Method development and validation for rapid identification of epigallocatechin gallate using ultra-high performance liquid chromatography.

Although Epigallocatechin gallate (EGCG) is the most available and beneficial catechin found in tea, its auto-oxidation property may lead to toxicity when consumed in large quantities. Thus, there is a need to quantify the EGCG, which enables to study the pharmacological characteristics of the compound. The study aimed to develop and validate a rapid and accurate analytical method for quantitative determination of EGCG. Standard EGCG was used to conduct trials for the optimization of the analytical method using Ultra-High Performance Liquid Chromatography (UHPLC). Tests for validation (specificity, linearity, accuracy, system suitability, method precision, robustness, and ruggedness) were performed. The preliminary trials yielded an analytical method with good peak shape and acceptable system suitability which was further validated. The method was shown to be specific, with a linear correlation coefficient of > 0.9996 and accurate with acceptable recovery rate (99.1% to 100.4%). Acceptable system suitability and method precision were confirmed with a relative standard deviation (less than 2%). Further, robustness and ruggedness experiments also demonstrated the suitability of the present analytical method. The method developed for determination of EGCG was validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and thus can be used in routine compliance tests in the laboratory for further studying/characterizing the properties of EGCG.

Introduction

Epidemiological studies have reported the favourable benefits of tea consumption in varied population [1]. The benefits mainly can be attributed to the polyphenols, especially Epigallocatechin 3-gallate (EGCG), for its antioxidant properties [2]. EGCG is known to alleviate chronic diseases such as obesity, type-2 diabetes, lipid metabolism abnormity, atherosclerosis, cardiovascular diseases, and cancer when consumed in safe doses [3,4]. The pro-oxidation action of EGCG is regarded as the crucial mechanism for its protective functions such as induction of adaptive responses and detoxifying capacities [5].

Intriguingly, studies have reported that EGCG in a higher dose can lead to health risks, majorly by inducing hepatotoxicity in both animals and human beings. The studies postulate that EGCG may also experience auto-oxidation, which leads to the generation of Reactive Oxygen Species (ROS) and thereby induce toxicity [6]. However, research is still being carried out to weigh the benefits and against toxicity to assess the safe physiological dose of EGCG. Therefore, this calls for assessing its pharmacological properties, which necessitates elucidating and comprehensively understanding the structure of EGCG. Few earlier studies across the literature, focused on ameliorating the potential side-effects of these compounds by various therapies and also how to alleviate these side effects by enhancing its bioavailability.

Previously, methods have been developed to estimate the EGCG using Spectrophotometry [7], High Performance Liquid Chromatography (HPLC) [8,9], Reverse Phase (RP) HPLC [10,11] and Ultra-Performance Liquid Chromatography (UPLC) [12,13], Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) [14], Ultra-Performance Liquid Chromatography/Electrospray Ionization–Mass Spectrometry (UPLC/ESI-MS), Ultra-Performance Liquid Chromatography-Diode Array Detector-Mass Spectroscopy (UPLC-DAD-MS) [15] and Ultra-Performance Liquid Chromatography—Time-of-Flight—Mass Spectrometry (UPLC-TOF-MS) [16]. Also keeping in view of the low bioavailability of the compound and the limited studies to identify EGCG alone from the samples, there is a strong need to develop and validate a simple and rapid method to identify EGCG.

Therefore, the study aimed to develop an analytical method to detect EGCG using a simple chromatography technique of UHPLC and also to validate the developed method by establishing the characteristic parameters and validation criteria were specificity, linearity, accuracy, precision (system suitability and method precision), robustness and ruggedness (intermediate precision). The acceptable criterion for each parameter was carried out under the guidelines given by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). [17].

Materials and methods

2.1 Instrumentation

Agilent 1290 Infinity II—UHPLC system (Agilent, California, US) was utilized for method development and validation. The instrument was provisioned with a High-Speed Pump, Multicolumn Thermostat (MCT) pump, an Ultra-Violet (UV) detector, an autosampler, and a control module. The chromatographic separation was executed on an AcquityC18 (50 mm x2.1 mm ID) 1.8μm of Waters (Milford, Massachusetts, USA). The software installed in the system was Openlab CDS Version 2.3.4 for data analysis and evaluation. An AB Sciex triple quadrupole mass spectrophotometer with analyst software was utilized for mass identification of the compound.

2.2 Chemicals and reagents

Pharmaceutical grade standards of EGCG (purity > 96%) were procured from M/s Cayman Chemicals Company (Ann Arbor, Michigan, US). Solvents such as methanol, acetonitrile, water of HPLC grade were purchased from M/s J.T.Baker Avanator (Radnor, Pennsylvania, USA), and potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) were procured from M/s Sigma Aldrich (St. Louis, Missouri, USA).

2.3 Chromatographic conditions

The mobile phase used for the study was phosphate buffer: methanol in the ratio of 70: 30 and was set in isocratic mode. The phosphate buffer solution was prepared by adding 3.394 gram of sodium phosphate monobasic and 20.209 gram of sodium phosphate dibasic to 800 mL of distilled water in a 1000 mL beaker. The pH was adjusted to 6.8 using HCl or NaOH and distilled water was used to make up the volume. Subsequently, the buffer solution was subjected to filtration using 0.45 μm membrane filter and then degassed by sonication for 15 minutes. The mobile phase was maintained at a flow rate of 0.5 mL/min. The chromatographic analysis was carried out on Agilent 1290 Infinity II—UPLC system, which was fitted with a UV detector set to 272 nm for obtaining chromatographic data. Waters Acquity C18 (50 mm x 2.1 mm ID) 1.8 μm Column was used for the chromatographic separation, which was maintained at 25°C. The injection volume was 20 μL. The total run time of the analytical method was set to 5 minutes in the UHPLC system.

2.4 Preparation of standard solution

The standard stock solution of EGCG (obtained from M/s Cayman chemicals) was prepared by dissolving 100 mg of EGCG in 100 mL of diluent (mobile phase). Subsequently, the filtration of the solution was done using a 0.45-micron syringe filter and sonication was done for 5 minutes before using for chromatographic analysis.

2.5 Extraction of EGCG from green tea

The procedure for extraction of EGCG from green tea is explained below in Fig 1. About 1 gram of green tea leaves (collected from the market) was dissolved in 10 mL methanol. Further, the solution was subjected to continuous vortex for 30 minutes and subsequently centrifuged at 2000 rpm (15 minutes), and the supernatant solution was collected for further processing. The supernatant was then extracted using QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique to remove the potential pigments from the green leaf solution. The obtained supernatant was taken into the Agilent Dispersive SPE 15mL [Fat + Pigments Association of Analytical Communities (AOAC)] tubes and was subjected to vortex for about 20 minutes. The tube was again centrifuged (2000 rpm, 10 minutes) to obtain the supernatant, which serves as the final solution. This solution was then filtered through a 0.2 micron syringe filter (Nylon) before analysis on the UHPLC instrument for the identification of EGCG.

Fig 1

Extraction Procedure of EGCG from green tea leaves.

2.6 Method validation

2.6.1 Specificity

The specificity of the analytical method is its ability to differentiate between the analyte and the other substances in the sample matrix and thereby generate signals which are free from interference [18]. In a UHPLC method, it is confirmed by complete separation of the analyte peak from other peaks, which may originate from the sample matrix and also quantify the analyte in complex matrices. Evaluation of specificity was performed by injecting separately 10 μL of blank (mobile phase) and placebo (phosphate buffer solution) into the system.

2.6.2 Mass Spectrometry (MS) analysis

This analysis was performed on Mass Spectrometry (AB Sciex 4000 Q Trap) installed with Analyst software. The standard and sample extracted solutions (100μg/mL) were run to ensure that both the analytes were chemical identical with similar molecular structures. To obtain optimum sensitivity and selectivity, Electrospray Ionization (ESI) technique was operated in the positive ion mode which was used for MS/MS Multiple Reaction Monitoring (MRM) analysis. Further, the optimized compound parameters viz., Declustering Potential (DP), Collision Energy (CE), and Cell Exit Potential (CXP) were evaluated.

2.6.3 Linearity

Linearity was evaluated by carrying out the chromatographic analysis of several EGCG standard solutions with increasing concentrations and by establishing the calibration plot of the response vs. concentration, which visually approximates a straight line. Additionally, linear regression analysis was performed to assess the linearity of the calibration curve by employing the least square linear regression method to obtain the slope, intercept and correlation coefficient [18]. To determine the linearity of the evaluated method, standard solutions of EGCG stock solution was diluted in mobile phase, yielding standard solutions with concentrations of 50, 75, 100, 125 and 150 μg/mL. Subsequently, the solution was filtered using a 0.45micron syringe filter, which was later sonicated for 5 minutes before injecting into the UHPLC system to obtain the chromatograms. Further, linearity studies were performed for EGCG in green tea by spiking the matrix at different concentrations (20, 40, 60, 80, 100 μg/mL) and extracting the samples as mentioned above and subjected for UHPLC analysis.

2.6.4 Accuracy

The accuracy of the analytical method is determined as the nearness of the obtained value to the true value. In the present study, accuracy was determined by spiking the reference standards of the EGCG which were administered into the blank matrix, yielding solutions containing analyte with concentration levels of 50%, 100%, 150%. At every concentration level, about three samples were prepared and later analyzed. The recovery studies were performed in triplicates (for each sample), and the percentages of recovery and mean recovery were calculated for EGCG.

2.6.5 Precision

Precision is the degree of agreement/closeness among the results of each test when the method is subjected to multiple homogenous samples [18]. It can be explained as the reproducibility of measurement, and to determine the same, series of measurements (of similar quantities) were carried out. Thus, the system and method precision were evaluated using a standard solution of 100 μg/mL of EGCG, which was administered six times and the chromatograms were recorded for the same. Further, to determine the precision, statistical analysis such as Standard Deviation (SD) and the percent Relative Standard Deviation (% RSD) of variables such as Retention Time (RT), peak area, number of Theoretical Plates (TP) and Tailing Factors (TF) were measured for the test results of multiple aliquots.

2.6.6 Limit of Detection (LOD)

The limit of detection (LOD) or detection limit (DL) can be defined as the lowest concentration of an analyte which can be reliably detected in the test sample. The LOD was calculated by taking 3.3 times of standard deviation of the response (σ) and the slope of the calibration curve (S) as given in Eq 1.

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

2.6.7 Limit of Quantification (LOQ)

The lowest concentration level at which a measurement is quantitatively meaningful is called the limit of quantitation (LOQ) or quantification limit (QL). This is most often defined as 10 times the signal-to-noise ratio. Thus, the LOQ is 10 times the standard deviation (σ) of the response and slope (S) of the calibration curve as given in Eq 2.

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

2.6.8 Robustness

The robustness of the method determines the susceptibility of a method to small changes such as pH values, temperature, mobile phase composition, etc. which might occur during routine analysis at the laboratory [18]. In the present study, robustness was investigated by deliberately making the following alterations in the analytical method: (i) Flow rate: ± 0.1 mL/min, (ii) Column Temperature: ± 10°C

At each condition, the standard solution of EGCG was administered into the chromatographic system in triplicates. The robustness of the method was assessed by calculating the % RSD of the peak area after three consecutive injections of the standard solution.

2.6.9 Intermediate precision (ruggedness)

Intermediate precision (ruggedness) is the measure of precision evaluated using pre-determined conditions: similar measurement procedure, similar measuring system, similar location, and replicate measurements on the same objects over a prolonged period of time. In the present study, the ruggedness was investigated using six individual sample preparations and % RSD was calculated.

Results and discussion

3.1 Method development and optimization

Information on the physiochemical properties of EGCG was reviewed from the literature search. Further, the UHPLC method was developed to optimize the chromatographic conditions such as mobile phase, column, and wavelength for recording the chromatograms. Preliminary trials were carried out by altering the variables mentioned above and a set of chromatographic conditions were tested. Trail 1 yielded chromatogram with broad and asymmetric peaks; therefore, to minimize this, the acidity of the mobile phase was increased by adding glacial acetic acid as mentioned in trial 2. Additionally, in trial 2, only the column size was decreased as the retention time of the compound was around 2 minutes and all other specifications were maintained. However, this yielded chromatograms with split peaks, and hence in trial 3, Triethylamine was added. This also yielded unsatisfied results with multiple peaks. Further, to address the issue of multiple/splitting peaks, the buffer was introduced (trail 4) into the mobile phase, which usually helps to reduce the peak splitting and helps to maintain the pH of the solution. Since fronting of peaks was observed in this trial, the volume of buffer was increased in the mobile phase (trail 5). The results obtained from the preliminary optimization are given in Table 1 and it was observed that trial-5 was found to result in good peak shape, met the system suitability requirements, and thus was accepted and evaluated. The chromatogram of EGCG obtained using the studied condition is given in Fig 2A. In the present study, the RT of EGCG peak was 2.1 minutes, which is found to be rapid as compared to previous studies in which RT was reported as 9 minutes and 5 minutes [10, 14].

Table 1

Details of the preliminary trials and the results of the optimization.

	Mobile Phase composition	Column features	Flow Rate & Run Time	Observation	Result
Trial 1	Methanol: Water70: 30	Phenomenex C18 (150x2.1 mm ID) 1.8 μm	0.5 mL/min10 Min	• Broad peak and asymmetric factor do not meet the system suitability	Rejected
Trial 2	Water: Methanol: Glacial acetic acid75: 25: 0.7	Waters AcquityC18 (50 mm x2.1 mm ID) 1.8 μm	0.5 mL/min15 Min	• Peak splitting found and the resolution was too low• The asymmetry factor for EGCG does not meet the system suitability requirements	Rejected
Trial 3	Water: Methanol: Triethylamine75: 25: 0.7	Waters AcquityC18 (50 mm x2.1 mm ID) 1.8 μm	0.5 mL/min10 Min	• Multiple peaks were found, and the baseline was found to be unstable.• The efficiency of the method is very l ow	Rejected
Trial 4	Phosphate buffer: Methanol30: 70	Waters AcquityC18 (50 mm x2.1 mm ID) 1.8 μm	0.5 mL/min10 Min	• Fronting of peaks was observed and asymmetric factor does not meet the system suitability• The baseline was found to be unstable	Rejected
Trial 5	Phosphate buffer: Methanol70: 30	Waters AcquityC18 (50 mm x2.1 mm ID) 1.8 μm	0.5 mL/min8 Min	• All the system suitability requirements were met• The peak Asymmetry factor was less than 2• The efficiency was more than 2000	Accepted & Optimized

Fig 2

A. Chromatogram of EGCG by the developed method. B. Chromatogram of EGCG identification in green tea leaves matrices. C. Chromatogram of Placebo.

The extracted solution from green tea was analysed on UHPLC under the selected conditions, and the identification of EGCG was obtained which can be seen in the chromatogram (Fig 2B).

3.2 Method validation

3.2.1 Specificity

The specificity of the method was determined by comparing the chromatograms attained from the blank (mobile phase) and placebo solution (phosphate buffer solution). For this purpose, analysis was conducted on placebo and the chromatogram is given in Fig 2C. The chromatograms of blank and placebo solutions were eluted before the peak of EGCG. It can also be noticed from the chromatograms that at the retention times of EGCG, no other peaks were found, confirming that the analyte was pure as there are no co-eluting peaks. Thus, it was observed that diluent or excipient peaks do not intervene with the EGCG peak, and this confirmed the specificity of the method developed in the present study.

3.2.2 Mass Spectrometry (MS) analysis

The mass of the standard EGCG was found to be 459.0. Using similar conditions, the sample extracted from green tea was also analyzed and the chromatograph is given in Fig 3, the mass was also found to be 459.0 which is similar to that of EGCG. Further, the optimized compound parameters viz., DP, CE and CXP were found to be 66 volts, 17 volts and 8 volts respectively.

Fig 3

Mass Spectra of EGCG in green tea.

3.2.3 Linearity

The standard stock solutions of EGCG were prepared and diluted to obtain five concentrations of 50, 75, 100, 125 and 150 μg/mL. The prepared solutions of EGCG were injected into the system, and the chromatograms were recorded. Peak areas of the analyte were obtained for each corresponding concentration and noted. A graph was plotted for EGCG against the concentrations of the solutions and the peak areas (Fig 4A). From the regression analysis, the linear equation obtained was y = 7.0638x - 169.54 and the correlation coefficient (R2) was ≥ 0.9991 for EGCG, indicating a linear relationship between concentration of the analyte and the area under the peak. Thus, the present analytical method was found to be linear in the specified range.

Fig 4

A. Calibration curve obtained between peak area and concentration obtained for standard EGCG solutions upon linearity determination. B. Calibration curve obtained between peak area and concentration obtained for EGCG solutions from green tea upon linearity determination.

Similarly, different concentrations of EGCG (20, 40, 60, 80, 100 μg/mL) were spiked into various aliquots of green tea, and the matrix was extracted to study the linearity. Chromatograms were obtained using UHPLC, and the graph was plotted between the concentrations and the peak areas (Fig 4B). The satisfactory correlation coefficient (R2) of ≥ 0.9988 was obtained using the linear equation y = 20.797x + 47.83, which indicates the linear relationship between the concentrations obtained from the green tea matrix and the areas.

3.2.4 Accuracy

In the present study, recovery studies are performed to check the accuracy of the method. The reference standards of the EGCG were made at the concentration levels of 50%, 100%, 150%. The studies for recovery were performed; percentage recovery and percentage mean recovery were calculated for EGCG (Table 2). The results of the recovery studies observed the recovery rate from 99.1% to 100.4% at all three levels. The results were within the acceptable criteria for recovery studies for an analytical method.

Table 2

Results for determination of recovery of the analytical method.

Spiked weight of Standard (mg)	Replicate number	Concentration of the solution(μg/mL)	Area	Concentration recovered(μg/mL)	Recovery (%)
50	1	50.0	281.9	50.1	100.1
	2	50.0	281.6	50.0	100.0
	3	50.0	281.8	50.0	100.1
100	1	100.0	564.4	100.2	100.2
	2	100.0	564.5	100.3	100.3
	3	100.0	565.6	100.4	100.4
150	1	150.0	840.2	149.2	99.5
	2	150.0	837.2	148.7	99.1
	3	150.0	837.3	148.7	99.1

3.2.5 System suitability and method precision

To certify that the analytical system is running correctly and can report accurate and precise, results were assessed by injecting samples of EGCG (100 μg/mL) to record the chromatograms. The % RSD of retention time (RT) was found to be 0.1% and the % RSD for peak area was 0.1%. While the number of theoretical plates (TP) was higher than 30000 (with a range of 30521 to 30581) for all analyte peaks, the tailing factors (TF) were all less than 2.0% (with a range of 1.22 to 1.27).

Further, the method precision was obtained by injecting sample solutions of EGCG six times at concentration (100 μg/mL), prepared separately. The chromatograms were reported and the results are as follows. The % RSD of assay for 6 samples determinations of EGCG was found to be 0.1% and the % assay was also within the limits (95 to 105). Since the results obtained of both system and method precision showed that the variables obtained were within the acceptable limits, it can be considered that the system and method is precise.

3.2.6 LOD and LOQ

The LOD and LOQ were determined based on the signal to noise ratio of 3.3 and 10, respectively. The standard deviation (σ) was found to be 0.364, and the slope (S) of the calibration curve (obtained from the linearity) was observed to be 7.06. Therefore, the LOD was calculated as 0.170 μg/mL, and the LOQ was 0.515 μg/mL. The limits in the present study were found to be lower than the previous study, which reports LOD as 0.528 μg/mL and LOQ as 1.600 μg/mL, thus suggesting the present method was more sensitive [13]. Similarly, the LOD and LOQ with regard to the green tea leaf extract were calculated. The standard deviation (σ) was obtained as 0.364 and slope (S) as 20.797 (from the calibration curve of the green leaf extract samples). The LOD and LOQ were found to be 0.05 μg/mL and 0.17 μg/mL respectively.

3.2.7 Robustness

The Robustness of the investigated analytical method was tested to evaluate the impact of minor changes in the U-HPLC system conditions on parameters like system suitability of the new method. The results due to deliberate variation in the method conditions are summarized below in Table 3, which includes minor change of flow rate and column temperature. For all the modifications and the three consecutive injections, the % RSD values for RT, Tailing Factor, Theoretical Plates, and peak areas were determined. The results obtained showed that the minor modifications applied in the robustness test had no significant change, and the variables determined in the test were found to be within the acceptable limits.

Table 3

Results for determination of Robustness of the analytical method.

Chromatographic changes		Retention Time (min)	Peak Area	Tailing Factor	Theoretical Plates
Flow rate(mL/min)	0.4	2.820	746.77	1.32	39481.87
		2.826	746.00	1.30	39439.75
		2.827	746.27	1.33	39190.81
	Mean	2.824	746.35	1.31	39370.81
	SD	0.003	0.3906	0.015	157.3
	RSD (%) (n = 3)	0.1	0.05	1.1	0.3
	0.6	1.705	449.05	1.22	27869.63
		1.702	449.43	1.20	27829.76
		1.7	449.31	1.24	27700.70
	Mean	1.702	449.26	1.22	27800.03
	SD	0.002	0.194	0.02	88.301
	RSD (%) (n = 3)	0.1	0.04	1.6	0.3
Column Temperature(°C)	25°C	2.400	562.20	1.24	30348.93
		2.404	562.16	1.27	3015.96
		2.405	562.09	1.24	30255.85
	Mean	2.403	562.15	1.25	30372.58
	SD	0.002	0.055	0.017	130.17
	RSD (%) (n = 3)	0.1	0.009	1.3	0.4
	35°C	1.937	615.62	1.28	32980.61
		1.913	618.37	1.26	33285.38
		1.899	616.53	1.28	33197.77
	Mean	1.9163	616.84	1.273	33154.59
	SD	0.019	1.400	0.011	156.90
	RSD (%) (n = 3)	1.002	0.2	0.9	0.4

3.2.8 Intermediate precision (ruggedness)

The measure of intermediate precision/ruggedness was performed using six preparations of the EGCG individually into the chromatography system to determine the % assay of individual samples. The average % assay was found to be 99.0% with a range of 97.7% to 99.4 and the RSD (%) for six preparations assay values was 0.7. From the above results, it can be observed that the SD of % assay was 0.67 and RSD (%) was 0.7, which were within the acceptance criteria, indicating that the present method is rugged.

Conclusion

This study was carried out to develop and evaluate a method for the estimation of EGCG using UHPLC. The results of the validation study deduced that this method was simple, rapid, accurate, precise, robust and rugged. The method was validated in accordance with the guidelines laid down by ICH and found to be acceptable. The method could provide accurate and precise quantitative results under minor changes of chromatographic conditions.

29 Oct 2019

PONE-D-19-27318

Method Development and Validation for rapid identification of Epigallocatechin Gallate using Ultra-Performance Liquid Chromatography

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Reviewer #1: This manuscript describes the method development and validation for EGCG using UHPLC. The goals of the study are straightforward. However, there are several issues with the manuscript that need to be addressed. These points are listed below in order of appearance in the manuscript.

Page 2, abstract: The abstract needs to be rewritten and proofread for grammar. The are issues with the first two sentences. "Specificity" is list twice in line 7.

Page 2, abstract and throughout the paper. UPLC should be replaced by UHPLC as it is the proper term. UPLC is a trademark of Waters Corp.

Page 2, line 3 of introduction: EGCG is know to help alleviate some symptoms, but does not prevent all the diseases mentioned here.

Page 6, end of specificity section: The contents of the blank and placebo solutions must be described.

Page 7 and 8: Equations should be numbered.

Page 8, Robustness section: The authors state the temperature is varied +/- 10 degrees. What temperature is varied? Is it the cinematographic column temperature? the solution temperature? It needs to be more specific.

Page 9, Table 1: "The efficiency was very less." does not make sense. What efficiency is being described? Efficiency is not described as a criterion in the Methods section.

Page 10, Figure 2: All the chromatograms should have the same time axis some that the comparison is easier to see by the reader.

Page 11, top of the page: The difference observed by mass spectrometry must be explained. A difference of 0.8 Da is significant. What is the cause of the difference?

Page 13, LOD and LOQ section: The slope used in the calculation is form the standard curve. The slope from the tea leaves is different and should be used here if the authors are showing how their method works in leaves. Or both analyses should be presented. The last sentence on this page does not agree with the authors calculations presented and are the same as reference 13.

Reviewer #2: An Ultra-Performance Liquid Chromatography method for single-analyte quantification of Epigallocatechin gallate (ECGC) in green tea was developed. The idea of the work is clear and the method was properly validated, as it seems. However, there are several language inconsistencies and terminology mistakes in some points that must be clarified and corrected for a better understanding and evaluation of the scientific content. Hence, I would consider the paper for publication in PLOSONE provided that the authors conduct major revisions on the manuscript, as detailed in the pdf file attached. Needless to say, that a major revision of the English and the analytical language is highly advised for acceptance of the final manuscript.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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Submitted filename: Revision PONE-D-19-27318.pdf

Click here for additional data file.

21 Nov 2019

Review Comments to the Author

Reviewer #1:

Comment 1: Page 2, abstract: The abstract needs to be rewritten and proofread for grammar. The are issues with the first two sentences. "Specificity" is list twice in line 7.

Response: As per the reviewer’s suggestions, necessary corrections were made in the abstract (Page number 2).

Comment 2: Page 2, abstract and throughout the paper. UPLC should be replaced by UHPLC as it is the proper term. UPLC is a trademark of Waters Corp.

Response: The term UPLC has been replaced by UHPLC throughout the revised manuscript.

Comment 3: Page 2, line 3 of introduction: EGCG is know to help alleviate some symptoms, but does not prevent all the diseases mentioned here.

Response: As suggested, appropriate word has been replaced in the revised manuscript.

Comment4: Page 6, end of specificity section: The contents of the blank and placebo solutions must be described.

Response: As desired, the contents of both blank and placebo solutions are described in the revised manuscript (Page numbers 6 and 11).

Comment 5: Page 7 and 8: Equations should be numbered.

Response: The equations given in page 8 are duly numbered in the revised manuscript.

Comment 6: Page 8, Robustness section: The authors state the temperature is varied +/- 10 degrees. What temperature is varied? Is it the cinematographic column temperature? the solution temperature? It needs to be more specific.

Response: The temperature of the column was varied to evaluate the robustness of the developed method. The same is now incorporated in the revised manuscript (Page numbers 9 and 15).

Comment 7: Page 9, Table 1: "The efficiency was very less." does not make sense. What efficiency is being described? Efficiency is not described as a criterion in the Methods section.

Response: The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travels through the UHPLC system and column. Ideally, the chromatographic peaks would be pencil thin lines; however, due to dispersion effects the peaks take on their familiar ‘Guassian’ shape. Therefore, in this context, efficiency of chromatograms means asymmetric and unacceptable shape along with split in peaks and disturbance in the baseline which was unstable.

Comment 8: Page 10, Figure 2: All the chromatograms should have the same time axis some that the comparison is easier to see by the reader.

Response: In Figure 2, since the time of identification was found to be around 2 minutes, run time was decreased to 5 minutes instead of 8 minutes and hence the difference in the time scale was observed.

Comment 9: Page 11, top of the page: The difference observed by mass spectrometry must be explained. A difference of 0.8 Da is significant. What is the cause of the difference?

Response: Generally, in mass spectra molecular ion is obtained as protonated and deprotonated ions. And deprotonation may have been observed in the green tea. We have used 4000 Qtrap which is not high-resolution MS.

Comment 10: Page 13, LOD and LOQ section: The slope used in the calculation is form the standard curve. The slope from the tea leaves is different and should be used here if the authors are showing how their method works in leaves. Or both analyses should be presented. The last sentence on this page does not agree with the authors calculations presented and are the same as reference 13.

Response: Since the aim of the study was to develop a method and validate a method for identification of EGCG, considering the slope from the standard curve obtained between peak area and concentration of standard EGCG solutions to calculate LOD and LOQ is sufficient and serves the purpose. Similarly, the LOD and LOQ with regard to the green tea leaf extract were calculated using the σ as 0.364 and S as 20.797 (obtained from the calibration curve of green tea leaf extract) and was found to be 0.05 and 0.17 respectively and is now incorporated in the revised manuscript (Page number 14).

Reviewer #2:

An Ultra-Performance Liquid Chromatography method for single-analyte quantification of Epigallocatechin gallate (ECGC) in green tea was developed. The idea of the work is clear and the method was properly validated, as it seems. However, there are several language inconsistencies and terminology mistakes in some points that must be clarified and corrected for a better understanding and evaluation of the scientific content. Hence, I would consider the paper for publication in PLOSONE provided that the authors conduct major revisions on the manuscript, as detailed in the pdf file attached. Needless to say, that a major revision of the English and the analytical language is highly advised for acceptance of the final manuscript.

Response: The revised manuscript was proof-read by a native English speaker and then crosschecked using Grammarly software for any grammatical and technical errors in the language.

Major Revisions

Question 1: Please revise the short title for “UPLC determination of EGCG” as quantitative analysis is presented in the manuscript.

Response: As per the suggestion of the reviewer, the short title is edited.

Question 2: Please revise all units according to the International System and make them consistent across the entire manuscript text and figures (especially figure 1). All units must be separated from values by a space, except for percentage in which the number must be close to the value. Beware that “1 gr” and “micron” are not the correct way of expressing units (Figure 1 and respective manuscript text). In this sense, make the formatting consistent and correct throughout the whole manuscript and the figures. Please include values of n in brackets, as the number of determinations, whenever a relative standard value is given.

Response: In the revised manuscript, all the units (both in figure and manuscript) are revised. Further, the number of determinations (n) is also included at the appropriate places.

Question 3: Authors seemed to have been rather careless about significant figures. All inaccuracies must be corrected. For instance, LOD and LOQ should bare a single or two significant figures at the most.

Response: The method employed to calculate the LOD and LOQ values was by using the standard deviation and slope of the calibration curve. Since, these values were already given while representing the linearity in Figure 4a, additional figures were avoided.

Question 4: Please revise the term “therapeutic doses of EGCG” (first paragraph of the introduction section) and clarify the sentence that follows “EGCG’s pro-oxidation action is regarded as a mechanism for its protective functions including its antioxidant properties”. This presents to me as an inconsistency to me as dose distinguished pro and antioxidant actions.

Response: As per the suggestion, the term and the sentence mentioned by the reviewer were edited (Page numbers 2 and 3).

Question 4: A more solid explanation must be given concerning the purpose of developing of a single-analyte method for EGCG detection by chromatography. Accordingly, authors must revise the third paragraph of the introduction section and link it with the first sentence of the following, on page 3: “Despite previous studies, there is a need to optimize a simple and rapid method to identify the EGCG compound using a simple chromatography technique using UPLC system”. Also, remove “compound” and “using UPLC system” from the sentence.

Response: The purpose of the present study was edited and the appropriate changes suggested by the reviewers were made (Page number 3). Also, the terms “compound” and “using UPLC system” were removed in the revised manuscript.

Question 5: Still on page 3 (last paragraph), please write “to validate the method by establishing the characteristic analytical parameters” and “validation criteria were”. Also, the ICH guideline that has been applied must be referenced here.

Response: The appropriate changes in terms of re-phrasing were made (Page number 3). The ICH guidelines which was considered in the present study was also referred (Reference number 17).

Question 6: In section 2.2, clarify what do authors mean by “pharmaceutical grade samples of EGCG”. Do they refer to calibration standards? In section 2.4, please clarify what the “diluent” was.

Response: Pharmaceutical grade samples of EGCG was corrected to Pharmaceutical Grade Standards (Page number 4). Mobile phase was used as the “diluent” and is mentioned in the revised manuscript (Page number 5).

Question 7: Before chromatographic analysis, a three-step sample preparation was employed. Please explain the need for this cumbersome procedure to extract EGCG. Also, I am afraid that some of the analyte may be lost in the filter immediately before analysis. Recovery percentages are quite high for such a multi-step procedure. Could the authors address this issue and clarify? Also, please add information on the syringe filter material.

Response: Most polyphenols including EGCG are soluble in solvents like methanol, water etc. Hence, green tea was dissolved in methanol to extract EGCG. Further, since the green tea is rich in pigments and may interfere/block the column, therefore to isolate these pigments SPE tubes were used. Additionally, to separate any undissolved large particles, filtration was done using 0.2 µ syringe filters (Nylon).

Question 8: In section 2.6.2, please re-phrase”: Linearity was evaluated by carrying out the chromatographic analysis of several EGCG standard solutions with increasing concentrations and by establishing the calibration plot of the response vs. concentration, which visually approximates a straight line.” Also, “To determine linearity (…), EGCG stock solution was diluted in mobile phase, yielding standard solutions with concentrations of (…).”.

Response: The suggested re-phrasing was done in the revised manuscript (Page numbers 6 and 7).

Question 9: In section 2.6.3, please restrict the description to the strategy that has been used in this work for accuracy assessment. It is rather confusing how this determination was performed. Please re-phrase starting by “In the present study, accuracy was determined by spiking (…)”.

Response: As per the reviewer’s suggestion, the phrase was revised and incorporated (Page number 7).

Question 10: In section 2.6.5, please re-phrase as follows “the LOD is defined as the lowest concentration of analyte which can be reliably detected in the test sample.”

Response: The sentence re-phrasing was done as per the reviewer’s suggestion (Page number 8).

Question 11: Please replace the word “optimise” by “studied”, “evaluated” or “investigated” whenever appropriate as no design of experiments was undertaken.

Response: As suggested by the reviewer, the term “optimise” was revised to appropriate word and included in the revised manuscript.

Question 12: In section 3.1, replace “five such trials were conducted” by “set of chromatographic conditions were tested”. The authors must elaborate further on the options taken to establish the five trials as a poor discussion is provided. For instance, many variables have been changed from trial 1 to trial 2, preventing the isolation of the motive for rejection. Was it the column length of the mobile phase composition? Also, in table 1, please write “Mobile phase composition” and “Column features” in the appropriate column. What do authors mean by “Baseline is not proper”? Was it unstable, too much noise?

Response: As suggested by the reviewers, the selected were replaced in the section 3.1 (Page number 9). Further, elaborate discussion is now provided on the five trials taken up during the method development (Page numbers 9 and 10). The appropriate words were revised in table 1 (Page numbers 10 and 11). Further, as suggested by the reviewers, the terms were revised in the appropriate column of the table 1 as ‘baseline was found to be unstable’.

Question 13: Please eliminate section 3.2 and include comments in section 3.1. Renumber the rest of the section, accordingly.

Response: As per the suggestion of the reviewer, section 3.2 was eliminated and the rest of the sections were renumbered accordingly.

Question 14: Concerning section 3.3.1, define “blank” and “placebo” appropriately. Please re-phrase the second sentence, as the idea is simply unclear.

Response: In the present, mobile phase was used “blank” and the phosphate buffer solution was used as the placebo. These are defined in the manuscript (Page numbers 6 and 11). Further, the second sentence has now been re-phrased in the revised manuscript.

Question 15: Please explain the purpose of performing a mass spectrometry analysis (page 11). Also, I do not recall reading the specifics of the determination in the methods section, particularly the m/z values of the main transitions used for EGCG identification. Please add this information wherever appropriate.

Response: The primary function of mass spectrometry is to detect a chemical compound based on its mass-to-charge (m/z) ratio. In the present study, standard EGCG was initially run on mass spectrometry to identify its mass. Later, sample extracted from the green tea leaves was also run on mass spectrometry to identify its mass and to correlate the same with the standard. This ensures that both the analytes have chemical identity and structure of molecules. The purpose of performing this analysis was to confirm that both of them are similar chemical compounds. Further, the details of the mass spectrometry analysis are now added in the methods section (Page number 6).

Question 16: The first three sentence of section 3.3.2 is just a repetition of the information of the respective experimental section. Please re-phrase them appropriately as matter of discussion. Please write R2 ≥ 0.9998. Also, in Figure 4a caption, re-phrase as “Calibration curve obtained between peak area and concentration obtained for standard EGCG solutions upon linearity determination.” The same phrasing should be applied for figure 4b. Please add error bars to the figures and the uncertainty obtained the slope and the intercept values. Finally, authors should explain the criteria to select the test concentration in light of the biological contents of EGCG.

Response: The sentences mentioned by the reviewer were re-phrased. Further, “≥” was added at the appropriate places. The captions for Figures 4a and 4b were changed according to the suggestions and error bars were added to the Figures 4a and 4b. With regard to the criteria for selecting the test concentrations, since the working linearity range is predefined by the purpose of the method and it is known that concentration of EGCG is around 30-40% in green tea, we have considered the linearity range from the lowest concentration of 20 µg/mL.

Question 17: Regarding Robustness, I do not see how temperature effect was evaluated. Please clarify.

Response: For the purpose of the determination of robustness, the column temperature was changed and evaluated (mentioned in page numbers 9 and 15 of the revised manuscript).

Question 18: Re-write the first sentence 3.3.4. it is just cumbersome.

Response: The sentence mentioned by the reviewer was revised and incorporated (Page number 14).

Minor revisions:

Question 1: Please revise the whole text for typos and make sure the references are in the correct format and numbered throughout the text.

Response: The whole manuscript was thoroughly checked for typos and the references were edited and numbered through the text.

Question 2: Please replace “same developed conditions” by “selected conditions” on page 10 (section 3.2) and whenever appropriate.

Response: The necessary corrections were made and incorporated (Page number 11).

Question 3: Abstract: Please correct “its auto-oxidation property may lead to toxicity”, write “linear correlation coefficient > 0.9996”, and remove average recovery.

Response: The appropriate changes mentioned by the reviewer with regard to the abstract were made (Page number 2).

Question 4: Remove capital letters from buffer salts, reagents, laboratory material (except for brands or equipment models), and analytical parameters within sentences.

Response: The suggested changes were made and incorporated (Page numbers 4 and 5).

Question 5: Figures and Tables: - In figure 1, remove all capital letters within the sentences and the respective caption. Add the meaning of AOAC to the respective caption and to the manuscript text. Correct “supernate” to “supernatant”, also in the main text.

- Erase the column for average recovery on Table 2. Please write “Recovery (%), recovery values, and RSD (%)” in all tables instead of % Recovery, percentage recovery, and % RSD respectively.

- Please revise significant figures in Table 3 as those are not consistent and in agreement with method precision. Also, express RSD values with a single decimal figure in all cases.

Response: The corrections mentioned with respect to the Figure 1 were duly made and incorporated in the revised manuscript. Table 2 and 3 were revised as suggested by the reviewer.

2 Dec 2019

PONE-D-19-27318R1

Method Development and Validation for rapid identification of Epigallocatechin Gallate using Ultra-High Performance Liquid Chromatography

PLOS ONE

Dear Dr. Sinha,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

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Reviewer #1: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript is improved over the previous version. However, the authors still have several issues that they need to address. The concerns are listed below.

1. The third line of the abstract is worded poorly and needs to be rewritten. Many of the previous wording concerns have been addressed, but a thorough proofreading is needed in any revision.

2. From the previous review "Comment 9: Page 11, top of the page: The difference observed by mass spectrometry must be explained. A difference of 0.8 Da is significant. What is the cause of the

difference?"

Response: Generally, in mass spectra molecular ion is obtained as protonated and

deprotonated ions. And deprotonation may have been observed in the green tea. We

have used 4000 Qtrap which is not high-resolution MS.

This response is not adequate. A difference of 0.8 Da is easily observed on a QTrap 4000. (I agree a QTrap 4000 is not a high resolution instrument.) If the authors think there is a protanation/deprotanation reaction, it needs to be demonstrated and explained. Why is there a Dalton shift between standard solutions and real samples?

3. Figure 2: All the chromatograms should have the same time axis so that the comparison is easier to see by the reader. I understand the change in run times, but the data should all have the same time scale.

4. Page 14, Section 3.2.6: The last part of this section is not clearly written. Further the calculated LOQ and LOD need to have units with the values.

5. There are still issues with significant figures in Tables 2 and 3. For example in Table 2, the concentration of a solution is listed as 50 ug/mL (1 significant figure), yet the recovered amount is listed as 50.06 ug/mL (4 significant figures). The recovered amount can not have more significant figures than the amount you started with, 1 significant figure. In Table 3, the number of theoretical plates is given to 0.01, this is beyond the precision of the measurement. Both tables need to be revised further.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Dec 2019

Review Comments to the Author

Reviewer #1:

Response 1: As has been suggested by the reviewer, the third line of the abstract has been rewritten (Page number 2) and in-depth proofreading of the whole manuscript was done in the revised manuscript.

Response 2: The authors are thankful to the reviewer for the comment. We have repeated the MS analysis for green tea extraction sample for the identification of EGCG several times and the mass was found to be 459.0 which is similar to mass of the standard EGCG which was also found at 459.0. Hence we could confirm that the developed method is suitable for identification of EGCG (Page number 11).

Response 3: As suggested by the reviewer, all chromatograms were given with the same time axis (Figure 2a, 2b and 2c).

Response 4: As suggested by the reviewer, the last part of the section 3.2.6 has been rewritten and also units were given along with the LOD and LOQ values (Page number 14).

Response 5: As per the suggestion of the reviewer, table 2 and table 3 were revised in the revised manuscript. With respect to table 3, all insignificant figures in the tables were corrected and the SD of all the factors were found within the significant ranges as suggested by the reviewer.

Submitted filename: PONE-D-19-27318 R2 Responses.docx

Click here for additional data file.

23 Dec 2019

Method Development and Validation for rapid identification of Epigallocatechin Gallate using Ultra-High Performance Liquid Chromatography

PONE-D-19-27318R2

Dear Dr. Sinha,

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Additional Editor Comments (optional):

Reviewers' comments:

31 Dec 2019

PONE-D-19-27318R2

Method Development and Validation for rapid identification of Epigallocatechin Gallate using Ultra-High Performance Liquid Chromatography

Dear Dr. N:

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