Quantitative determination of zopiclone and zolpidem in whole blood by liquid–liquid extraction and UHPLC-MS/MS (original) (raw)
Related papers
Journal of Analytical Toxicology, 1996
A 26-year-old black female was found dead at home. Her mouth was covered with a fluid containing chalky particles. Empty strips of Imovane | (zopiclone) and an empty bottle of Fortal | (pentazocine) were also found. No urine was available at autopsy. Screening of postmortem blood and stomach contents with enzyme-multiplied immunoassay technique (EMIT) detected only caffeine. Further screening using routine high-performance liquid chromatography (HPLC) with diode-array detection and gas chromatography (GC) with mass spectrometric detection revealed the presence of large amounts of pentazocine in the blood and stomach contents. In the HPLC chromatogram, a second peak that was only partially resolved from the solvent front was observed. Thin-layer chromatography demonstrated the presence of zopiclone, but optimized HPLC and GC conditions had to be used for proper identification and quantitation. This case illustrates the fact that zopiclone can be easily overlooked during routine forensic screening.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2017
A simple, fast, sensitive and stability-indicating derivative spectrofluorimetric method is presented for the assay of zopiclone (ZOP), a drug with hypnotic effect, and its main degradation product and major contaminant, 2-amino-5-chloropyridine (ACP). The method is based on measuring the inherent fluorescence intensity of both drugs at λex = 300 nm in methanol, then differentiation using D1 (first derivative technique). The developed method was found to be rectilinear over a range of 0.2-4 µg/mL of ZOP and 4-100 ng/mL of ACP. The limits of detection were 0.05 µg/mL of ZOP and 0.2 ng/mL of ACP with the limit of quantitation of 0.17 µg/mL of ZOP and 0.7 ng/mL of ACP. The out coming results of the proposed method were compared to those obtained by a reference method showing no significant statistical difference between them concerning precision and accuracy. Additionally, the developed method was applied for detecting ACP in spiked human urine and plasma specimens as a tool of clinical evidence of zopiclone intake that can be easily implemented in forensic laboratories. The proposed method was validated as per ICH guidelines.
Journal of Mass Spectrometry, 2006
Fast gas chromatography/negative-ion chemical ionization mass spectrometric (GC/NICI-MS) assay combined with rapid and nonlaborious sample preparation is presented for the simultaneous determination of benzodiazepines and a-hydroxy metabolites, zaleplon and zopiclone in whole blood. The compounds were extracted from 100 µl of whole blood by simultaneous multitube, microscale liquid-liquid extraction (LLE) and derivatized by N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), without the need for the time-consuming concentration stage. In the analytical separation, various parameters of fast GC/NICI-MS were applied, e.g. the use of hydrogen as a GC carrier gas, a high carrier gas velocity, a small film thickness of the analytical column, fast MS data acquisition, fast temperature ramping, and high initial and final temperatures of GC column. Sensitive identification, screening and quantitation of 18 compounds of interest were achieved in chromatographic separation in only 4.40 min. Accurate and reproducible results were obtained by using five different and carefully selected deuterated analogues on the basis of the chemical properties of the target analytes. Nevertheless, for a-OH-midazolam, and for bromazepam and flunitrazepam at low concentrations, the results can be considered only semiquantitative on the basis of the validation data. The extraction efficiencies ranged from 74.3 to 105.7% and the limits of quantitation (LOQ) from 1 to 100 ng ml −1. Rapid sample preparation and fast chromatographic separation allowed cost-efficient, reliable and high sample-throughput analyses with a low amount of manual work. The method was fully validated and accredited according to EN ISO/IEC 17025 standards and is applicable for sensitive, reliable and quantitative determination of benzodiazepines, zaleplon and zopiclone, e.g. in clinical and forensic toxicology.
Journal of Chromatography B, 2012
A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 L whole blood were added 500 L of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut ® columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepamd 5 , flunitrazepam-d 7 and nitrazepam-d 5 , respectively. The analytes were separated using UPLC with a 2.1 × 100 mm BEH C 18 -column, 1.7 m particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 M for flunitrazepam to the highest of 20 M for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R 2 values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 M and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.
Zopiclone poisoning: tissue distribution and potential for postmortem diffusion
Forensic Science International, 1994
Zopiclone is the first cyclopyrrolone hypnotic and is chemically unrelated to any existing drug. The authors studied the tissue distribution and postmortem redistribution of zopiclone in a fatal suicidal overdose. A 29-year-old female weighing 64 kg had cardiac blood ethanol 153 mg% and zopiclone blood concentrations in the range 0.9-2.0 &ml in 10 distinct sampling sites. ,4fter 40 h at room temperature the range was 0.9-1.8 &ml in 15 samples. Portal venous blood and urine concentrations were 3.0 and 10.5 &ml, respectively. Tissue concentrations (pg/g) were spleen 5.8, peri-renal fat 5.0, psoas muscle 3.3, brainstem 2.8, gastrocnemius muscle 1.9, myocardium 1.6, and kidney 1.7. Eight liver samples had concentrations in the range 0.5-4.9 &g, with highest concentrations in the left lobe and adjacent to the gall-bladder, probably reflecting postmortem diffusion from gastric residue (700 ml, 55.1 &ml) and bile (14.1 &/ml). Of six lung samples, paired upper and middle samples had concentrations in the range 2.1-2.3 pg/g, the right postero-basal 1.3 &g and the left posterobasal 3.4 &g. Drug concentration in putrefactive pleural fluid was also higher on the left (2.1 &ml) than the right (1.4 &ml), probably reflecting postmortem diffusion from gastric residue. The authors conclude that zopiclone showed little preferential concentration in solid organs and consequently has relatively stable postmortem blood concentrations, with little drug redistribution artefacts. Postmortem diffusion from gastric drug residue elevates drug levels in the left lobe of the liver and left lung lower lobe.
Biomedical Chromatography, 2006
A simple and robust method for quantification of zolpidem in human plasma has been established using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Es-citalopram was used as an internal standard. Zolpidem and internal standard in plasma sample were extracted using solid-phase extraction cartridges (Oasis HLB, 1 cm 3 /30 mg). The samples were injected into a C 8 reversed-phase column and the mobile phase used was acetonitrile-ammonium acetate (pH 4.6; 10 mm) (80:20, v/v) at a flow rate of 0.7 mL/min. Using MS/MS in the selected reaction-monitoring (SRM) mode, zolpidem and Es-citalopram were detected without any interference from human plasma matrix. Zolpidem produced a protonated precursor ion ([M+H] +) at m/z 308.1 and a corresponding product ion at m/z 235.1. The internal standard produced a protonated precursor ion ([M+H] +) at m/z 325.1 and a corresponding product ion at m/z 262.1. Detection of zolpidem in human plasma by the LC-ESI MS/MS method was accurate and precise with a quantification limit of 2.5 ng/mL. The proposed method was validated in the linear range 2.5-300 ng/mL. Reproducibility, recovery and stability of the method were evaluated. The method has been successfully applied to bioequivalence studies of zolpidem.
Analele Universitatii "Ovidius" Constanta - Seria Chimie, 2014
A rapid high performance liquid chromatography method, using a monolithic column, was developed for quantitative determinations of benzodiazepines (diazepam, clonazepam, lorazepam, midazolam) in whole blood. A liquid-liquid extraction step with n-chlorobutane isolates the drugs from alkalinized blood. The separation was carried out in reversed phase conditions using a Chromolith Performance (RP-18 100x4.6 mm) column. For the mobile phase, a mixture of a phosphate buffer (pH= 2.5)/acetonitrile (65/35 v/v), in isocratic mode at 2 mL/min. An ultraviolet spectrophotometer was used as the detector at the wavelength of 220 nm. The total run time of the analytical method is less than 4-6 minutes. The calibration curves showed linearity and the correlation coefficient of each individual curve was greater than 0.995. The method was linear over a concentration range of 0.03-0.6 μg/mL for clonazepam, lorazepam and midazolam. For diazepam of linearity was over the range 0.04-5.0μg/mL. Quantific...