A Comprehensive Study on Triacylglycerols in Tobacco Leaves Using Liquid Chromatography and Atmospheric- Pressure Chemical-Ionization Mass Spectrometry

Abstract

Triacylglycerols in tobacco leaves were investigated to further understand their compositional and metabolic differences among various tobacco leaves and their changes during the curing processes. Non-aqueous reverse-phase chromatography for efficient separation of triacylglycerols and atmospheric-pressure chemical-ionization mass spectrometry for their detection were selected for quantitative analysis. For measuring their absolute amounts, authentic triacylglycerols comprised of the same fatty acids were introduced to generate calibration curves and to quantify the analytes. A validation study using the combination of appropriate separation and authentic triacylglycerols produced very good results in terms of linearity (> 0.999), limit of detection (3-4 ng), limit of quantification (10-15 ng) and recovery ratio (80-120%). The quantification of triacylglycerols in tobacco leaves and a subsequent principal component analysis clarified for the first time their compositional and metabolic differences among various tobacco leaves.

A Simultaneous Analytical Method to Profile Non-Volatile Components with Low Polarity Elucidating Differences Between Tobacco Leaves Using Atmospheric Pressure Chemical Ionization Mass Spectrometry Detection

“…Actual analysis for tobacco samples was replicated three times on each extract without replicating extraction of each tobacco sample. This was because previously executed quantification of solanesols (13), triacylglycerols (15) and phytosterols (17) showed smaller variance among replicated extractions than among replicated analyses on one sample.…”

“…Since tobacco leaves were extracted in the same manner and the extracts were separated by the same chromatography as reported (13,15,17), only the parameters applied to MSD for detection were optimized to obtain desirable ion intensity of solanesols (13), triacylglycerols (15) and phytosterols (17). Although chlorophylls in tobacco leaf were originally extracted by acetone (14), as the compositions of both extracts did not show a large difference, n-hexane was used for the analysis to consolidate extraction procedures.…”

“…Components for identification were purchased and prepared by separation and synthesis as described in previous reports (13)(14)(15)17).…”

“…Compared to EI and ESI, atmospheric pressure chemical ionization (APCI) has still remained a rare interface due to its specificity in low polar components (21,29). Nevertheless the usefulness of APCI-MSD has already been presented by target analysis of non-volatile components with low polarity (13)(14)(15)17) and the application to metabolomics was expected to provide another comprehensive method and new correlation of differences in components with differences in tobacco leaves. The objective of this study was therefore to develop a comprehensive analytical method using LC/APCI-MSD hyphenated with non-aqueous reversed-phase chromatography (NARPC) to correlate differences in chemical profiles with differences in tobacco leaves.…”

“…However, the methodologies applied to those studies has employed only gas chromatography mass spectrometry detection (GC/MSD) oriented to volatile components (8,9), or liquid chromatography electrospray ionization mass spectrometry detection (LC/ESI-MSD) for components with middle to high polarity (11). Since non-volatile components with low polarity such as solanesols (12,13), chlorophyll derivatives (14), triacylglycerols (15), solanachromene (16) and phytosterols (17) (Figure 1) are also included in tobacco leaf and have been considered unsuitable for detection in using the previously employed instruments (8)(9)(10)(11), the metabolomics applied to tobacco leaves has yet to cover all the components in tobacco leaves. In the first place, comprehensive analyses for metabolomics consist of a separation and detection part which are hyphenated so as to maximize comprehensiveness of method (7,(18)(19)(20).…”