Abstract
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). DBS and DBMS were prepared from 50 and 30μL 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. The assay was validated over the concentration range of 16.6-5000ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2-1250ng/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 AUC0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165-6057) and 6050 (5217-6417)ngh/mL for 3TC, 3312 (2259-4312) and 4853 (4124-6691)ngh/mL for FTC and 1559 (930-1915) and 56 (45-80)ngh/mL for TFV. 3TC and FTC were quantifiable (>16.6ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants. 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.
Highlights
It is internationally recommended that HIV-positive women receive triple antiretroviral therapy (ART) throughout pregnancy until the end of breastfeeding or for life irrespective of clinical disease stage or CD4 count [1]
The LC–MS system consisted of a Synergi polar-RP column (80A, 150 *2.0 mm and 4 μ; Phenomenex, Macclesfield, UK) with a 2 μm C18 Quest column-saver (Thermo Electron Corporation, Hemel Hempstead, Hertfordshire, UK) on a HPLC connected to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Electron Corporation, Hemel Hempstead, Hertfordshire, UK) equipped with a heated electrospray ionisation source (H-ESI)
International guidelines recommend that HIV positive women who are pregnant and breastfeeding receive triple antiretroviral therapy with EFV, TFV and 3TC or FTC [1]
Summary
It is internationally recommended that HIV-positive women receive triple antiretroviral therapy (ART) throughout pregnancy until the end of breastfeeding or for life irrespective of clinical disease stage or CD4 count [1]. It is important to understand the breast milk transfer of these drugs since low infant levels predispose to HIV-drug resistance should HIV transmission occur [3,4], and there is conflicting data regarding the effects of tenofovir (TFV) on developing bone [5]. The pharmacokinetic profiles in paired maternal and infant plasma (calculated from dried blood spots [DBS]) and breast milk (BM) have been reported for EFV [6] and 3TC [7], but only three studies have sought to measure TFV [8,9,10] and a single study FTC in the BM of HIV-positive mothers [8], and these did not present intensive pharmacokinetic profiles and paired mother and infant data. We report the DBS and DBMS LC–MS/MS method for accurate simultaneous quantitation of TFV, 3TC and FTC, with application of the method in breastfeeding mother-infant pairs
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