1. INTRODUCTION:

Number of individuals affected by diabetes is continuing to increase worldwide therefore; the need for effective management assumes ever greater urgency. Combination therapies are found to be effective as it is well tolerated, convenient to take with few contraindications. Pharmacists should take various factors into consideration, for example drug stability, possible degradation products and potential interactions with the excipients in concern with safety and efficacy of drug products.

Sitagliptin phosphate monohydrate (SIT) is chemically {(3R)-3-amino-1-[3-(trifluoromethyl)-6,8-dihydro-5H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl) butan-1-one} (Fig.1). SIT is well known oral hypoglycemic drug of the dipeptidyl peptidase-4 (DPP-4) inhibitor class¹. DPP-4 inhibitors represent a new therapeutic approach to the treatment of type II diabetes that functions to stimulate glucose-dependent insulin release and reduce glucagon level. After extensive literature survey several methods have been found for determination of SIT including UV-Visible spectrophotometry², LC-MS/MS in human plasma using protein precipitation method³, in human plasma using liquid-liquid extraction method⁴ and in human urine and hemodialysate using turbulent flow online extraction method⁵.

Fig. 1. Chemical structure of sitagliptin phosphate

Metformin hydrochloride (MET) is chemically, 1:1 dimethyl biguanidine mono hydrochloride (Fig.2) is an anti-diabetic drug from the biguanide class of oral hypoglycaemic agents, given in the treatment of non–insulin-dependent diabetes mellitus⁶. MET is effective in patients who lack functioning of islet cells as it act by simulations of glycolysis in peripheral tissues ^7,8. Several analytical methods based on potentiometry, spectrofluorimetry and UV-Visible spectrophotometry⁹, HPLC^10,11 and HPTLC¹²are reported for determination of metformin in pure form.

Fig. 2. Chemical structure of metformin hydrochloride

Several methods have been reported for determination of metformin with other antidiabetic drugs such as UV-Visible spectroscopy with glibenclamide¹³, with pioglitazone and glimepiride¹⁴, RP-HPLC method with pioglitazone hydrochloride and glimepiride¹⁵, with gliclazide and pioglitazone hydrochloride¹⁶, with pioglitazone^17,18and glipizide, gliclazide, glibenclamide or glimperide in plasma¹⁹ and LC/(APCI)MS with glibenclamide in human plasma²⁰.

Several chromatographic methods have been reported for simultaneous estimation of MET and SIT in combination including RP-HPLC^21-26and UPLC²⁷ in bulk as well as in pharmaceutical formulation.

The advantage of High Performance Thin Layer Chromatography (HPTLC) is that, a large number of samples can be simultaneously analysed in a shorter time period. Unlike HPLC, this method utilizes less quantity of solvents, thus lowering the cost of analysis. An ideal stability indicating chromatographic method should estimate the drug and also be able to resolve the drug from its degradation products. It was found that one HPTLC method²⁶have been reported in bulk drug and dosage form. However there is no any method was stability indicating one; hence an attempt has been made to develop an alternate accurate, rapid, specific and reproducible method for the determination of sitagliptin phosphate monohydrate (SIT) and metformin hydrochloride (MET) in presence of their degradation products for the content analysis during stability studies.

2. MATERIAL AND METHODS:

2.1. Chemicals and reagents

Sitagliptin phosphate monohydrate (SIT) and metformin hydrochloride (MET) were obtained as a gift sample from Zydus Cadilla, Ahmadabad, India. Combined dose tablet formulation containing sitagliptin phosphate monohydrate (50 mg) and metformin hydrochloride (500 mg), Sitar-M, manufactured by Oclare Labs Ltd., was purchased from local market. The solvents and chemicals used in the study were of HPLC grade (MERCK).

2.2. Preparation of standard stock solutions

2.2.1. Standard stock solution A

Accurately weighed quantity of SIT (10 mg) was transferred to 10.0 mL volumetric flask, dissolved with 5.0 mL DMSO, sonicate for 10 min and diluted to 10.0 mL with methanol. The solution was centrifuged at 2000 rpm for 5 min and 2.0 mL of supernatant was diluted to 10.0 mL with methanol. (Concentration 200 μg/mL of SIT)

2.2.2. Standard stock solution B

Accurately weighed quantity of MET (100 mg) transferred to 10.0 mL volumetric flask, dissolved and diluted to the mark with methanol. The solution was centrifuged at 2000 rpm for 5 min and 2.0 mL of supernatant was diluted to 10.0 mL with methanol. (Concentration 2000 μg/mL of MET)

2.2.3. Standard stock solution C

Accurately weighed quantity of SIT (10 mg) and MET (100 mg) were transferred to 10.0 mL volumetric flask, dissolved with 5.0 mL DMSO, sonicate for 10 min and diluted to 10.0 mL with methanol. The solution was centrifuged at 2000 rpm for 5 min and 2.0 mL of supernatant was diluted to 10.0 mL with methanol. (Concentration 200 μg/mL of SIT and 2000 μg/mL of MET)

2.3. Selection and optimization of mobile phase

Aliquot portion (10 µL) of standard stock solution A, B and C were diluted to 1.0 mL with methanol and 10 µL of resultant solution was applied on the TLC plates in the form of band and run in different solvent systems. Different solvent systems using individual solvents and in combinations were initially tried in order to determine the best condition for the effective separation of SIT and MET. The mobile phase comprising of acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02 v/v/v) gave high resolution of SIT and MET with improved peak shapes and hence the mobile phase was selected for further analysis. Glacial acetic acid was added to improve the peak shape of SIT.

2.4. Selection of wavelength for densitometric evaluation of separated bands

Aliquot portion (10 µL) of standard stock solution C was diluted to 1.0 mL with methanol and 10 µL of resultant solution was applied on the TLC plates in the form of band using LINOMAT-IV automatic sample applicator. The plates were scanned densitometrically over the wavelength range of 200-400 nm and their overlain spectra were carried out (Fig. 3). From overlain spectra, it was observed that both SIT and MET exhibited significant absorbance at 216 nm, which was selected as the analytical wavelength for further analysis.

Fig. 3. Overlain spectra of sitagliptin phosphate and metformin hydrochloride

2.5. Instrumentation and optimized chromatographic conditions

Chromatography was carried out on 10 cm x 10 cm aluminium-backed HPTLC plates coated with 250nm layer of silica gel G 60F₂₅₄ (E. Merck, Darmstadt, Germany, supplied by Merck India, Mumbai, India). The plates were prewashed with methanol and activated at 110⁰C for 30 min prior to chromatography. The samples were applied as bands of 6 mm length, under a continuous flow of nitrogen, using CAMAG LINOMAT-IV sample applicator. Samples were applied with a 100 µL syringe (Hamilton, Switzerland) at a constant application rate and the distance between adjacent bands was 8mm. The mobile phase consisting of acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02 v/v/v) was used for the development of the chromatograms. For linear ascending development, a twin-trough glass chamber 10 cm × 10 cm (CAMAG , Muttenz, Switzerland) previously saturated with mobile phase for 10 min at room temperature (25 ± 2°C) was employed. The length of each chromatogram run was 70 mm. After chromatographic development, plates were air dried and densitometric scanning was performed at 216 nm with a CAMAG TLC scanner-III operated in reflectance–absorbance mode and controlled by WinCATS software (Version 1.4.3.6336). The slit dimensions were 5 x 0.45 mm and the scanning speed was 20 mm/s. The source of radiation used was a deuterium lamp emitting continuous UV spectra between 190-400 nm. Concentrations of the compounds chromatographed were determined from the intensity of the diffused light. Evaluation was done by peak areas with linear regression.

2.6. Development of calibration curves

Aliquots portions, 20, 40, 60, 80, 100, 120, 140 and 160 μL of standard stock solution C (200 μg/mL of SIT and 2000 μg/mL of MET) were serially diluted to 2.0 mL with methanol in different micro centrifuge tube and vertex for 1.0 min. 4 μL of each solution was applied on the HPTLC plate to deliver 8, 16, 24, 32, 40, 48, 56 and 64 ng/band of SIT and 80, 160, 240, 320, 400, 480, 560 and 640 ng/band of MET. This was done in triplicate. For each concentration, the applied band bands were evenly distributed across the plate to minimize possible variation along the silica layer.

Given considerable extent of the calibration range (two orders of magnitude), the homoscedasticity of the analytical method was evaluated with Cochran’s test. In order to achieve homoscedasticity, the Cochran C of 4 standards with 3 replicates of each standard should be less than the critical values of 0.768²⁸. Since the largest and smallest values of variance usually appear at the extremities of the calibration curve in the heteroscedastic case, the two lowest concentrations (8 and 16 ng/band of SIT and 80 and 160 ng/band of MET) and the two highest concentration (56 and 64 ng/band of SIT and 560 and 640 ng/band of MET) standards were included in the tests.

2.7. Assay

2.7.1. Preparation of standard solution

Aliquots portions, 100 μL of standard stock solution C (200 μg/mL of SIT and 2000 μg/mL of MET) was diluted to 2.0 mL with methanol in micro centrifuge tube and vertex for 1.0 min. (10 µg/mL of SIT and 100 µg/mL of MET)

2.7.2. Preparation of sample solution

Twenty tablets were weighed accurately; average weight was calculated, and crushed to obtain fine powder. Accurately weighed quantity of tablet powder equivalent to about 10 mg of SIT was transferred to 10.0 mL volumetric flask, dissolved with 5.0 mL DMSO, sonicate for 10 min, diluted to 10.0 mL with methanol, mixed well and filtered through Whatmann filter paper No.42. The solution was centrifuged at 2000 rpm for 5 min and 2.0 mL of supernatant was diluted to 10.0 mL with methanol. 100 μL of resultant solution diluted to 2.0 mL with methanol in micro centrifuge tube and vertex for 1.0 min (10 µg/mL of SIT and 100 µg/mL of MET). This solution was used as sample solution.

On the TLC plate, 4 µL each, two bands of standard solution and four bands of sample solution were applied and the plate was developed and scanned under the optimized chromatographic condition. Content of SIT and MET were calculated by comparing peak areas of sample with that of the standard. The densitogram of tablet formulation is shown in Fig. 4.

Fig. 4. Densitogram of marketed formulation, Peak 1-SIT and Peak 2-MET

2.8. Method validation

Validation of optimized HPTLC method was done with respect to ICH guidelines.

2.8.1. Accuracy

To ascertain accuracy of the method recovery studies were performed by the standard addition method. Pre-analyzed tablet powder equivalent to about 5mg of SIT was weighed and transferred to 10.0 mL volumetric flask, added 3mg, 5mg and 7mg of pure SIT and 30 mg, 50 mg and 70 mg of pure MET to the tablet powder for 80%, 100% and 120% level of recovery. Extraction and dilutions were performed as described in sample solution. Solutions were prepared in triplicate and analyzed. The procedure was repeated for three consecutive days. Accuracy was determined and expressed as percent recovery.

2.8.2. Precision

To ascertain repeatability and reproducibility of the method precision studies were performed. tablet sample solution was prepared and analyzed in the similar manner as described under analysis of the tablet formulation.

2.8.3. Repeatability

Six replicates of SIT-40 ng/band and MET-400 ng/band were applied on silica gel 60F₂₅₄ plate and analysed by the proposed method for system precision studies to determine variations due to the instrument.

Intermediate precision

For method precision were carried out at three different concentration levels (24, 40 and 56 ng/band of SIT and 240, 400 and 560 ng/band of MET). Intra-day precision was determined by repeating the assay six times at different time interval on same day and on three consecutive days for inter-day precision studies. Results of intermediate precision are expressed as percent relative standard deviation.

2.8.4. Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ were separately determined based on the standard deviation of the y-intercept and mean slope of the calibration curves.

2.8.5. Robustness

Robustness of the proposed method was studied by small but deliberate variations in the optimized method parameters. Variation in composition of the mobile phase (± 0.1 mL), volume of the mobile phase (± 10 %), chamber saturation time (± 20 %), time from bandting to development (5 min, 20 min and 1 hrs) and time from development to scanning (5 min, 20 min and 1 hr) was involved in this study. The effect of these changes on both the Rf values and peak areas were evaluated by calculating the relative standard deviation for each parameter.

2.8.6. Solution stability

Sample solution was prepared and was kept at room temperature (25 ± 2°C) on a shelf protected from direct light. The solution was analyzed after 20 min, 1 hrs, 3 hrs, 8 hrs and 24 hrs. The % label claim and the relative standard deviation were calculated.

2.9. Forced degradation studies

Forced degradation study was carried out by attempting deliberate exposing the drugs to different stress conditions. A mixed stock solution of SIT (10 mg) and MET (100 mg) was prepared in 10.0 mL methanol with the aid of DMSO. This mixed stock solution was used for forced degradation to provide an indication of the stability indicating property and specificity of the proposed method.

2.9.1. Acid and base induced degradation product

To 2.0 mL of mixed stock solution, 8.0 mL of 1N HCl and 8.0 mL of 1N NaOH were added separately. These mixtures were reflux separately for 3 h at 80⁰C. The forced degradation study in acidic and basic media was performed in the dark in order to leave out the possible degradative effect of light. 100 µL of sample solution was diluted to 2.0 mL with methanol and 4µL (40 ng/band of SIT and 400 ng/band of MET) was applied on TLC plate.

2.9.2. Hydrogen peroxide induced degradation product

To 2.0 mL of mixed stock solution, 8.0 mL of hydrogen peroxide (30% v/v) was added. This solution was heated in boiling water bath for 10 min to remove completely the excess of hydrogen peroxide and reflux for 3 h at 80°C. 100 µL of sample solution was diluted to 2.0 mL with methanol and 4µL was applied on TLC plate.

2.9.3. Dry heat induced degradation product

Dry heat degradation was performed by exposing the powdered SIT (10 mg) and MET (100 mg) to 60°C for 24 h under dry heat condition to study the inherent stability of the drugs. Dry heat exposed drugs was dissolved in 10.0 mL methanol with the aid of DMSO. 2.0 mL of this solution was diluted to 10.0 mL. 100 µL of resultant sample solution was diluted to 2.0 mL with methanol and 4µL was applied on TLC plate.

2.9.4. Wet heat induced degradation product

2.0 mL of mixed stock solution was diluted to 10.0 mL with methanol and reflux for 3 h at 80°C. 100 µL of sample solution was diluted to 2.0 mL with methanol and 4µL was applied on TLC plate.

2.9.5. Photolytic induced degradation product

2.0 mL of mixed stock solution was diluted to 10.0 mL with methanol. For photochemical stability study, 5.0 mL of resultant diluted stock solution was exposed to direct sunlight for 8 days on a wooden plank and kept on terrace. For UV radiation degradation study 5.0 mL of resultant diluted stock solution was exposed to UV radiations at 254 nm for 24 h in UV chamber. 100 µL of each sample solution were diluted to 2.0 mL with methanol and 4µL was applied on TLC plate.

2.9.6. Neutral hydrolysis

2.0 mL of mixed stock solution was diluted to 10.0 mL with distilled water and was reflux for 8h. 100 µL of each sample solution were diluted to 2.0 mL with methanol and 4 µL was applied on TLC plate.

All the exposed samples were analyzed as discussed in analysis of marketed formulation.

3. RESULT AND DISCUSSION:

Among the different mobile phase combinations acetonitrile-methanol-glacial acetic acid (7.0:3.0:0.02 v/v/v) gave better resolution and sharp peaks with Rf values of 0.21 ± 0.035 and 0.53 ± 0.029 for SIT and MET, respectively on densitometric scanning at 216 nm.

3.1. Linearity

Peak areas were found to have good linear relationship with the concentration than the peak heights. The r² values were found to be 0.9992 and 0.9994 for SIT and MET respectively. Calibration graphs were constructed in the concentration range of 8-64 ng/band for SIT and 80-640 ng/band for MET. The correlation coefficients, y-intercepts and slopes of the regression lines of the two compounds were calculated (Table-1).

Table 1: Optical Characteristics and Validation Data of SIT and MET

Parameters	SIT	MET
Linearity (ng mL^-1)	8-64	80-640
Slope	529.12	448.46
Intercept	362.87	225.56
Regression coefficient (r²)	0.9992	0.9994

*Average of six determinations

The homoscedasticity (homogeneity of variance) of the calibration standards was verified using a Cochran’s test. The C_calc values were 0.482 and 0.503 for SIT and MET, respectively. These test statistics were smaller than the critical value, C_{tab(α=0.05; k=4, n=3)}= 0.768. The two calibration curves pass the homoscedasticity test since the C_calc values were less than the critical value. Thus, straight lines were considered adequate to describe the relationships between the peak area and the concentrations for each compound (Table-2).

Table 4: Results of Recovery Study

Level of Recovery %	Amount of pure drug added (mg)		Amount of pure drug recovered (mg)		% Mean Recovery*		SD (±)*		% C.O.V.
Level of Recovery %	SIT	MET	SIT	MET	SIT	MET	SIT	MET	SIT	MET
80	3	30	3.008	29.817	100.28	99.39	0.926	1.611	0.931	1.596
100	5	50	4.994	49.805	99.87	99.61	1.256	1.236	1.245	1.229
120	7	70	6.978	69.594	99.68	99.42	1.056	1.325	1.089	1.331

*Denotes average of three estimations at each level of recovery.

Table 5: Results of Precision Study

Parameters	SIT				MET
Parameters	Theoretical amount (ng)	Amount estimated (ng)	% RSD	SE	Theoretical amount (ng)	Amount estimated (ng)	% RSD	SE
Repeatability	40	38.96	0.7258	0.2568	400	398.51	1.2541	0.4045
Intra-day precision	24	23.68	0.9854	0.2368	240	238.69	0.2358	0.5849
	40	39.55	0.2568	0.3568	400	399.84	0.2568	0.8457
	56	55.26	1.0458	0.3568	560	557.23	0.8544	0.5658
Inter-day precision	24	23.78	0.8845	0.3565	240	237.68	0.8457	0.5124
	40	39.11	0.3568	0.2548	400	398.60	0.5625	0.3325
	56	55.59	0.9854	0.1245	560	561.54	0.4859	0.2566

*Mean of six determinations

Table 6: Robustness Testing for HPTLC method

Chromatographic Changes
Factor	Level	Peak area*		Rf Value*
Mobile phase composition (± 0.1 ml)		SIT	MET	SIT	MET
7.1:3.1:0.02	+ 0.1	3186.66	7562.12	0.20	0.53
7.0:3.0:0.02	0	3233.45	7610.11	0.21	0.53
6.9:2.9:0.02	- 0.1	3262.15	7596.16	0.19	0.54
	RSD	1.056	1.368
Amount of mobile phase (v/v) (± 1 ml)		SIT	MET	SIT	MET
11	+ 0.1	3112.15	7711.15	0.22	0.53
10	0	3285.68	7574.55	0.20	0.54
9	- 0.1	2968.62	7682.18	0.22	0.55
	RSD	0.986	1.458
Duration of Chamber Saturation (± 20 %)		SIT	MET	SIT	MET
12 min	+ 20%	3256.54	7722.36	0.22	0.54
10 min	0	3189.22	7614.14	0.21	0.53
8 min	- 20%	3289.81	7645.28	0.23	0.51
	RSD	1.515	1.688
Time from Spotting to development		SIT	MET	SIT	MET
5 min	--	3389.54	7825.44	0.23	0.55
20 min	--	3412.13	7749.45	0.22	0.55
60 min	--	3378.38	7811.14	0.19	0.53
	RSD	1.378	1.712
Time from development to scanning		SIT	MET	SIT	MET
5 min	--	3216.22	7516.14	0.23	0.54
20 min	--	3128.65	7719.14	0.21	0.51
60 min	--	3287.11	7577.88	0.23	0.52
	RSD	0.996	1.145

*Mean of three determinations

Table 7: Result of solution stability study

Sr. No.	Time	Amount of drug Estimated (mg/tablet)*		% Label Claim*		S.D. (±)		% C.O.V.
Sr. No.	Time	SIT	MET	SIT	MET	SIT	MET	SIT	MET
1	20 min	49.58	494.63	99.16	98.93	0.875	1.323	0.869	1.328
2	1 hr	49.12	503.15	98.24	100.63	1.255	1.458	1.246	1.449
3	3 hr	49.57	496.87	99.14	99.37	1.781	0.983	1.763	0.971
4	8 hr	50.48	496.36	100.96	99.27	0.568	1.054	0.576	1.052
5	24 hr	49.91	492.58	99.82	98.52	0.659	1.563	0.652	1.568

*Mean of three determinations

4. CONCLUSION:

Results closed to 100% for repeatability and intra-day and inter-day precision with standard deviation <2% conclude that the developed method is precise. The percentage recovery at three levels (80%, 100% and 120%) with acceptable criteria < 2 %. gives indication of accuracy method. The proposed method was studied to check its robustness property and was found insignificant change in percent estimation and Rf values compare with the standard, concluded that the method is robust and stability indicating.

Validation studies indicate that the proposed method is suitable for simultaneous estimation of SIT and MET in pharmaceutical formulation without any interference from excipients. As the HPTLC method was able to quantitate Sitagliptin phosphate and Metformin hydrochloride, in presence of degradation products, it can be employed as a stability indicating assay method for determination of these drugs in fixed dose combination tablets. As there is no HPTLC method reported till date, the comparable data of the previously published method are not available. Hence, it is concluded that the proposed HPTLC method can be used for routine analysis of combined dose tablet formulation containing Sitagliptin phosphate and Metformin hydrochloride.

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Received on 19.05.2015 Modified on 01.07.2015

Asian J. Research Chem. 8(7): July- 2015 ; Page 472-480

DOI: 10.5958/0974-4150.2015.00076.0

Drug	Concentration in ng/ml	SD	SD²	Σ SD²	Cochran’s C
SIT	8	561.84	315664.18	20604313.43	0.482
	16	1008.92	1017919.57
	56	2087.62	4358157.26
	64	3861.68	14912572.42
MET	80	338.62	114663.50	12271577.21	0.503
	160	992.92	985890.12
	560	1545.52	2388632.07
	640	2963.51	8782391.52

Sr. No	Exposed stress conditions	% drug estimated ± S.D.		R_f value of degradation product	Figure
Sr. No	Exposed stress conditions	SIT	MET	R_f value of degradation product	Figure
1	Acid, 8 ml (1N HCl, reflux for 3 h at 80^OC)	98.26 ± 3.01	96.66 ± 2.88	Not detected	------
2	Base, 8 ml (1N NaOH, reflux for 3 h at 80^OC)	71.54 ± 4.25	96.65 ± 2.98	0.02, 0.03	Fig. 5
3	Oxide,8 ml,30%v/v H₂O₂(reflux for 3 h at 80^OC)	79.38 ± 1.98	98.55 ± 4.51	0.42	Fig. 6
4	Dry heat (24 h at 60°C)	102.52 ± 3.29	103.25 ± 3.11	Not detected	-------
5	Wet heat (reflux for 3 h at 80^OC)	98.16 ± 3.24	96.33 ± 3.24	Not detected	-------
6	Photo stability (Daylight, 8 days)	97.66 ± 3.59	102.35 ± 2.45	Not detected	-------
7	UV (254 nm for 24 h)	98.21 ± 3.33	82.54 ± 3.25	0.42	Fig. 7
8	Neutral hydrolysis (reflux for 8 h)	102.22 ± 3.12	98.26 ± 2.98	Not detected	-------