Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland.
Department of Medical Biology, National Institute of Cardiology, Alpejska 42, 04-628 Warsaw, Poland.
Molecules. 2021 Nov 29;26(23):7252. doi: 10.3390/molecules26237252.
The aim of the work was to prepare a simple but reliable HPLC-UV method for the routine monitoring of mycophenolic acid (MPA). Sample preparation was based on plasma protein precipitation with acetonitrile. The isocratic separation of MPA and internal standard (IS) fenbufen was made on Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) using a mobile phase: CHCN:HO:0.5M KHPO:HPO (260:700:40:0.4, /). UV detection was set at 305 nm. The calibration covered the MPA concentration range: 0.1-40 µg/mL. The precision was satisfactory with RSD of 0.97-7.06% for intra-assay and of 1.92-5.15% for inter-assay. The inaccuracy was found between -5.72% and +2.96% (+15.40% at LLOQ) and between -8.82% and +5.31% (+19.00% at LLOQ) for intra- and inter-assay, respectively, fulfilling acceptance criteria. After a two-year period of successful application, the presented method has been retrospectively calibrated using the raw data disregarding the IS in the calculations. The validation and stability parameters were similar for both calculation methods. MPA concentrations were recalculated and compared in 1187 consecutive routine therapeutic drug monitoring (TDM) trough plasma samples from mycophenolate-treated patients. A high agreement (r = 0.9931, < 0.0001) of the results was found. A Bland-Altman test revealed a mean bias of -0.011 μg/mL (95% CI: -0.017; -0.005) comprising -0.14% (95% Cl: -0.39; +0.11), whereas the Passing-Bablok regression was y = 0.986x + 0.014. The presented method can be recommended as an attractive analytical tool for medical (hospital) laboratories equipped with solely basic HPLC apparatus. The procedure can be further simplified by disapplying an internal standard while maintaining appropriate precision and accuracy of measurements.
本工作旨在制备一种简单但可靠的 HPLC-UV 方法，用于常规监测麦考酚酸（MPA）。样品制备基于用乙腈进行血浆蛋白沉淀。MPA 和内标（IS）芬布芬的等度分离在 Supelcosil LC-CN 柱（150×4.6mm，5μm）上进行，流动相为：CHCN:HO:0.5M KHPO:HPO（260:700:40:0.4，/）。UV 检测设定在 305nm。校准涵盖 MPA 浓度范围：0.1-40μg/mL。日内精密度的 RSD 为 0.97-7.06%，日间精密度的 RSD 为 1.92-5.15%，均令人满意。内、日间准确度分别为-5.72%至+2.96%（LLOQ 时为+15.40%）和-8.82%至+5.31%（LLOQ 时为+19.00%），满足接受标准。在成功应用两年后，使用不考虑计算中 IS 的原始数据对所提出的方法进行了回顾性校准。两种计算方法的验证和稳定性参数均相似。在 1187 例连续的麦考酚酯治疗患者的治疗药物监测（TDM）谷血浆样本中，重新计算并比较了 MPA 浓度。结果高度一致（r=0.9931，<0.0001）。Bland-Altman 检验显示平均偏差为-0.011μg/mL（95%CI：-0.017；-0.005），包括-0.14%（95%Cl：-0.39；+0.11），而通过 Bablok 回归为 y=0.986x+0.014。本方法可推荐作为一种有吸引力的分析工具，适用于仅配备基本 HPLC 仪器的医疗（医院）实验室。通过不使用内标，同时保持测量的适当精密度和准确度，可以进一步简化该程序。
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