Determination of vitamins K 1 1 , MK MK ‐4, and MK MK ‐7 in human serum of postmenopausal women by HPLC HPLC with fluorescence detection

摘要

Background New 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 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 K 1 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, K 1 , 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, K 1 , 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 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.