RP-HPLC method for estimation of miglitol and metformin hydrochloride in pharmaceutical formulation

A simple, rapid and sensitive RP-HPLC method was developed for the quantitative determination of metformin hydrochloride and miglitol in combined tablet dosage form. The chromatographic analysis was carried out on Phenomenex Gemini C18 (250 x 4.6 mm, 5 μm) with mobile phase containing methanol: TEA buffer (50:50 v/v). The flow rate of mobile phase was 1.0 mL/min and effluents were monitored at 272 nm. The retention times of miglitol and metformin hydrochloride were 2.121 min and 3.643 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity and robustness.The method was found to simple, rapid and sensitive and was successfully applied to the estimation of metformin hydrochloride and miglitol in combined dosage form.


Introduction
Metformin HCl [figure1] is an oral antidiabetic drug in the biguanide class. It is most widely prescribed antidiabetic drug in the world used to treat type 2 diabetes. Metformin(MET) helps to control the amount of glucose (sugar) in blood. It decreases the amount of glucose and also increases body's response to insulin, a natural substance that controls the amount of glucose in the blood. It is not used to treat type 1 diabetes. It is also used for treatment of gestational diabetes, polycystic ovary syndrome (PCOS). It works by decreasing hyperglycemia primarily by suppressing glucose production by the liver (hepatic gluconeogenesis). It helps to reduce LDL cholesterol and triglyceride levels, and is not associated with weight gain. Metformin comes as a liquid, as a tablet, and as an extended-release (long-acting) tablet taken orally. It is used alone or with other medications . Very rare but serious side effect with Metformin is lactic acidosis. Other than that common side effect are gastrointestinal irritations, including diarrhea, cramps, nausea, vomiting and increased flatulence. Literature survey revealed The HPLC methods for estimation of metformin in Bulk, human plasma and pharmaceutical dosage forms [1][2][3][4][5]. LC-MS-MS method was reported for the determination of Metformin in human plasma [6]. Literature survey reveals several Analytical and Bioanalytical methods for the analysis of Metformin. These methods reported with Metformin alone or in combination with other drug. These include, HPLC [7][8][9][10][11] and spectrophotometric analysis of Metformin in tablets [12][13].
Miglitol [figure2] belongs to a class of drug called alpha-glucosidase inhibitors used to control blood glucose (sugar) levels in type 2 diabetes (non-insulin-dependent diabetes). It is approved by FDA in December 1996. Miglitol(MIG) inhibits glycoside hydrolase enzymes called alpha-glucosidases thereby slowing the appearance of sugar in the blood after meal. It works by slowing down the absorption of carbohydrates from diet, so that blood sugar does not rise as much after meal. Alpha-glucosidase inhibitors are used to help control blood sugar levels that are not controlled by diet and exercise alone. It is believed that strict control of blood sugar in people with diabetes decreases the risk of eye, kidney and nerve damage. Controlling high blood sugar helps to decreases the risk of eye, kidney, nerve damage, loss of limbs and sexual function problems. It is used alone or in combination with a sulfonylurea such as glyburide (Diabeta).
It is an oral administrative drug available in form of tablet. The most common side effects of miglitol are Gastrointestinal symptoms such as abdominal pain, diarrhea, flatulence and skin rash. Rare but possible side effects include low serum iron. Literature survey revealed that several Analytical and Bioanalytical methods for its estimation using Reversed Phase-High Performance Liquid Chromatography [RP-HPLC] with UV detection, HPLC -electrospray tandem mass spectrometry, LC-MS, liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry [14] and RP-HPLC method [15][16]. The developed method has various advantages over the above mentioned methods, as it is simple, economical, faster, precise, accurate and specific for quantitative determination of Miglitol in pharmaceutical dosage form. As per our detailed literature survey as on date, there are very few reports using UV [17] & RP-HPLC [18] for the simultaneous quantitative estimation of Metformin and Migllitol in Bulk & Pharmaceutical dosage forms. The RP-HPLC [19][20][21][22][23][24] methods have superiority over the UV spectrophotometric methods [25][26][27][28] in terms of selectivity and sensitivity. We here in reported a simple, sensitive, precise, accurate, linear and isocratic RP-HPLC method for the simultaneous quantitative estimation of Metformin and Migllitol in bulk & Formulation as per ICH Guilences.

Instrumentation
The technique was developed using an Acquity HPLC system (Waters, Milford, MA, USA) with a model 2996 PDA detector. A Rheodyne micro-litre syringe fitted with a 20 µl loop was used for injection of sample into the column and data were recorded evaluated by use of empower software.

Materials
Metformin and Migllitol pure samples were procured as gift samples. MIGNAR MF tablets were procured from the local market. Label claim of MIGNAR MF tablets for MET and MIG were 500 mg and 50 mg respectively. Methanol, acetonitrile and glacial acetic acid and water of HPLC grade were purchased from E. Merck and used throughout the experiment.

Mobile phase preparation
Triethyl amine buffer and methanol in the ratio of 35:65 v/v was used as mobile phase. The mobile phase was sonicated for 15 min in an ultrasonic bath and filtered through nylon membrane disc filter of 0.45 µm pore size using a vacuum pump before pumping into the HPLC system. .

Preparation of stock standard solution
Metformin hydrochloride (500 mg) and miglitol (50 mg) were accurately weighed and transferred into 100 ml volumetric flask separately. They were dissolved in 100 ml methanol to obtain 5000 µg/ml and 500 µg/ml concentration of stock solutions respectively. From these stock solution 1 ml each of MET and MIG were taken into 10 ml volumetric flasks separately and further diluted with a mobile phase to get 500 µg/ml and 50 µg/ml concentrations of MET and MIG respectively. The solutions were then filtered through 0.45 µm Nylon filter.

Preparation of sample solution
For analysis of drugs, 20 tablets were weighed and triturated in glass mortar and quantity of powder equivalent to 500 mg of metformin hydrochloride was transferred to 100 ml volumetric flask and dissolved in sufficient quantity of methanol. It was sonicated for 10 min and volume was made up to 100 ml to obtain a stock solution of 5000 μg/ml of metformin hydrochloride and 500 μg/ml of miglitol. This solution was then filtered through nylon 0.45 µm membrane filter. The solution was further diluted with mobile phase to get a concentration of 100 μg/ml and 10 μg/ml of metformin hydrochloride and miglitol respectively. This solution was injected 6 times in to the column and chromatograms were recorded and respective peak areas were measured. The contents of MET and MIG were calculated by using the regression.

Chromatographic conditions
 Column: Phenomimex Gemini C18 (250 × 4.6 mm, 5 µm particle size)  Flow rate: 1 ml/min. Several HPLC methods were reported for the estimation of metformin hydrochloride and miglitol using methanol, water, acetonitrile and phosphate buffer as mobile phases.With a intention to develop a HPLC method with less retention time for MET and MIG we tried with methanol and TEA buffer using Phenomimex Gemini C18 (250 × 4.6 mm i.d., 5µ ) column. Different trails were performed using different proportions of methanol and TEA buffer. The mobile phase containing methanol and TEA buffer in the composition of 65:35 v/v was found to be satisfactory and gave symmetric and well resolved peak for MET and MIG. The retention time of miglitol and metformin hydrochloride was found to be 2.152 min and 3.646 min. The standard chromatogram was shown in Figure 3.

Method validation
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines [29].

System suitability
System suitability is used to verify, whether the resolution and reproducibility of the chromatographic system are adequate for analysis to be done. % RSD on five replicate injections of standards solution was calculated. The results of system suitability for MET and MIG are shown in Table 1.

Figure 4 Calibration curve for metformin hydrochloride
Calibration graphs were constructed by plotting peak area vs concentration for metformin hydrochloride and miglitol. The calibration graphs were plotted concentrations in the range of 50-250 µg/ml and 5-25 µg/ml for metformin and miglitol respectively. The regression line obtained was linear. From the data obtained, co-relation coefficient, slope and y-intercept were calculated. The linearity of the method was excellent as evidenced by the correlation coefficient of 0.999 for both drugs. The regression equation and other parameters obtained from the plot are mentioned in Table 1.

Precision
Precision was evaluated by injecting six replicate injections of metformin hydrochloride and miglitol of sample solution under the same chromatographic conditions and calculated by the % RSD. The intraday and interday precision study were conducted for both metformin HCl and miglitol. The % RSD indicates that the developed method is repeatable. The % RSD for assay of metformin hydrochloride and miglitol was found to be 1.72 and 0.36. The results are shown in Table  3. Both inter-day and intra-day R.S.D. were less than 2 %, indicating a sufficient precision of the developed method.

Accuracy
In order to judge the quality and applicability of method the recovery analysis was performed at three levels 50 %, 100 %, and 150 % by standard addition method. The % recoveries for Metformin Hydrochloride and Miglitol were calculated and it was found to be within the limits; the results are given in Table 4.

Specificity
Since bulk and tablet formulations are made of different components and excipients, the specificity was carried out through the comparison of the peak retention time of the formulations with paracetamol and tramadol hydrochloride standard drug sample and blank solution. No interference of the excipients was detected since no peak was detected in the same retention time of miglitol and metformin hydrochloride.

Robustness
The robustness as a measure of method capability to remain unaffected by small, but deliberate changes in chromatographic conditions was studied by testing influence of small changes in mobile phase composition (10 % absolute change in organic phase) and flow rate (±0.2 mL/min) and wavelength (± 2 nm). The theoretical plate count and tailing were within the limits. So, the method was found to be robust with respect to variability in all robust conditions.The results are shown in Table 5 and Table 6.

LOD and LOQ
The LOD and LOQ of tramadol hydrochloride and paracetamol were determined by using the signal to noise approach as defined in ICH guidelines. The results are given in Table 7.

Ruggedness
Ruggedness of the developed method was determined by analyzing six sample solutions of by two analysts in the same laboratory to check the reproducibility of the test result. The % recovery and standard deviation were calculated in both cases.The result was shown in Table 8.

Assay of pharmaceutical formulation
The proposed validated method was successfully applied to determine metformin hydrochloride and miglitol in its tablet dosage form. The result obtained for metformin hydrochloride and miglitol was comparable with the corresponding labelled amounts and they are given in Table 9.

Conclusion
The present work refers to the fact that the most accurate, precise, and robust HPLC method was developed and validated for estimation of metformin hydrochloride and miglitol in pharmaceutical dosage form in accordance with the ICH parameters. The method was validated and found to be simple, rapid, accurate, and precise. Percentage of recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of metformin hydrochloride and miglitol in its dosage form.