Development and validation of a method for the simultaneous quantitative analysis of the active forms of vitamins B1, B2, and B6 using 31P qNMR

With increasing demand for active vitamins in functional foods and supplements, accurate quantification is crucial to ensure that quality and safety standards are met. This study presents the development of a phosphorous-31 quantitative nuclear magnetic resonance (31P qNMR) spectroscopy method for the simultaneous analysis of the active forms of vitamins B1, B2, and B6 in multivitamin supplements. The 31P NMR signals of the analytes and internal standard were effectively resolved and easily distinguishable. The method was validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and stability. The curves for all three vitamins exhibited a high degree of linearity, with correlation coefficients (R2) exceeding 0.999. The LOD (0.04–––0.07 mg mL−1) and the LOQ (0.14–––0.24 mg mL−1) were adequate for analysis of multivitamin supplements, and the accuracy (99.2–––102.0 %) was excellent. The relative standard deviations for repeatability (1.06–––1.89 %) and intermediate precision (2.51–––3.25 %) were considered acceptable. The developed 31P qNMR method was successfully applied to commercial multivitamin supplements, demonstrating its practicality. Therefore, the 31P qNMR method is expected to be suitable for the simultaneous quantification of active vitamins B1, B2, and B6 and an effective analysis method for multivitamin preparations.