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 con sistently 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  4.4029×10-5 molar  to  7.8081×10-4 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  7.8081 x 10-4 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 amou nts 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 th is 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.

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