| Objectives1.To establish a method which can accurately measure the metabolite of heroin (including morphine, morphine-3-glucuronide and O6-Monoacetylmorphine) in urine and plasma by LC-MS/MS with solid-phase extraction.2.To complete the study of preserve stability of the main metabolites of herion in blank added blood and herion poisoned blood through animal experiments.3.To complete the study of preserve stability of the main metabolites of herion in blank addde urine of health people and herion addicted urine through experiments on human being.Methods1.The method of solid phase extraction of main metabolites of herion.After pretreatment of samples,The BAKERBONDTM spe Octadecyl (C18)3cc500mg solid phase extraction column was extracted, Analysis of the influence of different pH buffer conditions,different detergent and different elution solvent to the recovery of main metabolites of heroin, complete the optimization of solid phase extraction methods.2.To optimize the methods of LC-MS/MS for determining main metabolites of herion.To optimize the methods of LC-MS/MS of liquid mobile phase, chromatographic column and acquisition time etc. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for qualitative and quantitative analysis of main metabolites of herion. 3.Study on stability of heroin metabolites(including morphine, morphine-3-g lucuronide and O6-Monoacetylmorphine) in blood and urine.Take fresh rabbit blank blood and blank health human urine as samples,Copy respectively for all contain M3G,morphine and O6that concentration are200ng/mL of blood and urine samples,detect the content of M3G,morphine and O6in nine different time points,three different preservative temperature (20degrees,4degrees and-20degrees) through extracting and purifying the samples,to study the preserve stability.①Experiments on toxicity animal Toxic animal model:select three male rabbits which weight are similar (big ear and pure rabbit from Japan),24hours’fast later, carotid artery intubation and draw blood with negative pressure valve after injecting5mg/kg of heroin physiological saline in the ear marginal vein and keeping for2hours.②Experiments on human being Samples which are five different urines of herion addict that have been identified in daily Case.③Copy three blood and four urine samples as200ng/mL concentration of blood and urine samples which contain M3G,morphine and O6, detect the content of M3G,morphine and O6in nine different time points,three different preservative temperature (20degrees,4degrees and-20degrees) through extracting and purifying the samples,to study the preserve stability.Confirmatory experiment Take fresh rabbit blank blood and blank health human urine as samples, an unknown concentration of standard solution of O6which has been decomposed into morphine. Copy blood and urine as someof appropriate concentration of blood and urine samples later, detect the content of morphine and O6of them from first day to fourteen days which store at three different preservative temperature (20degrees,4degrees and-20degrees). Resultsl.The minimum detection limit (LOD) of M3G,morphine and O6in urine are1.24pg,6.71pg and0.47pg; the minimum detection limit (LOD) of M3G,morphine and O6in blood are1.50pg,8.21pg and0.52pg.2.The solid phase extraction method established in this paper is suitable for both blood and urine samples and very good purification and enrichment. The method of LC-MS/MS established in this paper has good peak shape of target material and good repeatability, testing time is8.5min.3.Stability results of main metabolites of heroin (including M3G, morphine and O6) in blood and urine.Blank added group of bloodO6:Component percentage of O6by initial34.37%to0.97%at the fourteenth day at20degrees, component percentage of O6by initial34.37%to1.10%at the fourteenth day at4degrees, component percentage of O6by initial34.37%to14.26%at the fourteenth day at-20degrees. The contents of O6are fell by97.51%,96.78%and49.73%on average.Morphine:Component percentage of morphine by initial38.02%to56.73%and66.49%at the one hundred and eightieth day respectively at4and-20degrees.M3G:Component percentage of M3G by initial27.61%to41.04%,40.29%and33.88%at the fourteenth day respectively at20,4and20degrees.Toxic blood group of animalO6:Group of B and C, component percentage of O6by initial5.90%and1.66%to0.06%,0.14%1.66%and0.10%,0.19%,0.22%at the fourteenth day respectively at20,4and-20degrees.Morphine:Group of A,B and C, component percentage of morphine by initial8.64%,6.07%and38.02%to2.10%,4.19%5.36%;0.63%,1.52%,3.34%and0.88%,2.62%,4.68%at the fourteenth day respectively at20,4and-20degrees.The contents of morphine are fell by84.28%,63.10%and38.85%on average.M3G:Group of A,B and C, component percentage of M3G by initial89.18%,88.02%and91.29%to97.52%,95.56%,94.42%;99.31%,98.34%,95.00%and99.01%,97.20%.95.10%. at the fourteenth day respectively at20.4and-20degrees.Blank added group of urineO6:Component percentage of O6by initial36.81%to0.08%at the twenty-eighth day at20degrees, component percentage of O6by initial36.81%to10.39%at the twenty-eighth day at4degrees, component percentage of O6by initial36.81%to28.29%at the fourteenth day at-20degrees. The contents of O6are respectively fell by99.78%,71.77%and23.15%.Morphine:Component percentage of morphine by initial42.62%to64.21%,56.26%and41.19%at the twenty-eighth day respectively at20,4and-20degrees. The contents of morphine are respectively by45.96%,32.00%and3.36%.M3G:Component percentage of M3G by initial20.57%to24.22%and20.33%at the one hundred and eightieth day respectively at4and20degrees.Group of heroin addict of urineO6:Group of A, B and D,component percentage of O6by initial0.04%,0.989%and2.74%to0at the twenty-eighth day.Morphine:Group of A,B,C,D and E,component percentage of morphine by initial8.32%,7.22%,5.57%,5.79%and11.07%respectively to15.65%,12.41%13.59%,14.22%;13.04%,12.41%;20.99%,16.37%and21.88,19.88%at the one hundred and eightieth day respectively at4and-20degrees.M3G:Component percentage of M3G by initial91.64%,91.79%,94.41%,91.47%and87.67%respectively to90.95%,94.05%,94.76%;91.29%,91.72%,91.81%;94.26%,94.24%,93.94%;93.81%,92.16%,92.73%and86.90%,86.14%,87.53%at the seventh day respectively at20,4and20degrees.Group of confirmatory experiment①Blank added group of blood:Component percentage of morphine by initial 11%to100%and29%at the fourteenth day at20and-20degrees,component percentage of O6by initial89%to0and71%at the fourteenth day at20and-20degrees.(2)Blank added group of urine:Component percentage of morphine by initial1%to22%at the fourteenth day at20degrees, component percentage of O6by initial99%to78%at the fourteenth day at20degrees; component percentage of morphine and O6have no obvious change at-20degrees.Conclusions1.①Use an Atalantis(?) HILIC Silica liquid column(2.1mm×150mm×3um)which connected with phenomenex protective column,the effect of peak is batter than C18column,so using the former chromatographic column; To add0.1%acid into liquid mobile phase can make the shape of target of peak symmetrical;Acquisition time shortest can be reduced to8.5minutes.The established method after LC-MS/MS optimization, the test results were proved to be accurate, sensitive, good reproducibility and to achieve qualitative and quantitative analysis requirements.②C18,MCX and CSGAU column were used In this study as solid phase extraction column, parallel operation, found that the C18column with high recovery and good reproducibility. So this paper uses the C18column for solid phase extraction column to extract and purify samples.③The method of LC-MS/MS we established, samples for extraction and purification by solid phase extraction technology, detection of M3G,morphine and O6in blood and urine achieved requirements of high recovery, range of linearity and detective sensitivity, It can be applied in forensic medicine inspection to identify whether heroin poisoned and died of heroin,also applied in forensic toxicity dynamics and stability study.2.Stability regular of main metabolites of heroin in blood and urine. group of blood The content of O6reduced; the content of morphine increased; the content of M3G increased slightly.group of urine The content of O6reduced; the content of morphine increased; the content of M3G is relative stability.Group of confirmatory experiment①Blank added group of blood:The content of morphine increased and the content of O6decreased both at20and-20degrees, the amount of morphine increased equaled with the amount of O6reduced,the amplitude of O6reduced and morphine increased is small and relatively stable.②Blank added group of urine:same as Blank added group of blood. To sum up, the content of O6reduced because its decomposition of morphine and irreversible. |
PDF Full Text Request