Chemical fingerprinting strategies based on comprehensive two-dimensional gas chromatography combined with gas chromatography-olfactometry to capture the unique signature of Piemonte peppermint essential oil (Mentha x piperita var Italo-Mitcham).

Accurate, reliable, and informative mapping of untargeted and targeted components across many samples is here performed by combining off-line GC-Olfactometry (GC-O) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry with variable ionization energy (TOF MS featuring Tandem Ionization™). In particular, untargeted and targeted (UT) features patterns are processed by chromatographic fingerprinting, giving differential priority to potent odorants' retention-times regions. Distinguishing peppermint essential oil (EO) from Piedmont (Italy - Mentha × piperita L. var. Italo-Mitcham - Menta di Pancalieri EO), with its unique sensory fingerprint (i.e., freshness and long-lasting sweetness), from high-quality peppermint EOs produced in other areas poses a great challenge. Chromatographic UT fingerprinting provided a great chemical dimensionality by mapping more than 350 peak-regions at 70 eV and 135 at 12 eV. From them, 95 components were identified and responses compared to available literature. Then, potent odorants, detected by GC-O using the aroma extraction dilution analysis (AEDA), were tracked over the chromatographic space and tentatively identified. With the highest flavor dilution (FD), 1,8-cineole (eucalyptus, fresh, camphoraceous); menthone (minty, herbaceous); and menthofuran (minty, musty, petroleum-like) were highlighted. Responsible for creamy and coumarinic notes were the diasteroisomers of (3,6)-dimethyl-4,5,6,7-tetrahydrobenzo[b]-furan-2(3H)-one (i.e., menthofurolactones), detected in higher relative abundance in Pancalieri EOs. By prioritizing the investigation of volatiles on higher LogFD retention regions, including 131 untargeted/targeted features, Pancalieri EOs were separately clustered from United States samples. Besides pre-targeted analytes, additional untargeted features were post-processed for identification within marker chemicals. Myrtenyl methyl ether, ethyl 3-methyl butanoate, propyl-2-methylbutanoate, and (E)-2-hexenal were putatively identified. Of the "unknown - knowns" with diagnostic roles, all metadata were collected including low energy spectra at 12 eV, which were found to be highly complementary to 70 eV spectra.

Glandular trichomes and essential oils in developing peppermint leaves: I. Variation of peltate trichome number and terpene distribution within leaves.

The trichome number and the variability in mono and sequisterpene distribution of peltate trichomes within a leaf was investigated during development of peppermint (Mentha×piperita L.) leaves. A larger number of peltate trichomes was initiated on adaxial epidermises than abaxial epidermises, but during leaf development a greater gland number was produced on abaxial epidermises. Evidence showed that the trichome number was not fixed at the time of leaf emergence. On both epidermises, the trichome distribution was found to change with its position on the leaf. A striking monoterpene composition variability was present among individual trichomes and between different parts of the leaf. Different rates of peltate trichome development and distribution as well as significant differences in trichome number and density were found between the epidermises. A clear and opposite trend was found between menthone and menthol percentages during development. Menthone and menthol were also found to be present in larger amounts on adaxial epidermises, whereas neomenthol and isomenthol had highest percentages on abaxial epidermises. Of the total oil collected from peltate trichomes, 60%, was composed of menthone. isomenthol, menthol and neomenthol and of the remaining 40 %, more than half was represented by other mono and sesquiterpenes while the final percentage was made up of a few unidentified early eluting compounds of high Volatility.