Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, 30 S. 2000 E. Rm 3861, Salt Lake City, UT 84108, USA.
Designer Drug Research Unit, IRP, NIDA, NIH, DHHS, 333 Cassell Drive, Suite 4400, Baltimore, MD 21224, USA.
J Anal Toxicol. 2021 Feb 6;45(1):21-27. doi: 10.1093/jat/bkz096.
Alpha-pyrrolidinovalerophenone (alpha-PVP), a novel psychoactive substance, has widespread recreational use. This with interest in its pharmacological effects creates a need for methods that measure alpha-PVP concentrations. We therefore developed a LC-MS/MS method that can quantitate alpha-PVP and 2-oxo-PVP in rat plasma using a 0.1-mL sample volume. Addition of internal standards (2.5 ng/mL alpha-PVP-d8/2-oxo-PVP-d6) was followed by liquid-liquid extraction with 1-chlorobutane:acetonitrile (4:1), evaporation and reconstitution with 0.1% formic acid. Extracts were analyzed by LC-MS/MS using an Agilent 1100 HPLC and a Thermo Scientific TSQ Quantum Access MS/MS, with a YMC ODS-AQ, 50 mm × 2 mm, 3 μm column. The mobile phase was 0.1% formic acid:acetonitrile gradient at a 0.2-mL/minute flow rate with positive ion electrospray. SRM was used for the analysis with transitions: alpha-PVP, 232 → 91; alpha-PVP-d8, 240 → 91; 2-oxo-PVP, 246 → 91; 2-oxo-PVP-d6, 252 → 91. Alpha-PVP and 2-oxo-PVP eluted at 6.4 and 8.9 min. Calibrators range from 0.25 to 500 ng/mL. Accuracy and precision evaluated quality control samples prepared at 0.75, 10 and 400 ng/mL. The intra-assay evaluation also included the 0.25-ng/mL LOQs prepared in six different blank plasma sources. The intra-assay accuracy ranged from 88.9 to 117.8% of the target, and the intra-assay precision ranged from 0.9 to 16.0%. The inter-assay accuracy ranged from 98.7 to 110.7% of the target, and the inter-assay precision ranged from 4.5 to 12.0%. Extraction recovery was at least 52% for alpha-PVP and 67% for 2-oxo-PVP. Ionization recoveries were at least 64% for alpha-PVP and 82% for 2-oxo-PVP. These losses did not adversely affect assay performance. Alpha-PVP and 2-oxo-PVP controls were stable at room temperature for up to 24 h and frozen for at least 36 days. Alpha-PVP and 2-oxo-PVP were also stable in processed samples (extracts) stored at room temperature for at least 24 days. The procedure was used to analyze rat plasma samples from a pharmacokinetic study.
阿尔法-吡咯烷酮基戊基苯（alpha-PVP）是一种新型精神活性物质，在娱乐场所被广泛使用。由于人们对其药理作用产生了兴趣，因此需要开发一种能够测量 alpha-PVP 浓度的方法。因此，我们开发了一种 LC-MS/MS 方法，可使用 0.1mL 样品体积定量测定大鼠血浆中的 alpha-PVP 和 2-氧代-PVP。加入内标物（2.5ng/mL alpha-PVP-d8/2-氧代-PVP-d6）后，用 1-氯丁烷：乙腈（4：1）进行液-液萃取，蒸发后用 0.1%甲酸重新溶解。提取物通过 Agilent 1100 HPLC 和 Thermo Scientific TSQ Quantum Access MS/MS 进行分析，使用 YMC ODS-AQ，50mm×2mm，3μm 柱。流动相为 0.1%甲酸：乙腈梯度，流速为 0.2mL/min，采用正离子电喷雾。SRM 用于分析，转换为：alpha-PVP，232→91；alpha-PVP-d8，240→91；2-氧代-PVP，246→91；2-氧代-PVP-d6，252→91。alpha-PVP 和 2-氧代-PVP 的洗脱时间分别为 6.4 和 8.9min。校准器范围为 0.25 至 500ng/mL。在 0.75、10 和 400ng/mL 制备的质控样品的准确度和精密度进行了评估。还对 0.25ng/mL 的 LOQ 进行了 6 种不同空白血浆来源的日内评估。内标物测定的准确度在目标值的 88.9%至 117.8%之间，精密度在 0.9%至 16.0%之间。在不同时间测定的准确度在目标值的 98.7%至 110.7%之间，精密度在 4.5%至 12.0%之间。alpha-PVP 的提取回收率至少为 52%，2-氧代-PVP 的提取回收率至少为 67%。alpha-PVP 的离子化回收率至少为 64%，2-氧代-PVP 的离子化回收率至少为 82%。这些损失并没有对测定结果产生不利影响。室温下 alpha-PVP 和 2-氧代-PVP 对照品稳定至少 24 小时，冷冻至少 36 天。处理后的样品（提取物）中 alpha-PVP 和 2-氧代-PVP 也稳定，至少 24 天。该方法用于分析药代动力学研究中的大鼠血浆样品。
