Simultaneous quantification of mycophenolic acid and its glucuronide metabolites in human plasma by an UPLC–MS/MS assay

Abstract

The aim of this study was to develop a reliable UPLC-MS/MS assay for accurate quantification of mycophenolic acid (MPA) and its glucuronide conjugates in human plasma. Plasma proteins were precipitated with acetonitrile and the chromatographic separation was achieved on a C18 column with a gradient elution. The detection was performed by a triple quadrupole mass spectrometer in the positive electrospray ionization and multiple reaction monitoring mode. Linearity of the assay was demonstrated over the range of 20-10,000 ng/mL for MPA and MPA glucuronide (MPAG), and 2-1000 ng/mL for acyl MPA glucuronide in human plasma. The assay was precise and accurate with coefficient of variation and bias <15%. MPA and MPAG were stable at 25 °C up to 1 day in both heparin- and EDTA-treated blood. In heparin- and EDTA-plasma, MPA and MPAG were stable for at least 1 week at 25 and 4 °C, and 1 month at -20 °C. However, 99% acyl MPA glucuronide degraded in both heparin- and EDTA-blood as well as plasma when stored at room temperature for 1 day. All the analytes remained stable for at least 3 months in acidified EDTA-plasma at -80 °C. The assay was successfully applied on patients post hematopoietic stem cell transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

“…The equations for MPA, MPAG and AcMPAG were y = 6.0395 x + 0.1258, y = 3.0525 x + 0.0559 and y = 4.0896 x + 0.0127, respectively. To our knowledge, in comparison with other published methods that have simultaneously quantified MPA, MPAG, and AcMPAG (Benoit‐Biancamano et al, ; Delavenne et al, ; Figurski et al, ; Iboshi et al, ; Kawanishi et al, ; Klepacki et al, ; Zegarska et al, ; Zhang et al, ), no other assays have obtained better sensitivity for MPA and MPAG. On the other hand, only Figurski et al () reported a lower LLOQ (0.0002 μg/mL) for free AcMPAG concentration in plasma compared with 0.00467 μg/mL obtained in our assay.…”

“…We have developed a sensitive LC–MS/MS assay for the simultaneous quantification of MPA, MPAG and AcMPAG in HepaRG cell culture medium, using MPA‐d 3 and MPAG‐d 3 as internal standards. LC–MS/MS assays for MPA and/or metabolites have been described in human plasma (Delavenne, Juthier, Pons, Mariat, & Basset, ; Figurski, Korecka, Fields, Waligorska, & Shaw, ; Kawanishi et al, ; Zegarska et al, ; Zhang, Chow, & Renbarger, ), urine (Benoit‐Biancamano et al, ; Klepacki et al, ), dried blood spots (Iboshi et al, ) and human liver microsomes (Mohamed, Harvey, & Frye, ). In contrast to other biological fluids (i.e.…”

“…In contrast to the frequently used ESI for the quantification of MPA and metabolites (Benoit‐Biancamano et al, ; Delavenne et al, ; Figurski et al, ; Iboshi et al, ; Kawanishi et al, ; Klepacki et al, ; Zegarska et al, ; Zhang et al, ), a DUIS approach combining both ESI and APCI ionizations was utilized in our assay. The ESI probe and the APCI corona needle are positioned in a 90° angle in the ion source and have distinctive ionization zones.…”

“…Similar to other studies (Syed & Srinivas, ), the ammonium adduct precursor ions were also identified but ultimately not utilized in our method owing to their relatively weak responses in our experimental conditions. The glucuronide moieties can be subjected to in‐source fragmentation as a result of high temperature or collision energy (Zhang et al, ); therefore, ion source temperature and voltage were further optimized manually to minimize this effect. The final conditions and mass transitions are presented as follows: the Q 1 pre‐bias, collision energy, and Q 3 pre‐bias were −12, −23, −14 V (MPA); −24, −18, −21 V (MPAG); −26, −20, −11 V (AcMPAG); −11, −21, −13 V (MPA‐d 3 ); and − 36, −21, −26 V (MPAG‐d 3 ), respectively.…”

Determination of mycophenolic acid in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its pharmacokinetic application in kidney transplant patients

“…With regard to the method presented in this work, the total run time of the analysis has a standard value of 3 min in comparison with total run time for other UPLC–MS/MS methods, which varies from 2 (Delavenne et al, ; Klepacki et al, ; Kuhn, Gotting, & Kleesiek, ; Upadhyay et al, ) to 5 min (Zhang, Chow, & Renbarger, ).…”