Development of a sequential extraction strategy for native B vitamins from milk using protein precipitation, solid‐phase extraction, and magnetic molecularly imprinted polymers for RP‐HPLC analysis

Background, Context or Rationale B‐vitamins are essential micronutrients that are necessary for metabolic activities in the human body. Most analytical methods are tailored for individual B vitamins to target fortified B‐vitamins in supplements and beverages, with limited focus on native B‐vitamins. Further, the low concentration of these vitamins challenges their accurate quantification. Aim(s) In this study, a systematic approach was adopted to develop a protocol for extracting native B‐vitamins from milk. The method involved comparing different protein‐precipitating reagents, followed by the use of a pre‐concentration method. Methods For simultaneous extraction of B vitamins, three different protein‐precipitating reagents, 5% acetic acid, 3% phosphoric acid and 10% trichloroacetic acid (TCA), were evaluated. For pre‐concentration, solid‐phase extraction (SPE) and magnetic molecularly imprinted polymers (MMIP) were used. Quantification was done using reverse‐phase HPLC (RP‐HPLC). Major Findings It was found that TCA was optimum for precipitating proteins and recovering native B‐vitamins from milk. Further, SPE has enhanced recovery. This process enabled the recovery of native vitamins B1, B2, B3, B5, B6 and B9; however, B7 and B12 were not detected at this stage. To address this, MMIPs were used to selectively extract B7 and B12. All B‐vitamins were subsequently quantified using RP‐HPLC. The method demonstrated recovery rates between 75 and 110%, with intra‐day and inter‐day relative standard deviations ranging from 1.12–6.93% and 2.21–5.49%, respectively, indicating high precision and reliability. The developed method was successfully applied for the quantification of native B‐vitamins in cow, buffalo and goat milk. Scientific or Industrial Implications This study provides a systematic method for simultaneous extraction and quantification of native B‐vitamins in milk, a significant advancement over existing single‐vitamin protocols. The integration of SPE and MIP allowed recovery of all B‐vitamins, including B7 and B12, with accuracy and reproducibility. This method has strong industrial relevance for quality assessment, nutritional labelling and functional product development.