Simultaneous determination of mycophenolate and its metabolite mycophenolate-7-o-glucuronide with an isocratic HPLC-UV-based method in human plasma and stability evaluation

Abstract

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords:

• Drug monitoring
• drug stability
• high pressure liquid chromatography
• immunosuppresants
• immunosuppressive agents
• mycophenolic acid
• validation studies

Acknowledgements

We are grateful to Susanne Nieser and Beate Unger from the Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Germany for sample measurement with the reference method for method comparison.

Disclosure statement

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.