Analysis of Urinary Metabolites of Tea Catechins by Liquid

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Chem. Res. Toxicol. 2001, 14, 702-707

Analysis of Urinary Metabolites of Tea Catechins by Liquid Chromatography/Electrospray Ionization Mass Spectrometry Chuan Li,† Xiaofeng Meng,† Bozena Winnik,‡ Mao-Jung Lee,† Hong Lu,† Shuqun Sheng,§ Brian Buckley,‡ and Chung S. Yang*,†,‡ Laboratory for Cancer Research, College of Pharmacy, and Department of Chemistry, Rutgers, The State University of New Jersey, and Environmental and Occupational Health Sciences Institute, Piscataway, New Jersey 08854 Received December 15, 2000

Tea has been proposed to have beneficial health effects which have been attributed to the polyphenolic compounds known as catechins. The bioavailability and biotransformation of these compounds, however, are not clearly understood. In this study, we used liquid chromatography/ electrospray ionization-mass spectrometry (LC/ESI-MS) to determine urinary glucuronidated and sulfated tea catechins and their metabolites (including methylated and ring-fission metabolites) based on the detection of deprotonated molecular ions and aglycone fragment ions. The compound resolution was achieved both chromatographically and mass spectroscopically. After green tea administration, the major conjugates appeared in human, mouse, and rat urine samples were identified as monoglucuronides and monosulfates of (-)-epigallocatechin (EGC) and (-)-epicatechin. We also found O-methyl-EGC-O-glucuronides and -O-sulfates and O-methyl-epicatechin-O-sulfates in human urine. (-)-5-(3′,4′,5′-Trihydroxyphenyl)-γ-valerolactone (M4) and (-)-5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (M6), the ring-fission metabolites of EGC and (-)-epicatechin, respectively, were also predominantly in monoglucuronide and monosulfate forms in the urine. In comparison to rats, the urinary metabolite profiles of tea catechins in mice resemble more closely to those in humans. This is the first report describing direct simultaneous analysis of multiple tea catechin conjugates in urine samples. This method will allow more thorough investigations of the biotransformation of tea polyphenols. Tea, a popular beverage, is a significant source of flavonoid antioxidants which may play a role in the prevention of cancer and cardiovascular disease (1-5). The major tea flavonoids are flavan-3-ols, commonly known as catechins, including (-)-epigallocatechin (EGC),1 (-)-epicatechin, (-)-epigallocatechin-3-gallate (EGCG), and (-)-epicatechin-3-gallate (ECG) (Figure 1). The oral bioavailability of these catechin derivatives following tea ingestion has been demonstrated in this laboratory (6-8) and by others (9, 10). The peak plasma concentrations were observed in humans between 1.5 and 2.5 h for these catechins following ingestion of a single dose of green tea (11). The renal excretion rates of EGC and (-)epicatechin reached peak values at 2 h after ingestion of tea, but neither EGCG nor ECG was detected in the human urine (12). Furthermore, we recently identified two ring-fission metabolites of EGC and (-)-epicatechin (Figure 1), i.e., (-)-5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone (M4) and (-)-5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (M6), respectively, in human urine and plasma

Figure 1. Chemical Structures of EGC, (-)-Epicatechin, and their Metabolites

* To whom correspondence should be addressed. Phone: (732) 4455360. Fax: (732) 445-0687. E-mail: [email protected]. † Laboratory for Cancer Research, Rutgers. ‡ Environmental and Occupational Health Sciences Institute. § Department of Chemistry, Rutgers. 1 Abbreviations: EGC, (-)-epigallocatechin; M4, (-)-5-(3′,4′,5′-trihydroxyphenyl)-γ-valerolactone; M6 (-)-5-(3′,4′-dihydroxyphenyl)-γvalerolactone; 4′-O-Me-EGC, 4′-O-methyl-epigallocatechin; -G, monoglucuronide; -2G, diglucuronide; -3G, triglucuronide; -S, monosulfate; -2S, disulfate; -3S, trisulfate; ESI, electrospray ionization; CID, collisionally induced dissociation; TIC, total-ion chromatogram; SIM, selected ion monitoring; PDA, photodiode array; tR, retention time.

following ingestion of tea (12). These two metabolites exhibited maximum renal excretion rates and peak plasma concentrations around 13 h after tea intake. Another major metabolite was characterized as 4′-Omethyl-epigallocatechin (4′-O-Me-EGC) (13). In these studies, the majority of the tea catechins and their metabolites were found in their conjugated forms and the samples were treated with β-D-glucuronidase (EC 3.2.1.31) and sulfatase (EC 3.1.6.1) before HPLC analysis.

10.1021/tx0002536 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/03/2001

LC/ESI-MS Analysis of Metabolites of Tea Catechins

Whereas glucuronides and sulfates are generally assumed to be rapidly excreted and pharmacologically inactive, it has been increasingly recognized that these conjugates may be more pharmacologically active than some of the parent compounds. Morphine-6β-glucuronide, a major metabolite of morphine, possesses more potent analgesic activity than the precursor itself (14-16). Minoxidil and cicletanine are bioactivated upon sulfation (17-19). In addition, glucuronides of a drug often accumulate during long-term therapy, particularly in those patients with renal failure (20). The structures, pharmacokinetics, and bioactivities of the conjugated forms of tea catechins and their metabolites are not known. It is possible that these conjugates are transported into target tissues and then hydrolyzed to their corresponding free forms by human β-D-glucuronidase and/or sulfatase (21, 22). Thus, it is important to examine their bioactivities and biological fates. Developing a direct analytical method to determine the major catechin conjugates in biological fluids is a prerequisite to these studies. The recent development of soft ionization methods, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), have made mass spectrometry (MS) an important tool for biological research. Many investigators have used LC/ESI-MS or LC/ APCI-MS for analysis of tea extracts (23-27). We have previously used LC/ESI-MS to identify tea catechin metabolites in urine and plasma samples (12, 13). This report describes the direct analysis of multiple glucuronides and sulfates of catechins and their metabolites in urine samples using a new LC/ESI-MS method.

Materials and Methods Materials and Chemicals. Decaffeinated green tea solids (dehydrated water extract of green tea, 1 g of powder was derived from 6 g of dry leaves) were obtained from Thomas J. Lipton Inc. (Englewood Cliffs, NJ). EGC, (-)-epicatechin, β-Dglucuronidase (EC 3.2.1.31, G-7896, from Escherichia coli with 9 000 000 units/g solid), and sulfatase (EC 3.1.6.1, S-9754, from Abalone entrails with 23 000 units/g solid) were purchased from Sigma (St. Louis, MO). M4 and M6 were isolated from human urine (12). In brief, the urine samples collected between 9 and 23 h after ingestion of green tea were treated with β-Dglucuronidase/sulfatase, and the M4 and M6 were purified by HPLC. The purity of M4 and M6 was checked by rechromatography. 4′-O-Me-EGC was chemically synthesized from EGC reacting with methyl iodide and K2CO3 in acetone (13). MicroHematocrit heparinized capillary tubes (1.1-1.2 mm i.d., 0.2 mm wall), other reagents, and HPLC-grade solvents were obtained from VWR Scientific (South Plainfield, NJ). HPLCgrade water (18 mΩ) was prepared using a Millipore Milli-Q purification system (Bedford, MA). Urine Samples. The Institutional Review Board approved the protocol for human experiment for the Protection of Human Subjects in Research (IRB no. 92-034M) at Rutgers University (Piscataway, NJ). Five healthy male volunteers (31-35 years old, weighing 55-80 kg, nonsmokers) participated in the study. The subjects did not consume tea, tea-related products, or other herbal products for at least 2 weeks before the experiments. Human urine samples were collected just before ingestion of a cup of tea (containing 1.2 g of green tea solid in 300 mL of warm water), and at 1-3 and 12-15 h thereafter. Aliquots of 20 mL of each urine sample were transferred into plastic bottles containing 20 mg of ascorbic acid (as a preservative) and stored at -80 °C until use. Rodent experiments were carried out according to the protocol approved by the Institutional Review Board for the Use and

Chem. Res. Toxicol., Vol. 14, No. 6, 2001 703 Care of Animals (IRB-UCA no. 91-024) at Rutgers University. Female SKH-1 mice with average body weights of ∼20 g (8 weeks old) were obtained from Jackson Lab (Bar Harbor, ME) and male Sprague-Dawleg rats with average weight of ∼250 g (6 weeks old) were purchased from Taconic Farm (Germantown, NY). The mice and rats were maintained in an air-conditioned animal quarters with an alternating 12-h light/dark cycle at a room temperature of 22 ( 2 °C and a relative humidity of 50 ( 10%. The rodents were given AIN-76A semipurified diet (Research Diet, New Brunswick, NJ) and water ad libitum. The experiments were started after acclimation to the facilities for 10 days. Green tea solution (0.6%, w/v) was prepared fresh and administered i.g. to the mice (0.1 mL/10 g body weight/d) and rats (0.5 mL/100 g body weight/d) at 9:00 a.m. Urine samples were collected using two mouse metabolic cages (4 mice/cage) or four rat metabolic cages (1 rat/cage) before oral administration of tea and 0-24 h thereafter. The urine collection containers for mice and rats contained 0.2 and 0.6 mL of 20% ascorbic acid aqueous solution, respectively. The rodent urine samples were frozen at -80 °C until use. HPLC/ESI-MS Detection of Tea Catechin Glucuronides and Sulfates in Urine. The thawed urine sample (300 µL) was first treated with 300 µL of dichloromethane. Following centrifugation at 14000g for 5 min, 200 µL of the aqueous phase was filtered in a 0.2 µm Nanosep disposable centrifugal filter (Pall Filtron, Northborough, MA). The resultant filtrant (10 µL) was injected onto the HPLC/ESI-MS system which consisted of a Waters 2690 LC separation module (Milford, MA) coupled to a Waters 996 photodiode array (PDA) UV-vis detector, followed by a Finnigan LCQ mass detector (San Jose, CA) fitted with an electrospray ionization (ESI) source. To achieve good ESI of test compounds, a Hitachi L-6200 intelligent pump (Tokyo, Japan) was used to deliver Solvent C, methanol-water (80:20, v/v), with a flow rate of 0.3 mL/min into postcolumn eluent flow. The mobile phases were: methanol-water-acetic acid (5:490:5, v/v) for solvent A, and methanol-water-acetic acid (250:245:5, v/v) for solvent B. The LC binary gradient elution system consisted of an initial 5-min isocratic segment (100% A and 0% B). Then, the linear gradient was changed to 22% B at 11 min and 60% B at 42 min. B was maintained at 100% from 42 to 46 min. Then it was immediately changed back to 100% A and maintained for 19 min. A 5 µm Supelcosil LC-18 column (100 mm × 3.0 mm i.d., Supelco, Bellefonte, PA) was maintained at 33 °C. An autosampler in the 2690 LC separation module was maintained at 6 °C and a 0.2 µm filter (Upchurch Scientific, Oak Harbor, WA) was used before the analytic column. The PDA-UV-vis detector was set to monitor UV absorption from 200 to 395 nm. The entire flow from the UV-vis detector at 0.5 mL/min was directed to the ESI source without splitting. The LCQ ion trap mass detector was operated in negative ion polarity mode and ESI mode for all our analytical experiments. The atmospheric pressure ionization (API) source consisted of ESI probe assembly and API stack. Sample solution entered the ESI probe through the sample tube that extended to within 1 mm from the end of the ESI needle, to which -4.5 kV was applied. The needle delivered the sample solution into MS interface with a sheath gas (inner) arbitrary flow rate of 90 arb and auxiliary gas (outer) arbitrary flow rate of 30 arb. The sheath gas and the auxiliary gas were dry nitrogen gas (99% pure, Air Products, Allentown, PA). The API stack under vacuum consisted of heated capillary, tube lens, and skimmer. The heated capillary was set at 230 °C and held at a -30 V potential. An offset voltage of -20 V was applied to the tube lens to accelerate the ions into background gas present in the capillary-skimmer region. Helium gas (99.999% pure, Air Products, Allentown, PA) at a partial pressure of about 0.1 Pa was used as a damping gas and a collision activation partner. The divert valve was set to introduce the eluent flow during 6.0-42.0 min to the MS detector with the other eluent flows to waste. The settings for the MS detector were optimized (in full scan mode) for detecting compounds with the same molecular mass as the possible glucuronides and sulfates from tea, in all

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subject urine samples. For LC/MSn (n ) 2-4), analysis conditions were optimized (also in full scan mode) for identifying the analytes through collisionally induced dissociation (CID) using the typical urine samples. The MS detector acquisition time was set for 65 min, and divided into three segments, i.e., 0-5.5, 5.542.5, and 42.5-65 min. The tea conjugates were found in segment 2. The flow rates of the sheath gas and the auxiliary gas in segments 1 and 3 were decreased to 80 and 10, respectively. The polarity was set for negative in full scan, and the mass range was set for m/z 150-1000. The deprotonated molecules exhibiting the same molecular mass as the target tea catechin conjugates were selected with an isolation width of m/z 2.0 and stored in the mass analyzer. These parent ions were dissociated with 30% relative collision energy to produce fragment ions. If a deprotonated aglycone ion of tea catechins or their metabolites appeared in the product-ion mass spectrum, the assigned molecule was identified as a certain tea catechin glucuronide or sulfate. The deprotonated aglycone ions for conjugated EGC, (-)-epicatechin, M4, M6, O-Me-EGC, and O-Me-epicatechin are at m/z 305, 289, 223, 207, 319, and 303, respectively.

Results and Discussion Glucuronidation and sulfation are important phase II metabolic reactions in which many xenobiotics and endobiotics are converted to more hydrophilic compounds. The glucuronides and sulfates of tea polyphenols are generally less retentive than their parent compounds on conventional octadecylsilane (C18) bonded-phase column with aqueous/organic mobile phase. Ascorbic acid (used as a preservative in the urine sample), urinary inorganic ions, and some other water-soluble substances are detrimental to the ESI process of the analytes. Therefore, the volume fraction of organic modifier (methanol) in the aqueous mobile phase was decreased to facilitate separation between these interfering substances and the analytes. The eluent flow (0-6 min) containing the interfering substances was diverted electronically to waste via the software-controlled valve. The volume fraction of methanol in the mobile phase used for analyte elution during 6-42 min of the chromatographic separation was too low (1-29%) to form an effective electrospray. Therefore, a second pump, delivering 80% methanol, was used to increase the volume fraction of methanol in the eluent flow to 48-60% to enhance and stabilize the ESI process for the analytes. The ESI source was optimized by using 10 ng/µL standard solutions of EGC, (-)-epicatechin, EGCG, and ECG. Before being introduced into the MS detector, the tuning solution, delivered at 10 µL/min by a syringe pump, was combined through a Peek tee union with 50% methanol eluent delivered at 0.5 mL/min by the Waters separation module and the Hitachi pump. Effective and stable ESI process in the ion source was achieved to give maximum sensitivity for the characterization of tea glucuronides and sulfates in urine. Before the test urine samples were analyzed, we used a solution containing standard EGC and (-)-epicatechin and a known human urine sample collected after ingestion of green tea to evaluate the efficacy and reliability of our analytical method. The urine sample was digested with β-D-glucuronidase and sulfatase to release free EGC and (-)-epicatechin before analysis. Figure 2 depicts the chromatograms obtained from the LC/MS and LC/MS/ MS (LC/MS2) experiments of these two samples. Panels S1 and U1 represent the total-ion chromatograms (TIC) and selected-ion monitoring (SIM) chromatograms of the standard and the urinary EGC and (-)-epicatechin,

Li et al.

respectively. Panels S2, U2, S3, and U3 show the respective LC/MS2 chromatograms and fragment ion mass spectra. As shown in this figure, urinary EGC and (-)epicatechin exhibited not only the same chromatographic retention time (tR) and molecular masses as their corresponding standard compounds, but also had the same fragment ion mass spectra. On the basis of the analytical method described above, a typical sample of human urine collected 1-2 h after ingestion of green tea was analyzed by HPLC/PDA-UV/ ESI-MS. The UV absorption chromatogram and TIC were obtained simultaneously (data not shown). The SIM chromatograms derived from the TIC are shown in Figure 3. The mono-, di-, and triglucuronides (-G, -2G, and -3G) as well as mono-, di-, and trisulfates (-S, -2S, and -3S) of the catechins and their metabolites (A) should have their deprotonated ions at m/z of (A + 176)-1, (A + 352)-1, (A + 528)-1, (A + 80)-1, (A + 160)-1, and (A + 240)-1, respectively. The peaks appearing in the SIM chromatograms at m/z 481, 385, 465, 369, 495, 399, and 383 had the same molecular masses as EGC-O-G, EGCO-S, epicatechin-O-G/EGC-O-2S, epicatechin-O-S, O-MeEGC-O-G, O-Me-EGC-O-S, and O-Me-epicatechin-O-S, respectively. Trace amounts of compounds corresponding to EGC-O-2G, EGC-O-3S, epicatechin-O-3S were also found in the urine sample (data not shown). Human urine samples collected between 12 and 15 h after ingestion of tea were used for the detection of the conjugates of catechin metabolites M4 and M6 (12). As shown in Figure 4, the peaks in the SIM chromatograms had the same molecular masses as M4-O-G, M4-O-S, M6O-G/M4-O-2S, and M6-O-S. Trace amount of M4-O-2G was also detected in human urine and identified by LC/ MSn. With LC/MS2, deprotonated fragment ion [(M4 O - G) - H]- (m/z 399) was detected by setting the parent deprotonated ion [(M4 - O - 2G)-]- at m/z 575. Fragment ion m/z 399 was further dissociated in LC/MS3, and [M4 - H]- (m/z 223) was observed as the base peak in the spectrum (data not shown). Further structural evidence for the assignment of these chromatographic peaks as the corresponding tea catechin conjugates was provided by the LC/MS2 analysis. The deprotonated aglycone ions of conjugated tea polyphenols were always the most predominant peaks in the fragment ion mass spectra. In one LC/MS2 analysis, the same human urine sample as used for Figure 3 was used and the precursor ion was set at m/z 481 for EGC-O-monoglucuronides. The product-ion mass spectra of the chromatographic peaks with retention times (tR) of 7.7, 9.4, 16.3, 17.3, 21.1, 27.2, and 29.5 min were observed, with the exception of the peak at 29.5 min, all of which exhibited a base deprotonated fragment ion peak at m/z 305 (derived from EGC) (Figure 5). The results suggested that these peaks represented various EGC-O-monoglucuronides. In another study, compounds corresponding to those eluted at 16.3 and 21.1 min (based on retention time and molecular mass) were enzymatically synthesized using EGC, UDP-glucuronic acid, and mouse liver microsomes as the enzyme source; these metabolites were identified as EGC-7-O-β-G and EGC-3′-O-β-G, respectively, by NMR. The detailed results will be reported elsewhere. Glucuronides of EGC corresponding to peaks at 17.3, 21.1, and 27.2 min were produced in incubation with mouse small intestinal microsomes. Glucuronides of EGC corresponding to peaks at 21.1 and 27.2 were produced

LC/ESI-MS Analysis of Metabolites of Tea Catechins

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Figure 2. LC/MS and LC/MS2 analysis of standard EGC and (-)-epicatechin and a known human urine sample collected between 1 and 2 h after ingestion of green tea. The urine sample was pretreated with β-D-glucuronidase and sulfatase. The relative collision energy for dissociation of the deprotonated EGC or (-)-epicatechin was set at 30%. Panels S1 and U1 represent the TIC and SIM chromatograms of standard and urinary EGC and (-)-epicatechin, respectively. Respective LC/MS2 chromatograms and fragment ion mass spectra of EGC (panels S2 and U2) and (-)-epicatechin (panels S3 and U3) are also shown.

Figure 3. SIM chromatograms derived from the TIC of a typical sample of human urine collected between 1 and 2 h after green tea ingestion for detecting the conjugates of EGC, (-)epicatechin, O-methyl-EGC and O-methyl-epicatechin. The separate [M - H]- ion chromatograms selected at m/z 481, 385, 465, 369, 495, 399, and 383 show urinary compounds exhibiting the same molecular masses as EGC-O-G, EGC-O-S, epicatechinO-G/EGC-O-2S, epicatechin-O-S, O-Me-EGC-O-G, O-Me-EGCO-S, and O-Me-epicatechin-O-S. The relative abundance is normalized to the most abundant peak in each chromatogram.

in human liver microsomes. Although different phenolic groups on EGC could be conjugated with glucuronic acid

in humans, EGC-3′-O-β-G (tR 21.1 min) was the major metabolite in human urine. Other EGC-O-monoglucuronides were present in less than 2% of the major one. Some urinary compounds with the same molecular mass as the -2G, -3G, -2S, or -3S were also detected, but their peak abundance in SIM chromatograms was very low. Although deprotonated fragment peaks from some of these compounds following the loss of a 176 or 80 moiety could be found in full scan LC/MS2 experiments, it was difficult to detect the deprotonated aglycone ions, when the fragment ions were further dissociated. Combining the data of LC/MS and LC/MSn analyses, listed in Table 1 are the major tea polyphenol conjugates detected in human urine after ingestion of green tea. The major EGC and (-)-epicatechin conjugates found in human urine were the monoglucuronides and monosulfates, but only trace amount of EGC-O-S was detected. Both O-methyl-EGC glucuronide and sulfate were present, whereas only O-methyl-epicatechin sulfates were found. For M4 and M6, the monoglucuronides and monosulfates are the major conjugates. No catechin metabolite was detected in the human urine samples collected before the ingestion of tea. Mouse and rat urine samples collected after oral administration of green tea were also analyzed similarly. The results are also included in Table 1. The monoglucuronides and monosulfates were the major tea catechin

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Table 1. Major Glucuronides and Sulfates of Tea Polyphenols Detected in Urine retention time of the conjugate in min metabolite

m/za

human

mouse

rat

EGC-O-G EGC-O-S Epicatechin-O-G Epicatechin-O-S O-m-EGC-O-G O-m-EGC-O-S O-m-epicatechin-O-G O-m-epicatechin-O-S M4-O-G M4-O-S M6-O-G M6-O-S

481 385 465 369 495 399 479 383 399 303 383 287

21.1 16.1 (trace),b 19.5 (trace) 29.2 (15.2), (17.9), 20.0 (18.8), (23.4), 25.4 (14.2), 20.7, 21.2 ND 18.6, 24.6 17.0, 22.2 12.3 (22.6), 23.5 15.2

16.3, 17.3, 21.1 13.4, 16.1 21.8, (29.2)c (17.9), 20.0 (19.5), 24.3, 25.4, (26.2) (16.9), 20.7, 22.2, 22.7 (24.4), 29.4, 30.8 (20.1), 24.6, (26.7) 17.0 (trace) 12.3, (14.1) ND 17.6

17.3 19.5 (trace) (16.6), 21.8 NDd (18.8), 23.4, 24.3, 26.2 18.3 (trace) 24.4, 30.8 ND 17.0 (trace) ND 23.5 (trace) ND

a For deprotonated ions. b The “trace” in parentheses is for the minor conjugates isomers, the relative peak abundance of which was less than 2% of that of the base peak. c The retention times in parentheses are for the minor conjugate isomers, the relative peak abundance of which was around 5-15% of that of the base peak. d ND, denotes “not detected”.

Figure 4. SIM chromatograms of a typical sample of human urine collected between 12 and 15 h after ingestion of green tea for detecting the conjugates of M4 and M6. The separate [M - H]- ion chromatograms selected at m/z 399, 303, 383, and 287 show urinary compounds exhibiting the same molecular masses as M4-O-G, M4-O-S, M6-O-G/M4-O-2S, and M6-O-S. The relative abundance is normalized to the most abundant peak in each chromatogram.

conjugates found in mouse and rat urine samples. Many of the major glucuronides and sulfates found in human urine were also detected in mouse urine, suggesting that the metabolite profiles of tea polyphenols in mice were similar to those in humans. Nevertheless, M4 and M6 conjugates in mouse urine were mainly their sulfates and only trace amounts of the glucuronides were found. In rats, the major conjugates detected in the urine samples were quite different from those in human urine samples. In an LC/MS3 experiment, EGC-O-2G with a tR of 17.7 min was detected in rat urine (data not shown). In the rat urine samples, only trace amounts of M4 and M6 conjugates were detected. No tea catechin metabolite was found in the urine samples collected before treatment. Without the standard compounds, it is difficult to detect directly the conjugated tea polyphenols in biomatrices by conventional HPLC coupled with UV or EC detection. The LC/ESI-MS method described in this report is sensitive and reliable for direct detection of glucuronides and sulfates of tea catechins and their metabolites in urine. This is thought to be the first report describing the direct analysis of multiple tea polyphenol conjugates in urine samples. This analytical method will allow more thorough investigation of the biotransformation of tea polyphenols. With the use of the purified conjugate compounds and an appropriate internal standard, the LC/ESI-MS technique described herein can be used for accurate quantification of the tea polyphenol metabolites in biomatrices. The method will also be useful for studies on other phenolic compounds.

Figure 5. Full scan MS/MS chromatogram of the human urine sample from Figure 3 and the product-ion mass spectra of the chromatographic peaks. The parent ion [M - H]- was set at m/z 481 for detecting EGC-O-monoglucuronides. The relative abundance is normalized to the most abundant peak in the chromatogram or in each MS spectrum.

Acknowledgment. We thank all volunteers for their participation in the tea experiment and Dorothy Wong for her assistance in the preparation of the manuscript. This study was supported by NIH Grant CA-56673 and carried out in facilities supported by NIEHS Grant ES05022.

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LC/ESI-MS Analysis of Metabolites of Tea Catechins

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