabolic pathway of peptidoarninobenzophenone.
As compound 1 underwent pyrolysis during gas
chromatography, it was converted into a thermally
stable aminoquinolone (5) by heating with alkali.
1
5
CCC-0306-042X/82/0009-0546$03.00
546
BIOMEDICAL MASS SPECTROMETRY, VOL. 9, NO. 12, 1982
@ Wiley Heyden Ltd, 1982
DETERMINATION OF PEPTIDE-AMINOBENZOPHENONE
Internal standards for these five compounds were
their respective deuterium labelled compounds (*H3or
2
H4).
pounds: C, ('H3)li D, ('H4)2+ ('H4)3
('H4)2+ ('H3)5; F, ( H4)3+ ('H4)4.
+ ('H4)4;
E,
Calibration curves
EXPERIMENTAL
Chemicals and reagents
Compounds 1-5 and their deuterium labelled compounds were synthesized by our group and the method
will be reported elsewhere in the near future. Extracting
solvents were used HPLC grade (Kanto Chemical Co.
Inc., Tokyo, Japan) and other chemicals and solvents
of reagent grade were used without further purification.
Instruments and parameters
The gas chromatography mass spectrometry (GC/MS)
system was a Varian-MAT 4 4 s coupled to a Varian
Model 3700 gas chromatograph with a split-splitless
injector (Varian-MAT, Bremen, FRG). The glass
capillary column was wall-coated with SE-54
(25 m x 0.3 mm i.d.). Operating temperature were injector, 270 "C; transfer line, 235 "C; ion source, 180 "C;
column, group A (compounds 2 and 5 (TFA) were
analysed in the split mode 1:20): 250 "C isothermal;
group B (compounds 3 (TMS) and 4 (2-TMS) were
analysed in the splitless mode): 175 "C isothermal for
1 min followed by temperature programming at 30 "C
per min to 260 "C. After a delay of 60 s following the
injection, the split exit vent was opened to give a splitting ratio of 1:20. Carrier gas: He; inlet pressure, 1 4
psi and linear velocity 65 cm s-'. The quantification was
carried out by the MIS program of this instrument in
the negative ion chemical ionization mode and the parameters were set as follows: dwell time for group A
(compounds 2 and 5 (TFA)) = 328 ms, and dwell time
for group B (compounds 3 (TMS) and 4 (2-TMS))=
169 ms. When compounds of group A were analysed,
the GC/MS system was deactivated by injection of a
solution of dipalmitoyl phosphatidylcholine. The typical
ion source operating conditions were: ion box potential
10 V; pusher potential 0 V; draw-out plate potential
OV; lens potential 1 8 0 V; electron energy 180eV;
emission current, 0.25 mA; voltage of secondary electron multiplier, 1700 V. Reagent gas, isobutane (c.
500 p.bar). The ion source voltages were set to give the
maximum signal response consistent with satisfactory
ion peak shape and unit mass resolution.
Stock solutions
Stock solutions (1.00 mg per 10.00 ml, as the free compound, except peptidoaminobenzophenone (1)which
was quantified as monohydrate) of compounds 1-5 were
prepared in ethanol and stored in a refrigerator at
- 20 "C. These solutions were diluted to the appropriate
concentration (1.00 or 0.10 ng pl-l) before use. These
solutions were stable over half a year.
Internal standard solutions (1.00 ng p.1-I) were prepared with the following combinations of labelled com-
Two calibration curves were prepared, the first to
confirm the validity of this analytical method without
the extraction and purification processes and the second
one for use as the actual working calibration curve.
The first curve (without extraction) was prepared as
follows: stock solutions of compounds 2 and 5 (1, 2, 4,
10,20,40 and 100 ng) and the internal standard solution
E (100 p.1= 100 ng) were placed in minivials (1ml,
Wako Pure Chemicals; Osaka, Japan). After evaporation of the solvent under a stream of nitrogen, MBTFA
(30 pl, Pierce: Rockford, Illinois, USA) was added and
the resulting solution was heated at 80 "C for 0.5 h on
a Reacti-Therm (Pierce). The excess reagent was evaporated under a stream of nitrogen, and the residue was
dissolved in AcOEt (5Opl). The solution (2 p1) was
injected into the gas chromatograph mass spectrometer.
Compounds 3 and 4 (1, 2, 4, 10, 20, 40 and 100ng)
and the internal standard solution F (100 p1) were also
placed in minivials (1ml). After evaporation of the
solvent under a stream of nitrogen, BSA (50 pl, Pierce)
was added, and the resulting solution was heated at
80 "C for 0.5 h. The solution (3 p.1) was injected into
the gas chromatograph mass spectrometer.
The second curve (with extraction) was prepared as
follows: To Hyland control serum (1.0m1, Div.
Travenol Laboratories, Inc. Bannockburn, Illinois,
USA) in a centrifugal tube (12 ml) were added 1, 2, 4,
10, 20,40 and 100 ng aliquots of the standard solution
of 1 and 100 p.1 of the internal standard solution C.
These mixtures were vortexed briefly, then 10 N KOH
(0.1 ml) was added to the spiked serum and the tubes
were heated on a boiling water bath for 1 h. After
cooling, the serum was vortexed twice with benzene
(5 ml) and centrifuged, then the two benzene extracts
were combined. Another control serum (1.0 ml) containing the standard of solution 2, 3 and 4 (1, 2, 4, 10,
20, 40 and 100ng each) and the internal standard
solution D (100 1.1) was extracted with benzene by the
same procedure as above, and then both benzene
extracts were combined and evaporated under reduced
pressure using a rotary evaporator. The residue was
applied to a T L C plate (Art 5715, E. Merck, Darmstadt,
FRG) and developed in a solvent system (CH'C12CH30H = 6 : 1).Compounds 2 and 5 (Rr= 0.59-0.77)
and compounds 3 and 4 (R,
= 0.29-0.55) were extracted
from the plate with AcOEt, respectively.
The mixture of 2 and 5 was placed in the minivial
(1ml), dissolved in MBTFA (30 pl, Pierce) and heated
at 80 "C for 0.5 h on the Reacti-Therm. After evaporation of the excess reagent, the residue was dissolved
in AcOEt (50 pl). The mixture of 3 and 4 was placed
in the minivial (1ml), dissolved in BSA (50 pl, Pierce)
and heated at 80 "C for 0.5 h on the Reacti-Therm.
Reproducibility test
The standard solutions (1,2, 3 and 4) (5, 10 and 50 ng)
in the control serum (1.0 ml) were analysed by the same
procedure as above.
BIOMEDICAL MASS SPECTROMETRY, VOL. 9,
NO. 12,
1982 547
S. HASHIMOTO, E. SAKURAI, M. MIZOBUCHI, S . TAKAHASHI, K-I. YAMAMOTO AND T. MOMOSE
TMS
I
CI
1
1
250
CI
450
350
m/z
m/z
9
CI
TMS
I
CI
250
&$
350
OTM S
4 50
m/z
Figure 1. lsobutane NI CI mass spectra of aminoquinolone derived from peptidoaminobenzophenone and its metabolites.
Analyses of human plasma
Internal standards
Ten patients were administered orally the peptidoaminobenzophenone twice; 5 mg at night and
another 5 mg the next morning. Blood samples were
obtained from the antecubital vein with heparinized
syringes and centrifuged. The plasma samples (2 ml)
were refrigerated at - 70 "C immediately, and stored
at this temperature until analysis.
The plasma were analysed by the same procedure
described for the second calibration curve.
Internal standards of compounds 2, 3 and 4 were the
deuterium labelled compounds of the benzene ring at
the 5-position while those of 1 and 5 were deuteriumlabelled methyl derivatives. Of course, 1 and 5 can also
be labelled with deuterium at the 5-position like 2, 3
and 4, but the amount of the deuteriobenzene derivative
was very small and thus, the deuteriomethyl derivative
was used.
Selected ion monitoring
RESULTS AND DISCUSSION
Derivatization of compounds
As peptidoaminobenzophenone (1) was thermally
unstable and decomposed during gas chromatography,
it was converted into thermally stable aminoquinolone
(5) by the same procedure reported by Agoh et a1.' The
trifluoroacetyl derivative of 5 (5-TFA) was found more
suitable for gas chromatographic analyses with respect
to peak shape and retention time.
Chlorodesmethyldiazepam (3) was strongly absorbed
on the column as was reported in the case of desmethyldiazepam," and lorazepam (4) was known to decompose on gas chromatography. l1 Trimethylsilyl derivatives of both 3 and 4 gave very sharp peaks with
appropriate retention times.
548
BIOMEDICAL MASS SPECTROMETRY, VOL. 9, NO. 12, 1982
Masses and retention times of selected ions for selected
ion monitoring of compounds 2, 3, 4 and 5 are listed
in Table 1. Mass spectra of these compounds are shown
in Fig. 1, the selection chromatograms in Fig. 2, and
the monitored fragments in Scheme 2.
Table 1. Retention times and monitored peaks of peptidoaminobenzophenone and its metabolites
Compounds
2
('H4)2
3 (TMS)
('H4)3 (TMS)
4 (2-TMS)
('H4)4 (2-TMS)
5 (TFA)
('H315 (TFA)
Retention time
Monitored peak
(S)
(m/z)
329
327
389
387
427
426
376
373
284
288
268
272
302
306
414
41 7
DETERMINATION OF PEPTIDE-AMINOBENZOPHENONE
.2
Y
4- 2TMS
3-TMS
lllllllllllllllllllllllllllllllllllnllllllllllll
I*"n*rn*L
I
I
I
m/z
= 284
m/z
m / z = 288
m/z
:414
= 268
m/z = 272
m / z = 417
m/z
= 306
m/z
306
:
Figure 2. Selected ion chromatograms of group A (chlorodiazepam (2) and aminoquinolone (5-TFA)) and group B (desmethylchlorodiazeparn (3-TMS) and lorazepam (4-2TMS))in the NI CI mode.
The base peak of compound 2 is of m / z 282, but the
selected ion chromatogram of this ion showed strong
interfering peaks, thus the ion of m / z 284 (37CI)was
monitored.
amount of the internal standard are Std H and Std 'H,
and the respective amounts of the sample and the standard are W,,
and Wstd, respectively, then the
measured ratio R is given by
R=
Calibration curves
When a sample is monitored at m f z = M H and the
standard at m f z = MzH, the intensities of peaks at MH
and M2H from the sample are Samp H and Samp 2H,
the intensities of peaks at MH and MzH from an equal
Monimwd
peak
[MI7= 414
-
Samp H * Wsamp
+ Std H W s t d
Samp 2H . W,,,, + Std 'H . W s t d
This means R will not be linear if both Samp 'H and
Std H are not zero.
Correction of these items by the method proposed
by Tohno et al." may give a linear regression line. The
corrected calibration curves obtained from the nonextracted standard of the compound (2-5) gave a
straight line through the origin with the theoretical slope
(0 = 45") over 1-100 ng range. But, actual determination of these compounds was carried out with the uncorrected calibration curves.
The equations for the corrected regression lines mentioned above are given in Table 2.
Reproducibility
Replicate analyses over three days of three of each
sample a day containing 5,lO or 50 ng of the compounds
C
I
IMf
T
c
l
-TMS
-CI
268
= 376
-1MS
-0TMS
-
302
Table 2. Equations for the corrected regression lines of
compounds 2 (chlorodiazepam), 3 (desmethyl(lorazepam)
and
5
chlorodiazepam), 4
(aminoquinolone derived from peptidoaminobenzophenone (1))
Compound
2
IM1: = 4 M
Scheme 2. Monitored fragments of aminoquinolone and the
metabolites used.
3
4
5
Equation
y = 0.9555~
+0.0036
y = 0.9917~
-0.0019
v = 1.0343~- 0.0057
y = 0.9686~
-0.0081
Correlation
coefficient
0.9997
0.9997
0.9993
0.9998
BIOMEDICAL MASS SPECTROMETRY, VOL. 3,
Coefficient of
variation (%)
3.4
3.2
5.1
2.5
NO. 12, 1382 549
S. HASHIMOTO, E. SAKURAI, M. MIZOBUCHI, S. TAKAHASHI, K-I. YAMAMOTO AND T. MOMOSE
Table 3. Day-to-day reproducibility of compounds 1-4 in
control serum over 3 days (n = 9)
Spiked amount
Observed amount
Compound
Peptidoaminobenzophenone
Chlorodiazepam
Desmethylchlorodiazepam
Lorazepam
C.V.
long
obs.
C.V.
5 ng
obs.
50 ng
obs.
C.V.
lng)
(%)
(ng)
(%)
(ng)
(%)
4.9
6.7
9.4
6.8
48.1
2.9
(2) 5.1
(3) 4.8
4.5
4.1
9.8
10.3
3.1
2.0
50.8
51.2
2.5
3.8
4.8
4.9
9.8
1.7
48.8
1.5
(1)
(4)
per 1ml of the control serum showed the procedure to
be within an acceptable range of precision and accuracy
as shown in Table 3.
Deactivation of the column
Figure 4. Interferences of endogenous substances in the El
mode; selected ion chromatogram of group A (chlorodiazepam
and aminoquinolone derived from the peptidoaminabenzophenone).
Deactivation of the column by frequent injection of a
5 g 1-l solution of dipalmitoyl phosphatidylcholine
(Sigma, Kingston-upon-Thames, Great Britain) was
reported by Rutherford.” This deactivation method
was effective for the analysis of group A (compounds
2 and 5-TFA) and was done two or three times a day.
However, this method was ineffective for the analysis
of group B (3-TMS and 4-2.TMS).
individuals who had twice received 5 mg of peptidoaminobenzophenone orally were determined by this
method and one typical example of plasma concentraion-time curves are shown in Fig. 3. Details of the
relationship of the plasma level and pharmacological
acitivity will be discussed elsewhere.
Determination with human plasma
Negative ion chemical ionization
The concentrations of the peptidoaminobenzophenone
and its metabolites in plasma samples from ten
The response of these compounds in the NICI mode
were nearly the same as those in the EI mode and the
highest response were obtained in the (PI) CI mode
(about 5 times); an injection of a few picograms in the
pure state could be determined. However, the background from endogenous substances is very low in the
NICI mode compared with the E I and CI modes, thus,
the NICI mode gave the highest signal to noise ratio.
Interference in the E I mode is shown in Fig. 4,and Fig.
2 shows the low background in the NICI mode.
Analysis by gas chromatography NICI mass spectrometry offers much higher sensitivity and selectivity
than either gas chromatography E I mass spectrometry
or gas chromatography positive ion CI mass spectrometry. Garland and Min’ reported that the sensitivity
in the NICI mode is highly dependent on the cleanliness
of the ion source and/or quadrupoles, and the high
sensitivity lasts for approximately two weeks. However,
in our case, the sensitivity of the determination over
two months decreased by only about half.
Selected ion monitoring in the NICI mode is thought
to be the best method for quantifying compounds in
biological fluids, if the compounds display a high negative ion response.
50.0.
Patient
It
B
U”
0-0 Peptidoominabenzophenane
0-0Chlorodiozepom
6.4 Desmethylchlorodionpom
D-GLorozeparn
20
t
5w
K.S
#Q.,
I,
0
t
5
22
6
(h)
(h)
5w
Figure 3. Plasma concentration-time curve of peptidoaminobenzophenone and its metabolites after oral administration
(5 mg x 2).
550
BIOMEDICAL MASS SPECTROMETRY, VOL. 9, NO. 12, 1982
Acknowledgements
The authors are deeply grateful to Dr H. Otsuka, Director of these
Laboratories, for his encouragement in this work. Thanks are due to
Drs Y. Mori and Y. Nakagawa for their helpful discussion and to the
member of the Analysis Room for the sampling of the standard
compounds.
DETERMINATION OF PEPTIDE-AMINOBENZOPHENONE
REFERENCES
1. K. Hirai, T. Ishiba, H. Sugimoto, K. Sasakura, T. Fujishita, Y.
Tsukinoki and K. Hirose, Chem. fharm. Bull. 26,1946 (1978).
2. M. R. Hackman, M. A. Brooks, J. A. F. da Silva and T. S. Ma,
Anat. Chem. 46, 1075 (1974).
3. J. A. F. da Silva and N. Strojny,J. fharm. Sci. 60,1303 (1971).
4. B. Peskar and S. Spector, J. fharmacol. Exp. Ther. 186,167
(1973).
5. P. Haefelfinger, Chromatographia 11, 10 (1978).
6. (a) J. A. F. da Silva, I. Bekersky, C. V. Puglisi, M. A. Brooks
and R. E. Weinfeld, Anal. Chem. 48, 10 (1976); (b) R. E.
Weinfeld, H. N. Posmanter, K.-C. Khoo and C. V. Puglisi, J.
Chromatogr. 143,581 (1977); (c) J. E. Wallace, H. A. Schwertner and E. L. Shimek Jr, Clin. Chem. 25, 1296 (1979).
7. (a) B. H. Min and W. A. Garland, J. Chromatogr. 139, 121
(1977); (b) S. Higuchi, H. Urabe and Y. Shiobara, J.
Chromatogr. 164, 55 (1979).
8. T. Agoh, M. Konishi and Y. Mori, J. Chromatogr. 182, 171
(1980).
9. W. A. Garland and B. H. Min, J. Chromatogr. 172,279 (1979).
10. D. M. Rutherford, J. Chrornatogr. 137,439 (1977).
11. (a) W. Sadee and E. Van der Kleijn, J. Pharm. Sci. 60, 135
(1971); (b) A. Forgione, P. Mattilli, F. Marcucci, R. Fanelli, E.
Mussini and G. C. Jommi, J. Chromafogr. 59,163 (1971).
12. M. Tohno, Y. Matsumura, T. Ofuji. A. Tatematsu, M. Suzuki,
H. Yoshizumi, K. Harada and T. Nadai, Mass Spectrosc. 26,
343 (1978).
Received (revised) 12 April 1982
BIOMEDICAL MASS SPECTROMETRY, VOL. 9, NO. 12, 1982 551
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