Optimization and validation of HRLC-MS method to identify and quantify triacylglycerol molecular species in human milk

Abstract

Study of the determination of triacylglycerols (TAG) molecular species in human milk is necessary for understanding the absorption of human milk fat as well as designing milk fats for infant formulas. The aim of the present study was to optimize fat extraction and validate a high resolution liquid chromatography-mass spectrometry (HRLC-MS) method to identify and quantify TAG in human milk. Intensity, repeatability, intermediate reproducibility, and recovery values were calculated, and a large sample set of human milk was analyzed. Each value for matrix effect of internal standard (IS) or standard solution (STD) in human milk during fat extraction ranged from 78 to 106% and from 56 to 107%, respectively, indicating that no matrix effect was found except for with CCC. For linearity of the method, correlation coefficient (r²) values were found to range from 0.9991 to 0.9999. Recovery values were 88 and 116% for each STD at three different concentrations. Except for c′c′c′ and LLL, the repeatability and intermediate reproducibility values were within 20% and 30%, respectively, indicating that the method was precise. The validated HRLC-MS method was applied to quantify TAG molecular species from human milk as a quality control (QC). Among the 21 quantified TAG, POL, PPO, PLS and OOP were predominant, ranging from 0.01 to 11.0 mg L⁻¹. TAG with short chain acyl, such as c′c′c′, MOB, and LLL, were quantified with low amounts in QC (between 0.01 and 0.06 mg L⁻¹). The results from the current study validate the fat extraction method followed by HRLC-MS for the efficient identification and quantification of TAG molecular species in human milk samples.