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Abstract：
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.
摘要：
这项研究的目的是开发和验证一种快速，灵敏的生物分析方法，以准确定量人前列腺组织中的磷霉素浓度。样品制备方法仅需要毫克的组织样品。将每个样品与两倍于其重量的水混合并均化。加入三倍于内标(磷霉素-13C3)体积的甲醇溶液,然后涡旋混合和离心。从匀浆的前列腺组织中提取后，通过在负电喷雾电离和多反应监测检测模式下运行的液相色谱-串联质谱（LC-MS/MS）三重四极杆系统对磷霉素进行定量。该分析程序在特异性方面得到了成功验证，灵敏度,线性度精度,准确度,基体效应，提取回收，定量极限，和稳定性,根据EMA指南。验证结果,相对于三个QC水平，日内和日间准确度（BIAS％）均为9.9％；日内精度为9.8％；日间精度为9.9。在测量中观察到显著的基质效应,但通过用内标归一化来校正。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0.1-20μg/g（R2为0.999）。在注射高度浓缩的样品后观察到可忽略的携带。样品匀浆提取物中的F在10°C和4°C下稳定至少24小时。在组织样品冻融实验中，从-80°C到室温仅两个循环后，观察到F浓度显着降低。该新方法已成功应用于从105例接受前列腺切除术的患者收集的前列腺组织样品中的磷霉素的测量。
