The antiretroviral drug tenofovir is in formulation given as the prodrug tenofovir disoproxil fumarate (TDF) in combination with the nucleoside reverse-transcriptase inhibitor emtricitabine. Here we show the impurity profiling of an active pharmaceutical ingredient with the Thermo Scientfic Vanquish Flex UHPLC system. The simultaneous separation of early-eluting polar compounds and later-eluting nonpolar compounds is achieved by applying a ternary gradient with the quaternary Vanquish F pump using a Thermo Scientific Accucore aQ column.