Validation and Clinical Application of a Liquid Chromatography-Ultraviolet Detection Method to Quantify Dolutegravir in Dried Blood Spots

Abstract

Background: Dolutegravir is currently the preferred component of first-line antiretroviral therapy. To facilitate clinical pharmacology studies in key populations, quantitative analytical methods compatible with microsampling and adaptable to resource-limited settings are desirable. The authors developed and validated a liquid chromatography-ultraviolet detection method to quantify dolutegravir in dried blood spots (DBS).

Methods: Calibration standards and quality control samples were prepared by spotting 50 μL of dolutegravir-spiked whole blood on each circle of DBS cards. Three spots (two 6-mm punches/spot) were extracted with methanol. Chromatographic separation was achieved with gradient elution of acetonitrile/potassium phosphate monobasic buffer (pH 5) on a reverse-phase C18 column (flow rate, 1 mL/min) using pioglitazone as the internal standard. UV detection was performed at 260 nm. In the clinical pharmacokinetic study, DBS from finger prick was collected from participants (n = 10) at 8 time points over 12 hours postdosing, with paired plasma at 1 and 12 hours. The method was used to quantify dolutegravir, estimating pharmacokinetic parameters. Agreement between DBS and plasma concentrations was evaluated using linearity and Bland-Altman plots.

Results: The method was validated over the concentration range of 0.4-10 mcg/mL, accuracy was 102.4%-114.8%, and precision was 3.4%-14.7%. The mean recovery was 42.3% (%CV: 8.3). The mean (±SD) dolutegravir concentration in DBS was 37.5% (±3.8%) lower than that in the plasma. DBS-derived and measured plasma concentrations showed strong correlation with linearity (R2 = 0.9804) and Bland-Altman plots. Means (%CV) of area under curve, Cmax, and C24 from the DBS-derived plasma concentration were 37.8 (23.2) mcg·h/mL, 2.7 (24.7) mcg/mL, and 1.34 (31.6) mcg/mL, respectively.

Conclusions: The application of this simple, accurate, and precise method will expand opportunities for clinical assessment of dolutegravir in resource-limited settings.