INTRODUCTION:

In recent years, the pharmaceutical industry and regulatory agencies have placed a greater focus on dissolution testing.¹The dissolution test is a commonly used method by the pharmaceutical industry to determine the effectiveness of formulations and the quality of products. The test is a required practice by many regulatory authorities for solid orally administered dosage forms, transdermal patches, stents, and oral suspensions.² Dissolution testing of immediate-release solid oral dosage forms, such as tablets, used to evaluate the batch wise quality of drug products which ensure consistent product quality and performance.³In the treatment of newly diagnosed diabetes, Metformin, an insulin sensitizer, is used. Metformin has minimal hypoglycaemic risks, and making minimal drug interactions and it is an acceptable first-line drug for the treatment of type-2 diabetes mellitus as well as Teneligliptin is the drug used for the treatment of type 2 diabetes mellitus.^4-5

High performance liquid chromatography (HPLC) method provides a wide dynamic linear range, selectivity via separation and superior sensitivity that has been used to solve a variety of analytical problems encountered during dissolution test of complex drug delivery systems. For the solid dosage form the characteristics of dissolution under physiological condition inﬂuence the in vitro dissolution. Solubility and permeability of drug products and release products are the major factors which affect the dissolution of development and quality control of synthetic drugs. The value of dissolution test enhances signiﬁcantly when performance of drug substance is evaluated as a function of time. Dissolution test useful in QC and production batch to ensure similarities, so the dissolution Test remains similar and is crucial for clinical trial batches; further dissolution proﬁling is used to support bioavailability and bioequivalence of a new pharmaceutical product.^6-8

The literature survey reveals that both drugs Metformin and Teneligliptin have been analyzed individually and in combination by many analytical methods like HPLC, HPTLC and spectroscopic method, but no method have been reported for the estimation of Metformin and Teneligliptin for dissolution in combined pharmaceutical dosage form. This paper presents is simple, accurate and reproducible dissolution method for estimation of Teneligliptin and Metformin and its combined dosage form by using RP-HPLC method and validation of the developed methods was performed according to ICH guidelines.^9-16

EXPERIMENTAL:

Materials:

Metformin as well as Teneligliptin were obtained as gift sample from Cadila Pharmaceuticals Limited, Dholka, Ahmedabad. Methanol, Water, Ortho phosphoric acid, Acetonitrile of HPLC grade, and potassium Dihydrogen phosphate is of analytical grade reagent was purchased from Merck, Rankem which were used for the present study. The commercially available Teneligliptin and Metformin HCl in combined tablet formulation Tenalifine M (Healing Pharma India ltd.) with label claim Metformin HCl 500 mg and Teneligliptin 20mg was procured from local pharmacy for use.

Instrumentation:

Lab India private limited dissolution apparatus, Shimadzu LC-20 AT HPLC chromatographic system, Systronic119 UV visible spectrophotometer, Shimadzu digital weighing balance (ATX 224), Frontline Ultrasonic Cleaner ultra-Sonicator, Ana lab Scientific Pvt. Ltd pH meter used for the method development.

Methods:

Selection and detection of wavelength¹⁷

UV detection which determined by proper selection of detection wavelength which is used for interpretation of sensitivity of HPLC method. It is an ideal wavelength gives good response for the both drugs i.e. Metformin and Teneligliptin.

Preparation of Mobile Phase:

Prepared 0.05M Potassium Dihydrogen Phosphate solution by dissolving accurately taken 6.8g of Potassium Dihydrogen Phosphate in 1000ml HPLC grade water in a 1Liter volumetric flask and pH was adjusted to pH 7.0 with 0.1N NaOH solution. The prepared buffer pH was checked by using pH meter. For 5 min by ultra sonicating method the solution was degassed and the resulting solution was filtered through 0.45µ Millipore filter. The mobile phase Prepared with the ratio of Buffer (pH 7.0): Acetonitrile (80:20 v/v).

Preparation of dissolution medium:

Phosphate buffer pH 6.8:

Accurately weigh 6.8g of Potassium Dihydrogen o-phosphate and 0.94g of NaOH and diluted with 1000mL of water in 1L volumetric flask. The pH of phosphate buffer checked using calibrated pH meter and phosphate buffer shows pH 6.8.

Acetate buffer pH 4.5:

Accurately weigh Sodium acetate 2.99g and 1.66mL of glacial acetic acid and diluted with 1000mL of water in 1L volumetric flask. The pH of Acetate buffer checked using calibrated pH meter adjusted with glacial acetic acid to get pH 4.5.

0.1N HCl:

Accurately taken 8.5mL of concentrated HCl and diluted with water to make 1000mL in 1Lvolumetric flask.

Standard Solutions Preparation:

Preparation of Standard Stock Solution of Metformin (5550µg/mL):

Accurately taken 555mg of Metformin bulk drug into a 100ml volumetric flask and dissolved with dissolution medium Phosphate buffer pH 6.8 up to the mark to get 5550μg/ml of Metformin Standard Stock Solution and then 5.0ml of this solution was transferred into a 50ml volumetric flask and finally volume was made up with dissolution medium phosphate buffer pH 6.8 to get 550.0μg/ml of Metformin working standard solution.

Preparation of Standard Stock Solution of Teneligliptin (220 µg/mL):

Accurately taken 22mg of Teneligliptin bulk drug into a 100ml volumetric flask and it dissolved in dissolution medium Phosphate buffer with pH 6.8 up to mark to obtain 220μg/ml of standard stock Solution of Teneligliptin and then 5.0ml of this solution was transferred into a 50ml graduated flask and the final volume was made up with dissolution medium phosphate buffer pH 6.8 up to the mark to get 22.0μg/ml of Teneligliptin working Standard Solution.

Preparation of Test Solution:

An intact tablet of Tenalifine M dissolved in dissolution media and set dissolution condition. Withdraw 10mL of sample by syringe at an interval of 10, 20, 30, 45 and 60 minutes and filter the solution with 0.45µ filter and the final filtrate collected as test solution. This test solution injected into the HPLC system to get the peak.

Chromatographic Separation:

Both the standard solutions of Metformin and Teneligliptin were injected 20μl in column with micro-syringe. And for appropriate minutes the chromatogram was run with mobile phase. And at 225nm the detection was performed. The chromatogram was stopped after achieved completely separation and recorded the data related to peak like retention time, area, resolution, height, etc. by using software.

Development and Optimization of Dissolution test method:

Determination of solubility in different Dissolution Medium:

At room temperature the solubility for Metformin in different dissolution medium which determined by taking Metformin and taken required quantity of solvent and it shake well for few minutes and for similarly solubility of Teneligliptin in different dissolution medium which determined by taking Teneligliptin and taken necessary quantity of solvent at room temperature and it shaken well for few minutes.

Optimization of dissolution medium at different conditions:

USP Dissolution Apparatus:

USP Dissolution Apparatus 2 (Paddle) was selected to calculate % release of Metformin and Teneligliptin drug in tablet dosage form. The result of % dissolution Metformin and Teneligliptin drug in tablet dosage form was observed and recorded.

Dissolution Medium of USP Dissolution Apparatus:

The Phosphate Buffer (pH 6.8) was selected as dissolution medium due to high solubility of Metformin and Teneligliptin drug in Phosphate Buffer (pH 6.8). A media volume of 900ml was kept constant and % release of Metformin and Teneligliptin drug in tablet dosage form was calculated using Phosphate Buffer (pH 6.8).

Speed of USP Dissolution Apparatus:

The USP Dissolution Apparatus 2 (Paddle) was rotated at 50, 75 and 100RPM to calculate % release of Metformin and Teneligliptin drug in tablet dosage form. It can be concluded that the % release of Metformin and Teneligliptin drug using USP Dissolution Apparatus 2 (Paddle) at 100RPM was found to be optimum as compared to other Revolution per Minute conditions. Therefore 100RPM was selected as one of the optimized dissolution apparatus speed.

Temperature of USP Dissolution Apparatus:

The USP Dissolution Apparatus 2 (Paddle) was maintained at temperature 37±0.5°C to calculate % release of Metformin and Teneligliptin drug in tablet dosage form.

Optimization of dissolution test:

The dissolution studies was performed by using a USP dissolution apparatus 2 by subjecting tablets in dissolution medium which containing 900mL of dissolution media of phosphate buffer with pH 6.8 using paddle dissolution test apparatus with it stirring speed of 100rpm at temperature of 37±0.5°C. and aliquots of 10 ml were withdrawn manually at an intervals of 10, 20, 30, 45 and 60 minutes and its same volume of fresh medium was replaced at 37±0.5°C to maintain the constant volume. And finally sample was filtered through filter paper and which was analyzed by RP-HPLC method.

Method Validation^18-19

The developed RP-HPLC method was validated for Metformin and Teneligliptin according ICH guidelines. The parameters validated are Specificity, System suitability, Precision, range, Linearity, Robustness, Limit of quantitation (LOQ), Limit of detection (LOD).

System suitability:

It is used to authenticate the reproducibility and resolution of the system for the analysis being performed. The System Suitability was calculated from different parameters like theoretical plates for the analyte peak and tailing factor for analyte peak.

Linearity and Range:

The linearity of a method is measured to see how well a calibration plot of response vs. Concentration approximates a straight line. The linearity of Teneligliptin and Metformin were evaluate by analysis of combined standard solution in range of 22.5 – 67.5μg/ml of Teneligliptin and 7.0-21.0μg/ml of Metformin respectively. Suitable aliquots of the standard stock solutions of Metformin transferred into series of 10ml volumetric flasks respectively to get concentration levels of 7.0μg/ml, 10.5μg/ml, 14.0μg/ml, 17.5μg/ml and 21.0μg/ml. Similarly concentration levels of 22.5μg/ml, 33.75μg/ml, 45.0μg/ml, 56.25μg/ml and 67.5μg/ml from standard stock solution of Teneligliptin prepared. The obtained graph of peak area verses respective concentration was plotted.

Precision:

System precision was performed by injecting five replicates of a standard solution of Metformin (14.0µg/ml) as well as standard solution of Teneligliptin (45.0µg/ml) and chromatograms were recorded and areas of peaks were measured to calculate results of repeatability. Method precision was performed by injecting sample solution of Metformin and Teneligliptin six times and areas of peaks measured % dissolution and % RSD was calculated.

LOQ and LOD:

Limit of quantitation and Limit of Detection was estimated from the 3 set of calibration curves which was used to determine method linearity. The LOQ and LOD were calculated with below formula.

LOQ=10 * SD/slope of calibration curve

LOD= 3.3 * SD/slope of calibration curve

Robustness:

The robustness study was carried out to evaluate the effect of small but considered variations in the chromatographic conditions, which have been described in the Chromatographic conditions section. The factors chosen for this study, which were critical sources of variability in the operating procedures such as pH of mobile phase changed (±0.2) and Temperature was changed (±2.0ºC) were identified. In all these experiments, the mobile-phase components were not changed and their effect observed on system suitability for standard preparation.

Analysis of Market formulation:

An intact tablet dissolved in dissolution media and set dissolution condition. After interval of 10 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes 10mL sample withdrawn from the cylinder by syringe and sample filtered with 0.45 micron membrane filter and the final filtrate collected as test solution. The test solution was injected concentration of 20µL and area of resulting which measured at 225nm.

RESULTS AND DISCUSSION:

Wavelength determination:

UV spectra of Metformin and Teneligliptin were taken in Methanol solvent and its λmax was observed using Systronic 119

Figure No.1 Overlay UV Spectrum of Metformin and Teneligliptin showing Selection of Wavelength Detection.

At 225nm detection wavelength Metformin and Teneligliptin both drug give higher absorbance. Hence λ max 225nm has been selected as detection wavelength.

Optimised chromatographic conditions:

The chromatographic trials have been taken for the effect of different mobile phase compositions on the separation of Metformin and Teneligliptin. Method development process was carried out by examining different conditions like mobile phase compositions like Water: Methanol, Water: Acetonitrile, phosphate buffer pH 7.0: Acetonitrile with different ratios were used. The Metformin and Teneligliptin were found showing a significant UV absorbance at 225 nm, so this wavelength was chosen for UV detection. By use of a C18 column it was found that the mobile phase consisting of Buffer (Phosphate Buffer, pH 7.0): Acetonitrile provided well defined peak shape with good resolution. The peaks with retention time (RT) 3.197 minutes and 6.543 minutes for Metformin and Teneligliptin. The representative chromatograms (Figure No. 2) which show significant amount of resolution and the good peak shapes with selected mobile phase.

Figure No.2: Chromatogram of Metformin and Teneligliptin in Buffer (pH 7.0): Acetonitrile

The final optimized chromatographic condition for Metformin and Teneligliptin and having Stationary phase used BDS Hypersil C18column with size of 250 mm×4.6 mm, 5 μm particle size, and utilized mobile phase was Phosphate Buffer (pH 7.0): Acetonitrile (80:20 v/v) with flow rate is 1 ml/min, at 225 nm wavelength detection for 10 min run time and its injection volume is 20μl.

Optimised dissolution conditions:

Solubility of Metformin and Teneligliptin in different Dissolution Medium:

The data for Solubility of Metformin and Teneligliptin in different Dissolution Medium is given in below table,

The solubility of Metformin and Teneligliptin in different dissolution media concludes that the Phosphate Buffer pH 6.8 was the Dissolution Medium for Metformin and Teneligliptin dissolution test and also ensured Sink Condition.

Table No. 1: Solubility of Metformin and Teneligliptin in different Dissolution Medium

Dissolution Medium	Solubility (mg/ml)	Sink Condition (Solubility>0.25mg/ml)	Solubility (mg/ml)	Sink Condition (Solubility>0.033mg/ml)
Dissolution Medium	Metformin		Teneligliptin
Water	0.025mg/ml	No	0.00225mg/ml	No
0.1N HCl	0.1mg/ml	No	0.001mg/ml	No
pH 4.5	0.025mg/ml	No	0.0225mg/ml	No
pH 6.8	≥ 0.25mg/ml	Yes	≥ 0.033mg/ml	Yes

Table No. 2: % dissolution with paddle apparatus at various speed of rotation (rpm) in dissolution medium pH 6.8

Time (Minutes)	% Dissolution at pH 6.8
	50 RPM		75 RPM		100 RPM
	Metformin	Teneligliptin	Metformin	Teneligliptin	Metformin	Teneligliptin
10	10.095	41.638	19.460	51.062	33.386	66.539
20	22.765	57.659	33.761	66.683	56.278	83.064
30	45.406	68.663	54.894	82.157	82.116	97.922
45	62.018	75.919	67.053	88.835	98.114	100.026
60	69.875	89.012	82.770	99.636	97.048	98.726

Optimization of dissolution parameters:

Different dissolutions parameters were performed for optimized dissolution parameter. Like maximum % release of drug its trials were taken by using USP Apparatus II i.e. paddle type at different speed of rotation (rpm) 50, 75 and 100. Based on the solubility of Metformin and Teneligliptin, phosphate buffer pH 6.8 was selected as suitable dissolution media (900mL).

It was observed that drug release in USP type-II (Paddle) apparatus shows maximum release for both drugs at 100 rpm. Hence dissolution parameters have been optimized. The optimized condition for dissolution method development includes USP dissolution apparatus II (paddle), 900 mL of phosphate buffer with pH 6.8 as dissolution medium at 100 rpm dissolution apparatus paddle speed at temperature of 37±0.5^oC.

Figure No.3: Chromatogram of % dissolution of Metformin andTeneligliptin with Paddle Apparatus at 100 RPM after 60 minutes time interval

Method Validation:

The proposed method was validated with the aspect of Specificity, Precision, System suitability, Limit of quantitation (LOQ), Limit of detection (LOD), Robustness, Linearity and range.

System Suitability:

The System Suitability was calculated from different parameters like retention time, theoretical plates, resolution, tailing factor. System suitability parameters observed for Metformin has retention time as 3.197, theoretical plates per column 6989 and tailing factor 1.333. The system suitability parameters observed for Teneligliptin has retention time as 6.543, theoretical plates per column 7321 and tailing factor 1.357. The resolution observed 14.587.

Specificity:

The specificity of the chromatographic method was determined to ensure separation of Metformin and Teneligliptin. The Chromatograms of Metformin and Teneligliptin sample did not show any interference with the Chromatogram of Metformin and Teneligliptin blank solution, So that the Method was developed.

Linearity and Range:

For Teneligliptin and Metformin the linearity was evaluated by analysis of combined standard solution in range of 22.5-67.5μg/ml and 7.0-21.0μg/ml respectively and its Correlation co-efficient for calibration curve of Metformin and Teneligliptin was found to be NLT 0.999 respectively.

Table No.3: Linearity Data for Metformin and Teneligliptin

Sr. No.	Linearity Level	Concentration(µg/ml)		Area
Sr. No.	Linearity Level	Metformin	Teneligliptin	Metformin	Teneligliptin
1	50%	7	22.5	1904.649	566.417
2	75%	10.5	33.75	2788.585	830.302
3	100%	14	45	3768.176	1122.257
4	125%	17.5	56.25	4649.475	1357.72
5	150%	21	67.5	5609.384	1670.547

Figure No. 4 Plots of calibration curves of Metformin and Teneligliptin

Precision:

System Precision:

Performed system precision by injecting five replicates of a standard solution of Metformin (14.0µg/ml) as well as standard solution of Teneligliptin (45.0µg/ml) and chromatograms were recorded and areas of peaks were measured to calculate results of repeatability. The data for system precision of peak area measurement for Metformin and Teneligliptin shows that the %RSD values observed within acceptance limit of NMT 5%.

Method Precision:

Performed the method precision by injecting sample solution of Metformin and Teneligliptin six times and areas of peaks measured % dissolution and % RSD was calculated. The data for method precision of peak area measurement for Metformin and Teneligliptin shows that the % RSD values observed within acceptance limit of NMT 5%. Hence the method is precise.

LOQ and LOD:

The limit of quantitation and Limit of detection for both the drugs were estimated using the linearity data. For five times Calibration curve was repeated and intercepts of the standard deviation (SD) was calculated. The Limit of detection for Metformin observed 6.857µg/ml and for Teneligliptin observed 4.939µg/ml. However the limit of quantitation for Metformin observed 20.779µg/ml and for Teneligliptin observed 14.968µg/ml.

Robustness:

The robustness study was performed to evaluate the effect of small but deliberate variations. And chromatographic factors as mobile phase pH (± 0.2) and temperature of Mobile phase was changed (±2ºC) without changing the mobile phase components and their effect observed on system suitability for standard preparation. The results shows the effect of changes was found to be the % RSD and within the acceptance criteria values observed within standard limit of not more than 5%. Hence the method is robust.

Table No. 4 system precision data and Method precision data for Estimation of Metformin and Teneligliptin

System Precision Data
Conc. (µg/ml)	Area	Mean Area ± S.D. (n=5)	% R.S.D	Conc. (µg/ml)	Area	Mean Area ± S.D. (n=5)		% R.S.D
Metformin				Teneligliptin
14.00 µg/ml	3712.142	3721.172±64.769	1.741%	45.00 µg/ml	1105.530	1101.791±13.387	1.215%
	3816.331				1122.861
	3674.121				1094.218
	3653.333				1088.023
	3749.935				1098.322
Method Precision Data
Area	%Dissolution	Mean Area ± S.D. (n=5)	% R.S.D	Area	%Dissolution	Mean Area ± S.D. (n=5)	% R.S.D
Metformin				Teneligliptin
3700.028	99.332	100.350±1.222	1.218%	1130.191	101.552	100.892±0.937	0.928%
3768.248	101.164			1110.091	99.746
3680.896	98.819			1124.390	101.031
3787.406	101.678			1134.993	101.983
3753.132	100.758			1114.590	100.150
3708.747	99.566			1132.861	101.792

Table No.5: Robustness data for Metformin and Teneligliptin

Sr. No.	Area at Temp (+2.0ºC)	% Dissolution at Temp (+2.0 ºC)	Area at Temp (-2.0 ºC)	% Dissolution at Temp (-2.0 ºC)	Area at pH (+2.0)	% Dissolution at pH (+2.0)	Area at pH (-2.0)	% Dissolution at pH (-2.0)
Metformin
1	3589.158	96.590	3826.410	102.057	3700.028	100.481	3741.040	99.179
2	3603.464	96.975	3941.757	105.134	3788.763	102.891	3810.711	101.026
3	3695.041	99.440	3850.007	102.686	3633.296	98.669	3600.894	95.464
4	3545.130	95.405	3732.659	99.557	3745.924	101.727	3830.488	101.551
5	3619.526	97.408	3861.731	102.999	3693.315	100.299	3700.802	98.113
6	3611.020	97.179	3902.781	104.094	3726.371	101.196	3733.746	98.986
%RSD	1.357	1.517	1.858	1.956	1.419	1.578	2.208	2.468
Teneligliptin
1	1096.354	98.178	1168.725	103.730	1130.191	102.127	1142.669	100.809
2	1100.783	98.575	1203.986	106.859	1157.324	104.579	1163.934	102.685
3	1128.772	101.081	1175.958	104.372	1119.618	101.172	1106.403	97.609
4	1061.727	95.077	1112.875	98.773	1144.278	103.400	1169.974	103.218
5	1105.684	99.014	1179.598	104.695	1137.137	102.755	1130.308	99.718
6	1103.125	98.785	1192.103	105.805	1138.292	102.859	1140.497	100.617
%RSD	1.970	2.197	2.700	2.881	1.120	1.252	2.023	2.259

Analysis of Market formulation and % dissolution of Metformin and Teneligliptin:

Relevancy of the proposed method was estimate by analyzing the commercially available Tablet formulation of Tenalifine M. During The results of assay and % dissolution are calculated. The average area of Metformin observed 3649.02 however % dissolution of Metformin observed 99.67%. The average area of Teneligliptin observed 1129.04 however % dissolution of Teneligliptin observed 101.61%. The results indicate that the developed method is simple, precise, accurate, and rapid. It can be used in the regular quality control test of formulation in industries.

CONCLUSION:

There is no analytical work has been available regarding dissolution RP-HPLC method for Metformin and Teneligliptin in a literature. It is the new efforts in a area of research has been made to validate and develop dissolution method via RP- HPLC. Conclusively, the dissolution method via RP HPLC-method described in this paper is specific, sensitive, rapid and easy to perform. The proposed dissolution test method was successfully validated in terms of specificity, precision, linearity and robustness as per ICH guidelines. It can be concluded that the proposed method can be employed for routine dissolution analysis of Teneligliptin and Metformin in pharmaceutical tablets.

ABBREVIATIONS:

RP-HPLC: Reverse Phase High Performance Liquid Chromatography; pH: Potential of Hydrogen; mm: millimetre; ml: Milliliters; M: Molar; µm: Micrometer; nm: nanometer; LOQ: Limit of quantitation; LOD: Limit of detection; ICH: International Conference on Harmonization; NLT: Not less than; NMT: Not more than; % RSD: Relative standard deviation; min: Minutes; Rs: Resolution; SD: Standard deviation;°C: Degree Celsius; mg: Milligrams; µg: Microgram; v/v: Volume/volume; %: Percentage; MET: Metformin; UV: Ultra violet; pvt: private; g: gram; ppm: parts per million; fig: figure.

COMPETING INTERESTS:

The authors declare no conflicts of interest.

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Received on 31.10.2021 Modified on 26.01.2022

Research J. Pharm. and Tech 2023; 16(1):133-139.

DOI: 10.52711/0974-360X.2023.00025