Development and Validation of Q-absorbance Ratio method for Simultaneous Estimation of Teniligliptin hydrobromide and Metformin HCl in Multicomponent Dosage Form

 

Sonali S. Gadge^1*, Ashish D. Nakod², Vaishali P. Wasnik², Tilottama M. Gatkine²,

Shrutika S. Zile², Najuka H. Mohije², Vikrant L. Salode²

¹Assistant Professor, P. R. Patil Institute of Pharmacy, Talegaon (S.P.), Wardha- 442202

 

ABSTRACT:

The main aim of this study was focuses on the development and validation of q - absorbance ratio method for simultaneous estimation of Teniligliptin hydrobromide and Metformin HCL in multicomponent dosage form with the main and important objective was to develop and validate the method for accuracy results. A simple, accurate, sensitive and precise UV- spectrophotometric (Q-Absorbance ratio) method   for the simultaneous estimation of Teniligliptin Hydrobromide and Metformin hydrochloride in multicomponent dosage form was developed and validated for linearity and range, accuracy, precision, limit of detection and limit of quantitation. Q-Absorbance ratio method is employed for simultaneous estimation of Teniligliptin hydrobromide and Metformin hydrochloride from multi component dosage forms. In this method, the absorbance was measured at 241.63 nm for Teniligliptin hydrobromide and 231.65 nm for Metformin hydrochloride. Linearity for detector response was observed in range of 0.5–2.5 µg/ml and 12.5–62.5µg/ml for Teniligliptin hydrobromide and Metformin HCl respectively. Recovery studies confirmed accuracy of proposed method and results were validated as per ICH guidelines. The % RSD values were found to be less than 2 indicates good precision and reproducibility of the method. The method can be used for routine quality control of pharmaceutical formulation containing Teniligliptin hydrobromide hydrate and Metformin hydrochloride.

 

 

 

INTRODUCTION:

Teniligliptin hydrobromide hydrate (TENI) is chemically described as {(2S, 4S)-4-[4-(-3-methyl-1phenyl-1H-pyrazol-5-yl)] pyrrolidin-2-yl]} (1, 3-thiazolidin-3-yl) methanone hemipentahydrobromide hydrate is a dipeptidyl peptidase 4 inhibitor. It’s molecular formula is C₁₇H₂₇NO₄ and molecular weight is 309.40 g/mol [1]. TENI shows the inactivation of incretin harmones, thereby increasing bloodstream concentrations and reducing fasting and postparandial glucose concentrations in a glucose – dependant manner in patients with type 2 diabetes mellitus. The amount of active plasma incretins inhibited by DPP-4, which helps with glycemic control. The incretins hormone is inactivated by DPP-4 which secrets insulin. There are incretin hormones like glucagon- like peptide -1(GLP-1) and insulinotropic polypeptide which is glucose dependent, which are released by the entero endocrine cells and stimulates the secreation of insulin [2,3,4,5].

 

Fig No. 1: chemical structure of Teniligliptin

 

Chemically Metformin (MET) is 3-(diaminomethylidene)-1, 1-dimethylguanidine, an oral antidiabetic drug, used for the treatment of type 2 diabetes, particularly in overweight and obese people; It is also useful in the treatment of ovarian hypothecosis and investigated for other diseases where insulin resistance may be an important factor. Therapeutic effect of Metformin is to reduce insulin resistance [6,7,8]. It comes under the category of Biguinides [1]. Hyperglycemia is decreased by metformin, suppressed glucose production by the liver called hepatic gluconeogenesis. Intestinal absorption of glucose is retarded by the Metformin [9].

 

Fig No. 2: chemical structure of Metformin

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MATERIALS AND METHODS:

Apparatus:

A Shimadzu model 1601 double beam UV/Visible spectrophotometer with fixed slit width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 1cm matched quartz cells were used for the measurement of absorbance of all the solutions. UV-Probe system software gave the spectra automatically. The instruments, an Electronic analytical balance and an ultrasonic bath were used for study.

 

Reagents and Material:

TENI as reference standard was obtained from zydus cadila (Ponda, Goa) as gift sample, Metformin was obtained from Cipla Laboratories as a gift sample, where as their formulations obtained from local market. Analytical grade Methanol obtained from college laboratory.

 

 

Preparation of Standard Stock solution:

Accurately weighed quantity of TENI (50mg) and MET (50mg) were transferred to separate 50ml volumetric flask and dissolved and diluted to the mark with Methanol (1mg/ml). Measure accurately 5ml portion from above solutions and diluted to 50ml with Methanol to obtained standard solution having concentration of TENI (100µg/ml) and MET (100µg/ml). This solution was used as working standard solution. From this 1 ml of each transferred to 10 ml volumetric flask volume made up to 10ml, solutions obtained having concentration 10 µg/ml of TENI and 10 µg/ml of MET.

 

Method:

In Q-Absorbance method, five working standard solutions having concentration 0.5, 1, 1.5, 2, 2.5µg/ml for TENI and 12.5, 25, 37.5, 50, 62.5 µg/ml for MET were prepared in Methanol and measured the absorbance at 231.65nm (λmax of MET) and 258.86nm (iso-absorptive point), absorptivity coefficients were calculated using calibration curve. The following equations are used for the calculation of concentration of two drugs in the mixture.

 

                Qm-Qx   ×  A1

C MET= ––––––––   –––……………………………..(1)

                Qy-Qx         ay1

 

 

                Qm-Qy   ×   A1

C TEN1= ––––––––   –––…………………………….(2)

                Qx-Qy         ax1

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Where,

A_{1 =} Absorbance of lab mixture at 231.65nm

A_{2 =} Absorbance of lab mixture at 258.86nm

ax_{1 =} Absorptivity value of TENI at 231.65nm

ax_{2 =} Absorptivity value of MET at 258.86nm

ay_{1 =} Absorptivity value of TENI at 231.65nm

ay₂ = Absorptivity value of MET at 258.86nm

and Qm = A_2/A_1, Qy = ay₂/ay₁ & Qx = ax₂/ax₁

 

METHOD VALIDATION:

Linearity:

The calibration curves were plotted over a concentration range 0.5 - 2.5µg/ml for TENI and 12.5 - 62.5µg/ml for MET. Accurately measured standard stock solutions of each TENI (0.5, 1, 1.5, 2 and 2.5ml) and MET were transferred to a series of 10 ml volumetric flask separately and diluted up to the mark with Methanol. The absorbance of solutions was then measured at 231.65nm and 258.86nm. The calibration curves were constructed by plotting absorbance versus concentration the calibration curves were plotted the regression equations were calculated.

 

Precision:

Intraday Precision:

Mixed solution containing 0.5–2.5µg/ml TENI and 12.5 –62.5µg/ml MET was analyzed 3 times on the same day and % RSD was calculated.

 

Interday Precision:

Mixed solutions containing 0.5–2.5µg/ml TENI and 12.5–62.5µg/ml

 

Accuracy:

The accuaracy of the method was determined by calculating recoveries TENI and MET in mixture by standard addition method. Known amount of standard solutions of TENI (0, 0.5, 1 and 1.5µg/ml) and MET (0, 12.5, 25 and 37.5µg/ml) were added to pre quantified sample of 1µg/ml TENI + 25µg/ml MET mixture. The absorbance of TENI and MET were recorded at λ₁ and λ_2. The percentage recovery was calculated by measuring the absorbance of both drug at 231.65nm and 258.86nm respectively and fitting these values into equation. Each response was average of three determinations.

 

Limit of Detection and Limit of Quantitation:

The limit of Detection (LOD) and limit of quantitation (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e. 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

 

LOD = 3.3 × σ/S

 

Where,

σ = standard deviation of the response and

S = slope of calibration curve.

Analysis of Tablet Dosage Form:

Twenty tablets were accurately weighed, average weight was calculated. Tablets were finely powdered and mixed thoroughly. An accurately weighed tablet powder equivalent to about 12.5mg of MET was transferred to 100ml volumetric flask then 0.5mg of TENI and 12.5mg of MET were added, dissolved in 5-10ml of methanol and volume was further made up to the mark with the same. The solution was then filtered through Wateman filter paper No.1 and 1.25ml portion taken out from these diluted to 10ml. the final dilutions were measured at 231.65nm and 258.86nm in 1cm cell against Methanol as blank. Five replicate estimations were done in similar way. Amount of each drug was estimated using equations as per laboratory mixtures and percentage estimation of labeled claim was calculated as follows

 

 

                      Amount of drug estimated×Average weight of tablet

% Labelled= ---------------------------------------------------------------×100

claim                     Weight of sample taken ×Labelled claim

 

 

RESULTS AND DISCUSSION:

In Q-Absorbance ratio method, the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s law at all the wavelength, which was fulfilled in case of both these drugs. The two wavelengths were used for analysis of the drugs were 231.65 nm (λmax of MET) and 258.86 nm (iso-absorptive point) at which calibration curves were prepared for both the drugs. The overlain UV absorption spectra of TENI (241.63 nm) and MET (231.63 nm) and iso- absorptive point (258.86nm) in Methanol is shown in fig. No. 3.

 

 

Fig no.3: Plot of overlain spectra of Teniligliptin and Metformin

 

Table no. 1: Observation, results and statistical data for recovery studies

Sr. No.	Weight of tablet powder(mg)	Amount added (µg/ml)		Absorbance		% Recovery
		TENI	MET	231.65nm	258.86nm	TENI	MET
1.	242.75	1.6	40	2.605	0.133	99.94	99.0
2.	242.73	2	50	2.890	0.152	100	100
3.	242.75	2.4	60	3.468	0.167	99.95	99.68
					Mean	99.96	99.56
					± SD	0.03214	0.5106
					RSD	0.032	0.5128

 

Table no 2: Analysis of TENI and MET by proposed method

Formulation	Lable claim (mg)		Amount found (mg)		% Lable claim(±S.D)
Ziten-M(Glenmark)	TENI	MET	TENI	MET	TENI	MET
	20	500	19.9	500	99.2±1.10	100±0.48

 

At all the wavelengths, the validation parameters were studied for the proposed method. Accuracy was determined by calculating the recovery and the mean, accuracy was determined (Table 1). The method was successfully used to determine the amounts of TENI and MET present in the tablet dosage forms. The results obtained were good agreement with the corresponding labeled amount (Table 2). As repeatability and inter and intraday variations (% RSD) for both the drugs, precision was calculated.

 

CONCLUSION:

The developed Q-Absorbance method is found to be simple, sensitive, accurate and precise and can be used for routine analysis of TENI and MET. The method was developed method was validated as per ICH guidelines. Statistical analysis proved that the method is repeatable and selective for the analysis of TENI and MET in their combined pharmaceutical formulations.

 

ACKNOWLEDGEMENT:

The authors are gratefully thankful to Zydus cadila and Cipla laboratories for providing Teneligliptin and Metformin as gift samples. The authors are also gratefully thankful to P. R. Patil, Institute of Pharmacy, Talegaon (SP), Wardha for providing necessary facilities for the research work.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest.

 

SYMBOLS AND ABBREVIATIONS:

 

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Received on 25.09.2019                 Accepted on 17.10.2019

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Asian J. Pharm. Ana. 2019; 9(4):215-218.