Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit

Background: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has be en found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-re activity of AcMPAG in the Emit assay for MPA was investigated. Methods: The procedure used simple sample preparation, separation with a Zo rbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. Results: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0 .04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 9 9-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-d ay imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and less than or equal to 14% for AcMPAG (0.1-5 mg/L). The between-d ay imprecision was <6.2%, <4.5%, and less than or equal to 14% for MPA, 7-O -MPAG, and AcMPAG, respectively. When isolated from microsomes, purified Ac MPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentratio ns determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at - 20 OC. Conclusions: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects a ssociated with mycophenolate mofetil therapy. (C) 2000 American Association for Clinical Chemistry.