Single-laboratory validation of a liquid chromatographic/tandem mass spectrometric method for the determination of free and total carnitine in infant formula and raw ingredients

A reversed-phase liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of free carnitine (FC) and total carnitine (TC) in infant formula and raw ingredients. Preparation of samples for FC determination required only nonvolumetric dilution in water, followed by filtration. Determination of TC in whey-based samples required a common saponification procedure, which hydrolyzed the acylcarnitines to FC. L-Carnitine standards were prepared in water at 3 concentrations (7.5, 30, and 60 ng/mL). L-Carnitine-d3 was incorporated in the sample and standard preparations as a stable isotope internal standard (ISTD), which helped to normalize signal suppression due to fouling of the ionization source, or matrix effects. A short C8 column (50 x 2.1 mm id) was used for liquid chromatography, with mobile phases consisting of 0.1% heptafluorobutyric acid in water and methanol. L-Carnitine and the ISTD eluted at about 2 min, and the injection-to-injection cycle time was about 11 min. Multiple-reaction monitoring was used for quantitation by monitoring the ion transitions m/z 162 > 103 and 162 > 85 for L-carnitine, and m/z 165 > 103 and 165 > 85 for the ISTD. The values for overall method precision for a whey-based infant formula, a protein hydrolyzate infant formula, and a water-soluble vitamin/amino acid premix were relative standard deviations of 1.32, 1.86, and 3.33%, respectively. The overall mean recoveries were between 97.3 and 102.8%. Additionally, the method results showed good correlation with those obtained for a radioenzymatic assay done in-house.