大鼠血浆中胸腺素β4 UPLC-MS/MS检测方法的建立及验证

doi:10.19803/j.1672-8629.20250333

摘要/Abstract

摘要： 目的探究超高效液相色谱-串联质谱法（UPLC-MS/MS）用于定量检测大鼠血浆中胸腺素β4,为后续药代动力学研究、药物疗效评估或相关疾病模型的机制研究等提供参考。方法采用蛋白沉淀法（PPT）处理血浆样品,流动相为纯水-乙腈（均含0.1%甲酸）,柱温为45°C,流速为0.25 mL·min^-1,色谱柱为ACQUITY UPLC Peptide BEH C₁₈（2.1 mm×50 mm,1.7μm）,梯度洗脱分离。质谱采用电喷雾离子化（ESI）,正离子多重反应监测模式（MRM）检测母离子和子离子,分析物胸腺素β4为m/z 709.9→810.6,内标物赖脯胰岛素为m/z 1 162→217,并对该方法进行方法学验证,包括选择性、标准曲线及定量下限、精密度与准确度、提取回收率与基质效应、稳定性。结果大鼠血浆中胸腺素β4在50~5 000 ng·mL^-1线性良好,胸腺素β4的低、中、高浓度（120、800、3 800 ng·mL^-1）质控样本批内和批间准确度（RE）为-1.6%~3.1%,精密度（RSD）为5.4%~9.9%。低、中、高浓度质控样本的提取回收率为81.94%~110.23%,基质效应为100.30%~108.08%,变异系数（CV）在±15%范围以内。低、中、高浓度（120、800、3 800 ng·mL^-1）质控样本在室温放置24 h、反复冻融3次和-20℃冻存7 d后,各浓度均值与标示浓度的偏差在±15%范围内,表明样品稳定性良好。结论本研究建立了一种适用于大鼠血浆基质中胸腺素β4定量分析的UPLC-MS/MS方法,该方法操作简单、分析效率较高。

关键词: 胸腺素β4, 超高效液相色谱-串联质谱法（UPLC-MS/MS）, 方法学验证, 蛋白沉淀法, 定量分析, 大鼠

Abstract: Objective To establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantitative determination of thymosin beta 4 in rat plasma in order to provide a reference for subsequent pharmacokinetic studies. Methods Plasma samples were pretreated using a protein precipitation method. The mobile phase consisted of pure water and acetonitrile (both containing 0.1% formic acid), with a column temperature of 45°C, a flow rate of 0.25 mL·min^-1 and a chromatographic column of ACQUITY UPLC Peptide BEH C₁₈ (2.1 mm×50 mm,1.7μm) under gradient elution. Thymosin beta 4 and the internal standard (lispro insulin) were detected and quantified using the multiple reaction monitoring (MRM) mode, with precursor-to-product ion transitions of m/z 709.9→810.6 for thymosin beta 4 and m/z 1 162→217.1 for the internal standard. Methodvalidation involved assessments of selectivity, the calibration curve and lower limit of quantification (LLOQ), precision and accuracy, extraction recovery, matrix effect, and stability. Results Thymosin beta 4 showed a good linearity in rat plasma within the range of 50 to 5000 ng·mL^-1. The intra- and inter-batch accuracy (RE) for thymosin beta 4 in low-, medium-, and high-concentration quality control (QC) samples ranged from -1.6% to 3.1%, and the precision (RSD) ranged from 5.4% to 9.9%. Extraction recoveries of quality control (QC) samples at low, medium, and high concentrations（120、800、3 800 ng·mL^-1) were between 81.94% and 110.23%, with matrix effects of 100.30%~108.08% (coefficient of variation, CV, within ± 15%). The deviation between the mean measured concentrations and the designated concentrations of quality control samples (120、800、3 800 ng·mL^-1) was within ± 15% after 24 hours of storage at room temperature, three freeze-thaw cycles, or 7 days of storage at -20°C, suggesting the good stability of the samples. Conclusion A simple and efficient UPLC-MS/MS method has been established and validated for quantitative determination of thymosin beta 4 in rat plasma.

Key words: Thymosin Beta 4, UPLC-MS/MS, Method Validation, Protein Precipitation, Quantitative Analysis, Rat

中图分类号:

R994.11

汪建英, 汪电雷. 大鼠血浆中胸腺素β4 UPLC-MS/MS检测方法的建立及验证[J]. 中国药物警戒, 2025, 22(11): 1241-1245.

WANG Jianying, WANG Dianlei. Establishment and Validation of a UPLC-MS/MS Detection Method for Thymosin β4 in Rat Plasma[J]. Chinese Journal of Pharmacovigilance, 2025, 22(11): 1241-1245.

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