Establishment and Validation of a UPLC-MS/MS Detection Method for Thymosin β4 in Rat Plasma

doi:10.19803/j.1672-8629.20250333

Abstract

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

CLC Number:

R994.11

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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URL: https://zgywjj.magtechjournal.com/EN/10.19803/j.1672-8629.20250333

https://zgywjj.magtechjournal.com/EN/Y2025/V22/I11/1241

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