Miller Eleanor I, Torrance Hazel J, Oliver John S
Forensic Medicine and Science Department, University of Glasgow, University Place, Glasgow G12 8QQ, Scotland.
J Anal Toxicol. 2006 Mar;30(2):115-9. doi: 10.1093/jat/30.2.115.
The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.
本研究的目的是验证免疫分析丁丙诺啡微孔板酶联免疫吸附测定法（ELISA）用于检测尿液样本中的丁丙诺啡。从接受舒倍生治疗方案的志愿者以及提交至实验室进行丁丙诺啡检测的常规样本中获取了69份尿液样本。对于ELISA分析，样本用K₂HPO₄（0.1M，pH 7.0）以1:10的比例稀释。计算得出丁丙诺啡的检测限为0.5 ng/mL。在丁丙诺啡浓度为1 ng/mL时，批内精密度和日间精密度分别为3.8%（n = 10）和8.6%（n = 50）。在去甲丁丙诺啡低浓度（1 ng/mL）时，免疫测定显示交叉反应率为78%。在去甲丁丙诺啡较高浓度（10 ng/mL）时，观察到交叉反应率更高，为116%。对二氢可待因、可待因、曲马多、吗啡、丙氧芬、美沙酮和乙二胺二苯酮在浓度为10 ng/mL和10,000 ng/mL时进行了检测，结果显示它们与该测定法无交叉反应。对于液相色谱 - 串联质谱法（LC-MS-MS），向标准品和样本中加入氘代内标混合物、1M乙酸盐缓冲液（pH 5.0）和β - 葡萄糖醛酸酶，然后在60℃下孵育3小时。孵育后，加入3 mL K₂HPO₄（0.1M，pH 6.0），并用1M KOH将pH调节至pH 6.0。随后通过固相萃取丁丙诺啡和去甲丁丙诺啡。通过LC-MS-MS确认21份样本为阳性，48份样本为阴性。使用丁丙诺啡的截断值为0.5 ng/mL时，免疫测定显示灵敏度和特异性均为100%。
