ANALYSIS OF METHYLBENACTYZIUM BROMIDE IN HUMAN URINEANALYSIS
THIN-LAYER
CHROMATOGRAPHYPYROLYSISBY THIN-LAYER CHROMATOGRAPHY AND PYROLYSIS GASCHR OMATO GRAPHY
OMATO
M. NISHIKAWAa, M. TATSUNOa, S. SUZUKIb,* and H. TSUCHIHASHIb
Forensic Science Laboratory, Osaka Prefectural Police H.Q. 3-1-16, Otemae, Chuo-ku, Osaka 540 andb
Identification Center, Kinki Regional Police Bureau, 2-1-22, Otemae, Chuo-feu, Osaka 540 (Japan)(Received May 25th, 1990)(Revision received December 18th, 1990)

a(Accepted January 28th, 1991)

Summary
A rapid and simple method of utilizing thin-layer chromatography (TLC) and pyrolysis gas
chromatography (PyGC) for the identification and determination of methylbenactyzium bromide in
human urine was studied in this report. Methylbenactyzium bromide was extracted from urine with
ODS-cartridge (Sep-Pak C18 ), then spotted onto a silica gel 60 F 254 TLC plate. After development,
the separated spot of methylbenactyzium bromide was scraped and wrapped with a ferromagnetic
foil without extraction by any organic solvents. The sample was applied into PyGC analysis. The optimum temperature for pyrolysis was 590°C. The main degradation product of methylbenactyzium
bromide 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 variation obtained for eleven replicate analyses of the 3ìg /spot of standard methylbenactyzium bromide
was 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

Introduction
Methylbenactyzium bromide (2-diethylaminoethyldiphenylglycolate
methylbromide, C21H28BrNO3; 422.37) has been used as a spasmolytic for thetreatment of gastrointestinal ulcer. The structure of methylbenactyzium is shown
inFig.1.
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, theseparation of this drug from several biological fluids is quite difficult. Furthermore, the drug can not be extracted from scraped silica gel in preparative thin-layer chromatograph and gas chromatographic analysis is difficult because of its
low volatility.
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 wereanalyzed with reversed phase ion pair chromatography [1].
For the analysis of other quaternary ammonium compounds, Ellin et al. reported
high performance liquid chromatography (HPLC) of pyridostigmine in serum after purification with Sep-Pak C18 cartridge [2]. Charles et al. also reported theion pair extraction of propantheline by perchloric acid and the analysis of it by
HPLC [3]. Stevens et al. reported that suxamethonium in urine was extractedwith 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)[5]. However, these methods were difficult to be conducted in forensic laboratories becauseof their handling difficulty.
Tsuchihashi et al. have reported the simultaneous extraction of the eight kindsof quaternary ammonium salt by ODS-cartridge; they also reported the analysisof these compounds with thin-layer chromatography (TLC). They also identified
the pyrolyzed products by Curie point pyrolysis gas chromatography/mass spectrometry (PyGC/MS) [6]. Simple and easy extraction method was carried out, butlarge amounts of urine were required for analysis of these drugs and the interference of urine components were observed on its pyrogram [6]. 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 applied 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 tobe useful in forensic science field.

Materials and Methods

Materials
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.

Apparatus
Pyrolyzer. This was a JHP-3 type model Curie point pyrolyzer (Nippon Bunseki
Kougyo, Tokyo, Japan), and the gas chromatograph a Shimadzu GC-7AG equipped with an CBP-10 fused silica capillary column with a film thickness of 0.25
ìm 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, N2; linear velocity, 30 cm/sec;
split ratio, 1:60. Obtained data was processed with Shimadzu C-R3A data processor. TLC plate was Silica Gel 60 F254(Merck, Darmstadt, FRG) and the ODScartridge was a Sep-Pak C18 obtained from Waters Assoc. (Milford, MA, U.S.A.).
Analytical procedures
One milliliter of urine sample was adjusted to pH 7.5 by saturated trisodiumphosphate solution, and was added to the ODS-cartridge pre-activated by introducing 5 ml of water, 5 ml of methanol and 20 ml of water into the cartridge, andthe drug was retained in the cartridge. The cartridge was washed with 3 ml ofwater then eluted with 3 ml of methanol. The methanol fraction was dried in vacuum and 1 ml of methanol was added to the residue. An aliquot (10 ìl) of thissolution was spotted onto the silica gel plate and developed with 5% formic acid/methanol/tetrahydrofuran (6:6:7, by vol.). The observed spot (RF value was0.55) under UV irradiation (254 nm) was scraped and the scraped fraction corresponding to methylbenactyzium was wrapped with ferromagnetic foil (590 °C,
9 x 23 mm) and pyrolyzed for 3 s in a Curie point pyrolyzer.
Results and Discussion

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 peaksof pyrolyzed products of methylbenactyzium were observed on its pyrogram under
this condition. These products were assigned to benzophenone anddiphenylmethane by gas chromatography/mass spectrometry (GC/MS). GC/MS
spectra showed that the major product of purification followed by pyrolysis wasdiphenylmethane, and the second product being benzophenone. Contrary to thisresult, we have already reported that the major product of pyrolysis of this drug
without PTLC purification was benzophenone [6J. The pyrograms obtained fromt h e s e
e x p e r i m e n t sa r es h o w ni n
F i g .
2 .
The amount of diphenylmethane, the major degradation product of this procedure, was compared with that of the benzophenone, major pyrolyzed productproduced without PTLC purification [6]. The detection limit determined bymajor pyrolyzed products after PTLC purification was twice higher than thatwithout PTLC treatment.

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 thecase of pyrolysis of methylbenactyzium. From these results, diphenylmethane andbenzophenone were presumed to be produced from benzilic acid as an intermediate.

Optimum pyrolysis period and temperature
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 pyrolyzed. The amount of major product, diphenylmethane was measured by its peak areaon the pyrogram. The results are shown in Fig. 3.
The higher the temperature of pyrolysis, the more abundant was the amountof major degradation product observed; however, the background from scraped
silica gel also became higher. Overall, the most suitable pyrolysis temperature
was 590°C.
Also the pyrolysis period was observed for 2, 3 and 4 s. The peak areas of thetwo degradation products on its pyrogram did not increase even with longer period of pyrolysis. Therefore, the pyrolysis of methylbenactyzium was conducted for3s.
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 ofdegradation products were not effected by the amount of silica gel.

Calibration curve
According to this procedure, the calibration curve of methylbenactyzium wasprepared 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 detection limit of methyibenactyzium by this method was 0.3 ìg, and was twice as highas that of direct pyrolysis of effluent from the cartridge without PTLC purification. 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 solution of methylbenactyzium were conducted, the calibration coefficients were 3.8%.
Another quaternary ammonium salt drug
The urinary solution of other quaternary ammonium salts were applied to ODScartridge 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 productson their mass fragmentogram. The peak area ratio of major pyrolyzed productof each compound (Smethod 2 /Smethod 1 ) of both methods were calculated [6]. Thesevalues were in distigmine (150%), pancuronium (807%), propantheline (341%),
suxamethonium (65%), neostigmine (76%) and benzethonium (25%).
These results are summarized in Table 1.

Conclusions
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 wasassigned to diphenylmethane. The calibration curve was prepared using this peakarea on its pyrogram. The optimum temperature and period of pyrolysis was590°C, 3 s, respectively. Under these conditions, the amounts of degradation products were abundant, and the interference by background was not observed. The
detection limit of methylbenactyzium under this condition was 0.3 ìg and the calibration graph showed linearity between 1 and 75 ìg. Furthermore, by this method,
6 other quaternary ammonium salts could be detected with good sensitivity.

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