Favretto Donata, Frison Giampietro, Vogliardi Susanna, Ferrara Santo Davide
Forensic Toxicology and Antidoping, University Hospital of Padova, Via Falloppio 50, I-35121 Padova, Italy.
Rapid Commun Mass Spectrom. 2006;20(8):1257-65. doi: 10.1002/rcm.2444.
A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4-buprenorphine (d4-BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with beta-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 x 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1-10 ng/mL in urine and blood, in the range 10-160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death.
已开发出一种液相色谱/电喷雾电离串联质谱法（LC/ESI-MS/MS），用于分析生物体液中的丁丙诺啡（BUP）和去甲丁丙诺啡（NBUP）。加入d4-丁丙诺啡（d4-BUP）作为内标后，通过固相萃取从尿液和血液中分离分析物。毛发的制备包括外部去污、机械粉碎、在酸性介质中过夜孵育以及萃取前的中和。可进行β-葡萄糖醛酸酶的酶促水解，以区分游离BUP和总BUP。通过在氰丙基2.1×150 mm柱上进行梯度洗脱完成色谱分离。在离子阱质谱仪中进行正离子ESI和MS分析。使用这种质量分析仪可对ESI产生的MH+物种进行有效的碰撞实验。产生大量产物离子，可与前体离子一起监测而不损失灵敏度。因此，相对于仅监测前体离子的LC/ESI-MS/MS方法，该测定法的选择性肯定有所提高。该方法具有良好的线性（尿液和血液中的校准曲线在0.1-10 ng/mL范围内呈线性，毛发中的校准曲线在10-160 pg/mg范围内呈线性），血液和尿液样本中BUP和NBUP的检测限均为0.05 ng/mL，毛发中两种分析物的检测限均为4 pg/mg。在所研究的三个浓度下，批内和批间精密度及准确度均令人满意：尿液中的相对标准偏差<13.7%，血液中的相对标准偏差<17.3%，毛发中的相对标准偏差<17.8%；尿液和血液中平均值与真实值的偏差百分比始终<10.5%，毛发中的偏差百分比<16.1%。该方法可用于测定接受替代治疗的吸毒者尿液和毛发中的两种分析物，以及在怀疑与药物相关死亡时测定尸检血液样本中的分析物。
