Federal Public Justice Service, National Institute of Criminalistics and Criminology, 1120 Brussels, Belgium.
J Anal Toxicol. 2009 Nov-Dec;33(9):578-87. doi: 10.1093/jat/33.9.578.
An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2) > 0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.
建立并验证了一种用于血液和尿液中苯丙胺类药物分析的自动化在线固相萃取-液相色谱-串联质谱（SPE-LC-MS-MS）方法。采用 Nucleodur Sphinx RP 柱和 LC 梯度（10mM 甲酸铵缓冲液和乙腈的混合物）进行色谱分离，确保在 11 分钟内洗脱苯丙胺、甲基苯丙胺、MDMA、MDA、MDEA、PMA 和麻黄碱。该方法完全按照国际指南进行了验证，仅使用 100 和 50μL 的血液和尿液即可。该方法对两种基质的两个外部质控样品（QC）以及血液和尿液的三个和两个“内部”QC 均表现出极好的内和日间精密度（相对标准偏差 < 11.2%，偏差 < 13%）。响应在研究范围内呈线性（r(2)>0.99，血液为 2.5-400μg/L，尿液为 25-1000μg/L）。定量限分别确定为血液和尿液的 2.5 和 25μg/L。血液的检测限范围为 0.05-0.5μg/L，尿液的检测限范围为 0.25-2.5μg/L，具体取决于化合物。此外，分析物和处理后的样品在自动进样器中至少稳定 72 小时，且经过三个冻融循环后也稳定，所有化合物均未观察到基质干扰。此外，在分析高浓度样品（15000μg/L）后也未观察到拖尾现象。该方法随后应用于从法医案例中获得的真实血液和尿液样本，涵盖了广泛的浓度范围。验证结果和实际样品分析表明，该方法具有坚固性、精确性、准确性，并且非常适合常规分析，因为在 24 小时内可以不停歇地分析超过 72 个样品，且样品处理量最少。在线 SPE 的高通量与 MS-MS 保证的灵敏度和选择性的结合消除了与样品制备要求相关的瓶颈，提高了灵敏度、准确性和精密度。
