LC-MS/MS method for determination of r-RGD-hirudin in human plasma and its application in pharmacokinetic study

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS Analytical biochemistry Pub Date : 2023-01-15 DOI:10.1016/j.ab.2022.114987

Ai Peng , Li Xiao , Lin Yang , Xiaoqin Yi , Jin Xiang

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Abstract

Objective

This study aimed to develop a simple, sensitive, and selective Liquid chromatography with a Mass spectroscopic method for simultaneous quantification of a recombinant bifunctional hirudin (r-RGD-Hirudin, Bifunctional Hirudin, BFH) in human plasma and verify its effectiveness.

Methods

The analytes and the internal standards from human plasma were extracted using the solid-phase extraction technique. The reconstituted samples were chromatographed on Waters C₁₈ column (BEH 50 × 2.1 mm, 1.7 μm) using a mixture of 0.1% formic acid/acetonitrile (85%/15%, v/v) with gradient elution as the initial mobile phase at a flow rate of 0.3 mL/min.

Results

The effectiveness of the proposed method was verified over the concentration range of 10–2000 ng/mL for r-RGD-Hirudin. A linear calibration curve was obtained. The precision and accuracy of BFH in the intra- and inter-day runs fell within the range of ±15% at LQC, GMQC, MQC and HQC concentrations. The extraction recoveries and matrix effect at two quality control (QC) levels for BFH were confirmed to conform to the relevant requirement.

Conclusion

The proposed method was successfully adapted to examine the pharmacokinetics of BFH in 40 Chinese healthy volunteers, respectively.

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LC-MS/MS法测定人血浆中r- rgd -水蛭素及其药动学研究

目的本研究旨在建立一种简单、灵敏、选择性的液相色谱-质谱法同时定量人血浆中重组双功能水蛭素（r-RGD-hirudin，bifunctional hirudin，BFH），并验证其有效性。方法采用固相萃取技术提取人血浆中的分析物和内标物。重组样品在Waters C18柱（BEH 50×2.1 mm，1.7μm）上进行色谱分离，使用0.1%甲酸/乙腈（85%/15%，v/v）的混合物作为初始流动相，流速为0.3 mL/min。结果在10–2000 ng/mL的浓度范围内验证了所提出方法对r-RGD-Hirudin的有效性。获得了线性校准曲线。在LQC、GMQC、MQC和HQC浓度下，日内和日间运行中BFH的精密度和准确度均在±15%范围内。BFH在两个质量控制（QC）水平下的提取回收率和基质效应均符合相关要求。结论所提出的方法分别适用于40名健康志愿者体内BFH的药代动力学检测。

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.