Ranmadugala Dinali, Grainger Megan, Manley-Harris Merilyn, Berenjian Aydin
Faculty of Science and Engineering, University of Waikato, Hamilton, New Zealand.
National Aquatic Resources Research and Development Agency, Colombo, Sri Lanka.
Curr Pharm Biotechnol. 2018;19(8):664-673. doi: 10.2174/1389201019666180828090637.
An efficient and accurate HPLC method was developed for the determination of menaquinone-7 (MK-7) in microbial fermentation using 2-propanol and n-hexane as extraction solvents as well as the eluent.
Extraction was carried out with 2-propanol and n-hexane (2:1, v/v) after enzymatic hydrolysis with 1% (w/v) lipase and ethanol water treatment prior to quantification in order to remove interfering lipids and denatured proteins. Chromatographic separation of MK-7 was accomplished isocratically on a C 18 Gemini column using a mobile phase mixture of 2- propanol: n-hexane (2:1, v/v) with a flow rate of 0.5 mL/min. UV detection was carried out from 200-400 nm and the chromatogram was extracted at a wavelength of 248 nm. A linear response was shown by the method with a coefficient of determination (R2) value of 0.9982.
The recoveries of MK-7 were greater than 94% and the intra and inter day R.S.D values were less than 2%, demonstrating the accuracy of the method. The lower limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/mL and 0.29 µg/mL, respectively.
The general usefulness of the described method is demonstrated by the application of this method in the analysis of MK-7 from Bacillus species. Under these conditions, the analysis of MK-7 was achieved in less than 8 minutes with a retention time of 7.19 ± 0.1 minutes.
开发了一种高效准确的高效液相色谱法，用于测定微生物发酵中的甲基萘醌-7（MK-7），该方法使用异丙醇和正己烷作为萃取溶剂及洗脱剂。
在定量前，先用1%（w/v）脂肪酶进行酶解，再经乙醇水处理，然后用异丙醇和正己烷（2:1，v/v）进行萃取，以去除干扰性脂质和变性蛋白质。MK-7的色谱分离在C18 Gemini柱上采用等度洗脱，流动相为异丙醇:正己烷（2:1，v/v），流速为0.5 mL/min。紫外检测在200 - 400 nm范围内进行，色谱图在248 nm波长处提取。该方法呈现线性响应，测定系数（R2）值为0.9982。
MK-7的回收率大于94%，日内和日间相对标准偏差值均小于2%，证明了该方法的准确性。检测下限（LOD）和定量下限（LOQ）分别为0.1 µg/mL和0.29 µg/mL。
该方法在芽孢杆菌属MK-7分析中的应用证明了其普遍实用性。在这些条件下，MK-7的分析在不到8分钟内完成，保留时间为7.19 ± 0.1分钟。
