Lamotrigine analysis in serum by high-performance liquid chromatography.

Lamotrigine is an anticonvulsant drug soon to be introduced to the North American market. It is chemically unrelated to any currently available antiepileptic drug. The objective of this study was to develop a quantitative high-performance liquid chromatography assay for lamotrigine in serum. Lamotrigine was extracted from serum at alkaline pH into ethyl acetate after addition of the internal standard (BW725C78). After mixing, the organic layer was evaporated to dryness before dissolving the residue in methanol for isocratic separation on a RP-8 column (5 microns) with a mobile phase of water/0.5 M phosphate buffer at pH 6.5/acetonitrile (790/10/200) with eluant monitoring at 306 nm. Calibration was performed with five serum standards (2-32 microM and recovery averaged 88% at 25 microM. Between-run precision was 4.1 and 2.5% C.V. at 13.6 and 31.6 microM, respectively. At room temperature, lamotrigine was stable for a minimum of 7 days. Interference studies were performed on serum specimens containing commonly monitored drugs. The only potentially interfering drug was carbamazepine, which elutes 2.5 times longer than lamotrigine. We conclude that this is a reliable method for quantitation of lamotrigine in serum.

Urinary screening for adinazolam and its major metabolites by the Emit d.a.u. and FPIA benzodiazepine assays with confirmation by HPLC.

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 alpha-OH-alprazolam. The objective of this study was to characterize the reactivity of adinazolam, N-desmethyladinazolam, and estazolam in the Emit d.a.u. benzodiazepine assay and the Abbott TDx urine (FPIA) benzodiazepine assay. N-desmethyladinazolam and 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-desmethyladinazolam and adinazolam had equivalent net polarization values as the 300-ng/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-desmethyladinazolam (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.

Urinary screening for alpha-OH triazolam by FPIA and EIA with confirmation by GC/MS.

Triazolam is a very short-acting triazolobenzodiazepine with sedative-hypnotic properties. Approximately 2% of an oral dose is excreted unchanged in the urine. The major urinary metabolite is alpha-hydroxytriazolam glucuronide (70% of the dose). The objective of this study was to characterize the reactivity of alpha-hydroxytriazolam in the urine benzodiazepine assay by fluorescence polarization immunoassay (FPIA; Abbott TDx) in comparison with enzyme immunoassay (EIA; Syva EMIT d.a.u. benzodiazepine assay). alpha-OH triazolam at 300 ng/mL gave a response equivalent to the 200-ng/mL nordiazepam Abbott calibrator. In the EMIT assay, alpha-OH triazolam gave a response equivalent to the 300-ng/mL calibrator (Syva) at 100-200 ng/mL. Both immunoassays gave positive results in 9 out of 9 urine specimens collected from individuals receiving triazolam. Confirmation was performed by analyzing for alpha-OH triazolam after enzymatic hydrolysis and formation of a TMS derivative for GC/MS. All urine specimens were positive for alpha-OH triazolam. In conclusion, both the FPIA and EIA immunoassay screening assays are acceptable for detecting the presence of alpha-OH triazolam in the urine of patients receiving therapeutic doses of triazolam.

Toxicological analysis of a fatal baclofen (Lioresal) ingestion.

A fatality following ingestion of the drug baclofen (Lioresal) is described. Baclofen was identified in urine by gas chromatography/mass spectrometry. After derivatization with trinitrobenzene sulfonic acid, baclofen was quantitated in serum and urine by high-performance liquid chromatography. The concentration of baclofen was 17 mg/L in serum and 760 mg/L in urine collected approximately 12 h after the overdose. To our knowledge, this is only the second reported fatality involving a baclofen overdose. The previous case did not include quantitation of baclofen in any biological fluid.

Urinary screening for alprazolam and its major metabolites by the Abbott ADx and TDx analyzers with confirmation by GC/MS.

Alprazolam is a short-acting triazolobenzodiazepine with anxiolytic and antidepressant properties. It has a half-life of 10-15 hours after multiple oral doses. Approximately 20% of an oral dose is excreted unchanged in the urine. The major urinary metabolites are alpha-OH alprazolam glucuronide and 3-HMB benzophenone glucuronide. The objective of this study was to characterize the reactivity of alprazolam and three metabolites in the Abbott ADx and TDx urinary benzodiazepine assays compared with the EMIT d.a.u. benzodiazepine assay. Alprazolam (at 300 ng/mL) gave an equivalent response as the 300 ng/mL low control (nordiazepam). alpha-OH alprazolam gave an equivalent response to this control between 300-500 ng/mL and 4-OH alprazolam between 500-1000 ng/mL. The 3-HMB benzophenone was not positive even at 10,000 ng/mL. The ADx screening assay was positive in 26 of 31 urine specimens collected from alprazolam-treated patients. All 31 of these specimens were confirmed positive for alpha-OH alprazolam by GC/MS after enzymatic hydrolysis and formation of a TMS derivative. For the TDx, 27 of 31 specimens were positive for benzodiazepines and all 31 were confirmed by GC/MS. All 5 of the negative ADx specimens and 4 of 5 TDx specimens contained 150-400 ng/mL of alpha-OH alprazolam. In conclusion, both the ADx and TDx urine benzodiazepine assays are acceptable screening assays for alprazolam use when the alpha-OH alprazolam concentration is greater than 400 ng/mL.

Urinary screening for midazolam and its major metabolites with the Abbott ADx and TDx analyzers and the EMIT d.a.u. benzodiazepine assay with confirmation by GC/MS.

Midazolam is a short-acting 1,4-imidazole benzodiazepine with sedative-hypnotic, anxiolytic, and amnestic properties. It is administered orally for sleeping disorders and intravenously for sedation during surgery. This drug has a short half-life (1.5-3.5 h), with less than 1% of a midazolam dose being excreted unchanged. The major urinary metabolite is alpha-hydroxy midazolam glucuronide. The objective of this study was to characterize the reactivity of midazolam and its two major metabolites in the EMIT d.a.u. benzodiazepine assay and in the Abbott TDx and ADx urine benzodiazepine assays. Midazolam and alpha-OH midazolam gave an equivalent response to the EMIT low calibrator at 200 ng/mL. On both Abbott analyzers, midazolam and alpha-OH midazolam gave an equivalent net polarization at 500 ng/mL to the Abbott low control. All three screening assays were positive in all of 21 random urine specimens collected from midazolam-treated patients. Confirmation testing was performed by analyzing for alpha-OH midazolam after enzymatic hydrolysis and formation of a TMS derivative for GC/MS. All urine specimens were confirmed positive for alpha-OH midazolam. In conclusion, all three immunoassay screening assays are acceptable for detecting the presence of midazolam metabolites in urine.

Evaluation of the EMIT amitriptyline and nortriptyline assays for the determination of serum clomipramine and desmethylclomipramine.

Homogeneous enzyme immunoassay reagents (EMIT) developed for the measurement of amitriptyline and nortriptyline in serum were modified to allow quantitation of clomipramine and desmethylclomipramine. The method was compared to a high-performance liquid chromatographic method. Between-run precision [coefficient of variation (CV)] for clomipramine in the EMIT assay for amitriptyline ranged from 2.6 to 3.2%. For desmethylclomipramine in the nortriptyline assay, the between-run CV ranged from 1.4 to 1.9%. Serum specimens from 43 patients (desmethylclomipramine) and 59 patients (clomipramine) were analyzed by both methods, with good correlation between methods. For clomipramine, recovery ranged from 100 to 102% (0-600 ng/ml range) and was 95-103% for desmethylclomipramine (0-600 ng/ml). The modified EMIT assays offered sufficient reproducibility, accuracy, and correlation with an established method for routine analysis of clomipramine and desmethylclomipramine.

Tissue distribution of ethosuximide and clobazam in a seizure related fatality.

The case of a six-year-old male who died in a hospital while receiving several anticonvulsant drugs is described. Phenytoin, desmethyldiazepam, clobazam (an experimental 1,5 benzodiazepine), and desmethylclobazam were quantitated in serum, liver, and brain tissue by high performance liquid chromatography. Ethosuximide was quantitated by gas chromatography. To our knowledge, this is one of few reports describing tissue concentrations of ethosuximide collected at autopsy and the first report of clobazam/desmethylclobazam tissue distribution in man.

Modification of the EMIT tox benzodiazepine assay for screening of alprazolam in serum.

Alprazolam (Xanax) is a relatively new triazolobenzodiazepine which is anxiolytic in man and is also prescribed in the treatment of panic attacks. The objective of this study was to optimize the reactivity of the EMIT tox serum benzodiazepine assay for toxicologic screening of alprazolam. The standard EMIT procedure was extensively modified so that therapeutic concentrations of alprazolam could be detected. Modifications for Methods A and B included a single dilution of specimens, increasing incubation temperature to 37 degrees C, dilution of Reagents A and B, and increasing specimen volume to 100 microL. Method B also included supplementing Reagent A with additional glucose-6-phosphate and beta-nicotinamide adenine dinucleotide. Using serum standards, the modified EMIT assays have detection limits of approximately 25 ng/mL (Method A) and 20 ng/mL (Method B). The patient specimens analyzed were considered positive by Method B and 24/28 positive by Method A. All 28 patient specimens tested by Method B were confirmed positive by gas chromatography/electron capture detection. The day-to-day precision of both methods was less than 2% CV with diluted EMIT calibrators (N = 6). In conclusion, the modified EMIT assay can be used to screen for alprazolam in serum at therapeutic concentrations.

Distribution of trimipramine and its major metabolites in a fatal overdose case.

A fatality following ingestion of the tricyclic antidepressant trimipramine (Surmontil) is described. Quantitation was performed by high-performance liquid chromatography. Trimipramine and desmethyltrimipramine concentrations were 4.8 and 2.1 mg/L, respectively, in postmortem blood. The concentration of trimipramine, desmethyltrimipramine, and their respective 2-hydroxy metabolites were also measured in liver and urine. Analysis of gastric contents revealed a tricyclic antidepressant drug. These findings are compared to previously published reports of trimipramine-related fatalities.

A carpipramine related fatality.

A fatality following ingestion of the tricyclic antidepressant carpipramine (Prazinil) and ethyl alcohol is described. Carpipramine was quantitated by high performance liquid chromatography. The concentration of carpipramine was 2.0 mg/L in blood and 0.44 mg/L in urine. Ethyl alcohol was measured by headspace gas chromatography and found to be 105 mg/dL in blood and 55 mg/dL in the urine. Quantitative analysis of stomach contents was positive for carpipramine by thin-layer chromatography. To our knowledge, this is the first reported fatality involving carpipramine.

Analysis of 2-hydroxyimipramine in an imipramine-related fatality.

A fatality following ingestion of the tricyclic antidepressant imipramine (Novopramine), acetaminophen, and ethyl alcohol is described. Imipramine, desipramine, acetaminophen, and 2-hydroxyimipramine were quantitated by high performance liquid chromatography, and ethyl alcohol by gas liquid chromatography. Concentrations of imipramine, desipramine, 2-hydroxyimipramine, and acetaminophen were: in blood--9.0, 1.1, 3.9, and 11 mg/L; in urine--92, 14, and 42 mg/L (acetaminophen not quantitated in urine). Ethyl alcohol concentration in blood was less than 10 mg/dL and 105 mg/dL in the urine by headspace gas chromatography. These findings are compared to previous reports of imipramine-related fatalities. To our knowledge, this is the first fatality reported involving imipramine where analysis included quantitation of 2-hydroxyimipramine in blood and urine.

A fatality involving clomipramine.

A fatality following ingestion of the tricyclic antidepressant clomipramine (Anafranil), alprazolam (Xanax), and ethyl alcohol is described. Clomipramine and N-desmethylclomipramine were quantitated by high performance liquid chromatography and alprazolam by gas liquid chromatography. Concentrations of clomipramine and N-desmethylclomipramine were: in blood--0.84 and 1.4 mg/L; in urine--0.56 and 0.62 mg/L. Alprazolam concentration in blood was 0.069 mg/L. Ethyl alcohol was measured by headspace gas chromatography and found to be 375, 385, and 435 mg/dL in blood, urine, and vitreous humor, respectively. These findings are compared to previous reports of clomipramine related fatalities and alprazolam toxicity combined with ethyl alcohol.

Toxicologic studies in a fatal overdose of 2,4-D, mecoprop, and dicamba.

A suicidal poisoning committed by a 61-year-old woman, who ingested an unknown quantity of Killex, containing in aqueous solution 100 g/L of (2,4-dichlorophenox)acetic acid (2,4-D), 50 g/L of mecoprop, and 9 g/L of dicamba as amine salts is described. Quantitation of chlorophenoxy acids was performed by extraction from an acidified mixture and concentration before high performance liquid chromatography analysis. All three herbicides were separated in a phosphate buffer/acetonitrile mixture at 280 nm on a RP-8 column. Concentrations of herbicides found were: in blood--520-mg/L 2,4-D, 530-mg/L mecoprop, and 170-mg/L dicamba; in urine--670-mg/L 2,4-D and 520-mg/L mecoprop; in bile--340-mg/L 2,4-D, 530-mg/L mecoprop, and 140-mg/L dicamba; and in liver--540-mg/Kg 2,4-D, 500-mg/Kg mecoprop, and less than 100-mg/Kg dicamba. Liquid chromatography was found to be a reliable method for herbicide quantitation in biological tissues and fluids. The technique offered definite advantages over ultraviolet spectrophotometry and avoids the derivatization requirement for gas chromatography.