Modified graphene-silica as a sorbent for in-tube solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry. Determination of xanthines in coffee beverages.

Given the increasing need for analyzing natural or contaminating compounds in complex food matrices in a simple and automated way, coupling miniaturized sample preparation techniques with chromatographic systems have become a growing field of research. In this regard, given the low extraction efficiency of conventional sorbent phases, the development of materials with enhanced extraction capabilities is of particular interest. Here we present several synthesized graphene-based materials supported on aminopropyl silica as sorbents for the extraction of xanthines. The synthesized materials were characterized by infrared spectroscopy and scanning electron microscopy. Aminopropyl silica coated with graphene oxide and functionalized with octadecylsilane/end-capped (SiGOC18ecap) showed the best performance for xanthines extraction. Hence, this material was employed as an in-tube solid phase microextraction (in-tube SPME) device coupled online with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and applied for the analysis of xanthines in roasted coffee samples. Extraction parameters and detection conditions were optimized. The method showed low limits of quantification (0.3-1.0 µg L-1), precision as relative standard deviation (RSD) values lower than 10%, recoveries between 73 and 109%, and pre-concentration factors from 5.6 to 7.2. Caffeine was determined in all ground roasted and instant coffee samples, in a wide range (0.9 to 36.8 mg g-1), and small amounts of theobromine and theophylline were also detected in some samples. This work demonstrated that functionalized graphene-based materials represent a promising new sorbent class for in-tube SPME, showing improved extraction capacity. The method was efficient, simple, and fast for the analysis of xanthines, demonstrating an excellent potential to be applied in other matrices.

Mass spectrometry imaging of thin-layer chromatography plates using laser desorption/low-temperature plasma ionisation.

Thin-layer chromatography (TLC) is a classic method for the separation and analysis of complex mixtures. Biological assays, chemical derivatisation and spectroscopy techniques are compatible with TLC and provide extra information about isolated compounds. However, coupling TLC to mass spectrometry is hampered by the difficulty to desorb the analytes from the silica surfaces. In this study, we used a multimodal ion source for laser desorption (LD) and low-temperature plasma (LTP) post-ionisation. Efficient desorption was reached by covering the TLC plates with activated carbon. Regions of interest can be analysed by spots, by lines or by area. We show the separation of methylxanthines from coffee, tea and cocoa preparations by TLC, with subsequent mass spectrometry imaging (MSI). Using a lateral resolution of 400 µm × 400 µm, allowed the acquisition of 21 895 spectra in 2.4 h (2.5 pixels per s). Further, we demonstrate the possibility of direct mass fragmentation studies and quantification. We mounted the system on an Open LabBot with a theoretical lateral resolution of 12.5 µm and performed the visualisation of ions of interest and the pixel-wise review of mass spectra with our free software RmsiGUI (). This non-proprietary and modular platform enables the cost-efficient adaption of the system and further development by the community.

LC-MS/MS-based metabolomic analysis of caffeine-degrading fungus Aspergillus sydowii during tea fermentation.

Natural microorganisms involved in solid-state fermentation (SSF) of Pu-erh tea have a significant impact on its chemical components. Aspergillus sydowii is a fungus with a high caffeine-degrading capacity. In this work, A. sydowii was inoculated into sun-dried green tea leaves for SSF. Metabolomic analysis was carried out by using UPLC-QTOF-MS method, and caffeine and related demethylated products were determined by HPLC. The results showed that A. sydowii had a significant (P < 0.05) impact on amino acids, carbohydrates, flavonoids, and caffeine metabolism. Moreover, A. sydowii could promote the production of ketoprofen, baclofen, and tolbutamide. Along with caffeine degradation, theophylline, 3-methylxanthine, 1,7-dimethylxanthine, 1-methylxanthine, and 7-methylxanthine were increased significantly (P < 0.05) during inoculated fermentation, which showed that demethylation was the main pathway of caffeine degradation in A. sydowii secondary metabolism. The absolute quantification analysis showed that caffeine could be demethylated and converted to theophylline and 3-methylxanthine. Particularly, about 93.24% of degraded caffeine was converted to theophylline, 27.92 mg/g of theophylline was produced after fermentation. PRACTICAL APPLICATION: Aspergillus sydowii could cause caffeine degradation in Pu-erh tea solid-state fermentation and produce theophylline through the demethylation route. Using a starter strain to ferment tea leaves offers a more controllable, reproducible, and highly productive alternative for the biosynthesis of theophylline.

Extraction of methyl xanthines and their UHPLC-DAD determination in consumable beverages used in Eastern province of Saudi Arabia.

Coffee and tea are the most widely consumed beverages worldwide. However, the consumer may be unaware of the exact amount of methyl xanthine (MX, i.e. caffeine [C], theobromine [TB] and theophylline [TH]) consumed, as most of the products do not list the proper amounts. This may lead to serious risks including cardiovascular, kidney and stimulant effects. The aim of the study was to determine the MX amount in ready-to-use beverages (coffee and tea) collected from various outlets in the city of Al-Khobar, Saudi Arabia. Forty different samples of espresso, black coffee and red tea were collected. A fast, reliable and efficient UHPLC-DAD method was developed and validated for MX determination. Total lipids were extracted and fractionated in order to determine glycolipids, phospholipids and neutral lipids. The r2 value for the method was 0.980-0.988 in a linearity range of 0.5-200 ppm. The range for MX (C [0.02-2.39 mg/ml], TB [0.00-0.10 mg/ml] and TH [0.00-0.004 mg/ml]) and total lipids was 1-5 g. The amount of glycolipids (3.1 g) was higher among the lipid fractions followed by phospholipids (1.8 g) and neutral lipids (0.25 g). In general, espresso beverages (20-30 ml) contained high amounts of MX whereas black coffee beverages contained high amount of lipids. Most of the beverages expressed C, TB, TH, lipids or their fractions; however, the product with high amounts of MX and lipids at the same time was espresso (brands Chemistry and Wogard). Although the MX and lipid levels in these beverages well below the allowed limits, care must still be taken, especially when using the beverages with high serving volumes (200-250 ml) or coffee prepared via the filter method i.e. black coffee, using a high temperature for a longer time.

Solvent and temperature effect of accelerated solvent extraction (ASE) coupled with ultra-high-pressure liquid chromatography (UHPLC-PDA) for the determination of methyl xanthines in commercial tea and coffee.

BACKGROUND: Methyl xanthines (MX), known for its psychostimulant effect, occurs mostly in tea and coffee samples. However most of the market available products does not mention the proper amount and quality of MX present where, its consumption in high amount may pose health risks. AIM OF THE STUDY: To develop and validate a fast, efficient and reliable method of MX extraction along with a sensitive, rapid and precise method for simultaneous analysis of MX i.e. Theobromine (TB), Theophylline (TH) and Caffeine (C), with application in commercial tea and coffee samples. MATERIALS AND METHODS: Accelerated Solvent Extraction (ASE) was utilized for the first time to extract MX, whereas UHPLC-DAD was applied in order to quantify MX. RESULTS: ASE resulted a high extract yield (940.22 ± 192.28 mg/g) with optimized conditions of temperature (100 °C) and solvent (MeOH). UHPLC-DAD showed retention time (min) of 1.51 (TB), 1.81 (TH), 2.30 (C) with r2 values (0.980-0.988). Average MX (µg/mL) was as; TB (14.73 ± 20.9), TH (32.05 ± 55.5), C (121.87 ± 32.3). The method application in commercial samples showed a high extract yield with MX concentration (mg/g) as; TB (0.13-0.38), TH (0-0.55), C (7.14-11.20). Temperature and solvent variation showed important correlation with samples in terms of extraction yield. CONCLUSION: ASE-UHPLC/DAD revealed a fast and sensitive method of MX extraction, quantification and quality determination in market available tea and coffee samples.

Effects of hydroalcoholic and enzyme-assisted extraction processes on the recovery of catechins and methylxanthines from crude and waste seeds of guarana (Paullinia cupana).

Catechins and methylxanthines are natural molecules in guarana (Paullinia cupana) that are associated with antioxidant and stimulatory effects in the human body. There are few natural sources of these antioxidants. The most popular molecule used in foods and beverages is caffeine, which, most of the time, is derived from synthetic sources. In this work, cold hydroalcoholic (CHM), hot hydroalcoholic (HHM), and aqueous enzymatic maceration (AEM) were applied to crude (CG) and waste guarana seeds (WG) to process these materials into natural added-value products with enhanced levels of catechin, epicatechin, epicatechin gallate, caffeine, theobromine and theophylline. The highest level of catechins and methylxanthines was extracted with HHM. Nevertheless, AEM enhanced the global yield in the extract, probably due to the solubilization of other substances. The maceration procedures applied to guarana contributed to the valorization of this plant crop by providing antioxidant sources with clear applications in food and nonfood industries.

Multivariate analysis of the composition of bioactive in tea of the species Camellia sinensis.

This study deals with the development of a separation and quantification method by high performance liquid chromatography with a diode array detector (HPLC- DAD), for the determination of phenolic acids and methylxanthines in tea (Camellia Sinensis) samples. Six phenolic acids (Gallic, trans-cinnamic, caffeic, ferulic and p-coumaric acids) and two methylxanthines (caffeine, theobromine) were studied, according to the principles of green chemistry, with the goal of comparing teas with different processing level. After development and validation of separation and extraction methods was applied to 11 extracts obtained by infusions of green and black tea samples in commercial areas of the city of Salvador-BA, showing feasible and efficient. Was employed multivariate data analysis and the Tukey test to correlate the chromatographic profile with the different samples. Principal component and analysis and hierarchical cluster analysis were used for the chromatographic analysis, allowing the visualization of two groups, formed by green and black tea samples.

Green coffee seed residue: A sustainable source of antioxidant compounds.

Oil extraction from green coffee seeds generates residual mass that is discarded by agribusiness and has not been previously studied. Bioactive secondary metabolites in coffee include antioxidant phenolic compounds, such as chlorogenic acids. Coffee seeds also contain caffeine, a pharmaceutically important methylxanthine. Here, we report the chemical profile, antioxidant activity, and cytotoxicity of hydroethanolic extracts of green Coffea arabica L. seed residue. The extracts of the green seeds and the residue have similar chemical profiles, containing the phenolic compounds chlorogenic acid and caffeine. Five monoacyl and three diacyl esters of trans-cinnamic acids and quinic acid were identified by ultra-performance liquid chromatography/electrospray ionization-quadruple time of flight mass spectrometry. The residue extract showed antioxidant potential in DPPH, ABTS, and pyranine assays and low cytotoxicity. Thus, coffee oil residue has great potential for use as a raw material in dietary supplements, cosmetic and pharmaceutical products, or as a source of bioactive compounds.

Ilex paraguariensis: Potential antioxidant on aluminium toxicity, in an experimental model of Alzheimer's disease.

Ilex paraguariensis, yerba mate is a native plant from the southern region of Brazil. Studies showed that yerba mate has an antioxidant potential, which could help to reduce the risk of developing neurodegenerative diseases, as Alzheimer's Disease (AD). It's known that I. paraguariensis grows in acid soils with aluminium (Al), which is bioavailable in these soils. Al has a neurotoxic potential related with the progression of neurological disorders. This study aim was to evaluate the potential of I. paraguariensis in the etiology of AD using strains of Caenorhabditis elegans and the concentration of Al and antioxidants in the yerba mate extract. The results of the I. paraguariensis infusions made at 65°C and at 75° C show that there was no significant difference between both temperatures when preparing the tea infusion in relation to the presence of Al, methylxanthines, phenolic compounds and flavonoids. Additionally, in the case of Al, there was no difference between the extracts prepared at both temperatures. The behavioral parameters of C. elegans were altered after a long-term exposure to both factors: I. paraguariensis extract and Al. Through the antioxidant levels results along with the Al content on the Acetylcholinesterase (AChE) activity it is possible to observe that the acute and chronic exposure to Al and I. paraguariensis leaves extract are very similar to wild-type worms. Moreover, we can observe that the results in both the transgenic strains long-term exposed to I. paraguariensis leaves extract and to the Al concentrations presented an increase in the AChE activity.

Daily Intake of Chlorogenic Acids from Consumption of Maté (Ilex paraguariensis A.St.-Hil.) Traditional Beverages.

The main objective of this study was to investigate the daily intake of chlorogenic acids (CGAs) and methylxanthines by consumers of maté traditional beverages (MTBs), terere and chimarrão (Ilex paraguariensis A.St.-Hill). In the studied population (450 citizens from Toledo, PR, Brazil), 63% consume the chimarrão and 37% terere, with weighted mean daily intakes estimated at 648-2160 and 244-746 mL, respectively. For every 100 mL of beverage consumed, the average amount of total phenol compounds extracted was 673.6 mg for chimarrão and 1184.9 mg for terere. Regarding CGAs composition, caffeoylquinic acids (CQAs) constitute about 38.4% for chimarrão and 55.3% for terere, and dicaffeoylquinic acids (diCQAs) represent 61.6 and 44.7% of the extracted compounds, respectively. The daily intake of phenolic compounds by MTB consumers was estimated for chimarrão (512.5-1708.5 mg/day) and terere (583.0-1779.7 mg/day). These results allow us to conclude that MTBs are important dietary sources of CGAs, mainly CQAs and di-CQAs.

Study of nutritional value of dried tea leaves and infusions of black, green and white teas from Chinese plantations

Background: The processing of tea leaves determines the contents of bioactive ingredients, hence it should be expected that each variety of tea, black, red or green, will represent a different package of compounds of physiological importance. Taste and aroma, as well as price and brand are the main factors impacting consumers' preferences with regard to tea of their choice; on the other hand consumers less frequently pay attention to the chemical composition and nutritional value of tea. Objective: The purpose of the study was assessment of the nutritional value of black, green and white high-quality tea leaf from Chinese plantations based on the chemical composition of the dried leaves as well as minerals and caffeine content in tea infusions. Material and methods: The research material included 18 high-quality loose-leaf teas produced at Chinese plantations, imported to Poland, and purchased in an online store. The analyses included examination of the dried tea leaves for their chemical composition (contents of water, protein, volatile substances and ash) and assessment of selected minerals and caffeine contents in the tea infusions. Results: High-quality Chinese green teas were found with the most valuable composition of minerals, i.e. the highest contents of Zn, Mn, Mg, K, Ca and Al and the highest contents of protein in comparison to the other products. Chinese black teas had the highest contents of total ash and caffeine and white teas were characterized with high content of volatile substances, similar to the black teas, and the highest content of water and the lowest content of total ash. Conclusions: The three types of tea brews examined in the present study, in particular green tea beverages, significantly enhance the organism's mineral balance by providing valuable elements

Variation of theanine, phenolic, and methylxanthine compounds in 21 cultivars of Camellia sinensis harvested in different seasons.

This is the first study to use chemometric methods to differentiate among 21 cultivars of Camellia sinensis from China and between leaves harvested at different times of the year using 30 compounds implicated in the taste and quality of tea. Unique patterns of catechin derivatives were observed among cultivars and across harvest seasons. C. sinensis var. pubilimba (You 510) differed from the cultivars of C. sinensis var. sinensis, with higher levels of theobromine, (+)-catechin, gallocatechin, gallocatechin gallate and theasinensin B, and lower levels of (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), respectively. Three cultivars of C. sinensis var. sinensis, Fuyun 7, Qiancha 7 and Zijuan contained significantly more caffeoylquinic acids than others cultivars. A Linear Discriminant Analysis model based on the abundance of 12 compounds was able to discriminate amongst all 21 tea cultivars. Harvest time impacted the abundance of EGC, theanine and afzelechin gallate.

Genetic and Phytochemical Analysis to Evaluate the Diversity and Relationships of Mate (Ilex paraguariensis A.St.-Hil.) Elite Genetic Resources in a Germplasm Collection.

The aim of this study was to evaluate the phytochemical and genetic diversity, relationships and identification of mate (Ilex paraguariensis A.St.-Hil.) elite genetic resources belonging to the Brazilian germplasm collection and mate breeding program. Mate has been studied due to the presence of phytochemical compounds, especially methylxanthines and phenolic compounds. The samples were collected from the leaves of 76 mate elite genetic resources (16 progenies × 5 localities). Total DNA was extracted from mate leaves and 20 random primers were used for DNA amplification. Methylxanthines (caffeine and theobromine) and phenolic compounds (chlorogenic, neochlorogenic, and criptochlorogenic acids) were quantified by HPLC. The genetic divergence estimated was higher within (92%) than among (8%) the different populations. Analysis of genetic distance between origins provided the formation of two groups by UPGMA cluster analysis, with higher polymorphism (94.9%). The average content of caffeine ranged from 0.01 to 1.38% and theobromine of 0.10 - 0.85% (w/w). The caffeoylquinic acids concentrations (1.43 - 5.38%) showed a gradient 3-CQA > 5-CQA > 4-CQA. The coefficient of genetic variation (CVg) was of low magnitude for all mono-caffeoylquinics acids. Significant correlations (positive and negative) were observed between the phytochemical compounds. Genetic diversity analysis performed by RAPD markers showed a greater intra-populational diversity; genetic resources with low caffeine and higher theobromine content were identified and can be used in breeding programs; the correlation between methylxanthines and phenolic compounds can be used as a good predictor in future studies.

Nutritional composition, bioactive compounds and volatile profile of cocoa beans from different regions of Cameroon.

Analysis of the complex composition of cocoa beans provides fundamental information for evaluating the quality and nutritional aspects of cocoa-based food products, nutraceuticals and supplements. Cameroon, the world's fourth largest producer of cocoa, has been defined as "Africa in miniature" because of the variety it habitats. In order to evaluate the nutritional characteristics of cocoa beans from five different regions of Cameroon, we studied their polyphenolic content, volatile compounds and fatty acids composition. The High Performance Thin Layer Chromatography (HPTLC) analysis showed that the Mbalmayo sample had the highest content of theobromine (11.6 mg/g) and caffeic acid (2.1 mg/g), while the Sanchou sample had the highest level of (-)-epicatechin (142.9 mg/g). Concerning fatty acids, the lowest level of stearic acid was found in the Mbalmayo sample while the Bertoua sample showed the highest content of oleic acid. Thus, we confirmed that geographical origin influences the quality and nutritional characteristics of cocoa from these regions of Cameroon.

Comparative characterisation of green tea and black tea cream: physicochemical and phytochemical nature.

Tea cream is prevalent in various types of tea, yet a comparison of the mechanism of creaming in different teas remains uncertain. Here, we compared physicochemical characteristics, phytochemical composition, and simulated digestive profiles of green tea and black tea cream, looking to exploit their concentration and structure based mechanisms and in vitro bioaccessibility. Green tea cream particles were roughly one order of magnitude larger than those of black tea in size. Moreover, creaming concentrations of catechins, proteins and methylxanthines of green tea were dramatically higher than black tea. As major creaming components, gallated catechins, theaflavins, thearubigins, theabrownines, proteins and methylxanthines also exhibited high creaming affinities. Green tea cream particles, which were completely destroyed by simulated digestion, had few impacts on digestive recoveries of catechins and methylxanthines. In comparison, black tea cream particles were more stable under mimicking digestion, and clarification remarkably decreased the in vitro bioaccessibility of catechins and methylxanthines.

Methods of biological fluids sample preparation - biogenic amines, methylxanthines, water-soluble vitamins.

In recent years demands on the amount of information that can be obtained from the analysis of a single sample have increased. For time and economic reasons it is necessary to examine at the same time larger number of compounds, and compounds from different groups. This can best be seen in such areas as clinical analysis. In many diseases, the best results for patients are obtained when treatment fits the individual characteristics of the patient. Dosage monitoring is important at the beginning of therapy and in the full process of treatment. In the treatment of many diseases biogenic amines (dopamine, serotonin) and methylxanthines (theophylline, theobromine, caffeine) play an important role. They are used as drugs separately or in combination with others to support and strengthen the action of other drugs - for example, the combination of caffeine and paracetamol. Vitamin supplementation may be also an integral part of the treatment process. Specification of complete sample preparation parameters for extraction of the above compounds from biological matrices has been reviewed. Particular attention was given to the preparation stage and extraction methods. This review provides universal guidance on establishing a common procedures across laboratories to facilitate the preparation and analysis of all discussed compounds.

Effects of extreme climate events on tea (Camellia sinensis) functional quality validate indigenous farmer knowledge and sensory preferences in tropical China.

Climate change is impacting agro-ecosystems, crops, and farmer livelihoods in communities worldwide. While it is well understood that more frequent and intense climate events in many areas are resulting in a decline in crop yields, the impact on crop quality is less acknowledged, yet it is critical for food systems that benefit both farmers and consumers through high-quality products. This study examines tea (Camellia sinensis; Theaceae), the world's most widely consumed beverage after water, as a study system to measure effects of seasonal precipitation variability on crop functional quality and associated farmer knowledge, preferences, and livelihoods. Sampling was conducted in a major tea producing area of China during an extreme drought through the onset of the East Asian Monsoon in order to capture effects of extreme climate events that are likely to become more frequent with climate change. Compared to the spring drought, tea growth during the monsoon period was up to 50% higher. Concurrently, concentrations of catechin and methylxanthine secondary metabolites, major compounds that determine tea functional quality, were up to 50% lower during the monsoon while total phenolic concentrations and antioxidant activity increased. The inverse relationship between tea growth and concentrations of individual secondary metabolites suggests a dilution effect of precipitation on tea quality. The decrease in concentrations of tea secondary metabolites was accompanied by reduced farmer preference on the basis of sensory characteristics as well as a decline of up to 50% in household income from tea sales. Farmer surveys indicate a high degree of agreement regarding climate patterns and the effects of precipitation on tea yields and quality. Extrapolating findings from this seasonal study to long-term climate scenario projections suggests that farmers and consumers face variable implications with forecasted precipitation scenarios and calls for research on management practices to facilitate climate adaptation for sustainable crop production.

AG-690/11026014, a novel PARP-1 inhibitor, protects cardiomyocytes from AngII-induced hypertrophy.

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme, as a sensor of DNA damage, could convert nicotinamide adenine dinucleotide (NAD) into long poly(ADP-ribose) chains and regulate many cellular processes, including DNA repair, gene transcription, cell survival and chromatin remodeling. However, excessive activation of PARP-1 depletes its substrate NAD and leads to cell death. Mounting evidences have shown that PARP-1 overactivation plays a pivotal role in the pathogenesis of cardiac hypertrophy and heart failure. In present study, a novel PARP-1 inhibitor AG-690/11026014 (6014) was identified based on virtual screening and validated by bioassay. Our results further showed that 6014 prevented the cardiomyocytes from AngII-induced hypertrophy, accompanying attenuation of the mRNA and protein expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), and reduce in the cell surface area. Additionally, 6014 reversed the depletion ofcellular NAD and SIRT6 deacetylase activity induced by AngII in cardiomyocytes. These observations suggest that anti-hypertrophic effect of 6014 might be partially attributed to the rescue of NAD depletion and subsequent restoring of SIRT6 activity by inhibition of PARP-1. Moreover, 6014 attenuated the generation of oxidative stress via suppression of NADPH oxidase 2 and 4, which might probably contribute to the inhibition of PARP-1.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Development and validation of a rapid ultra high pressure liquid chromatographic method for the determination of methylxanthines in herbal infusions.

An ultra high pressure liquid chromatographic method coupled with diode array detector (UHPLC-DAD) has been developed and validated for the fast separation and determination of three major methylxanthines, i.e., caffeine, theophylline and theobromine, in various herbal beverages. Isocratic elution using 0.1vol% formic acid/CH3OH (92.5:7.5, v/v) enabled the completion of the separation cycle in less than 3min using a flow rate of 0.7mL/min and a column temperature of 50°C. Validation of the method included linearity (0.5-50mg/L), limits of detection (12-35µg/L) and quantification (40-120µg/L), precision, matrix effect and accuracy. The percent recoveries ranged between 90 and 108%.