Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for quantification of five phospholipid classes in various matrices

19 January 2023, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


Hydrophilic interaction liquid chromatography (HILIC), coupled to tandem mass spectrometry, can be used to separate and determine various polar lipid classes. The development of an HILIC chromatographic separation of several molecular species among five phospholipid classes (PC, PE, PG, PI and PS) is reported here. In this method, a gradient with acetonitrile and 40 mM ammonium acetate buffer was employed. The initial composition was 95 % of acetonitrile, then this proportion was decreased to 70 % in order to elute all the compounds of interest for a total running time of 11 mins. Furthermore, mobile phase pH can affect the ionizable character of the compounds, according to their pKa values, and also the stationary phase charge state. The influence of such a parameter on both retention times and resolution was evaluated. Besides, the response of different kinds of internal standards (post-extraction standard addition) was evaluated in four different biological matrices, two microalgae extracts and two marine fish extracts. This study found that the recovery rates were between 70-140 % of the expected value, with relative standard deviations between 10 and 35 %, and then limited matrix effects.


mobile phase pH

Supplementary materials

MRM transitions
MRM transitions for HILIC-ESI-MS/MS phospholipid analysis


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.