Determination of vitamins K1, MK‐4, and MK‐7 in human serum of postmenopausal women by HPLC with fluorescence detection

Background

New high‐performance liquid chromatography (HPLC) method was developed for the determination of vitamin K₁ and two forms of vitamin K₂ (MK‐4 and MK‐7) in human serum, and the levels of vitamin K were determined in 350 samples of postmenopausal women.

Methods

Vitamin K was determined by HPLC with fluorescence detection after postcolumn zinc reduction. The detection was performed at 246 nm (excitation) and 430 nm (emission). The internal standard and 2 mL of ethanol were added to 500 μL of serum. The mixture was extracted with 4 mL of hexane, and solid phase extraction was then used.

Results

The 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 K₁ 0.03 ng/mL, and for MK‐7 0.03 ng/mL. The mean recoveries of the corresponding compounds were 98%‐110%. Serum levels of MK‐4, K₁, and MK‐7 in postmenopausal women with osteoporosis were 0.890 ± 0.291 ng/mL, 0.433 ± 0.394 ng/mL, and 1.002 ± 1.020 ng/mL, respectively (mean ± SD). Serum levels of MK‐4, K₁, and MK‐7 in postmenopausal women without osteoporosis were 0.825 ± 0.266 ng/mL, 0.493 ± 0.399 ng/mL, and 1.186 ± 1.076 ng/mL, respectively (mean ± SD).

Conclusion

New HPLC method for the determination of vitamins K₁, MK‐4, and MK‐7 in serum was evaluated and validated. This method is highly specific and sensitive with the low limit of quantification.