Urinary Screening for Adinazolam and Its Major Metabolites by the ...

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Adinazolam is a triazolobenzodiazepine, currently under clinical investigation, that possesses antidepressant and anxiolytic activity. It has a short half-life (less ...

Journal of Analytical Toxicology, Vol. 17, November/December 1993

Urinary Screeningfor Adinazolamand Its Major Metabolitesby the Emit d.a.u:"and FPIA BenzodiazepineAssayswith Confirmationby HPLC A l b e r t D. F r a s e r 1,2, A r t h u r F. I s n e r 1, a n d W e n d y B r y a n 1

'Toxico/ogy Laboratory, Victoria General Hospital and eDepartment of Pathology, Dalhousie University, Halifax, Nova Scotia Canada B31-12Y9

Abstract Adinazolam is a triazolobenzodiazepine,currently under clinical investigation, that possesses antidepressant and anxiolytic activity. It has a short half-life (less than 3 h), and less than 2% of an oral dose is excreted unchanged. The major urinary metabolite is N-desmethyladinazolam, and minor metabolites are estazolam and or The objective of this study was to characterize the reactivity of adinazolam, N-desmethyladinazolam,and estazolam in the Emit| d.a.u." benzodiazepineassay and the Abbott TDx| urine (FPIA) benzodiazepineassay. N-desmethyladinazolamand estazolam gave an equivalent response to the Emit cutoff calibrator (300 ng/mL) at 100-200 ng/mL, and adinazolam gave an equivalent response at 200 ng/mL. By FPIA, N-desmethyiadinazolam and adinazolam had equivalent net polarization values as the 300ng/mL low control at 500-1000 ng/mL, and estazolam gave a positive response at 300 ng/mL. Six volunteers received single oral doses of 10, 30, and 50 mg of adinazolam. Urine specimens (N=7) were collected from 0 to 36 h post-administration. By Emit, all urine specimens at all doses were positive from 2 to 36 h, and all FPIA analyzed specimens were positive from 2 to 24 h. Confirmation testing was performed by HPLC by analyzing for N-desmethyladinazolam.All urine specimens were confirmed positive for N-desmethyladinazoiam(greater than 200 ng/mL) except for the blank specimens (time = 0) and 7 of 18 specimens collected 36 h post-administration. In conclusion, both immunoassay screening assays are acceptable for detecting the presence of adinazolam in human urine for up to 24 h after a single oral dose of 10-50 mg.

Emit | d.a.u. T M benzodiazepine assay and the Abbott TDx | urine benzodiazepine assay (FPIA) to cross-react with adinazolam and metabolite standards prepared in blank human urine. These systems were also used to analyze urine specimens obtained from human volunteers who received single oral doses of 10-50 mg adinazolam in an Upjohn Company pharmacokinetic study (7). The reactivities of adinazolam, N-desmethyladinazolam, and estazolam (Figure l) were assessed in the Abbott TDx assay and the Emit d.a.u, assay over a wide concentration range (100-10,000 ng/mL). The reactivity of tx-OH-alprazolam has been reported previously (8). The clinical specimens were analyzed by both immunoassay techniques and by a quantitative reversed-phase highperformance liquid chromatography (HPLC) procedure for Ndesmethyladinazolam. Some urine specimens were also analyzed for ct-OH-alprazolam by gas chromatography/mass spectrometry (GC/MS). Toxicology laboratories require reliable screening assays for recently approved benzodiazepines and benzodiazepines under investigation, such as adinazolam, where limited urinary excretion data and few analytical method reports are available.

Methods and Materials

Adinazolam, tx-OH-alprazolam, and N-desmethyladinazolam were obtained from the CNS Diseases Research Office at the

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Introduction Adinazolam is a triazolobenzodiazepine with the same ring structure as alprazolam and triazolam. In clinical trials, adinazolam has been shown to possess anxiolytic, antipanic, and antidepressant activity (1-4). Adinazolam is metabolized in the liver to Ndesmethyladinazolam (Figure 1), N,N-didesmethyladinazolam, estazolam, and ~-OH-alprazolam. The absolute bioavailability of adinazolam is approximately 40%, with concentrations of Ndesmethyladinazolam higher than the parent drug in blood after oral administration. The relative amounts of adinazolam and metabolites excreted in the urine are shown in Table I (5-6). The objective of this study was to evaluate the capability of the






Figure 1. Metabolismof adinazolamin humans.

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JournalofAnalyticalToxicology, Vol17, . November/December 1993 Upjohn Company. Estazolam and 2-OH-imipramine were obtained from Abbott Laboratories and Ciba-Geigy Canada, respectively. (x-OH-alprazolam-d 5 was purchased from Radian Corporation. Reagents for the TDx analyzer were obtained from Abbott Laboratories, and Emit reagents were obtained from Syva Canada. N-O-bis(trimethylsilyl)trifluoroacetamide- 1% trimethylchlorosilane (BSTFA-TMCS) was obtained from Pierce Chemical. The [3-glucuronidase (5000 units/mL, #G-0215) was from Sigma Chemical. Standard solutions of adinazolam, N-desmethyladinazolam, o~-OH-alprazolam, and estazolam were prepared in methyl alcohol at 1 mg/mL. Appropriate dilutions with drugfree urine resulted in eight standard solutions, ranging from 100 to 10,000 ng/mL, o~-OH-alprazolam was prepared in three concentrations from 200 to 1000 ng/mL for GC/MS analysis. Enzyme immunoassay. The standard solutions and urine specimens collected from individuals in the single dose Upjohn study were analyzed according to the standard procedure provided by Syva. Analysis was performed on a Gilford Stasar III spectrophotometer attached to a Syva autocarousel and an LP-6500 data processor. Emit negative, low, and medium calibrators were analyzed in each run along with the adinazolam and metabolite standards, or urine specimens. Fluorescence polarization immunoassay. The Abbott TDx analyzer was operated according to the manufacturer's specifications, as outlined in the operator manuals for the TDx instrument. The Abbott low and high controls were analyzed in each run along with the adinazolam and metabolite standards, or urine specimens. High-performance liquid chromatography. To 2.0 mL of standard, control, or unknown specimen was added 0.1 mL of 2OH-imipramine (internal standard), 0.1 mL of saturated sodium carbonate solution, and 5 mL of toluene. After extraction, the organic layer was taken to dryness at 70~ The residue was reconstituted in 0.1 mL methyl alcohol and 0.5 mL water, prior to being transferred into silanized autosampler vials. Analysis was performed at ambient temperature on a 250- • 4.6-mm RP-8 column with 5 lam particle size (Brownlee Column, Applied Biosystems) using a mobile phase of 0.01M phosphate bufferacetonitrile-nonylamine (800:200:0.5) at a final pH of 3.2. The flow rate was 1.6 mL/min and the eluent was monitored at 230 rim. Liquid chromatography was performed on a Spectra Physics SP8770 pump, SP8780XR autosampler (50-~L injection), SP4270 integrator, and a Hewlett-Packard 1050 variable wavelength UV detector. Five standards were run at concentrations ranging from 200 to 4000 ng/mL. Gas chromatography/mass spectrometry was used for quantitative analysis of tx-OH-alprazolam in the urine specimens of one volunteer receiving a 50-rag dose of adinazolam. A HewlettPackard 5890 gas chromatograph, operated in the splitless injection mode (valves off for 1.5 min), was attached to an HP5970B mass selective detector. A DB-1 column (15 m • 0.26-mm i.d.)

was operated at 200~ for 1.5 min, increased at 30~ to 300~ and held at that temperature. Quantitation was performed on ion 381 and qualifier ions were m/z 383 and 396. For deuterated t~OH-alprazolam, the quantitation ion was m/z 386 and qualifier ions were m/z 388 and 401. For GC/MS analysis, 2 mL of standard or urine specimen was added to 20 laL of deuterated internal standard and heated with 0.5 mL ~-glucuronidase at 37~ for 12-15 h. After cooling, the solutions were treated with 0.1 mL of a saturated solution of sodium bicarbonate and extracted with methylene chloride (5 mL). The organic layer was evaporated to dryness at 50~ dissolved in 20 I.tL ethyl acetate, and derivatized at 70~ for 0.5 h with 20 I.tL BSTFA-TMCS; 2 pL was injected onto the GC/MS.


N-desmethyladinazolam and adinazolam were positive in the Emit assay (Figure 2) at 200 ng/mL, whereas estazolam, positive between 100 and 200 ng/mL, was more reactive. For the FPIA ass a y s (Abbott TDx), the adinazolam and metabolite reactivity w a s compared to the Abbott low control (300 ng/mL), which is equivalent in concentration to the Emit low calibrator. By FPIA (Figure 3), e s t a z o l a m w a s equally reactive at 300 ng/mL as the low control. Adinazolam and N-desmethyladinazolam were less reactive than the estazolam standard and did not give net polarization readings less than the low control until their concentrations were between 500 and 1000 ng/mL. Cross-reactivity of adinazolam by FPIA ranged from 68% at 100 and 200 ng/mL to 11% at 10,000 ng/mL. For N-desmethyladinazolam, cross-reactivity was maximal at 200 ng/mL (72%) and was lowest at 10,000 ng/mL (13%). Estazolam was most reactive, with 125% cross-reactivity at 100 ng/mL and 80% at 2000 ng/mL. The instrument read HI (i.e., beyond the quantitative limits of the assay) for estazolam standard concentrations greater than 2000 ng/mL. Table II shows the between-run precision of the Abbott controls. Urine specimens collected from six volunteers receiving single

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Table I. Urinary Elimination of Adinazolam and Metabolites Compound N-desmethyladinazolam Adinazolam ce0H-alprazolam Estazolam N,N-didesmethyladinazolam


Percent recovery in urine (24 h) >50

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