Study On A Rapid Method Of Discriminating Heroin Abusers By Quanlitative Analysis Of SPE/LC-MS/MS In Urine

Objectives1.To establish a method of solid phase extraction(SPE) and high-performance liquid chromatography-tandem mass spectrometry(LC-MS/MS) for determination of morphines in human urine.2.To establish a rapid qualitative method of LC-MS/MS to discriminate heroin abuser.3.To study metabolism of morphine,codeine and heroin in urine from Compound Liquorice Tablets user,Codeine and Guaifenesin Syrup user,pericarpium papaveris eater and heroin abuser.4.To identify urines from Compound Liquorice Tablets user,Codeine and Guaifenesin Syrup user,pericarpium papaveris eater and heroin abuser when the result of immunoassay is positive.Methods1.The method of solid phase extraction of opioids:Opioids were extracted in urine using Oasis(?)MCX 3cc(60mg) extraction cartridges.The SPE method was optimized in terms of choices of different pH value,conditioning solution and eluent.2.To optimize the methods of LC-MS/MS for determining morphines in urine:The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring(MRM) was used for qualitative and quantitative analysis of drug and its metabolites.3.The metabolism of opioids3.1 The designs of experimentTwenty-six healthy volunteers were randomly divided into three groups.3.1.1 The first group(ten persons) took tablets of Compound Liquorice Tablets.A:Two persons took the tablets for three days,three times per day,three tablets per time;Eight persons took four tablets only once.3.1.2 The second group(eight persons) took 10ml Codeine and Guaifenesin Syrup only once.3.1.3 The third group(eight persons) ate normal appetite soup with pericarpium papaveris.3.2 Collection and deposition of samplesUrines of control samples were collected before taking drugs half an hour and the test samples were collected 0.5h,1.0h,1.5h,2.0h,2.5h,3.0h,3.5h,4.0h,6.0h...46.0h and 48.0h after taking drugs.All samples were stored at -20℃.3.3 Sample pre-treatment and Auto-SPEThe urine samples were thawed and 3ml was transferred into tubes.All tubes were centrifuged at 4000rpm for 3min.Then the supernatant fluid was transferred to a new tube.4.0ml of phosphate buffer(pH4.0) were added.Samples were extracted using the method of SPE.3.4 The method of LC-MS/MS.The qualitative analysis was based on retention time and 2-3 pair ion fragmentation.The quantitative analysis was on exterior standard method of 1 pair ion fragmentation which has the highest reponse.4.Statistics:SPSS11.5 statistic software was used for data analysis and output was mean±standard deviation((?)±S).Results1.In the condition of pH4.0 phosphate buffer,the average recoveries for heroin, 6-monoacetylmorphine(6-MAM),codeine,morphine,morphine-3-glucuronide(M3G), codeine-glucuronide(C6G),papaverine,thebaine,noscapine,natriline were 66.52%,109.51%, 99.18%,78.47%,101.92%,91.62%,88.88%,6.09%,88.35%,91.36%,respectively.2.The method of LC-MS/MS.This method of LC-MS/MS had better separation and sensitivity of opioids by MRM(Multiple Reaction Monitoring) mode.Limits of Detection(LOD) were 5pg for M3G,10pg for morphine,5pg for codeine,5pg for 6-MAM,2pg for heroin,5pg for M3G,0.2pg for C6G,0.5pg for papaverine,10pg for thebaine,5pg for noscapine,10pg for natriline.The linear range is 0.01μg/ml～10μg/ml.The correlation coefficient of heroin,6-MAM, codeine,morphine,M3G,C6G,papaverine,thebaine,noscapine,natriline were 0.9997,0.9994, 0.9992,0.9958,0.9998,0.998 2,0.999 9,0.994 8,0.999 9,0.996 7,respectively.3.The results of urines from antitussive drugs user.Morphine,codeine,M3G and C6G,et al were detected in all samples,and arrived at peak concentration in 0.5～3.0h.But the ratio of total morphine to total codeine(Mor/Cod) is different.The ratio of Mor/Cod in urine is between 1.61±0.67 and 3.07±1.85 after taking Compound Liquorice Tablets.However the value Mor/Cod is between 0.09±0.07 and 0.24±0.12 after taking Codeine and Guaifenesin Syrup.4.The results of urine samples from pericarpium papaveris eater.Morphine,codeine,M3G and C6G,et al were detected in all samples,and arrived at peak concentration in 0.5～3.0h.The ratio of Mor/Cod is between 2.48±2.08 and 6.41±2.65.And papaverine,natriline,noscapine sometimes could be detected sometimes. 5.The results of urine samples from heroin abuser.6-MAM was detected sometimes.A few samples contained heroin or acetyl-codeine prototype.6-MAM was detected in ninty urine samples among one hundred sixty-two cases.The ratio of Mor/Cod is below 1 in nine samples among ninty urine samples,and the ratio of others is over 1.Conclusions1.This study established the method of SPE/LC-MS/MS for determination opioids.This method was simple,rapid and accurate for quantitative analysis of opioids and their metabolites in human urine.2.The analysis of urine samples of heroin abuser.If 6-MAM,heroin and acetyl-codeine were detected,we can make sure that this person must have taken in heroin.But if we can only detect morphine,codeine,M3G and C6G,it is necessary for us to consider the ratio of Mor/Cod.3.The comparison analysis of urine samples of Compound Liquorice Tablets user and heroin abuser.When morphine,codeine,M3G and C6G,et al can be detected,but 6-MAM,heroin and acetyl-codeine cannot be founded,and the ratio of Mor/Cod in urine is over 1,we are still unable to confirm that the urine sample from a heroin abuser.However,it is feasible for us to test enoxolone in urine by negative ion mode.If enoxolone is detected,this person probably has taken Compound Liquorice Tablets recently.4.The comparison analysis of urine samples of Codeine and Guaifenesin Syrup user and heroin abuser.The ratio of Mor/Cod is between 0.09±0.07 and 0.24±0.12,and 6-MAM cannot be detected.we can confirm the person who has taken antitussive drugs which contain more codeine,for example,Codeine and Guaifenesin Syrup.When Mor/Cod is below 1,and 6-MAM can be detected in urine,we can confirm that the person is a heroin abuser.5.The comparison analysis of urine samples of pericarpium papaveris user and heroin abuser.For the former,morphine,codeine,et al.were detected in urine.Sometimes papaverine, thebaine and noscapine,et al.were also detected,and the ratio of Mor/Cod in urine is between 2.48±2.08 and 6.41±2.65.If 6-MAM cannot be detected,we cannot differentiate pericarpium papaveris user from heroin abuser,and further analysis is necessary.6.The analysis of impurity in drug.Guaiphenesin can be detected in urine of 59.9%Heroin abusers,but seldom detected in urine of opiods user.Whether this discovery can become an evidence which can distinguish heroin abuser from opiods user,Further research is necessary.