Analytical Methods to Quantify Free Fatty Acids

Fatty acids are a class of carboxylic acid compounds consisting of hydrocarbon groups attached to carboxyl groups. Fatty acids are usually present in esterified form as components of lipids such as neutral lipids, phospholipids and glycolipids (also known as fatty acid esters or total fatty acids). Esterified fatty acids are hydrolyzed by phospholipases to obtain free fatty acids (FFA), which are non-esterified fatty acids and are an important component of lipid metabolites. On the one hand, exogenous fat enters adipocytes as free fatty acids through plasma transport and then synthesizes fat for storage, while endogenous fat in the body is mainly synthesized in the liver and also enters adipocytes for storage through plasma transport. On the other hand, the stored fat is constantly degraded and enters the tissues in the form of free fatty acids to be oxidized and utilized, so that the fat metabolism is in dynamic balance. Studies have shown that free fatty acids are closely associated with abnormal glucose and lipid metabolism and other cardiovascular diseases, such as obesity, hypertension, hyperinsulinemia, and type 2 diabetes.

In addition, free fatty acids in edible oils are mainly the hydrolysis products of triglycerides. Free fatty acids are unstable and can easily lead to oxidation and rancidity of the oil, which affects the quality and functionality of the oil. Free fatty acid composition in crude oils can be used to characterize the quality of pressed oils and to assess the degree of oil damage. The free fatty acid composition can be used as a parameter for degradation of the oil at different moisture, temperature, oxygen content, light storage and during frying.

Currently, liquid chromatography-mass spectrometry (LC -MS) techniques are mainly used for the detection of free fatty acids, found at extremely low levels in organisms and oils. The lack of easily ionizable functional groups in their structures can lead to low sensitivity in mass spectrometry analysis. Their structural modification using chemical derivatization techniques is an effective means to improve chromatographic behavior and enhance the mass spectrometric response.

Chemical derivatization technique

A compound to be measured is reacted in a quantitative and rapid manner with a derivatization reagent having a specific group to produce a derivatization product that meets the requirements. The amount of the derivatization product is then measured indirectly to determine the amount of the analyte. Chemical derivatization can change the detection characteristics of the analyte, increase its response to the detector, and generate stable derivatization products to improve the instability of the analyte. Derivatization techniques can improve ionization efficiency, reduce ionization suppression during complex sample analysis, and thereby, improving the detection sensitivity and selectivity of electrospray mass spectrometry. The derivatization technique is suitable for the accurate and relative quantification of metabolites with target functional groups in real samples.

Free fatty acid detection method

LC-MS is currently the most widely used method for the determination of fatty acids. The separation of free fatty acids by LC requires the addition of organic acids such as formic acid to the mobile phase to enable the retention of the analytes in the reversed-phase column and to ensure chromatographic separation. However, weak acids in electrospray droplets can carry many negative charges and inhibit the formation of negative ions of analyte carboxylic acids. This inhibits the ionization efficiency of the analyte and is detrimental to the mass spectrometric detection of the analyte. Therefore, conversion of carboxylic acid compounds into cationic compounds by chemical derivatization reaction and analysis in positive ion detection mode of mass spectrometry can avoid ion suppression of acidic mobile phase and improve ionization efficiency. Thus, the detection sensitivity of electrospray ionization mass spectrometry (ESI-MS) can be improved.