Analytical Investigation and Validation of Sitagliptin and Metformin in Their Pure and Combined Dosage Form using Spectrophotometry

Diabetes mellitus is one of the most prevalent chronic metabolic disorders among worldwide individuals creating a challenge on healthcare facilities of any nation ^{1, 2}. Combination therapy has become the preferred treatment strategy for patients with Type 2 diabetes mellitus (T2DM), as it improves glycemic control through complementary mechanisms of action while reducing the risk of treatment failure  . Sitagliptin (STG) chemically known as (3R)-3-purcino-l-[3- (trifiuoromethyl)-5,6-dihydro[1,2,4] triazolo [4,3-a]pyrazin-7 (8H)-yl] -4-(2,4,S-trifiuorophenyl) butan-lone phosphate monohydrate is a selective and orally active dipeptidyl peptidase-4 (DPP-4) inhibitor that enhances endogenous incretin activity by preventing the degradation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Owing to its favourable efficacy and safety profile, Sitagliptin is extensively used either as monotherapy or in combination with other oral antidiabetic agents ^{4, 5 6}. Metformin Hydrochloride (MFT), chemically known as N,N-dimethyl imidodicarbonimidic diamide hydrochloride, is a traditionally used oral antihyperglycaemic agent which primarily acts by suppressing hepatic gluconeogenesis, improving peripheral insulin sensitivity, and enhancing glucose uptake in skeletal muscle without causing significant hypoglycemia ⁷. The complementary pharmacological actions of Sitagliptin and Metformin make their fixed-dose combination an effective therapeutic option for the long-term management of Type 2 diabetes mellitus ⁸. A reliable analytical method is essential to ensure the quality, safety, and efficacy of pharmaceutical products throughout their development, manufacturing, and quality control processes. Among the available analytical techniques, UV-visible spectrophotometry remains one of the most widely employed methods because of its simplicity, rapidity, cost-effectiveness, and suitability for routine analysis in quality control laboratories⁹.

The simultaneous equation (Vierordt's) method is particularly advantageous for the quantitative estimation of multicomponent formulations, as it enables the determination of individual drug concentrations without prior separation by utilizing the additive nature of absorbance at selected wavelengths¹⁰. An extensive review of the published literature indicates that several analytical methods, including high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS), have been reported for the estimation of STG and MFT in combination dosage form. Although a few spectrophotometric methods have been described for the simultaneous estimation of Sitagliptin and Metformin in combined dosage forms, hence there remains a need for a simple, economical, accurate, and validated analytical procedure that can be conveniently employed for routine quality control analysis without the requirement for sophisticated instrumentation.

Therefore, the present study was undertaken to develop and validate a simple UV-visible spectrophotometric method based on the simultaneous equation approach for the quantitative estimation of Sitagliptin and Metformin in bulk drugs and combined tablet dosage forms. The proposed method was validated in accordance with the International Council for Harmonisation (ICH Q2(R1)) guidelines by evaluating its linearity, accuracy, precision, ruggedness, limit of detection, and limit of quantification. The validated method is intended to provide a reliable, rapid, and cost-effective analytical tool for routine pharmaceutical quality control.

MATERIALS AND METHODS

Materials and Method: The relation between the concentration of analyte and the amount of light absorbed based on Beer–Lambert law is the basis used here¹¹. All spectrophotometric tracing were performed using double beam Uv-Visible spectrophotometer (Shimadzu UV-1900i, Kyoto, Japan), equipped with matched 1cm quartz cells. Precise masses of all the chemicals were weighed by using electronic balance (Kerro BL3003KE) with precision upto 1mg, while all the glasswares employed were calibrated before the study. Chemicals and reagents used are of analytical grade. Pharmaceutical grade Sitagliptin Phosphate (purity: 99.99%) and Metformin Hydrochloride (purity: 99.99%) were procured from Dhamtech Pharma Supplies, Mumbai, India, and were used as reference standards without further purification. Commercially available tablet formulation containing Sitagliptin Phosphate equivalent to 50 mg of Sitagliptin and Metformin Hydrochloride 500 mg per tablet (Batch No. 0426580) was purchased from the local market for analytical evaluation. Freshly prepared 0.1 N sodium hydroxide (NaOH) was employed as the solvent throughout the investigation.

Preparation of Standard Stock Solutions: Standard stock solutions of Sitagliptin and Metformin were prepared separately by accurately weighing 100 mg of each drug and diluting with 100 mL of 0.1 N NaOH in volumetric flasks. Further working standard solutions (100 µg/mL) were prepared by titering 10 mL of the respective stock solution with separate 100 mL 0.1 N NaOH.

Determination of Maximum Absorption Wavelength (λmax): Aliquots of the working standard solutions were appropriately diluted with 0.1 N NaOH to obtain concentrations ranging from 5–25 µg/mL for Sitagliptin and Metformin. Each solution was scanned over the wavelength range of 200–400 nm against 0.1 N NaOH as the blank. The wavelength corresponding to the maximum absorbance (λmax) was selected for quantitative estimation. Sitagliptin exhibited maximum absorbance at 234 nm, whereas Metformin showed maximum absorbance at 269 nm.

Preparation of Sample Solution: Twenty tablets of the marketed formulation weighed individually, and average tablet weight was calculated. The tablets were finely powdered, and powder equivalent to 100 mg of Sitagliptin and in-situ of Metformin was diluted upto 100ml with 0.1 N NaOH and sonicated for 20 minutes to ensure complete dilution. An appropriate aliquot was diluted with 0.1 N NaOH to obtain the required working concentration for analysis.

Simultaneous Estimation: The absorbance of both standard and sample solutions was measured at 234 nm and 269 nm. The concentrations of Sitagliptin and Metformin present in the sample solution were calculated using the simultaneous equation (Vierordt's). The concentrations were determined by applying Cramer's rule to the simultaneous equations ^{12, 13}.

Concentration of Drug (Cx) = A2 x  ay1 -A1 x ay2   ax2   x   ay1  - (ax1   x   ay2)  

Concentration of Drug (Cy)= A1 x   ax2 -A2    x    ay1   ax2   x   ay1  - (ax1   x   ay2)  

Method Validation: The proposed analytical method was validated in accordance with the International Council for Harmonisation (ICH Q2(R1)) guideline for analytical method validation.^{14, 15}

Linearity and range: Linearity was evaluated by analyzing a series of standard solutions at concentration range of 5–25 µg/mL for Sitagliptin and 50–250 µg/mL for Metformin. Calibration curves were constructed by plotting absorbance against concentration, and the corresponding regression equations and correlation coefficients were determined.

Accuracy: The accuracy of the proposed method was evaluated by the standard addition technique at three concentration levels (80%, 100%, and 120%). Known quantities of standard drug were added to pre-analysed sample solutions, and the percentage recovery of each analyte was calculated.

Precision: Method precision was assessed by analyzing replicate measurements (n = 3) of homogeneous sample solutions under identical experimental conditions. The results were expressed as mean, standard deviation (SD), and percentage relative standard deviation (%RSD).

Limit of Detection and Limit of Quantification: The limit of detection (LOD) and limit of quantification (LOQ) were calculated according 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 = 3.3σ/S

LOQ = 10σ/S

where σ represents the standard deviation of the response and S represents the slope of the calibration curve.

Ruggedness: Ruggedness of the developed method was evaluated by performing intra-day and inter-day precision studies. The results were expressed as percentage relative standard deviation (%RSD), and values below 2% were considered indicative of acceptable ruggedness and reproducibility.

RESULT AND DISCUSSION

The UV-Visible spectrophotometer was employed easily to assess the concentration of component drugs in combined dosage form using a simultaneously equation method. In this method, the diluted solution of STG and MFT was scanned from 200-400nm. Two wavelengths 234 nm and 269nm are selected for STG and MTZ respectively as λmax as shown in spectrum figure.

Figure 1: Calibration curve of STG and MFT.

Table 5: Showing result of Combined Evaluation of Pure drug and in their Dosage form

Drug Name	Concentration (µg/ml) (c)	Absorbance of STG at 234 nm (abs)	Absorbance of STG at 269 nm(abs)	Absorptivity at 234 nm (abs/c)	Absorptivity at 269 nm (abs/c)
STG (x)	5	0.050	0.097	0.0100	0.0194
	10	0.026	0.132	0.0026	0.0132
	15	0.046	0.147	0.0030	0.0098
	20	0.037	0.175	0.0018	0.0087
	25	0.031	0.189	0.0012	0.0075
Mean	--	0.038	0.148	0.0037 (ax1)	0.01172 (ax2)
MFT (y)	5	0.532	0.077	0.1064	0.0154
	10	0.084	0.093	0.0084	0.0093
	15	1.186	0.086	0.0790	0.0057
	20	1.901	0.065	0.0950	0.0032
	25	2.444	0.073	0.0977	0.0029
Mean	--	1.2294	0.0788	0.0773(ay1)	0.0073(ay2)
Dosage Form		2.651 (A1)	3.214  (A2)	--	--

c) LOD & LOQ:

Table showing  results of LOD & LOQ determinations.

Sr. No	Volume taken in ml	Diluted upto	Concentration µg/ml		Abs  of STG at λmax 234 nm	Abs  of MFT at λmax 269 nm
			STG	MFT
F	0.1	10	1	10	0.159	0.014
2	0.2	10	2	20	0.018	0.017
3	0.3	10	3	30	0.022	0.018
4	0.4	10	4	40	0.006	0.022
5	0.5	10	5	50	0.007	0.025
Standard Deviation					0.065546	0.004324
Slope					0.316	0.027
LOD					0.6845	0.528532
LOQ					1.07425	1.601611

Discussion: The RSD as a percentage was verified using intra- and inter-day analysis. The findings of precision studies conducted within day showed % RSD 1.574 for STG and 1.032 for MFT and between days showed % RSD 1.564 for STG and 1.012 for MFT are presented.

CONCLUSION

The UV-Spectrophotometric method was developed and validated as per ICH guidelines using simultaneous equation method for the estimation of STG and MFT using 0.1 N NaOH as the solvent system. The developed method was found to be more simple, accurate, precise, and economic, thus can be used routinely for the analysis of Sitagliptin and Metformin in the bulk and combined dosage forms. The proposed Vierodt’s method offers a sensitive, accurate, and precise framework for simultaneous drug analysis, provided the linearity parameters are optimized.

LIST OF SYMBOLS:

λ- Lanbda

µg- Microgram

LIST OF ABBREVIATIONS:

ICH- International Council for Harmonisation

N –Normality

NaOH-Sodium Hydroxide

nm- Nanometer

RSD- Relative Standard Deviation

S.D. - Standard Deviation

Λmax- Absorption maxima

CONFLICT OF INTEREST

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors wants to acknowledge the Dean Academics, Principal and Supporting staff of Mata Gujri College of Pharmacy, A constituent unit of Mata Gujri University for providing necessary facilities for carrying out this research.

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