The water-soluble vitamins are gaining rapid importance not only for human health but also being widely recommended for animal health care because of theirs wide spectrum of activities ranging from deficiency, pathogenic condition to nutritional supplements. A plethora of articles are published for simultaneous evaluation of B-Complex Vitamins, but most of their methods are not selective and time consuming which involve pre-treatment of sample due to complexities involved in the placebo matrix. In this study, we propose a simple, specific, precise, accurate and linear method to separate vitamin B1 (thiamine), vitamin B2 (riboflavin sodium phosphate), vitamin B3 (niacin amide), vitamin B6 (pyridoxine) and vitamin C (ascorbic acid) by using Zorbax XDB C18 column (250 mm x 4.6 mm i.d., 5µm). The flow rate was kept as 1.3 mL/min and monitored at 270 nm with ambient temperature. Effective separations and quantification were obtained in 30 minutes with 10 µl injection volume. The current reverse phase gradient method resulted in significant resolution between interested vitamins and placebo matrix. Analytical method development, followed by analytical method validation was accomplished according to International Conference of Harmonization guidelines (ICH). Statistical evaluation of validation parameters proved that the results were highly specific, precise, accurate, robust and linear over a wide concentration range. This method has been validated and found to be applicable in routine analysis of vitamin B1, vitamin B2, vitamin B3, vitamin B6 and vitamin C in oral powder for veterinary consumption.
___
[1] Ortega-Barrales P, Fernandes de Cordova ML, Molina-Diaz A. Micro determination of vitamin B1 in the presence of vitamins B2, B6 and B12 by solid-phase UV spectrophotometry. Anal Chem. 1998; 70(2): 271-275.
[2] Morelli B. Determination of ternary mixtures of vitamins by ratio-spectra zero-crossing derivative spectrophotometry. Anal Lett. 1994; 27(14): 2751-2768.
[3] Morelli B. High-resolution/higher-order derivative spectrophotometry for determination of ternary mixtures of Bcomplex vitamins in pharmaceuticals. Fresenius J Anal Chem. 1996; 354(1): 97–102.
[4] Bhushan R, Parshad V. Separation of vitamin B complex and folic acid using TLC plates impregnated with some transition metal ions. Biomed Chromatogr. 1994; 8(4): 196-198.
[5] Dinelli G, Bonetti A. Micellar electro kinetic capillary chromatography analysis of water-soluble vitamins and multivitamin integrators. Electrophoresis. 1994; 15(8-9): 1147-1150.
[6] Tallaksen CM, Bøhmer T, Karlsen J, Bell H. Determination of thiamin and its phosphate esters in human blood, plasma, and urine. Methods Enzymol. 1997; 279: 67-74.
[7] Poongothai S, Ilavarasan R, Karrunakaran CM. Simultaneous and accurate determination of vitamins B1, B6, B12 and alphalipoic Acid in multivitamin capsule by reverse–phase high performance liquid Chromatographic method. Int J Pharm Pharm Sci. 2010; 2(4):133-139.
[8] Jin P, Xia L, Li Z, Che N, Zou D, Hu X. Rapid determination of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and ascorbic acid in vitamins with minerals tablets by high-performance liquid chromatography with diode array detector. J Pharm Biomed Anal. 2012; 70: 151–157.
[9] Amidzic R, Brboric J, Cudina O and Sote. RP-HPLC determination of vitamins B1, B3, B6, Folic acid and B12 in multivitamin tablets. J Serb Chem Soc. 2005; 70(10): 1229-1235.
[10] Abano EF, Dadzie RG. Simultenous detection of water-soluble vitamins using the high performance liquid chromatography (HPLC) - A review. Croat J Food Sci Technol. 2014; 6 (2): 116-123.
[11] Kucukkolbasi S, Bilber O, Ayyildiz HF, Kara H. Simultenous and accurate determination of water and fat soluble vitamins in multivitamin tablets by using an RP-HPLC method. Quim Nova. 2013; 36(7): 1044-1051.
[12] Markopouloua CK, Kagkadisa KA, Koundourellis JE. An optimized method for the simultaneous determination of vitamins B1, B6, B12, in multivitamin tablets by high performance liquid chromatography. J Pharm Biomed Anal. 2002; 30(4): 1403-1410.
[13] Soni H, Singhai AK, Mishra K, Sharma S. Simultaneous determination of vitamins B1, B2 and B6 in multivitamin tablet and biological fluid by RP-HPLC. Int J Pharm Sci Res 2012; 3(7): 2163–2167.
[14] ICH (International Conference on Harmonization), Validation of analytical procedures: text and methodology Q2 (R1). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Gui deline.pdf (accessed October 18, 2017).
[15] European Pharmacopeia, Chromatographic separations techniques (2.2.46). http://online6.edqm.eu/ep902/, (accessed October 18, 2017).
[16] United States Pharmacopeia, Chromatography 621. http://app.uspnf.com/uspnf/login, (accessed October 18, 2017).