A liquid chromatography-mass spectrometry workflow for in-depth quantitation of fatty acid double bond location isomers

Abstract

Tracing compositional changes of fatty acids (FAs) is frequently used as a means of monitoring metabolic alterations in perturbed biological states. Given that more than half of FAs in the mammalian lipidome are unsaturated, quantitation of FAs at a carbon-carbon double bond (C=C) location level is necessary. The use of 2-acetylpiridine (2-acpy) as the charge-tagging PB reagent led to a limit of identification in the subnanomolar range for mono- and polyunsaturated as well as conjugated FAs. Conjugated free FAs of low abundance such as FA 18:2 (n-7, n-9) and FA 18:2 (n-6, n-8) were quantified at concentrations of 0.61 ± 0.05 and 0.05 ± 0.01 mg per 100 g in yak milk powder, respectively. This workflow also enabled deep profiling of eight saturated and 37 unsaturated total FAs across a span of four orders of magnitude in concentration, including ten groups of C=C location isomers in pooled human plasma. A pilot survey on total FAs in plasma from patients with type 2 diabetes revealed that the relative compositions of FA 16:1 (n-10) and FA 18:1 (n-10) were significantly elevated compared with that of normal controls. In this work, we have developed a workflow for global quantitation of FAs, including C=C location isomers, via charge-tagging Paternò-Büchi (PB) derivatization and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Keywords: 2-acetylpiridine; Fatty acids; Paternò-Büchi; charge-tagging; double bond location isomers; lipidomics; liquid chromatography; quantitation; tandem mass spectrometry; type 2 diabetes.

Fig. 1

A: Schematic presentation of the charge-tagging PB reaction and C=C diagnostic ions formed from PB-MS² CID. Different orientations of the PB reagent relative to the C=C produce two types of PB products, P₁ and P₂. For PB-MS² CID only C=C diagnostic ions are shown, one containing methyl end (^n-xf_O) while the other containing carboxylic group (^n-xF_O). Superscript n-x denotes the location of C=C counting from the methyl terminus. Subscript “O” defines that the fragment contains olefin functional group at the cleavage site. B: A list of acetylpyridine derivatives tested as the charge-tagging PB reagents for FA analysis.

Fig. 2

A: EICs of 5 μM intact FA 18:1(n-9Z) ([FA-H]⁻, m/z 281.3, green trace) and 2-acpy modified FA ([FA+2-acpy+H]⁺, m/z 404.3, orange trace). B: PB-MS² CID of FA 18:1(n-9Z) (m/z 404.3 eluted from 6.2 min to 6.4 min). C: PB-MS² CID of the PB product from 2-acpy and FA 20:4(n-6Z, n-9Z, n-12Z, n-15Z) (m/z 426.3 eluted from 4.9 min to 6.0 min).

Fig. 3

A: Fragmentation schemes of FA 16:1 C=C location isomers (synthetic standards): n-7Z, n-9Z, and n-10Z; (B) EICs of the diagnostic ions: ^n-7f_O in yellow trace, ^n-9f_O in red trace, and ^n-10f_O in blue trace. C: EICs of intact C=C location isomers ([FA 16:1-H]⁻ at m/z 253.2).

Fig. 4

A: Ratio plots of ion abundances of the diagnostic ions against molar ratios of corresponding C=C location isomers of FA 16:1: n-7/n-9 in black and n-9/n-10 in green. B: Fragmentation schemes of FA 18:2(n-7E, n-9Z), FA 18:2(n-6Z, n-8E), and FA 18:2(n-6Z, n-9Z); (C) PB-MS² CID of CLA: FA 18:2(n-7E, n-9Z); (D) PB-MS² CID of LA: FA 18:2(n-6Z, n-9Z); (E) ratio plots of ion abundances of the unique C=C diagnostic ions against molar ratios of the corresponding isomers of FA 18:2: FA 18:2(n-6, n-9)/FA 18:2(n-7, n-9) in orange and FA 18:2(n-7, n-9)/FA 18:2(n-6, n-8) in blue. Error bars represent the standard deviation of the mean (N = 3).

Fig. 5

A: Relative quantitation of FFAs at sum composition level in yak milk powder. B: Relative compositions (%) of C=C location isomers in eight groups of FAs. Error bars represent standard deviation of the mean (N = 3). C: Quantitation of three C=C location isomers of FA 18:2 in yak milk powder. Numbers in parenthesis represent the concentration of each isomer in units of mg per 100 g yak milk powder.

Fig. 6

A: Relative quantitation of total FAs at sum composition level in pooled human plasma. B: Relative composition (%) of C=C location isomers in eight groups of FAs. Error bars represent standard deviation of the mean (N = 3). C: Relative composition (%) of C=C location isomers of FA 16:1 in pooled human plasma.

Fig. 7

Analysis of total FAs in human plasma samples, normal control (N, n = 6) versus T2D (n = 6). Relative quantitation of (A) FA 16:1 and (B) FA 18:1 at sum composition level. Relative composition (%) of n-10 C=C location isomers in (C) FA 16:1 and (D) FA 18:1. Statistical difference between the two groups was evaluated using the two-tailed student's t test (∗∗P < 0.01, ∗∗∗P < 0.001). Error bars represent standard deviation of the mean (n = 6).