Simultaneous determination of PUFA-derived pro-resolving metabolites and pathway markers using chiral chromatography and tandem mass spectrometry

Abstract

Lipid mediators play an important role as biological messengers involved in inflammatory processes. Deriving from different polyunsaturated fatty acids, endogenously built mediators featuring both pro- and anti-inflammatory properties as well as pro-resolving lipid mediators and their biological precursors have been investigated. A newly developed method using chiral chromatography-tandem mass spectrometry on human plasma has demonstrated its suitability for the simultaneous determination of prostaglandins, lipoxins, D-series derived resolvins as well as protectins, maresin 1, leukotriene B4 and several precursors of them in order to yield information about metabolic pathways. Due to the matrix complexity, a solid phase extraction method using an octadecyl-modified silica gel cartridge was carried out. The developed method allows the determination of 34 analytes in 25 min showing enough selectivity as well as precision and accuracy (≤15% relative standard deviation, ≤15% relative error) in the calibration range of 0.1-10 ng mL or 0.2-20 ng mL depending on the analytes. Stability of the analytes in plasma has been demonstrated for at least 3 h at room temperature, 72 h in the autosampler and 60 days in the freezer at -80 °C. This method has been validated and shown its suitability for the determination of all studied analytes in human plasma samples.

Development of an Optimized LC-MS Method for the Detection of Specialized Pro-Resolving Mediators in Biological Samples

“…For RvD2 the most sensitive fragment ( m/z 375.3 → 175.0) is unlikely to be formed by an α-cleavage and may be formed by a γ-cleavage toward the hydroxyl group or another mechanism (Figure 3C). However, as this fragment is the most sensitive with our instrument and is also used by other groups for RvD2 (Barden et al, 2014; Homann et al, 2014; Toewe et al, 2018), it was chosen as primary transition. These backbone fragments [“chain-cut ions” (Hong et al, 2007)] are specific allowing to discriminate between regioisomers, whereas fragments referred to as “peripheral-cut ions” (Hong et al, 2007) that result from the unspecific loss of water (hydroxyl group) and/or carbon dioxide (carboxylic group) are not selective and do not allow to draw conclusions on the position of the hydroxyl groups being essential for the selective detection of e.g., RvD5, PD1, and MaR1 (DHA-derived dihydroxy-FA, Q1 mass: m/z 359.1) and other isobaric autoxidation products which could be formed from PUFA.…”

“…Enzyme linked immunoassays can be used for the detection of single compounds (Chiang et al, 1998; Kirkby et al, 2013), though their specificity might be limited with respect to a large number of possible regio- and stereoisomers formed. Nowadays, methods used for identification and quantification of SPMs and other oxylipins are mostly based on reversed phase liquid chromatography (RP-LC) (Masoodi et al, 2008; Mas et al, 2012; Le Faouder et al, 2013; Colas et al, 2014; Jónasdóttir et al, 2015; Skarke et al, 2015), chiral LC (Oh et al, 2011; Homann et al, 2014; Toewe et al, 2018), or both (Massey and Nicolaou, 2013; Barden et al, 2015) hyphenated via electrospray ionization (ESI) to tandem mass spectrometric (MS/MS) detection. However, despite application of state-of-the-art LC-MS/MS based methodology, SPM detection in biological samples remains challenging.…”

Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids

“…We had to perform analytical runs on two different chiral phases in order to resolve distinct subsets, comprising most of the targeted monoepoxides on Lux Cellulose-3 and all monohydroxides as well as several mid-chain monoepoxides on Lux Amylose-1. Other investigators used Chiralpak AD-RH, Lux Amylose-2, or Lux Amylose-1 with 250 × 4.6 mm size to run chiral-LC coupled with ESI-MS/MS for the targeted lipidomics of resolvins and other specialized pro-resolving lipid mediators (8)(9)(10). Relatively long equilibration times needed before and in between successive analytical runs (see Technical notes and conditions for the analysis of biological samples in the Results section) are another problem concerning the moderate throughput in our chiral approach.…”

“…This approach allows performing reversed-phase chiral-LC coupled with ESI-MS/MS detection and quantitation of the resolved stereoisomers. Reversed-phase chiral-LC-ESI-MS/MS has been successfully used for the determination of enantiomers as well as diastereomeric epimers of pro-resolving lipid mediators (8)(9)(10).…”

Measurement of Enzymatic and Nonenzymatic Polyunsaturated Fatty Acid Oxidation Products in Plasma and Urine of Macular Degeneration Using LC‐QTOF‐MS/MS

“…Liquid chromatography‐mass spectrometry (LC–MS), on the other hand, is a powerful alternative that provides high precision and comparable sensitivity, and reduces the complexity in analytical method due to derivatization. Moreover, previous efforts have successfully demonstrated the applications of liquid chromatography tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in monitoring nonenzymatic PUFA oxidation products (Dupuy et al, 2016; Janicka et al, 2013; Rund et al, 2018; Sanchez‐Illana et al, 2017) or enzymatic PUFA oxidation products (Thakare et al, 2018; Toewe et al, 2018), in both plasma and urine samples. Analysis of urinary PUFA oxidation products is of interest because it is noninvasive and provides additional information on the excretory system ( e.g .…”