Aroma components of tobacco powder from different producing areas based on gas chromatography ion mobility spectrometry

2.1 Instruments

The gas chromatography-ion mobility spectrometry (GC-IMS) (FlavourSpec^®, Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) uses an Agilent 490 GC (Agilent Technologies, USA) with a MXT-WAX column (30 m × 0.53 mm i.d., 0.1 µm film thickness, RESTEK). The GC was equipped with an autosampler (CTC Analytics AG, Zwingen, Switzerland) with a headspace sampling unit and a 1 mL gastight syringe (Gerstel GmbH, Mühlheim, Germany).

2.2 Materials

Ten tobacco samples of four grades from three places were used as analysis materials. GDNX C3L (GuangDong NanXiong C3L), GDNX C3F (GuangDong NanXiong C3F), GDNX C4F (GuangDong NanXiong C4F), and GDNX B2F (GuangDong NanXiong B2F) were obtained from Nanxiong in Guangdong province; FJNP C3L (FuJian NanPing C3L), FJNP C3F (FuJian NanPing C3F), FJNP C4F (FuJian NanPing C4F), and FJNP B2F (FuJian NanPing B2F) were obtained from Nanping in Fujian province; JLCC C3L (JiLin ChangChun C3L) and JLCC C3F (JiLin ChangChun C3F) were obtained from Changchun in Jilin province.

Tobacco samples were crushed by a pulverizer and sieved with 80 mesh sieve. The moisture content of tobacco powder was 5.0%. The weight of tobacco powder used in the analysis process was 1.0 g.

2.3 Analysis methods

Aroma substances in tobacco samples from different regions were analyzed by GC-IMS. GC-IMS is equipped with an automatic headspace sampler, which does not require pretreatment of tobacco samples. Accurately weigh 1.0 g tobacco powder and place it in the sample inlet bottle. The injection volume was 500 µL, incubation time was 15 min, and incubation temperature was 80°C. The GC-IMS conditions were: FS (fused silica)-SE-54 (the stationary phase is composed by methyl silicone: 5% phenyl, 1% vinyl silicone) – CB-0.5 (the thickness of stationary phase is 0.5 μm) – 15 m (Column length) – ID (internal diameter): 0.53 mm; 60°C transfer line temperatures; nitrogen carrier gas at a flow rate of 0–2 min, 2 mL/min; 2–10 min, 2–10 mL/min; 10–20 min, 10–100 mL/min; 20–25 min, 100–150 mL/min; the MS conditions were: 45°C ion source and 85°C injector temperature.

1. Ethical approval: This article does not contain any studies with human participants performed by any of the authors.

3.1 GC-IMS 3D spectra analysis

Figures 1 and 2 are GC-IMS spectrum of tobacco powder and GC-IMS spectrum difference of tobacco powder, respectively. The background of the entire figure is blue, and the left red vertical line is the RIP peak (Reactant Ions Positive peak, drift time is around 7.81 ms). Each point on both sides of the RIP peak represents a volatile organic compound. GC-IMS spectrum was referred to as one spectrum. The colors of substances with the same concentration in another spectrum cancel out as white. White implies low concentration, and red is a high concentration. A darker color implies a higher concentration. The blue area in the referenced sample indicated that the concentration of the substance was lower than that of the reference sample. A darker blue implies a lower concentration. The red area in the referenced sample indicated that the concentration of the substance was higher than that of the reference sample. A darker red implies a higher concentration.

Figure 1

Ion mobility spectrum of tobacco samples.

Figure 2

Difference diagram of ion mobility spectrum of tobacco samples.

GC-IMS technology can detect a variety of aroma substances from tobacco leaves as shown in the ion mobility spectrum. The difference diagram of the ion mobility spectrum shows that GC-IMS technology can compare and analyze the differences of aromatic substances in different tobacco samples. The GC-IMS spectrum results show that the two strip peaks to the right of the RIP peak (indicated by the red arrow) were monomers and dimers of acetic acid because the high content in the sample led to the peak throughout. Comparing GC-IMS spectrum of each sample, we show that the composition and proportion of aroma substances in different regions and different grades of tobacco were similar. At the same time, the red part in the atlas of JNCC and FJNP is significantly higher than the blue part, and the red part was very dark, which indicated that the content of aromatic substances in Jilin Changchun (JNCC) and Fujian Nanping (FJNP) was higher than that in Guangdong Nanxiong (GDNX).

3.2 Fingerprint analysis of aroma substances

All of the peaks analyzed in the GC-IMS spectrum were selected to generate fingerprints to compare the differences of volatile organic compounds among the different samples (Figure 3).

Figure 3

Fingerprints of volatile organic compounds peaks.

Each row was a sample, and each line was a signal peak of organic matter (the same substance in different samples) with the same retention time and drift time. The fingerprint shows that the contents of aroma substances in tobacco in JNCC and FJNP were more abundant than those in GDNX; their aromatic contents varied. Aromatic substances with a higher concentration in GDNX tobacco samples were n-decyl aldehyde, 1-hydroxy-2-acetone, 2-methyl-butanol, and 2-methylpropionic aldehyde. Aromatic substances with a higher concentration in FJNP tobacco samples were n-hexanol, cyclohexanone, and n-valeric acid. The aromatic substances with higher concentration in JNCC tobacco samples are 2-acetylfuran, 2-octanol, isopentanol, 3-methyl-pentanoic acid, phenylacetic acid, and ethyl acetate. The higher concentrations of aromatic substances in GDNX and FJNP were dimethyl disulfide and ethyl propionate. The aromatic substances with higher concentrations in FJNP samples and JNCC samples were 2-amylfuran, 2-acetopyrrole, benzyl alcohol, linalool, furfural, methyl-5-furfural, acetaldehyde, phenylacetaldehyde, 6-methyl-5-hept-2-ketone, and isoamyl acetate. The higher concentrations of aromatic substances in the samples of GDNX tobacco and JNCC tobacco were 2-3-butanediol, 3-hydroxy-2-butanone, and 3-methylbutyraldehyde.

The aromatic substances with similar concentrations in three tobacco samples from different regions were γ-butyrolactone, acetone, and methyl acetate. The concentrations of 2-phenyl alcohol, 2-hexenol, (trans)-2-hexene-1-alcohol, 2-heptanol, heptanal, benzaldehyde, 2-methyl-butyric acid, ethyl butyrate, and toluene in tobacco samples from GDNX, FJNP, and JNCC increased successively. The concentration of aromatic substances in tobacco samples in GDNX varies greatly. This is mainly reflected in the other three grades of C3L where the concentrations of aroma substances such as 2-hexenol, valeraldehyde, hexanaldehyde, heptanal, octanal, n-nonaldehyde, and propionic acid in C3L grade samples were higher than the other three grades. The 2-3-butanediol, dimethyl disulfide, and ethyl propionate in C3L samples were lower than the other three grades. Table 1 is the peak volume of aroma substances in tobacco. The fingerprint can compare and analyze the concentration of aroma substances among different samples by comparing the fingerprint with the peak volume of aromatic substances.

3.3 Principal component analysis and similarity analysis of different samples

Figure 4 is a principal component analysis of all volatile aroma substances in tobacco samples in which the contribution rate of the first principal component was 59%, the contribution rate of the second principal component was 20%, and the contribution rate of the two principal components was 79%. The results show that tobacco samples from three regions could be clearly distinguished by only the first and second principal components. The dispersion degree of tobacco samples from GDNX and JNCC was relatively high, but the dispersion degree of tobacco samples of C3F and C4F grades in GDNX was relatively low. In other words, the samples were quite similar. The dispersion of tobacco samples in FJNP was relatively low. Samples C3F, B2F, and C4F were quite similar, and they had no obvious distinction. For a more accurate analysis of different grades of tobacco samples from different producing areas, the similarity of each sample was calculated using information on all volatile substances as the object of calculation (Table 2).

Figure 4

PCA plots of all tobacco samples.

We took the information on volatiles as the calculation object to determine the similarity of each sample (Table 2); two parallel samples were used for each sample. Table 2 shows that the similarity of the same grade tobacco samples from the same producing area in GDNX was higher than 93, and the similarity of C3F and C4F was 94. Therefore, to judge the grade of tobacco samples in GDNX, the similarity degree with known samples was supposed to be higher than 94. The similarity between the same grade of tobacco samples from the same producing area in FJNP and JLCC was the lowest at 94. The similarity between samples of different grades of tobacco was the highest at 92. Therefore, to judge the grade of tobacco samples from the two places, the similarity of known samples ought to reach 93. The similarity between tobacco samples from GDNX and those from the two other regions was the highest at 80, and the lowest at 72. Therefore, the similarity between samples from GDNX and those from GDNX ought to reach above 80 to determine whether the tobacco samples were produced in GDNX. The similarity between tobacco samples from FJNP and those from other two regions was 81, while the similarity between tobacco samples from FJNP was 85. Therefore, to judge whether the tobacco sample was produced in FJNP, the similarity between the sample and Nanping should reach 85. The similarity between the tobacco samples from JLCC and the other two regions was the highest at 82 and the lowest at 89. Therefore, the similarity between the tobacco samples from JLCC should be at least 89.