Study On The Qualitative Indicators In Results Based On LC-MS/MS In Forensic Toxicological Analysis

Objective:1.To establish a method of liquid chromatography-tandem mass spectrometry(LC-MS/MS)for detection 8 analytes of drugs(methamphetamine,ketamine and morphine),benzodiazepines(estazolam,midazolam,diazepam and clonazepam)and barbiturates(phenobarbital)in human blood.2.To study the maximum allowable deviation of retention time and ion ratio in qualitative results of LC-MS / MS for 8 analytes,and compare them with relevant guidance documents at home and abroad.3.Through the study of the qualitative indicators in actual cases,the reliability of the indicators in qualitative results is verified.Method:1.Optimizing chromatographic and mass spectrometry conditions.Liquid-liquid extraction was used to optimize the extraction method for blood.Establish an extraction and detection method by analyzing the influence of different extraction solvents and pH on drug recoveries.2.Prepare different concentrations of blood samples and the corresponding standard samples according to a certain order of injection.Study the qualitative parameters such as tolerance of retention time and ion ratios by processing data.3.Collect actual cases to study the deviation of the qualitative parameters in actual cases.4.Statistical methods and other software: SPSS 20.0 software was used for data analysis and Graphpad Prism 6 was used for plotting.Results:1.Methamphetamine,ketamine,morphine,estazolam,midazolam,diazepam,clonazepam and phenobarbital can be detected by LC-MS/MS.The results showed that they all had a good linear in a certain concentration and the limit of detection were 3ng/mL,1ng/m L,20ng/m L,0.5ng/mL,0.5ng/m L,0.3ng/mL,5ng/mL and 150ng/m L,respectively.The absolute recoveries were higher than 60%,the accuracy were between 85-115%,intra-day precision and daytime precision were less than 15%,the absolute matrix effects were between 85-115%,and the relative matrix effect were less than 15%,so it could be considered that there were almost no matrix interference.2.The deviations of RT and RRT were very small,with RSD were less than 1% for every analyte,which was injected at different time,whether they were bloods or standards.The RT and RRT of these analytes were stable both in standards and blood samples.For the deviation of retention time,the peak time and concentration of each substance affect the deviation.The later the peak time,the greater the deviation;and the deviation of the retention time slightly decreased with concentration increases,but the concentration effect small.Statistical results show that the maximum allowable deviations of ΔRT(absolute)and ΔRT(relative)of the 8 drugs(poisons)are ±0.05 min and ±1.0%,respectively;and the maximum allowable deviation of ΔRRT(relative)is ±0.5%.3.The ion ratios of the 8 drugs showed a trend of stabilization with the increasing of concentrations regardless of whether they were in the standard or in the blood-spiking samples,and the deviation of ion ratio itself was relatively larger at lower concentrations.In the study of the relative deviation of ion ratios,it is not necessary to discuss between partitions.A standard can be adopted,but the deviation is highly related to the concentration.The deviation range should be discussed in terms of concentration.When the drug content is low below the LOQ,the relative maximum allowable deviation of ΔR is ±60%;at higher concentrations above LOQ,the relative maximum allowable deviation of ΔR is ±40%.4.In the actual cases collected,100% data was in the reference range obtained in this experiment for the maximum allowable deviation of the qualitative index.Conclusion:1.Liquid-liquid extraction and LC-MS/MS qualitative and quantitative analysis of 8 drugs in human blood established in this experiment have high recovery,high sensitivity and repeatability,which can be used in detection of methamphetamine,ketamine,morphine,estazolam,midazolam,diazepam,clonazepam and phenobarbital in forensic toxicological analysis.2.The maximum allowable deviations of ΔRT(absolute)and ΔRT(relative)for the 8 drugs were ±0.05 min and ±1.0%,respectively;and the maximum allowable deviation of ΔRRT(relative)was ±0.5%.But this result was only for the LC-MS/MS instrument and extraction method used in this experiment.3.The deviation of the ion ratios of the 8 drugs(poisons)is highly related to the concentration.When the drug is lower than LOQ,the relative maximum allowable deviation of ΔR is ±60%;when the drug is at higher concentrations above the LOQ,the relative maximum allowable deviation of ΔR is ±40%.But this result was only for the LC-MS/MS instrument and extraction method used in this experiment.4.The reference range of the qualitative index in the experiment can meet the qualitative analysis of cases in forensic toxicology analysis and provide experimental data for qualitative analysis.