Rapid quantification of phenobarbital and barbital in human whole blood by liquid–liquid extraction combined with DART-orbitrap-HRMS,Forensic Toxicology

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Rapid quantification of phenobarbital and barbital in human whole blood by liquid–liquid extraction combined with DART-orbitrap-HRMS
Forensic Toxicology ( IF 2.8 ) Pub Date : 2022-11-27 , DOI: 10.1007/s11419-022-00650-w
Shi Ke _{1,

2,

3} , Ru Lian ₃ , Rong Wang ₃ , Yulan Rao ₄ , Chen Liang ₃ , Jianying Liang ₁ , Yurong Zhang ₃

Affiliation

Purpose

This study aims to develop and validate a rapid, simple, and efficient bioanalytical method for the simultaneous quantification of phenobarbital and barbital in human whole blood using liquid–liquid extraction combined with direct analysis in real time (DART) and high-resolution mass spectrometry (HRMS).

Method

Phenobarbital-d5 and aprobarbital were selected as internal standards (ISs) of phenobarbital and barbital, respectively. A mixed solvent of o-xylene and ethyl acetate at a ratio of 1:6 was used to extract analytes of interest and ISs from 100 μL of human whole blood samples. Phenobarbital and barbital were detected by DART-HRMS. The proposed method has been validated in accordance with United States Food and Drug Administration Guidelines for Bioanalytical Method Validation in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery, stability, and dilution integrity.

Results

The lower limits of quantification (LLOQs) of phenobarbital and barbital were both 10 ng/mL. The linearities were in the range of 10–1000 ng/mL (R² ≥ 0.99). The mean recovery values of phenobarbital and barbital were 99.7% and 88.1%, respectively. The interday and intraday precision values were less than 10.4%, and the interday and intraday accuracy values ranged from 87.6 to 106.7%. Furthermore, the validated method was applied to four cases of phenobarbital poisoning at the Shanghai Institute of Forensic Science.

Conclusion

The developed and fully validated method enabled the simultaneous quantification of phenobarbital and barbital in human whole blood and was successfully applied to authentic cases.

中文翻译：

液-液萃取结合 DART-orbitrap-HRMS 快速定量人全血中的苯巴比妥和巴比妥

目的

本研究旨在开发和验证一种快速、简单、高效的生物分析方法，使用液-液萃取结合实时直接分析 (DART) 和高分辨率质谱同时定量人全血中的苯巴比妥和巴比妥。人力资源管理系统）。

方法

分别选择苯巴比妥-d5 和阿普巴比妥作为苯巴比妥和巴比妥的内标 (IS)。使用邻二甲苯和乙酸乙酯按 1:6 的比例混合溶剂从 100 μL 人全血样本中提取目标分析物和内标。通过 DART-HRMS 检测苯巴比妥和巴比妥。所提出的方法已根据美国食品和药物管理局生物分析方法验证指南在选择性、线性、准确度、精密度、基质效应、回收率、稳定性和稀释完整性方面进行了验证。

结果

苯巴比妥和巴比妥的定量下限 (LLOQ) 均为 10 ng/mL。线性范围为 10–1000 ng/mL ( R ² ≥ 0.99)。苯巴比妥和巴比妥的平均回收率分别为 99.7% 和 88.1%。日间和日内精密度值均小于10.4%，日间和日内准确度值范围为87.6%至106.7%。此外，经验证的方法还应用于上海法医学研究所的四起苯巴比妥中毒案件。