Supramolecular Solvents(SUPRASs) Extraction-Gas Chromatography-Tandem Mass Spectrome Try For Detection Of Benzodiazepines And Zolpidem In Urines And Blood

Objective:A high-throughput and sensitive method using supramolecular solvents(SUPRASs)for detecting 9 benzodiazepinesand zolpidem in human urines 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.Method:Urine and blood samples containing nine benzodiazepine and zolpidem drug controls were liquid-liquid extracted with a supramolecular solvent consisting 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;deuterium isotopeinternal standard method was employed for quantification of each targeted compound.Results:(1)When the dosage of tetrahydrofuran is 1m L,1-hexanol is selected as the alkyl alcohol and the dosage is 200 μL,the supramolecular solvent composed of adding to urine or blood and vortexing for 5 minutes has better extraction effect for 10 compound.The fluctuation of extraction efficiency is relatively stable and the pretreatment time was shorter.(2)In urine samples,when the range of mass concentration was 0.20-100 ng/m L for estazolam,1-100 ng/m L for diazepam,midazolam,flunazepam,clozapine and zolpidem,2-100 ng/m L for nitrazepam and clonazepam,5-100 ng/m L for lorazepam and alprazolam,respectively,good linearities obtained,correlation coefficients were 0.999 1-0.999 9,the lower limits of the quantification ranged from 0.20 to 5 ng/m L,the extraction recovery rates for all tested compounds were not lower than 81.12%,,which reached to 102.76%.The intra-day precision [(Relative standard deviation,RSD)] and accuracy(Bias)for this method were 9.86 and 9.51%,respectively;the inter-day precision(RSD)and accuracy(Bias)were 9.53 and 9.99%,respectively.Ten drugs in urine samples showed good stabilities under different storage temperatures within 15 days and after3 freeze-thaw cycles.In blood samples,when the range of mass concentration was1-100 ng/m L for diazepam,midazolam,zolpidem,estazolam and clozapine,2-100ng/m L for flunitrazepam,5-100 ng/m L m L for alprazolam,50-200 ng/m L for lorazepam,nitrazepam and clonazepam,respectively,good linearities obtained,correlation coefficients were 0.999 1-0.999 8,the lower limits of the quantification ranged from 1 to 50 ng/m L,the extraction recovery rates for all tested compounds were not lower than 80.74%,which reached to 95.84%.The intra-day precision(RSD)and accuracy(Bias)for this method were 9.69% and 9.73%,respectively;the inter-day precision(RSD)and accuracy(Bias)were 9.04% and 9.64%,respectively.Ten drugs in blood samples showed good stabilities under different storage temperatures within 15 days and after 3 freeze-thaw cycles.(3)This experimental method was applied to 16 healthy volunteers who divided into 2 groups with a single dose of 5 mg diazepam and zolpidem respectively.After taking the drug,the urine and blood drugs were measured.The concentration of diazepam in urine of volunteers in the first group was 1.07 to 5.03 ng/m L,at each determination during 8-120 h after ingestions,the concentration of diazepam in blood was 1.43 to 49.19 ng/m L,at each determination during 24-312 h after ingestions.The concentration of zolpidem in the urine of volunteers in the second group was 1.07 to 54.86 ng/m L,at each determination during 8-24 h after ingestions,the concentration of zolpidem in the blood was 1.87 to 72.56 ng/m L,at each determination during 8-24 h after ingestions.Conclusion:The method based on supramolecular solvent extraction composed of tetrahydrofuran and 1-hexanol,combined with GC-MS/MS determination,was first applied to the analysis of benzodiazepines and zolpidem drugs in urine and blood.The method provided by this institute is simple,fast,accurate and sensitive.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,public security organs and for judicial identification.