Fig. 6.

Selection of solvents for liquid–liquid extraction. Human serum was spiked with deuterated 1α,25(OH)₂D₃ and prepared as described in the “Experimental” section. Extraction was performed in two steps (st) with methyl tert-butyl ether (MTBE), dichloromethane (DCM) and ethyl acetate (EtAc). No second extraction was performed for DCM, which formed an emulsion that was difficult to separate. Data are normalized to MTBE recoveries in the first step. No d₆ 25(OH)D₃ was available at the time of the experiment, and so native 25(OH)D₃ was used to measure the relative recovery. Four samples were analyzed independently in each sample group (black bars, d₆ 1α,25(OH)₂D₃; gray bars, d₆ 25(OH)D₃). Error bars represent standard deviations