Determination of ertugliflozin in pharmaceutical formulations by RP-HPLC method

A novel, specific, accurate, rugged, precise reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative determination of Ertugliflozin in active pharmaceutical ingredients and in its Pharmaceutical dosage form by using Symmetry ODS C18 (4.6×250mm, 5µm) column with a mobile phase containing a mixture of Methanol: Phosphate Buffer pH-3.6 in the ratio of 35:65%v/v. The flow rate was 1.0 ml/min and effluent were monitored at 235 nm and a peak eluted at 2.552 min and column oven temperature was maintained ambient. Calibration curve was plotted with a range from 6-14 µg/ml. The LOD and LOQ values of Ertugliflozin were found to be 1.2µg/ml and 3.6µg/ml respectively. The percentage recovery of the Ertugliflozin was found to be within the limits. The developed RP-HPLC method was validated according to the current International Conference on Harmonization (ICH) guidelines for specificity, LOD, LOQ, linearity, accuracy, precision, intermediate precision and robustness. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Ertugliflozin in bulk drug and in its pharmaceutical dosage form. The proposed method was applied for the analysis of tablet formulations, to improve QC and assure therapeutic efficacy.


Introduction
Ertugliflozin belongs to the class of potent and selective inhibitors of the sodium-dependent glucose cotransporters (SGLT), more specifically the type 2 which is responsible for about 90% of the glucose reabsorption from glomerulus.(Figure .No.1).Ertugliflozin is an oral, selective inhibitor of sodium glucose co-transporter-2 (SGLT2) that results in urine glucose excretion (UGE) and decreases in plasma glucose and haemoglobin A1c (A1C) in individuals with type 2 diabetes mellitus (T2DM).[7] Literature review revealed that only few HPLC method [8][9][10][11] have been reported for the determination of ertugliflozin and a pharmacokinetic study which used LC-MS method for the determination of ertugliflozin.2][13][14] Existing literature reveals that there are only few methods for the assay of ertugliflozin in bulk and dosage forms.Hence an attempt has been made to develop a new simple, reliable, and reproducible, isocratic RP-HPLC methods to estimate the ertugliflozin in bulk and pharmaceutical formulation with good precision, accuracy, linearity and reproducibility respectively.The proposed method was validated as per ICH guidelines [15][16][17] .

Equipment
Chromatographic separation was performed on Waters HPLC system consist of model 2695 having PDA detector and Rheodyne injector with 20µl loop volume.Waters Empower software was applied for data collecting and processing.

Reagents and chemicals
Acetonitrile, Methanol and water of HPLC grade were procured from Rankem lab ltd.ertugliflozin was received as gift samples from Reddys Labs Ltd., Hyderabad, India, respectively.Steglatro ® and tablets were purchased from local market.Nicolan Healthcare Pvt Ltd

HPLC conditions
Symmetry ODS C18 (4.6×250mm, 5µm) column was used as the stationary phase.A mixture of Methanol: Phosphate buffer adjusted to pH-3.6 in the ratio of 35:65%v/v was used as the mobile phase.It was filtered through 0.45µ membrane filter and degassed.The mobile phase was pumped at 1.0 ml/min.The eluents were monitored at 235 nm.The injection volumes of samples and standard were 20µl.

Standard solutions
A stock solution containing 1000µg/ml of Ertugliflozin was prepared by dissolving Ertugliflozin in mobile phase.A working standard solution contain in 10 -30µg/ml of EGZ was prepared from the above stock solution.All the stock solutions were covered with aluminum foil to prevent photolytic degradation until the time of analysis.20 tablets (each tablet contains 5 mg of Steglatro ® tablets) were accurately weighed and calculated their average weight.Then it was taken into a mortar and crushed to fine powder and uniformly mixed.A quantity of powder equivalent to10 mg of EGZ was weighed and transferred to a 10 ml standard flask.The drug was initially dissolved in diluent and sonicated for 10 minutes.The volume was made up to 10 ml with mobile phase.Then the solution was filtered using 0.45-micron syringe filter.After that 0.1ml of the above filtrate was diluted to 10 ml with the diluent so as to give a concentration of 10µg/ml of Ertugliflozin.Then 20µl of this solution was injected in to the column and chromatogram was recorded and shown in Fig. 2.Each concentrations of EGZ in the tablet formulation were calculated by comparing area of the sample with that of standard.The percentage assay of individual drug was calculated and presented in table1.

Preparation of mobile phase
Accurately measured 350 ml (35%) of Methanol, 650 ml of Phosphate buffer (65%) were mixed and degassed in digital ultra sonicater for 15 minutes and then filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation
The Mobile phase was used as the diluent.
System suitability studies The system suitability test was carried out on freshly prepared stock solution of EGZ to check various parameters such as column efficiency, tailing factor and number of theoretical and presented in Table 2.The values obtained were demonstrated the suitability of the system for the analysis of the drug.System suitability parameter may fall within  2% standard deviation range during routine performance of the method.

Linearity and Range
Linearity was studied by preparing standard solution at five different concentration levels.The linearity range was found to be 6-14 µg/ml.20µl of each solution was injected into chromatograph.Peak areas were recorded for all the chromatogram.Calibration curve was constructed by plotting peak areas (Y axis) against the amount of drug in µg/ml (X axis).Peak area of linearity range and the parameters were calculated and presented in table 3 respectively.The linearity curve of Ertugliflozin was shown in Figure .4.

Limit of detection and Limit of quantification
The limit if detection (LOD) was calculated from the linearity curve using the formula LOD = 3.3Χ {Residual Standard deviation/Slope}.
The LOD for Ertugliflozin was confirmed to be 1.2g/ml.
The Limit of quantification (LOQ) was calculated from the linearity curve using the formula.LOQ = 10 Χ {Residual Standard deviation/Slope}.
The LOQ for Ertugliflozin was confirmed to be3.6µg/ml

Accuracy
The accuracy of the method was determined by recovery experiments.Placebo was spiked with known quantities of standard drugs at levels of 50 to 150% of label claim.The recovery studies were carried out 3 times and the percentage recovery and standard deviation of the percentage recovery were calculated and presented in table 4. The mean recovery is well within the acceptance limit, hence the method is accurate.

System precision
The system precision of the method was established by six replicate injections of the standard solution containing Ertugliflozin The percentage RSD were calculated and presented in Table 5.From the data obtained, the developed RP-HPLC method was found to be precise.

Method precision
The method precision of the method was established by carrying out the analysis of Ertugliflozin (n=6) using the proposed method.The low value of the relative standard deviation showed that the method was precise.The results obtained were presented in table 5.

Specificity
Specificity of the method was determined by injecting the diluted placebo.There was no interference of placebo with the principle peak, hence the developed analytical method was specific for Ertugliflozin in tablet dosage form.

Standard and sample solution stability
Standard and sample solution stability was evaluated at room temperature and refrigerator temperature for 24h.The relative standard deviation was found below 2.0%.It showed that both standard and sample solution were up to 24h at room temperature and refrigerator temperature.

Ruggedness and robustness
The ruggedness of the method was determined by carrying out the experiment on different instruments like Shimadzu HPLC (LC2010 A4T), Water Alliance HPLC 2695 by different operators using different columns of similar type like Hypersil C18 column and Phenomenex C18 column.Robustness of the method was determined by making slight change in the chromatographic condition.It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed is rugged and robust.The results of ruggedness were presented in table 6.The results of robustness were presented in table 7.

Conclusion
The proposed RP-HPLC method for the estimation of Ertugliflozin in tablet dosage forms is accurate, precise, linear, rugged, robust, simple and rapid.The developed method offers several advantages in terms of simplicity in mobile phase, isocratic mode of elution and sample preparation steps and comparative short run time makes the method specific, repeatable and reliable for its intended use in determination of Ertugliflozin in bulk form and pharmaceutical dosage form.Hence the present RP-HPLC method is suitable for the quality control of the raw material, formulation and dissolution studies.The method validation shows satisfactory data for all the method validation parameter tested.

Table 1
Table for Assay T1 and T2 are two different brands of tablet formulations.EGZ denotes Ertugliflozin respectively.*Eachvalue is average of six determinations .

Table 2
System suitability results of Ertugliflozin

Table 3
Analytical performance parameters of linearity curve

Table 4
Recovery studies of Ertugliflozin *Average of six or three determinations, Mean  Standard Deviation

Table 6
Method ruggedness of Ertugliflozin in dosage forms *Average of six determinations, mean  Standard Deviation

Table 7
Method robustness of Ertugliflozin in dosage FormsParameter used for sample analysis Peak Area Retention Time Theoretical plates Tailing factor