Assay of Vitamin B6 (Pyridoxine Hydrochloride) Utilizing Isocratic Reversed Phase High Performance Liquid Chromatography

Aims: To demonstrate an analysis for vitamin B6 from commercial aqueous nutritional drinks and
solid tablets, utilizing isocratic conditions with high performance liquid chromatography (HPLC) and
UV detection at 290 nm.
Study Design: Vitamin B6 in the form of pyridoxine hydrochloride is assayed by HPLC from various
samples.
Place and Duration of Study: Department of Chemistry, Durham Science Center, University of
Nebraska, Omaha Nebraska from May to August 2016.
Methodology: Utilizing a reversed-phase C-18 column with eluent solvent (19% ethanol, 77% water,
4% acetic acid), the samples were prepared in sample solvent (19% ethanol, 81% distilled water).
Detection of vitamin B6 was accomplished at 290 nm. Analysis of samples was done following
solubilizing in aqueous conditions having ethanol at 10% to 20% (v/v). Column pressure at 1900 psig,
rise time 0.1 with flow rate 1.0 mL/minute. Elution peak for vitamin B6 occurred consistently at 1.6
minutes. Nutritional samples, aqueous samples, and solid pills were prepared in aqueous solvent with
various levels of ethanol.
Results: Levels of vitamin B6 detected were as low as 4.4029x10-5 molar to 7.8081x10-4 molar.
Sensitivity for vitamin B6 was highest at 290 nm. Reverse phase isocratic conditions is shown to be
effective for determination of vitamin B6 in aqueous based samples. Standard curves applied are
highly linear in range from zero to 7.8081x10-4 molar (y = 112,521,145.5x + 2,818.6), having
coefficient of determination (R2 = 0.9948) with very strong positive correlation coefficient (r= 0.9974).
Percent recovery of vitamin ranged from 95% to 105%. Amounts of vitamin present in drinks from
same manufacturer were consistent.
Conclusion: Utilizing reversed phase column, isocratic solvent conditions with ethanol in water, and a
UV detector set at 290 nm is effective for determination of vitamin B6. Ethanol-water solvent system
is effective. Vitamin B6 was found in various amounts in nutritional drinks tested. Percent recovery of
vitamin averaged 101% with a standard deviation of 2.4%. Pyridoxine hydrochloride was effectively
assayed from aqueous samples, vitamin preparations, and vitamin tablets. The methodology
presented in this study will be useful for quality control analysis for commercial production. Analysis
methods for vitamin assay are a necessary objective to ensure quality control of commercial products
and medicinal applications.