Development, validation and clinical application of a method for the simultaneous quantification of lamivudine, emtricitabine and tenofovir in dried blood and dried breast milk spots using LC–MS/MS

Objectives To present the validation and clinical application of a LC–MS/MS method for the quantification of lamivudine (3TC), emtricitabine (FTC) and tenofovir (TFV) in dried blood spots (DBS) and dried breast milk spots (DBMS). Methods DBS and DBMS were prepared from 50 and 30 μL of drug-spiked whole blood and human breast milk, respectively. Following extraction with acetonitrile and water, chromatographic separation utilised a Synergi polar column with a gradient mobile phase program consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection and quantification was performed using a TSQ Quantum Ultra triple quadrupole mass spectrometer. The analytical method was used to evaluate NRTI drug levels in HIV-positive nursing mothers-infant pairs. Results The assay was validated over the concentration range of 16.6–5000 ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2–1250 ng/mL. Intra and inter-day precision (%CV) ranged from 3.5–8.7 and accuracy was within 15% for all analytes in both matrices. The mean recovery in DBS was >61% and in DBMS >43% for all three analytes. Matrix effect was insignificant. Median AUC 0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165–6057) and 6050 (5217–6417) ng h/mL for 3TC, 3312 (2259–4312) and 4853 (4124–6691) ng h/mL for FTC and 1559 (930–1915) and 56 (45–80) ng h/mL for TFV. 3TC and FTC were quantifiable (>16.6 ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants. Conclusions DBS and DBMS sampling for bioanalysis of 3TC, FTC and TFV is straightforward, robust, accurate and precise, and ideal for use in low-resource settings.