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 (P-31 qNMR) spectroscopy method for the simultaneous analysis of the active forms of vitamins B-1, B-2, and B-6 in multivitamin supplements. The P-31 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 (R-2) 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 P-31 qNMR method was successfully applied to commercial multivitamin supplements, demonstrating its practicality. Therefore, the P-31 qNMR method is expected to be suitable for the simultaneous quantification of active vitamins B-1, B-2, and B-6 and an effective analysis method for multivitamin preparations.