Determination and validation of mycophenolic acid by a UPLC-MS/MS method: Applications to pharmacokinetics and tongue tissue distribution studies in rats

Highlights

• A sensitive, specific and reproducible UPLC-MS/MS method for mycophenolic acid.

• Method for rat plasma and tongue tissue samples.

• Method validation according to FDA’s Guidelines.

• Application to pharmacokinetic studies in rats.

• Tongue tissue distribution and blood diffusion studies with transmucosal patch.

Abstract

Mycophenolic acid (MPA) has being used clinically for organ rejection prophylaxis. Recent studies have revealed that MPA can also act as a chemo-sensitizing agent when used in combination with various chemotherapeutic agents in a cancer type-specific manner, including with oxaliplatin on oral squamous cell carcinoma (OSCC) cells. To prepare for the analysis of a novel drug delivery route for MPA absorption via oral mucosa as a potential therapeutic product, it is essential to develop and validate a highly sensitive analytical method for the quantification of MPA in biological samples for pharmacokinetic and tissue distribution studies. Herein, we report a sensitive, specific and reproducible UPLC-MS/MS method to do so. Blank rat plasma or tongue tissue homogenates coupled with griseofulvin, as internal standard, was used for generating standard curves ranging from 0.5 to 1000 ng/mL (r > 0.9990) for both plasma and tongue tissue homogenates. The chromatographic separation was achieved by a reverse phase ACE Excel 2 Super C₁₈ column with a flow rate of 0.4 mL/min under gradient elution. Mass detection was performed under positive ionization electrospray. Inter- and intra-day accuracy and precision of the assay were ≤15% in both plasma and tongue tissue homogenates. The matrix effect was non-significant and extraction recovery rates were within 87.99% and 109.69% in plasma and tongue homogenates, respectively. The validity of this assay has been confirmed by measuring MPA in rat plasma for pharmacokinetics following intravenous administration of 0.5 mg/kg of mycophenolate sodium, as well as monitoring MPA in rat tongues for tissue distribution and detecting MPA that diffused into systemic circulation following a 4-h transmucosal delivery of 357 μg/cm² of mycophenolate sodium.

Introduction

Mycophenolic acid (MPA) is used to prevent organ rejection after transplant in conjunction with other immunosuppressive agents by oral administration in clinic [1]. MPA acts by inhibiting inosine-5-monophosphate dehydrogenase activities, which is critical in the de novo synthesis of purine nucleotides [2]. MPA has also been shown to activate p53 gene, inhibit cyclin D3 and p27, and block Ras-MAPK and mTOR pathways, leading to the inhibition of tumor growth. Interestingly, when combined with oxaliplatin, MPA showed a strong synergistic inhibition effect on oral squamous cell carcinoma (OSCC-25) cells [3]. This finding suggests a potential use of MPA for treating oral precancerous lesions and the possibility of a targeted drug delivery and absorption of MPA via oral mucosa. Thus, there is a need to develop a sensitive and specific analytical method for the quantification of MPA in plasma and tongue tissues for preclinical evaluation of the drug delivery system.

Determination of MPA concentrations in different human biological samples has been reported using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Upadhyay et al. [4] and Kawanishi et al. [5] developed LC-MS/MS methods in human plasma with linear range from 15 to 15,000 ng/mL and 5–200 ng/mL, respectively. Dom et al. [6] reported MPA quantification method with 0.6 ng/mL lower limit of quantification (LLOQ) in human kidney and rat kidney. Only a few high performance liquid chromatography (HPLC) methods are available in literature for the analysis of MPA in rat biological samples. Jia et al. [7] developed a HPLC-UV method for quantifying MPA in rat plasma, stomach, duodenum, jejunum, ileum, colon and rectum with LLOQ at 500 ng/mL. Gao et al. [8] reported a HPLC-UV method measuring MPA in rat plasma and bile with LLOQ at 1000 ng/mL and 250 ng/mL, respectively. Ishizaki et al. [9] reported a better assay sensitivity of MPA in rat plasma, bile and urine with 90 ng/mL LLOQ. Liu et al. and Jiao et al. [10], [11] was able to accomplish LLOQ of MPA in rat plasma and urine at 2 ng/mL and 100 ng/mL, respectively. However, all of the reported MPA assays in rat biological samples were analyzed by ultra-violet (UV) detection with relatively low sensitivity, at least 20 μL injection volume and more than 12 min running time.

In this study, we report a sensitive, specific and reproducible Ultra Performance LC-MS/MS (UPLC–MS/MS) method for the quantitation of MPA concentration in the rat plasma and tongue tissue samples. The method has a significantly better LLOQ of 0.5 ng/mL in both plasma and tongue homogenates with 5 μL sample injection volume and 4.6 min running time. Furthermore, this method has been proven to be suitable to characterize MPA pharmacokinetics and tissue distribution following the drug administration.