Validated RP-HPLC Method for Estimation of Ranitidine Hydrochloride, Domperidone and Naproxen in Solid Dosage Form

Md. Ahsanul Haque¹, Mohammad Shahriar¹, Most. Nazma Parvin² and S. M. Ashraful Islam^1*

¹Department of Pharmacy, University of Asia Pacific, Dhanmondi, Dhaka-1209, Bangladesh

²Department of Pharmacy, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka-1217, Bangladesh *Corresponding Author E-mail: ashraf@uap-bd.edu

ABSTRACT:

In the present study, a simple, sensitive and specific liquid chromatography (RP-HPLC) method has been developed and validated for the quantification of ranitidine hydrochloride, domperidone and naproxen in solid dosage form. A shim-pack CLC-ODS column (250 mm X 4.6 mm, 5μ and a mobile phase constituting 0.1 M orthophosphoric acid solution (pH 3.0): methanol (35:65 v/v) were used. The flow rate was 1.0 ml/min and detection was carried by using ultraviolet (UV) detector at a wavelength of 280 nm. The retention times of ranitidine hydrochloride, domperidone and naproxen were 2.702 min, 3.666 min and 9.842 min respectively. The peaks were well separated (resolution 4.55 and 20.2). The calibration curves were linear over the concentration range of 80% to 120% of target concentration (R² > 0.999 for ranitidine and naproxen and 0.998 for domperidone). The method is accurate with 99.5% to 100.04% recovery (% RSD < 1.23). The proposed method was successfully applied for the estimation of ranitidine hydrochloride, domperidone and naproxen and potency was found within limit. Therefore, this method can be used for the analysis of ranitidine hydrochloride, domperidone and naproxen in single or combine dosage form.

KEYWORDS: Ranitidine hydrochloride, domperidone, naproxen, method validation, HPLC, quantitative analysis.

INTRODUCTION:

Ranitidine hydrochloride is a H₂- receptor antagonist and is widely used for the short-term treatment of duodenal ulcer and for the management of hypersecretory conditions. Chemically Ranitidine HCl is N-[2-[[[-5-[(Dimethylamino) methyl]-2-furanyl] methyl] thio] ethyl]-N- methyl-2-nitro-1, 1-ethenediamine hydrochloride.

Domperidone is a D₂ receptor antagonist. It increases gastrointestinal peristalsis and motility that prevent reflux esophagitis and it is used to prevent nausea and vomiting. Chemically domperidone is 5- chloro- 1- [1- [3- (2- oxo- 2, 3- dihydro- 1H-benzimidazol- 1- yl) propyl]- piperidin- 4- yl]- 1, 3- dihydro- 2H benzimidazol- 2- one.

Naproxen is a well-known non-steroidal anti-inflammatory drug which is clinically used in treatment of rheumatoid arthritis and other painful musculoskeletal disorders. It works by inhibiting both the COX-1 and COX-2 enzymes. Chemically naproxen is 2-Naphthaleneacetic acid, 6-methoxy-α-methyl-,(s)-(+)-(s)-6-Methoxy-α-methyl-2- naphthaleneacetic acid.

These three drugs are widely prescribed either in single or in combination for various disease conditions. Naproxen and ranitidine combination is widely prescribed by the physicians in order to avoid NSAID induced ulcers. On the other hand domperidone is widely prescribed with ranitidine to control reflux esophagitis, nausea and gastritis. So it is highly needed to develop a simple method for simultaneous determination of these drugs in combination dosage form.

All the three drugs, ranitidine hydrochloride, domperidone and naproxen, are official in BP¹. But only ranitidine hydrochloride and naproxen are official in USP². BP describes HPLC method for ranitidine tablet and UV method for naproxen tablet. On the other hand, USP describes HPLC method both for ranitidine and naproxen tablet. But none describes any method for simultaneous determination of ranitidine hydrochloride, domperidone and naproxen. Literature survey reveals that HPLC methods have been reported for the assay of ranitidine hydrochloride³, domperidone⁴ and naproxen⁵ in individual formulations. HPLC methods for the analysis of these three drugs in combination with other drugs are also reported^6-9. But not a single method is reported for the simultaneous determination of ranitidine hydrochloride, domperidone and naproxen. So, the present work was undertaken with the aim to develop and validate an economic, rapid reversed-phase high performance liquid chromatographic method with high resolution according to ICH and Global Quality guideline^10-11. The method will be helpful to determine these three drugs form either single or combine pharmaceutical dosage form.

MATERIALS AND METHOD:

Materials:

Ranitidine hydrochloride and domperidone were provided by General Pharmaceuticals limited Dhaka, Bangladesh and naproxen was a gift from Eskayef Bangladesh Ltd. Methanol was of HPLC grade and was purchased from E. Merck, Darmstadt, Germany. Orthophosphoric acid and other reagents were of analytical-reagent grade and purchased from E. Merck, Darmstadt, Germany. Water was deionised and double distilled. Ranitidine tablet 150 mg, domperidone tablet 10 mg and naproxen tablet 250 mg were purchased from local drug store in Dhaka city after checking their manufacturing license numbers, batch numbers, production and expiry dates.

Instrumentation and chromatographic conditions:

A Shimadzu (Japan) HPLC system consisting of a CMB-20 Alite system controller, two LC-20AT pumps, SIL-20A auto-sampler and CTO-10ASVP column oven were used. Ultraviolet detection was achieved at 280 nm with a SPD-20A UV-VIS detector (Shimadzu, Japan). The drug analyses data were acquired and processed using LC solution (Version 1.3, Shimadzu, Japan) software running under Windows XP on a Pentium PC. The mobile phase, a mixture of 0.1 M orthophosphoric acid solution (pH 3.0): methanol (35:65 v/v) pumped at a flow rate of 1.0 ml/min through the column (C₁₈; 250 mm X 4.6 mm, 5μ shim-pack, Japan) at 30⁰C. The mobile phase was filtered through a 0.2μ nylon membrane filter and degassed prior to use under vacuum. Elusions were analyzed by UV detector at a sensitivity of 0.0001.

Preparation of standard solution:

Stock solution of ranitidine, domperidone and naproxen were prepared as per their dose ratio. Stock solution of ranitidine (600µg/ml), domperidone (200µg/ml) and naproxen (1000µg/ml) were prepared by dissolving 60 mg ranitidine (as ranitidine hydrochloride), 20 mg domperidone and 100 mg naproxen in 100 ml mobile phase separately. Several aliquots of standard solutions of ranitidine, domperidone and naproxen were taken in different 100 ml volumetric flasks and diluted up to the mark with mobile phase to get five different concentrations (80%, 90%, 100% 110% and 120% of target concentration). Solution containing mixture of ranitidine, domperidone and naproxen of five different concentrations (80%, 90%, 100% 110% and 120% of target concentration) were prepared by diluting aliquots of standard solutions of ranitidine domperidone and naproxen in 100 volumetric flasks.

Preparation of sample solution:

Average weight of ranitidine tablet 150 mg, domperidone tablet 10 mg and naproxen tablet 250 mg were calculated. Then the tablets were grinded separately to a fine powder with the help of mortar and pestle. Then, powder containing 75 mg ranitidine, 5 mg domperidone and 125 mg naproxen was transferred to a volumetric flask, dissolved in mobile phase, shaken for about 10 minutes and filtered through filter paper. The filtered solution was further diluted in the mobile phase to make the final concentration of working sample equivalent to 100% of target concentration.

Validation of HPLC method:

Present study was conducted to obtain a new, affordable, cost-effective and convenient method for HPLC determination of ranitidine hydrochloride, domperidone and naproxen in solid dosage form. The method was validated for the parameters like system suitability, selectivity, linearity, accuracy, precision and robustness.

The system suitability was assessed by six replicate analyses of standard solution at a 100% level to verify the resolution and reproducibility of the chromatographic system. This method was evaluated by analyzing the repeatability of retention time, tailing factor, theoretical plates (Tangent) of the column and resolution between the drug peaks.

To determine the selectivity of the method, standard samples of ranitidine, domperidone and naproxen were injected first. Then solution containing mixed components, commercial product, blank and excipients solution were run in the instrument one after another. The chromatograms were analyzed for retention time, peak area and peak shape to determine selectivity of the method.

The linearity of an analytical method is its ability to elicit that test results are proportional to the concentration of analyte in samples within a given range. This was determined by means of calibration graph using increasing amounts of standard solutions (80%, 90%, 100%, 110% and 120% of target concentration). These standards were tested six times in agreement to the International Conference on Harmonization (ICH)¹⁰. Calibration curves were constructed and the proposed method was evaluated by its correlation coefficient and intercept value, calculated in the corresponding statistical study (ANOVA) (p < 0.05).

Table-1: Results of system suitability study

Parameters	Ranitidine			Domperidone			Naproxen
	Average	SD	%RSD	Average	SD	%RSD	Average	SD	%RSD
Retention time	2.702	0.001	0.033	3.666	0.001	0.024	9.842	0.018	0.179
Area	1046302.33	3218.493	0.308	108585.333	468.352	0.431	1682585	103.811	0.006
Theoretical plates	2752.333	3.141	0.114	4596.000	10.733	0.234	10106	79.679	0.788
Tailing factor	1.175	0.003	0.288	1.322	0.004	0.295	1.702	0.059	3.466
Resolution				4.55	0.026	0.577	20.2	0.022	0.110

Characteristic parameters for regression equation (y = a + bx) of the HPLC method were obtained by least squares treatment of the results and these parameters were used to confirm the good linearity of the method.

Accuracy indicates the deviation between the mean value found and the true value. Accuracy was determined by means of recovery experiments, by the addition of active drugs to placebo formulations. The accuracy was calculated from the test results as the percentage of the analyte recovered by the assay.

The precision of the method was investigated with respect to repeatability, ruggedness (intermediate precision) and reproducibility (by means of an inter laboratory trial). Repeatability was determined by performing four repeated analysis of the three standard solutions (90%, 100% and 110% of target concentration) on the same day, under the same experimental conditions. Ruggedness (intermediate precision) of the method was assessed by carrying out the analysis of standard solutions on three different days (inter-day) in the same laboratory. For reproducibility the procedure repeated in the Quality Control Laboratory of another lab. The relative standard deviation (% RSD) was determined in order to assess the precision of the method.

The robustness of the method was assessed by altering the some experimental conditions such as, by changing the flow rate from 0.9 to1.1 ml/min, amount of methanol (63% to 67%) the temperature of the column (28 °C to 32 °C) and P^H (2.9-3.1) of the mobile phase.

RESULTS AND DISCUSSION:

Methods development and optimization:

This isocratic mode method with UV detection was developed for the determination of the ranitidine hydrochloride, domperidone and naproxen. The mobile phase was chosen after several trials with 0.1 M ortho phosphoric acid and methanol in various proportions like 70: 30, 65:35, 50:50, 40: 60, 30:70 and 35:65 at different pH values. When aqueous phase was increased in mobile phase tailing factor and retention time of domperidone and naproxen were also increased. So, mobile phase containing 0.1 M ortho phosphoric acid solution (pH 3.0) and methanol (35:65 v/v,) was selected to achieve maximum separation and sensitivity.

Different flow rates in between 0.50 to 2.0 ml /min were studied. A flow rate of 1.0 ml /min gave an optimal signal to noise ratio with a reasonable separation time.

Wave length for UV detection was determined by scanning individual and combined solution of ranitidine hydrochloride, domperidone and naproxen. Then HPLC analysis of individual and combined standard was measured at 228, 240, 280, 314, and 332 nm. Finally, all the analysis were done at 280 nm as at this wavelength, all the three drugs absorbed light better and peaks could be distinguished properly.

Method validation:

The experiment was carried out according to the official specifications of USP, ICH- 1995, and Global Quality Guidelines.^{2, 10-11}

System Suitability:

The system suitability tests were carried out to evaluate the resolution and reproducibility of the system for the analysis. Table 1 represents system suitability results of this method. The system is found suitable in respect of retention time (% RSD 0.001-0.179) mean theoretical plate count (% RSD 0.114-0.788) and resolution between peaks.

Selectivity:

Selectivity is the ability of the method to assess the analyte in the presence of excipients, impurities, degraded products, matrix etc. Peaks from individual sample solutions and mixed sample solution were on same time (Figure 1). On the other hand no other peaks other than drugs were found within 20 min run time from the commercial products. Blank and excipients did not change the retention time or interfere the analysis results. So the method is highly selective for ranitidine hydrochloride, domperidone and naproxen.

Figure 1.Chromatogram of combined (C) and single sample of ranitidine (R), domperidone (D) and naproxen (N).

Linearity:

Linearity of the method was evaluated from the correlation coefficient of calibration curves that were constructed from average peak area of drugs at different concentration level (80%, 90%, 100%, 110% and 120%). Correlation coefficient was 0.999 for ranitidine hydrochloride and naproxen and 0.998 for domperidone (Table 2 and Figure 2).

Figure 2.Calibration curve of ranitidine hydrochloride (R), domperidone (D) and naproxen (N).

Accuracy and Precision:

Results of accuracy and precision (repeatability, ruggedness and reproducibility) are summarized in table 2 along with linearity results. Accuracy is generally assessed by analyzing samples with known concentration and comparing the measured value with the true value .The measured value was obtained by recovery test. % recovery was found 99.5% to 100.04% with % RSD value 0.37 to 1.23. All the results indicate that the method is highly accurate.

The measurement for repeatability was done from 9.00 am to 9.00 pm. Four determinations of three concentrations across the intended range (90%, 100% and 110% of target concentration) were included in the study. % RSD of peak areas was calculated for various run. The method is highly precise as % RSD of peak area was 0.35%.

Test results for ruggedness were obtained by analyzing three concentrations (90%, 100% and 110% of target concentration) with 4 runs over 3 days. The average peak area obtained at different levels and different days indicate that the method is precise. % RSD values were found slightly higher for all the drugs in this study than repeatability study. Samples analysis results in another lab (reproducibility) were also within limit (% RSD was less than 2%).

Table 2: Results of a linearity, accuracy and precision study

Validation parameters		Ranitidine	Domperidone	Naproxen
Linearity (regression coefficient-R^2) (*Y = mX+C)	R^2(mean±SD)	0.9997±0.0002	0.9980±0.0005	0.9993±0.0002
	%RSD **	0.021	0.056	0.021
	Slope (mean±SD)	22987.87±38.79	33871.77±470.05	22782.91 ±78.77
Accuracy	% Recovery	100.04 ± 0.37	100.01± 1.23	99.5± 0.52
	%RSD	0.37	1.23	0.53
Precision (%RSD)	Repeatability	0.35	0.27	0.095
	Ruggedness	0.59	0.43	0.25
	Reproducibility	1.32	0.94	0.83

*Y = mX+C; where Y = peak area, m = slope, X = concentration (μg/ml) and C = intercept.

**%RSD = Relative standard deviation = (Standard deviation X 100)/mean, R² = Correlation coefficient

Table 3: Analysis of marketed tablet and new formulated capsule by proposed HPLC method

Run No.	Commercial tablet			New formulated capsule (ranitidine 150 mg, domperidone 10 mg and naproxen 250 mg)
	Ranitidine tab (150 mg)	Domperidone tab (10 mg)	Naproxen tab (250 mg)	Ranitidine	Domperidone	Naproxen
	% potency
R-1	99.01	102,16	103.95	99.57	99.93	100.36
R-2	98.93	104.62	100.46	99.23	99.23	100.00
R-3	100.05	102.97	102.81	99.85	98.82	98.97
Average	99.33	103.80	102.41	99.55	99.33	99.78
SD	0.625	1.167	1.780	0.310	0.561	0.723
% RSD	0.629	1.124	1.738	0.312	0.565	0.725

Robustness:

Robustness study was performed by making slight variations in flow rate, amount of methanol, temperature and pH of the mobile phase. No significant effect was observed in the recovery of drugs. % recovery was 98% to 102%. On the other hand changes in retention time, theoretical plate, resolution were also negligible. So we can say that the method is robust.

Analysis of market products:

The proposed method was used to determine the potency of commercially available tablets (Ranitidine tablet 150 mg, domperidone tablet 10 mg and naproxen tab 250 mg). Capsule containing 150 mg ranitidine, 10 mg domperidone and 250 mg naproxen were prepared using common excipients and analyzed. Three replicate determinations (n=3) were carried out and the results are summarized in Table 3.

CONCLUSION:

The validation study shows that the developed method is accurate, rapid, precise, reproducible and inexpensive with acceptable correlation co-efficient, RSD (%) and standard deviations which make it versatile and valuable for simultaneous determination of ranitidine hydrochloride, domperidone and naproxen in bulk or pharmaceutical dosage form (individual or combine). The advantages lie in the simplicity of sample preparation and the low cost of reagents used. The proposed method is simple and do not involve laborious time-consuming sample preparation. So this RP-HPLC method can be used in the quality control department.

ACKNOWLEDGEMENTS:

The authors are thankful to General Pharmaceuticals limited Bangladesh, for providing gift samples of ranitidine hydrochloride and domperidone. Authors are also thankful to Eskayef Bangladesh Ltd. for providing gift samples of naproxen.

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Received on 22.08.2011 Accepted on 26.08.2011

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Asian J. Pharm. Ana. 1(3): July-Sept. 2011; Page 59-63