DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF DICYCLOMINE HYDROCHLORIDE AND FAMOTIDINE IN BULK AND TABLETS

 

SHAILESH T. DONDA, VISHAL B. BAVISKAR, PRASHANT K. DESHMUKH, SANJAY B. BARI, PRAVIN O. PATIL^*

Department of Quality Assurance, H. R. Patel Institute of Pharmaceutical Education and Research, KarwandNaka, Shirpur. Dist. Dhule 425 405 (M.S.)
* e-mail: rxpatilpravin@yahoo.co.in

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ABSTRACT

In the present study an isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous estimation of dicyclomine hydrochloride (DCY) and famotidine (FAM) in bulk and pharmaceutical dosage forms. The separation was achieved on a Phenomex Gemini C column (4.6 x 250 mm, 5μ particle size) using a mixture of methanol and 0.1% triethylamine (TEA) pH 3.0 (adjusted with orthophosphoric acid) in the ratio of 40:60 (v/v) as a mobile phase. The flow rate was 1.0 mL min^-1, column temperature was kept at 28 ⁰ C and detection was monitored at 270 nm. The retention time of dicyclomine hydrochloride and famotidine was found to be 10.72 and 2.72 min respectively. Recovery studies were satisfactory and the correlation coefficient for FAM and DCY were 0.9947 and 0.9935 respectively, indicates linearity of the methods within the limits. The developed method can be used for routine quality control simultaneous analysis of DCY and FAM in tablet dosage form without any interference of excipients.

Keywords: Dicyclomine hydrochloride, Famotidine, RP-HPLC

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INTRODUCTION

Dicyclomine (Also called dicycloverine, DCY) hydrochloride (Figure 1) is chemically, 2-(diethyamino) ethyl 1-cyclohexylcyclohexane-1-carboxylate, is a muscarinic antagonist and used as an antispasmodic as well as in urinary incontinence. In addition it does have some local anesthetic properties and is used in gastrointestinal, biliary, and urinary tract spasm ^{1, 2}. Famotidine (FAM) is chemically 3-[({2-[(diaminomethylidene) amino] -1, 3-thiazol-4-yl} methyl) sulfanyl] -N'-sulfamoylpropanimidamide (Figure 2). The FAM is a competitive H₂-receptor antagonist. This competitive inhibition results in reduced basal and nocturnal gastric acid secretion and a reduction in gastric volume, acidity, and amount of gastric acid released in response to stimuli including food, caffeine, insulin, betazole, or pentagastrin ^{3, 4}. Combination of Dicyclomine and Famotidine is used widely for various clinical complications like Gl spasm, irritable bowel syndrome, hyper-peristalsis, peptic ulcer, flatulence, diarrhoea, benign duodenal and gastric ulcer, reflux oesophagitis, acid peptic disease etc.

 

Fig. 1: Chemical structure of Dicyclomine hydrochloride.

 

Fig. 2: Chemical structure of Famotidine.

 

Several analytical methods have been reported for quantification of DCY and FAM in biological specimen as well as in formulations. Most of them were used for the determination of FAM alone ⁵, in binary combination with, Mefenamic acid-DCY ⁶, nimesulide-DCY ⁷, moxifloxacin-FAM ⁸, diclofenac sodium-FAM ⁹, FAM-ibuprofen ¹⁰, tertiary combination lock, DCY-Mefenamic acid-paracetamol ², FAM-potential degradates-preservatives ¹¹ by means of HPLC. Few spectrophotometric methods were mentioned for the estimation of DCY and FAM, FAM alone ¹² and combinations with other drugs viz. DCY-mefenamic acid ¹³, FAM-domperidone ¹⁴, FAM-ibuprofen ¹⁵, FAM- diclofenac potassium ¹⁶. Estimation of DCY and FAM alone was also mentioned in United State Pharmacopoeia using HPLC ¹⁷.

A combination of DCY and FAM is commercially available in solid (Tablet) dosage form. The literature reveals that no analytical methods were existing for simultaneous determination of these two drugs in combination. Therefore, the main objective of the present investigation was to develop simple, fast, accurate and sensitive method which could be applied to simultaneous analyses of DCY and FAM in bulk and tablets.

EXPERIMENTAL

Material and Reagents

A gift sample of dicyclomine was obtained from Alaric Healthcare Pvt. Ltd. (India) and FAM from Lincoln Pharmaceutical Ltd. (India). HPLC grade methanol purchased from Merck, Darmstadt, Germany and was used throughout experimentation. Double distilled water was used to prepare all solutions and filtered through a 0.45 μm filter. Analytical grade orthophosphoric acid (OPA) was purchased from S. D. Fine Chemicals, Mumbai. Pharmaceutical dosage form containing DCY and FAM was purchased from local markets. Each tablet was labelled to contain DCY 10 mg and FAM 20 mg.

Instrumentation

Analysis was performed on a Shimadzu HPLC system with inbuilt PDA detector was utilized for method development and validation. The chromatography software LC solution was used for data collection and processing. The analytical column was Phenomex Gemini C₁₈ column (4.6 x 250 mm, 5μ particle size).

Chromatographic Conditions

Simultaneous separation of DCY and FAM was performed using an isocratic mobile phase consisted of a mixture of methanol and 0.1% triethylamine (TEA) pH 3.0 (adjusted with OPA) in the ratio of 40:60 (v/v). The well resolved, sharp peak for FAM and DCY was obtained at a retention time of 2.37 min and 10.72 min respectively. The flow rate was maintained at 1.0 mL min^-1, UV detection was performed at 270 nm.

Preparation of standard stock and sample solution

An accurate weighted FAM (10 mg) reference standard was transferred to a 10 ml volumetric flask and was dissolved in the mobile phase to make a solution of 1000 μg mL^-1. From this stock solution, by the serial dilution, required concentration of 20, 40, 60, 80, 100, 120 μg mL^-1 were prepared.

An accurate weighted DCY (10 mg) reference standard was transferred to a 10 ml volumetric flask and was dissolved in the mobile phase to make a solution of 1000 μg mL^-1. From this stock solution, by the serial dilution, required concentration of 10, 20, 30, 40, 50, 60 μg mL^-1 were prepared.

Each tablet containing 10 mg DCY and 20 mg FAM were powdered finely. A composite of 20 tablets was prepared by grinding them to a fine, uniform size powder. A quantity of powder which equivalent to 10 mg DCY, and 20 mg FAM were transfered in 10 mL volumetric flask and dilute up to 10 mL of mobile phase and sonicate for 15 minute then filtered through 0.45 μιη filter paper. From this solution 0.2 ml was transferred in 10 ml volumetric flask and diluted up to the mark with mobile phase to obtain a sample solution containing 20 μg mL^-1 of DCY and 40 μg mL^-1 of FAM.

Method validation

The proposed method was validated as per International Conference Harmonization guidelines for various parameters like accuracy, precision, linearity, robustness, system suitability ¹⁸. The replicate of the drug were carried out to assess the system suitability. It was further evaluated by analyzing the peaks, tailing factor, repeatability, theoretical plates of the column, resolution between the peaks, peak symmetry and relative retention time.

RESULT AND DISCUSSION

Method development and Optimization

A RP-HPLC method was optimized with an intention to develop an accurate and reproducible method for simultaneous estimation of DCY and FAM. Isocratic elution is simple, requires only one pump and flat baseline separation for easy and reproducible results. Both drugs showed absorbance at 270 nm. Therefore the wavelength selected for the determination of DCY and FAM was 270 nm. The final chromatographic conditions were set for stationary phase giving satisfactory resolved peak and run time with reversed phase Phenomex Gemini C₁₈ column (4.6 x 250 mm, 5μ particle size) column. A series of mobile phases consisted of varying pH and volume fractions of methanol and water were tested and the best results were obtained using the mobile phase consisted of methanol: 0.1% triethylamine, pH 3 (adjusted with OPA), in 40:60 giving well resolved, sharp peak for FAM and DCY with a retention time (tR) of 2.37 min and 10.72 min respectively (Fig. 3). The flow rate of 1.0 mL min^-1 at 270 nm and ambient temperature for column oven (28 ⁰ C) was found to be the best for analysis.

 

Figure 3. A typical chromatogram of a mixture of FAM and DCY.

 

This isocratic-mode method with PDA detection was developed for the determination of the active ingredients, DCY and FAM. Initially, the reversed-phase column was checked and tested. The system suitability carried out as per specified ICH guidelines ¹⁸.

In trial 1, the mobile phase consisted of methanol and water in a ratio of (70:30 v/v, pH 3.0 adjusted with OPA) with a flow rate 1.0 ml min^-1 was tried and chromatogram obtained from combination DCY and FAM had showed a broad peak of DCY with lesser number of theoretical plates. While in trial 2, the mobile phase consisted of acetonitrile and 0.1% triethylamine in the ratios of (60:40 v/v, pH 3.0 adjusted with OPA) with a flow rate 1.0 mL min^-1 was attempted. A chromatogram revealed much lesser retention time for FAM. On the other hand in trial 3, the mobile phase consisted of methanol and 0.1% triethylamine in the ratios of (60:40 v/v, pH 3.0 adjusted with OPA) with a flow rate 1.0 ml min^-1 was utilized and chromatogram obtained again showed the less retention time for FAM and theoretical plate was within the limit. In trial 4, the mobile phase consisted of methanol and 0.1% triethylamine in the ratios of (40:60 v/v, pH 3.0 adjusted with OPA) with a flow rate 1.0 mL min^-1 was utilized and chromatogram obtained showed the retention time for FAM 2.37 min, DCY 10.72 min and all other criteria's were found to be within the limit. Chromatogram for FAM and DCY were shown in Figure 3. The system suitability parameter is an important part of chromatographic method and is used to verify that the resolution and reproducibility of the system were adequate for the analysis to be performed (Table 1).

 

Table 1. System suitability parameters for DCY and FAM.

 

Method validation

Linearity

Standard curves of DCY and FAM were linear (r² = 0.9935 and r² = 0.9947 respectively) over the concentration range of 10-60 μg mL^-1 and 20-120 μg mL^-1 respectively. The linearity equation was y=11705x + 377435 for DCY and y=25074x + 1675106 for FAM where 'y' is the peak area and 'x' is concentration in μg mL^-1.

Accuracy

Accuracy was determined by evaluating the percentage of recovery of known analytes to the pharmaceutical formulation. Each sample was injected three times and accuracy was determined at 80%, 100 and 120%. The accuracy was expressed as the percentage of the analyte recovered. The result of accuracy studies were shown in Table 2.

 

Table 2. Results of Accuracy study.

 

Precision

The precision of this method was determined by inter-day, intra-day and repeatability precision. It was expressed as % R.S.D. of a series of measurement. The experimental values obtained for the intra-day and inter-day precision of DCY and FAM in sample were shown in Table 3. The results obtained for the repeatability of DCY and FAM in sample was shown R.S.D. less than 2, indicating good repeatability. The % RSD was less than 2 in intraday, interday precision and all parameters of robustness were within the limit. Thus the proposed method is precise and accurate. All the value and data obtained were within the acceptance criteria.

 

Table 3. Results of Precision study.

 

Limit of detection (LOD) and Limit of quantification (LOQ) The LOD and LOQ were calculated by using the equations LOD = (3.3 χ σ)/S and LOQ = (10 × σ)/S respectively, where 'σ' is the standard deviation of the peak areas of the drug (n=3) and 'S' is the slope of the corresponding calibration plot. The LOD were 2.43 μg mL^-1 and 4.33 μg mL^-1 and LOQ were 7.38 μg mL^-1 and 13.13 μg mL^-1 for DCY and FAM respectively.

Robustness

To evaluate robustness, various parameters like mobile phase, flow rate, detection wavelength were deliberately varied for estimation of DCY and FAM (Table 4).

 

Table 4. Results of Robustness study.

 

Assay

The proposed method was evaluated in the assay of commercially available tablet containing DCY (10mg) and FAM (20mg) six replicate determination (n=6) were carried out on an accurately weighted amount of the pulverized tablet equivalent to 10mg of DCY and 20mg of FAM. The results are shown in Table 5.

 

Table 5. Assay of tablet formulation containing FAM and DCY.

 

Specificity and selectivity

The specificity of the RP-HPLC method was determined by comparison of the chromatogram of mixed standards and sample solutions. The specificity test of the proposed method demonstrated that the excipients from tablets do not interfere in the drug peak around the retention time of DCY and FAM and also baseline showed no any significant noise. The studying the effect of excipients on quantification of DCY and FAM, a placebo was analyzed. The results obtained indicate that excipients had no interference with proposed quantification of DCY and FAM. The results obtained indicate that excipients had no interference with proposed quantification of DCY and FAM. The method is quite selective.

CONCLUSION

A new RP-HPLC method has been developed for the simultaneous estimation of DCY and FAM in bulk and pharmaceutical formulations. The method gave good resolution for both drugs with a short analysis time less than 12 minutes. The good percentage recoveries in the tablet dosage form suggest that the excipients present in the dosage form had no interference in the determination of active constituents. The proposed method can be used for routine analysis of DCY and FAM in tablet dosage form. The method was validated as per ICH guidelines.

Declaration of Interest: The authors report no declarations of interest.

ACKNOWLEDGEMENTS

The authors are thankful to H.R. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India for providing facilities to carry out this research work and also thankful to the Alaric Healthcare Pvt. Ltd. (India) for providing a gift sample of dicyclomine as well as Lincoln Pharmaceutical Pvt. Ltd. (India) for providing an authentic sample of famotidine.

 

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