Mapping the regioisomeric distribution of fatty acids in triacylglycerols by hybrid mass spectrometry

Abstract

This study describes the use of hybrid mass spectrometry for the mapping, identification, and semi-quantitation of triacylglycerol regioisomers in fats and oils. The identification was performed based on the accurate mass and fragmentation pattern obtained by data-dependent fragmentation. Quantitation was based on the high-resolution ion chromatograms, and relative proportion of sn-1(3)/sn-2 regioisomers was calculated based on generalized fragmentation models and the relative intensities observed in the product ion spectra. The key performance features of the developed method are inter-batch mass accuracy < 1 ppm (n = 10); lower limit of detection (triggering threshold) 0.1 μg/ml (equivalent to 0.2 weight % in oil); lower limit of quantitation 0.2 μg/ml (equivalent to 0.4 weight % in oil); peak area precision 6.5% at 2 μg/ml concentration and 15% at 0.2 μM concentration; inter-batch precision of fragment intensities < 1% (n = 10) independent of the investigated concentration; and averaged accuracy using the generic calibration 3.8% in the 1-10 μg/ml range and varies between 1-23% depending on analytes. Inter-esterified fat, beef tallow, pork lard, and butter fat samples were used to show how well regioisomeric distribution of palmitic acid can be captured by this method.

Fig. 1.

Typical results obtained from a beef tallow sample using the described hybrid MS approach. (A) High-resolution base peak ion chromatogram obtained in the Orbitrap. (B) Mass tag between ammoniated and sodiated ions that serves as an additional triggering criterion. (C) High-resolution ion chromatogram of ion 883.77275 Da that serves as a basis for quantification. (D) Averaged product ion mass spectrum of parent ion 878 Da obtained in the linear ion trap in parallel to the high-resolution chromatogram; only monoisotopic product ions are present, representing the loss P, S, O, and one ammonia unit.

Fig. 2.

(A) Chromatogram of the TAG standard mixture using the optimal conditions as described in the text. The numbering corresponds to the numbers in the tables. (B) Ion chromatograms of all TAG that were detected in butter fat using same scale as in (A). Letters stand for the following TAG: (a) 16:0-16:0-4:0/4:0-14:0-18:0/4:0-18:0-14:0; (b) 4:0-16:0-18:1/4:0-18:1-16:0; (c) 4:0-12:0-18:0/4:0-14:0-16:0; (d) 14:0-14:0-4:0/4:0-12:0-16:0/4:0-16:0-12:0; (e) 16:0-16:0-18:1/16:0-18:1-16:0/18:0-14:0-18:1; (f) 18:1-18:1-16:0/18:1-16:0-18:1; (g) 16:0-16:0-16:0/16:0-14:0-18:0; (h) 18:0-16:0-18:1/16:0-18:1-18:0/16:0-18:0-18:1; (i) 14:0-16:0-18:1/16:0-14:0-18:1/18:0-12:0-18:1/16:0-16:1-16:0; (j) 12:0-16:0-18:1/16:0-12:0-18:1/14:0-18:1-14:0/16:0-14:1-16:0/16:0-14:0-16:1/18:0-14:0-14:1/10:0-18:0-18:1; (k) 16:0-16:0-12:0/16:0-12:0-16:0/14:0-16:0-14:0/14:0-12:0-18:0/12:0-18:0-14:0/10:0-18:0-16:0/16:0-10:0-18:0/10:0-16:0-18:0; (l) 16:0-16:0-6:0/6:0-14:0-18:0/6:0-18:0-14:0; (m) 4:0-16:0-18:0/4:0-18:0-16:0; (n) 10:0-14:0-18:0/10:0-18:0-14:0/12:0-18:0-12:0/16:0-10:0-16:0/8:0-18:0-16:0; (o) 16:0-14:0-16:0/16:0-12:0-18:0/14:0-18:0-14:0/12:0-18:0-16:0. An extraction window of 10 ppm was used for all analytes.

Fig. 3.

Results of chromatographic robustness analysis are shown by varying the amount of n-hexane in solvent B. The shift of retention times are depicted as percentage compared with the retention time obtained using the optimal composition (50% n-hexane). Each data point stands for a specific TAG eluted in the same order as in Fig. 2A. 55% n-hexane, open circles; 50% n-hexane, open triangles; 45% n-hexane, open squares.

Fig. 4.

Schematic diagram of TAG analysis, identification, and quantitation.

Fig. 5.

Results of fatty acid regiodistribution are shown for beef tallow (A) and pork lard (B). The absolute quantity of fatty acids in the sn-1(3) and sn-2 positions can be read on the vertical axis. In the case of pork lard sample, 83% of P is esterified at the sn-2 position, whereas in the beef tallow sample, only 17% of P is esterified at the sn-2 position.

Fig. 6.

(A) Regiodistribution of fatty acids in the butter fat sample. (B) Size distribution of 565 intact TAG detected in the same analysis and expressed as function of carbon number of acyl chains. Note that TAG with even acyl chain carbon numbers are greater than ten times more abundant than TAG with odd acyl chain carbon numbers.