Chemicals & Reagents

All tea polyphenol standards used were purchased from Sigma-Aldrich, USA. The Assam and Darjeeling black tea leaves were purchased from Goodricke Group Ltd. an internationally reputed tea merchant. Every experiment described herein was conducted with tea of the same lot for both the Darjeeling and Assam black tea. All other reagents used for the study were procured from Merck, Germany. A stock solution of the polyphenol standards (each 1 mg/ml) was prepared with 0.2% ascorbic acid and 0.005% EDTA and stored in small aliquots at -80°C till further use.

Preparation of Tea Infusions

0.5 g of Assam and Darjeeling varieties of black tea were separately added to 10 ml of boiling water and brewed for 5 min with continuous stirring before being filtered into a hermetically sealed container [6]. The filtrates were designated as the Assam Black Tea (ABT) infusion and Darjeeling Black Tea (DBT) infusion respectively.

Treatment of Animals

Male short-hair guinea pigs weighing 350–450 g were used for our experiments. All animal treatment procedures were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), USA. The treatment protocol was approved by the Institutional Animal Ethics Committee (IAEC), Department of Biochemistry, University of Calcutta [Permit Number: IAEC/CU/BIOCHEM/DJC-KP (5)]. Throughout the animal treatment, all efforts were made to minimize stress and suffering of the animals. The guinea pigs were administered a polyphenol free diet (wheat bran 700 g, casein 200 g, sucrose 80 g, USP XVII salt mixture 10 g, AOAC vitamin mixture 10 g/kg of feed) and was orally administered water ad libitum along with suboptimal levels of vitamin C (2 mg/kg animal body weight) from seven days prior to the initiation of the experiment, to prevent scurvy [6]. The animals were finally divided into the following experimental groups (6 animals per group); Control Group: Orally administered water (20 ml/kg body wt) instead of black tea in two equal dosages daily for 14 days; Experimental Group I: Orally administered 20 ml/kg body weight of 5% Assam Black Tea (ABT) infusion in two equal dosages daily for a period of 14 days; Experimental Group II: Orally administered 20 ml/kg body weight of 5% Darjeeling Black Tea (DBT) infusion in two equal dosages daily for a period of 14 days. The time interval between the two dosages was maintained at 4 hours with the tea infusions freshly prepared before each administration. After being fed with the polyphenol-free diet for seven days, the guinea pigs belonging to the two experimental groups described above, were orally administered either ABT or DBT infusion for additional 14 days after which they were euthanized through intravenous ketamine hydrochloride injection around 120 min after the final oral administration of tea infusion (6 hours after the first dose and 2 hours after the second tea dose, on the 14^th day of treatment). Blood was immediately collected in vacutainer tubes containing ethylene-diamine-tetraacetate (EDTA) as anticoagulant and was centrifuged at 1000 g for 15 min at 4°C for the separation of plasma. The plasma was collected carefully and immediately mixed with an ascorbate-EDTA solution (20% ascorbic acid and 0.1% EDTA (w/v) in 0.4 M NaH₂PO₄ buffer, pH 3.6) in the ratio of 10:1 (plasma: ascorbate-EDTA solution) and stored at -80°C for subsequent use. The lung, kidney, liver and heart tissues were excised and collected from the experimental animals, washed in cold saline solution and stored at -80°C for further use. The urine was directly collected from the urinary bladders of the animals during their sacrifice, mixed with relevant volume of ascorbate-EDTA buffer (pH 5.0) and then stored at -80°C for subsequent use.

Preparation of Samples for Analyses

Polyphenols were extracted from plasma and organs by the liquid-liquid extraction method described by Lee et. al. with slight modifications [19]. 200 μl of the thawed plasma was mixed with 20 μl of ascorbate-EDTA solution and 20 μl of phosphate buffer (pH 7.4). To this, 2.0 ml of ethyl acetate was added, vortexed for 5 min and centrifuged at 4500 rpm for 20 minutes. The upper ethyl acetate layer was then collected and after a second extraction, the combined supernatant was evaporated under nitrogen at 35°C to dryness. The dried sample was reconstituted with 100 μl of 15% acetonitrile containing 0.2% acetic acid. This was centrifuged at 13,000 rpm for 10 min, and 30 μl of the supernatant was loaded in the UPLC auto-sampler for relevant analysis of polyphenols.

For processing the tissue samples, 500 mg of lung, kidney, liver and heart tissues were homogenised using a mechanical homogenizer in 2.5 ml of the ascorbate-EDTA solution. The tissue lysates were then precipitated with 200 μl of ethanol to which 500 μl of dichloromethane was added. The mixture was then vortexed and centrifuged at 16,000 g for 5 min. The upper aqueous phase was collected, and after a second extraction with water, the pooled aqueous phase was again extracted with ethyl acetate similar to that done with the plasma samples. The spiked plasma and organ lysates were prepared by adding definite amounts of authentic standard mixture of polyphenols to the blank plasma and organ lysates collected from the control guinea pigs that were orally administered water instead of tea infusion, and were similarly processed by ethyl acetate extraction along with their blanks.

For identification of metabolites in the urine, 200 μl of the collected urine was centrifuged and the supernatant was diluted with equal volume of reconstitution solution consisting of 15% acetonitrile and 0.2% acetic acid and thereafter used for metabolite analyses.

Instrumentation

Liquid chromatography (LC) coupled with tandem quadrupole mass spectrometry (LC/MS/MS) and operated in ‘multiple reaction monitoring’ (MRM) mode, was used for determination of catechin and theaflavin levels in our study, considering the potential of this technique to accomplish high selectivity and sensitivity in quantitative bioanalysis.

The liquid chromatography (LC) system consisted of a binary solvent delivery system comprising of two pumps and a refrigerated auto-sampler with fixed-loop injector (Acquity Binary UPLC system, Waters, USA). Chromatographic conditions were standardized through several trials to achieve best possible sensitivity and resolution by efficient separation of the analytes from possible matrix interferences. The chromatography was optimized on an Atlantis T3 (4.6 mm x 50 mm x 3 μm) C18 column (Waters, USA) that was maintained at 40°C. Separation was achieved using a mobile phase consisting of (A) aqueous solution of 0.2% acetic acid and (B) acetonitrile containing 0.2% acetic acid, at a flow rate of 0.8 ml/min. For analysis, 30 μl of the sample was injected under a programmed gradient which was initiated with 98% A, then ramped down to 70% A within 4 min, and was maintained at 70% A up to 7 min, before being changed to 2% A by 7.10 min, in order to accomplish efficient separation of the polyphenols. This was kept unchanged till 8 min following which the system was re-equilibrated for a fresh run.

A tandem quadrupole mass spectrometer, Xevo-TQD MS system (Waters, USA) with the capability of electrospray ionisation (ESI) switching between positive and negative polarity conditions was used. ESI negative mode was used for catechins while positive mode was used for detection of theaflavins. The capillary voltage was maintained at 3 KV, cone voltage at 30 V, and extractor voltage at 4 V for both positive and negative mode to achieve the best sensitivity for the analytes. The source temperature and desolvation temperatures were maintained at 150°C and 625°C, respectively. The cone gas flow rate was kept at 50 lit/hr and that of the desolvation gas flow at 1100 lit/hr. Catechins and theaflavins were identified based on their respective retention times as well as MRM transitions. The metabolites in urine were studied by precursor ion scan by monitoring the parent ions based on an earlier report [27].

Software Used and Statistical Analysis

Data acquisition was performed with MassLynx 4.1 from Waters Corporation, USA, and was processed with TargetLynx application manager. The MassLynx software is commercially and globally available with UPLC systems coupled to Waters Xevo TQD Mass Spectrometers as acquisition control software, while TargetLynx is deployed for the purpose of quantitation of the acquired data. Certain statistical analyses were also performed with Origin Pro 8, scientific graphing and data analysis software from Origin Lab Corp. USA.

Regression analysis was used to evaluate the linearity of the developed method for individual catechins and theaflavins, and also to estimate their concentrations in the unknown samples. The paired t-test (α = 0.05) was used to determine whether the observed difference in the levels of the catechins and theaflavins between the 5% Assam black tea (ABT) and the Darjeeling black tea (DBT) infusions as well as the plasma and tissue lysates of the animals administered water against tea infusions, was statistically significant. The same test was also used to determine the significance of difference between the polyphenol levels detected in the lung and kidney lysates of animals orally administered black tea infusions.

Method Development

Our preliminary analysis, which included detection of tea polyphenols separately in positive and negative electro-spray ionisation (ESI) modes, indicated that catechins were best detected in negative mode whereas theaflavins in positive mode. This permitted higher selectivity in detection without compromising the detection sensitivity and hence could render the simultaneous detection of catechins and theaflavins possible in complex tissue matrices. This also mandated the use of an effective mass-spectrometric (MS) system that could accomplish equally efficient detection of both catechins and theaflavins despite accommodating the required switching between the two polarities in a single run. We were precisely able to accomplish this using the Xevo-TQD MS triple quadrupole mass spectrometer (Waters, USA) which is capable of switching between positive and negative polarities within 20 milli seconds. The data acquisition in MS/MS mode was optimized for the best possible selectivity and sensitivity for each analyte with collision energy of 20 for TF, TF3G and ECG, 18 for TF33'diG and EGC, 16 for EC and 15 for EGCG. Both the quadrupoles were operated at unit resolution for such runs. The MRM transitions which showed the highest response and signal-to-noise (s/n) ratio for each analyte were 565 to 139 for TF, 717 to 139 for TF3G; 869 to 139 and 869 to 744 for TF33'diG, 289 to 245 and 289 to 179 for EC, 305 to 125, 305 to 139 and 305 to 179 for EGC, 441 to 289 and 441 to 169 for ECG and 457 to 169 for EGCG. Other parameters, like those related to the nebulizer and the heater gases, were optimized to obtain a better spray shape for improved ionization. For the extraction of the polyphenols, a simple and widely used liquid-liquid extraction technique was employed as described under the ‘Materials and Methods’ section [19].

The present method was initially developed with the aqueous standards of the polyphenols and was subsequently modified for the matrix extracted samples. The aqueous linearity of the method, which was examined by running mixtures of polyphenol standards from 5 to 320 ng/ml, (EC analyzed from 10–640 ng/ml), showed linear and reproducible curves, with correlation coefficients of 0.996119, 0.9920, 0.998915, 0.999207 respectively for EGC, EC, EGCG, and ECG and 0.998375, 0.996340, 0.997688, 0.988960 for TF, TF3G, TF3'G, TF33'diG. The precision and accuracy of the method, determined by running six injections of different quality control standards, showed that they were well within the acceptable limits [28, 29] (Table 1). Table 1 also delineates the limit of detection (LOD), the lower limit of quantification (LLOQ), correlation coefficient (r) and linear regression (LR) of our developed method. Such aqueous linearity data was used to determine the relative content of polyphenols in the ABT and DBT infusions (S2 Fig). For achieving improved accuracy in the simultaneous detection and quantification of the polyphenols, we determined detection and quantification limits in consonance with the Clinical and Laboratory Standards Institute (CLSI) EP17 and the US-FDA guidelines while taking into consideration several parameters including the blank response, LOB (limit of blank), accuracy-precision as well as % R.S.D (percentage relative standard deviation) of analyte responses at low concentrations [28–30]. A uniform LLOQ (lower limit of quantification) of ~5 ng/ml was determined for all the polyphenols across all the analyzed matrices which helped us to accomplish a signal to noise (s/n) ratio well above the conventional s/n ratio of 1:10 (S1 Table).

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Table 1. Precision (R.S.D in %), Accuracy (%), Limit of Detection (LOD), the Lower Limit of Quantification (LLOQ), Linear Regression (LR) and Correlation Coefficient (r), for the Analyzed Catechins and Theaflavins¹.

https://doi.org/10.1371/journal.pone.0163498.t001

The linearity, sensitivity and selectivity of the method with respect to different biological matrices were initially determined through analyses of the plasma and organ lysates spiked with individual polyphenols. Such linearity data was subsequently used for quantifying catechins and theaflavins in the tissues of tea consuming guinea pigs (Fig 1, S3 and S4 Figs). The supplementary S1 Table (S1 Table) delineates the correlation coefficient (r) linear regression (LR), signal to noise ratio (s/n) at LLOQ as well as the LLOQ accuracy of our method for simultaneous detection of catechins and theaflavins in the plasma as well as lung and kidney tissue lysates spiked with polyphenols.

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Fig 1. Detection of Catechins and Theaflavins in Plasma Spiked with Individual Polyphenols.

Representative MRM chromatograms of epigallocatechin (EGC), epicatechin (EC), epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), theaflavin (TF), theaflavin-3-monogallate (TF3G), theaflavin-3,3'-digallate (TF33'diG), theaflavin-3'-monogallate (TF3'G), in blank plasma (control) spiked with the polyphenol standards to the final concentration of their LLOQ, showing the retention time (labelled below the analyte name) and response (labelled below the retention time) of the analytes (analyte names highlighted against their corresponding chromatograms). The right-hand top labels on the chromatograms indicate their respective MRM transitions and peak heights. Data are representative of three independent experiments done under similar conditions.

https://doi.org/10.1371/journal.pone.0163498.g001

The fact that our method was able to achieve a lower limit of quantification of ~ 5 ng/ml in the analysed matrices clearly indicated that it was almost ten to hundred times more sensitive than prevalent HPLC-UV and capillary electrophoresis-based methods [17, 18], and closely comparable to the HPLC-ECD [19] as well as most existing LC/MS-MS protocols [3, 23, 25, 26], notwithstanding deploying polarity switching to accomplish the goal of simultaneous determination of catechins and theaflavins. Notably, no endogenous components were found to significantly interfere with the analytes around their recorded retention times establishing the high selectivity of our method.

Using the chromatographic conditions described above, the retention times for elution were uniformly observed to be 3.02 ± 0.035 min for EGC, 3.70 ± 0.029 min for EC, 3.78 ± 0.032 min for EGCG, 4.48 ± 0.027 min for ECG, 5.74 ± 0.057 min for TF, 6.22 ± 0.049 min for TF3G, 6.48 ± 0.048 min for TF33'diG and 6.62 ± 0.053 min for TF3'G for all the analysed matrices. Compared to previously reported HPLC-UV protocols [16–18] or the single reported HPLC-ECD method [19] our procedure was up to seven times faster in accomplishing complete separation of the individual polyphenols. In fact, our method was also faster or comparable to the existing LC/MS-MS protocols used for such analyses even though such methods have been largely limited to the detection of catechins alone [23, 25].

Simultaneous Determination of Catechins and Theaflavins in Pure Black Tea Infusion as well as Plasma, Lung and Kidney of Tea Consuming Animals

The major objective of our study was to develop a broadly applicable, rapid and sensitive method for simultaneous detection of catechins and theaflavins across different matrices. To examine whether we can accomplish this task with our developed method, we applied it to pure black tea infusions as well as different tissues of tea-consuming guinea pigs, and found that it could indeed simultaneously detect catechins and theaflavins in most of the analyzed samples. Our method successfully assessed the relative content of such polyphenols in 5% ABT infusion as 51.5 ± 6.2 μg/ml EGC, 56.3 ± 5.5 μg/ml EC, 310.8 ± 10.5 μg/ml EGCG, 104.7 ± 8.8 μg/ml ECG, 30.3 ± 5.1 μg/ml TF, 54.8 ± 5.2 μg/ml TF3G, 37.1 ± 4.1 μg/ml, TF3'G, 125.9 ± 10.2 μg/ml TF33'diG compared to 5% DBT infusion which had 75.3 ± 9.4 μg/ml EGC, 91.6 ± 11.8 μg/ml EC, 893.2 ± 25.2 μg/ml EGCG, 210.3 ± 11.0 μg/ml ECG, 22.6 ± 4.1 μg/ml TF, 15.2 ± 5.0 μg/ml TF3G, 11.5 ± 4.1 μg/ml TF3'G and 28.6 ± 5.1μg/ml TF33'diG (Table 2, S2 Fig). The catechin content of the 5% DBT infusion was therefore found to be over two-fold higher than that of 5% ABT infusion, while the latter had significantly higher levels of total theaflavins (P < 0.05) compared to DBT (S2 Fig). Such observation was also in agreement with earlier reports [31, 32]. Consequently, DBT-treated animals received significantly higher amounts of catechins in comparison to their ABT-administered counterparts who in turn received relatively higher amounts of theaflavins compared to their DBT-treated counterparts. This was indeed reflected in the relative content of these polyphenols detected in the plasma, lung and kidney tissues of the ABT and DBT treated animals (Tables 3 and 4).

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Table 2. Bioactive Polyphenol Content of Assam and Darjeeling Black Tea infusion^1,2.

https://doi.org/10.1371/journal.pone.0163498.t002

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Table 3. Levels of Tea Polyphenols Detected in Guinea Pig Plasma after Oral Administration of ABT and DBT^1,2

https://doi.org/10.1371/journal.pone.0163498.t003

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Table 4. Levels of Tea Polyphenols Detected in Guinea Pig Lung and Kidney after Oral Administration of ABT or DBT¹.

https://doi.org/10.1371/journal.pone.0163498.t004

Oral administration of both 5% ABT and DBT black tea infusion to guinea pigs for 14 days resulted in detectable accumulation of all major polyphenols in the plasma of the treated animals as evidenced through our UPLC-MS/MS analysis (Table 3 & S3 Fig). The mean level of EGC in the plasma of the 5% DBT-treated animals was found to be approximately 12.9 ng/ml while that of EC, EGCG and ECG, 11.6 ng/ml, 130.8 ng/ml and 34.6 ng/ml respectively (Table 3). Plasma of animals consuming 5% ABT contained approximately 8.4 ng/ml EGC, 7.6 ng/ml EC, 40.2 ng/ml EGCG and 19.3 ng/ml ECG (Table 3). As expected, relative levels of plasma catechins were more in the guinea pigs administered DBT infusion compared to the ABT infusion administered animals. Although, the plasma content of individual catechins was found to increase with increasing levels of catechins in the administered black tea, such dose-dependent increase was found to be statistically significant only for the gallated catechins, namely, EGCG and ECG (P < 0.05) (Table 3). Apparently due to the low abundance of theflavins in DBT, we were unable to detect these polyphenols in the plasma of the DBT-treated animals (Table 3 & S3C Fig). However, we could detect modest levels of all the theaflavins in the plasma of the ABT-treated animals, using our method (Table 3 & S3B Fig). Whether this difference in the quantified levels of plasma catechins and theaflavins (Table 3) is attributable only to the lower abundance of theaflavins compared to catechins in black tea (Table 2), or also to a possible difference in bioavailability or bio-retention of the two polyphenols, needs to be further investigated.

While the plasma levels of the polyphenols help to provide valuable insights on their relative rates of absorption in vivo, it does not necessarily furnish accurate information on their precise organ distribution. Considering the projected health benefits of these polyphenols in various human diseases, it is imperative to determine their distribution in the organs involved in such diseases. Thus our present animal study is a step forward towards the future elucidation of the accurate tissue levels as well as the possible protective role of these polyphenols in such diseases.

The polyphenol content in the lung and kidney tissues of the ABT and DBT-treated animals is summarised in Table 4. Catechin levels in both the lung and kidney tissues of the DBT-treated animals were approximately 1.6 times that of the ABT-treated animals (Table 4). This can be perhaps explained by the relatively higher catechin content of the DBT infusion compared to the ABT infusion (Table 2). The total catechin level in the lung was significantly higher (P < 0.05) than the kidney irrespective of whether the animals were administered DBT or ABT infusion (Table 4 & S4 Fig). Although modest levels of theaflavins could be detected both in the lung and kidney tissues in the ABT-treated animals, they could not be quantified as they apparently fell below the LLOQ threshold of our method. Besides the lung and kidney, we also analyzed the liver and heart tissues for their polyphenol content. While free polyphenols (non-metabolised) could not be detected in the liver, low levels of polyphenols were found in the heart tissues. However, such levels were too low (below LLOQ thresholds of our method) for accurate quantification.

While a number of reports have indicated that the peak plasma concentration of polyphenols is achieved within 1–2 hours of consumption of such polyphenols, the exact time taken by them to reach various tissues is still unknown [16, 24]. In our study, we administered the animals with a relatively high dose of 20 ml 5% ABT or DBT infusion per kg body weight in two equal dosages daily for a continuous period of 14 days before evaluating the tissue levels of tea polyphenols. The difference in tissue distribution of the polyphenols observed between different organs in our study could be explained at the level of differential uptake, retention, bioconversion and excretion as is observed with respect to the uptake of any orally administered biochemical including drug molecules. Indeed, differences in vascularisation and blood perfusion, tissue binding, tissue accumulation, local pH, and permeability of cell membranes between the different organs might contribute to the observed dissimilarity in tissue uptake and content of the administered polyphenols. Moreover, after absorption, tea catechins (EC, ECG, EGC, and EGCG) may undergo extensive bioconversion in vivo mainly through glucuronidation, sulfation as well as methylation. Such metabolic conversions are already known to be catalyzed by various enzymes like UDP-glucuronosyltransferases (UGT), Catechol-O-methyltransferases (COMT), Cytosolic sulfotransferases (SULT) typically present in such tissues [33–37]. As liver is the primary site of such bio-transformation reactions, it is not surprising that we were unable to detect any free polyphenols in this organ. Moreover, both glucuronidation and sulfation have been reported in other tissues like lung and kidney [38–41]. The differential expression and activity of these enzymes in the lung and the kidney tissues may also contribute to the observed disparity in their polyphenol content besides the factors that have been delineated above. The precise factors contributing to our failure to detect these polyphenols in the heart tissues, however mandates a more detailed study. In all, elucidation of the exact factors responsible for the differential content of tea polyphenols in the different organs analyzed in our study is admittedly a complex exercise, and indeed requires to be addressed through a more extensive and carefully designed experimental approach that is beyond the purview of our study, that is primarily dedicated to the development of a method for the simultaneous detection of catechins and theaflavins.

Moreover, we strategically did not subject our samples to glucuronidase and sulfatase digestion, as that would have potentially converted the polyphenol metabolites to their free forms. This enabled us to successfully identify the true levels of the biologically active free polyphenols as well as several catechin metabolites in the urine using our protocol. The catechin metabolites were identified using precursor ion scan, by monitoring the negatively charged molecular ions ([M-H]^-) corresponding to the parent ion m/z, daughter ion m/z and calculating the associated loss of mass (m/z) during fragmentation (S2 Table). After administration of 5% DBT infusion for 14 days, glucuronides and sulfates of EGC and EC were identified in the urine of the treated animals along with the methylated forms of EGC glucuronide, EGC sulfate and EC glucuronide (Fig 2). None of the metabolites of the gallated catechins could however be detected in the urine, consistent with previous reports [20, 27, 42, 43]. This is probably because they are not excreted through the urine as several studies using rat models indicate that EGCG might be instead excreted through the bile [44–46]. This perhaps may also be applicable for our guinea pig model and needs further investigation.

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Fig 2. Detection and Identifiction of Catechin Metabolites in the Urine of Tea-Consuming Guinea Pigs.

Representative chromatograms for the identification of catechin metabolites in the urine of tea consuming guinea pigs analysed by precursor ion scan showing epigallocatechin glucuronide (EGC-glucuronide), epicatechin glucuronide (EC-glucuronide), o-methyl epigallocatechin glucuronide (o-methyl EGC glucuronide), epigallocatechin sulphate (EGC-sulphate), epicatechin sulphate (EC-sulphate), o-methyl epigallocatechin sulphate (o-methyl EGC-sulphate), o-methyl epicatechin sulphate (o-methyl EC-sulphate) as the primarily identifiable metabolites. The chromatograms also depict the retention times (RT) (highlighted corresponding to the monitoring ions) and the intensity count (highlighted at the right hand side of the chromatograms) of the identified metabolites. Data are representative of three independent experiments done under similar conditions.

https://doi.org/10.1371/journal.pone.0163498.g002

Recognizing the fact that there can be significant variations in the composition of the tea infusions depending on the varieties, sources and conditions of the tea leaves used, as was also observed in our analyses of ABT and DBT infusions (Table 2, S2 Fig), our entire study was carried out with tea leaves from a single lot (single source) of both ABT and DBT. The ABT and DBT used in our study were both purchased from an internationally reputed tea merchant, Goodricke Group Ltd. Evidently, while the DBT infusion was found to be rich in catechins, the tea infusion prepared from ABT was comparatively rich in theaflavins, revealing a significant difference in the composition of these two varieties of black tea (Table 2, S2 Fig). This observation also agrees well with existing reports [31, 32, 47]. A previous study by Henning et.al., has already shown that variations in flavanol content among tea bags procured from different lots at different times and different stores are smaller compared to differences observed with tea bags representing different brands [48]. However, our primary aim was to develop a method for simultaneous determination of major bioactive tea polyphenols in tea liquor as well as various biological tissues of animals consuming the above two varieties of black tea. Notwithstanding the above-mentioned variations, our method can be effectively utilized for simultaneous detection of different catechins and theaflavins from any source of such polyphenols with high reproducibility.

An important limitation of our study is perhaps the absence of a suitable internal standard which has admittedly restricted proper optimization of the method developed by us for simultaneous detection of catechins and theaflavins. Since an internal standard was not used in our analysis, we had to keep our quantification limits comparatively high, with elevated signal to noise ratio (S1 Table), in order to make adequate provisions for matrix effects and analytical errors. While internal standards for chromatographic analysis of catechins are well characterized, a reliable internal standard is yet to be identified for the analysis of theaflavins. This might partly explain why there are virtually no reported methods for simultaneous detection of catechins and theaflavins with high sensitivity along with the existence of only a few methods for theaflavin analysis compared to the large number of established methods for catechin quantification alone [3, 19]. The use of an internal standard is however not mandatory for relative quantification of polyphenols [49], that could help in the elucidation of the most bio-available and efficacious polyphenol(s) for potential formulation of tea based therapeutic products. Further, our method had an LLOQ of approximately 5 ng/ml, that fell short of allowing dependable quantification of the detected polyphenols in some tissues. This certainly can be circumvented through future improvement of the overall sensitivity of our method. Here again, the non-availability of an internal standard for theaflavins was the main constraint behind our failure to lower the LLOQ of our method to such qualifying levels. In addition, although several catechin metabolites were detected in the urine of the tea consuming animals in our present study, we could not quantify them due to the apparent non-availability of reliable reference standards for the detected metabolites.

It thus needs to be accepted that a method optimization for simultaneous determination of catechins and theaflavins has its own share of challenges and would perhaps take further work to address with due perfection. As our principal aim of the present study was to develop a universal UPLC-MS/MS-based protocol for simultaneous detection of catechins and theaflavins that can be used for all biological matrices, our present method admittedly has the potential to accomplish this goal.