生物样品中常见鸦片类毒品的检测技术研究
摘要
吸毒和药物滥用是目前广受全球关注的社会问题。近年来，受境外毒品活动的影响，吸毒、贩毒已再次殃及我国，而且各类毒品案件呈现快速上升趋势，过境贩毒和国内消费共存的现象已经形成。在各类毒品中，鸦片类毒品的消费量最大，因此，对生物样品中鸦片类毒品的分析检测就显得更加重要和迫切。
本文对生物样品中常见鸦片类毒品的分析检测技术进行了综述，并建立了利用金标免疫分析技术、TOXI—LAB毒物分析实验室系统对尿液中常见鸦片类毒品进行快速分析和复核的方法；尿液中吗啡的自动固相萃取—GC／NPD、GC／MS分析检测方法；毛发中常见鸦片类毒品的液—液萃取—GC／MS分析检测方法。
第一，概述了生物样品中鸦片类毒品的检验情况，主要包括：常见鸦片类毒品的种类和结构；常见鸦片类毒品在体内的代谢；生物样品中常见鸦片类毒品的提取净化方法，如液—液萃取法(LLE)、固相萃取法(SPE)和超临界流体萃取(SFE)等；生物样品中常见鸦片类毒品的检测方法，如免疫分析法(Immunoassay)、气—质联用法(GC／MS)、液相色谱法(HPLC)、液—质联用法(LC—MS)和毛细管电泳法(CE)等。
第二，利用新型混合模式(反相吸附和阳离子交换)的Oasis MCX固相柱，配合Zymark自动固相萃取仪，联合GC／NPD、GC／MS对尿液中的吗啡进行了检测研究。建立了快速、批量分析尿液中吗啡的方法，具有较高的回收率(＞88％)和重现性(日间精密度RSD％为：3.0-5.5)标准曲线回归方程为：y=2594.15x+3.11(r=0.9993，SD=7.0，n=9)，定量检出限为0.11μg／mL，检出限为0.050g／mL(S／N=3)。利用建立的方法，考察了尿样储存初始pH对其中总吗啡稳定性的影响，结果发现：尿样初始pH较低，其中总吗啡的含量则较稳定。
第三，利用金标免疫分析技术、TOXI—LAB毒物分析实验室系统和前述GC／NPD、GC／MS检测方法，探讨了TOXI—LAB毒物分析实验室系统对尿液中吗啡进行快速复核检验的可行性。通过对118例吸毒嫌疑人尿样的分析发现，用金标免疫试剂盒检测，有7例为阴性；用TOXI—LAB一般方法检测，73例为阳性，45例为阴性，对其中45例阴性样品再用TOXI—LAB酸水解方法检测，有17例为阳性，28例为阴性；而用GC／NPD、GC／MS检测，102例为阳性，16
首都师范大学硕士学位论文
生物样品中常见鸦片类毒品的检侧技术研究
例为阴性。TOXI一LAB系统检测吸毒嫌疑人尿样中吗啡的特异性为100%，该
方法可对吸毒嫌疑人尿样中的吗啡进行快速复核检验。
第四，从水解方法、提取方法等角度，研究了毛发中常见鸦片类毒品的检测
技术。建立了液一液萃取，GC胭S分析毛发中常见鸦片一类毒品的检测方法，吗
啡、可待因和6一单乙酞吗啡的回收率分别为:82%、80%和87%，检测限(SIM)
分别为:0.o52ng、0.o4ng和0.037ng，线性回归方程分别为
Y=0 .02913x+0.01406(厂0.9997)、Y=0.03255x一0.004468(r=09992)
Y=0 .0302 lx+0.01632(r=0.9993)。
Drug abuse is the focus of the society in all over the world .In recent years, drug abuse have come back to our country , and the phenomena of drug abuse and drug selling exist together. The amount of abusing opiates is biggest in all kinds of illicit drugs. As a result of it, the determination of opiates in biological samples is of great importance and necessity.
In this paper, a review on the determination of usual kinds of opiates in biological samples was given. A method for rapid identification of morphine in urine applying colloid golden immunoassay and TOXI-LAB system has been developed. A combination of automated solid phase extraction (SPE) and GC/NPD,GC/MS has been developed for the determination of morphine in urine. In addition, A method for determination of opiates in hair using liquid-liquid extraction(LLE) and GC/MS has been developed
Firstly, a review was given on the determination of usual kinds of opiates in biological samples , including the brief introduction of opiates and their structures, the metabolize of opiates in human body, techniques of extraction and cleaning and methods of detection. There were three main techniques of extraction and cleaning , such as liquid-liquid extraction(LLE), solid phase extraction(SPE) and supercritical fluid extraction(SFE). There were five main methods of detection, such as Immunoassay, GC/MS, HPLC, LC/MS and CE.
Secondly, a combination of automated solid phase extraction (SPE) and GC/NPD, GC/MS has been developed for the determination of morphine in urine. SPE with mixed mode(reverse phase absorb and cation-exchange) cartridges Oasis?MCX was fully automated with a Zymark Rapid Trace?SPE robot. GC/NPD and GC/MS analysis was performed after derivatization with MSTFA. High recovery(>88%) and good reproducibility (RSD% between day and day: 3.0-5.5 ) were found.The equation of linearity was y=2594.15x+3.11(r=0.9993),with limit of quantity (LOQ) 0.1ug/mL and limit of detection (LOD) 0.05ug/mL.In addition, improved total
morphine stability was found in samples with lower initial pH .
Thirdly, the feasibility of TOXI-LAB system for identification of morphine in urine of the suspect drug addict has been evaluated. The urine of 118 cases was analyzed by colloid golden immunoassay, TOXI-LAB, GC/NPD and GC/MS respectively. By colloid golden immunoassay, there were only 7 negatives .By general TOXI-LAB, 73 cases were positive, and 45 cases were negative. The 45 negative cases were analyzed by acid hydrolyzed TOXI-LAB again, and 17 positive cases were found. By GC/NPD and GC/MS, there were 16 negatives. The specificity of the TOXI-LAB for identification of morphine in urine of the suspect drug addict was 100%, and applying TOXI-LAB system for identification of morphine was feasible.
Finally, a method for determination of opiates in hair using liquid-liquid extraction(LLE) and GC/MS has been developed. The recovery of morphine ,codeine and 6-monoacetylmorphine were 82%,80% and 87% respectively.The limit of detection(SIM,LOD) for morphine ,codeine and 6-monoacetylmorphine were 0.052ng, 0.04ng and 0.037ng respectively.The equation of linearity of morphine ,codeine and 6-monoacetylmorphine were Y=0.02913x+0.01406(r=0.9997),Y=0.03255x-0.004468 (r=0.9992) and Y=0.03021x+0.01632 (r=0.9993) .
本文对生物样品中常见鸦片类毒品的分析检测技术进行了综述，并建立了利用金标免疫分析技术、TOXI—LAB毒物分析实验室系统对尿液中常见鸦片类毒品进行快速分析和复核的方法；尿液中吗啡的自动固相萃取—GC／NPD、GC／MS分析检测方法；毛发中常见鸦片类毒品的液—液萃取—GC／MS分析检测方法。
第一，概述了生物样品中鸦片类毒品的检验情况，主要包括：常见鸦片类毒品的种类和结构；常见鸦片类毒品在体内的代谢；生物样品中常见鸦片类毒品的提取净化方法，如液—液萃取法(LLE)、固相萃取法(SPE)和超临界流体萃取(SFE)等；生物样品中常见鸦片类毒品的检测方法，如免疫分析法(Immunoassay)、气—质联用法(GC／MS)、液相色谱法(HPLC)、液—质联用法(LC—MS)和毛细管电泳法(CE)等。
第二，利用新型混合模式(反相吸附和阳离子交换)的Oasis MCX固相柱，配合Zymark自动固相萃取仪，联合GC／NPD、GC／MS对尿液中的吗啡进行了检测研究。建立了快速、批量分析尿液中吗啡的方法，具有较高的回收率(＞88％)和重现性(日间精密度RSD％为：3.0-5.5)标准曲线回归方程为：y=2594.15x+3.11(r=0.9993，SD=7.0，n=9)，定量检出限为0.11μg／mL，检出限为0.050g／mL(S／N=3)。利用建立的方法，考察了尿样储存初始pH对其中总吗啡稳定性的影响，结果发现：尿样初始pH较低，其中总吗啡的含量则较稳定。
第三，利用金标免疫分析技术、TOXI—LAB毒物分析实验室系统和前述GC／NPD、GC／MS检测方法，探讨了TOXI—LAB毒物分析实验室系统对尿液中吗啡进行快速复核检验的可行性。通过对118例吸毒嫌疑人尿样的分析发现，用金标免疫试剂盒检测，有7例为阴性；用TOXI—LAB一般方法检测，73例为阳性，45例为阴性，对其中45例阴性样品再用TOXI—LAB酸水解方法检测，有17例为阳性，28例为阴性；而用GC／NPD、GC／MS检测，102例为阳性，16
首都师范大学硕士学位论文
生物样品中常见鸦片类毒品的检侧技术研究
例为阴性。TOXI一LAB系统检测吸毒嫌疑人尿样中吗啡的特异性为100%，该
方法可对吸毒嫌疑人尿样中的吗啡进行快速复核检验。
第四，从水解方法、提取方法等角度，研究了毛发中常见鸦片类毒品的检测
技术。建立了液一液萃取，GC胭S分析毛发中常见鸦片一类毒品的检测方法，吗
啡、可待因和6一单乙酞吗啡的回收率分别为:82%、80%和87%，检测限(SIM)
分别为:0.o52ng、0.o4ng和0.037ng，线性回归方程分别为
Y=0 .02913x+0.01406(厂0.9997)、Y=0.03255x一0.004468(r=09992)
Y=0 .0302 lx+0.01632(r=0.9993)。
Drug abuse is the focus of the society in all over the world .In recent years, drug abuse have come back to our country , and the phenomena of drug abuse and drug selling exist together. The amount of abusing opiates is biggest in all kinds of illicit drugs. As a result of it, the determination of opiates in biological samples is of great importance and necessity.
In this paper, a review on the determination of usual kinds of opiates in biological samples was given. A method for rapid identification of morphine in urine applying colloid golden immunoassay and TOXI-LAB system has been developed. A combination of automated solid phase extraction (SPE) and GC/NPD,GC/MS has been developed for the determination of morphine in urine. In addition, A method for determination of opiates in hair using liquid-liquid extraction(LLE) and GC/MS has been developed
Firstly, a review was given on the determination of usual kinds of opiates in biological samples , including the brief introduction of opiates and their structures, the metabolize of opiates in human body, techniques of extraction and cleaning and methods of detection. There were three main techniques of extraction and cleaning , such as liquid-liquid extraction(LLE), solid phase extraction(SPE) and supercritical fluid extraction(SFE). There were five main methods of detection, such as Immunoassay, GC/MS, HPLC, LC/MS and CE.
Secondly, a combination of automated solid phase extraction (SPE) and GC/NPD, GC/MS has been developed for the determination of morphine in urine. SPE with mixed mode(reverse phase absorb and cation-exchange) cartridges Oasis?MCX was fully automated with a Zymark Rapid Trace?SPE robot. GC/NPD and GC/MS analysis was performed after derivatization with MSTFA. High recovery(>88%) and good reproducibility (RSD% between day and day: 3.0-5.5 ) were found.The equation of linearity was y=2594.15x+3.11(r=0.9993),with limit of quantity (LOQ) 0.1ug/mL and limit of detection (LOD) 0.05ug/mL.In addition, improved total
morphine stability was found in samples with lower initial pH .
Thirdly, the feasibility of TOXI-LAB system for identification of morphine in urine of the suspect drug addict has been evaluated. The urine of 118 cases was analyzed by colloid golden immunoassay, TOXI-LAB, GC/NPD and GC/MS respectively. By colloid golden immunoassay, there were only 7 negatives .By general TOXI-LAB, 73 cases were positive, and 45 cases were negative. The 45 negative cases were analyzed by acid hydrolyzed TOXI-LAB again, and 17 positive cases were found. By GC/NPD and GC/MS, there were 16 negatives. The specificity of the TOXI-LAB for identification of morphine in urine of the suspect drug addict was 100%, and applying TOXI-LAB system for identification of morphine was feasible.
Finally, a method for determination of opiates in hair using liquid-liquid extraction(LLE) and GC/MS has been developed. The recovery of morphine ,codeine and 6-monoacetylmorphine were 82%,80% and 87% respectively.The limit of detection(SIM,LOD) for morphine ,codeine and 6-monoacetylmorphine were 0.052ng, 0.04ng and 0.037ng respectively.The equation of linearity of morphine ,codeine and 6-monoacetylmorphine were Y=0.02913x+0.01406(r=0.9997),Y=0.03255x-0.004468 (r=0.9992) and Y=0.03021x+0.01632 (r=0.9993) .
引文
1. Moriya F.;Hashimoto Y. J.Forensic Sci.42(4):736-740(1997).
2. Burt M.J,;Kloss J,;Apple F.S.J.Forensic Sci.46(5):1138-1142(2001).
3. Romberg R.W,;Lee L. J.Anal.Toxicol.19:157-162(1995).
4. Smith M.L,;Shimomura E.T, Summers J. et al. J.Anal.Toxicol.25:504-514(2001).
5. Kintz P,;Mangin P.Forensic Sci.Int.73:93-100(1995).
6. Meatherall B. J.Anal.Toxicol.23:177-186(1999).
7. Fogerson B,;Schoendorfer D,;Fay J. et al. J.Anal.Toxicol.21:451-458(1997).
8. Rothe M.;Pragst F. J.Anal.Toxicol. 19:236-240(1995).
9. Tebbrtt I.R. Chromatographia23:377-378(1987).
10. Chen X.H.;Hommerson A.L.C,Zweipfenning P.G,M. et al. J.Forensic Sci.38:668-676(1993).
11. Gerostamoulos J.; Drummer O.H. Forensic Sci.Int.77:53-63(1995).
12. Tatsuno M.;Nishikawa M.;Katagi M. et al. J.Anal.Toxicol.20:281-286(1996).
13. Diamond F.X.;Vickery W.E.;Kanel J. J.Anal.Toxicol.20:587-591(1996).
14. Bogusz M.J.; Maier R.D.;Driessen S. J.Anal.Toxieol.21:346-355(1997).
15. Cingolani M.;Froldi R.;Mencarelli R. et al. J.Anal.Toxicol.25:31-34(2001).
16. Weinmann W.;Renz M.;Vogt S. et al. Int.J.Legal.Med. 113:229-235(2000).
17. Gaillard Y.;Pepin G. Forensic Sci.Int.86:49-59(1997).
18. Salem M.Y.;Ross S.A.;Murphy T.P. et al. J.Anal.Toxicol.25:93-98(2001).
19. Dietzen D.J.;Koenig J.;Turk J. J.Anal.Toxicol. 19:299-303(1995).
20. Zuccaro P.;Ricciareilo R.;Pichini S. et al. J.Anal.Toxicol.21:268-277(1997).
21. Fuller D.C.J.Forensic Sci.42(4):685-689(1997).
22. Staub C. Forensic Sci.Int.84:295-304(1997).
23. Allen D.L.;Scott K.S.;Oliver J.S.J.Anal.Toxicol.23:216-218(1999).
24. Kadehjian L.J. J.Anal.Toxicol.25:671-679(2001).
25. Peace M.R.;Tarnai L.D.;Poklis A. J.Anal.Toxicol.24:589-594(2000).
26．苏木金；张豫；刘静飞等．放射免疫学杂志，Vol．11，1，56-57，1998
27. Jehanli A.;Brannan S.;Moore L. et al. J.Forensic Sci.46(5): 1214-1220(2001).
28. Barrct C.;Good C.;Moore C. Forensic Sci.Int. 122:163-166(2001).
29. Huestis M.A.;Cone E.J.;Wong C.J.et at. J.Anal.Toxicol. 24:509-521(2000).
30. Moeller M.R.; Mueller C.Forensic Sci.Int.70:125-133(1995).
31. Montagna M.;Stramesi C.;Vignali C. et al. Forensic Sci.Int. 107:157-167(2000).
32. Ricossa M.C;Bernini M.;Ferrari F.D. Forensic Sci.Int. 107:301-308(2000).
33. Tsatsakis A.M.;Tzatzarakis M.N.;Psaroulis D. et al. J.Forensic Med.Pathol.22(1):73-77(2001).
34. Potsch L.;Skipp G. Forensic Sci.Int.81:95-102(1996).
35. Balikova M.;Maresova V.Forensic Sci.Int.94:201-209(1998).
36. Pelders M.G;Ros J.J.W. J.Forensic Sci.41 (2):209-212(1996).
37. Skopp G.;Ganssmann B.Cone E.J. et al. J.Anal.Toxicol.21:105-111(1997).
38. Lemos N.P;Anderson R.A;Valentini R. et al. J.Forensic Sci.45(2):407-412(2000).
39. Skopp G.;Potsch L.;Klingmann A. et al. J.Anal.Toxicol.25:2-7(2001).
40. Weinberge R, Luriel S. Anal Chem,1991, 63:823-827.
41．孟品佳，孙毓庆，姜兆林，等．分析测试学报 1999；18(1)：17
42．余兆楼、陈义、常理文．分析化学，Vol．24(4)，444，1996
43．姜廷福，陆豪杰，李菊白 等．分析科学学报．Vol．18 No．5，385，2002
44. Tagliaro, F.; Manetto, G.; Crivellente, F.et al. Forensic Sci. Int., 92(2-3), 201-211,1998
45. Wu, W.-S.; Tsai, J.-L. Biomed. Chromatogr., 13(3), 216-219 (English) 1999
46. Zhang, X.-X.; Li, J.; Gao, J.et al. J. Chromatogr., A, 895(1+2), 1-7 (English) 2000
47. Wey, A. B.; Caslavska, J.; Thormann, W. Journal of Chromatography, A, 895(1+2), 133-146 (English) 2000
48. Tsai, Jin L.; Wu, Wen-S.; Lee, Hei-H. Electrophoresis, 21(8), 1580-1586(English) 2000
49. Wey, A. B.; Thormann, W. Journal of Chromatography, A, 916(1-2), 225-238 (English) 2001
2. Burt M.J,;Kloss J,;Apple F.S.J.Forensic Sci.46(5):1138-1142(2001).
3. Romberg R.W,;Lee L. J.Anal.Toxicol.19:157-162(1995).
4. Smith M.L,;Shimomura E.T, Summers J. et al. J.Anal.Toxicol.25:504-514(2001).
5. Kintz P,;Mangin P.Forensic Sci.Int.73:93-100(1995).
6. Meatherall B. J.Anal.Toxicol.23:177-186(1999).
7. Fogerson B,;Schoendorfer D,;Fay J. et al. J.Anal.Toxicol.21:451-458(1997).
8. Rothe M.;Pragst F. J.Anal.Toxicol. 19:236-240(1995).
9. Tebbrtt I.R. Chromatographia23:377-378(1987).
10. Chen X.H.;Hommerson A.L.C,Zweipfenning P.G,M. et al. J.Forensic Sci.38:668-676(1993).
11. Gerostamoulos J.; Drummer O.H. Forensic Sci.Int.77:53-63(1995).
12. Tatsuno M.;Nishikawa M.;Katagi M. et al. J.Anal.Toxicol.20:281-286(1996).
13. Diamond F.X.;Vickery W.E.;Kanel J. J.Anal.Toxicol.20:587-591(1996).
14. Bogusz M.J.; Maier R.D.;Driessen S. J.Anal.Toxieol.21:346-355(1997).
15. Cingolani M.;Froldi R.;Mencarelli R. et al. J.Anal.Toxicol.25:31-34(2001).
16. Weinmann W.;Renz M.;Vogt S. et al. Int.J.Legal.Med. 113:229-235(2000).
17. Gaillard Y.;Pepin G. Forensic Sci.Int.86:49-59(1997).
18. Salem M.Y.;Ross S.A.;Murphy T.P. et al. J.Anal.Toxicol.25:93-98(2001).
19. Dietzen D.J.;Koenig J.;Turk J. J.Anal.Toxicol. 19:299-303(1995).
20. Zuccaro P.;Ricciareilo R.;Pichini S. et al. J.Anal.Toxicol.21:268-277(1997).
21. Fuller D.C.J.Forensic Sci.42(4):685-689(1997).
22. Staub C. Forensic Sci.Int.84:295-304(1997).
23. Allen D.L.;Scott K.S.;Oliver J.S.J.Anal.Toxicol.23:216-218(1999).
24. Kadehjian L.J. J.Anal.Toxicol.25:671-679(2001).
25. Peace M.R.;Tarnai L.D.;Poklis A. J.Anal.Toxicol.24:589-594(2000).
26．苏木金；张豫；刘静飞等．放射免疫学杂志，Vol．11，1，56-57，1998
27. Jehanli A.;Brannan S.;Moore L. et al. J.Forensic Sci.46(5): 1214-1220(2001).
28. Barrct C.;Good C.;Moore C. Forensic Sci.Int. 122:163-166(2001).
29. Huestis M.A.;Cone E.J.;Wong C.J.et at. J.Anal.Toxicol. 24:509-521(2000).
30. Moeller M.R.; Mueller C.Forensic Sci.Int.70:125-133(1995).
31. Montagna M.;Stramesi C.;Vignali C. et al. Forensic Sci.Int. 107:157-167(2000).
32. Ricossa M.C;Bernini M.;Ferrari F.D. Forensic Sci.Int. 107:301-308(2000).
33. Tsatsakis A.M.;Tzatzarakis M.N.;Psaroulis D. et al. J.Forensic Med.Pathol.22(1):73-77(2001).
34. Potsch L.;Skipp G. Forensic Sci.Int.81:95-102(1996).
35. Balikova M.;Maresova V.Forensic Sci.Int.94:201-209(1998).
36. Pelders M.G;Ros J.J.W. J.Forensic Sci.41 (2):209-212(1996).
37. Skopp G.;Ganssmann B.Cone E.J. et al. J.Anal.Toxicol.21:105-111(1997).
38. Lemos N.P;Anderson R.A;Valentini R. et al. J.Forensic Sci.45(2):407-412(2000).
39. Skopp G.;Potsch L.;Klingmann A. et al. J.Anal.Toxicol.25:2-7(2001).
40. Weinberge R, Luriel S. Anal Chem,1991, 63:823-827.
41．孟品佳，孙毓庆，姜兆林，等．分析测试学报 1999；18(1)：17
42．余兆楼、陈义、常理文．分析化学，Vol．24(4)，444，1996
43．姜廷福，陆豪杰，李菊白 等．分析科学学报．Vol．18 No．5，385，2002
44. Tagliaro, F.; Manetto, G.; Crivellente, F.et al. Forensic Sci. Int., 92(2-3), 201-211,1998
45. Wu, W.-S.; Tsai, J.-L. Biomed. Chromatogr., 13(3), 216-219 (English) 1999
46. Zhang, X.-X.; Li, J.; Gao, J.et al. J. Chromatogr., A, 895(1+2), 1-7 (English) 2000
47. Wey, A. B.; Caslavska, J.; Thormann, W. Journal of Chromatography, A, 895(1+2), 133-146 (English) 2000
48. Tsai, Jin L.; Wu, Wen-S.; Lee, Hei-H. Electrophoresis, 21(8), 1580-1586(English) 2000
49. Wey, A. B.; Thormann, W. Journal of Chromatography, A, 916(1-2), 225-238 (English) 2001