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Abstract

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

2.1. LC-ESI-MS Data

Nineteen SHFAs were used in the present study. Eight of them were regio-isomers of hydroxy palmitic acid [2-hydroxypalmitic acid (2HPA), 3-hydroxypalmitic acid (3HPA), 7-hydroxypalmitic acid (7HPA), 8-hydroxypalmitic acid (8HPA), 9-hydroxypalmitic acid (9HPA), 10-hydroxypalmitic acid (10HPA), 11-hydroxypalmitic acid (11HPA), 16-hydroxypalmitic acid (16HPA)], and seven were regio-isomers of stearic acid [2-hydroxystearic acid (2HSA), 3-hydroxystearic acid (3HSA), 7-hydroxystearic acid (7HSA), 8-hydroxystearic acid (8HSA), 9-hydroxystearic acid (9HSA), 10-hydroxystearic acid (10HSA), 12-hydroxystearic acid (12HSA)]. In addition, four 3-hydroxy FAs [3-hydroxycapric acid (3HCA), 3-hydroxylauric acid (3HLA), 3-hydroxymyristic acid (3HMA), 3-hydroxypentadecanoic acid (3HPDA)] were included in the set of compounds tested. The high-resolution mass spectra of these HFAs were recorded in electrospray ionization (ESI) negative mode. The structures of the SHFAs used along with the exact masses of the deprotonated molecules and the retention times observed for each one of them in the chromatographic method are presented in . The MS/MS spectra of all the 19 analytes in negative ESI mode are also presented in the .

2.3. Analysis of Samples

Seventeen cow milk samples and 12 goat milk samples were purchased from the local market and analyzed. The extracted ion chromatograms (EICs) of a cow milk sample (A) and a goat milk (B) sample are shown in . The simultaneous determination of 19 SHFAs was achieved in a single 10-min run. The contents of the 19 analytes in milk samples (in triplicate) are summarized in , and they are expressed as the µg of HFA per mL of milk. The integration of the peak areas was performed manually using MultiQuant 3.0.2. As Hill et al. discussed [], in particular cases a manual baseline may be employed for the quantification of the peak areas. The same integration parameters were used in all cases.