Investigation of Continuous Flow Methods for Determining the Content of Reducing Sugar in Tobacco *

Flue-cured tobacco (A), Burley tobacco (B), Oriental tobacco (C), flue-cured tobacco cigarettes (D), and blended cigarettes (E) were provided by the China National Tobacco Quality Supervision and Test Center; the reference cigarette 3R4F was provided by the Center for Tobacco Reference Products, and CM7 (G) was the CORESTA monitor cigarette.

The reagents used were as follows: D-glucose ($ 98%, Sigma-Aldrich, America); acetic acid ($99.5%, Shandong Haozhong Chemical Technology Co., Ltd., Jining, China); hydrochloric acid (36–38%, Yantai Shuangshuang Chemical Co., Ltd., Yantai, China); sodium hydroxide (98%, Alfa Aesar, America); calcium chloride ($ 95.0%), p-hydroxybenzoic acid; citric acid ($ 99.5%, Tianjin Ruijinte Chemical Co., Ltd., Tianjin, China); polyethoxy lauryl ether (Brij-35, Shanghai Anyun Biological Co., Ltd., Shanghai, China); ultrapure water (resistivity $ 18.2 MΩ@cm, China National Tobacco Quality Supervision and Test Centre, Zhengzhou, China).

In this study, we used the following equipment: AA3 continuous flow analyzer (Seal Analytical Ltd, Wrexham, UK); BSA224S-CW Electronic Balance (inductance 0.0001G; Sartorius, Göttingen, Germany); FD115 oven (Binder GmbH, Tuttlingen, Germany); HY-8 adjustable speed oscillator (Changzhou Guohua Electric Appliance Co., Ltd., Jiangsu, China); Elga PURELAB P water purifier (Veolia Group, Beijing, China).

D-Glucose (10.0 g; accurate to 0.0001 g) was weighed and added to a 5% acetic acid solution to obtain the standard reserve solution of 1 L. The standard working solution was obtained by pouring 0.5, 1.0, 2.5, 3.0, and 5.0 mL of the standard reserve solution into a volumetric flask (50 mL) and diluting the solutions with a 5% acetic acid solution as required.

The tobacco cigarette was stripped, and the cut tobacco was crushed and sieved (No. 40 Mesh sieve, 380 μm), thus, ensuring that the weight of each sample was at least 10 g. The A, B, and C samples were prepared by crushing the tobacco leaves and passing them through the No. 40 mesh sieve. The moisture content of the sample was determined by the oven method (i.e., baking at 100 °C for 2 h).

The method for preparing the ameliorative solution to determine the reducing sugar content is shown in Table 1. The remaining solutions were prepared according to ISO 15154:2003.

Approximately 0.2500 g (accurate to 0.0001 g) of the sample was added to 25 mL of 5% acetic acid solution and shaken for 30 min. The solution was then filtered using quality filter paper, and the filtrate obtained was used to conduct further tests.

The reducing sugars were analyzed by ISO 15154:2003 and the revised method (pipeline diagrams are shown in Figure 1).

Figure 1.

The content of reducing sugars was calculated using the following equation: 1α=n×c×Vm×(1−W)×1000×100$$\alpha = {{n \times c \times V} \over {m \times (1 - W) \times 1000}} \times 100$$

α: Reducing sugar content on a dry basis (%, as in glucose);

n: Dilution multiple;

c: Instrumental determination of reducing sugars in the extract (mg/mL);

V: Volume of the extract (mL);

m: Sample mass (g);

W: Moisture content of the sample (as a mass fraction).

The ultraviolet-visible spectroscopy (UV-Vis) absorption spectra of standard solutions of different concentrations are shown in Figure 3. According to the reaction principle, the standard curve was established by the test results of the standard solution. The maximum absorption wavelength was between 400 nm and 410 nm. The wavelength of the filter was maintained at 410 nm.

Figure 3.

The standard working solution was determined by the revised method, and the peak height x of the standard working solution was linearly regressed by the concentration of the target substance y. The regression equation was and the correlation coefficient (R) was 0.9999. The standard deviation (SD) was calculated. The detection limit (3 SD) and the quantitative limit (10 SD) were 0.0057% and 0.0190%, respectively.

The reducing sugar contents of A, B, and F samples were determined five times a day for five consecutive days following the revised method. The results showed that the Relative Standard Deviation (RSD) of the three samples was less than 5%, indicating that the method was highly precise (Table 6).

To verify the repeatability (r) and reproducibility (R) of the method, samples A (high), B (low), and F (middle) with relatively high, medium, and low reducing sugar content were prepared to conduct a collaborative study involving 10 tobacco detection laboratories in China. Each sample was measured 20 times, and the r and R values were calculated according to the test results. The results showed that the r and R values of the revised method were similar to those of ISO 15154:2003 (Table 7).

The reducing sugar contents of samples A, F, and G were determined according to the revised method. The standard solutions with high, middle, and low concentrations were added, respectively, and each sample was repeatedly tested five times to calculate the recovery rate. The results showed that the recovery of the revised method ranged from 98.22% to 103.65% (Table 8), which indicated the high precision of the revised method that might meet the requirements of quantitative analysis.

To determine the differences between ISO 15154:2003 and the revised method, samples A, B, C, D, E, F, and G were prepared, and the results were compared. The results showed that the F values of the two methods were below the critical value, indicating that there was no significant difference between the two methods (Table 9).