DETERMINATION OF PHOSPHATIDYLETANOL IN DRIED BLOOD SPOT BY UHPLC-MS/MS: EVALUATION OF ALCOHOL CONSUMPTION IN CHEMICAL DEPENDENTS

We developed and validated an UHPLC-MS/MS method for the quantification of phosphatidylethanol (PEth) in dried blood spots (DBS). Sample preparation was a liquid extraction and chromatographic separation was performed in an Acquity C8 column. The mobile phases were 4 mM ammonium acetate in water (A) and acetonitrile (B) at 24:76 (v/v). The total analytical run time was 6 minutes, with retention time of 3.36 min for PEth and 4.13 min for phosphatidylpropanol. The method was linear from 10 to 3000 ng mL-1, specific, with, precise and accurate. The analyte was stable in DBS stored from -20 to 45 °C for 21 days. Matrix effect was compensated with the internal standard (-5.21% to + 6.09%). The method was applied in the evaluation of alcohol consumption in DBS from 25 chemical dependents. The PEth concentrations ranged from 14.5 to 2380.8 ng mL-1, with significant correlation with the self-report alcohol consumption AUDIT scores (r=0.41).

本研究建立并验证了一种超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，用于定量检测干血斑（Dried Blood Spots, DBS）中的磷脂酰乙醇（phosphatidylethanol, PEth）。样品前处理采用液液萃取法，色谱分离使用Acquity C8色谱柱。流动相为含4 mmol·L⁻¹乙酸铵的水溶液（流动相A）与乙腈（流动相B），二者体积配比为24:76。总分析运行时长为6分钟，其中PEth的保留时间为3.36 min，磷脂酰丙醇的保留时间为4.13 min。该方法在10~3000 ng·mL⁻¹范围内线性良好，且具备良好的专属性、精密度与准确度。待测物在-20℃至45℃条件下保存的干血斑中可稳定存在21天。通过内标法可有效补偿基质效应，基质效应偏差范围为-5.21%~+6.09%。将该方法应用于25名物质依赖者的干血斑样本酒精摄入水平评估，结果显示样本中PEth浓度范围为14.5~2380.8 ng·mL⁻¹，且与受试者自我报告的酒精使用障碍识别测试（Alcohol Use Disorders Identification Test, AUDIT）得分呈显著相关（r=0.41）。