Quantitative analysis of cholesterol in infant formula by isotope dilution liquid chromatography-tandem mass spectrometry

동위원소희석 액체크로마토그래피 질량분석법에 의한 분유 내 콜레스테롤의 정량

  • Ahn, Eun Jeong (Department of chemistry, Choongnam National University) ;
  • Lee, Hwa Shim (Center for bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Kim, Byung Joo (Center for bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Lee, Gae Ho (Department of chemistry, Choongnam National University)
  • 안은정 (충남대학교 대학원 화학과) ;
  • 이화심 (한국표준과학연구원, 삶의질측정표준본부, 바이오임상표준센터) ;
  • 김병주 (한국표준과학연구원, 삶의질측정표준본부, 바이오임상표준센터) ;
  • 이계호 (충남대학교 대학원 화학과)
  • Received : 2015.12.11
  • Accepted : 2015.12.15
  • Published : 2015.12.25


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 ℃, 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.


Cholesterol;Saponification;Infant formula;ID LC-MS/MS;Primary method


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