SIMULTANEOUS ESTIMATION AND ANALYTICAL METHOD DEVELOPMENT, VALIDATION FOR THE TENELIGLIPTIN AND METFORMIN BY RP-UFLC

SIMULTANEOUS ESTIMATION AND ANALYTICAL METHOD DEVELOPMENT, VALIDATION FOR THE TENELIGLIPTIN AND METFORMIN BY RP-UFLC

R. Maruthi ^*, R. S. Chandan and Prachi Raikar

Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru - 570015, Karnataka, India.

ABSTRACT: The study aims to develop an analytical method for the simultaneous estimation of Teneligliptin and Metformin using RP-UFLC. A simple, sensitive and accurate method was developed for Teneligliptin and Metformin using the chromatographic conditions of C18 Phenomenex Kinetex (250 mm × 4.6 mm i.e., 5 μm particle size) column in gradient elution mode with the mobile phase consisting of methanol, acetonitrile and potassium dihydrogen orthophosphate adjusted to pH 4.6 using orthophosphoric acid (40:20:40) with a flow rate of 1.0 mL/min, injection volume 10 µl and the eluent was detected at 250 nm using PDA and UV detector. The retention time of Teneligliptin and Metformin were found to be 5.2 min and 2.5 min respectively. The above method was validated concerning system suitability, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ), accuracy (recovery) and robustness according to ICH guidelines. The linearity of the above methods was found to be 2-10 µg/mL for Teneligliptin and 25-125 µg/mL for Metformin. Hence, these methods can be used for routine analysis in quality control laboratories.

RP-UFLC, Teneligliptin, Metformin, ICH guidelines

INTRODUCTION: Analytical chemistry is the science ^{1, 2} of obtaining, processing and communicating information about the composition and structure of matter. It deals with the separation of materials into their components and identifying each one and how much there is of each one. Pharmaceutical analytical chemistry ³ is defined as the branch of practical chemistry which deals with the resolution, separation, identification, determination, and purification of a given sample of a medicine or a pharmaceutical, the detection and estimation of impurities that may be present therein is also included.

The use of analytical sciences in the discovery, development, and manufacture of pharmaceuticals is wide-ranging. From the analysis of minute amounts of complex biological materials to the quality control of the final dosage form, the use of analytical technology covers an immense range of techniques and disciplines.

Gliptins are ordinarily known as DPP-IV inhibitors have turned out to be a new class of potential medication applicant and are being sought as a lasting eraser after sort 2 diabetes. Consequently, gliptins have been a focal point of innovative work. Teneligliptin hydrobromide is a dipeptidyl peptidase 4 inhibitors is an uncommonly persuading in chopping down blood glucose levels. Teneligliptin hydrobromide hydrate is a very strong, focused, and durable DPP-4 inhibitor (1, 2, 3, and 4). Glucagon-like peptidase (GLP-1) a peptidase discharged from the GIT because of sustenance consumption upgrades insulin emission and stifles glucagon emission from the pancreas, consequently assuming a critical part in controlling postprandial blood glucose level. The peptide is quickly inactivated by debasement by DPP-4 inhibitor; a chemical generally conveyed in the body. DPP-4 inhibitor debasement, expanding the centralization of dynamic GLP-1 in the blood, which fortifies glucose subordinate insulin discharge and in the meantime, smothers glucagon emission, subsequently displaying glucose bringing down impact. It is successfully used to treat type 2 diabetes mellitus.

The most typically detailed unfavorable responses incorporate hypoglycemia, clogging, and feeling of the developed stomach area, stomach distress, sickness, stomach torment, meteorism, stomatitis, skin irritation, rash, pruritus, dermatitis, and disquietude. Composing study reveals RP-UFLC techniques have been represented the assessment of Teneligliptin hydrobromide pure and tablet dosage forms ^{4, 5, 6, 7}.

Teneligliptin:

Class: Anti-diabetic drugs known as dipeptidyl peptidase-4 inhibitors gliptins.

Chemical (IUPAC) Name: [(2S, 4S) -4 -[4-(5-methyl -2 –phenylpyrazol -3 -yl) piperazin-1-yl] pyrrolidin-2-yl]-(1, 3-thiazolidin-3-yl) methanone.

Physical State: White to off-white powder

Chemical Formula: C₂₂H₃₀N₆OS

Chemical Structure:

FIG. 1: CHEMICAL STRUCTURE OF TENELIGLIPTIN

Molecular Mass: 426.6 g/mol

Melting Point: 211ºC

pKa: 8.7

Solubility: Soluble in water, DMSO and methanol

Brand Name: Tenepride 20 mg

Applications: Used in the treatment of type 2 diabetes mellitus.

Metformin hydrochloride is 1, 1-Dimethyl-biguanide hydrochloride [1] and is used in the treatment of diabetes mellitus. It is completely different from the hypoglycemic sulfonamides [2] both in its structure and its mode of action. It possibly interferes with mitochondrial respiratory chains and promotes peripheral glucose utilization by enhancing anaerobic glycolysis, or it enhances the binding of insulin to its receptors and potentiates its action. Another explanation is that it suppresses hepatic gluconeogenesis and inhibits intestinal absorption of glucose. It causes little or no hypoglycemia in non-diabetic patients.

Metformin:

Class: Metformin is a biguanides anti-hyperglycemic agent.

Chemical (IUPAC) Name: 1-carbamimidamido-N, N-dimethylmethanimidamide.

Physical State: white crystalline powder.

Chemical Formula: C₄H₁₁N₅

Chemical Structure:

FIG. 2: CHEMICAL STRUCTURE OF METFORMIN

Molecular Mass: 129.16 g/mol

Melting Point: 223-226 ºC

pKa: 12.4

Solubility: Freely soluble in water, methanol.

Brand name: Exermet 500 mg

Application: First-line medication for the treatment of type 2 diabetes

There is no Pharmacopoeia method for simultaneous analysis of Trelagliptin and Metformin. A literature review revealed that chromatographic methods for determination of Trelagliptin and Metformin alone or in a combination of others are available. Literature search also revealed LC methods for simultaneous determination of Trelagliptin and Metformin in bulk forms with different mobile phase composition and using wavelength programming technique ⁹. The objective of the present study is to develop a simple, accurate, precise and selective reverse phase UFLC method for simultaneous determination of Trelagliptin and Metformin from bulk forms available in the market ^{8, 9, 10, 11, 12}.

Preparation of Mobile Phase: To a 1000 ml volumetric flask, add 1.36 g of potassium dihydrogen orthophosphate was taken and diluted with Millipore water and made up to the mark, and the pH was adjusted to 4.6 using orthophosphoric acid. The solution was then filtered through a 0.45µ membrane filter.

Preparation of Diluent: The diluent is a mixture of 50 parts of methanol and 50 parts of acetonitrile mixed thoroughly.

Preparation of Standard Stock Solution for Teneligliptin: 100 mg of Teneligliptin was taken into 100 ml volumetric flask. To this add 50 ml of diluent and sonicate to dissolve and the volume was made up to the mark with diluent (1000 µg/mL). Pipette 1 ml of the above solution into 10 ml volumetric flask and make up the volume using diluent (100 µg/mL).

Preparation of Solutions for Linearity: The solutions for linearity were prepared from the stock solution by diluting with diluent. The concentration ranging from 2, 4, 6, 8, 10 μg/mL were prepared for Teneligliptin. Pipette 0.2, 0.4, 0.6, 0.8, 1.0 ml in 10 ml volumetric flasks and make up the volume using diluent to get the above concentrations.

Preparation of Standard Stock Solution for Metformin: 100 mg of Metformin was taken into 100 ml volumetric flask. To this add 50 ml of diluent and sonicate to dissolve and the volume was made up to the mark with diluent (1000 µg/mL).

Preparation of Solutions for Linearity: The solutions for linearity were prepared from the stock solution by diluting with diluent. The concentration ranging from 25, 50, 75, 100, 125 μg/mL were prepared for Metformin. Pipette 0.25, 0.50, 0.75, 1.0, and 1.25 in 10 ml volumetric flasks and make up the volume using diluent to get the above concentrations.

Chromatographic Conditions:

TABLE 1: CHROMATOGRAPHIC CONDITIONS FOR TENELIGLIPTIN AND METFORMIN

Method Validation:

Linearity and Range: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of an analyte in samples within a given range.

Procedure: 2-10 µg/mL for Teneligliptin and 25-125 µg/ml for Metformin was prepared. The regression line obtained was linear. From the data obtained, co-relation coefficient, slope, and Y-intercept were calculated. Ideally co-relation coefficient should be not less than 0.99 and statistical Y- should be not more than 2.0.

Accuracy: Accuracy is performed in three different levels for Teneligliptin and Metformin using standard addition method at 50%, 100%, and 150%. A known quantity of sample was spiked to the standards. Samples are analyzed in triplicate for each level. From the results, % recovery was calculated. % Recovery at each spike level shall be not less than 98.0 % and not more than 102.0%, % RSD for the duplicate observations shall be not more than 2.0. Overall % RSD for the % Recovery shall be not more than 2.0.

% Recovery = Amount of drug recovered/ Amount of drug added× 100

Procedure:

Accuracy at 50%: A known amount of standard drug solution of Teneligliptin and Metformin (25µg/mL) from the stock solution was added to the sample solution of a determined concentration (50 μg/mL) the solutions were taken in a 10 ml volumetric flask and the volume was made up with diluent and filtered through 0.2 μ syringe filter. The solutions were injected, analyzed and the recovery was calculated.

Accuracy at 100%:  A known amount of standard drug solution of Teneligliptin and Metformin (50μg/mL) from the stock solution was added to the sample solution of a determined concentration (50μg/mL). The solutions were taken in a 10 mL volumetric flask, and the volume was made up with diluent and filtered through a 0.2 μ syringe filter. The solutions were injected, analyzed and the recovery was calculated.

Accuracy at 150%: A known amount of standard drug solution of Teneligliptin and Metformin (75 μg/mL) from the stock solution was added to the sample solution of a determined concentration (50 μg/mL) the solutions were taken in a 10 ml volumetric flask and the volume was made up with diluent and filtered through 0.2 μ syringe filter. The solutions were injected, analyzed and the recovery was calculated.

Precision:

System Precision: The system precision is checked by injecting 6 sample injections and checking the reproducibility in the retention time and area. The % RSD calculated must be less than 2%.

Method Precision:

Intraday Precision: The intraday precision is checked by using standard Teneligliptin and Metformin samples to ensure that the analytical system is precise. The retention time and area of three determinations was measured, and RSD was calculated. % RSD of the assay value for three determinations shall not be more than 2.0%.

Interday Precision: The interday precision is checked by using the same standard Teneligliptin and Metformin samples analyzed for intraday precision on an alternate day to ensure that the analytical system is precise.

The retention time and area of three determinations was measured, and RSD was calculated. % RSD of the assay value for three determinations shall not be more than 2.0%.

Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ were calculated using the mathematical equations:

LOD = 3.3 σ/S

Where, σ = the standard deviation of the response, S = the slope of the calibration curve

LOQ = 10 σ/S

Where, σ = the standard deviation of the response, S = the slope of the calibration curve

Robustness: The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Procedure: Robustness was done by changing the flow rate (± 0.1 ml), and mobile phase ratio (± 2) and pH (± 0.1). All the system suitability parameters must meet as per the method. The flow rate of the mobile phase was increased to 1.1mL/min and decreased to 0.9 mL/min from 1.0mL/min. The final variation was done by changing the mobile phase (methanol: acetonitrile: potassium dihydrogen orthophosphate) ratio to 38: 18: 44 and 42: 22: 36 from 40: 20: 40. The pH was changed to 4.5 and 4.7 from 4.6.

System Suitability: Five replicate injections of standard solutions were injected, and the chromatograms were recorded. The system is suitable for analysis if

• The theoretical plates in five replicate injections should be not less than in
• USP tailing factor for Teneligliptin and Metformin peaks should be not more than 2.0.
• The % relative standard deviation for five replicate injections should not be more than 2%.

Procedure: The standard solution (10 μg/mL) was prepared and injected into the UFLC system six times. The tailing factor and theoretical plate count were recorded.

Assay of Marketed Formulation:

Sample Preparation: In case of marketed formulations, twenty tablets were taken, weighed, finely powdered and an accurate amount equivalent to 100 mg of drug powder was transferred into a 100 ml volumetric flask. The stock solution was further diluted with diluents, and it was filtered through a 0.45µ nylon filter to obtain a concentration of 100 µg/mL of Teneligliptin and Metformin, and then the resultant solution is analyzed.

Method Validation:

System Suitability: System suitability tests are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions.

TABLE 2: SYSTEM SUITABILITY RESULTS FOR TENELIGLIPTIN AND METFORMIN

Data Interpretation: It was observed from the data tabulated above that the method complies with system suitability parameters.

Hence, it was concluded that the system suitability parameter met the requirement of method validation.

Linearity and Range:

Teneligliptin: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of an analyte in samples within a given range of 2-10µg/mL.

Data Interpretation: The method for Teneligliptin was found to be linear in the concentration range of 2 µg/mL to 10 µg/mL and the correlation coefficient obtained is 0.997.

Metformin: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of an analyte in samples within a given range of 25-125 µg/mL.

TABLE 3: RESULTS FOR LINEARITY OF TENELIGLIPTIN

Data Interpretation: The method for Metformin was found to be linear in the concentration range of 25 µg/mL to 125 µg/mL and Correlation coefficient obtained is 0.999.

TABLE 4: RESULTS FOR LINEARITY OF METFORMIN

Precision: The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of a homogeneous sample.

System Precision: The system precision was carried out to ensure that the analytical system was working correctly. The system precision is performed by 6 sample injections and checking the reproducibility in the peak area.

TABLE 5: SYSTEM PRECISION FOR TENELIGLIPTIN

TABLE 6: SYSTEM PRECISION FOR METFORMIN

Data Interpretation:  It is observed from the data tabulated above, that the area responses are consistent as evidenced by the values of relative standard deviation. Hence, it can be concluded that the system precision parameter meets the requirement of method validation.

Method Precision:

Intraday Precision: The precision express reliability of the method, where it defines the extent for the individual test results can agree to repeated test result on the same operating conditions at a short period. Repeatability of the method is accepted.

Interday Precision: Repeatability method procedure was repeated on the next day. The method passed the test, as both retention time (<1%) and response peak areas (<2%), % RSD obtained were in the limits.

 Data Interpretation: From the above results, it was concluded that the method is precise.

TABLE 7: METHOD PRECISION FOR TENELIGLIPTIN

TABLE 8: METHOD PRECISION FOR METFORMIN

Data Interpretation: From the above results, it was concluded that the method is precise.

Limit of Detection and Limit of Quantitation: Limit of detection is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Limit of quantitation is the lowest amount of analyte in an example that can be quantitated with acceptable accuracy and precision, under the stated experimental conditions.

Limit of detection (LOD) and Limit of quantitation (LOQ) was calculated based on residual standard deviation of response and slope.

TABLE 9: RESULTS OF LOD AND LOQ OF TENELIGLIPTIN AND METFORMIN

 Accuracy: The accuracy of an analytical method is the closeness of test results obtained by that method to the true value (Standard value).

% Recovery = Amount of drug recovered / Amount of drug added × 100

Robustness: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage ^13-26.

TABLE 10: RECOVERY RESULTS FOR TENELIGLIPTIN AND METFORMIN

TABLE 11: RESULTS FOR ROBUSTNESS OF TENELIGLIPTIN

TABLE 12: RESULTS FOR ROBUSTNESS OF METFORMIN

CONCLUSION: A novel, simple, rapid and cost-effective RP-UFLC method was successfully developed for simultaneous estimation of Teneligliptin and Metformin present in the formulation. The proposed method was optimized and validated for the various experimental parameters. Influence of pH of the mobile phase, mobile phase ratio and flow rate on the analysis of Teneligliptin and Metformin was evaluated. All the analytes were well resolved and separated in less than 10 min. The developed method can be conveniently used by quality control outfits to determine the contents of Teneligliptin and Metformin simultaneously in routine and stability samples. This method could be used for the analysis of the drugs in pharmaceutical preparations and routine laboratory analysis. Overall, the proposed method provides high throughput for determination of Teneligliptin and Metformin with excellent accuracy, precision, selectivity, and reproducibility.

ACKNOWLEDGEMENT: The authors express their sincere thanks to the Principal, JSS College of Pharmacy, and Mysuru for providing the necessary facilities to carry out the research work.

CONFLICT OF INTEREST: No

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    How to cite this article:

    Maruthi R, Chandan RS and Raikar P: Simultaneous estimation and analytical method development, validation for the Teneligliptin and Metformin by RP-UFLC. Int J Pharm Sci & Res 2019; 10(4): 1811-19. doi: 10.13040/IJPSR.0975-8232.10(4).1811-19.

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