Volumetric Absorptive Microsampling (VAMS) for assayingimmunosuppressants from venous whole blood by LC–MS/MS using anovel atmospheric pressure ionization probe (UniSprayTM)

Abstract

Therapeutic drug monitoring (TDM) of immunosuppressants (IMS) is crucial to prevent rejection or toxicity after solid organ transplantation. Microsampling techniques (sampling <50 μL of blood) can be a good alternative to conventional venous sampling for TDM, due to their numerous advantages, including its easy and low-invasive sampling, enabling self-collection, and cost-saving shipment and storage. Furthermore, volumetric absorptive microsampling (VAMS) enables the collection of precise and accurate blood volumes, overcoming the hematocrit (Hct) effect related to dried blood spots, while offering the same benefits. In this work, an LC-MS/MS method for the determination of the 5 most common IMS (mycophenolic acid -MPA-, tacrolimus -TAC-, sirolimus -SIR-, everolimus -EVE- and cyclosporin A -CsA-) in venous blood collected with Mitra™ VAMS devices was developed and validated, employing a novel LC-MS/MS interface, Unispray™. The method was fully validated including linearity, limits of detection (LOD) and quantification (LLOQ), accuracy, precision, selectivity, carry-over, matrix effect, recovery, impact of Hct on recovery and autosampler and short-/long-term stability, satisfying acceptance criteria in all cases. LLOQs were 0.5 ng/mL for TAC, SIR and EVE, 20 ng/mL for CsA and 75 ng/mL for MPA. No impact of the Hct (range: 0.2 to 0.62 L/L) on recovery was found for any analyte. All compounds were stable in VAMS for at least 8 months at -20 °C. In addition, as part of the VAMS analytical method validation, we performed for the first time a broad statistical study to compare liquid venous blood concentrations from patients under TAC (n = 53) and MPA (n = 20) treatment to those observed when the same specimens were absorbed into VAMS. Our results showed that venous blood VAMS concentrations were correlated to those found in the original liquid venous blood, proving that the VAMS material itself will not bias blood drug concentrations. Therefore, the present method could be applied to evaluate possible correlations between venous blood and capillary blood collected with VAMS.