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

Abstract

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 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.

Rapid Determination of L-carnitine in Infant and Toddler Formulas by Liquid Chromatography Tandem Mass Spectrometry

“…A method for sample preparation was based on the previous study ( Starkey et al ., 2008 ), however, additional techniques were used for protein removal through the acid hydrolysis and lipid removal through the organic solvent extraction. Samples containing 0.3 g was placed in 50 mL conical tubes and dissolved in 9 mL of distilled water and 1 mL of 0.1 M HCl.…”

“…And simultaneous analysis method utilizing liquid chromatography-mass spectrometry (LC-MS) of free carnitine and total choline content was published in AOAC official journal ( Andrieux et al ., 2008 ). Besides, a method through liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrumental analysis was also presented ( Starkey et al ., 2008 ).…”

Current Methods for the Analysis of Selected Novel Nutrients in Infant Formulas and Adult Nutritionals

“…A microwave-assisted acid hydrolysis technique was developed for the analysis of free carnitine and total choline in four food-based certified reference materials including infant formula (33). A simplified technique for the analysis of free carnitine by dilution and filtering makes use of the high degree of selectivity and sensitivity of modern LC-MS/MS instruments (34).…”

“…Under reversed-phase conditions, chromatographic separation of carnitine is challenging because of its ionic nature. Ion pair (IP) chromatography using heptafluorobutyric acid has been used to retain carnitine on C 8 (34) and C 18 (29) stationary phases. Alternative column chemistries can offer significant advantages for the retention of polar and ionic analytes without the need for IP reagents and hydrophilic interaction liquid chromatography (HILIC) columns (36), and mixed-mode reversedphase and ion-exchange columns (33,35) have been used.…”

“…Mixed-mode columns allow for a high degree of selectivity by modification of mobile-phase pH, ionic strength, and organic solvent, which may be beneficial, particularly in the analyses for which MS/MS is not used. Electrospray ionization (ESI) in positive mode with either MS (29,33) or MS/MS (34)(35)(36) has been applied to the quantitative analysis of carnitine.…”