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Quantitative analysis of cholesterol in infant formula by isotope dilution liquid chromatography-tandem mass spectrometry
Analytical Science and Technology / Analytical Science and Technology, (P)1225-0163; (E)2288-8985
2015, v.28 no.6, pp.460-466
https://doi.org/10.5806/AST.2015.28.6.460
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(2015). Quantitative analysis of cholesterol in infant formula by isotope dilution liquid chromatography-tandem mass spectrometry. Analytical Science and Technology, 28(6), 460-466, https://doi.org/10.5806/AST.2015.28.6.460
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Abstract
An isotope dilution liquid chromatography tandem mass spectrometry was developed as a primary method for the quantitative analysis of cholesterol in infant formula. Cholesterol-d4 was used as an internal standard and spiked into the infant formula sample. In order to release cholesterol out of cholesteryl ester, which is cholesterol bound to fatty acids in infant formula, saponification was carried out. Saponification conditions were optimized with heating temperature, reaction time and the concentration of KOH. The optimum conditions were as follows; heating temperature was 70 oC, reaction time was 180 min and the concentration of KOH was 0.8 mL of 8 M KOH for about 0.1 g infant formula sample. Extraction of cholesterol out of sample solution was carried out with hexane uisng liquid-liquid extraction. Chromatographic analysis was carried out using Phenomenex Kinetex C18 column. Mobile phase was 0.1% acetic acid in methanol/water (v/v, 99/1) and flow rate was 0.3 mL/min. Cholesterol and cholesterol-d4 were monitored at mass transfer m/z 369/259 and 373/ 263 respectively. Reproducibility of the method was evaluated to be 0.23% of the measurement result. The expanded uncertainty of the measurement result of cholesterol in infant formula was approximately 1.9% at a 95% confidence level. NIST standard reference material having certified values of cholesterol in infant formula, was analyzed in order to verify this method. The ID-LC/MS/MS results were well agreed with the certified values of NIST SRM within the uncertainty.
- keywords
- Cholesterol, Saponification, Infant formula, ID LC-MS/MS, Primary method
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