INTRODUCTION:

Metformin [1] is a biguanide antihyperglycemic agent used for treating non-insulin- dependent diabetes mellitus (NIDDM). It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated glucose uptake. Metformin is the only oral antihyperglycemic agent that is not associated with weight gain.

Gliclazide ^[1]is an oral antihyperglycemic agent used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). It belongs to the sulfonylurea class of insulin secretagogues, which act by stimulating β cells of the pancreas to release insulin. Sulfonylurea’s increase both basal insulin secretion and meal-stimulated insulin release. Medications in this class differ in their dose, rate of absorption, duration of action, route of elimination and binding site on their target pancreatic β cell receptor. Sulfonylurea’s also increase peripheral glucose utilization, decrease hepatic gluconeogenesis and may increase the number and sensitivity of insulin receptors [2-3]. Sulfonylurea’s are associated with weight gain, though less so than insulin. Due to their mechanism of action, sulfonylureas may cause hypoglycemia and require consistent food intake to decrease this risk. The risk of hypoglycemia is increased in elderly, debilitated and malnourished individuals. Gliclazide has been shown to decrease fasting plasma glucose, postprandial blood glucose and glycosolated hemoglobin (HbA1c) levels (reflective of the last 8-10 weeks of glucose control). Gliclazide is extensively metabolized by the liver; its metabolites are excreted in both urine (60-70%) and feces (10-20%). Several analytical methods based on UV [4-6], Spectroflourimetry [6], RP-HPLC [7-8], LC-MS/MS [9-11] was reported for the determination of Sitagliptin phosphate monohydrate in plasma and urine of humans, rats and dogs. Metformin hydrochloride (MTF) (C₄H₁₁N₅.HCl) is 1: 1 dimethylbiguanidine monohydrochloride is an anti-diabetic drug from the biguanide class of oral Hypoglycaemic agents, given orally in the treatment of non –insulin-dependent diabetes mellitus[12] .Major action of Metformin HCl in increasing glucose transport across the cell membrane in skeletal muscle[13-14]. Several analytical methods based on UV [15-18], Spectroflourimetry [15], Reverse Phase-HPLC[19-27], HPTLC [28] and LC-MS/MS [29-30] was reported for the determination of Metformin. Although literature survey reveals that various methods were reported for Metformin (MTF) and Gliclazide (GZ) both for single estimation and in combination with others drugs, but no method was reported for the analysis of these drugs in combination. The chemical structure of the drugs was represented in Fig. no. 1 and 2.

Fig. No. 1 Chemical Structure of Metformin

Fig. No. 2 Chemical Structure of Gliclazide

MATERIALS AND METHOD[31-32]

Chemicals and Reagents Used:

The following chemicals were procured for the process: Water [HPLC Grade], Metformin and Gliclazide [Working Standard], Methanol [HPLC Grade], Ortho phosphoric acid [HPLC Grade] all the chemicals are procured from STANDARD SOLUTIONS and RECLEMET brand name of the tablet [Metformin-500mg+Gliclazide-80mg] was collected from local market and the manufacturing company was Dr. Reddy’s ^®.

Apparatus and Chromatographic Conditions:

Equipment : High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

Column : XTERRA C₁₈(4.8 X 150mm, 5 μm)

Flow rate : 0.8 mL per min

Wavelength : 223 nm

Injection volume : 20 ml

Column oven : Ambient

Run time : 6min

Retention time : Metformin-1.741

Gliclazide-3.274

Detector : Photo diode array

Soft ware : Empower 2

Preparation of buffer [33]:

The buffer was prepared by weighing accurately and transferred 0.1gm of OPA into a 100ml beaker and the volume was made up to the mark with water [HPLC grade].

Preparation of mobile phase:

The mobile phase was prepared by making a mixture of 350 mL (35%) buffer and 650 mL Acetonitrile [HPLC grade] (65%) and degassed in ultrasonic water bath for 5 minutes. Then the resultant solution was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation:

The mobile phase was used as diluent.

Preparation of the Metformin and Gliclazide Standard and Sample Solution:

Standard Solution Preparation:

The Standard solution was prepared by weighing accurately and transferred 10 mg of Metformin and Gliclazide [working standard] into a 100mL clean dry volumetric flask. About 70 ml of the diluent was added and sonicated to dissolve it completely and further the volume was made up to the mark with the same solvent. From the above prepared Stock Solution pipette out 4.0 ml and 1.0ml of Metformin and Gliclazide into a 10ml volumetric flask and the volume was made upto the mark with diluent

Sample Solution Preparation:

The Sample solution was prepared by weighing accurately and transferred 10 mg of Metformin and Gliclazide into a 100ml clean dry volumetric flask. About 70mL of diluent was added and sonicated to dissolve it completely and further the volume was made up to the mark with the same solvent. From the above prepared stock solution pipette out 4.0 ml and 1.0 ml of Metformin and Gliclazide into a 10ml volumetric flask and the volume was made up to the mark with diluent .

The standard solution and sample solution [20µl] was injected into the chromatographic system. The area was measured for the individual drug and the % Assay was calculated by using the suitable formulae.

System Suitability results: The Tailing factor for the peaks due to Metformin and Gliclazide in Standard solution should not be more than 1.5. The Theoretical plates for the Metformin and Gliclazide peaks in Standard solution should not be less than 2000.

Formula for calculation of Assay:

Assay % =

Where:

AT = Average area counts of sample preparations

AS = Average area counts of standard preparation.

WS = Weight of working standard taken in mg.

P = Percentage purity of working standard

LC = Label Claim of Metformin mg/ml,

Label Claim of Gliclazide mg/ml.

System Suitability Results for Metformin:

1) The Tailing factor obtained from the standard injection was 1.86

2) The Theoretical Plates Obtained from the standard injection was 2069

Assay Result for Metformin:

System Suitability Results for Gliclazide:

1) The Tailing factor obtained from the standard injection was 1.18

2) The Theoretical Plates obtained from the standard injection was 2342

Assay Result for Gliclazide:

VALIDATION METHOD [34-38]

1. Precision: The precision of an analytical procedure express the closeness of agreement between a series of measurements from multiple sampling of the same homogenous sample under prescribed conditions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 1 and 2)

Table no.1The Precision results for Metformin.

Injection	Area
Injection-1	4542582
Injection-2	4539827
Injection-3	4554820
Injection-4	4569427
Injection-5	4555492
Average	4552430
Standard Deviation	11830.0
%RSD	0.26

Table no.2The Precision results for Gliclazide.

Injection	Area
Injection-1	242568
Injection-2	240889
Injection-3	243268
Injection-4	246276
Injection-5	242438
Average	243088
Standard Deviation	1982.9
%RSD	0.82

Acceptance Criteria: The % RSD for the area of five standard injections results should not be more than 2%.

2. Intermediate Precision/Ruggedness: To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 3 and 4)

Table no.3The Intermediate Precision/Ruggedness results for Metformin

Injection	Area
Injection-1	4573255
Injection-2	4566860
Injection-3	4585475
Injection-4	4596757
Injection-5	4589101
Average	4582289
Standard Deviation	12099.1
%RSD	0.26

Table no.4The Intermediate Precision/Ruggedness results for Gliclazide

Injection	Area
Injection-1	243638
Injection-2	243929
Injection-3	245239
Injection-4	243873
Injection-5	245102
Average	244356
Standard Deviation	753.2
%RSD	0.31

Acceptance Criteria: The % RSD for the area of Five standard injections results should not be more than 2%.

3. Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. Standard solutions with Accuracy -50%, 100% and 150% were injected into chromatographic column and calculated the Amount found and Amount added for Metformin and Gliclazide. Same time the Individual recovery and Mean recovery values were also calculated (Table no. 5 and 6).

4. Linearity: The linearity of the analytical procedure is its ability (within a given range) to obtain the test results which are directly proportional to the concentration (amount) of analyte in the sample. Different levels were prepared and injected into the chromatographic system and measured the peak areas. A graph was plotted between peak area versus concentration and correlation coefficient value was calculated (Table no 7 and 8).

Table no.7The Linearity results for Metformin

S.No	Linearity Level	Concentration	Area
11	I	20ppm	2425172
2	II	30 ppm	3550954
3	III	40 ppm	4610109
4	IV	50 ppm	5790066
5	V	60 ppm	6894512
Correlation Coefficient			0.999

Table no.8The Linearity results for Gliclazide

S.No	Linearity Level	Concentration	Area
1	I	3.2 ppm	106223
2	II	4.8 ppm	146862
3	III	6.4 ppm	197967
4	IV	8ppm	243698
5	V	9.6 ppm	293773
Correlation Coefficient			0.999

Acceptance Criteria: The Correlation coefficient should be not less than 0.999.

5. Limit of detection: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non instrumental or instrumental.

Limit of Detection for Metformin:

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 42 µV

Signal Obtained from LOD solution (3.9% of target assay concentration): 125 µV

S/N = 125/42 = 2.97

a. Limit of Detection for Gliclazide:

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 42 µV

Signal Obtained from LOD solution (0.4% of target assay concentration): 126 µV

S/N = 126/42 = 3.0

Acceptance Criteria: The S/N Ratio value shall be 3 for LOD solution.

6. Limit of quantification: The Quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The Quantification limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/ or degradation products. Several approaches for determining the Quantification limit are possible, depending on whether the procedure is a non- instrumental or instrumental.

Limit of quantification for Metformin:

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 42 µV

Signal Obtained from LOQ solution (6.5% of target assay concentration): 418µV

S/N = 418/42 = 9.95

Limit of quantification for Gliclazide:

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 42 µV

Signal Obtained from LOQ solution (1.6% of target assay concentration): 423µV

S/N = 423/42 = 10.07

Acceptance Criteria: The S/N Ratio value shall be 10 for LOQ solution.

Robustness: As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

a) The flow rate was varied at 1.1 ml/min to 1.3ml/min.: The Standard solution was prepared and analysed using the varied flow rates along with developed flow rate. On evaluation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it indicates that the method is robust even by change in the flow rate ±10%.

b) The Organic composition in the Mobile phase was varied from 75% to 85%: The standard solution of was prepared and analysed using the varied Mobile phase composition (in the organic phase composition) along with the actual mobile phase composition in the method. On evaluation of the above results, it was concluded that the variation in 10% Organic composition in the mobile phase did not affected the method significantly. Hence it indicated that the method was robust even by change in the Mobile phase ±10. (Table No.9 and 10)

Table no. 9The System suitability results for Metformin

S.No	Flow Rate (ml/min)	System Suitability Results
S.No	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.7	2126	1.78
2	0.8	2069	1.86
3	0.9	2043	1.91

Table no. 10The System suitability results for Gliclazide

S.No	Flow Rate (ml/min)	System Suitability Results
S.No	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.7	2194	1.38
2	0.8	2342	1.18
3	0.9	2142	1.71

RESULTS AND DISCUSSION:

The present study was carried out to develop a sensitive, precise and accurate HPLC method for the analysis of in pharmaceutical dosage forms. In order to method development under isocratic conditions, mixtures of 0.1% Orthophosporic Acid and Acetonitrile in the ratio of 35:65 v/v was proved to be the most suitable of all combinations since the chromatographic peaks were better defined and resolved and almost free from tailing. Flow rate was selected 0.8 ml/min in Isocratic Mode. The run time was maintained 6mins. The detection was monitored at 230 nm. The retention time was 1.7min for [MFH] and 3.2min for [GZ]. A model chromatogram was represented in fig .no.3.

Fig. No.3 The typical chromatogram for the Optimized method

Fig. No.4 The typical chromatogram for Blank

Fig. No. 5 The typical chromatogram for Standard

Fig. No. 6The typical chromatogram for Sample

For Precision Studies the standard solution was injected for five times and measured the area for all the five injections in HPLC system. The % RSD for the area of five standard injections results should not be more than 2%.The %RSD for the area of the above five standard injections of both the drugs was found to be within the limits. So, that the method was said to be precise. The data was represented in table no. 1 and 2. A model chromatogram is represented in Fig. no. 7

Fig. No. 7The typical chromatogram for Precision

For Intermediate Precision/Ruggedness studies the standard solution was injected for five times and measured the area for all the five injections in HPLC system. The %RSD for all the area of five replicate injections was found to be within the specified limits. The data was represented in Table 3 and 4. A model chromatogram was represented in Fig.No.8.

Fig. No.8 The typical chromatogram for intermediate precision/ruggedness

The % RSD for the area of five standard injections results should not be more than 2%.The %RSD for the area of the above five standard injections of both the drugs was found to be within the limits. So, that the method was said to be precise. For Accuracy, the standard solution of different concentrations was injected, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions. The amount found was calculated and the amount added for Metformin and Gliclazide was calculated. The individual recovery and mean recovery values were also calculated. The data were represented in Table 5, and 6. A model chromatogram was represented in Fig.No.9, 10 and 11.

Fig. No.9The typical chromatogram for Accuracy (50% concentration)

Fig. No.10The typical chromatogram for Accuracy (100% concentration)

Fig. No. 11 The typical chromatogram for Accuracy (150% concentration)

The % Recovery for each level was found to be in between 98.0 to 102.0%. The % recovery for each level was found to be within the limits. So, that the method was said to be accurate.

In order to test the linearity of the method, the solutions of each level were injected into the chromatographic system and measured the peak area. A graph was plotted, Peak area versus Concentration (on X-axis concentration and on Y-axis Peak area) and the correlation coefficient was calculated. The data was represented in table 7 and 8. The Correlation coefficient was found 0.999.The correlation coefficient was found to be within limits. So, that the method was said to be linear. The data were represented in Table no. 7 and 8. The Linearity Curve was represented in Fig. No.12 and 13

Fig.No.12The Linearity Curve for Metformin

Fig. No.13 The Linearity Curve for Gliclazide

Limit of detection and limit of quantification of the method were calculated basing on standard deviation of the response and the slope (s) of the calibration curve at approximate levels of the limit of detection and limit of quantification. The limit of detection and limit of quantification for [MFH] and [GZ] were found to be 0.018 µg/ml, 0.03 µg /ml and 0.005 µg /ml, 0.11µg /ml respectively. A model chromatogram was represented in Fig. no. 14 and 15.

Fig. No. 14The typical chromatogram for Limit of Detection

Fig. No. 15The typical chromatogram for Limit of quantification

Robustness of the method was found out by testing the effect of small deliberate changes in the chromatographic conditions in the chromatographic conditions and the corresponding peak areas. The factors selected for this purpose were flow rate and percentage composition variation in organic phase of the mobile phase (Acetonitrile). The method was found to be robust enough that the peak area was not apparently affected by small variation in the chromatographic conditions. The data were represented in Table no. 8 and 9. A model chromatogram was represented in Fig. No.16 and 17.

Fig. No. 16The typical chromatogram for robustness with change in flow rate

Fig. No. 17The typical chromatogram for robustness with change in the composition of Mobile Phase (Organic phase)

CONCLUSION:

Development of new analytical methods for the determination of drugs in pharmaceutical dosage forms is more important in pharmacokinetics, toxicology and biological studies. Pharmaceutical analysis occupies a pivotal role in industry, the quality assurance and quality control department play major role in bringing out a safe and effective drugs or dosage form. The current good manufacturing practices (CGMP) and the food drug administration (FDA) guidelines insist for adoption of sound method of analysis with great sensitivity and reproducibility. Therefore, the complexity of problems encountered in pharmaceutical analysis with the importance of achieving the selectivity , speed , low cost, simplicity, sensitivity, specificity, precision and accuracy in estimation of drugs. It was concluded that the proposed RP-HPLC method developed for the quantitative determination of Metformin and Gliclazide in bulk and in its formulations was simple, selective, sensitive, accurate, precise and rapid. The method was proved to be superior to most of the reported methods. The mobile phases were simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence this method can easily be adopted as an alternative method to reported ones for the routine determination of Metformin and Gliclazide depending upon the availability of chemicals and nature of other ingredients present in the sample. The method will also find use in clinical, biological and pharmacokinetic studies of Metformin and Gliclazide at future.

FUTURE ASPECTS:

The proposed method can be used as alternative methods to the reported ones for the routine determination of selected drugs under the study in pharmaceutical dosage forms. Thus the purpose of the present investigation was successfully achieved.

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Research J. Pharm. and Tech. 7(2): Feb. 2014; Page142-150

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	4590938	5.0	5.09	101.72%	100.70%
100%	9006381	10.0	9.98	99.78%
150%	13620813	15.0	15.09	100.6%

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	198827	5.0	5.03	100.5%	100.4%
100%	392707	10.0	9.93	99.2%
150%	601972	15.0	15.2	101.4%