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A simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous analysis enmetazobactam (also known as AAI101) and cefepime in human plasma. Sample preparation was based on protein precipitation with acetonitrile. Separation was performed on Acquity BEH HILIC column (50 mm × 2.1 mm, 1.7 μm) with a mobile phase containing ammonium formate in water and acetonitrile. The analytes were analyzed with the corresponding isotopically labeled internal standards and were detected in multiple reactions monitoring (MRM) using API 5000 triple-quadrupole mass spectrometer with electrospray (ESI) source operating in positive ion mode. The calibration curves were linear over the selected ranges (r > 0.9970 for both analytes). The intra and inter-assay precision of the Quality Control samples showed CV ≤ 15% and the accuracy was within 85 and 115% in all cases for both compounds. The lower limit of quantification was 0.05 μg/mL for enmetazobactam and 0.5 μg/mL for cefepime., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A precise and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of zidebactam (ZID) and cefepime (FEP) in dog plasma. Ceftazidime was used as an internal standard. Protein precipitation method was used as sample preparation approach. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range 0.156-80 μg/mL for ZID and 0.312-160 μg/mL for FEP. The method was validated as per US Food and Drug Administration guidelines and the results met the acceptance criteria. A run time of 3.5 min for each sample made it possible to analyze the maximum number of samples per day. The proposed method was successfully applied for pharmacokinetic study in beagle dogs., (Copyright © 2018 John Wiley & Sons, Ltd.)

As a new hypnotic, zopiclone is widely used in clinical treatment. There are many methods for determination of zopiclone, including spectrophotometry, chromatography and chromatography mass spectrum, etc. Present paper reviews different kinds of biological samples associated with zopiclone, extraction and purification methods, and determination and analysis methods, which aims to provide references for the relevant research and practice., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Aim: Avibactam, a novel non-β-lactam β-lactamase inhibitor co-administrated with the β-lactam antibiotic ceftazidime, is in clinical development. The need to evaluate its PK and PD requires accurate and reliable bioanalytical methods., Methods: We describe LC-MS/MS methods for the determination of avibactam and ceftazidime in human plasma. Avibactam was extracted using weak anionic exchange solid-phase extraction and analyzed on an amide column. Ceftazidime was extracted using protein precipitation and analyzed on a C18 column. Calibration curves were established over 10-10,000 ng/ml (avibactam) and 43.8-87,000 ng/ml (ceftazidime)., Results & Conclusion: Method validation, cross-validation between three laboratories and incurred sample re-analysis demonstrated method robustness. The methods were successfully applied to multiple clinical studies.

An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid-liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), with a flow rate of 0.5mL/min, 1μL injected and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R(2) values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4-12.9% RSD and from -5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50nmol/L (0.19ng/mL) or 0.05pg injected on column, and 3.5nmol/mL (1.10ng/mL) for zolpidem, or 0.27pg injected on column. The limit of detection (LOD) was 0.2nmol/L (0.08ng/mL) for zopiclone and 0.3nmol/L (0.09ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This paper reports an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method to quantitate 21 benzodiazepines, zolpidem and zopiclone in serum and plasma. After liquid-liquid extraction, an Acquity UPLC with a TQ Detector and BEH C18 column was used (Waters, Milford, MA). The injection-to-injection run time was 7.5 min. Forty-eight authentic serum and plasma patient specimens were analyzed and results compared to those obtained using a previously published method. Average r(2) values for linearity (1 to 1,000 ng/mL over five days) were all above 0.995, except α-hydroxytriazolam (0.993). Intra-day and inter-day relative standard deviation values were within ± 15% and the percent deviation from the expected concentrations were within ± 11%. Recovery ranged from 62 to 89%. Matrix effects ranged from -28% to +6%. The limits of detection were 1 ng/mL, except for lorazepam, nordiazepam, oxazepam and temazepam (5 ng/mL). Ion ratios were ± 15% for all analytes. For authentic patient specimens (n = 48, 76 positive results), there was excellent correlation between the UPLC-MS-MS results and the previous method. The best least-squares fit had an equation of y = 1.0708x + 1.6521, r(2) = 0.9822. This UPLC-MS-MS method is suitable for the quantification of benzodiazepines and hypnotics in serum and plasma, and offers fast, reliable and sensitive results.

For fast and reliable screening, identification, and quantification of as many analytes as possible, multi-analyte approaches are very useful in clinical and forensic toxicology. Using ultra high performance liquid chromatography-tandem mass spectrometry, such an approach has been developed for blood plasma analysis after simple liquid-liquid extraction. In the present paper, validation and application is described for 31 neuroleptics, 28 benzodiazepines, and Z-drugs (zaleplone, zolpidem, and zopiclone). The validation parameters included recovery, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes, selectivity, crosstalk, accuracy and precision, stabilities, and limits of quantification and detection. The results showed that the approach was selective, sensitive, accurate, and precise for 24 neuroleptics and 21 benzodiazepines and Z-drugs. The remaining analytes were unstable and/or too low dosed. Cost- and time-saving one-point calibration was applicable only for half of the analytes. The applicability was successfully shown for most of the drugs by analyzing authentic plasma samples and external quality control samples.

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min(-1). Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL(-1). The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

Traditional chiral chromatographic separation method development is time consuming even for an experienced chromatographer. This paper describes the application of computer software ACD Lab to facilitate the development of chiral separation for the quantitation of eszopiclone using LC-MS/MS technology. Assisted by ACD/Chrom Manager and LC Simulator software, the optimal chiral chromatographic development was completed within hours. The baseline chiral separation was achieved with a total cycle time of 3 min. For sample extraction method development, a Waters Oasis Sorbent Selection Plate containing four different sorbents was utilized. Optimal conditions were determined using a single plate under various load, wash and elution conditions. This was followed by a GLP validation which demonstrated excellent intra- and inter-day accuracy and precision for the quantitation of eszopiclone in human plasma at 1.00-100 ng/mL range using LC/MS/MS technology. This method was utilized to support multiple clinic bioequivalence studies., (Copyright (c) 2010. Published by Elsevier B.V.)

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for detection of 23 benzodiazepines and related compounds in whole blood was developed and validated. The method is used for screening and quantitation of benzodiazepines in whole blood received from autopsy cases and living persons. The detected compounds were alprazolam, bromazepam, brotizolam, chlordiazepoxide, demoxepam, clobazam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, estazolam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, midazolam, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, triazolam, zaleplon, and zopiclone. Whole blood from drug-free volunteers was used for all experiments. Blood samples (0.200 g) were extracted with ethyl acetate at pH 9. Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole in positive electrospray ionization, multiple reaction monitoring mode. The use of deuterated internal standards for most compounds verified that the accuracy of the method was not influenced by matrix effects. Extraction recoveries were 73-108% for all analytes. Lower limits of quantification ranged from 0.002 to 0.005 mg/kg. Long-term imprecision (CV%) ranged from 6.0 to 18.7%. We present a fully validated UPLC-MS-MS method for 23 benzodiazepines in whole blood with a run-time of only 5 min and using only 0.200 g of whole blood.