Determination Of Zaleplon And Its Metabolites In Bio-samples

The non-Benzodiazepines drugs is an inhibitor which has direct role in the central nervous system,belongs to the third generation of sedative hypnotics.The representative drugs are zolpidem,zopiclone and zaleplon,which have a good function of sedative hypnotic,antianxiety,and muscle relaxation since them appeared in the late 1980 s.The zaleplon is commonly used for clinical treatment of sedative hypnotic,which is widely used.At the same time,some criminals also use the zaleplon commit suicide,anesthesia robbery,anesthesia rape or other crimes.So it is necessary to establish a rapid and effective identification method.Given that situation,this article studied the determination methods of zaleplon and its two metabolites in bio-samples.The detailed steps as follows: 1.Establish the analyzing method of zaleplon.2.Establish the pretreatment of zaleplon and its two metabolites in blood with protein precipitation,QuEChERS.3.Establish the pretreatment of zaleplon and its two metabolites in urine with protein precipitation,solid-phase extraction(SPE).4.Study on the Metabolism Regul of zaleplon by establish a model of rats.Results:1.Waters BEH C18 column by gradient elution with 0.1% formic acid-0.1% formic acidacetonitrile as mobile phase,ionized with positive electrospray(ESI+),detected under multiple reaction monitoring(MRM)mode.The parent ion for zaleplon was m/z 306.1.Two daughter ions were monitored at m/z 264.4 and 236.0.The parent ion for 5-oxozaleplon was m/z 322.2.Two daughter ions were monitored at m/z 252.2 and 280.2.The parent ion for desethylzaleplon was m/z 278.2.Two daughter ions were monitored at m/z 260.3 and 236.4.The limit of determination,based on a S/N ratio of 3:1,zaleplon was 0.01ng/mL,5-oxozaleplon was0.05ng/mL and desethylzaleplon was 0.02ng/mL respectively.The limit of quantitation,based on a S/N ratio of 10:1,zaleplon was 0.05ng/mL,5-oxozaleplon was 0.5ng/mL and desethylzaleplon was 0.1ng/mL respectively.The calibration curve was linear within the concentration range 0.5~100ng/mL.The correlation coefficient exceeds 0.999 that indicates an excellent linearity.2.The optimized QuEChERS(Quick,Easy,Cheap,Effective,Rugged and Safe)method was used for blood sample preparation.As a result,zaleplon and its two metabolites displayed excellent linearity in the concentration of 0.5~100ng/mL(R2>0.997).The average recoveries ranged from 92.0%to 100.1%.Inter-day precision was 2.1%~5.3% and intra-day precision was3.1%~10.3%,that indicates a good reproducibility.3.The solid-phase extraction(SPE)method was used for urine sample preparation.As a result,zaleplon and its two metabolites displayed excellent linearity in the concentration of0.5~100 ng/mL.The average recoveries ranged from 88.0%~95.8%.Inter-day precision was2.2%~5.3% and intra-day precision was 5.3%~10.4%,that indicates a good reproducibility.4.Establish a model of 12 female rats whose were given zaleplon by gavage.The rats were collected blood at the set point of time.Rats placed in the metabolic cage were collected natural urine in time,which accurately weighed.The experiment sustained 8 days.The time window of the test is sorted as: 5-Ozaleplon in the urine>zaleplon in the urine>desethylzaleplon in the urine>desethylzaleplon in the blood>zaleplon in the blood >5-Ozaleplon in the blood.This metabolic law is of great significance to the extraction of samples in forensic science and to improve the efficiency of inspection and identification.