Determination of vitamins K-1, MK-4, and MK-7 in human serum of postmenopausal women by HPLC with fluorescence detection
Abstract
BackgroundNew high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K-1 and two forms of vitamin K-2 (MK-4 and MK-7) in human serum, and the levels of vitamin Kwere determined in 350 samples of postmenopausal women. MethodsVitamin K was determined by HPLC with fluorescence detection after postcolumn zinc reduction.
The detection was performed at 246nm (excitation) and 430nm (emission). The internal standard and 2mL of ethanol were added to 500L of serum.
The mixture was extracted with 4mL of hexane, and solid phase extraction was then used. ResultsThe HLPC method was fully validated.
The intra- and interday accuracy and precision were evaluated on two QC samples by multiple analysis, and CV were less than 10%. The limit of quantification for MK-4 was found at 0.04 ng/mL, for K-1 0.03ng/mL, and for MK-7 0.03ng/mL.
The mean recoveries of the corresponding compounds were 98%-110%. Serum levels of MK-4, K-1, and MK-7 in postmenopausal women with osteoporosis were 0.8900.291ng/mL, 0.433 +/- 0.394ng/mL, and 1.002 +/- 1.020ng/mL, respectively (mean +/- SD).
Serum levels of MK-4, K-1, and MK-7 in postmenopausal women without osteoporosis were 0.825 +/- 0.266ng/mL, 0.493 +/- 0.399ng/mL, and 1.186 +/- 1.076ng/mL, respectively (mean +/- SD). ConclusionNew HPLC method for the determination of vitamins K-1, MK-4, and MK-7 in serum was evaluated and validated.
This method is highly specific and sensitive with the low limit of quantification.