The Relationship Between Morphine Glucuronide, Morphine And Codeine In Urine In Chinese Population

Drug abuse is a serious social problem around the world. It is regarded as a highly addictive drug and considered a serious crime act in China. Monoacetylmorphine (6-MAM) in alleged heroin users was regarded as the direct evidence for heroin abuse. However, the half life of 6-MAM was so short that we could only detect morphine in such cases. Not only heroin but slso codeine would have morphine as metabolites. Therefore, an effective criterion was required to distinguish heroin abuse from administration of codeine or other opioids drugs.In this thesis, we conducted a study to investigate the characteristics of urine samples collected from volunteers who have oral doses of codeine and alleged heroin users by detecting the content of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine. After systematically statistic analysis of all the data, a possible criterion which contained several parameters was finally given out for heroin abuse judgment. The thesis was composed by five major parts which were listed as below:1 Analysis method with UPLC-MS/MSAfter precipitation of protein by acetonitrile, the urine samples, with contained the morphine- d₃(MOR- d₃) and morphine-3-glucuronide- d₃ (M3G- d₃) as internal standards, were pre-treated by Sirocco ^TM protein precipitation plate, and then simultaneously detected for the codeine(COD), 6-monoacetylmorphine(6-MAM), morphine(MOR), morphine-3-glucuronide(M3G) and morphine-6-glucuronide(M6G) in human urine samples by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS).The limit of detection was 0.2 ng/mL for both COD and MAM while the limit of quantization was 0.5 ng/mL for both COD and MAM; The limit of detection was 0.5 ng/mL for MOR, M3G and M6G while the limit of quantization was 1 ng/mL for them. The linear correlation coefficients were no less than 0.9997. The inter-day precisions were below 5% while the intra-day precisions below 10%. The inter-day accuracies were above 97% while the intra-day accuracies above 94%. The recoveries were above 90% while the matrix effects were about 45% to 90.0%. Such established method is simple, rapid and accurate. It shows the great potential in the application of forensic toxicological analysis.2 Analysis of urine samples from alleged heroin abuser89 urine samples from heroin abusers of 2006 2009 were collected. The urine samples were analyzed with UPLC - MS/MS method and the experimental data were statistically analyzed by using SPSS16.0 software.Because of the distribution normality of the data, method of percentiles with median and standard IQR (interquartile range) was selected to analyze each set of data from heroin users'urine samples. The medians of MOR, COD, M3G and M6G concentration were 1130.00, 344.50, 9670.00 and 2215.00ng/mL, respectively; Lower limits of one-sided 95% confidence limits were 67.05, 20.40, 498.50 and 47.55ng/mL, respectively. The medians of MOR/COD, M3G/COD, M6G/COD, (M3G+M6G)/COD and (MOR+M3G+M6G)/COD were 2.98, 16.30, 3.74, 18.88 and 24.70, respectively; Lower limits of one-sided 95% confidence limits were 1.13, 1.14, 0.34, 1.48 and 5.24, respectively. The M3G/COD, M6G/COD, (M3G+M6G)/COD and (MOR+M3G+M6G)/COD would increased while the MOR/COD increase.3 Analysis of urine samples from codeine administrationUrine samples from 21 healthy volunteers ingesting different doses of compound codeine phosphate oral solution were collected at different time points. The samples were analyzed with UPLC-MS/MS method and the data were statistically analyzed by using SPSS16.0 software.Total 871 samples with effective data were analyzed. For the same reason above, method of percentiles was selected to analyze each set of data. The medians of MOR, COD, M3G and M6G concentrations were 11.10, 178.50, 199.00 and 50.55ng/mL, respectively; Upper limits of one-sided 95% confidence limits were 66.17, 1322.00, 1224.00, 342.70 ng/mL, respectively. The medians of MOR/COD, M3G/COD, M6G/COD, (M3G+M6G)/COD and (MOR+M3G+M6G)/COD were 0.07, 1.36, 0.32, 1.76 and 1.84, respectively; Upper limits of one-sided 95% confidence limits were 1.14, 29.82, 7.07, 36.01, 36.89, respectively. M3G/COD, M6G/COD, (M3G+M6G)/COD and (MOR+M3G+M6G)/COD would increased while the MOR/COD increase. The concentration of codeine would reached the first high point at 0.5h after single dose then decrease. It would reaced the second high point at 5h; The concentration of morphine would reach the high point at 0.5h then decrease; The concentration of morphine-3- glucuronide reached the first high point at 1h and maintain for 8 hours before decreasing; The concentration of morphine-6- glucuronide reached the first high point at 6h then decrease. The ratio of morphine to codeine (MOR/COD) would would reach the high point at 36 hour after codeine administration; The curve of M3G/COD is similar to MOR/COD, but a bigger rangeability. M6G/COD would reach a high point at 28h then decrease. In multiple dose experiments, concentrations of MOR, COD, M3G , M6G and value of MOR/COD, M3G/COD, M6G/COD fluctuate in a range during codeine administration. Afterwards, the trends were the same as that of single dose.4 Possible criterion for heroin abuse judgmentCombining the data from heroin abuse and codeine administration, we finally gave out one possible criterion for heroin abuse judgment using both concentration of morphine ([M]) and ratio of morphine to codeine (MOR/COD). [M] was observed above 67 ng/mL while MOR/COD was observed above 1.14, we make the judement of heroin abuse. Once both values were observed not qualified with our criterion, we make the judement of codeine administration.5 Validation of our criterion in heroin abuse judgment11 urine samples from flu patients who were taking cough mixture containing codeine and 238 urine examples from daily cases that only morphine and codeine can be detected in 2006 2009 were collected. The urine samples were analyzed with UPLC - MS method, and the data were statistically analyzed by using SPSS16.0 software.11 sets of data of [M] and MOR/COD which were obtained from two flu patients were analyzed. With this criterion, 11 samples are not considered from heroin abuse, which consistented with the fact.238 samples in which only morphine and codeine could be detected while monoacetylmorphine can not be observed were analyzed. According to the criterion from this study, 175 samples, 73.53% of the total number, can be confirmed for either heroin abuse or codeine administration while 63 samples, 26.47% of the total, cannot be confirmed.In summary, this thesis (1) established UPLC - MS method which can separate and detect M3G, MAM, MOR, M6G and COD in urine at single run; (2) gave out one possible criterion for heroin abuse judgment using [M] and MOR/COD in urine samples.