B-156 Extraction strategy for therapeutic drug monitoring of sirolimus and tacrolimus using volumetric absorptive microsampling
Authors: Guarino B , Pickett S , Garrett R , Kellogg M , Peake R W , Ptolemy A S 
Published: October 2024

Abstract

Background
Following organ transplantation, patients are placed on immunosuppressant drugs (ISDs) to minimize the risk of rejection. Sirolimus and tacrolimus are two of the most widely used ISDs, however, due to their narrow therapeutic indices, patients require frequent therapeutic drug monitoring (TDM). Recurrent venipuncture can become uncomfortable and disruptive, especially in the pediatric population. Therefore, we describe an extraction method using dried whole blood from volumetric absorptive microsampling (VAMS) devices for the detection of sirolimus and tacrolimus by turbulent flow liquid chromatography tandem mass spectrometry (TFC-MS/MS). The use of VAMS in the clinical laboratory will provide ISD patients with a convenient alternative to traditional blood draws.

Methods
MassTrak immunosuppressant calibrators based on a whole blood matrix (Waters) were applied to 30 μL VAMS Mitra devices (Neoteryx) and dried overnight. To extract the whole blood off of VAMS tip, we tested various methanol:water solvent ratios, of which 30 μL was added to the device. Tips were extracted using ultrasonication (30 min), vortexing (5 min), and centrifugation. Once extracted from the tip, 25 mM zinc sulfate was added to whole blood extract at a 1:1 (v/v) ratio. An acetonitrile and methanol extraction reagent was then added to the whole blood/zinc sulfate solution at a 2:1 (v/v) ratio. After brief vortexing, samples were centrifuged at 16,060 × g (10 min) and tested using an established TFC-MS/MS method which deploys a TLX-2 system for sample preparation coupled to a Q-Trap tandem mass spectrometer (AB Sciex 5500).

Results
A 80:20 (v/v) water:methanol extraction solvent yielded the greatest extraction efficacy for both sirolimus and tacrolimus from the VAMS tips compared to other tested ratios. We established a 5-point calibration curve to correlate the measured values of ISDs with known MassTrak calibration standard concentrations. The resulting average calibration curve from 3 independent experiments was highly linear for both sirolimus (R2= 0.992; slope= 1.1; intercept= 0.94) and tacrolimus (R2= 0.996; slope= 1.1; intercept = 0.8819). Our VAMS whole blood extraction strategy was correlated to the established neat whole blood protocol, which utilized all of the sample preparation steps outlined above except the initial extraction from the VAMS tip. The average extraction efficacy was 89.1% for sirolimus with an average coefficient of variation (CV%) of 12.1% across 3 independent experiments. For tacrolimus, we demonstrated an average extraction efficacy of 100.1% with an average CV% of 8.4%, again across 3 independent experiments.

Conclusions
We demonstrate the feasibility of using a VAMS approach to TDM of sirolimus and tacrolimus. Our results demonstrate favorable recoveries for both of the tested drugs, evidenced by high recoveries of the external calibration standards. Additionally, our method exhibited high extraction efficacy and low variability. Further validation studies, including the utilization of clinical samples is necessary to confirm our preliminary findings.