Establishment Of Analytical Method For Benzodiazepines And Study Of Pharmacokinetics In Rats By LC-MS

Objective To establish the analytical method of pyrazolam,clonazepam,diclazepam,meclonazepam,flubromazepam,flubromazolam in rat plasma and urine by LC-MS.And to study the pharmacokinetics of pyrazolam,diclazepam and its metabolites of diclazepam.Methods 1.The optimal conditions were obtained by optimizing the chromatographic and mass spectrometry conditions of pyrazolam,clonazepam diclazepam,meclonazepam,flubromazepam,flubromazolam six benzodiazepines(BZDs).And optimizing the pretreatment conditions of the six BZDs in the plasma and urine by comparing the effects of protein precipitation and liquid-liquid extraction on the extraction recovery rate.Finally,the method was validated by specificity,standard curve and lower limit of quantification,precision and accuracy,extraction recovery rate and matrix effect,and stability.2.Rats were given 0.09 mg/kg of pyrazolam,and the pharmacokinetics of pyrazolam in rats was studied by collecting plasma after administration to determine the blood concentration.3.Rats were given diclazepam 2.625 mg/kg,and the pharmacokinetics of diclazepam and its metabolites in rats were studied by collecting plasma and urine.4.An anesthesia case involving diclazepam and its metabolites was decteted and analyzed to prove whether the suspect committed a crime through diclazepam.Results 1.It could simultaneously detect pyrazolam,clonazepam,diclazepam,meclonazepam,flubromazepam,flubromazolam in rat plasma and urine by HPLC-Q-TOF-MS.All analytes had a good linear relationship within the linear range of 20 to 2000 ng/m L,and the lower limit of quantification was 20 ng/m L,the precision was between-15% and15%,and the accuracy was basically greater than 80% and less than 115%.The six BZDs in rat plasma and urine after protein precipitation method and liquid-liquid extraction method had better extraction recovery rate,and the matrix effect was small,respectively.It was stable under three conditions of 20-24 h at room temperature,freezing for three days,and repeated freezing and thawing for three times.2.After intragastric administration of pyrazolam(0.09 mg/kg)to rats,pyrazolam was only detecteded in the plasma 0.5-24 h after administration,but was not detected in the plasma on the 2nd and 3rd day.The half-life of pyrazolam in rats was about 1.5 h,and the highest plasma concentration was reached 30 min after administration.3.After intragastric administration of 2.625 mg/kg of diclazepam to rats,the half-life of diclazepam was about 93 h.In plasma,diclazepam and the three metabolites delorazepam,lometazepam and lorazepam all reached the highest plasma concentration 1 h after administration,and the detection time limits were 144 h,24 h,144 h,48 h,respectively.In urine,the peak time of diclazepam,delorazepam,lometazepam and lorazepam were all 8h,and the detection time limits were 144 h,144 h,81 h,81 h,respectively.4.Diclazepam,delorazepam,lometazepam and lorazepam were detected in the blood of the victim,and the contents were 16.94 ng/m L,345.33 ng/m L,33.81 ng/m L,30.56 ng/m L,respectively.Conclution This study established a rapid and sensitive detection method that can simultaneously detect pyrazolam,clonazepam,diclazepam,meclonazepam,flubromazepam,flubromazolam,and the metabolites of diclazepam in rat plasma and urine by LC-MS.There have been preliminary studies on the pharmacokinetics of pyrazolam,diclazepam and the metabolitesof diclazepam in rats.It can provide references for the clinical treatment of related drug poisoning and the detection of related cases.