Klapkova Eva, Cepova Jana, Dunovska Katerina, Prusa Richard
Department of Medical Chemistry and Clinical Biochemistry, Charles University, 2nd Faculty of Medicine and University Hospital Motol, Prague, Czech Republic.
J Clin Lab Anal. 2018 Jun;32(5):e22381. doi: 10.1002/jcla.22381. Epub 2018 Jan 15.
New high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K and two forms of vitamin K (MK-4 and MK-7) in human serum, and the levels of vitamin K were determined in 350 samples of postmenopausal women.
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.
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 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 , 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 , 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).
New HPLC method for the determination of vitamins K , MK-4, and MK-7 in serum was evaluated and validated. This method is highly specific and sensitive with the low limit of quantification.
开发了一种新的高效液相色谱（HPLC）方法，用于测定人血清中的维生素K及两种维生素K形式（MK-4和MK-7），并测定了350例绝经后女性样本中的维生素K水平。
采用柱后锌还原-高效液相色谱荧光检测法测定维生素K。检测在246nm（激发）和430nm（发射）波长下进行。向500μL血清中加入内标和2mL乙醇。混合物用4mL己烷萃取，然后进行固相萃取。
高效液相色谱法得到充分验证。通过多次分析对两个质量控制样品进行日内和日间准确度及精密度评估，变异系数小于10%。MK-4的定量限为0.04ng/mL，维生素K为0.03ng/mL，MK-7为0.03ng/mL。相应化合物的平均回收率为98%-110%。骨质疏松症绝经后女性血清中MK-4、维生素K和MK-7水平分别为0.890±0.291ng/mL、0.433±0.394ng/mL和1.002±1.020ng/mL（均值±标准差）。无骨质疏松症的绝经后女性血清中MK-4、维生素K和MK-7水平分别为0.825±0.266ng/mL、0.493±0.399ng/mL和1.186±1.076ng/mL（均值±标准差）。
对测定血清中维生素K、MK-4和MK-7的新型高效液相色谱法进行了评估和验证。该方法具有高特异性和高灵敏度，定量限低。
