LC–MS/MS System Developed for Fatty Acid Analysis

A recent study led by Hebei Medical University scientists from Shijiazuhang, China details the development of a sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for analyzing free fatty acids in human serum. The scientists published their findings in the Journal of Chromatography A (1).

Molecule model og Omega - 6 fatty acid. White is Hydrogen, black is Carbon, red is Oxygen, White is Hydrogen, black is Carbon, and red is Oxygen. | Image Credit: © Kim - stock.adobe.com

Fatty acids (FAs) are a type of carboxylic acids that contain fatty acid chains, are typically composed of three elements: carbon, hydrogen, and oxygen. FAs are composed of an acyl (hydrocarbon) chain with a methyl and a carboxyl group at either end (2). Depending on the length of the carbon chain, FAs can be classified as short-chain fatty acids (SCFAs; number of carbon atoms ≤ 6), medium-chain fatty acids (MCFAs; 7 ≤ number of carbon atoms ≤ 12) or long-chain fatty acids (LCFAs; number of carbon atoms ≥ 13). Further, FAs can be divided into essential or nonessential FAs based on whether the body can synthesize it. Essential FAs must be obtained through food because the human body cannot manually synthesize them. Polyunsaturated fatty acids (PUFAs), act as precursor substances to generate different eicosanoids through enzymatic oxidation or nonenzymatic free radical mechanisms in the human body and act as important bioactive signaling molecules in various physiological or pathological conditions, regulating a range of different homoeostatic and inflammatory processes in the organism. Additionally, PUFAs are sensitive indicators factors like lipid and glucose metabolism. Abnormal increases and decreases are commonly observed in individuals with obesity, diabetes, hyperthyroidism, pituitary insufficiency, and cardiovascular diseases. Accurately tracking these and other free fatty acids levels in human serum can enable timely detection of abnormal FFA levels, subsequently promoting prevention, early treatment, and health monitoring.

In this study, the scientists developed a stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatization strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine. With this system, they determined 11 FFAs in human blood samples. Serum samples were put through liquid-liquid extraction and centrifuged, with a supernatant being collected for a two-step derivatization reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution.

Eventually, the effects of the derivatization reaction time, temperature and concentration of derivatization reagents on the response values of the analytes were investigated. The optimal conditions were 1.0 mg mL⁻¹ CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL⁻¹ 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL⁻¹; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng m⁻¹; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The method was simple accurate, and reliable, the scientists wrote. It could also be applied to the sensitive determination of FFAs in human blood samples.

References

(1) Zeng, Y.; Li, Q.; Zhang, R.; Wei, M.; et al. Development and Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitative Analysis of 11 Free Fatty Acids in Human Serum Using a Derivatisation Strategy. J. Chromatogr. A 2024, 1728, 465019. DOI: 10.1016/j.chroma.2024.465019

(2) Fatty Acids. Elsevier B.V. 2024. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/fatty-acids (accessed 2024-6-13)