Determination of dolutegravir's unbound fraction in human plasma using validated equilibrium dialysis and LC-MS/MS methods

This study evaluated (i) dolutegravir's fu through equilibrium dialysis (ED), (ii) the pre-analytical parameters that influence fu, and (iii) fu's inter-individual variability in HIV patients. Validation of the LC-MS/MS method followed FDA guidelines. The results, based on coefficients of variation (results from nominal concentrations <15%), allowed accurate measurement of unbound and total dolutegravir concentrations. Equilibrium during ED was obtained in 4h. Sparse non-specific binding (9%) was observed, allowing results interpretation without interference. Steps before analysis (e.g., conservation at +4°C, freeze/thaw cycles) did not influence fu, allowing easy integration of fu analysis within laboratory routines. Anticoagulants from samples (citrated versus heparinized; p <0.001) and hemolysis (p =0.007) influenced fu and could lead to misinterpretation. Developed was then performed to the HIV-patients' plasma (n =54). Results, expressed as median InterQuartile Range [25%;75%] were 0.45% IQR [0.38; 0.55] for fu, 9.26μg/L IQR [4.62; 15.14] for unbound, and 2035μg/L IQR [878.5; 2640] for total concentration. The high inter-individual variability observed in the unbound form from HIV patients was a first step towards integrating dolutegravir TDM. Graphical abstract
Source: Clinica Chimica Acta - Category: Laboratory Medicine Source Type: research