Development and Validation of RP- HPLC Method for Simultaneous Estimation of Metformin and Miglitol in Bulk and Dosage Form

 

Sufiyan Ahmad¹*, Ansari Sajjad¹, Md. Rageeb Md. Usman², Mohammed Imran³,

Rashid Akhtar³

¹Department of Quality Assurance, Gangamai College of Pharmacy, Nagaon, Dist. Dhule (M.S.), India

²Department of Pharmacognosy, Smt. S. S. Patil College of Pharmacy, Chopda, Maharashtra, India

 

ABSTRACT:

Attempts were made to develop RP-HPLC method for simultaneous estimation of Metformin and Miglitol from tablet. For the RP ‑ HPLC method, Younglin (S.K.) Gradient system UV detector and C₁₈ column with 150mm x 4.6 mm i. d. and 5μm particle size Acetonitrile: ph. Buffer (40: 60v/v) pH 3.2was used as the mobile phase for the method. The detection wavelength was 235 nm and flow rate was 0.7 ml/min. In the developed method, the retention time of Metformin and Miglitol sodium were found to be 3.4667 min and 7.4833 min. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness was within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible.

A new, simple, accurate, precise, linear and rapid RP-HPLC method was developed and validated for the simultaneous estimation of Metformin and Miglitol in bulk drugs and formulations a s per ICH guidelines. Hence the method can be used for the routine and stability analysis in various pharmaceutical industries in bulk drugs and formulations.

 

 

 

 

INTRODUCTION:

Metformin (MET) [Figure 1] is an oral anti- diabetic drug in the biguanide class. It is most widely prescribed anti-diabetic drug in the world used to treat type 2 diabetes. Metformin 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 I diabetes.

 

It is also used for treatment of gestational diabetes, polycystic ovary syndrome (PCOS) [1]. 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. MET 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 [2–7]. LC-MS-MS method was reported for the determination of MET in human plasma [8]. Literature survey reveals several analytical and bio-analytical methods for the analysis of MET. These methods reported with Metformin alone or in combination with another drug. These include, HPLC [9-11] and spectrophotometric analysis of MET in tablets [12 -13].

 

Miglitol (MIG) [Figure 2] 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 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 de creases 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. Recent study on rats by Shrivastva et al showed that Miglitol has antioxidant effect and hypocholesterolemic effect [14]. 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 MIG 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 bio-analytical methods for its estimation using RP-HPLC with UV detection, HPLC- electrospray tandem mass spectrometry, LC-MS, liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry and RP -HPLC method [15-19]. 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 [20] using UV & RP-HPLC for the simultaneous quantitative estimation of MET and MIG in Bulk & Pharmaceutical dosage forms. We here in reported a new, simple, sensitive, precise, accurate, linear and isocratic RP -HPLC method for the simultaneous quantitative estimation of MET and MIG in bulk & Formulation as per ICH Guideline [21].

 

Figure 1: Structures of Metformin

 

Figure 2: Structures of Miglitol

 

MATERIALS AND METHODS:

Instruments:

The analysis of the drug was carried out on Youngline (S.K.) Gradient System UV Detector. Equipped with Reverse Phase (Premesil) C18 column (4.6mm x 150mm; 5µm), a SP930D pump, a 20µl injection loop and UV730D Absorbance detector and running autochro-3000 software.

 

Reagents and Materials:

MET and MIG in the form of gift samples were kindly supplied by R. S. I. T. C, Jalgaon respectively. HPLC grade potassium phosphate buffer from Avantor Performance material India Ltd. Thane, Maharashtra. Acetonitrile and water from Merck specialities Pvt. Ltd. Shiv Sager Estate ‘A’ Worli, Mumbai. A combination of Miglitol 50 mg and Metformin 500 mg in tablet formulation was procured from local pharmacy (Mignar 50-MF Glenmark Ltd).

 

Preparation of standard stock solution:

Preparation of std. Metformin solution: (Stock I):

From the freshly prepared standard stock solution (1000 μg/ml), 0.1ml stock solution was pipette out in 10 ml of volumetric flask and volume was made up to 10 ml with mobile phase to get final concentration of 10μg/ml [Figure 3].

 

Preparation of std. Miglitol solution: (Stock II):

From the freshly prepared standard stock solution (1000μg/ml), 0.1 ml stock solution was pipette out in 10 ml of volumetric flask and volume was made up to 10 ml with mobile phase to get final concentration 10μg/ml [Figure 4].

 

Preparation of std. Metformin and Miglitol solution: (Stock III):

From the freshly prepared standard stock solution (1000 μg/ml), 0.1 ml stock solution was pipette out in 10 ml of volumetric flask and volume was made up to 10 ml with mobile phase to get final concentration 10 μg/ml. In the standard mixture of MET and MIG theoretical plates were found above 2000 i.e. for MET and MIG 3671.2 and 10626.8 at minimum RT 3.4667 and 7.4833 respectively [Figure 5 and Table 1].

 

Figure 3: Chromatogram of standard Metformin

 

Figure 4: Chromatogram of standard Miglitol

 

Figure 5: Chromatogram of standard combination of MET and MIG

 

Table 1: Details of chromatogram of standard combination containing MET and MIG

Sr. No.	RT [min]	Area [mV*s]	Area%	TP	TF	Resolution
1	3.4667	2202.4653	95.31	3671.2	1.3002	0.0000
2	7.4833	163.3959	4.69	10626.8	1.3675	15.4716
Sum		2365.8613

 

Method development and validation:

Working standard of various concentrations was prepared by taking aliquots of standard

solution and diluted to get required concentration for calibration plot and which was injected.

 

Analysis of tablet formulation:

Weigh 20 MET and MIG combination tablets and calculated the average weight accurately weigh and transfer the sample equivalent to 12.47 mg MET and MIG into 10 ml volumetric flask. Add about 10ml ACN of diluents and sonicate to dissolve it completely and make volume up to the mark with diluents. Mix well and filter through 0.45 µm filter. Further pipette 0.4ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents. (40 µg/ml). The simple chromatogram of test VAL and HCTZ shown in [Figure 6]. The amounts of MET and MIG per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation Analysis of marketed formulation were also % Label Claim was found to be 99-101% Satisfactory are concluded [Table 2].

 

Figure 6: Chromatogram for marketed formulation

 

Table 2: Analysis of marketed formulation

 

 

RESULTS AND DISCUSSION:

Linearity and Range:

The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 10-50 µg/ml for MET and 1-5 µg/ml for MIG [Table 3 and 4] depict the calibration data of MET and MIG. The respective linear equation for Metformin was y = 103.0x + 123.0 and Miglitol equation y = 85.97 x-3.638. Where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of MET and MIG is depicted in [Figure 7 and 8].

 

Table 3: Linearity data for Metformin

           

Figure 7: Calibration curve of Metformin                                                         Figure 8: Calibration curve of Miglitol

 

Table 4: Linearity data for Miglitol

 

Accuracy:

It is defined as closeness of agreement between the actual (true) value and analytical value and obtained by applying test method for a number of times. Accuracy of the methods was determined at three different concentration levels i.e. 80%, 100% and 120% [Figure 9- 11] in triplicate for each drug as per ICH guidelines. From the total amount of drug found, the percentage recovery was fond in range of 99-101% [Table 5 and 6].

 

Figure 9: Chromatogram of accuracy 80%

 

Figure 10: Chromatogram of accuracy 100%

 

Figure 11: Chromatogram of accuracy 120%

 

Table 5: Result of recovery data for Metformin and Miglitol

*mean of each 3 reading for RP-HPLC method

 

Table 6: Statistical Validation of recovery studies MET and MIG

*Denotes average of three determinations for RP-HPLC method

 

Figure 12: Chromatogram of precision

 

Precision:

Precision was studied to find out intra and inter-day variations in the test method of MET and MIG. Intra‐day precision was determined by analyzing three concentrations in three replicate measurements of within linearity range of drugs on three different times in the same day. Inter‐day precision was conducted during routine operation of the system over a period of 3 consecutive days. Intraday and Inter Day Precision studies on HPLC method for MET and MIG which shows the high precision % amount in between 98% to 100% indicates to analytical method that concluded [Table 7 and Figure 12, 13 and 14].

 

 

Figure 13: Chromatogram Intra-day precision

 

Figure 14: Chromatogram Inter-day precision

 

Table 7: Result of Intra-day and Inter day precision studies on RP-HPLC method for MET and MIG

*Mean of each 3 reading for RP-HPLC method

 

Robustness:

The robustness is measure of its capacity to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability during normal usage hence the following are performed by slight variations in parameters. The changes were did

 

flow rate (±1 ml/ min^-1), PH of mobile phase composition (±1 ml/ min^-1), and Wavelength (±1 ml/ min^-1). %RSD for peak area was calculated which should be less than 2%.the result shown in analytical method that concluded [Table 8 and 9].

 

Table 8: Result of Robustness study of Metformin

 

 

System suitability parameters: (Repeatability):

Repeatability studies on RP-HPLC method for MET and MIG was found to be, The %RSD was less than 2%, which shows high percentage amount found in between 98% to 102% indicates the analytical method that concluded [Table 10].

 

Table 9: Result of Robustness study of Miglitol

Parameters	Conc.(µg/ml)	Amount of detected (mean ±SD)	% RSD
Chromatogram of flow change 0.6ml	50	429.97±3.80	0.88
Chromatogram of flow change 0.8 ml	50	482.52±3.97	0.82
Chromatogram of comp change 41mlACN+59ml Ph. Buffer	50	468.01±10.78	2.30
Chromatogram of comp change 39mlACN+61ml Ph. Buffer	50	449.86±4.00	0.89
Chromatogram of comp change wavelength change 234nm	50	476.34±5.73	1.20
Chromatogram of comp change wavelength change 236 nm	50	486.06±6.17	1.27

 

Table 10: Repeatability studies on RP-HPLC for MET and MIG

 

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD is the lowest amount of analyte in a sample that can be detected but not necessarily quantify under the stated experimental conditions. LOQ is the lowest concentration of analyte in a sample that can be determined with the acceptable precision and accuracy under stated experimental conditions.

 

CONCLUSION:

Simple, rapid, accurate and precise RP-HPLC as well as spectrophotometric methods have been developed and validated for the routine analysis of MET and MIG in API and tablet dosage forms. Both methods are suitable for the simultaneous determination of MET and MIG in multi-component formulations without interference of each other. The developed methods are recommended for routine and quality control analysis of the investigated drugs in two component pharmaceutical preparations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also, the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms.

 

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Received on 30.05.2017          Accepted on 20.06.2017        

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