Determination of Nonderivatized para-Hydroxylated Metabolites of ...

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16, July/August 1992. Determination of Nonderivatized para-Hydroxylated. Metabolites of Diazepam in Biological Fluids with a GC aegabore Column System.

Journal of Analytical Toxicology, Vol. 16, July/August 1992

Determination of Nonderivatizedpara-Hydroxylated Metabolites of Diazepam in Biological Fluids with a GC aegabore Column System T.V. Beischlag and T, Inaba Department of Pharmacology, Faculty of Medicine, University of Toronto, M5S 1A8 Canada

Abstract I A method was developed using gas chromatography (GC) and s Megabore column system capable of simultaneous detection of diazapam, N-desmethyldiazepam, temazepam, oxazepam, and their par~hydroxylated metabolltes. This method does not require derlvatlzation of para-hydroxylated metabolltes. Standard curves for pure reference compounds were linear, with the minimum detectable concentration of diazepam and its metabolites as low as 0.13 ng/Injection.

Introduction para-Hydroxylation of diazepam (DZP) and its metabolites (Figure 1) is the major biotransfbrmation pathway found in rat (1-3) and has been detected in man (3,4). Several HPLC assay methods (3,5-8) have been described for the measurement of these products, but no reliable gas chromatographic (GC) methods have been reported. We have developed a simple GC megabore column method capable of detecting the para-hydroxylated products of DZP metabolism. This method requires no derivatization of polar samples and detection in the subnanomolar concentration ranges for DZP and p-hydroxydiazepam (p-OHDZP) was adequate for in vivo and in vitro analysis of rat and human metabolites of DZE Diazepam metabolism and the disposition of DZP is known to be widely variable between individuals (4,9-12) and has been linked to the mephenytoin p-hydroxylation polymorphism (13,14). Figure 1 shows a schematic outline of the metabolic pathways of diazepam. This new GC method will aid in the study of diazepam metabolism, especially p-hydroxylation, in different species.

Biochemistry, University of Birmingham, U.K). Urethane (ethyl carbamate) was purchased from BDH Chemicals (Toronto, Ontario); beta-glucuronidase came from Sigma Chemicals. All other chemicals were of analytical reagent grade. Animals, drug administration, and collection. Nine male Wistar rats weighing 260-300 g (Charles River Canada, Lasalle, Quebec) were used and drug administration and collection were by the method of Umeda and Inaba (15). Briefly, three rats were anaesthetized with urethane (i.p. 1.2 g/kg) and their urethral orifices were tied off. Diazepam (25 mg/kg) was injected i.p. Two hours after injection, rats were sacrificed by cervical dislocation, and aliquots of urine were obtained by bladder puncture. Urine collected from three rats after administration of I0 mg/kg temazepam or oxazepam was also obtained. Analysis of diazepam and its metabolites. To a 0.5-mL aliquot of urine was added 0.45 mL acetate buffer (0.5 M at pH 5) and 50 laL ~-glucuronidase (approx. 2900 Fishman U). This mixture was then incubated for 2 h at 37~ The reaction was stopped by the addition of 2 mL ether/isopropanol mixture (v:v 7:3). The mixture was then vortexed vigorously and approximately 0.8 mL of the organic layer was aspirated from each tube and dried gently under nitrogen stream. The dried samples were then redissolved in 100-200 ~ acetone and a 1-2 laL aliquot was injected into the GC for analysis. A Shimadzu GC-9A gas chromatograph fitted with a 15-m DB-17 Megabore capillary column (J&W Scientific) was used for analysis of diazepam and its metabolites. Quantification of DZP ob





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Materials Drugs and chemicals. DZP, N-desmethyldiazepam (NDZ), and p-hydroxy-N-desmethyldiazepam (p-OHNDZ) were provided by Hoffman-La Roche; temazepam (TMZ) and oxazepam (OXZ) by Sandoz Canada (Dorval, Quebec) and Wyeth (Windsor, Ontario), respectively; p-DZP was from Dr. R.H. Waring (Department of 236



Figure 1. Postulated pathways of diazepam oxidation in humans. I = Ndemethylation; II = C3-hydroxylation; III = para-hydroxylation.

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Journal of Analytical Toxicology, Vol. 16, July/August 1992

metabolites was carded out using an electron capture detector


(63Ni, ECD). The injection port and detector temperature was 260~ and the column/oven temperature was 225~ Nitrogen served as the carrier gas. Gas chromatographic analysis of rat urine after DZP, TMZ, and OXZ dosing yielded characteristic GC tracings (see Figures 2-4). Standards for DZP, NDZ, TMZ, OXZ, p-OHDZP, and p-OHNDZ were spiked into blank urine separately and together to identify their characteristic retention times, as listed in Table I. From these tracings it was noted that retention times of DZP and NDZ increased by the same relative amount (3.4x) after hydroxylation at the para position. It was based on these data that theoretical retention times for p-OHTMZ and p-OHOXZ were calculated (see Table I), as these p-hydroxylated compounds could not be obtained. Calibration curves for the primary metabolites of diazepam, pOHDZP, NDZ, and TMZ were constructed by adding varying concentrations (0.5-5.0 of reference compounds to drugfree rat urine and by extracting with ether/isopropanol, as described above; flurazepam (0.5 lag) was used as internal standard and added before the extraction step.

Little has been done to investigate the extent ofp-hydroxylation of diazepam in humans in contrast to the rat or to characterize the enzymatic reactions involved. In order to achieve these ends, a reliable and sensitive method for the determination of these compounds was necessary. Several groups have investigated DZP metabolism but did not attempt to reveal the presence


Results A characteristic GC tracing of DZP and its metabolites is presented in Figure 2. The first four peaks correspond to standards of OXZ, DZP, NDZ, and TMZ, respectively. The largest peak, at 7.1 min, corresponding to the reference compound p-OHDZP, and a peak at 9.6 min for the reference compound p-OHNDZ eluted off the column at a time 3.4 times longer than DZP and NDZ, respectively. After analyzing rat urine extract, two additional peaks were observed, each one eluting at a time 3.4 times. greater than OXZ and TMZ. These two peaks, designated as Y and X, at 4.8 and 12.0 min are interpreted to be p-OHOXZ and p-OHTMZ, respectively. The identification of X and Y can be supported by the observation that after TMZ dosing, both X and Y were observed (Figure 3), while after OXZ only Y and the parent compound were observed (Figure 4). Quantification of DZP and its metabolites was attempted. With flurazepam as internal standard for urine spiked with 0.5-5.0 lag/mL ofp-DZP, NDZ, and TMZ, the standard curves were linear. The lowest concentration for these reference compounds was 125 ng/mL, or 0.13 ng/injection (Figure 5).

Table I. GC Retention Time Metabolites




of Diazepam and Its Relative retention






2.07 1.42 2.82 3.56 7.10 9.60

p-OH/parent 1.0 .69 1.4 1.7 3.4 4.6

1 3.4 -

1 3.4

1 -


Urinary metabolites peak Y* peakX*

TIIV~ (rain) 4.80 12.0

2.3 5.8



3.4 -


"No pure reference compounds available; X and Y from rat urine are proposed to be p-OHTMZ and p-OHOXZ, respectively (see text).

Figure 2. Characteristic gas chromatogram

of DZP and its metaholites in rat urine after i.p. injection of 25 mg/kg DZP. DZP = diazepam; NDZ = Ndesmethyldiazepam; TMZ = temazepam; OXZ = oxazepam; p-0H- = parahydroxy-; X (postulated to be p-OHTMZ); Y (postulated to be p-OHOXZ).


Journal of AnalyticalToxicology,Vol. 16, July/August 1992

of p-hydroxylated metabolites (16,17). St-Pierre and Pang (5) have addressed the problem and reported an HPLC method for simultaneous determination of DZP and all its metabolites. The GC method originally developed in our lab (18) required derivatization of the very polar p-hydroxy metabolites with diazomethane and utilized a packed GC column that lacked sensitivity. Reports that ring-hydroxylated metabolites of dextromethorphan and mephenytoin required no derivatization when measured with capillary columns in the GC assay (19) prompted us to improve the assay for the determination of DZP and its metabolites. The method described in this paper involves no derivatization of any of the polar metabolites. The use of a Megabore column provided improved sensitivity, to the extent that sub-picomolar quantities could be injected onto the GC column and still be recognized with ample sensitivity. With the use of pure reference standard of p-OHDZP and pOHNDZ, it was noted that p-hydroxylation of NDZ and DZP





Figure 4. Characteristic gas chromatogram of OXZ and its metabolite and compoundY (p-OHOXZ)in rat urineafter i.p. injectionof 10 mg/kg oxazepam.

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Figure3. Characteristicgas chromatogram of TMZ and its metabolites in rat urine after i.p. injection of 10 mg/kg temazepam.X = (p-0HTMZ) and Y = (p-OHOXZ).















Figure 5. Calibration curves for NDZ, TMZ, and p-OHDZP,the primary metabolites of diazepamextracted from urine: flurazepamwas added as an internal standard before the extraction step.

Journal of AnalyticalToxicology,Vol. 16, July/August1992

caused an equal relative change in retention time (3.4 x t~ of parent). Therefore it seemed likely, based on this observation, that the peaks Y and X detected with the same relative retention times relative to OXZ and TMZ were p-OHOXZ and p-OHTMZ. Further experiments were done to support the possibility that these compounds are p-OHTMZ and p-OHOXZ. Figure 1 details the biotransforrnation of DZP. By this scheme if TMZ were given alone, only TMZ, OXZ, and their p-hydroxylated counterparts should appear. Analysis of rat urine after TMZ dosing yielded two peaks tentatively identified as p-OHTMZ and p-OHOXZ, as well as TMZ and OXZ (Figure 3). Furthermore, Figure 4 shows that after OXZ dosing, only OXZ and the peak identified as pOHOXZ were present in substantial quantities, consistent with the observation of Sisenwine and Tio (20). The high doses of DZP, TMZ, and OXZ administered to the rats were useful for method development and are consistent with those found in the literature. This assay has detected small quantities ofp-OHDZP, as well as other metabolites in human urine from subjects after oral administration of 10 mg of DZP (21). The polymorphic oxidation of mephenytoin by cytochrome P450 involves p-hydroxylation of the phenyl ring. We plan to study the extent of p-hydroxylation of diazepam in humans and also to compare this pathway with the mephenytoin polymorphism.

Acknowledgment We wish to thank Agnes Bleiwas for critically reading the manuscript. This research was supported by the Medical Research Council of Canada.

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