Free mycophenolic acid determination in human plasma ultrafiltrate by a validated liquid chromatography–tandem mass spectrometry method

Abstract

The aim of this study was to develop and validate fully the liquid chromatography-tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA-d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB-C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA-d3, respectively. The method was selective, with no carry-over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1-500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63-110.46%, imprecision: 6.23-7.76%), as well as interday assay (accuracy: 98.81-110.63%; imprecision: 5.36-10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography-tandem mass spectrometry method for free MPA monitoring using MPA-d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.

Pharmacokinetics of free and total mycophenolic acid in adult lupus nephritis patients—implications for therapeutic drug monitoring

“…2). These results suggest that monitoring only total MPA concentrations might seem sufficient to conduct TDM, especially taking into account that free MPA concentrations should be determined by more demanding LC-MS/MS [16], which is not as easily available in every laboratory as HPLC-UV or even immunoassays [26]. However, our results have shown that there is a significant negative relationship between serum albumin concentration and MPA free fraction, similarly to the findings reported in in vitro [10], as well as in transplantation [9] studies.…”

“…Free MPA concentrations in plasma ultrafiltrate were determined after prior ultrafiltration with Centrifree Micropartition System® (Merck Millipore, Co. Cork, Ireland) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed, validated, and described in detail recently [16].…”

Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)

“…HPLC/MS/MS is probably the most commonly used method for MPA and MPAG quantification in the United States. Total MPA concentrations are commonly measured, but an assay of free MPA levels may be desirable in patients with renal failure and hypoalbuminemia (Butch, 2007;Luszczynska, Pawinski, Kunicki, Sikorska, & Marszalek, 2017;Meier-Kriesche et al, 2000). However, measurement of free MPA may be challenging, as only ß2% of the drug is in the free form.…”

Validated LC–MS/MS method for quantitation of total and free mycophenolic acid concentration and its application to a pharmacokinetic study in pediatric renal transplant recipients

“…Several methods for free drug quantification have been described previously, including the enzyme‐multiplied immunoassay technique (Rebollo, Calvo, Martin‐Suarez, & Dominguez‐Gil, 2011), high‐performance liquid chromatography coupled to ultraviolet detection (HPLC–UV) (Aresta, Palmisano, Zambonin, Schena, & Grandaliano, 2004; Smits et al, 2014; Zeng, Nath, Shaw, Earl, & McLachlan, 2009) and tandem mass spectrometry (LC–MS/MS) (Atcheson et al, 2004; Benoit‐Biancamano et al, 2007; Bittersohl et al, 2017; Figurski, Korecka, Fields, Waligorska, & Shaw, 2009; Heinig, Bucheli, Hartenbach, & Gajate‐Perez, 2010; Kawanishi et al, 2015; Laverdiere, Caron, Couture, Levesque, & Guillemette, 2012; Luszczynska, Pawinski, Kunicki, Sikorska, & Marszalek, 2017; Patel et al, 2004; Shen, Jiao, Yu, Zhang, & Zhong, 2005; Streit, Shipkova, Armstrong, & Oellerich, 2004; Willis, Taylor, Salm, Tett, & Pillans, 2000). Immunoassay is a commonly used assay in clinical therapeutic drug monitoring, but it is restricted to the use of expensive antibodies and single drug detection and shows the risk of 33.48% overestimation of MPA levels owing to cross‐reactivity with metabolites of MPA under the conditions of the assay (Kunicki, Pawinski, Boczek, Was, & Bodnar‐Broniarczyk, 2015).…”

“…In addition, gradient LC methods have been used in other reports. The complex composition of the mobile phase and time‐consuming preconditioning increased the complexity of detection and brought difficulties to clinical routine monitoring (Figurski et al, 2009; Kawanishi et al, 2015; Luszczynska et al, 2017; Streit et al, 2004).…”