RP-HPLC for simultaneous determination of water-soluble vitamins in leafy vegetables using ultrasonicated acid hydrolysis

Abstract

This study reports the development and application of a rapid and efficient reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous separation and identification of water-soluble vitamins (WSVs) such as B₁, B₂, B₃, B₆, and B₉ in green leafy vegetables (GLVs) from Chhattisgarh, India. A sample preparation method based on acid hydrolysis assisted by ultrasonication was optimized using 0.1 M HCl. Separation and detection of the vitamins were carried out using a C-18 column with a gradient elution of orthophosphoric acid (OPA) and methanol (MeOH), with UV detection at 270 nm. The method demonstrated excellent linearity (r² > 0.993), low limits of detection (LODs) ranging from 0.06 to 0.15 μg mL⁻¹, and high recovery rates (91.5–98.0%), confirming its sensitivity and accuracy. The analysis of 17 GLVs from the Chhattisgarh region demonstrated considerable variation in WSV content per 100 g. The concentration of WSV B₁ ranged from 0.12 to 0.85 mg, B₂ from 0.08 to 0.40 mg, B₃ from non-detectable (ND) to 2.64 mg, B₆ from 0.07 to 0.64 mg, and B₉ from ND to 0.08 mg. Furthermore, estimated daily intake (EDI) values were calculated to assess the contribution of these GLVs toward the recommended dietary intake for both men and women. The findings suggest that regular consumption of these vegetables can significantly enhance nutritional adequacy. This method provides a robust analytical tool for the simultaneous determination of five essential vitamins in food matrices.