. Author manuscript; available in PMC: 2018 Aug 15.

Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jun 9;1060:158–165. doi: 10.1016/j.jchromb.2017.06.017

Table 5.

Validation of the quantification of 25-OHD₃-3-sulfate and 25-OHD₃-3-glucuronide in human plasma. Plasma samples from 3 donors were spiked with a standard mixture containing 25-OHD₃-3-sulfate and 25-OHD₃-3-glucuronide. The native and spiked plasma samples were prepared and analyzed at the same time.

	Baseline (nM) ^a	Spiked^b

		Calculated (nM) ^c	Measured (nM)	Accuracy %
*25-OHD₃-3-glucuronide*
Donor 1	2.78 ± 0.12	10.8	11.6 ± 0.3	108
Donor 2	4.47 ± 0.17	12.5	13.2 ± 0.2	106
Donor 3	1.86 ± 0.07	9.86	10.6 ± 0.2	108
*25-OHD₃-3-sulfate*
Donor 1	70.3 ± 2.1	150	164 ± 5	109
Donor 2	77.1 ± 0.7	157	158 ± 9	101
Donor 3	53.5 ± 2.0	133	147 ± 3	111

Baselines represented the original metabolite concentrations in human plasma before spiking with standards.

Each plasma sample (200 µL) was spiked with metabolite standards which theoretically increased concentrations of 25-OHD₃-3-sulfate and 25-OHD₃-3-glucuronide by 8 and 80 nM, respectively.

Calculated concentration = baseline concentration + spiked concentration.