Analysis of triacylglycerol and phospholipid sn‐positional isomers by liquid chromatographic and mass spectrometric methodologies

Abstract

Analysis of triacylglycerol (TG) and phospholipid sn‐positional isomers can be divided into two main categories: (a) direct separation by chromatography or other means such as ion mobility mass spectrometry and (b) quantification of regioisomer ratios by structurally informative fragment ions with mass spectrometric methods. Due to long retention times and limited performance, researchers are moving away from direct chromatographic separation of isomers, using mass spectrometry instead. Many established analytical methods are targeting specific isomers of interest instead of untargeted analysis of comprehensive profiles of regioisomers. Challenges remain arising from the large number of isobaric and isomeric lipid species in natural samples, often overlapping chromatographically and sharing structurally informative fragment ions. Further, fragmentation of glycerolipids is influenced by the nature of the attached fatty acids, and the lack of available regiopure standards is still an obstacle for establishing calibration curves required for accurate quantification of regioisomers. Additionally, throughput of many methods is still quite limited. Optimization algorithms and fragmentation models are useful especially for analysis of TG regioisomers, as identification using calibration curves alone without proper separation is difficult with complex samples.

Advanced Tandem Mass Spectrometric Analysis of Complex Mixtures of Triacylglycerol Regioisomers: A Case Study of Bovine Milk Fat

“…Currently, we use ammonium acetate in the mobile phase as an additive, leading to the formation of ammoniated TAG ions [M + NH 4 ] + . Ammonium ions are the most widely used complexing agent for TAG regioisomer analyses across different studies . However, a recent study suggested that [M + NH 4 ] + ions might not be the ideal adduct type for establishing a fragmentation model, as the fragment ion ratios of different ACN:DB combinations are not predictable, and sodiated [M + Na] + adducts might be more suitable for creating a general fragmentation model .…”

“…Ammonium ions are the most widely used complexing agent for TAG regioisomer analyses across different studies. 42 However, a recent study 43 suggested that [M + NH 4 ] + ions might not be the ideal adduct type for establishing a fragmentation model, as the fragment ion ratios of different ACN:DB combinations are not predictable, and sodiated [M + Na] + adducts might be more suitable for creating a general fragmentation model. 43 Contrary to the findings of Makarov et al, we have established an accurate fragmentation model using [M + NH 4 ] + adducts using two different triple quadrupole instruments (Shimadzu LC-MS 8045 and Waters Quattro Premier) used in this and a previous study.…”

Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS

“…Six isobaric tags were made to 83.8% purity and 90.9% isotopic purity using selective placement of six total isotopes on either the quaternary amine (balancer as the neutral loss) or on the chain (constant mass reporter ion). 53 Deuterium and 15 N were used to generate the appropriate sixplex isobaric tags. The majority of the isotopic impurity is due to the <100% purity of the starting material, which is almost entirely nonisotopic.…”

“…The 15 N-containing tags were synthesized by a Gabriel synthesis. Phthalimide- 15 N potassium salt was mixed 2:1 with 0.5 g of dibromopropane isotopologes, followed by mono-Boc protection of the resulting diamine. 31,39 The protected diamines were methylated by (HCHO isotopologes), followed by reductive amination.…”

“…Phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) play a significant role in cell membrane signaling, fat absorption, cell permeability, blood coagulation, and lipoprotein formation. , Recently, PCs have gained importance as potential biomarkers for cancer, atherosclerosis, Alzheimer’s disease, and metabolic diseases. − Structurally, PCs are phospholipids (PLs) containing a central phosphoglycerol backbone with a quaternary amine-choline group and one (lyso-) or two acyl chains. The zwitterionic PCs contain anionic phosphate and cationic choline groups that can complicate mass spectrometric analyses through adduct formation or multidimension separation requirements. − …”