Ulrich H. Engelhardt

Chromatography of tea constituents

Modern chromatographic techniques such as high-performance liquid chromatography are currently the most helpful approach to the routine analysis of and research of non-volatile tea constituents. Using these techniques some errors in the more classical analytical techniques could be detected. Unfortunately, some of these methods of analysis are still in widespread use, even as official methods. However, knowledge of especially the polyphenols in tea is still lacking, and for many of the minor polyphenols no chromatographic methods for the determination exist.

Influence of catechins and theaflavins on the astringent taste of black tea brews

ZusammenfassungDie Korrelation der Gehalte an den fünf mengenmäßig bedeutendsten Catechinen und den vier Haupttheaflavinen zum sensorisch ermittelten adstringierenden Geschmack wurde untersucht. Catechine und Theaflavingehalte wurden mittels HPLC bestimmt. Es ergaben sich signifikante Korrelationen zum adstringierenden Geschmack für die Summe der Catechine und alle Einzelkomponenten außer Catechin. Zwischen Theaflavingehalten und adstringierendem Geschmack ergab sich keine Korrelation.SummaryThe concentrations of the five main catechins (flavanols), epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and four main theaflavins, theaflavin, theaflavin-3′-gallate, theaflavin-3-gallate, theaflavin-3,3′-digallate in black tea brew and their effects on astringency were investigated. The catechins and theaflavins were assayed by HPLC. Astringency, which was scaled from 1 to 9, was estimated by a trained panel. The correlations between astringency and content of total catechins and individual catechins, except catechin itself, were significant, whereas no correlation between theaflavins and astringency was observed.

Bioavailability and antioxidant potential of rooibos flavonoids in humans following the consumption of different rooibos formulations.

In a complete crossover design, a human study with twelve healthy male volunteers has been conducted using a placebo and different rooibos drinks (rooibos tea and an isolated active fraction) from unfermented rooibos (Aspalathus linearis). Blood and urine samples were collected before and up to 24h after consumption of the drinks. By HPLC-MS/MS, seven metabolites of aspalathin and nothofagin were identified in urine samples, as well as intact aspalathin and nothofagin. Moreover, sulphated, glucuronidated, methylated, both glucuronidated and methylated aspalathin, and glucuronidates of the aglycones of aspalathin and nothofagin were detected. The main metabolite excreted was methylated aspalathin. Most of the metabolites were detected after administration of both rooibos formulations. In plasma samples characteristic unchanged flavonoids derived from unfermented rooibos (e.g. aspalathin) were detected in trace quantities this is due to the changes in Table 5 after ingestion of both rooibos formulations. On average a total of 0.76nmol of flavonoids were detected during their peak concentration after intake of the rooibos tea, accounting for 0.26% compared to the total amount of flavonoids ingested. Despite the comparable intake of total flavonoids, only an overall 0.41nmol of flavonoids could be detected after ingestion of the isolated active fraction. No significant increase in plasma antioxidant capacity was observed using the ORAC assay giving rise to the assumption that the effects of rooibos flavonoids have to be detected using other endpoints.

Determination of Chlorogenic Acids with Lactones in Roasted Coffee

An HPLC method has been developed which allowed the determination of mono- and dicaffeoylquinic acids (CQA and di-CQA), corresponding lactones (CQL) and feruloylquinic acids (FQA) in roasted coffee within one chromatographic run. The elution order was verified by isolation of the individual compounds by preparative HPLC, chromatography of the fractions on the analytical HPLC system, NMR spectroscopy and thermospray LC-MS. At least 15 additional minor compounds had the spectra of hydroxycinnamic acids, some of them only occurring in roasted coffee. Caffeoyltryptophan, which has been identified some time ago by another working group could also be separated with this system. The average contents in commercial roasted coffee samples (n=12) were: 3-CQA, 5·0 g kg−1; 4-CQA, 6·2 g kg−1; 5-CQA, 11·4 g kg−1; 4-FQA, 0·7 g kg−1; 5-FQA, 1·4 g kg−1; 3-CQL, 2·1 g kg−1; 4-CQL, 1·0 g kg−1; 3,4-di-CQA, 0·7 g kg−1; 3,5-di-CQA, 0·4 g kg−1 and 4,5-di-CQA, 0·8 g kg−1 dry matter. There were only small differences between the different brands. Two series of samples with different degrees of roast produced from the same green coffee have also been analysed. One series was steam treated before roasting (a process which is commercially used to improve digestibility). Only in the initial stages of roasting (light roast) could a difference in the contents of the acids between both series be observed. Keeping coffee brews at an elevated temperature (4 h at 80°C) reduced the amounts of CQL to 60% of the initial value. The contents of 3-CQA and 4-CQA increased, whilst that of 5-CQA decreased. The overall contents of CQA decreased.

Determination of flavonol glycosides in green tea, oolong tea and black tea by UHPLC compared to HPLC

An UHPLC method for the determination of flavonol glycosides (FOG) from green and oolong tea vs. black tea has been developed for the first time. Sample clean-up method by means of polyamide column chromatography was optimized with multiple-step elution. Using UHPLC and HPLC with gradient elution and photodiode array detection, eighteen FOG compounds were determined with the aid of electrospray tandem mass spectrometry. These FOG compounds were qualified on both UHPLC and HPLC, and this UHPLC method successfully separated rutin (quercetin-3-O-rutinoside) and K-grg (kaempferol-3-O-glucorhamnoglucoside) while conventional HPLC method did not. The total amounts of FOG compounds in the tea samples were 2.32-5.67g/kg dry weight (calculated as aglycones), and there is no significant difference for the total FOG content among green tea, oolong tea and black tea. However, kaempferol glycosides are more abundant in green teas, while oolong tea has more quercetin and myricetin glycosides. In black tea quercetin glycosides were most abundant.

Residues of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites in plasma of dairy cows fed Fusarium contaminated maize and their relationships to performance parameters.

A feeding trial with dairy cows fed graded proportions of a Fusarium toxin contaminated maize containing mainly deoxynivalenol (DON) was carried out to relate the plasma levels of DON, zearalenone (ZEN) and their metabolites to the performance. German Holstein cows (n=30) were divided into three groups (n=10 in each): CON (0.02mgZEN and 0.07mgDON, per kg dry matter, DM), FUS-50 (0.33mg ZEN and 2.62mgDON, per kg DM), FUS-100 (0.66mgZEN and 5.24mgDON, per kg DM). The average performance level was characterised by daily DM intake, energy balance and milk yield which were not affected by the DON and ZEN levels in feed. DON, de-epoxy-DON (de-DON) and ZEN were detected simultaneously in all plasma samples. A linear relationship between toxin intake and plasma levels could be established. Moreover, a linear relationship between DON and de-DON concentration could be derived. It was concluded that DON and ZEN intake of 0.5mgZEN/kg and 5mgDON/kg (current guidance values) had no considerable effects on the performance parameter of dairy cows. Furthermore, increased plasma concentrations of ZEN, DON and de-DON may hint on toxin exposure through the diets.

Thermospray-LC-MS analysis of various groups of polyphenols in tea. II: Chlorogenic acids, theaflavins and thearubigins.

An account is given of the application of thermospray LC-MS in the analysis of caffeoyl- andp-coumaroylquinic acids, theaflavins and thearubigins in black tea. All compounds, except for the thearubigens, could be detected as the pseudo-molecular ion [M+H]+. In addition to [M+H]+, other species such as adducts with sodium, ammonium and potassium, as well as solvent clusters were observed. The formation of those adducts depended upon on the structure of the compound. A fragmentation of chlorogenic acids occurred at elevated temperatures yielding the constituents of the molecules (caffeic,p-coumaric and quinic acids).

Determination of flavanols, theogallin, gallic acid and caffeine in tea using HPLC

ZusammenfassungEs wird die Probenvorbereitung und HPLC-Bestimmung von Flavanolen (Catechinen), Theogallin, Gallussäure und Coffein beschrieben. Zur Reinigung der wäßrigen Aufgüsse wird eine Festphasenextraktion an RP-18-Kartuschen eingesetzt. Die HPLC-Trennung erfolgt an Hypersil-ODS mit einer Gradienten-Methode (Eluenten: 2% Essigsäure, Acetonitril). Die Methode ist für grüne, halbfermentierte (Oolong) und schwarze Tees sowie Tee-Extrakte geeignet. Die Gehalte in den untersuchten Tees lagen für Epigallocatechingallat (EGCG) zwischen 0,93 und 5,74 g/100 g i. Tr., für Epigallocatechin (EGC) zwischen 0,73 und 3,25 g/100 g, für Epicatechingallat (ECG) zwischen 0,17 und 1,16 g/100 g, für Catechin zwischen 0,04 und 0,48 g/100 g und für Epicatechin (EC) zwischen 0,1 und 0,8 g/100 g. Theogallin war zwischen 0,17 und 0,97 g/100 g, Gallussäure zwischen 0,06 und 0,62 g/100 g und Coffein zwischen 2,25 und 4,33 g/100 g enthalten. In Tee-Extrakten wurden 2–3fach höhere Gehalte gefunden. Die Gehalte an Catechinen waren erwartungsgemäß in den grünen Tees höher als in den schwarzen; für freie Gallussäure traf das Umgekehrte zu. Weiterhin wurde der Einfluß verschiedener Aufguß- bzw. Extraktionsmethoden auf die Gehalte der genannten Substanzen untersucht.SummaryAn RP-HPLC method for the determination of flavanols (catechins), theogallin, gallic acid and caffeine, including the sample preparation using a solid-phase extraction technique, has been developed. HPLC separation was performed on a Hypersil-ODS column using a gradient method with 2% acetic acid (aq) and acetonitrile as solvents. The method has been applied to green, oolong and black tea samples as well as tea extracts. The contents in different kinds of tea were 0.93–5.74 g/100 g dry mass for epigallocatechin gallate, 0.73–3.25 g/100 g dry mass for epigallocatechin, 0.17–1.16 g/100 g dry mass for epicatechingallate, 0.04–0.48 g/100 g dry mass for catechin, 0.1–0.8 g/100 g dry mass for epicatechin, 0.17–0.97 g/100 g dry mass for theogallin, 0.06–0.62 g/100 g dry mass for gallic acid and 2.25–4.33 g/100 g dry mass for caffeine. Instant tea contained 2–3-fold higher amounts. As expected the contents of flavanols in green teas were higher than in black teas, for gallic acid it was the opposite. Furthermore, the influence of different extraction methods on the contents of these compounds has been investigated.

HPLC analysis of chlorogenic acid lactones in roasted coffee

ZusammenfassungEs wird die Identifizierung von 3- und 4-Caffeoyl-γ-chinid (CQL) in Röstkaffee beschrieben. Die Lactone wurden mit Hilfe eines isokratischen RP-HPLC-Systems und Dioden-Array-Detektion von den übrigen Chlorogensäuren (CGA) getrennt und als Kaffeesäure-Derivate identifiziert. Thermospray-LC-MS-Spektren (Discharge- und Puffer-Ionisierung) zeigten das Quasi-Molekül-Ion und charakteristische Fragmente (z. B. Kaffeesäure, Chinid). Die Lactone wurden mittels Polyamid-Säulenchromatographie gefolgt von semipräparativer RP-HPLC aus dem Röstkaffee isoliert. Die Identifizierung erfolgte durch FTIR- und NMR-Spektroskopie. Es scheint ein Gleichgewicht zwischen den beiden Lactonen zu bestehen. Sowohl die Isolierung des 3- als auch die des 4-Caffeoylchinids liefert unter den experimentellen Bedingungen ein Gemisch aus den Substanzen. Die Gehalte der Lactone in Kaffeeproben des Handels reichten von 1.5 bis 3.5 g/kg bezogen auf Trockenmasse.AbstractThe identification of 3- (3-CQL) and 4-caffeoylquinic acid-γ-lactone (4-CQL) in roasted coffee is described. The lactones were separated from chlorogenic acids (CQA) by an isocratic reversed-phase (RP)-HPLC system and identified as caffeic acid derivatives using their ultraviolet spectra obtained on the fly with an diode array detector. Thermospray liquid-chromatography mass-spectrometry spectra (discharge ionisation, buffer ionisation) showed the quasi molecule ion and characteristic fragments (e.g. caffeic acid, quinide). The lactones were isolated from coffee by polyamide column chromatography followed by RP-HPLC on a semipreparative scale, and structures assigned using Fourier-transform infrared spectroscopy and various nuclear magnetic resonance techniques. There seems to be an equilibrium between the two lactones: isolation of 3-CQL and 4-CQL under the experimental conditions used yielded both lactones in the final product. The content of the caffeoylquinides in commercial coffee samples ranged from 1.5 to 3.5 g/kg dry matter.

Determination of flavone C-glycosides in tea

ZusammenfassungZur Bestimmung der Flavon-C-glykoside im Tee wurde eine HPLC-Methode ausgearbeitet. Bei der Probenaufarbeitung wurde zuerst ein wäßriger Aufguß durch Polyamid-Säulenchromatographie gereinigt. Anschließend erfolgte eine enzymatische Hydrolyse der O-glykosidischen Flavonoide und eine zweite Reinigung über Polyamid-Säulenchromatographie. Mit Hilfe der HPLC an einer Umkehrphase mit Gradientenelution und Dioden-Array-Detektion gelang die Trennung von insgesamt 8 Flavon-C-glykosiden. 7 Flavon-C-glykoside wurden mittels präparativer HPLC isoliert. Die Identifizierung erfolgte durch Cochromatographie, UV-Spektroskopie,1H- und13C-NMR-Spektroskopie (ein- und zweidimensional) und FAB(Fast Atom-Bombardment)-Massenspektrometrie. Die Isomerenpaare Apigenin-6-C-glucosyl-8-C-arabinosid (Schaftosid) und Apigenin-6-C-arabinosyl-8-C-glucosid (Isoschaftosid) sowie Luteolin-8-C-glucosid (Orientin) und Luteolin-6-C-glucosid (Isoorientin) konnten erstmals im Tee nachgewiesen werden. Bei den drei anderen Substanzen handelte es sich um Apigenin-8-C-glucosid (Vitexin), Apigenin-6-C-glucosid (Isovitexin) und Apigenin-6,8-di-C-glucosid (Vicennin-2), deren Vorkommen im Tee bereits bekannt war. Ein weiteres Flavon-C-glykosid konnte anhand des UV-Spektrums den Apigeninglykosiden zugeordnet werden. Die Flavon-C-glykoside wurden in 16 schwarzen und 2 grünen Tees verschiedener Anbäugebiete sowie in einem Oolong-Tee quantitativ bestimmt. Die Gesamtgehalte bewegten sich zwischen 0,48 und 2,69 g/kg i.Tr. Die Luteolinglykosidgehalte lagen in allen Fällen weit unterhalb der Gehalte der identifizierten Apigeninglyko-side. Bei den meisten schwarzen Tees wurde unabhängig von der Herkunft ein ähnliches Flavon-C-glykosid-muster beobachtet. Lediglich ein chinesischer und ein indonesischer schwarzer Tee sowie die beiden grünen Tees zeigten eine andere Verteilung der Flavon-C-glykoside. Herkunftsspezifische Charakeristika konnten anhand dieser Ergebnisse nicht festgestellt werden. In Hydrolysaten von höhermolekularen Polyphenolfraktionen (Mr>5000) aus Schwarzteeaufgüssen konnten geringe Mengen an Flavon-C-glykosiden (1,2–2,2 mg/g der isolierten Fraktion) nachgewiesen werden.AbstractAn HPLC method for the determination of flavone C-glycosides (FCG) from black tea has been developed. Sample clean-up was accomplished by means of polyamide column chromatography, followed by enzyme hydrolysis of interfering compounds such was flavonol glycosides and a second polyamide column Chromatographic step. Using HPLC with gradient elution and photodiode array detection eight FCG were separated. Seven FCG were isolated by means of preparative HPLC. Identification was carried out using co-chromatography, FAB(Fast Atom Bombardment)-mass spectrometry and various nuclear magnetic resonance techniques. Apigenin 6-C-glucosyl-8-arabinoside (schaftoside) and apigenin 6-C-arabinosyl-8-C-glucoside (isoschafto-side) as well as luteolin 8-C-glucoside (orientin) and luteolin 6-C-glucoside (isoorientin) have been detected in tea for the first time. Three of the other compounds have been identified as apigenin 8-C-glucoside (vitexin), apigenin 6-C-glucoside (isovitexin) and apigenin 6,8-di-C-glucoside (vicenin-2). Their occurrence in tea has been previously reported. From its UV spectrum another compound was concluded to be an apigenin glycoside. The FCG were quantified in a variety of teas of different origins (16 black, two green and one oolong). The total amounts of the FCG were 0.48–2.69 g/kg dry weight. The FCG pattern of teas of different origins were similar to each other and no origin-dependent characteristics have yet been observed. Small amounts of FCG (1.2–2.2 mg/g) were detected in hydrolysates of high relative molecular mass fractions (Mr>5000) of a black tea liquor.