| Objective1. To establish a solid phase extraction (SPE) and LC-MS/MS analysis of clozapine and its metabolites in blood, urine and tissues.2. To study the postmortem distribution and postmortem diffusion of clozp zine and its metabolites and provide experimental basis for the forensic identifi cation of clozapine pisoned death cases.Methods1. Solid phase extraction(SPE) and LC-MS/MS:After a pretreatment, CLP and its metabolites were extracted by a Waters OasiaoR(?)HLB3cc (60mg) SPE column, and determined by LC-MS/MS with an Agilent(?)XDB-C18(4.6mm×150mm×3.Sum) column.2. The postmortem distribution of CLP and its metabolites in rabbits:Six male New Zealand rabbits were divided randomly into LD50and4LD50groups, which were given an intragastric administration of clozapine with a dose of270mg/kg and1080mg/kg. As soon as the vital signs disappeared, the rabbits were dissected and the specimens such as heart blood, heart, liver, spleen, lung, kidney, brain, were sampled immediately for the determination of CLP and its metabolits. CLP, DMCLP, CLP-NO was detected qualitatively and quantitatively by LC/MS/MS after a solid phase extraction, CLP-G was detected qualitatively by LC/MS/MS after a solid phase extraction.3. Postmortem diffusion:Thirty three male New Zealand rabbits were divided into postmortem diffusion group(15rabbits), temperature affection group (6rabbits), dose affection group(6rabbits) and time affection group(6rabbits). Postmortem diffusion group:Fifteen rabbits were given a postmortem intragastric administration of LD50(270mg/kg) clozapine at1h after the death, and put at room temperature. Three rabbits were dissected and the specimens such as heart, liver, spleen, lung, kidney, brain, leg muscle, heart-blood, peripheral blood, bile, urine, were collected respectively at0.5d, Id,2d,4d, and6d after the postmortem administration. Clozapine and its metabolites in the specimens were detected by a SPE-LC/MS/MS.Temperature group:Nine dead rabbits were given a postmortem intragastric administration of clozapine with a LD50(270mg/kg) dose at lh after the death, three of which were put at room temperature,4℃or-20℃respectively. Rabbits were dissected and the specimens as postmortem diffusion group’s were collected at4d after the postmortem administration. Clozapine and its metabolites in the specimens were detected by a SPE-LC/MS/MS.Dose group:Nine dead rabbits were put at room temperature, three out of which were given an intragastric administration of clozapine with a dose of1/2LD50(135mg/kg), LD50(270mg/kg) or2LD5o (540mg/kg) respectively at1h after the death. Rabbits were dissected and the specimens as group1’s were collected at4d after the postmortem administration. Clozapine and its metabolites in the specimens were detected by a SPE-LC/MS/MS.Time group:Nine dead rabbits were put at room temperature, three out of which were respectively given an intragastric administration of clozapine with a dose of LD50(270mg/kg) at1h,3h or6h after the death. Rabbits were dissected and the specimens as group1’s were collected at4d after the postmortem administration. Clozapine and its metabolites were detected by a SPE-LC/MS/MS.4. Statistics:Data were processed by SPSS11.5and expressed as x±S.Results1. SPE and LC-MS/MS①The Chromatographic separation and SPE:The Chromatographic separation was performed on an Agilent(?)XDB-C18(4.6mm×150mm×3.5um)column equipped with a phenomenex guard column. Target chemicals(the internal standard, clozapine and its metabolites) showed a good separation. When2.5moL/L sodium carbonate was added to specimen and the pH was adjusted to11, SPE (OASIS(?)HLB3cc (60mg) column) showed a better recovery for CLP and its metabolits than the liquid-liquid extraction by ethyl acetate did.②LC/MS/MS:Data acquisition was performed in MRM mode with a positive electrospray ionization (ESI+), Quantitative ion pairs of CLP, DMCLP, CLP-NO and diazapam respectively are327/192,313/270.2,343/243.1and313/154.1. The linearity for CLP, DMCLP,CLP-NO detected in the urine, blood, liver were over ranges of5or10-1000ng/mL,2or5-1000ng/mL,50-1000ng/mL, and the LOQ were5or lOng/mL,2or5ng/mL,50ng/mL2. Postmortem distributionCLP, DMCLP and CLP-NO were detected in all collected specimens. CLP-G could be detected in the urine of poisoning death rabbits, and could not be detected in other collected specimens.LD50group:The order of CLP detected in rabbits:lung> liver, kidney, brain, spleen> bile, myocardium, heart blood> urine,vitreous humor, muscles of lower limb; The order of DMCLP detected in rabbits:lung, liver> kidney, brain, spleen> bile, myocardium, heart blood, urine, vitreous humor> muscles of lower limb; The order of CLP-NO detected in rabbits:lung, liver> kidney, brain, spleen>bile, myocardium> heart blood, urine, vitreous humor, muscles of lower limb.4LD50group:The order of CLP detected in rabbits is:lung, live>kidney, brain, spleen>bile, myocardium> heart blood, urine, vitreous humor, muscles of lower limb; The order of DMCLP detected in rabbits is:lung, liver> kidney, brain, spleen>bile, myocardium, heart blood, urine, vitreous humor> muscles of lower limb; The order of CLP-NO detected in rabbits:lung, liver>kidney, brain, myocardium> spleen, bile, heart blood, vitreous humor, urine, muscles of lower limb.3. Postmortem diffusionNo metabolites (DMCLP, CLP-NO, CLP-G) of CLP were detected in all a nimal models of postmortem diffusion. Postmortem diffusion group:At0.5d after the postmortem administration, CLP were detected in myocardium, heart blood, lung, liver, kidney, which was also detected in brain and leg muscle at1d, and was also detected in bile at2d. No CLP was detected in all vitreous humor and urine.Temperature group:At-20℃, CLP was only detected in liver, spleen and lung; At4℃, CLP was only detected in heart, liver, spleen, lung, kidney and brain; At room temperature, CLP was detected in all collected specimen except in urine and vitreous humor.Dose group:CLP was detected in all collected specimens except in leg muscles, urine and bile.Time group:CLP was not detected in urine, vitreous humor in1h group,in leg muscles urine, vitreous humor in3h group, and in leg muscles urine, vitreous humor heart and kidney in6h group.Conclusions1. A simultaneous qualitative and quantitative analysis of CLP, DMCLP, CLP-NO and qualitative analysis of CLP-G by SPE-LC/MS/MS was established, which was with a good recovery, reproducibility and high specificity and high sensitivity, and which can be used for the detection of CLP and its metabolites in the study on the forensic toxicokinetics and the forensic identification of CLP poisoning death.2. CLP and its metabolites showed auneven postmortem distribution in rabbits. In the LD50and4LD50group, CLP, DMCLP and CLP-NO were detected in all tissues and body fluids of the rabbits, CLP-G. was not detected in all tissues and body fluids except in urine.3. It was proved once again that there was a postmortem diffusion of CLP in postmortem administrated rabbits, which was related to storage time, storage temperature and postmortem administration time. The results indicated DMCLP, CLP-NO and CLP-G were not detected in all postmortem diffusion animal model in6d. 4. The postmortem distribution and postmortem diffusion of CLP and its metabolites in rabbits indicated that metabolites of CLP, especially CLP-G,can be used as the marker of antemortem administration of CLP, and provide experimental evidence for identifying antemortem taken CLP and postmortem given CLP. |
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