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.

UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets.

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for sample preparation. The 20 µL of 1-undecanol and 200 µL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSH™C18 column with mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in positive mode. The assay demonstrated good linearity in the range of 0.27-1000 ng mL-1 with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL-1, respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.

Identification of Suvorexant in Urine Using Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry (LC-Q/TOF-MS).

Suvorexant (Belsomra®) is a new hypnotic drug with a novel mechanism of action. In prescribed doses of 10 mg before bedtime, the drug produces rapid onset of sleep by inhibiting the orexin neurons of the arousal system, promoting decreased wakefulness. Suvorexant is a potent and highly selective dual orexin receptor antagonist. Sedative hypnotics are of forensic importance due to their widespread use, potential for additive effects with other central nervous system depressants, impairing effects and potential for misuse. In this report we describe a highly sensitive assay for the identification and quantification of suvorexant in urine. Suvorexant was isolated using liquid/liquid extraction (LLE) and identified using liquid chromatography-quadrupole/time-of-flight mass spectrometry. Suvorexant was quantified using a quadratic calibration model between 5 and 250 ng/mL (R2 = 1.000, n = 6). Processed sample stability was demonstrated for up to 24 h. The limit of detection was 0.5 ng/mL and the limit of quantification (LOQ) was 5 ng/mL. The accuracy, bias and precision of the assay at the LOQ were 99% (81-117%), -1% and 12% (n = 18). Intraassay (n = 5) and interassay (n = 15) precision (% CV) at 10, 50 and 200 ng/mL were ≤8%, and bias ranged from -2% to 4% (98-104% accuracy). No qualitative interferences were detected from matrix, internal standard or 50 common drugs. Matrix effects evaluated at low and high concentrations were -16% and -9%, respectively, and produced CVs of 11% and 5% (n = 20). Suvorexant is a new drug of forensic importance. In this report we describe how a simple acidic/neutral LLE can be used to isolate this lipophilic drug with high recoveries and sound analytical performance.

Quantification of suvorexant in urine using gas chromatography/mass spectrometry.

Suvorexant (Belsomra®) is a novel sedative hypnotic drug that is prescribed to promote sleep in patients with insomnia. It is the first of a new class of drugs classified as dual orexin receptor antagonists (DORAs). Sedative hypnotics with central nervous system depressant effects feature prominently in forensic toxicology investigations. For this reason, a new analytical method was developed to identify suvorexant in urine using liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Due to the absence of a commercially available isotopically labeled internal standard, estazolam-D5 was used due to its azepine, triazole and chlorinated functionality. The limit of detection and limit of quantitation was 10ng/mL and the linear range of the assay was 10-1000ng/mL. Accuracy and precision (%CV) were 98-101% and <11% at 30, 250 and 800ng/mL. Interferences from matrix and fifty common drugs were not present and processed samples were stable for 24h at room temperature. Suvorexant is a new drug of significant forensic interest due to its hypnotic and central nervous system depressant effects. The absence of commercially available metabolites and its chromatographic properties present some challenges in terms of identification. Nevertheless, a robust, reliable and sensitive assay was developed to identify suvorexant using GC/MS analysis.