[Determination of 25-hydroxyvitamin D in serum by pre-column derivatization-stable isotope labeling-ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry].

Abstract

Objective: To establish a rapid and accurate method for the determination of 25-hydroxyvitamin D_3(25(OH)D_3) and 25-hydroxyvitamin D_2(25(OH)D_2)in serum by pre-column derivatization with stable isotope labeling and ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry, which could be used to diagnose vitamin deficiency and to assess the nutritional status of vitamin D in the population.

Methods: The serum samples with isotopic internal standard were extracted by mixed extraction solvent(ethyl acetate∶hexane = 2∶1, V/V), centrifuged and dried by the nitrogen blowing, and derivatized with 4-phenyl-1, 2, 4-triazoline-3, 5-dione(PTAD). The reaction products were separated on a BEH C_(18) column(2.1 mm×50 mm, 1.7 μm), and eluted with a 0.1% formic acid/water solution-acetonitrile gradient. The mass spectrometry was performed in positive electrospray ionization(ESI~+) and parallel reaction monitoring(PRM) for the detection of the targets, and quantified by isotope internal standard.

Results: The limits of detection both for 25(OH)D_3 and 25(OH)D_2 were 0.01 ng/mL and the limits of quantification were 0.03 ng/mL. The concentration series of 25(OH)D_2 ranged from 0.5 to 40.0 ng/mL and the concentration series of 25(OH)D_3 ranged from 2.5 to 200.0 ng/mL. The recoveries(n=5) were 95.7%-101.3% for 25(OH)D_3, and 98.7%-108.6% for 25(OH)D_2, with the relative standard deviations(RSDs) of 0.5%-4.9% and 2.2%-3.4%, respectively. The accuracy of the determination of 25(OH)D_3 and 25(OH)D_2 in Level 2 serum of the 25(OH)D standard reference material NIST SRM 972a was 104.8% and 94.9%, respectively. The M(P25, P75)serum levels of 25(OH)D_3 and 25(OH)D_2 for 116 pregnant women at the first trimester from Beijing were 25.7(20.8, 32.6) ng/mL and 0.8(0.4, 1.1) ng/mL, respectively.

Conclusion: This method is highly sensitive, qualitatively accurate and suitable for evaluation and monitoring the nutritional status of vitamin D in the population.