Vitamin K2-4 and K2-7 Estimation in Nutraceutical Solid Dosage Forms by Post Column Derivatization with Fluorescence Detection

Abstract

A post column derivatization method for the estimation of fat soluble vitamins K 2 -4 and K 2 -7 was developed by reverse phase HPLC and validated as per ICH guidelines. The compounds were extracted by solvent extraction with acetone followed by evaporation in a rotary evaporator. The residue was dissolved in ethanol and injected in to a chromatograph consisting of C18 column (Waters symmetry, 150×4.6, 3.5 μ) and fluorescence detector. The derivatization reagent was prepared by dissolving 136 mg of zinc chloride, 40 mg of sodium acetate and 0.1 ml of glacial acetic acid in methanol. The mobile phase comprises of methanol:isopropyl alcohol:acetonitrile:zinc chloride buffer solution 850:90:50:10 with a flow rate of 1 ml/min. The overall percentage recoveries of five different levels were found to be 99.85 and 100.5%, respectively. The linearity of the analytical method was determined from 10% to 120% level and the linear regression coefficients were 0.9991 and 0.9995 which is well within the acceptance criteria of 0.999. The limits of detection and limits of quantification were determined based on signal to noise ratio. The established values were 0.050, 0.50 μg/ml and 0.005, 0.047 μg/ml which is much lesser than available literatue limits. The developed method can be more suitable for the estimation of K 2 -4 and K 2 -7 present in drug substances as well as drug product formulations.Key words: Post column derivatization, nutraceutical solid dosage forms, estimation of K 2 -4 and K 2 -7 by HPLC, zinc chloride buffer solution, analytical estimation Vitamin K 2 includes several subtypes; the two subtypes most studied are menaquinone-4 (menatetrenone, MK-4) and menaquinone-7 (MK-7). Vitamin K 2 -4 ( fig. 1a) is yellow coloured powder, molecular formula C 31 H 40 O 2, molecular weight 444.65 g/mol, soluble in acetone and ethyl alcohol [1] . Vitamin K 2 -7 ( fig. 1b) is light yellow microcrystalline powder, molecular formula C 46 H 64 O 2, molecular weight 648.99 g/mol, soluble in petroleum ether, acetone, ethyl alcohol and insoluble in water [1] . Vitamin K is used for the treatment of anticoagulantinduced prothrombin deficiency caused by warfarin [2][3][4] . The American Academy of Paediatrics recommends that vitamin K should be given to all new born babies as a single intramuscular dose to prevent vitamin K deficiency bleeding. In Japan, vitamin K 2 is used for the management of osteoporosis. The fermented soya product nattō is rich in menaquinone-7 [2][3][4] .Derivatization reaction with the reduction of zinc metal ions to isolate compounds of closely eluting peaks by chromatography is fast emerging tool for analytical separation. The dosage form formulated with two active ingredients such as K 2 -4 and K 2 -7 along with excipients is very challenging effort to separate from its moiety. In order to separate the K series vitamins with almost same structural properties, the post column derivatization technique with fluorescence detection was used. The reduction mechanism of components with zinc chloride derivatizatio...

An UFLC-DAD Method for the Quantification of Menaquinone-4 in Spiked Rabbit Plasma

“…Data on the dietary admission of menaquinones is constrained because of the absence of diet tables that rundown menaquinone-4 fixation in ordinary nourishment. The quantitative analyses of menaquinone-4 till date were done using HPLC-Fluorescence, [11][12][13][14][15][16] HPLC-PDA, [17][18][19] HPLC-MS 18,20 and HPLC-MS/MS 17,[21][22][23][24] techniques. Fluorescence detection for menaquinone-4 was carried in human serum, 11,14,15 food, 12 nutraceuticals 13 and human plasma 16 for which a post-column reduction with zinc or zinc added to mobile phase has been used.…”

“…The quantitative analyses of menaquinone-4 till date were done using HPLC-Fluorescence, [11][12][13][14][15][16] HPLC-PDA, [17][18][19] HPLC-MS 18,20 and HPLC-MS/MS 17,[21][22][23][24] techniques. Fluorescence detection for menaquinone-4 was carried in human serum, 11,14,15 food, 12 nutraceuticals 13 and human plasma 16 for which a post-column reduction with zinc or zinc added to mobile phase has been used. There is no specified pathway mentioned for post-column derivatization and a comparison of fluorescence detection with other HPLC detections.…”