Handling of highly coeluted chromatographic peaks by multivariate curve resolution for a complex bioanalytical problem: Quantitation of prednisolone, methylprednisolone and mycophenolic acid in human plasma

Corticosteroids are prescribed as a critical medication in immunosuppressive regimens after solid organ transplantation. Due to numerous adverse effects and long term complications of corticosteroids, different multi-drug regimes have been advised by physicians to limit exposure[1]. One of the critical co-medications in this field is mycophenolic acid (MPA) which is the primary biologically active form of mycophenolate mofetil. In multi-drug protocols, therapeutic drug monitoring (TDM) plays an important role in the optimal use of immunosuppressants in patients carrying an organ transplant. So, developing a fast, easy and reliable analytical methodology for simultaneous quantitation of medications is highly valuable[2]. In the present study, prednisolone (Predl), methylprednisolone (Mpredl) and MPA are quantified in plasma samples by a fast high performance liquid chromatography with a diode array detector (HPLC-DAD) followed by multivariate curve resolution-alternating least square (MCR/ALS) modeling. The LC method optimized at isocratic reverse phase over a symmetric C18 column using an acetonitrile–phosphate buffer mobile phase (pH=3.5). The most challenges in the present study were: the highly coelution of analytes of interest with the matrix interferences and highly spectral similarity of selected corticosteroids and thus causing rank deficiency. To circumvent these drawbacks, the whole chromatographic run was divided into two sections. Then, the row-wise and column-wise augmentation strategies were implemented for the first and second regions, respectively. Highly acceptable resolution and quantification results were obtained. The calibration concentration ranged from 19 ng/mL to 1.6 μg/mL for Predl, 25 ng/mL to 2.4 μg/mL for Mpredl and 5 μg/mL to 14 μg /mL for MPA. The average recoveries for all components were acquired between 90 to 110 percent. Accurate and precise results, elimination of expensive and time consuming sample pretreatment steps and a very short chromatographic runtime, are among the advantages of the presented method.