Proença Paula, Franco João Miguel, Mustra Carla, Marcos Mariana, Pereira Ana Rita, Corte-Real Francisco, Vieira Duarte Nuno
National Institute of Legal Medicine - Centre Branch, Largo da Sé Nova, 3000-213 Coimbra, Portugal.
J Forensic Leg Med. 2011 Oct;18(7):320-4. doi: 10.1016/j.jflm.2011.06.003. Epub 2011 Jul 7.
Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil(®) next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode. The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC(®) BEH (2.1 × 50 mm id, 1.7 μm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1-143.1 for valproic acid and m/z 296.1-205.0 for hydrochlorothiazide used as an internal standard (IS). The limit of quantification (LOQ) was 0.5 μg/mL and the method was linear in the concentration range of 0.5-100 μg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 μg/mL, 10 μg/mL and 50 μg/mL). Toxicological results showed high concentration of VPA (556 μg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L. A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.
丙戊酸（VPA）一直被用作抗惊厥药来治疗癫痫。作者报告了一例致命病例，一名45岁女性被发现死在床上，旁边有迪普莱西（Diplexil）® 的空药瓶。她是一名慢性酒精中毒者和癫痫患者，一直在接受精神治疗，曾多次表现出自杀意图。开发并验证了一种快速方法，通过超高效液相色谱（UPLC）与串联质谱（MS/MS）联用，采用电喷雾电离源负离子模式来测定血液中的VPA。该方法包括用磷酸处理样品，然后进行固相萃取。使用Acquity UPLC® BEH（2.1×50 mm内径，1.7μm）色谱柱和含有醋酸铵和乙腈的流动相，以0.5 mL/min的流速实现色谱分离。通过多反应监测（MRM）实现VPA的检测和定量。用于监测的MS/MS转换为：丙戊酸的m/z 143.1 - 143.1，用作内标（IS）的氢氯噻嗪的m/z 296.1 - 205.0。定量限（LOQ）为0.5μg/mL，该方法在0.5 - 100μg/mL的浓度范围内呈线性。所研究的三个浓度水平（5μg/mL、10μg/mL和50μg/mL）的准确度和精密度的变异系数均小于10%，平均回收率为95%。毒理学结果显示VPA浓度很高（556μg/mL），同时存在硫必利、米氮平、奥沙西泮和去甲西泮的治疗浓度。血液样本分析还显示乙醇浓度为1.34 g/L。开发了一种用于测定血液中VPA的特异性、选择性和灵敏性方法，可用于常规法医调查。毒理学结果使病理学家判定死亡原因是同时摄入高浓度VPA和酒精导致的中毒，具有自杀性法医学病因。
