Validation of a simple liquid chromatography coupled to tandem mass spectrometry method for the simultaneous determination of tacrolimus, sirolimus, everolimus and cyclosporin A in dried matrix on paper discs

Abstract

Introduction: Due to its high specificity and sensitivity, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard method for immunosuppressant quantification in therapeutic drug monitoring. In this context, dried blood spots (DBS) have become a promising strategy as a sample collection procedure. Although the advantages of DBS over venipuncture are well known, this approach has limitations that strongly influence the acceptance of analytical results. Among them, the most important is hematocrit (Ht). The easiest way of overcoming this problem is by analyzing complete spots. In this strategy, called dried matrix on paper discs (DMPD), blood is volumetrically applied on pre-punched discs. Objectives: To validate an LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. Methods: The procedure was validated according to international guidelines using a commercial kit. The following performance parameters were evaluated: selectivity, carryover, linearity, accuracy, precision, lower limit of quantitation, relative recovery, commutability and stability. In addition, a method comparison study was performed to evaluate the clinical influence of Ht on the results. Results: All performance parameters were within acceptance criteria and, hence, it was determined that the validated method is fit for the intended purpose. Likewise, calculated bias values on medical decision levels showed that there was no clinical influence of Ht on the results. Conclusion: Unlike other similar methodologies that have been published, here, a simple method has been fully validated. This is the first LC-MS/MS methodology adapting a commercial kit to use DMPD as a sampling strategy.

Keywords

• Dried matrix on paper discs (DMPD)
• Immunosuppressants
• Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)
• Hematocrit
• Method comparison