Position-Specific Isotope Analysis of Xanthines: A (13)C Nuclear Magnetic Resonance Method to Determine the (13)C Intramolecular Composition at Natural Abundance.

The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.

Isolation and identification of spermidine derivatives in tea (Camellia sinensis) flowers and their distribution in floral organs.

BACKGROUND: Recently, tea (Camellia sinensis) flowers have attracted increasing interest because of their content of bioactive compounds such as catechins. The aim of this study was to investigate the occurrence of some characteristic compounds in tea flowers. RESULTS: A principal component analysis of metabolites using ultra-performance liquid chromatography/time-of-flight mass spectrometry showed differences in metabolite profile between flowers and leaves of C. sinensis var. Yabukita. Four spermidine derivatives were isolated from tea flowers. One of them was determined as N(1) ,N(5) ,N(10) -tricoumaroyl spermidine based on NMR, MS and UV data. The other three were identified as feruoyl dicoumaroyl spermidine, coumaroyl diferuoyl spermidine and triferuoyl spermidine based on MS(n) data. Tricoumaroyl spermidine as the major spermidine conjugate was not detected in tea leaves. Furthermore, it decreased during floral development and mainly occurred in anthers. CONCLUSION: This study has provided the first evidence that spermidine-phenolic acid conjugates occur in tea flowers in considerable amounts. Their presence should prompt a reconsideration of the ecological role of tea flowers. From an economic point of view, tea flowers might be suitable as a raw material in the healthcare food and pharmaceutical industries.

Occurrence of glycosidically conjugated 1-phenylethanol and its hydrolase ß-primeverosidase in tea (Camellia sinensis) flowers.

A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE ß-d-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE ß-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase ß-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, ß-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time.

Purification and gas chromatography-combustion-isotope ratio mass spectrometry of aroma compounds from green tea products and comparison to bulk analysis.

A method for carbon isotope ratio (δ(13)C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ(13)C value from bulk isotope analyses. The δ(13)C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl ß-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl ß-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ(13)C analysis of volatile compounds. On the basis of the gas chromatography-combustion-isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (Z)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ(13)C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea.