INTRODUCTION:

Diabetes mellitus (DM) is a complex chronic illness associated with a state of high blood glucose level, or hyperglycemia, occurring from deficiencies in insulin secretion, action, or both.¹ The clinical diagnosis of diabetes is reliant on either one of the four plasma glucose (PG) criteria: elevated (i) fasting plasma glucose (FPG) (>126 mg/dL), (ii) 2 h PG during a 75-g oral glucose tolerance test (OGTT) (>200 mg/dL), (iii) random PG (>200 mg/dL) with classic signs and symptoms of hyperglycemia, or (iv) hemoglobin A1C level >6.5%.²,³

The major classes of oral antidiabetic medications include biguanides, sulfonylureas, meglitinide, thiazolidinedione (TZD), dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter (SGLT2) inhibitors, and α-glucosidase inhibitors.⁴^,⁵^,⁶

The present study were carried out with an aim to study the various HPLC methods developed for the determination of the antidiabetic drugs of SGLT2 class in bulk and various pharmaceutical dosage forms. Present study also envisaged upon the method developed for the estimation of drugs individually or in combination formulation with other oral hypoglycemics.

High Performance Liquid Chromatography (HPLC):

High Performance Liquid Chromatography (HPLC) was derived from the classical column chromatography and, is one of the most important tools of analytical chemistry today. The principle is that a solution of the sample is injected into a column of a porous material (stationary phase) and a liquid (mobile phase) is pumped at high pressure through the column. The separation of sample is based on the differences in the rates of migration through the column arising from different partition of the sample between the stationary and mobile phase.⁷^,⁸ High Performance Liquid Chromatography is more versatile than gas chromatography since (a) it is not limited to volatile and thermally stable samples, and (b) the choice of mobile and stationary phases is wider.⁹^,¹⁰^,¹¹

High performance liquid chromatography (HPLC) is an essential analytical tool in assessing drug product.¹²^,¹³ Validation is the process of establishing the performance characteristics and limitations of a method and identification of the influences which may change these characteristics and to what extent. This article reviews the use of HPLC for chromatographic analysis of antidiabetic drugs (Sodium-glucose co-transporter (SGLT2) inhibitor) Canaglifozine, Dapagliflozine, Empagliflozine, Ertugliflozine.

Method Validation:

Methods should be validated to include consideration of characteristics included in the International Conference on Harmonization (ICH) guidlines1, 2 addressing the validation of analytical methods. Analytical methods outside the scope of the ICH guidance should always be validated.¹⁴

HPLC Analysis of Dapagliflozin:

Dapagliflozin is a sodium-glucose co-transporter 2 inhibitor indicated for managing diabetes mellitus type 2. When combined with diet and exercise in adults, dapagliflozin helps to improve glycemic control by inhibiting glucose resorption the proximal tubule of the nephron and causing glycosuria.

In an experiment by Debata et al., (2017) a new, simple, selective, accurate, rapid and precise reversed-phase high-performance liquid chromatographic technique of Dapagliflozin was established as per ICH Guidelines. RP-HPLC was performed on a Waters C18, 5µm particle size, 25cm × 4.6mm i.d., with phosphate buffer and acetonitrile in the ratio of 60:40 v/v as a mobile phase and a flow rate of 1.0ml min-1. UV detection was performed at 237nm. The total run time was 6.0 min. The retention time of Dapagliflozin was found to be 3.461 minutes. Validation of the developed method was done as per USP and ICH guidelines. Method validation revealed that the method was rapid, accurate, precise, reliable, and reproducible. Linear calibration plots were obtained in the concentration range of 10-60µg/ml for Dapagliflozin. The limit of detection was 0.02µg/ml and the limit of quantification was 0.06µg/ml for Dapagliflozin. The high recovery and low coefficients of variation confirmed the effectiveness of the process in the dosage form. The results suggested that the developed method was a suitable method for assay, purity which could help in the analysis of Dapagliflozin in different formulations.¹⁵

In another study carried out by Basha and Sravanthi, (2017) the estimation of dapagliflozin using reversed-phase high-performance liquid chromatography (RP-HPLC) technique in bulk and tablet formulation was done to develop and validate a simple, selective, precise, and accurate method. The proposed method utilized chromatographic conditions hypersil BDS (250mm × 4.6mm, 5µ), mobile phase was buffer: acetonitrile (60:40) ratio, flow rate was maintained 1ml/minute, column temperature was set at 30°C, detection wavelength was 245nm, and diluent was mobile phase. Their results were found well under the acceptance criteria when they injected 5 times of the standard solution system and studied the suitability parameters. The linearity study was performed by taking 25-150% levels, and the R2 value was found to be 0.999, precision was found to be 0.5 for repeatability and 0.31 for intermediate precision. The % recovery was found to be 99.89%. Limit of detection and limit of quantitation were found to be 0.60µg/ml and 1.81µg/ml, respectively. The % purity was found to be 99.71%. Degradation study on dapagliflozin was performed and it was concluded that the purity threshold was more than purity angle and within the acceptable range.

The results of the forced degradation studies showed the major route of degradation is in acid hydrolysis followed by alkali, oxidation, thermal, photolytic, and neutral, respectively. The developed method concluded that dapagliflozin was stable in neutral, photolytic, thermal and oxidative stress conditions, and unstable in acidic and alkali conditions.¹⁶

Verma et al., (2017) in their study developed a precise, accurate and reproducible stability assay method by RP-HPLC for estimation of dapagliflozin in API and pharmaceutical dosage form. In the experiment, adequate separation was carried using Agilent C18 (4.6 ml (millimeter)*150,5µm (micrometer), mixture of acetonitrile: dipotassium hydrogen phosphate with pH-6.5 adjusted with OPA (40:60 %v/v) as a mobile phase with the flow rate of 1 ml/min (milliliter/minute) and the effluent was monitored at 222nm (nanometer) using photodiode array detector. The retention time of dapagliflozin API and dapagliflozin tablets was 3.160 min (minute) and 3.067 min (minute) respectively. According to their results, linearity for dapagliflozin was found in the range of 50-150µg/ml (microgram/ milliliter) (R2 = 0.99) respectively. The accuracy of the present method was evaluated at 50 %, 100% and 150%. The % recoveries of dapagliflozin API and tablet were found to be in the range of 99.00– 99.99 % and 98.50–99.99% respectively. Precision studies were carried out and the relative standard deviation values were less than two. The method was found to be robust. Overall, the proposed method was found to be specific, accurate, precise and robust and could be used for the estimation of dapagliflozin in API and Pharmaceutical dosage form.¹⁷

According to a study done by Mante et al., (2018) RP-HPLC method was developed for the determination of Dapagliflozin in the tablet dosage form. The method was validated and found to be simple, sensitive, accurate, and precise. The chromatographic separation was achieved by isocratic mode with a mixture of Acetonitrile: 0.1% Triethylamine (pH-5.0) in the ratio of 50:50v/v as mobile phase using Princeton C18 column at a flow rate of 1mL/min and detection wavelength of 224nm. Using optimized chromatographic conditions, the retention time of the drug was found to be 5.163min. The proposed method obeyed Beer’s-lambert’s law in the concentration range of 10-70μg/mL, with a correlation coefficient value 0.999. The mean percent amount of drug estimated was 100.57%, found to be good in agreement with the label claim of marketed tablet formulation. The validation parameters like accuracy, precision, ruggedness, robustness, linearity and range were studied for the proposed method and were found to be within limits. Stress testing under various conditions such as pH (acid/base), oxidation, temperature, light, humidity, etc. were also carried out.¹⁸

In another work done by Aswini e al., (2018) a rapid, specific, accurate and precise Reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous determination of Dapagliflozin and Saxagliptin in bulk and pharmaceutical dosage form. Successful chromatographic separation of Dapagliflozin and Saxagliptin was carried out with Inertsil-ODS, C18 column (250 × 4.6mm; 5μm) with mobile phase consisted of a mixture of Methanol and Potassium dihydrogen phosphate buffer in the ratio of 45:55 v/v delivered at a flow rate of 1.0 ml/min. The eluents were monitored by PDA detector and peaks values were measured at 210nm. The retention times for Dapagliflozin and Saxagliptin were 4.707 min and 6.684 min respectively. The analytical method was validated according to ICH guidelines (ICH, Q2 R1). The linearity study of Dapagliflozin and Saxagliptin was found in the concentration range of 20-70μg/ml and 20-70μg/ml respectively and the coefficient of variance was 0.999 for both drugs. The % recovery was found to be 100.37% and 100.16% for Dapagliflozin and Saxagliptin respectively. LOD was 0.109μg/ml and 0.58μg/ml and LOQ was 0.332μg/ml and 1.77μg/ml for Dapagliflozin and Saxagliptin respectively. It was inferred that the developed method was successfully applied for the simultaneous estimation of Dapagliflozin and Saxagliptin in bulk and its commercial pharmaceutical dosage forms and could be used for the routine analysis of the studied drugs in quality control laboratories¹⁹

HPLC analysis of Canagliflozin:

Canagliflozin is a C-glycosyl compound that is used (in its hemihydrate form) for the treatment of type II diabetes via inhibition of sodium-glucose transport protein subtype 2. It is a C-glycosyl compound, a member of thiophenes and an organofluorine compound.

In a research Kaur et al., (2016) attempted to develop a validate simple, authentic and stability-indicating HPLC method for estimation of Canagliflozin in bulk and tablet dosage forms. In methodology, the research investigated the use of a mobile phase consisting of Acetonitrile: orthophosphoric acid in a ratio of 55:45 v/v at a flow rate of 1ml/min with an injection volume of 20 μL was selected for this study. The method was validated as per the on the parameters Linearity, Precision, Accuracy, Robustness, Ruggedness, Limit of Detection (LOD) and Limit of Quantification (LOQ). The separation was achieved at a temperature of 30ºC and the eluents were observed by photodiode array detector set at 290 nm. A linear range of 1-6μg/ml with a correlation coefficient of 0.998 unfolds good linear relationship between area and concentration in the calibration curve. The retention time obtained was at 6.29 min. A recovery of Canagliflozin in tablet formulation was observed in the range of 99.6-99.8%. Percentage assay of Canagliflozin tablets (INVOKANA^®) was found to be 99.92%. As per their findings, the researchers proposed that the present method is definite, meticulous and reproducible and can be used for routine analysis of Canagliflozin in bulk and pharmaceutical dosage form.

The values of accuracy, precision, robustness, ruggedness, LOD, and LOQ were within the limits. Canagliflozin is very sensitive so it is unstable in alkaline, acidic, oxidative, thermal and photo light. Statistical analysis for the results clearly demonstrate that the method is suitable for the determination of Canagliflozin in bulk and tablet forms without any interference from the degradation products, and it is endorsed for routine use in quality control industry laboratories.²⁰ However the mobile phase used in the study employed have the ortho-phosphoric acid at the very high ration which can cause the clogging of the column through precipitation.

In another research Marella et al., (2017) planned their studies with an objective to develop a simple, specific and accurate reverse phase high performance liquid chromatographic method for the determination of Canagliflozin in bulk and pharmaceutical dosage forms. As a result of their effort, it was found that the Canagliflozin peak was eluted at 4.4 min using the mobile phase. The calibration curve was a linear combination of 0.02% Formic acid: Acetonitrile (40:60) at a flow rate of 1.2ml/min and the eluents were monitored at 230nm. In other chromatographic conditions, ODS-3(250×4.6mm, 5µ) column was used. The mean percentage assay was found to be 98.2. As per the ICH guideline, it was found to validate the method and can be used for the determination of Canagliflozin in bulk and pharmaceutical dosage form with LC-MS compatible mobile phase composition in short run time.²¹

Singh et al., (2019) develop, validate and compare a spectrophotometric and a high-performance liquid chromatography method for estimating canagliflozin in bulk and tablet dosage form. Spectrophotometry and high-performance liquid chromatography were carried out using standard instrumental parameters, which were optimized. The optimized ratio of the mobile phase in high-performance liquid chromatography under low-pressure gradient mode was 50:50 % v/v of acetonitrile: orthophosphoric acid (0.01 M), which provide a sharp peak with a short retention time of 4.732 minutes. In the spectrophotometric analysis, methanol as a solvent gave adequate molar absorptivity at a λmax of 280 nm. Results indicated that both spectrophotometric and high-performance liquid chromatography methods were linear, precise, accurate, rugged and robust with RSD values less than 2% and percent recovery were within the standard limits (90-110%). Both the methods were found to be statistically non-significant at 95% confidence intervals (p<0.05) with respect to each other. The proposed methods were found to be highly effective and could be used for quantification of canagliflozin in bulk and tablet formulations for routine analysis.²²

In the study performed by Parida et al., (2018) a method was developed for the estimation of Canagliflozin in pharmaceutical dosage forms using Reverse Phase High-Performance Liquid Chromatography. The analysis was performed using the C18 column having 250mm×4.6 mm ID dimension, 5μm particle size) and Agilent LC1220 HPLC machine with UV detection (VWD detector) at 293nm. The mobile phase was used as Methanol: Phosphate buffer (PH adjusted to 4 with orthophosphoric acid) (65:35 v/v) at a flow rate of 1ml/ min. As a result of the procedure, the Chromatogram gets separated and indicates the retention time of 2.980 min. The recovery of Canagliflozin was found to be in the range of 99.33-99.92%. The developed method was also validated as per the ICH guidelines. Linearity was done for Canagliflozin in the range of 10- 125μg/ml with a correlation coefficient of 0.999. The percentage recovery of the drug was achieved in the range of 98-102% which become within the acceptance criteria.²³

HPLC Analysis of Empagliflozin:

Empagliflozin is a C-glycosyl compound consisting of a beta-glucosyl residue having a (4-chloro-3-{4-[(3S)-tetrahydrofuran-3-yloxy] benzyl} phenyl group at the anomeric center. It is a C-glycosyl compound, aromatic ether, a tetrahydrofuran ether and a member of monochlorobenzenes. Empagliflozin works by helping the kidneys get rid of glucose from your bloodstream. Empagliflozin is used together with diet and exercise to improve blood sugar control in adults with type 2 diabetes mellitus.

In a study done by Siridevi et al., (2019) Empagliflozin was estimated in bulk and tablet dosage form by the HPLC method. Chromatography was carried out on Enable C18G (250 x 4.6mm i.d., 5µ) column. Methanol and water in the ratio of 70:30 % v/v as mobile phase at a flow rate of 1 ml/min and effluent was monitored at 233 nm. The retention time of the drug was found at 6.2 mins. The method was effectively applied to tablets of empagliflozin and the % recovery of empagliflozin from tablet formulation was found to be 99.65 -99.89%. The method was also validated as per the ICH guidelines and the method was found simple, accurate, precise and reproducible and hence can be applied for routine quality control analysis of empagliflozin in pure and tablet dosage form. Statistical results and low % RSD values indicate that the method is precise, accurate, robust, specific, and can be used across a wide range of concentrations. All these key features proposed that this method can be considered as advantageous over other methods.²⁴

Godasu and Sreenivas (2017) performed an HPLC analysis for the simultaneous estimation of Metformin and Empagliflozin. The chromatographic conditions were successfully developed and used Symmetry C18 column (4.6×150mm) 5µ, flow rate was 1ml/min, mobile phase ratio was (70:30 v/v) methanol: phosphate buffer (KH₂PO₄and K₂HPO₄) phosphate pH 3 (pH was adjusted with orthophosphoric acid), detection wavelength used was Waters HPLC Auto Sampler. The retention times were found to be 2.403 mins and 3.907 mins for Metformin and Empagliflozin respectively. The % purity of Metformin and Empagliflozin was found to be 99.87% and 100.27% respectively. The analytical method was validated according to ICH guidelines. Percentage recovery was found to be 99.56% and 99.48%. LOD value was 2.17 and 0.0372 and LOQ value was 6.60 and 0.1125 respectively. The method was found to be simple, precise, accurate and sensitive for the simultaneous estimation of Metformin and Empagliflozin in pharmaceutical dosage forms. ²⁵

Shyamala et al., (2016) in their work described the development and validation of a stability-indicating reverse phase HPLC (RP-HPLC) method for the analysis of Empagliflozin in its API. The proposed method utilizes Hypersil BDS column Mobile phase 0.1% OPA: Acetonitrile in the ratio of 70:30 and flow rate was maintained at 1ml/min, detection wavelength was 233nm, and the column temperature was set to 30^oC. The developed method was successfully validated for different validation parameters as per ICH guidelines. The stability of the drug was determined by studying the degradation of the drug under acidic, alkaline, peroxide, neutral, heat and UV conditions. Therefore, simple, selective, sensitive and accurate stability-indicating RP-HPLC method was developed and validated for the analysis of Empagliflozin API. Further, the method was found to be linear, precise, accurate and robust. The degradation studies reveal the stability of the drug. Hence the proposed method can be safely and successfully used for the estimation of Empagliflozin API in routine analysis²⁶

Analysis of Ertugliflozin by HPLC

Ertugliflozin belongs to the class of potent and selective inhibitors of the sodium-dependent glucose cotransporters (SGLT), more specifically type 2 which is responsible for about 90% of the glucose reabsorption from the glomerulus. Compounds that inhibit Sodium-Glucose Transporter 2. They lower blood sugar by preventing the reabsorption of glucose by the kidney and are used in the treatment of Type 2 Diabetes Mellitus.

In a research Babu et al., (2018) performed a simultaneous estimation of the Ertugliflozin (ETR) and Sitagliptin (SGT) in bulk and its dosage form using RP-HPLC method. A determination was carried on water's C18 column capacitate (250X4.6mm, 5μm particle size). The mobile phase was consisted mixture of 0.5mM potassium dihydrogen orthophosphate buffer: Methanol in the ratio of 55:45 v/v, pH 5.3 was adjusted with HCl and flow of mobile phase was maintained 1mL/min. As a result of the chromatographic analysis, the drugs were separated and retention times of Ertugliflozin and Sitagliptin were found to be 2.39 and 4.60 min. The method gives an option for the rapid and accurate determination of drugs in the combination. The developed method was also validated as per the ICH guideline and the calibration curve was linear and the regression coefficient (R 2 ) value found to be 0.999 and concentration ranging from 37.5-112.5 and 250-750 μg/mL for Ertugliflozin and Sitagliptin respectively. The quantization limit and detection limit of the method were found 0.1 and 0.3μg/ml and 0.4 and 1μg/ml for Ertugliflozin and Sitagliptin.²⁷

In a research Rao et al., (2019) performed a high-performance liquid chromatography (HPLC) method for the simultaneous estimation of metformin hydrochloride and ertugliflozin in pharmaceutical formulation. As per the analytical conditions C8 column (150mm × 4.6mm, 5μm) at room temperature. Phosphate buffer: acetonitrile in 55:45 v/v ratio was used as the mobile phase. The flow rate was maintained at 1.0 ml/min, and the analysis was carried out at 224nm. The method was found to be linear in the concentration range of 125–750 μg/ml and 1.875–11.25μg/ml for metformin hydrochloride and ertugliflozin with regression coefficient r2 = 0.999. The method was found to be precise with percentage relative standard deviation below 2%. The percentage recovery of the developed method was 100.15%. A simple, accurate, precise, and less time-consuming reversed-phase HPLC method for the simultaneous estimation of metformin hydrochloride and ertugliflozin has been developed and validated in accordance with the ICH guidelines.²⁸

In an investigation furnished by Laxmi et al., (2019) an analytical method on reverse phase high performance liquid chromatography with PDA detection method for the simultaneous determination ertugliflozin and sitagliptin in bulk and in its tablets. The separation and assay of ertugliflozin and sitagliptin was done using Cosmicsil C8 column (250mm × 4.6mm I.D., 5μm size particle) in isocratic mode of elution . The optimized mobile phase was 0.1 Molar dipotassium hydrogen phosphate and methanol (65:35, v/v). The eluted analytes are monitored at 225nm wavelength. The method separated ertugliflozin and sitagliptin within a 7 min run time. Linearity of ertugliflozin and sitagliptin was in concentration range of 7.5 -22.50µg/ml and 50-150µg/ml , respectively by the method . The developed method was assessed through analysis of sitagliptin and ertugliflozin in the available tablet dosage form. The percent recoveries (± RSD) were 99.60±0.027 and 99.83±0.017 for sitagliptin and ertugliflozin, respectively. The results proved the non interference from the tablet excipients with good recovery and precision . Hence the method can be suggested for the routine quality control analysis of sitagliptin and ertugliflozin.29

Han et al., (2019) developed a novel bioanalytical method using high-performance liquid chromatography (HPLC) coupled with fluorescence detection for quantitative determination of ERTU in rat plasma. Acetonitrile-based protein precipitation method was used for sample preparation, and chromatographic separation was performed on a Kinetex® C18 column with an isocratic mobile phase comprising acetonitrile and 10 mM potassium phosphate buffer (pH 6.0). The eluent was monitored by a fluorescence detector at an optimized excitation/emission wavelength pair of 277/320 nm.³⁰

In another research by Anjali et al., (2019) a simple, accurate, precise method was developed for the simultaneous estimation of the Sitagliptin and Ertugliflozin in Tablet dosage form. The method involved the solving of simultaneous equations (Vierodt's method), based on the measurement of absorbance at two wavelengths, 210nm and 221nm which were the λmax values of SGT and ETR respectively in Mixture of 0.1% OPA buffer and acetonitrile. Both SGT and ETR showed linearity at all the selected wavelengths and obeyed beer's law in the concentration range of between 7.0–42μg/ml and 4.2–6.3μg/ml at 210nm and 221nm respectively. Recovery studies for SGT and ETR were performed and the percentage recovery for both the drugs was obtained in the range of 100.34–99.95% respectively.³¹

CONCLUSION:

SGLT2 inhibitors are also known as gliflozins. These drugs inhibit the re-absorption of glucose from the blood that is filtered through the kidneys and therefore lower blood sugar by facilitating glucose excretion in urine. They act by inhibiting sodium-glucose transport protein 2 (SGLT2).

To date, the US FDA has approved four types of SGLT2 inhibitors to treat diabetes mellitus II: Canagliflozin, Dapagliflozin, Empagliflozin and Ertugliflozin. Table 2 summarizes various HPLC methods developed for the determination of this class of antidiabetic drugs.

Table 2: HPLC methods developed for the determination of various drugs belonging to SGLT2 category.

Drug	Authors	Retention Time	Mobile Phase
Dapagliflozin	Debata et al., (2017)	3.461 min	Phosphate buffer and acetonitrile (60:40)
Dapagliflozin	Basha and Sravanthi, (2017)	2.789 min	Orthophosphoric acid buffer and acetonitrile(60:40)
Dapagliflozin	Verma et al., (2017)	API: 3.160 min; Tablet: 3.067 min	Mixture of acetonitrile: di-potassium hydrogen phosphate with pH-6.5 adjusted with OPA (40:60 %v/v)
Dapagliflozin	Mante et al., (2018)	5.163 min	Acetonitrile: 0.1% Triethylamine (pH-5.0) (50:50)
Dapagliflozin	Aswini e tal., (2018)	4.707 min	Methanol and Potassium dihyrogen phosphate buffer (45:55)
Dapagliflozin	Game and Bopudi, (2018)	API: 6.744 min Tablet: 6.69 min	Methanol: water (80: 20)
Canagliflozin	Kaur et al., (2016)	6.29 min	Acetonitrile: orthophosphoric acid (55:45)
Canagliflozin	Marella et al., (2017)	4.423 min	0.02% Formic acid: Acetonitrile (40:60)
Canagliflozin	Singh et al., (2019)	4.732 min	Acetonitrile: orthophosphoric acid (0.01 M) (50:50)
Canagliflozin	Zaghary et al., (2017)	0.9 min	Methanol and 0.03M phosphate buffer (75: 25) at pH 3.2
	Kommineni et al.,(2017)	2.671 min	0.1% OPA (pH 2.8) and Acetonitrile (45:55)
	Parida et al., (2018)	2.980 min	Methanol: Phosphate buffer (PH adjusted to 4 with orthophosphoric acid) (65:35)
Empagliflozin	Siridevi et al., (2019)	6.2 min	Methanol and water (70:30)
Empagliflozin	Godasu and Sreenivas (2017)	3.907 min	Methanol: phosphate buffer (KH₂PO₄and K₂HPO₄) phosphate pH 3 (pH was adjusted with orthophosphoricacid) (70:30)
Empagliflozin	Shyamala et al., (2016)	3.215 min	0.1% OPA: Acetonitrile (70:30)
Ertugliflozin	Babu et al., (2018)	2.39 min	0.5 mM Potassium dihydrogen ortho phosphate buffer: Methanol (55:45), pH 5.3 adjusted with HCl
Ertugliflozin	Rao et al., (2019)	3.143 min	Phosphate buffer: acetonitrile (55:45)
Ertugliflozin	Laxmi et al., (2019)	3.035 min	0.1 M Dipotassium hydrogen phosphate and methanol (65:35)
Ertugliflozin	Han et al., (2019)	3.114 min	Acetonitrile and 10 mM potassium phosphate buffer (pH 6.0).
Ertugliflozin	Anjali et al., (2019)	2.413 min	0.1% OPA buffer and acetonitrile

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Received on 22.11.2019 Modified on 09.01.2020

Research J. Pharm. and Tech 2020; 13(9):4479-4483.

DOI: 10.5958/0974-360X.2020.00790.8