ANALYSIS OF METHYLBENACTYZIUM BROMIDE IN HUMAN URINEBY THIN-LAYER CHROMATOGRAPHY AND PYROLYSIS GASCHR OMATO GRAPHY
M. NISHIKAWAa, M. TATSUNOa, S. SUZUKIb,* and H. TSUCHIHASHIbaForensic Science Laboratory, Osaka Prefectural Police H.Q. 3-1-16, Otemae, Chuo-ku, Osaka 540 andbIdentification Center, Kinki Regional Police Bureau, 2-1-22, Otemae, Chuo-feu, Osaka 540 (Japan)(Received May 25th, 1990)(Revision received December 18th, 1990)
A rapid and simple method of utilizing thin-layer chromatography (TLC) and pyrolysis gaschromatography (PyGC) for the identification and determination of methylbenactyzium bromide inhuman urine was studied in this report. Methylbenactyzium bromide was extracted from urine withODS-cartridge (Sep-Pak C ), then spotted onto a silica gel 60 F TLC plate. After development,
the separated spot of methylbenactyzium bromide was scraped and wrapped with a ferromagneticfoil without extraction by any organic solvents. The sample was applied into PyGC analysis. The op-timum temperature for pyrolysis was 590°C. The main degradation product of methylbenactyziumbromide was identified as diphenylmethane in this procedure by gas chromatography/mass spectrometry(GC/MS). A calibration graph prepared by absolute calibration method showed a good linearity overthe concentration range of 1-75 ìg/spot for methylbenactyzium bromide. The coefficient of varia-tion obtained for eleven replicate analyses of the 3ìg /spot of standard methylbenactyzium bromidewas 3.8%. The detection limit of this compound by this procedure was 0.1ìg /spot.
Key words: Methylbenactyzium bromide; Thin-layer chromatography; Pyrolysis gas chromatography;ODS-cartridge
Methylbenactyzium bromide (2-diethylaminoethyldiphenylglycolate
methylbromide, C H BrNO ; 422.37) has been used as a spasmolytic for the
treatment of gastrointestinal ulcer. The structure of methylbenactyzium is shown
As with other quaternary ammonium salts, it is very soluble in water and can
not be extracted with any organic solvents. For the reason mentioned above, the
separation of this drug from several biological fluids is quite difficult. Further-
more, the drug can not be extracted from scraped silica gel in preparative thin-
layer chromatograph and gas chromatographic analysis is difficult because of its
There are few reports concerning the analysis of methylbenactyzium. Ohtsubo
et al. suggested in their report that this drug and other quaternary ammonium
compounds were extracted by Bond-Elute CBA extraction cartridge and were
analyzed with reversed phase ion pair chromatography .
For the analysis of other quaternary ammonium compounds, Ellin et al. reported
high performance liquid chromatography (HPLC) of pyridostigmine in serum af-
ter purification with Sep-Pak C cartridge . Charles et al. also reported the
ion pair extraction of propantheline by perchloric acid and the analysis of it by
HPLC . Stevens et al. reported that suxamethonium in urine was extracted
with ion pair extraction using bromothymol blue (BTB) and gas chromatography
(GC) could be performed using ester exchange of this compound [4 ]. Castagnoli
et al. also investigated the extraction of pancuronium in serum with potassium
iodide, and direct inlet/chemical ionization mass spectrometry (DI/CI-MS). How-
ever, these methods were difficult to be conducted in forensic laboratories because
Tsuchihashi et al. have reported the simultaneous extraction of the eight kinds
of quaternary ammonium salt by ODS-cartridge; they also reported the analysis
of these compounds with thin-layer chromatography (TLC). They also identified
the pyrolyzed products by Curie point pyrolysis gas chromatography/mass spec-
trometry (PyGC/MS) . Simple and easy extraction method was carried out, but
large amounts of urine were required for analysis of these drugs and the in-
terference of urine components were observed on its pyrogram . In this report,
the effluents from ODS-cartridge were purified by preparative thin-layer
chromatography (PTLC), and the separated spot was scraped, and this fraction
containing silica gel, was wrapped with ferromagnetic foil and was directly ap-
plied to PyGC analysis. This method was simple and the purification effect of PTLC
would be expected to improve the detection limit of this drug, and considered to
be useful in forensic science field.
Materials and MethodsMaterials Methylbenactyzium bromide was obtained from Yamanouchi Pharmaceutical Co. ,
Ltd. (Tokyo, Japan). Standard solution of this compound were prepared to add
to drug-free human urine (pH 7.2), giving a concentration of 0.01-100 mg/ml.
Other reagents used in this experiment were analytical grade.
Pyrolyzer. This was a JHP-3 type model Curie point pyrolyzer (Nippon Bunseki
Kougyo, Tokyo, Japan), and the gas chromatograph a Shimadzu GC-7AG equip-
ped with an CBP-10 fused silica capillary column with a film thickness of 0.25ìm and dimensions of 50 m x 0.2 mm i.d. The GC conditions were: column
temperature, 150-270°C (10°C/min); carrier gas, N ; linear velocity, 30 cm/sec;
split ratio, 1:60. Obtained data was processed with Shimadzu C-R3A data pro-
cessor. TLC plate was Silica Gel 60 F (Merck, Darmstadt, FRG) and the ODS-
cartridge was a Sep-Pak C obtained from Waters Assoc. (Milford, MA, U.S.A.).
One milliliter of urine sample was adjusted to pH 7.5 by saturated trisodium-
phosphate solution, and was added to the ODS-cartridge pre-activated by introduc-
ing 5 ml of water, 5 ml of methanol and 20 ml of water into the cartridge, and
the drug was retained in the cartridge. The cartridge was washed with 3 ml of
water then eluted with 3 ml of methanol. The methanol fraction was dried in vac-
uum and 1 ml of methanol was added to the residue. An aliquot (10 ìl) of this
solution was spotted onto the silica gel plate and developed with 5% formic ac-
id/methanol/tetrahydrofuran (6:6:7, by vol.). The observed spot (RF value was
0.55) under UV irradiation (254 nm) was scraped and the scraped fraction cor-
responding to methylbenactyzium was wrapped with ferromagnetic foil (590 °C,
9 x 23 mm) and pyrolyzed for 3 s in a Curie point pyrolyzer.
Degradation products of methylbenactyzium by pyrolysis
One milliliter of 1 mg/ml of standard solution of methylbenactyzium bromide
was added to pre-activated ODS-cartridge, the effluent was dried on ferromagnetic
foil, and the residue on the foil was applied to a Curie point pyrolyzer. Two peaks
of pyrolyzed products of methylbenactyzium were observed on its pyrogram under
this condition. These products were assigned to benzophenone and
diphenylmethane by gas chromatography/mass spectrometry (GC/MS). GC/MS
spectra showed that the major product of purification followed by pyrolysis was
diphenylmethane, and the second product being benzophenone. Contrary to this
result, we have already reported that the major product of pyrolysis of this drug
without PTLC purification was benzophenone [6J. The pyrograms obtained from
t h e s e e x p e r i m e n t s a r e s h o w n i n F i g . 2 .
The amount of diphenylmethane, the major degradation product of this pro-
cedure, was compared with that of the benzophenone, major pyrolyzed product
produced without PTLC purification . The detection limit determined by
major pyrolyzed products after PTLC purification was twice higher than that
One milligram of benzophenone or benzilic acid was spotted onto TLC plates
and each fraction was scraped and pyrolyzed by the method mentioned above;
diphenylmethane was not produced from benzophenone. On the other hand,
diphenylmethane and benzophenone were produced from benzilic acid as in the
case of pyrolysis of methylbenactyzium. From these results, diphenylmethane and
benzophenone were presumed to be produced from benzilic acid as an intermediate.
After PTLC of 10 /ìg of methylbenactyzium, the fraction of this drug with silica
gel was wrapped at 386, 445, 500, 590 and 650°C ferromagnetic foil and pyrolyz-
ed. The amount of major product, diphenylmethane was measured by its peak area
on the pyrogram. The results are shown in Fig. 3.
The higher the temperature of pyrolysis, the more abundant was the amount
of major degradation product observed; however, the background from scraped
silica gel also became higher. Overall, the most suitable pyrolysis temperature
Also the pyrolysis period was observed for 2, 3 and 4 s. The peak areas of the
two degradation products on its pyrogram did not increase even with longer peri-
od of pyrolysis. Therefore, the pyrolysis of methylbenactyzium was conducted for
Effect of amounts of silica gel on the detection limits
Methylbenactyzium bromide (10 ìg) was spotted on TLC plates, and was scraped
from the plate containing this area with 1-10 mg (2-10 mm diameter) of silica
gel, and PyGC was conducted. The results are shown in Fig. 4. Peak areas of
degradation products were not effected by the amount of silica gel.
According to this procedure, the calibration curve of methylbenactyzium was
prepared using the peak ea of diphenylmethane; the linearity was observed within
the range of 1-75 / ìg/spot. The calibration curve is shown in Fig. 5. The detec-
tion limit of methyibenactyzium by this method was 0.3 ìg, and was twice as high
as that of direct pyrolysis of effluent from the cartridge without PTLC purifica-
tion. Though methylbenactyzium was also detected by UV absorption (254 nm),
and several color reagents, such as Dragendorff reagent and iodo-platinate reagent
on TLC plate, the detection of limits of this drug by these procedures were lower
than present method. When 11 repeated analyses of 3 /ìg /spot of standard solu-
tion of methylbenactyzium were conducted, the calibration coefficients were 3.8%.
The urinary solution of other quaternary ammonium salts were applied to ODS-
cartridge and the effluents were (1) directly applied to PyGC and (2) after PTLC
purification, also analyzed by Pyr/GC, and the detection limits by both methods
were investigated by comparing with the peak area of major pyrolyzed products
on their mass fragmentogram. The peak area ratio of major pyrolyzed productof each compound (S
) of both methods were calculated . These
values were in distigmine (150%), pancuronium (807%), propantheline (341%),
suxamethonium (65%), neostigmine (76%) and benzethonium (25%).
These results are summarized in Table 1.
The analytical procedure for one of the quaternary ammonium drugs,
methylbenactyzium bromide, utilizing ODS-cartridge extraction, PTLC, and PyGC
was investigated. In this method, the major degradation product of pyrolysis was
assigned to diphenylmethane. The calibration curve was prepared using this peak
area on its pyrogram. The optimum temperature and period of pyrolysis was
590°C, 3 s, respectively. Under these conditions, the amounts of degradation pro-
ducts were abundant, and the interference by background was not observed. The
detection limit of methylbenactyzium under this condition was 0.3 ìg and the cal-
ibration graph showed linearity between 1 and 75 ìg. Furthermore, by this method,
6 other quaternary ammonium salts could be detected with good sensitivity.
1 K. Ohtsubo, S. Higuchi, T. Aoyama, N. Fujii and I. Goto, Sensitive determination ofambenonium chloride in serum from patients with myasthenia gravis using ion exchange resin extraction and reversed-phase ion pair chromatography. J. Chromatogr. , 496 (1989) 397-406. 2 R.I. Ellin, P. Zvirblis and M.R. Wilsom, Method for isolation and determination of pyridostigmine
and metabolites in urine and blood. J. Chromatogr., 228 (1982) 235-244.
3 B.G. Charles and P.J. Ravenscroft, Analysis of propantherine bromide in serum by high-
performance liquid chromatography. J. Chromatogr. , 306 (1984) 424-428.
4 H.M. Stevens and A.C. Moffat, A rapid screening procedure for quaternary ammonium compounds
in fluids and tissues with special reference to suxamethonium. J. Forensic Sci. Soc , 14 (1974)141-148.
5 K.P. Castagnoli, Y. Shinohara, T. Furuta, T.L. Nguyen, L.D. Gruenke, R.D. Miller and N.
Castagnoli, Jr. , Quantitative estimation of quaternary ammonium neuromuscular blocking agentsin serum by direct insertion probe chemical ionization mass spectrometry. Biomed. Environ. Mass Spectrom., 13 (1986) 327-332.
6 H. Tsuchihashi, M. Tatsuno and M. Nishikawa, The analysis of quaternary ammonium compounds
by pyrolysis gas chromatography/mass spectrometry and thin-layer chromatography. EiseiKagaku, 36 (1990) 28-35.
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