FACILE HPLC/UV METHOD FOR DETERMINATION OF METFORMIN AND 1-CYANOGUANIDINE USING NOVEL HALOGENATED STATIONARY PHASE

Metformin is the most prescribed drug for diabetes. We aimed to develop a new HPLC method for determination of metformin and its main impurity 1-cyanoguanidine in raw material and its pharmaceutical tablets. The separation was achieved on pentabromobenzyl column using a mixture of 20% 10.0 mM phosphate buffer (pH 5.5) and 80% methanol as a mobile phase at a flow rate of 1.0 mL min -1 and UV detection at 227 nm. The analysis time was less than 4.0 minutes. The method was linear through concentration ranges of 3.0 -80 µg mL -1 and 0.5-10 µg mL -1 and detection limits of 0.73 µg mL -1 and 0.11 µg mL -1 for metformin and 1-cyanoguanidine, respectively. The method was successfully applied for determination of metformin in bulk and pharmaceutical dosage forms. The high sensitivity of the developed method allowed for determination of 1-cyanoguanidine below its specified limits in metformin raw material and tablets.


INTRODUCTION
Metformin (MET) is chemically described as 1,1-dimethylbiguanide hydrochloride ( Figure 1). It is an orally administered and considered as the most prescribed drug in treatment of type-2 diabetes mellitus worldwide. It works through increasing insulin sensitivity and uptake of glucose by the cells and inhibits glucose production by the liver (gluconeogenesis) 1&2 .
The British Pharmacopeia (BP) and the United State Pharmacopeia (USP) listed six impurities (impurity A-F) in MET monograph. HPLC assay of impurity A (1-cyanoguanidine; CGN; Figure 1) in MET raw material and tablets is described in both pharmacopeias. The limit of unwanted CGN is not more than 0.02% and 0.1% in raw material and tablets, respectively 3&4 . MET is assayed in BP, European Pharmacopeia (Ph. Eur.) and USP using non-aqueous titration 3-5 . Many other analytical methods for determination of MET in pharmaceutical and biological matrices, either alone or in combination with other drugs were also reported. Recent reported methods include spectroscopy 6-8 , HPLC 9-11 , HPTLC 12-14 , LC/MS 15-19 , capillary electrophoresis (CE) 20&21 and voltammetry [22][23][24] . Most of these previously published methods were focusing on the determination of MET regardless its main impurity CGN. Only very few methods considered CGN in their determination, and these methods include HPLC [25][26][27] and CE methods 28 .

Fig. 1: Chemical structures of MET and CGN
MET is highly hydrophilic compound, therefore, MET cannot adequately retain on the conventional reversed-phase C 18 columns and eluted very rapidly 10 . Furthermore, MET strongly binds with polar normal-phase columns and consequently cannot be eluted easily 29 .
A pentabromobenzyl-bonded silica column known as Cosmosil PBr is a newly emerged halogenated stationary phase. It separates the analytes through dispersion force interactions which is useful for separation of structurally similar compounds, halogenated and polar compounds [34][35][36] . In this study, we aimed to develop a simple HPLC-UV method based on using PBr column as an alternative chromatographic approach for the determination of MET and its main impurity CGN in raw material and tablets with low cost, high sensitivity and short analysis time.

Materials and reagents
All chemicals used were of analytical grade and double distilled water was used during the whole experiments.  Metformin pure sample (99%) was kindly supplied by Chemical Industries Development (CID) (Giza, Egypt

Chromatographic conditions
The analysis was performed on a Cosmosil PBr (150 mm x 4.6 mm I.D., 5 µm particle size) column kept at 30 °C. The mobile phase was composed of methanol/phosphate buffer (10 mM, pH 5.5) (80:20 % v/v.), filtered through 0.45 µm membrane filter and sonicated for 30 minutes before use. The flow rate was adjusted at 1.0 mL/min and UV detection at 227 nm.

Preparation of stock solutions
Stock solution containing 1.0 mg mL -1 of each of MET and CGN were prepared in methanol. Working solutions were prepared by appropriate dilution of stock solutions with the same solvent. The stock solutions were kept in the refrigerator at 4°C.

Construction of calibration graphs
Accurately measured aliquots of each of MET and CGN working solutions were transferred separately into a series of 10.0 mL volumetric flasks, completed to the volume with the mobile phase to obtain final concentrations of 3.0-80 and 0.5-10 µg mL -1 of MET and CGN, respectively. 5 µl of each solution was injected in triplicate under the optimum chromatographic conditions. The peak area was plotted versus the concentration of each analyte in µg mL -1 to construct the calibration curves and the corresponding regression equations were derived.

Assay of pharmaceutical dosage forms
Twenty tablets were accurately weighted, finely ground and powdered. An amount of the powder equivalent to 10.0 mg was accurately transferred to 10.0 mL volumetric flask, sonicated with the mobile phase for 30.0 minutes and completed to the mark with the same diluent. The solution was centrifuged for 10.0 minutes and then an appropriate portion of the clear supernatant was further diluted with the same diluent for assay of MET in tables.
For analysis of CGN, the same procedures were followed but the final concentration of MET was adjusted to be 2.5 mg mL -1 and 0.5 mg mL -1 in MET raw material and MET tablets, respectively, in order to detect any trace amount of CGN.

RESULTS AND DISCUSSION
The developed assay permitted the separation of MET and its main impurity CGN in less than 4.0 minutes. The retention times for MET and CGN were 2.88 and 1.78, respectively. The new Cosmosil PBr column has achieved excellent separation and resolution of the studied polar analytes. Different factors that may affect the chromatographic behavior of MET and CGN were carefully studied and optimized to obtain optimum separation in reasonable time.

Method development and optimization Choice of detection wavelength
To determine the optimum wavelength for the assay, the UV spectra of MET and CGN were investigated, MET and CGN were found to exhibit λ max at 232 and 227 nm, respectively, as shown in Figure 2, UV detection at 227 nm was selected as the most suitable wavelength to obtain the highest possible sensitivity for the impurity CGN with adequate sensitivity for MET.

Mobile phase composition
Methanol, ethanol and acetonitrile in different ratios were investigated as the organic modifier, screening experiments revealed that the peak shapes are better upon using methanol or ethanol instead of acetonitrile, finally, methanol was selected as the organic modifier as it produces lower column backpressure than ethanol. Optimization experiments also revealed that, 80% was the optimum methanol content regarding geed separation and short analysis time.
Different buffers (acetate and phosphate buffer) at pH range of (3.5 and 6.0) were evaluated as an aqueous part of mobile phase. It was observed that, tailed broad peak of MET at pH lower than 5.0 was obtained, which may be attributed to the high ionization of the investigated drug at lower pH that cause high interaction with the stationary phase 25 . Optimum symmetric and sharp peaks with good resolution were obtained in the pH range of 5.0-6.0 in either of the studied buffers. Therefore, pH value of 5.5 of phosphate buffer was selected for further investigations.

Effect of flow rate and column temperature
The effect of flow rate of mobile phase on the separation of MET and CGN was investigated to select the optimum flow rate in term of good separation and minimal run time.
Flow rate of 1.0 mL/min was suitable for the separation of analytes in short analysis time. Flow rates more than 1.0 mL/min caused high column backpressure. Similarly, different column temperatures were studied, and best result was attained at 30 °C.
Under the optimized conditions, good separation between MET and its main impurity CGN was obtained as shown in Figure 3 which displays a typical chromatogram for the separation of MET and CGN at retention time (t R ) of 2.88 and 1.78 minute for MET and CGN, respectively.

Method validation
Validation of the developed method was performed by following the international council for harmonization (ICH) guidelines 37 . Different validation characteristics were investigated as follows:

Linearity and range
Under the optimized experimental conditions, a linear relationship was established by plotting the peak area versus concentrations of each analyte as indicated by high correlation coefficients of 0.9996 and 0.9995 for MET and CNG, respectively, indicating good linearity of the proposed method. The analytical data of the calibration plots are summarized in Table 1.

Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ values of MET and CGN were determined according to ICH Q2 (R1) recommendations.

LOD = 3.3 Sa/b LOQ = 10 Sa/b
Where, Sa is the standard deviation of the intercept and b is the slope of the calibration plot. The results are shown in Table 1. The LOD values were 0.73 and 0.11 µg mL -1 for MET and CGN, respectively, indicating high sensitivity of the proposed HPLC method.

Accuracy and precision
The accuracy and precision of the developed method were studied at three concentration levels for MET and CGN using three replicates for each concentration through the same day for intra-day precision and three consecutive days for inter-day precision ( Table  2). The obtained RSD values were less than 2% indicate high precision of the proposed method.

Robustness
Robustness of the developed method was assessed by studying the influence of small deliberate changes in the experimental conditions on system suitability parameters. The method was found to be robust to minor changes in chromatographic conditions including methanol concentration (± 1% v/v), flow rate (± 0.1 mL min -1 ) and pH (± 0.5).

System suitability test
According to ICH recommendations 37 , number of theoretical plates (N), resolution (R s ), capacity (K') and selectivity (α) factors were measured as the parameters for system suitability testing. Table 3 shows the system suitability parameters under the optimum chromatographic conditions. 3.88

Method applications Application of the proposed method to quality control of MET commercial tablets
The proposed method was successfully applied for the determination of MET in its commercial tablet formulations without interference from co-existing excipients, the results are shown in Table 4. The good percentage recoveries and small RSD values indicate suitability of the developed method for routine analysis of MET in commercial tablets. The obtained results were statistically compared with those obtained by the reference method using student's t test and variance ratio F test and the results indicate that there was no significant difference between the proposed method and reference method as shown in Table 4.

Application of the proposed method for determination of CGN in MET bulk and tablet dosage form
CGN was successfully determined in MEW raw material and tablet formulations by the proposed HPLC method, the results are presented in Table 5. The obtained results indicate the applicability of the proposed method for its use in QC laboratories.

Conclusion
A simple, sensitive and rapid HPLC method was developed for the determination of MET and CGN using pentabromobenzyl column. The method was validated according to ICH guidelines in terms of linearity, precision, accuracy, limit of detection, limit of quantitation and robustness. The method was successfully applied for determination of MET and CGN in raw material and tablet formulation. The short analysis time and simple analytical procedure for the developed method allow its use for cost-effective and routine analysis in quality control laboratories.