测量人类前列腺组织中磷霉素浓度的灵敏液相色谱-串联质谱法

Matteo Conti, Beatrice Giorgi, Rossella Barone, Milo Gatti, Pier Giorgio Cojutti, Federico Pea
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摘要

本研究旨在开发并验证一种快速、灵敏的生物分析方法,用于准确定量检测人类前列腺组织中的磷霉素浓度。样品制备方法只需毫克组织样品。将每个样本与两倍重量的水混合并匀浆。加入三倍于内标物(磷霉素-13C3)体积的甲醇溶液,然后涡旋混合并离心。从均质化的前列腺组织中提取磷霉素后,采用液相色谱-串联质谱(LC-MS/MS)三重四极杆系统,在负电喷雾电离和多反应监测检测模式下进行定量分析。根据 EMA 准则,分析程序在特异性、灵敏度、线性度、精密度、准确度、基质效应、提取回收率、定量限和稳定性等方面都得到了成功验证。相对于三个质控水平,验证结果为:日内和日间准确度(BIAS%)均为 9.9%;日内精密度为 9.8%;日间精密度为 9.9%。测量中观察到了明显的基质效应,但用内标进行归一化后得到了纠正。平均总回收率很高(在三个对照水平下约为 97%)。该方法的动态范围为 0.1-20 μg/g(R2 为 0.999)。在注入高浓度样品后观察到的迁移量微乎其微。样品匀浆提取物中的 F 在 10 ℃ 和 4 ℃ 下至少 24 小时内保持稳定。在组织样品冻融实验中,从 -80 ℃ 到室温仅两个循环后,F 浓度显著下降。这种新方法被成功应用于105名前列腺切除术患者的前列腺组织样本中磷霉素的检测。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue
The aim of this study was to develop and validate a fast and sensitive bioanalytical method for the accurate quantification of fosfomycin concentrations in human prostatic tissue. The sample preparation method only required milligrams of tissue sample. Each sample was mixed with two times its weight of water and homogenized. A methanol solution that was three times the volume of the internal standard (fosfomycin-13C3) was added, followed by vortex mixing and centrifugation. After its extraction from the homogenized prostatic tissue, fosfomycin was quantified by means of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) triple quadrupole system operating in negative electrospray ionization and multiple reaction monitoring detection mode. The analytical procedure was successfully validated in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability, according to EMA guidelines. The validation results, relative to three QC levels, were 9.9% for both the within-day and inter-day accuracy (BIAS%); 9.8% for within-day precision; and 9.9 for between-day precision. A marked matrix effect was observed in the measurements but was corrected by normalization with the internal standard. The average total recovery was high (approximatively 97% at the three control levels). The dynamic range of the method was 0.1–20 μg/g (R2 of 0.999). Negligible carry-over was observed after the injection of highly concentrated samples. F in the sample homogenate extracts was stable at 10 °C and 4 °C for at least 24 h. In the tissue sample freeze–thaw experiments, a significant decrease in F concentrations was observed after only two cycles from −80 °C to room temperature. The novel method was successfully applied to measure fosfomycin in prostatic tissue samples collected from 105 patients undergoing prostatectomy.
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