Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry

Background Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. Objective A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. Method Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography‒tandem mass spectrometry (LC-MS/MS), with quantitation accomplished by internal standard technique. Results The method was shown to be accurate, with acceptable recovery (thiamine, 99.3–101.1%; pantothenic acid, 99.2–108.6%). A certified reference material (NIST 1849a), showed no statistical bias (α = 0.05) for thiamine (P = 0.64); although a statistically significant bias (P < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC-MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (RSDr) (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. Conclusions This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.