Supramolecular solvent (SUPRASs) extraction method for detecting benzodiazepines and zolpidem in human urine and blood using gas chromatography tandem mass spectrometry.

OBJECTIVE: A high-throughput and sensitive method using supramolecular solvent (SUPRASs) for detecting 9 benzodiazepines and zolpidem in human urine and blood by gas chromatography-tandem mass spectrometry (GC-MS/MS) was newly established and applied to authentic human urine and blood samples in this study. METHODS: Urine and blood samples were subjected to liquid-liquid extractions with supramolecular solvent mixture which consists of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and subjected to analysis by GC-MS/MS in multiple reaction monitoring (MRM) mode; internal standard method was employed for quantifying of each targeted compound. RESULTS: The regression equation has a good linear relationship with correlation coefficients for all tested compounds were not lower than 0.9991. The lower limits of the quantification ranged from 0.20 to 5 ng/mL for tested compounds in urine; Meanwhile, the lower limits of the quantification in this method ranged from 1 to 50 ng/mL for tested compounds in blood. These results showed that excellent reproducibility and satisfactory extraction recovery rates could be obtained for the established analytical method for 10 drugs in both blood and urine samples. CONCLUSION: The established method in this study was high-throughput, simple and sufficiently sensitive for determining of benzodiazepinesand zolpidem in human urine and blood. Therefore, this newly established method could be of use for qualitative and quantitative determination of such drugs in urine and blood samples either for clinical poisoning monitoring or for forensic identification.

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.

Zolpidem and Zolpidem Carboxylic Acid Results from Medication Monitoring.

Zolpidem (Ambien®) is one of the "Z" drugs often used to improve sleep in older patients and those suffering from insomnia. Schwope, D.M., DePriest, A., Black, D.L., Caplan, Y.H., Cone, E.J., Heltsley, R. (2014) Determing zolpidem compliance: urinary metabolite detection and prevalence in chronic pain patients . Journal of Analytical Toxicology, 38, 513-518 reported that zolpidem in urine is not very prevalent being present <23% of the time in patient urine while the major metabolite, zolpidem 4-phenyl carboxylic acid (ZCA), is much more prevalent in urine with positive rates as high as 50% of the patient samples reviewed. Results from patient testing over a year's time are in agreement with the reported zolpidem results. However, the data observed herein for ZCA are not consistent with the earlier report. These data suggest that monitoring ZCA may result in even higher levels of positivity. Further, while the Food and Drug Administration has pointed out that female dosing should be half that given to males, results of this population testing indicate that the majority of patients (83% male and 73% female) receive 10 mg/day or 12.5 mg/day for Ambien CR® with females demonstrating statistically significantly higher levels of ZCA albeit zolpidem levels are not statistically significantly different between men and women. Estimates of patient positivity are dependent upon the value of the limit of quantification (LOQ) as demonstrated by the zolpidem results herein (LOQ = 50 ng/mL vs. 4 ng/mL). However, even with a much higher LOQ of 50 ng/mL for ZCA in this work, the positivity from ZCA results is significantly higher (e.g., 64.8%) than reported earlier (50.3%). Nevertheless, these data support the addition of ZCA for monitoring zolpidem in urine.