Method Development and its Validation for Simultaneous Estimation of Ecombination of Metformin Hydrochloride and Methylcobalamin as API and in Combination of Tablet Dosage Form by UV-Visible Spectrophotometry

Metformin Hydrochloride[MET] is chemically N,N-Dimethylimidodicarbonimidic diamide [Figure 1]. It is  hypoglycemic agent. It is used in the treatment of type 2 diabetes[1],[2],[3],[4] and is official in Indian           Pharmacopoeia 2010.

Figure1. Metformin Hydrochoride

Methylcobalamin[MEC]        is         chemically carbanide;cobalt(3+);[(2~{R},3~{S},4~{R},5~{S})5(5,6dimethylbenzimidazol1yl)4hydroxy2(hydroxymethyl)oxolan3yl]1[3[(1~{R},2~{R},3~{R},5~{Z},7~{S},10~{Z},12~{S},13~{S},15~{Z},17~{S},18~{S},19~{R})-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl) 3,5,8,8,13,15,18,19octamethyl2,7,12,17tetrahydro1~{H}corrin24id3yl]propanoylamino]propan-2-yl phosphate [Figure 2]. It is used in the treatment of peripheral neuropathy, megaloblastic anemia [6],[7] and is official in British Pharmacopoeia 2012, United States Pharmacopoeia 2011 and Indian Pharmacopoeia 2010.

Figure 2. Methylcobalamin

Literature survey reveals spectrophotmetric [10],[11],[12],[13], HPLC, HPTLC and other methods for the estimation of Metformin Hydrochloride and Methylcobalamin individually and in combination with other drugs. However, no method has been reported so far in any literature for the simultaneous estimation of these two drugs in combined dosage form. Therefore aim of the research was to develop and validate simple, accurate, precise and specific UV spectrophotometric method for simultaneous estimation of Metformin Hydrochloride and Methylcobalamin in pharmaceutical tablet dosage form.

MATERIALS AND METHODS    

Apparatus:

A double beam UV-VIS Spectrophotometer (UV 1800, Shimadzu, Japan) Spectral bandwidth of 1 nm and wavelength accuracy of ± 0.5 nm with a pair of 1 cm matched quartz cells was used to measure the absorbance of all the solutions.  Spectra were automatically obtained by UV- Probe system software (UV Probe version 2.31). All weights were taken on Digital electronic balance Sartorius, CP225D.

Reagents and chemicals:

All the chemicals used were of analytical grade. Methanol A.R. grade was procured from Loba Chem. Ltd., Mumbai. Metformin Hydrochloride was kindly supplied by Cadila Healthcare Ltd., Ahmedabad and Methylcobalamin was kindly supplied by Torrent Pharmaceuticals Ltd., Mehsana (Gujarat) as a gift sample.

Marketed formulation:

The commercial fixed dose combination tablet Sugament MC (Metformin Hydrochloride 250 mg and Methylcobalamin 750 mcg) was procured from local market.

Preparation of standard stock solutions:

An accurately weighed quantity of Metformin Hydrochloride (10 mg) and Methylcobalamin (10 mg) were transferred to a separate 100 ml volumetric flask, dissolved and sonicated in methanol and final volume was adjusted to the mark with methanol to get a standard solution containing 100 μg/ml of each drug.

Determination of wavelength of maximum absorbance (λ_max) for Metformin Hydrochloride:

Standard solution of Metformin Hydrochloride (1ml) was transferred to a 10 ml volumetric flask. The volume was adjusted to 10 ml with methanol as solvent. The absorbance of the final solution (10 mg/ml) was scanned in the range 800-200 nm against methanol as blank and λ_max was found to be 232.0 nm.

Figure 3:  UV scan for determination of ?max. of Metformin Hydrochloride

Determination of wavelength of maximum absorbance (λ_max­) for Methylcobalamin:

Standard solution of Methylcobalamin (1 ml) was transferred to 10 ml volumetric flask. The volume was adjusted to 10 ml with methanol as solvent. The absorbance of the final solution (5 mg/ml) was scanned in the range 800 to 200 nm against methanol as blank and λ_max was found to be 353.0 nm. [Figure 4].

Preparation of calibration curve of Metformin Hydrochloride and Methylcobalamin:

Standard solutions of Nebivolol Hydrochloride in the concentration range of 6 mg/ml to 20 mg/ml were prepared by transferring 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 ml of Metformin Hydrochloride stock solution (100 ppm) to a series of eight volumetric flasks of 10 ml and standard solutions of Methylcobalamin in the concentration range of 2 mg/ml to 7 mg/ml were prepared by transferring 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 ml of Methylcobalamin stock solution (100 ppm) to a series of six volumetric flasks of 10 ml. The volume in each volumetric flask was made up with methanol as solvent. The absorbance of the solutions were measured at both the wavelength 281.5 nm and 240.0 nm against the methanol as blank and calibration curves were plotted[Figure 5] [Figure 6]

Figure 5 :  Calibration curve for Metformin HCl at 232.0 nm

Figure 6 :  Calibration curve for Metformin HCl at 353.0 nm

Figure7 :  Calibration curve for Methylcobalamin at 232.0 nm

Figure 8 :  Calibration curve for Methylcobalamin at 353.0 nm

Preparation of synthetic API mixture of Metformin Hydrochloride and Methylcobalamin:

The synthetic API mixture of Metformin Hydrochloride and Methylcobalamin was prepared in ratio of 10:1. Accurately weighed 100 mg of Metformin Hydrochloride and 30 mg of Methylcobalamin were transferred to 100 ml volumetric flask, 100 ml of methanol as solvent was added to it upto quantity sufficient. Further 10 ml of prepared solution (1000 ?g per ml) was transferred to a 100 ml volumetric flask. The volume was adjusted to 100 ml with methanol as solvent. The final standard stock solution contain 100 ?g per ml of Metformin Hydrochloride and 30 ?g per ml of Methylcobalamin.

Estimation of Metformin Hydrochloride and Methylcobalamin in synthetic mixture:

The API synthetic mixture (0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 ml) was transferred to a series of seven volumetric flask of 10 ml and volume was made up to mark using methanol as solvent. The absorbances of these solutions were measured at 232.0 nm and 353.0 nm wavelength.

Amount of each drug was determined by using simultaneous equation as following formula^[21],[22]:

C_MET    =   (A2 ay1 - A1 ay2)

             (ax2ay1 - ax1 ay2)            ---------(1)

and                                  

C_MEC    =   (A1 ax2 - A2 ax1)

             (ax2 ay1 - ax1 ay2)            ---------(2)

where,

• C_MET = Concentration of Metformin Hydrochloride in g/L
• C_MEC = Concentration of Methylcobalamin in g/L
• A₁ = Absorbance of mixture at 232.0 nm
• A₂ = Absorbance of mixture at 353.0 nm
• a_x1 = Absorptivity of Metformin Hydrochloride at 232.0 nm
• a_x2 = Absorptivity of Metformin Hydrochloride at 353.0 nm
• a_y1 = Absorptivity of Methylcobalamin at 232.0 nm
• a_y2 = Absorptivity of Methylcobalamin at 353.0 nm

Validation of proposed method:

The proposed method was validated according to International Conference on Harmonization (ICH) guidelines:

Specificity:               

The synthetic mixture of Metformin Hydrochloride and Methylcobalamin was prepared in ratio of 10:1. Common excipients used in the tablet formulation were added into this mixture. The absorbance was measured before addition of excipients and after addition of excipients at 232.0 nm and 353.0 nm.

Linearity and Range:

Aliquots of standard stock solutions of Metformin Hydrochloride and Methylcobalamin were taken in 10 ml volumetric flasks and diluted with methanol to get final concentration in the range of 10-60 mg/ml for Metformin Hydrochloride and 1-6 mg/ml for Methylcobalamin. This calibration range was prepared six times and absorbances were measured at 232.0 nm and 353.0 nm for each drug separately.

Accuracy:

The accuracy of the method was determined by carrying out recovery studies using standard addition method. Known amounts of standard solutions of MET and MEC were added to prequantified sample solutions of MET and MEC (5 μg/ml MET and 15 μg/ml MEC). The amounts of MET and MEC were estimated by applying obtained values to the respective regression line equations. The experiment was repeated for three times.

Method Precision (Repeatability):

The precision of the method was checked by repeated scanning and measurement of absorbance of solutions (n = 6) for MET (5 μg/ml) and MEC (15 μg/ml) without changing the parameter of the proposed spectrophotometry method.

Intermediate Precision (Reproducibility):

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day at 2 hours interval and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of MET and MEC (4,5,6 μg/ml for MET and 12,15,18 μg/ml for MEC). The result was reported in terms of relative standard deviation (% RSD).

Limit of Detection and Limit of Quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines:

LOD = 3.3 × SD/S

LOQ = 10 × SD/S

Where,

• SD = the standard deviation of the response
• and S = slope of the calibration curve

LOD and LOQ were determined from the standard deviations of the responses for six replicate determinations.

Robustness:

Solution containing mixture of 5 μg/ml MET and 15 μg/ml MEC was prepared from the standard stock solution (100 ?g/ml of MET and 30 ?g/ml of MEC) through dilution. Prepared solution was analyzed as per proposed method with small but deliberate change in ?max. (?max. ± 1 nm) and scanning speed (slow, medium and fast).

Estimation of Nebivolol Hydrochloride and Indapamide in combined tablet dosage form:

Twenty tablets were weighed and average weight was calculated and finally powdered. An accurately weighed quantity of tablet powder equivalent to 10 mg of MET and 3 mg of MEC was transferred to 100 ml volumetric flask and mixed with 70 ml of methanol as a solvent and solution was sonicated for 20 minutes. There after volume was made up to 100 ml with the same solvent to produce the resultant solution of 100 ?g/ml and 30 ?g/ml of MET and MEC respectively.  The solution was filtered through Whatmann filter paper 42. From the filtrate, 1.3 ml was transferred to five different 10 ml volumetric flasks and volume in each was made up to 10 ml with methanol as a solvent. Absorbances of these solutions i.e. A1 and A2 were measured at 232.0 nm and 353.0 nm using methanol as blank. Values of the A1 and A2 were substituted in Eqn. 1 and 2 to obtain the concentration of MET and MEC respectively.

RESULTS AND DISCUSSION

Validation:

Specificity:

The developed method was found to be specific as percent interference obtained was 0.461% and 0.369% for MET and MEC respectively, which were less than prescribed limit (0.5%) as per ICH guidelines. Thus it was concluded that the addition of excipients had a very negligible change in the concentration of MET and MEC.

Linearity and Range:

Linearity range was found to be 10.0 - 60.0 µg/ml for MET at 232.0 nm and 353.0 nm. The correlation coefficient was found to be 0.999 & 0.998 which showed good linearity between ranges. The slope was found to be 0.049 & 0.005 and intercept was found to be 0.022 and -0.002.

For MEC at 232.0 nm and 353.0 nm, linearity range was found to be 1.0 – 6.0 µg/ml. The correlation coefficient was found to be 0.997 & 0.999 which showed good linearity between ranges. The slope was found to be 0.022 and 0.091 and intercept was found to be 0.003 and -0.006 [Table 1].

Accuracy

The results obtained for the accuracy study (recovery method) from three sample studies (n = 3) for each level indicated that the mean of the % recovery was 99.637% and 99.484% and % RSD was 0.828 % and 0.833 % for MET and MEC respectively in synthetic mixture. The method was found to be accurate [Table 2].

Method Precision (Repeatability)

Repeatability study showed a RSD of 0.597 % for MET and 0.529 % for MEC, which was less than 2%.

Intermediate Precision (Reproducibility)

The method was found to be precise as intra-day precision study showed a RSD of 0.08% for MET and a RSD of 0.60% for MEC and inter-day precision study showed a RSD of 0.33% for MET and a RSD of 0.74 % for MEC. The % RSD values were less than 2% [Table 3].

Limit of Detection and Limit of Quantification

The LOD was found to be 0.259mg/ml and 0.194 mg/ml and LOQ was found to be 0.786 mg/ml and 0.589 mg/ml for MET and MEC respectively which represents that sensitivity of the method was high.

Robustness

The robustness study for change in scanning speed showed a RSD of 0.195% and 0.825% for MET and MEC respectively. The robustness study for change in λ_max showed a RSD of 0.472% and 0.194% for MET and MEC respectively. The method was found to be robust at small variations in scanning speed and λ_max [Table 3].

Summary of all validation parameters is shown in [Table 3].

Table 1: Data showing recovery study

Table 2: Data showing Intra and inter day precision analysis

Table3 : Summary of validation parameters by UV-Visible spectrophometry method