A Study on the Spatial Distribution of the Chemical Composition of Flue-Cured Tobacco Leaves Using an Electrospray/Photochemical Ionisation Mass Spectrometry Imaging Source

Areas of application: the tobacco industry; plant metabolomics;

‌Introduction

This experiment utilised a combination of imprinting and electrospray/photochemical ionisation (DESI) mass spectrometry imaging source technology to visualise the spatial distribution of various chemical constituents within initially cured dry tobacco leaves. It comprehensively analysed the spatial distribution patterns of alkaloids, amino acids, sugars and other substances in both the longitudinal and transverse directions of the tobacco leaves, and conducted imaging analysis of the distribution of characteristic metabolites in areas affected by tobacco mosaic virus.

This method overcomes the issues of polarity discrimination and ion suppression associated with traditional DESI ionisation, enabling the intuitive visualisation of variations in compound concentrations within a single tobacco leaf. It provides a precise molecular imaging tool for tobacco cultivation, re-curing and processing, and research into the mechanisms of disease.

‌Materials and Methods

Instrumentation: Electrospray/Photochemical Ionisation Mass Spectrometry Imaging Source (DESI/PI), Agilent 6224 high-resolution time-of-flight mass spectrometer;

Samples: primary-cured dry tobacco leaves.

‌Results and Conclusions

1. Longitudinal distribution patterns (leaf tip → leaf base) of chemical constituents

Key quality-related compounds, such as alkaloids, amino acids and sugars, generally exhibited the highest concentrations at the leaf tip, with levels decreasing progressively towards the leaf base.

Plant alkaloids (nicotine, nicotene, dehydronicotine, etc.): Signal intensity peaks near the leaf tip, with levels declining gradually from the middle of the leaf towards the base.

Amino acids (L-proline, glutamic acid, 5-amino-hydroxyvaleric acid, etc.): Proline content is significantly higher than that of the other amino acids, and its distribution pattern is consistent with that of nicotine; α-aminohexanedioic acid decreases only from the leaf tip to the middle of the leaf, remaining essentially unchanged from the middle to the base.

Amadori compounds (Amadori compounds are a class of stable products formed during the Maillard reaction through condensation, dehydration and rearrangement of aldoses and various amino acids) and nicotinic acid-fructose complexes: their distribution is highly synchronised with that of free proline, demonstrating that the concentration of amino acid substrates during the roasting stage directly determines the content of Maillard reaction products.

 Figure 1 Schematic diagram of longitudinal sampling points on tobacco leaves (green boxes indicate sampling areas); (B) Mass spectrometry imaging of 12 representative chemical compounds in tobacco leaves detected in positive ion mode; (C) Longitudinal distribution of five plant alkaloids (a. nicotine; b. nicotene; c. dehydronicotine; d. N′-formyl-nicotinine; e. (R,S)-N-ethyl-nicotinine/N′-methyl-pseudo-equisapin); (D) Longitudinal distribution of five amino acids (f. L-proline; g. 5-amino-hydroxyvaleric acid; h. glutamic acid; i. α-aminohexanedioic acid; j. 3-(N-nitroso-N-methylamino)propionic acid/asparagine); (E) Longitudinal distribution of 1-deoxy-1-L-prolyloxy-D-fructose (k) and 1-[1-(2′S)-nicotine]-1-deoxy-β-D-furanfructose (I)

2. Lateral distribution patterns (left leaf margin → midrib → right margin) of chemical constituents

The concentrations of various characteristic compounds were highest at the left and right margins of the tobacco leaf and lowest in the midrib region; the trends in concentration were not entirely symmetrical relative to the leaf stalk (due to the effects of cultivation, curing and sampling practices designed to avoid the leaf veins).

Alkaloids, proline and Amadori products: concentrations rise steadily from the midrib towards the left margin; from the midrib towards the right margin, concentrations first decrease and then increase, peaking at both margins;

Glutamic acid and α-aminohexanedioic acid: concentrated at the left and right margins, with concentrations significantly lower at the midrib.

Figure 2 (A) Schematic diagram showing the transverse sampling points on a tobacco leaf; (B) Mass spectrometry imaging of 12 representative chemical compounds in tobacco leaves detected in positive ion mode; (C) Lateral distribution of five plant alkaloids (a. nicotine; b. nicotene; c. dehydronicotine; d. N′-formyl-nicotinine; e. (R,S)-N-ethyl-nicotinine/N′-methyl-pseudo-equisapin); (D) Spatial distribution of five amino acids (f. L-proline; g. 5-amino-hydroxyvaleric acid; h. 3-(N-nitroso-N-methylamino)propionic acid/asparagine; i. α-aminohexanedioic acid; j. glutamic acid); (E) Spatial distribution of 1-deoxy-1-L-prolyloxy-D-fructose (k) and 1-[1′-(2′S)-nicotine]-1-deoxy-β-D-furanfructose (I)

3. Region-specific imaging results for tobacco climate spot disease

Climate spots are induced by ozone, resulting in a significant overall reduction in total nitrogen, nicotine, sugars and free amino acids within the affected area:

In the black spot areas, there were virtually no mass spectrometry signals for alkaloids, amino acids or Amadori sugar complexes; these were detected only in the surrounding healthy tobacco leaves;

Disease-specific metabolites: the substance at m/z=193.05 is distributed only in a ring around the spot; those at m/z=203.15, 217.16, 291.22, etc., are enriched in a dotted pattern on the periphery of the spot. These are metabolic markers of ozone stress in tobacco leaves and can be utilised in research into the pathogenesis of the disease.

Figure 3 (A) Schematic diagram showing the location of climate spots on tobacco leaves; (B) Enlarged optical image of the climate spot region in Figure 5A; (C–U) Imaging of representative compounds detected near climate spots in cation mode

 Conclusions

Electrospray/photochemical ionisation mass spectrometry imaging technology offers advantages such as non-polarity discrimination, soft ionisation and high ionisation efficiency. As the sample requires no complex pre-treatment, ambient-pressure in-situ imaging enables the simultaneous spatial visualisation and semi-quantification of multiple components. This instrument is suitable for research in various scenarios, including growth stress in tobacco leaves, disease metabolism and the optimisation of processing formulations.

References

[1]. Lü Chunchun; Jiang Yuting; Hu Yonghua; Wu Liutian; Qi Keke; Liu Chengyuan; Pan Yang. Study on the spatial distribution of chemical components in flue-cured tobacco leaves using imprinting-analytical electrospray/photoionisation mass spectrometry imaging, Analytical Chemistry, 2024, 52, 876–884.