Esquire lc ms system

All reagents and solvents for the synthesis and separation were purchased from commercial sources and were used as received. Reactions of 5,7-diene structures were all carried out in the dark by wrapping the flasks with aluminum foil. Moisture- or air-sensitive reactions were performed under an argon atmosphere. All reactions were routinely monitored by TLC on silica gel using ethyl acetate and hexane as mobile phases and visualized by 5% phosphomolybdic acid in ethanol or UV light. Mass spectra of all compounds were obtained by a Bruker ESQUIRE-LC/MS system equipped with an ESI source. The purities of final D3 compounds, as analyzed by an Agilent 1100 HPLC system (Santa Clara, CA), were above 98%. Ethyl acetate was used for extraction of reaction mixtures and then dried over anhydrous Na₂SO₄, filtered, and removed by rotary evaporator under reduced pressure. For comparison of retention times of enzymatically^{6 (link),23 (link)} and chemically gererated 20S,24(OH)₂D3 isomers, HPLC was carried out with an acetonitrile in water gradient comprising 45–100% acetonitrile for 25 min, then 100% acetonitrile for 15 min or a methanol in water gradient comprising 64–100% methanol for 20 min, then 100% methanol for 25 min, using a flow rate of 0.5 mL/min and a C18 column (Grace Alltima, 25 cm × 4.6 mm, particle size 5 µm).