Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.