Aliquots of 3.0 ml fasting venous blood were collected in an SST-II vacuum tube (BD, USA) in the early morning and centrifuged at about 1500 × g for 10 min to separate serum for determining levels of 25(OH)D2, 25(OH)D3, and C3-epi-25(OH)D3. If not immediately determined, store the serum sample at 2 ~ 8℃ for less than seven days24 (link).
The lipid-soluble vitamin assay kits (Cat.No.:1001040, FINDBiotech, Chengdu FIND Medical laboratory Co., LTD, CHN) were used for sample pretreatment to obtain a supernatant for chromatographic analysis, as well as to provid the standards. On the day of measurement, 200 μl serum sample was mixed with 10 μl internal standard (a mixture solution of 25[OH]D3-d6 and C3-epi-25(OH)D3-[2H3]), vortexed with 1.0 ml tertbutyl methyl ether release agent (CNW, Germany) for 5 min, centrifuged at 12,000 × g for 5 min. The supernatant (800 μl) was dried using an MD200-1A Nitrogen Evaporator (Allsheng, China), and the resulting substance was redissolved in 100 μl methanol solution containing 0.1% formic acid. After vortex mixing for 2 min and centrifugation at 12,000 × g for 5 min, a sample of the supernatant (50 μl) was collected to measure levels of 25(OH)D metabolites.
The lipid-soluble vitamin assay kits (Cat.No.:1001040, FINDBiotech, Chengdu FIND Medical laboratory Co., LTD, CHN) were used for sample pretreatment to obtain a supernatant for chromatographic analysis, as well as to provid the standards. On the day of measurement, 200 μl serum sample was mixed with 10 μl internal standard (a mixture solution of 25[OH]D3-d6 and C3-epi-25(OH)D3-[2H3]), vortexed with 1.0 ml tertbutyl methyl ether release agent (CNW, Germany) for 5 min, centrifuged at 12,000 × g for 5 min. The supernatant (800 μl) was dried using an MD200-1A Nitrogen Evaporator (Allsheng, China), and the resulting substance was redissolved in 100 μl methanol solution containing 0.1% formic acid. After vortex mixing for 2 min and centrifugation at 12,000 × g for 5 min, a sample of the supernatant (50 μl) was collected to measure levels of 25(OH)D metabolites.