Rissling Olesja, Bauer Steffen, Shipkova Maria, Glander Petra, Mai Marco, Hambach Pia, Budde Klemens
a Department of Nephrology , Charité Universitätsmedizin , Berlin , Germany.
b Institute of Pharmacy, Freie Universitaet Berlin , Berlin , Germany.
Scand J Clin Lab Invest. 2016 Dec;76(8):612-619. doi: 10.1080/00365513.2016.1230775. Epub 2016 Sep 27.
Mycophenolic acid (MPA) is an immunosuppressive agent which is commonly used in a fixed dose regime in solid organ transplantation. For clinical trials and therapeutic drug monitoring measuring plasma concentrations is necessary. Also, stability issues have to be addressed.
We describe an isocratic, RP-based HPLC-UV method for simultaneous determination of MPA and its major metabolite Mycophenolic acid 7-o Glucuronide (MPAG) in human plasma. Pre-analytics included protein precipitation with acetonitrile. The method was validated according to EMA/FDA guidelines. Patient lithium-heparin plasma and blood was used for evaluation of short-term (72 hours at room temperature = RT) and long-term stability (2 years at -80 °C) without acidification.
Linearity was assessed in the concentration range of 0.5-40.0 μg/mL for MPA and 5.0-350.0 μg/mL for MPAG, respectively. For MPA coefficient of variation was <7.0% (lower limit of quantification = LLOQ: 10.8%), for MPAG <9.6% (LLOQ: 10.6%). Bias ranged between -1.9 and +1.5% for MPA and for MPAG between -4.3 and -0.3%. The method showed agreement with a reference method for both analytes. MPA remained stable for 7 h (-1.6 to +8.4% change to the initial concentration) and MPAG for 24 h (-1.8 to -11.5% change) at RT in lithium heparin blood. After 2 years of storage at -80 °C MPA, MPAG concentrations and 95% CIs remained within ±15% of the initial value.
The presented assay is applicable for clinical studies. Blood samples were stable for 7 hours at RT and plasma for 2 years stored at -80 °C.
霉酚酸（MPA）是一种免疫抑制剂，在实体器官移植中通常采用固定剂量方案使用。对于临床试验和治疗药物监测而言，测量血浆浓度是必要的。此外，还必须解决稳定性问题。
我们描述了一种基于反相高效液相色谱-紫外检测的等度洗脱方法，用于同时测定人血浆中的MPA及其主要代谢物霉酚酸7 - O - 葡萄糖醛酸（MPAG）。分析前处理包括用乙腈进行蛋白沉淀。该方法根据欧洲药品管理局（EMA）/美国食品药品监督管理局（FDA）指南进行了验证。使用患者的锂肝素血浆和血液评估了未酸化情况下的短期稳定性（室温72小时）和长期稳定性（-80°C下2年）。
MPA的线性评估浓度范围为0.5 - 40.0μg/mL，MPAG为5.0 - 350.0μg/mL。MPA的变异系数<7.0%（定量下限=LLOQ：10.8%），MPAG<9.6%（LLOQ：10.6%）。MPA的偏差在-1.9%至+1.5%之间，MPAG在-4.3%至-0.3%之间。该方法与两种分析物的参考方法具有一致性。在锂肝素血中，室温下MPA可稳定7小时（相对于初始浓度变化-1.6%至+8.4%），MPAG可稳定24小时（变化-1.8%至-11.5%）。在-80°C储存两年后，MPA、MPAG的浓度及95%置信区间仍在初始值的±15%范围内。
所提出的检测方法适用于临床研究。血液样本在室温下可稳定7小时，血浆在-80°C储存2年。
