Study On Chemical Fingerprinting Of Dark Teas From Different Regions

Dark tea is fermented in the later period, and it is the special category of tea produced in China. Hunan, Sichuang, Yunan, Guangxi, hubei and Shaanxi provinces are the main production regions of dark tea. The determination of the main index components and sensory analysis of dark tea were the main methods to the quality evaluation in the past. The production and trade volumes of dark tea have continuously increased in recent years, and the quality differences exist between dark teas from different regions. Chemical fingerprinting is an integral expression of the consitutents in dark tea and thus can properly express their properties of complexity and integrity, and it can supply a reference method to the identification and quality assessment of dark teas from different regions. In this study, 108 dark tea samples from six different regions were analyzed by chromatography and spectroscopy. A method was developed for simultaneous determination of eleven chemical components from tea by HPLC-DAD-ELSD. The chromatographic and spectral conditions, and the extraction and preparation conditions were optimized. The HPLC-DAD, HPLC-ELSD and FTIR fingerprinting of dark teas from different regions were established by HPLC and FTIR. The main results were as follows:1. The common pattern of chromatographic fingerprinting of dark teas from different regions respectively and in China were established by HPLC using DAD and ELSD. The dark tea simultaneously contains chromophoric and non-chromophoric components; for example, the amino acids in dark tea have poor UV absorptions; so ELSD was employed to establish HPLC-ELSD fingerprinting of dark tea. To establish the HPLC fingerprinting analysis method of dark tea, the extraction and chromatographic conditions were optimized. A suitable chromatographic system was established using a linear gradient elution with methanol and 0.1% trifluoroacetic acid as the mobile phase, the column temperature was at 30℃, the DAD detection wavelength was 280nm, the flow speed of mobile phase was 1.0mL/min, the drift tube temperature was at 110℃ and the flow speed of air was 2.8L/min. A suitable extraction condition was established using ultra pure water as the extraction solvent, the bath temperature was at 90℃, extracting time was 15min, and the ratio of solid to liquid was 1:70. The HPLC fingerprinting were established with good stability, precision and reproducibility. The average relative retention time, common peaks, average relative peak area and the peaks of the same chemical compound were assayed respectively. The results showed that 13 common peaks of dark tea samples in China were demarcated in the chromatographic fingerprinting by HPLC-DAD, and 10 common peaks were demarcated in the chromatographic fingerprinting by HPLC-ELSD; 31,29,19,27,26, 24 common peaks from Hunan, Sichuang, Yunan, Guangxi, hubei and Shaanxi provinces respectively were demarcated in the chromatographic fingerprinting by HPLC-DAD, and 16,16,12,16,16,18 common peaks were demarcated respectively in the chromatographic fingerprinting by HPLC-ELSD. Compared the average peak area and the average relative peak area of the common peaks of dark tea samples from different regions and the results indicated that there were certain differences on the average peak area and the average relative peak area. The fingerprinting was evaluated by the similarity evaluation software published by committee of pharmacopeia. The similarity analysis between 108 dark tea samples and the common pattern of HPLC fingerprinting of dark tea samples from different regions respectively showed that the similarity of 102 dark tea samples were more than 0.900. The similarity analysis between common pattern of dark tea samples in China and the control showed that the similarity of the control samples were less than 0.900. The cluster analysis of dark tea samples manufactured in 2012 was performed by SPSS software, and the results showed that the 36 dark tea samples from different regions could be divided into six types.2. Some common peaks in the HPLC chromatograms of dark tea samples from different regions were identified based on the comparation of their relative retention times to the 11 reference substances of main bioactive components from tea. This chromatographic method could be used for determination of catechins, caffeine, amino acids, gallic acid in tea simultaneously.3. Dark tea is a complex mixture. FTIR was used to compare the infrared absorption spectral differences of dark tea samples from different regions. FTIR reflected the overlying of the blending consititutive molecule absorbs in dark tea, as long as the quality and amounts of chemical components in dark tea are relatively stable and the consistent preparation conditions are to be used, therefore their infrared spectra ought to be relatively stable. To establish the FTIR fingerprinting analysis method of dark teas, the preparation and spectral conditions were optimized. A suitable preparation condition was established using the sieve size of 200 mesh, mass ratio of dark tea powder to KBr of 1:100 and the scan number was 32. The resolution of FTIR was 4 cm-1 and the scanning range was 4000cm-1～400cm-1. Infrared absorption spectrometry was used to compare the infrared absorption spectral differences of 108 dark tea samples from 6 different regions. The characteristic peaks of the FTIR fingerprinting of dark tea samples were recognized and compared. The FTIR fingerprinting was established with good stability, precision and reproducibility. The results showed that the FTIR fingerprinting of the 108 dark tea samples contained 14 common peaks and there were some differences in the number and intensity of the absorption peaks in the wave number range of 1800 cm-1～700 cm-1 because of different chemical composition and relative content. The infrared spectrograms of the dark tea samples in different years appeared to be similar, but there were some differences in the absorbance of the 14 common absorption peaks. The absorbance of the common absorption peaks decreased with the duration of storage time. The similarity analysis results of the second derivative IR spectrum of the dark tea samples from different regions indicated that the highest similarity was Sichuan and Shaanxi, and the similarity was 87.1%; the lowest similarity was Yunnan and Shaanxi, and the similarity was 61.7%. The similarity of the second derivative IR spectrum of the dark tea samples from different regions was above 90.0% compared with the average second derivative IR spectrum of the dark tea samples in China. The results from a cluster analysis of the absorbance in the wave number range of 1800 cm-1～700 cm-1 indicated that the 36 average infrared spectrograms of the dark tea samples from different regions could be divided into six types. The results from a cluster analysis of the absorbance and the similarity analysis results of the second derivative IR spectrum allowed the discrimination of dark tea samples from different regions.