Quantification of immunosuppressants from one 3.2 mm dried blood spot by a novel cold-induced phase separation based LC-MS/MS method

Abstract

Dried blood spots (DBS) have been regarded as a promising alternative for therapeutic drug monitoring (TDM) of immunosuppressants (ISDs) for over fifteen years. Nonetheless, there are still three main issues impeding its preference: (i) the requirement of relatively large disc; (ii) the controversial and intricate desorption approaches; (iii) the lack of feasible extraction strategies. For improvement, this work described a new LC-MS/MS method realizing quantification of four ISDs from one piece of 3.2 mm DBS. During sample pretreatment, a modified approach (infiltrating the DBS in pure water before adding acetonitrile and zinc sulfate as protein-precipitators) was developed to completely dissociate the targets from filter paper. Afterward, effective enrichment and purification of the targets were achieved by using cold-induced phase separation technique. Benefiting from these novelties, the method exhibited satisfying throughput (15 min for sample preparation), applicability (consuming only one 3.2 mm disc), reliability (82.3–107.8% for accuracy and <14.3% for precision) and sensitivity (lower limit of quantification of 0.5, 7.6, 0.7 and 0.8 ng mL−1 for tacrolimus, cyclosporine A, everolimus and sirolimus, respectively). Without hematocrit correction, the method showed favorable interchangeability to the certified whole blood method through analyzing 120 paired clinical samples. By taking ±20% of the mean as the limit of acceptance for cross validation, over 90% of the detection met the criterion. It can be expected the developed method is able to further promote the popularization of DBS-based TDM for ISDs in practice.