Application of the Ratio Subtraction Ultraviolet Spectrophotometry Method for Simultaneous Quantification of B1, B2, B3, B6, and B12 Vitamins in Health Supplement Syrups

Abstract

The present investigation aimed to simultaneously determine the concentrations of thiamine (B₁), riboflavin (B₂), nicotinamide (B₃), pyridoxine (B₆), and cobalamin (B₁₂) in health supplement syrups, commonly used as multivitamins. Precise quantification of these vitamins is essential to guarantee product efficacy, safety, and compliance with quality standards. The analytical approach employed was based on ratio subtraction ultraviolet (UV) spectrophotometry, utilizing methanol as a solvent to resolve the overlapping UV absorption spectra of the vitamin mixture. Carefully optimized divisor concentrations 12 µg.mL^–1 for B₁, 22 µg.mL^–1 for B₂, 18 µg.mL^–1 for B₃, 11.5 µg.mL^–1 for B₆, and 25 µg.mL^–1 for B₁₂ were selected to ensure accurate spectral differentiation of each analyte. The method was rigorously validated according to standard parameters, including linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ), all of which fulfilled established validation criteria. The quantified vitamin contents in health supplement syrup samples were 102.75% ± 2.20% for B₁, 101.03% ± 2.17% for B₂, 102.65% ± 2.21% for B₃, 102.11% ± 2.16% for B₆, and 101.26% ± 2.17% for B₁₂, all of which conformed to the specifications outlined in the United States Pharmacopeia 38 and National Formulary 33. The proposed ratio subtraction ultraviolet spectrophotometry method offers a straightforward, cost-effective, and selective solution for concurrent estimation of these vitamins in complex pharmaceutical matrices, without the need for sophisticated instrumentation or elaborate procedures.

HIGHLIGHTS

• A novel ratio subtraction UV spectrophotometry method was developed for simultaneous quantification of 5 B-complex vitamins (B₁, B₂, B₃, B₆, and B₁₂) in multivitamin syrup formulations.
• This method involves sequential division of the spectrum using selected vitamins as divisors, followed by mathematical subtraction and multiplication to reconstruct the 0-order absorption spectrum of each compound.
• Eliminates the need for chromatographic separation, enabling selective detection despite overlapping spectra in multicomponent mixtures.
• Validated according to ICH guidelines, demonstrating excellent linearity (r ≤ 1), high accuracy (recoveries between 90% - 110%), precision (RSD ≤ 2%), and achieves low LOD and LOQ values, ensuring sensitive and reliable detection of each vitamin component.
• Offers a rapid and selective alternative to HPLC method, eliminating the need for derivatization, software modeling, or high-cost instrumentation.

GRAPHICAL ABSTRACT