Development and Validation of an LC-MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction.

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 µg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified ß-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Detailed quantum mechanical studies on bioactive benzodiazepine derivatives and their adsorption over graphene sheets.

Estazolam (Z1) and related derivatives, adinazolam (Z2), alprazolam (Z3), 4-hydroxyalprazolam (Z4) and triazolam (Z5) have been studied by using various computational tools to analyze their geometry and spectral characteristics. The compounds were found to interact with graphene monolayer results shows that there is enhancement in various physico-chemical descriptors and surface enhanced Raman spectra (SERS). The various reactive descriptors obtained from the FMO analysis predict the reactive nature of the compound. The various lone pair/sigma to pi conjugation was analyzed using NBO formalism, which provides valuable information about intra molecular electron transfer which is vital in predicting the inherent stability of the molecule. Simulated electronic spectra using TD-DFT and CAM-B3LYP functional are discussed in detail with respect to electronic transitions and light harvesting efficiency. Suitability of candidates as a photo sensitizer in dye sensitized solar cells was studied and 4-Hydroxyalprazolam is identified as a suitable candidate. Nucleophilic and electrophilic regions of the molecules are identified using MESP, which adds to the reactivity information. It can be seen that the highest interaction energy has been obtained in the case of the Z5-graphene system, while the lowest interaction energy has been obtained in the case of the Z1-graphene system. Docking indicates that the ligands adsorbed over graphene also form stable complexes with the receptors as indicated by the high binding affinity energy values.

LC-MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium.

An LC-MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n = 5, limit of quantification (LOQ) to 400 ng/g], limits of detection (n = 6, 1-20 ng/g), LOQ (n = 9, 5-20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6-111.5%), imprecision (n = 15, 0-14.6%), matrix effect (n = 10, -73 to 194.9%), extraction efficiency (n = 6, 35.9-91.2%), process efficiency (n = 6, 20.1-188.2%), stability 72 h in the autosampler (n = 3, -8.5 to 9%) and freeze/thaw stability (n = 3, -1.2 to -47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography-tandem mass spectrometry.

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 µL) mixed with 80 µL of the IS solution were centrifuged. An aliquot (5 µL) of the sample solution was directly injected into the LC-MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 µm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5-20 ng/mL (zolpidem), 1.0-40 ng/mL (flurazepam and temazepam), 2.5-100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0-200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10-400 ng/mL (lorazepam and desalkylflurazepam) and 10-100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r(2)) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and -12.8 to 12.5%, respectively. The process efficiency for this method was 63.0-104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than -10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

Pharmacokinetics and physiologic effects of alprazolam after a single oral dose in healthy mares.

The objective of this study was to evaluate the pharmacokinetic properties and physiologic effects of a single oral dose of alprazolam in horses. Seven adult female horses received an oral administration of alprazolam at a dosage of 0.04 mg/kg body weight. Blood samples were collected at various time points and assayed for alprazolam and its metabolite, α-hydroxyalprazolam, using liquid chromatography/mass spectrometry. Pharmacokinetic disposition of alprazolam was analyzed by a one-compartmental approach. Mean plasma pharmacokinetic parameters (±SD) following single-dose administration of alprazolam were as follows: Cmax 14.76 ± 3.72 ng/mL and area under the curve (AUC0-∞ ) 358.77 ± 76.26 ng·h/mL. Median (range) Tmax was 3 h (1-12 h). Alpha-hydroxyalprazolam concentrations were detected in each horse, although concentrations were low (Cmax 1.36 ± 0.28 ng/mL). Repeat physical examinations and assessment of the degree of sedation and ataxia were performed every 12 h to evaluate for adverse effects. Oral alprazolam tablets were absorbed in adult horses and no clinically relevant adverse events were observed. Further evaluation of repeated dosing and safety of administration of alprazolam to horses is warranted.

A mixed MDPV and benzodiazepine intoxication in a chronic drug abuser: determination of MDPV metabolites by LC-HRMS and discussion of the case.

We report on a case of repeated MDPV consumptions that resulted in severe psychosis and agitation prompting the concomitant abuse of benzodiazepines. A 27-year-old man was found irresponsive in his apartment and was brought to the emergency department (ED) of a local hospital. When in ED, he rapidly recovered and self-reported to have recently injected some doses of MDPV that he had bought in the Internet. He left the hospital without medical cares. 15 days after, he was again admitted to the same ED due to severe agitation, delirium and hallucinations, and reported the use of MDPV and pharmaceutical drugs during the preceding week. He was sedated with diazepam and chlorpromazine. Urine samples collected in both occasions were sent for testing using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-high resolution multiple mass spectrometry (LC-HRMS/MS) on an Orbitrap. The LC-HRMS analysis revealed the presence of MDPV and its phase I and phase II metabolites (demethylenyl-MDPV, demethylenyl-methyl-MDPV, demethylenyl-methyl-oxo-MDPV, demethylenyl-hydroxy-alkyl-MDPV, demethylenyl-methyl-hydroxy alkyl-MDPV, demethylenyl-oxo-MDPV and their corresponding glucuronides), alprazolam and alprazolam metabolite at the first ED admission; at the time of the second ED access, the same MDPV metabolites, alprazolam, temazepam, and chlordiazepoxide were detected together with diazepam and metabolites. LC-HRMS/MS was use to determine the following concentrations, respectively on his first and second admission: MDPV 55ng/mL, alprazolam 114ng/mL, α-hydroxyalprazolam 104ng/mL; MDPV 35ng/mL, alprazolam 10.4ng/mL, α -hydroxyalprazolam 13ng/mL; chlordiazepoxide 13ng/mL, temazepam 170ng/mL, diazepam 1.3ng/mL, nordiazepam 61.5, oxazepam 115ng/mL. The toxicological findings corroborated the referred concomitant use of multiple pharmaceutical drugs and benzodiazepines. Confirmation of previous hypothesis on human metabolism of MDPV could be inferred by the analysis of urine.

Hydrophilic interaction liquid chromatography/positive ion electrospray ionization mass spectrometry method for the quantification of alprazolam and α-hydroxy-alprazolam in human plasma.

A hydrophilic interaction liquid chromatography/positive ion electrospray-mass spectrometry (HILIC-ESI/MS) has been developed and fully validated for the quantification of alprazolam and its main metabolite, α-hydroxy-alprazolam, in human plasma. The assay is based on 50µL plasma samples, following liquid-liquid extraction. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5µm) under isoscratic elution. The mobile phase was composed of a 7% 10mM ammonium formate water solution in acetonitrile and pumped at a flow rate of 0.20mLmin(-1). Running in positive electrospray ionization and selected ion monitoring (SIM) the mass spectrometer was set to analyze the protonated molecules [M+H](+) at m/z 309, 325 and 494 for alprazolam, α-hydroxy-alprazolam and tiamulin (ISTD) respectively. The assay was linear over the concentration range of 2.5-250ngmL(-1) for alprazolam and 2.5-50ngmL(-1) for α-hydroxy alprazolam. Intermediate precision was less than 4.1% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis benzodiazepines in human plasma. With a small sample size (50µL human plasma) and a run time less than 10.0min for each sample the method can be used to support a wide range of clinical studies concerning alprazolam quantification.

Metabolism of alprazolam (a marker of CYP3A4) in hemodialysis patients with persistent inflammation.

OBJECTIVE: To investigate the impact of persistent inflammation in hemodialysis (HD) patients on the pharmacokinetics of alprazolam, a cytochrome P450 (CYP) 3A4 substrate, and its metabolites and the role of HD in the impact of persistent inflammation in this clinical context. METHODS: The study population comprised 26 HD patients (mean age 64 years, range 27-79 years; 19 men, 7 women) who were given 1 mg of alprazolam orally in the evening before the day of HD. Unconjugated and conjugated alprazolam and its 4-hydroxy and α-hydroxy metabolites were measured by liquid chromatography-mass spectrometry at 10, 34 (start of HD) and 38 (end of HD) h after intake. C-reactive protein (CRP) was measured weekly beginning 2 months before study initiation, and alpha 1-acid glycoprotein and 4ß-hydroxycholesterol were measured at baseline. CYP3A4 activity was estimated as the ratio of unconjugated alprazolam to 4-hydroxyalprazolam between 10 and 34 h following alprazolam intake. RESULTS: After a single dose of alprazolam, plasma concentrations of unconjugated alprazolam and its metabolites decreased gradually, and unconjugated 4-hydroxyalprazolam was eliminated more rapidly than unconjugated alprazolam by HD. In contrast, the plasma concentrations of conjugated alprazolam and its conjugated metabolites increased during the 34 h following drug intake and the subsequent HD decreased their levels by almost 80%. The ratio of unconjugated alprazolam to 4-hydroxyalprazolam was correlated with CRP levels (r(s) = 0.49, P = 0.01). There was no significant correlation between CYP3A4 activity measured by alprazolam (4-hydroxylation) and alpha 1-acid glycoprotein or 4ß-hydroxycholesterol. Conjugated alprazolam was also found in the plasma. CONCLUSIONS: The correlation between CYP3A4 activity (assessed by alprazolam 4-hydroxylation) and CRP level suggests that inflammation may downregulate CYP3A4 activity. If confirmed, this could have major implications for drug dosing in persistently inflamed patients.

Use of conventional bioanalytical devices to automate DBS extractions in liquid-handling dispensing tips.

BACKGROUND: Conventional liquid-handling devices were employed, along with an improved punching device, to semi-automate dried blood spot (DBS) extraction of alprazolam, α-hydroxyalprazolam and midazolam from human whole blood. Liquid-handling devices were used to add internal standard to the DBS cards and to extract the analytes from the DBS, in order to be analyzed by HPLC-MS/MS. RESULTS: The technique was shown to be accurate (±12.0%) and precise (10.3%) across the dynamic range of the assay. CONCLUSION: The semi-automated extraction reduced sample preparation time by more than 50% when compared with more conventional DBS manual extraction methods.

Quantitation of benzodiazepines in blood and urine using gas chromatography-mass spectrometry (GC-MS).

The benzodiazepine assay utilizes gas chromatography-mass spectrometry (GC-MS) for the analysis of diazepam, nordiazepam, oxazepam, temazepam, lorazepam, alpha-hydroxyalprazolam, and alpha-hydroxytriazolam in blood and urine. A separate assay is employed for the analysis of alprazolam. Prior to solid phase extraction, urine specimens are subjected to enzyme hydrolysis. The specimens are fortified with deuterated internal standard and a five-point calibration curve is constructed. Specimens are extracted by mixed-mode solid phase extraction. The benzodiazepine extracts are derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSFTA) producing tert-butyldimethyl silyl derivatives; the alprazolam extracts are reconstituted in methanol without derivatization. The final extracts are then analyzed using selected ion monitoring GC-MS.

LC-MS/MS analysis of 13 benzodiazepines and metabolites in urine, serum, plasma, and meconium.

We describe a single method for the detection and quantitation of 13 commonly prescribed benzodiazepines and metabolites: alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam and 7-aminoclonazepam in urine, serum, plasma, and meconium. The urine and meconium specimens undergo enzyme hydrolysis to convert the compounds of interest to their free form. All specimens are prepared for analysis using solid-phase extraction (SPE), analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and quantified using a three-point calibration curve. Deuterated analogs of all 13 analytes are included as internal standards. The instrument is operated in multiple reaction-monitoring (MRM) mode with an electrospray ionization (ESI) source in positive ionization mode. Urine and meconium specimens have matrix-matched calibrators and controls. The serum and plasma specimens are quantified using the urine calibrators but employing plasma-based controls. Oxazepam glucuronide is used as a hydrolysis control.

Metabolic interaction between ethanol, high-dose alprazolam and its two main metabolites using human liver microsomes in vitro.

Alprazolam is widely used as a short-acting antidepressant and anxiolytic agent and its effect appears at very low doses while ethanol is used as a social drug worldwide. Sometimes, toxic interactions occur following combined administration of these two drugs. In this study we have investigated the interaction between ethanol and high-dose alprazolam using human liver microsomes in vitro. The interaction effects between ethanol and alprazolam were examined by a mixed-function oxidation reaction using a human liver microsomal preparation. Alprazolam and its two main metabolites (alpha-hydroxyalprazolam: alpha-OH alprazolam, 4-hydroxyalprazolam: 4-OH alprazolam) were measured by HPLC/UV. The production of 4-OH alprazolam, one main metabolite of alprazolam, was weakly inhibited by higher dose of ethanol, but not alpha-OH alprazolam. These results using a human liver microsomal preparation show that the production of 4-OH alprazolam is weakly inhibited by ethanol but not alpha-OH alprazolam. Toxic levels may be reached by simultaneous administration of ethanol and high-dose alprazolam.

Inhibition of CYP3A4 and CYP3A5 catalyzed metabolism of alprazolam and quinine by ketoconazole as racemate and four different enantiomers.

OBJECTIVE: The antifungal drug ketoconazole (KTZ) is known as an inhibitor of, especially, the CYP3A subfamily, which catalyzes the metabolism of a large variety of drugs. Interactions between KTZ and CYP3A substrates have been reported both in vivo and in vitro. Most of them, however, involved the KTZ racemate. KTZ racemate and the separate enantiomers, 2R,4R; 2R,4S; 2S,4S, and 2S,4R, were evaluated for their selectivity in inhibiting alprazolam and quinine metabolism. METHODS: The inhibition of alprazolam and quinine metabolism was studied in an in vitro system of human liver microsomes (HLM), recombinant of CYP3A4 and CYP3A5. The concentrations of formed 3-hydroxyquinine and 4- and alpha-hydroxyalprazolam were measured by HPLC and LC-MS, respectively. RESULTS: Quinine 3-hydroxylation was catalyzed to a similar extent by CYP3A4 and CYP3A5. The formation rate of 4-hydroxyalprazolam was higher than that of alpha-hydroxyalprazolam for each HLM, CYP3A4 and CYP3A5. KTZ racemate and enantiomers showed differential inhibitory effects of quinine and alprazolam metabolism. Quinine metabolism catalyzed by HLM, CYP3A4 and CYP3A5 was potently inhibited by the trans-enantiomer KTZ 2S,4S, with IC(50) value of 0.16 microM for HLM, 0.04 microM for CYP3A4 and 0.11 microM for CYP3A5. The same enantiomer showed the lowest IC(50) values of 0.11 microM for HLM and 0.04 microM for CYP3A5 with respect to alprazoalm 4-hydroxylation and also the same pattern for alprazolamalpha-hydroxylation, 0.13 microM for HLM and 0.05 microM for CYP3A5. Alprazolam metabolism (both alpha- and 4- hydroxylations) catalyzed by CYP3A4 was inhibited potently by the cis-enantiomer KTZ 2S,4R, with IC(50) values of 0.03 microM. CONCLUSIONS: Alprazolam and quinine metabolism is catalyzed by both CYP3A4 and CYP3A5. The present study showed that different KTZ enantiomers inhibit CYP3A4 and CYP3A5 to different degrees, indicating that structural differences among the enantiomers would be related to their inhibitory potency on these two enzymes.

Alprazolam as a probe for CYP3A using a single blood sample: pharmacokinetics of parent drug, and of alpha- and 4-hydroxy metabolites in healthy subjects.

OBJECTIVES: (1) To determine the pharmacokinetic parameters of alprazolam and its two metabolites in plasma from healthy volunteers; (2) to identify a suitable single time point to take a plasma sample for CYP3A phenotyping. METHODS: Twelve healthy Swedish volunteers received a single oral dose of 1 mg alprazolam. Blood samples were collected before drug intake and frequently up to 72 h thereafter. A liquid-chromatography/mass-spectrometry (LC/MS) method was used for the quantification of alprazolam, and 4- and alpha-hydroxyalprazolam. RESULTS: The interindividual variation in the area under the concentration-time curve (AUC) was two, three and fourfold for alprazolam, 4-hydroxyalprazolam and alpha-hydroxyalprazolam, respectively. Plasma concentration ratios collected between 1 h and 48 h for both alprazolam/4-hydroxyalprazolam and alprazolam/alpha-hydroxyalprazolam correlated significantly to the corresponding AUC0-infinity ratios. CONCLUSIONS: The metabolic ratios of alprazolam to respective metabolite in a single plasma sample at 3-24 h are suggested to reflect the alprazolam 4- and alpha-hydroxylation activities. In future, it will be important to study these activities in populations where CYP3A5, in addition to CYP3A4, is expressed at a high frequency and to clarify the relative importance of the two enzymatic pathways for in vivo clearance of alprazolam.

Simultaneous quantification of alprazolam, 4- and alpha-hydroxyalprazolam in plasma samples using liquid chromatography mass spectrometry.

A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4-hydroxyalprazolam and alpha-hydroxyalprazolam in plasma. The work up procedure was solid phase extraction. Liquid chromatography-mass spectrometry (LC-MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05 ng/mL for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within- and between-assay coefficients of variation were in the range of 1.9-17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1mg of alprazolam.

Quantitation of alprazolam and alpha-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS.

A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and alpha-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 microL/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL for both the parent drug and metabolite and linear to 50 ng/mL. The intra-assay percent coefficients of variation (%CV) for AL at 2, 5, and 20 ng/mL were all < or = 5.6. At these concentrations, and all OH-AL intra-assay %CVs were < or = 8.4. The interassay variabilities for AL were 11.8%CV, 8.7%CV, and 8.7%CV at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL interassay variabilities were 9.6%CV, 9.2%CV, and 7.8%CV at the same concentrations, respectively. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/mL 1 h after the dose and AL was detectable for 48 h. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples.

A fatality due to alprazolam intoxication.

Alprazolam is one of the most widely prescribed benzodiazepines in the United States. It is generally considered a safe and effective drug for the treatment of anxiety disorders and panic attacks. Few overdoses that are due to the sole ingestion of alprazolam have been reported. This paper documents a fatality due to alprazolam intoxication and describes the distribution of alprazolam and an active metabolite, alpha-hydroxyalprazolam, in tissues obtained at autopsy. Qualitative identification of the drugs was achieved by full-scan gas chromatography-mass spectrometry, and quantitative analysis was performed by high-performance liquid chromatography. High concentrations of alprazolam were found in all specimens analyzed, but the metabolite was detected only in subclavian blood, urine, bile, and liver. A postmortem heart blood alprazolam concentration of 2.1 mg/L is the highest reported in the literature to date.

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry.

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.

Comparative evaluation of five immunoassays for the analysis of alprazolam and triazolam metabolites in urine: effect of lowering the screening and GC-MS cut-off values.

This study included evaluation of five commercially available immunoassays for the detection of alprazolam and triazolam metabolites in urine following single oral doses of these drugs. The products investigated were the EMIT d.a.u. assay, EMIT II assay, Abbott TDx (FPIA) assay, Bio Site TRIAGE device, and the Boehringer Mannheim/Microgenics CEDIA assay for urinary benzodiazepines. Urine specimens were also analyzed quantitatively by gas chromatography-mass spectrometry. Percent cross-reactivity was assessed by analysis of drug free urine containing drug standards at concentrations ranging from 100 to 10,000 ng/mL. The drug standards analyzed were alpha-OH-alprazolam, alpha-OH-triazolam, and alpha-OH-alprazolam glucuronide. The effect of lowering the screening cut-off value to 100 ng/mL, lowering the confirmation cut-off value to 50 and 25 ng/mL and the use of beta-glucuronidase hydrolysis prior to analysis was also studied. Lowering the screening cut-off value and using enzymatic hydrolysis prior to screening increased the positive detection rate for benzodiazepines with the EMIT d.a.u. assay and fluorescence polarization immunoassay (FPIA). The TRIAGE device gave the lowest percent cross-reactivity in the analysis of the drug standards and gave negative results in all urine specimens analyzed following ingestion of alprazolam and triazolam.