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.