INTRODUCTION:

Sitagliptin (SGT) chemically is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazin-7(8H)-yl] - 4-(2,4,5-trifluorophenyl) butan-1-one phosphate monohydrate^1,2 an antidiabetic and pharmacologically inhibits dipeptidylpeptidase-4, an enzyme responsible for degradation of the incretin hormones glucagon like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptidase is used in the management of type 2 diabetes mellitus³. Chemical structure is shown in (Fig No 1).

Fig 1: Chemical structure of Sitagliptin and Glimepiride Drug molecule

Various analytical methods have been reported for the estimation of SGT alone or in combination with other anti-diabetic agents in pharmaceutical dosage form includes lonely UV spectroscopic methods^4-7, with other drug UV spectroscopic methods^8-10, stability indicating HPTLC and HPTLC methods^11-14, pharmacological study¹⁵, analytical HPLC methods^16-19, bio analytical MS method^20,21, efficacy and safety of drug^22-25 and review on analytical methods^26,27.

Glimepiride (GMP) is chemically 1- [[4- [2-(3-ethyl-4 methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulphonyl]-3-trans-(4-methyl cyclo hexyl) urea^1,2 and is oral antidiabetic sulfonylurea drug promotes insulin release at ATP-sensitive potassium channels on pancreatic β-cells via binding to a 65 kDa subunits of the sulfonyl urea receptor¹. It is used for the treatment of type 2 diabetes mellitus and has a duration of action of upto 24 Hrs³. Chemical structure is shown in (Fig No 1)

Various analytical methods have been reported for the estimation of GMP alone or in combination with other antidiabetic drugs includes UV spectroscopic method^28-31, stability indicating HPLC methods³², HPLC methods ^33-37, bio analytical HPLC chromatographic method^38-39, LC-MS/MS methods⁴⁰, bio analytical LC-MS/MS method^41-45, bio analytical LC-ESI-MS method^46-47 and articles of safety and efficacy of glimepiride along with sitagliptin or other antidiabetic drug^48-52. Both drugs are official in Indian pharmacopoeia and British pharmacopoeia^2,53.

For analytical method validation ICH Q2 (R1) has given various method performance characteristics^54,55.

MATERIALS AND METHODS:

Instrumentation:

Analysis was performed with a UV-1900i Shimadzu Double beam spectrophotometer (Shimadzu, Kyoto, Japan) with spectral bandwidth of 1 nm and wavelength accuracy of ±0.3nm with 10 mm matched Quartz cells was used. Drugs were weighed on electronic balance ‘Afcoset’ make by (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1mg Model No. ER 200A and for degassing the solutions digital Ultrasonic cleaner 1.8 Ltr (Labman scientific Instruments Chennai) was used.

Reagents and Chemicals:

Pharmaceutically pure samples of Sitagliptin from Akums Drugs and Pharmaceuticals Ltd Haridwar Uttarakhand and Glimepiride from Zim Laboratories, Kalmeshwar Dist. Nagpur were procured as gift samples and the commercial formulation were procured from local market. NaOH pellets AR grade and distilled water were utilised for preparation of solvent.

Solvent Selection:

Research article⁵⁶was focused on techniques to be adopted while selection of suitable solvent. SGT is freely soluble in water, DMSO and DMF slightly soluble in methanol, ethanol, acetone and acetonitrile insoluble in isopropanol¹ similarly GMP is freely soluble in DMF, DMSO, dichloromethane, chloroform, soluble in ethanol, slightly soluble in methanol, acetone, acetonitrile and dil alkali, dil acids, practically insoluble in water¹. Although solubility of the procured drug was studied in 0.1 N HCL, water and 0.1 N NaOH; and to understand characteristic nature of spectra each solution of known conc of analyte was scanned in UV range. The recorded spectra in these solvents are shown in (Fig No 2 and 3).

Fig 2: UV-VIS spectra of Sitagliptin in selection of solvent study

Fig 3: UV-VIS spectra of Glimepiride in selection of solvent study

Preparation of stock solutions and standard solutions:

10mg of pure drug SGT and GMP were accurately and separately weighed; and transferred into separate 25ml volumetric flask. Dissolved both the drugs SGT into 0.1 N NaOH separately and volume was made to 25ml with respective solvent. Working standard solution of SGT 100μg/ml and GMP 10μg/ml was obtained by diluting aliquot of stock solution.

Selection of Wavelength and Conc Range:

From UV spectra (Fig No 4) it was found that SGT and GMP have measurable absorbance at 267.5 and 228nm in 0.1 N NaOH respectively. Also, drug solution was prepared simulated to marketed formulation. Above discussed observations was guided to select critical parameters listed in Table No 1 and significant utility of these parameters was revealed during method development and validation as per ICH guidelines and for analysing marketed preparations.

Fig 4: UV-VIS overlaid spectra of Sitagliptin and Glimepiride in selected solvent

Table No 1: Selected critical parameter for UV-VIS analytical method of SGT and GMP

Parameter	Selected variables for SGT	Selected variables for GMP
Wavelength range	400-200 nm	400-200 nm
Wavelength	267.5 nm	228 nm
Solvent	0.1 N NaOH	0.1 N NaOH
Scan speed	Fast	Fast
Sampling interval	± 0.2 nm	± 0.2 nm

Experimental Method for Estimation:

Selection of suitable method among many approaches of multicomponent analysis is based on nature of spectra for simultaneous estimation of both these drugs. Among of this simultaneous equation method and absorption ratio method were applied for estimation of SGT and GMP from the dosage form.

Method-I: Simultaneous Equation Method:

SGT was shown absorbance at (λ_max)267.5 nm and GMP has maximum absorbance (λ _max) at 228 nm. The wavelength 228 and 267.5 nm were considered as 1 (λ₁) and 2 (λ₂) respectively. The equation A= abc was applied for x (GMP) and y (SGT) determination. On rearranging the 2 generated equations, the conc of x and y was calculated by following formula. Working standard solutions of SGT of conc 160μg/ml and GMP of conc 12μg/ml were separately prepared and used for the method.

Where Cx and Cy = Conc of GMP and SGT in sample solution

A₁and A₂ = absorbance of sample solution at 1 and 2 wavelengths

ay₁anday₂= absorptivity of SGT at 1 and 2 wavelengths of standard solution

ax₁andax₂= absorptivity of GPE at 1 and 2 wavelength of standard solution

Method-II Absorbance Correction Method:

At 267.5nm λ_max of SGT negligible absorbance of GMP was found hence this wavelength is suitable for precise measurement of SGT by absorbance correction of GMP; and at 228 nm λ_max of GMP where SGT shows interference, which was corrected by absorbance correction. The equation A= abc was applied for X (GMP) and Y (SGT) determination. Working standard solutions of SGT and GPE containing 120 μg/ml and 12 μg/ml conc were separately prepared and used for the method.

At 267.5nm A _s = A₁ + A₂

A _S = a X_2.b.C_X + aY₂. b.C_Y

On rearranging equation and b=1 cm

A_S = absorbance of sample containing SGT and GMP at 267.5 nm

aX₂ = absorptivity of GMP at 267.5 nm

aY₂ = absorptivity of SGT at 267.5 nm

b = Pathlength of solution 1 cm

C_Y = Conc of SGT in sample

Similarly, equation was derived for Glimepiride

A_S = absorbance of sample containing SGT and GMP at 228 nm

aX₁ = absorptivity of GMP at 228nm

aY₁ = absorptivity of SGT at 228nm

b = Pathlength of solution 1cm

C_Y = Conc of SGT in sample

Validation of the Method:

To attain analytical target profile of the method, selected critical parameters should meet the performance characteristics of the analytical method. In order to implement this ICH guideline Q2 R1 was applied to study methods performance with critical parameters. The method was validated as per ICH guidelines^54,55.

System Suitability:

System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc 120μg/ml for SGT and 12μg/ml for GMP were prepared separately and absorbance was recorded; SD and %RSD of the response was calculated.

Linearity:

The linearity of an analytical method is its capability to obtain response i.e. absorbance which is in spectroscopy directly proportional to the conc of analyte. Working standard solutions in series order were prepared in conc. range of 20 to 250μg/ml (SGT) and 1 to 24μg/ml (GMP) and scanned in 400-200nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at 228 and 267.5nm for GMP and SGT respectively in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis to attain slope, intercept, and correlation coefficient. From the nature of spectra working conc range 20 to 250μg/ml and 1 to 24 mcg/ml (μg/ml) was selected in solvent for SGT and GMP respectively.

Assay of Combined Drug:

Assay was carried out by proposed methods and method was validated by obtaining statistical parameters.

Estimation of Admixture by Simultaneous Equation/Absorbance Correction Method:

Tablets were weighed, powdered and tablet powder equivalent to 100mg SGT and tablet powder equivalent to 2mg GMP was weighed and transferred into 100ml volumetric flask. Dissolved into 0.1 N NaOH solvent shaking for 15 mins and volume was made to 100.0ml. Solution was filtered through whatman filter paper No 40 and aliquots of solution were diluted to obtain tablet solution. Solution was scanned in the range of 200 to 400nm in spectrum mode to obtain absorbance of tablet solution at 228 and 267.5nm in spectrum order. A set containing 5 samples were prepared and analysed for assay study.

Obtained absorbance was utilised to assess unknown conc of tablets; and statistically results were validated to obtain % of nominal conc, standard deviation and % of RSD.

Accuracy and Precision:

The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e., standard addition method; diluted standard solution of SGT and GMP was prepared and standard solutions added in 50,100 and 150% proportionate to the tablet solution. At each of these three levels three replicates were prepared, measured for response and % of conc, SD and RSD of replicates were calculated.

The precision study was supported by performing assay of dosage form six times; also the reproducibility in results were studied by interday / intraday precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of SGT and GMP by the proposed method were obtained using calibration graph method and calculated as 3.3σ/s for LOD and 10 σ/s for LOQ where σ is the standard deviation of calibration curve and s is the slope of regression line.

Robustness and Ruggedness:

It is measure of capability of analytical procedure to persist unaffected by small but deliberate variations in method parameter.

RESULTS AND DISCUSSION:

Method development involves numerous steps; of which solvent selection, method selection for measurement are significant one. Uses of environmental solvents have got significant weightage due to low cost, readily available and ecologically sound. Drugs underlying method must have appreciable solubility in the selected solvent. Chemical structure of the drug molecule, physico-chemical characteristics available in the literature guides about use of appropriate solvent in the method. Solubility of SGT and GMP was studied in each solvent; and in 0.1 N NaOH solvent both drugs were shown maximum and consistent absorbance as compare to other solvent.

System Suitability:

Absorbance of prepared six replicate of standard solutions (120 and 12μg/ml) are reported in Table No 2. The SD was found for SGT and GMP within tolerable limit and meets the system suitability requirements indicates method was suitable for analysis.

Table No 2: System suitability study of SGT and GMP

Sr No	Conc in μg /ml	Absorbance of SGT	Conc in μg /ml	Absorbance of GMP
1	120 μg /ml	0.691	12 μg /ml	0.582
2	120 μg /ml	0.667	12 μg /ml	0.571
3	120 μg /ml	0.692	12 μg /ml	0.593
4	120 μg /ml	0.686	12 μg /ml	0.569
5	120 μg /ml	0.682	12 μg /ml	0.571
6	120 μg /ml	0.679	12 μg /ml	0.597
	SD	0.00843	SD	0.01113

Linearity:

The overlay spectra obtained in linearity study was shown in Fig No 5 and 6 and the obtained calibration curve of both analytes was seem to be linear in the selected conc range as shown in Fig No 7. The regression equation of line and its parameters slope, r²value and intercept are tabulated in Table No 3, which evidenced the linear relationship between conc and obtained response.

Fig 5: UV-VIS overlay spectra of GMP in linearity study

Fig 6: UV-VIS overlay spectra of SGT in linearity study

Fig 7: Calibration curve of Sitagliptin and Glimepiride

Table No 3: Parameters of regression equation obtained in Microsoft excel office

Parameters	SGT	GMP
Detection wavelength	267.5 nm	228 nm
Solvent	0.1 N NaOH	0.1 N NaOH
Beer’s law limit (μg/ml)	20-250 μg/ml	1-24 μg/ml
Correlation coefficient (r²)	0.9985	0.9957
Regression equation (y = mx + c)	y = 0.005 x + 0.0109	y = 0.0479 x + 0.0149

Assay

The assay was supported out by calibration curve method. The spectra of formulation was obtained and calculated % of nominal conc and SD, data was seen within tolerable limits are summarized in Table No 4. The results directed applicability of the method for estimation of Formulation.

Accuracy and Precision:

The results obtained in accuracy study are shown in Table No 5 and 6, the obtained results were within satisfactory limit; and methods accuracy was supported by calculating % drug content. The precision parameter was performed by carry out assay of solutions; further the reproducibility in result was studied by interday / intraday precision. The values obtained SD and % RSD was shown methods precision and are summarised in Table No 5 and 6.

Table No 4: Results of assay of combined drug by proposed method

Method	Drug	Label Claim (mg/Tablet)	Amount found/mg; n=6	Drug Content %	Std Deviation	% RSD
Method-I	SGT	100 mg	99.54 mg	99.54 %	1.7031	1.7107
Method-I	GMP	2 mg	1.912 mg	95.61 %	0.5044	0.5276
Method-II	SGT	100 mg	99.16 mg	99.16 %	2.1088	1.7011
Method-II	GMP	2 mg	1.9232 mg	96.16 %	1.8711	1.9664

Table No 5: Results of accuracy and precision of Method-I

S N	Parameter	Level of study	Data Title	Obtd. Data	S.D.	RSD
1	Precision study of SGT	Intraday Precision	Mean of Abs n= 6	99.54	1.7030	1.7107
1	Precision study of SGT	Interday precision	Mean of Abs n= 6	103.31	2.1088	1.7011
2	Accuracy study of SGT	80 %	% Purity	101.22	0.7524	0.7433
		100 %		99.66	0.0541	0.0318
		120 %		98.42	2.1638	1.6001

Table No 5: Cont

S N	Parameter	Level of study	Data Title	Obtd Data	S.D.	RSD
1	Precision study of GMP	Intraday Precision	Mean of Abs n= 6	95.61	0.5442	0.5276
1	Precision study of GMP	Interday precision	Mean of Abs n= 6	96.16	1.8712	1.9664
2	Accuracy study of GMP	80%	% Purity found	97.02	0.4287	0.4419
		100%		102.50	2.3451	3.0501
		120%		101.56	3.4801	3.4264

Table No 6: Results of accuracy and precision of Method-II

S N	Parameter	Level of study	Data Title	Obtd. Data	S.D.	RSD
1	Precision study of SGT	Intraday Precision	Mean of Abs n= 6	106.08 %	5.4982	5.1827
1	Precision study of SGT	Interday precision	Mean of Abs n= 6	105.05 %	1.2055	1.1689
2	Accuracy study of SGT	80 %	% Purity	99.28	1.9218	1.9357
		100 %		101.03	3.0515	3.0203
		120 %		98.98	1.1717	1.1838

Table No 6: Cont…….

S N	Parameter	Level of study	Data Title	Obtd Data	S.D.	RSD
1	Precision study of GMP	Intraday Precision	Mean of Abs n= 6	102.428	0.9843	0.9612
1	Precision study of GMP	Interday precision	Mean of Abs n= 6	94.374	0.6442	0.6821
2	Accuracy study of GMP	80%	% Purity found	101.19	3.0148	2.9779
		100%		103.28	2.2045	1.1343
		120%		99.86	2.6684	2.6722

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of GMP and SGT by the proposed method were found within acceptable limit.

Robustness and Ruggedness:

Robustness was studied and capability of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method parameter like variation in the wavelength ± 1nm, variation in the solvent strength by ±0.1 %. The analytical process was found rugged during development; similarity in the result was obtained by performing the analysis by different analyst.

CONCLUSION:

The method was developed with eco-friendly and easily available aqueous 0.1 N HCl solvent. Sitagliptin and Glimepiride were estimated from the formulation by the method and satisfactory results were obtained. The simultaneous equation method was given reproducible results as compare to absorption correction method; however, obtained results of the methods were within acceptable limits given in the pharmacopoeia. The validated method is economical, precise, accurate, robust and reproducible hence can be routinely used for estimation of both the drugs from the dosage form.

CONFLICT OF INTEREST:

All Authors declared that there is no conflict of interest.

ACKNOWLEDGEMENT:

Authors are thankful to Akums Drugs and Pharmaceuticals Ltd Haridwar Uttarakhand for providing sitagliptin and Zim Laboratories, Kalmeshwar Dist. Nagpur for providing glimepiride as a gift sample. Authors are thankful to Management, Principal SVPM’S College of Pharmacy Malegaon BKII Baramati Dist- Pune, Maharashtra, India for providing facilities for research.

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Received on 27.12.2024 Revised on 28.02.2025

Accepted on 14.04.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):91-98.

DOI: 10.52711/2231-5675.2025.00015

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