A novel UHPLC-ESI-MS/MS method and automatic calculation software for regiospecific analysis of triacylglycerols in natural fats and oils

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

“…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 . 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 . We used argon as the collision gas for CID with both instruments, whereas helium was used in the study of Makarov et al While it is difficult to evaluate the full effect without access to an instrument using different collision gases, the collision gas type could possibly explain at least some of the differences observed in the positional sensitivity (differences in the fragment ion ratios between the two regioisomers) of [M + NH 4 ] + adducts with different instruments.…”

“…In our earlier studies, the Waters Quattro Premier triple quadrupole MS/MS instrument was used to analyze the regiopure reference TAGs for constructing the calibration curves and subsequently the first fragmentation model of the TAG Analyzer software. , In the current study, to establish the updated fragmentation model, we analyzed the reference compounds using a Shimadzu LC-MS 8045 triple quadrupole MS/MS instrument. In addition to the fragmentation model, individual calibration curves were also constructed for easier comparison of the calibration data from two different instruments for evaluating inter-instrument robustness of the two different instrument platforms (Figure S1).…”

“…Various analytical methods have been used in numerous studies to investigate the FA distribution of TAGs in natural fats and oils. The methods used for such purposes can be categorized into enzymatic methods, − chemical methods, − chromatographic methods, − nuclear magnetic resonance (NMR) spectrometric methods, − and mass spectrometric methods. − Typically enzymatic and chemical methods are more time-consuming and produce less reliable results due to possible acyl migration. , More importantly, the enzymatic methods only provide information about the overall positional distribution of FAs instead of individual molecular species or regioisomers of TAGs. NMR, on the other hand, is a spectroscopic method based on characteristic chemical shifts, which provides both qualitative and quantitative information about positional distribution of FAs in TAGs.…”

“…Previously, we have developed a UHPLC-MS/MS method for the analysis of TAG regioisomers based on the relative abundances of structurally informative diacylglycerol (DAG) fragments after fragmentation with CID . For data processing and automatic calculation of complex samples, we recently developed TAG Analyzer software to address the challenge brought by chromatographically overlapping isobaric TAGs and their fragments …”

“… 25 For data processing and automatic calculation of complex samples, we recently developed TAG Analyzer software to address the challenge brought by chromatographically overlapping isobaric TAGs and their fragments. 19 TAG Analyzer uses a unique optimization algorithm to interpret the fragment ion data. Utilizing a fragmentation model calibrated with numerous reference standards, the algorithm finds the optimal TAG regioisomer composition that produces the best matching fragment spectrum, which can comprise regioisomers of different molecular species, including isobaric fragments from multiple sources.…”