CH3 Clan 0. [ Abstract ] Introduction

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1 Journal of Analytical Toxicology, Vol. 23, January/February 1999 The Urinary Elimination Profiles of and Its Metabolites,, Temazepam, and Oxazepam, in the Equine after a 10-mg Intramuscular Dose* Amanda Marland, Pratibha Sarkar ~, and Randy teavitt Maxxam Analytics, Inc., 5540 McAdam Road, Mississauga, Ontario, Canada L4Z I PI [ Abstract ] A method for the extraction of diazepam and its metabolites (nordiazepam, temazepam, and oxazepam) from equine urine and serum and their quantitation and confirmation by liquid chromatography-tandem mass spectrometry is presented. Valium a formulation of diazepam, was administered at a dose of 10 mg intramuscularly to four standard-bred mares. is extensively metabolized in the horse to nordiazepam, temazepam, and oxazepam. urinary concentrations were found to be less than 6 ng/ml. was found to be mainly in its glucuronide-conjugated form and was measured out to a collection time of h. Oxazepam and temazepam were entirely conjugated, and their urinary concentrations were measured out to collection times of 121 h and h, respectively. and nordiazepam were measured in equine postadministration serum out to collection times of 6 and 54 h, respectively. Oxazepam and temazepam were not detected in postadministration serum. Introduction (Valium) is a member of the benzodiazepine class of drugs which exhibit the clinical properties of tranquilizers, anticonvulsants, muscle relaxants, and antianxiety agents. In horses, diazepam has been used as a restraining agent and found to be a useful muscle relaxant (1). Previous studies of diazepam in the horse have shown that diazepam undergoes extensive metabolism to nordiazepam, oxazepam, and temazepam (1,2). was not detected in postadministration urine in this study. Urinary oxazepam and temazepam were completely conjugated, whereas nordiazepam was detected in both its free and conjugated forms., oxazepam, and temazepam are also commercially available drugs. The chemical structures of diazepam and its metabolites are shown in Figure 1. and its * Copyright held by Her Majesty the Queen in the Right of Canada represented by the Minister of Agriculture and Agri-Food, Canada, ~" Author to whom correspondence should be addressed. Current address: Eli Lilly Canada, 3650 Danforth Ave., Scarborough, ON, Canada M1N 2E8. metabolites are also metabolites of medazepam (Nobrium another benzodiazepine (3). The purpose of this study was to obtain elimination data on diazepam and its metabolites after administration of Valium. This work describes the quantitation and confirmation of diazepam metabolites in equine urine by liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods. An N-Demethylation; partial conjugation H I Hydroxylation; conjugation l CH3 Clan 0 Clio Oxazcpaln Hydroxylatlon; conjugation Cl cn~ Tcmazepam N-Demethylation; conjugation Figure I. and ils metabolites, nordiazepam, oxazepam, and temazepam. H tt Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 29

2 Journal of Analytical Toxicology, Vol. 23, lanuary/february 1999 Lorazepam (Internal Standard) LC-MS-MS method was chosen because low detection limits were required for the detection of diazepam in equine urine. Thermal degradation and derivatization of diazepam metabolites that may be required with gas chromatography methods are not factors of LC-MS-MS, and all analytes could be measured in a single analytical run. There have been previous reports on the analysis of glucuronide metabolites of benzodiazepines by LC-negative-chemical-ionization MS (4), high-performance liquid chromatography-thermospray MS of benzodiazepines (5), capillary electrophoresis-ms (6), and use of bench-top LC-MS for the analysis of benzodiazepines (7). Experimental - 101,6 P4,1 '1 " " - ~]~ 7-I I~I llm ~.;; 1.1o ~.II 2,1S rim Temazepam 5633 Reagents and chemicals Analytical standards of diazepam, nordiazepam, oxazepam, temazepam, and lorazepam were obtained from Radian International LLC (Austin, TX). 13-Glucuronidase was obtained from Sigma (St. Louis, MO). Solvents were glass distilled (OmniSolv, HPLC grade) and obtained from BDH (Toronto, ON, Canada). All other chemicals were reagent grade. Drug administration and sample collection Valium was administered intramuscularly to four standardbred mares at a dose of 10 mg at the Equine Drug Evaluation Center in Jerseyville, ON, Canada by Dr. Mike Weber. Urine and serum samples were collected for 120 h. 9, o.72 o t.43 Oxazepam ~ 2483 ~C IC 7~ 6~ g~ 3~ zo 1o n 1~ eo 1o 6o! zo 10 s~ Figure 2. LC-MS-MS multiple reaction monitoring chromatograms of a composite spike at l O ng/ml urine extract. "tim. Urine extraction for quantitation by LC-MS-MS Duplicate standard curves were prepared in blank equine urine at concentrations of 0, 5, 10, 20, 50, 100, 200, and 300 ng/ml for nordiazepam, oxazepam, and temazepam. spikes were prepared at 0, 1, 2, 5, 10, and 20 ng/ml. Quality control (QC) spikes were prepared in duplicate at 50 ng/ml for nordiazepam, oxazepam, and temazepam. QC spikes were prepared at ] ng/ml with each set of samples analyzed. Internal standard (lorazepam, 50 pl of a 10 ng/ljl standard to give 100 ng/ml) was added to urine samples (5 ml), and the ph was adjusted to 5 with 1N acetic acid (200 pl) and 1N sodium acetate buffer at ph 5 (1 ml). 13-Glucuronidase from Helix pomatia (Sigma catalog number G 7017, 97,200 units/ml, 20 IJL) was added to the urine, and the urine was heated at 65~ for 2 h. The ph of the urine was adjusted to 9.5 with ammonium hydroxide buffer at ph 9.5 (1 ml), and dichloromethane (DCM) was added (5 ml). The tubes were rotated for 15 rain, centrifuged at approximately 2000 rpm for 10 rain, and then filtered through phase-separating filters into 50-mL tubes. Hydrochloric acid (6N, 5 ml) was added to the DCM layer; the tubes were rotated for 15 rain, centrifuged, and the DCM layer was discarded. The ph was re-adjusted to 9.5 with 6N sodium hydroxide (5 ml) and ammonium hydroxide buffer at ph 9.5 (0.5 ml). DCM (5 ml) was added, and the tubes were rotated, centrifuged, and filtered through phase-separating filters into 15-mL tubes. The DCM layer was concentrated under nitrogen, and the 30

3 Journal of Analytical Toxicology, Vol. 23, January/February 1999 urine stage II [ Average of four horses,ool i 1 I~l I I I I I I I I I time (h) Figure :igu e 3. Elimination Eli1 nin ttion profiles of diazepam, )am, nordiazepam, c oxazepam, and temazepam after administration of 10 mg of valium (diazepam) intramuscularly as a semi-log plot. 9 -A- Oxazepam 9 Temazepam Table I. Concentrations (ng/mt) in Postadministration Urine after a lo-mg Administration of Valium () Intramuscularly 0-I I I.I * Below LOD (0.6 ng/ml). Table II. Concentrations (nglmt) in Postadministration Urine after a lo-mg Administration of Valium () Intramuscularly 0-I I "i * Below LOD (I.3 nglml). residue was reconstituted in methanol (1 ml) for LC-MS-MS analysis. Serum extraction for quantitation by LC-MS-MS Duplicate serum standard curves were prepared in blank equine serum at concentrations of 0, 1, 2, 5, 10, 20, and 50 ng/ml for all analytes. QC spikes were prepared at 2 ng/ml in duplicate for all analytes with each set of samples analyzed. Serum samples (2 ml) were spiked with internal standard (lorazepam, 20 I.tL of a 10 lag/ml standard to give 100 ng/ml). The ph was adjusted to 5 with 1M sodium acetate buffer at ph 5 (1 ml) and 1N acetic acid (100 I~L). 13-Glucuronidase (15 lal) was added, and the sample was vortex mixed to mix the contents and then heated at 65~ for 2 h. The proteins in the serum precipitated during the heating process to give a thick white mixture. The ph was re-adjusted to 9.5 with ammonium hydroxide buffer at ph 9.5 (1 ml), and DCM (5 ml) was added. The mixture was rotoracked for approximately 10 min, centrifuged for 10 min at approximately 2000 rpm, and then filtered through phase-separating filters into glass tubes. The DCM layer was dried under nitrogen and reconstituted in methanol (1 ml) for LC-MS-MS analysis. Instrumentation A Perkin Elmer Sciex atmospheric pressure ionization (API) III MS with a heated nebulizer inlet at 400~ in the positive ion mode was used for quantitation of all analytes in the multiple reaction monitoring (MRM) mode. The MRM mode monitors the transitions of parent to product ions. The transitions monitored were 285 to 154 for diazepam, 271 to 140 for nordiazepam, 287 to 241 for oxazepam, 301 to 275 for temazepam, and 321 to 275 for lorazepam. For confirmation purposes, the product ion mass spectra of each analyte were obtained in separate analytical,runs of the same extract. Product ion mass spectra are the mass spectra obtained from fragmentation of a single parent ion. A Spectra Physics P200 LC pump and AS3500 autosampler were connected to the MS. A 20-1aL aliquot of extract was injected onto the column (60 RP Select B, mm, 5-1am particle size, Merck). The mobile phase was methanol/0.6% acetic acid (80:20) in 5raM ammonium acetate, and the flow rate was 1 ml/min. 31

4 Journal of Analytical Toxicology, VoL 23, January/February 1999 Results and Discussion Urine Quantitation of diazepam and its metabolites in postadministration urine were calculated based on relative area counts of the analyte to internal standard lorazepam. The 10 ng/ml Table III. Oxazepam Concentrations (ng/ml) in Postadministration Urine after a lo-mg Administration of Valium () Intramuscularly lime (h) Horse C9 Horse D6 Horse 15 Horse L6 0-I I O * Below LOD (I.4 nglml). Table IV. Temazepam Concentrations (ng/mt) in Postadministration Urine after a lo-mg Administration of Valium () Intramuscularly time (h) Horse C9 Horse D6 Horse 15 Horse L6 composite spike extract MRM chromatogram is shown in Figure 2. The limit of detection (LOD) and limit of quantitation (LOQ) calculations were based on eight replicate spikes at 1 ng/ml for nordiazepam, temazepam, and oxazepam, and at 0.5 ng/ml for diazepam. The LODs were calculated based on the addition of three standard deviations to the spike concentration, and LOQs were based on the addi- tion of ten standard deviations to the spike concentration. The diazepam LOD was calculated at 0.6 ng/ml, and the LOQ was 1.0 ng/ml. The other analytes had LODs lower Average than 1.5 ng/ml and LOQs at approximately 2 ng/ml. Seven replicate urine spikes of _ diazepam, nordiazepam, oxazepam, and - temazepam were prepared at 8, 10, and 12 3o ng/ml and analyzed on 3 consecutive days. 80 The inter- and intraday average relative relo6 sponse variances (CV) varied less than 15% 115 for all analytes. 99 The urinary elimination profiles of I00 diazepam and its metabolites as the aver-age 95 of four horses are shown in Figure 3. These 76 results are presented in Tables I-IV. The preadministration urine of each horse 49 was analyzed to show no drug present pre-ad- 45 ministration. The diazepam urinary concen- 19 trations were too low to be confirmed as 15 daughter ion mass spectra; however, a ng/ml spike was easily confirmed. A lar-ger 8.9 sample volume and other modifications to - the method would be required for confirmation of diazepam in postadministration urine. Temazepam and nordiazepam show elimination profiles that peak together; however, Average 0-I I * Below LOD (1.2 nglml). 32 temazepam is detected at later collection times and at higher concentrations than nordiazepam. Oxazepam is detected atthe latest collection times and has a gradual decrease in concentrations with collection times. Oxazepam and temazepam were not de- tected in nonenzyme-hydrolyzed urine extracts. and nordiazepam concentrations were increased in enzyme hydrolyzed urine. The comparison of diazepam and nordiazepam concentrations in enzyme-hydrolyzed and nonhydrolyzed urine is shown in Table V. Serum Serum LODs and LOQs were calculated using the same criteria as for urine based on 1-ng/mL spikes for diazepam and its metabolites. The LODs were calculated to be 1.1 ng/ml, and the LOQs were less than 1.5 ng/ml for all analytes. The inter- and intraday variances based on seven replicate spikes at 8, 10, and 12 ng/ml over 3 days were less than 5% for all analytes. Oxazepam and temazepam were not de-

5 Journal of Analytical Toxicology, Vol. 23, January/February 1999 Table V. Comparison of and Urinary Concentrations (ng/ml) With and Without Enzyme Hydrolysis after a 10-ms Administration of Valium () Intramuscularly to Horse D6 Non- Enzyme Non- Enzyme time (h) hydrolyzed hydrolyzed hydrolyzed hydrolyzed Pre -* * Below LOD. Table VI. Concentrations (ng/mt) in Postadministration Serum after a 10-ms Administration of Valium () Intramuscularly Pre -* min rain rain rain h h h h.... * Below LOD (1.1 ng/ml). Table VII. Concentrations (ng/mt) in Postadministration Serum after a 10-ms Administration of Valium () Intramuscularly Pre -* - 5 rain rain rain rain h h h h h h h h * Below LOD (1.1 ng/ml) tected in the postadministration serum. and nordiazepam postadministration serum concentrations are presented in Tables VI and VII and shown in Figures 4 and 5. Conclusions is only observed at very low concentrations in the urine and is partially conjugated. was quantitated at concentrations less than 6 ng/ml in all postadministration urines. The total peak urinary diazepam concentration (5.5 ng/ml) was observed at the 1-2-h collection time, and the last diazepam detection (0.7 ng/ml) was at h. The peak serum diazepam concentration (17.7 ng/ml) was detected at 40 min postadministration, and the latest detection was at 6 h for all horses. The average peak urinary total nordiazepam concentration was measured at 246 ng/ml at a collection time of 1-2 h. The last detection (2.6 ng/ml) was at h postadministration. Urinary nordiazepam is mainly conjugated. Nonhydrolyzed urine showed a peak concentration of only 12 ng/ml, in comparison to 209 ng/ml for the same horse. was quantitated at concentrations less than 6 ng/ml in postadministration serum. The peak serum concentration was measured at 5.9 ng/ml at 3 h postadministration. The last detection occurred at 54 h postadministration. The average peak urinary oxazepam concentration was measured at 115 ng/ml at the collection time of 8-10 h. The latest detection was at the last collection time of h for two of the four horses. The elimination of oxazepam peaked later than nordiazepam or temazepam, and it was detected at much later collection times. Oxazepam was eliminated only as its glucuronide conjugate, and no oxazepam was detected in nonhydrolyzed urine. Oxazepam was not observed in any postadministration serum with an LOD of 1.1 ng/ml. The average peak temazepam concentration was measured at 192 ng/ml at a collection time of 2-4 h. The latest detection (2.8 ng/ml) was observed at a collection time of h. Temazepam was eliminated only as its glucuronide conjugate. Temazepare, with a limit of detection of 1.1 ng/ml, was not observed in any postadministration serum. 33

6 Journal of Analytical Toxicology, Vol. 23, January/February 1999 I serum stage II 20 ~ (pre-12 h) i authors are grateful for helpful discussions of their colleague Mr. Soo Beng Tan regarding benzodiazepines and their metabolism. ~E 15 C9,~ 10 s time (rain) i ol6 Average LOD l.i ng/ml Figure 4. serum elimination profiles after administration of 10 mg of valium (diazepam) intramuscularly. o = c 4 2 serum stage II (pre-54 h) i i i! w time (rain) II C9.e. O6.dr 15 Q. L6 n Average LOD 1.1 ng/ml Figure 5. serum elimination profiles after administration of 10 mg of valium (diazepam) intramuscularly. Acknowledgments This work was fully funded by the Canadian Pari-Mutuel Agency, Agriculture Canada. Drug administration and sampiing were carried out by Dr. M. Weber and the staff of the Equine Drug Evaluation Center (Jerseyville, ON, Canada). The References 1. W.W. Muir, R.A. Sams, R.H. Huffman, and J.S. Noonan. Pharmacodynamic and pharmacokinetic properties of diazepam in horses. Am. J. Vet. Res. 43: (1982). 2. A.J. Stevenson, M.P. Weber, D. Hopkins, and S. Kacew. Analytical Methodology for Detection and Confirmation of Drugs in Equine Body Fluids. Volume VI: Muscle Relaxants, Sedatives and Tranquilizers. Canadian Pari-Mutuel Agency, Ottawa, ON, Canada, 1997, p J.A.F. de Silva and C.V. Puglisi. Determination of medazepam (Nobrium), diazepam (Valium) and their major biotransformation products in blood and urine by electron capture gas-liquid chromatography. Anal. Chem. 42.' (1970). 4. S. Dragna, C. Aubert, and J.P. Cano. Identification of some glucuronides by direct liquid-inlet liquid chromatography/negative chemical-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 3:17-20 (1989). 5. I.S. Lurie, D.A. Cooper, and R.F.X. Klein. High-performance liquid chromatographic analysis of benzodiazepines using diode array, electrochemical and thermospray mass spectrometric detection. J. Chromatogr. 598:59-66 (1992). 6. I.M. Johansson, R. Pavelka, and J.D. He- nion. Determination of small drug molecules by capillary electrophoresis-atmospheric pressure ionization mass spectrometry. J. Chromatogr. 559: (1991). 7. K.L. Duffin, T. Wachs, and J.D. Henion. Atmospheric pressure ionsampling system for liquid chromatography/mass spectrometry analyses on a benchtop mass spectrometer. Anal. Chem. 64: (1992). Manuscript received January 21, 1998; revision received March 26,