Rapid screening and quantitative analysis of 26 soluble sugars in tobacco by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry

Sugars are one of the important chemical components in tobacco. They serve as key precursor substances for various aromatic compounds in tobacco. Furthermore， they also have a significant impact on the taste and burning characteristics of tobacco. Thus， obtaining accurate information on the composition and content of soluble sugars in tobacco can provide a scientific basis for quality assessment of tobacco leaves. Moreover， it offers critical data support for quality control and process optimization. Traditional techniques for soluble sugar analysis include continuous flow analysis， ion chromatography， gas chromatography， and liquid chromatography， etc. However， these methods still have limitations such as tedious sample pretreatment， poor detection sensitivity， and low analysis efficiency. In contrast， ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry （UPLC-Q-TOF-MS） is a highly effective analytical technology. It integrates the excellent separation capability of UPLC and the superior sensitivity of MS. This combination offers great potential for qualitative and quantitative analysis of target compounds. Nonetheless， the types of soluble sugars detectable in tobacco by UPLC-Q-TOF-MS remain limited. This defect restricts the comprehensive evaluation of sugar components in tobacco. Herein， a rapid and accurate analysis method was established for high-throughput screening and quantification of 26 soluble sugars in tobacco leaves. The tobacco leaf samples were ultrasonically extracted with 30 mL 40% acetonitrile aqueous solution at 120 W for 30 min. Soluble sugar extract solution was prepared using a Waters Sep-Pak C18 solid-phase extraction cartridge. With the help of self-built sugar library， the tobacco leaf samples were screened by UPLC-Q-TOF-MS. Then matrix-matched standard calibration curves were employed to accurately quantify the target analytes. The established method was verified. The 26 soluble sugars exhibited good linear relationships with correlation coefficients （R2） ranging from 0.999 1 to 0.999 9. The limits of quantification （LOQs） were in the range of 0.03-20 mg/L. The spiked recoveries ranged from 92.39% to 111.75%， and relative standard deviations （RSDs） were ≤4.65%. Finally， this method was applied to analyze 59 tobacco leaf samples from different origins， grades and years. Six soluble sugars including erythritol， fructose， sorbitol， glucose， sucrose and inositol were successfully identified. The content of each soluble sugar varied significantly among samples from different origins， grades， and years. Even among samples from the same origin， grade， and year， the soluble sugar contents showed certain fluctuations. This is speculated to be related to factors such as the growth environment， maturity， processing， and harvest time of the tobacco leaves. Among them， the contents of fructose and glucose were at relatively high levels in all tobacco leaf samples. The soluble sugar content from Heilongjiang was generally higher than that from Guizhou， Hunan， and other regions. Additionally， the overall soluble sugar content in samples from 2021 and 2022 was higher than that from 2020. Principal component analysis （PCA） and hierarchical cluster analysis （HCA） were conducted on tobacco leaf samples from different positions， respectively. These analyses demonstrated significant differences in soluble sugar contents among tobacco leaves from different positions. It confirmed that soluble sugars could serve as effective indicators for distinguishing positions of tobacco leaves. In summary， the established method enables the qualitative and quantitative detection of the 26 soluble sugars without relying on standard substances. Moreover， the method exhibits high throughput， simplicity， rapidity， and accuracy. Meanwhile， it is suitable for the qualitative and quantitative detection of soluble sugars in tobacco leaf samples. It provides robust technical support for in-depth research on tobacco chemical components and the digital formulation design of cigarette products.