A SIMPLE RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF RANITIDINE HYDROCHLORIDE AND DOMPERIDONE IN COMBINE DOSAGE FORM

Online Published (2011) ISSN: 0976-7908 Ashraful et al

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A SIMPLE RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF

RANITIDINE HYDROCHLORIDE AND DOMPERIDONE IN COMBINE

DOSAGE FORM

Gauri Rani Das, Md. Ahsanul Haque, Mohammad Shahriar and S.M. Ashraful Islam*

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

ABSTRACT A simple High Performance Liquid Chromatographic (RP-HPLC) method for the analysis of ranitidine hydrochloride and domperidone has been developed and validated. The chromatographic system consisted of a LC-20 AT pump, SPD-20A UV detector, SIL-20A auto-sampler and CTO-10ASVP column oven. The Separation was achieved by C18 column (5μ, 4.6 X 250 mm, Waters, USA) at 300C temperature with a mobile phase consisting of methanol: water (55:45 v/v, PH=3.00 adjusted with 80% orthophosphoric acid) at a flow rate of 1ml/min. Retention time was 2.74 minutes for ranitidine hydrochloride and 5.05 minutes for domperidone. The peaks of ranitidine hydrochloride and domperidone were well separated (Resolution 9.74). The calibration curves were linear over the concentration range of 80% to 120% of target concentration (r2 = 0.999 for both the drugs). The proposed method is accurate with 101.60% recovery for ranitidine hydrochloride and 99.61% recovery for domperidone and precise (%RSD of Intra day and Inter day variation were less than 2%). The method has been used to test marketed tablets and potency was found within 98.7%-100.14%. This method can be used as for the analysis of ranitidine hydrochloride and domperidone in combine dosage form. Keywords: Ranitidine hydrochloride, domperidone, method validation, HPLC, quantitative analysis INTRODUCTION

Combination of drugs is widely used now a days. So it is essential to develop

simple, precise and accurate method for simultaneous determination of drugs in combine

dosage from. Reversed-phase high performance liquid chromatography (RP-HPLC) is a

well-known technique for the qualitative and quantitative analysis of drugs. It is very

useful for simultaneous determination of pharmaceutical dosage forms. This technique is

extensively used in different analytical lab for higher sensitivity and selectivity than other

methods such as titration or UV spectrophotometric method. This paper describes a

simple reverse phase high performance liquid chromatographic (RP-HPLC) method for

estimation of ranitidine hydrochloride and domperidone in tablet dosage form.



Ranitidine hydrochloride is a H2- receptor antagonist and is widely used for the

short-term treatment of duodenal ulcer and for the management of hyperacidity

conditions [1]. Chemically Ranitidine hydrochloride (Fig. 1), is N-[2-[[[-5-

[(Dimethylamino) methyl]-2-furanyl] methyl] thio] ethyl]-N- methyl-2-nitro-1, 1-

ethenediamine hydrochloride.

Figure 1. Ranitidine Hydrochloride Domperidone

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

[1]. Chemically Domperidone (Fig. 1) 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.

Ranitidine Hydrochloride is official in BP and USP [2-3]. BP describes non

aqueous titration method for estimation of ranitidine hydrochloride in bulk and HPLC

method for ranitidine tablet. On the other hand, USP describes HPLC method both for

ranitidine bulk and tablet dosage form. HPLC methods for analysis of ranitidine along

with other drugs are also reported [4-5].

Domperidone is official in BP. BP describes non aqueous titration method for

estimation of domperidone in bulk. Rajendra et al, 2009 reported spectrophotometric

method for the estimation of domperidone in bulk and in pharmaceutical formulations [6].

Varalakshmi et al, 2011 developed a RP –HPLC method for the determination of

domperidone maleate [7]. Spectroscopic and HPLC methods for the analysis of

domperidone along with other drugs are also available [8-16].

Now a days domperidone is widely prescribed by the physicians with ranitidine to

control reflux esophagitis, nausea and gastritis. So an initiative was taken to develop and

validate a RP-HPLC method to estimate domperidone and ranitidine in combine dosage

form.

A Spectroscopic method is reported previously for the simultaneous

determination of Ranitidine and Domperidone [17]. Spectroscopic methods are not suitable



for combination drug products as excipients present in the formulations may interfere the

estimation. On the other hand HPLC method that is reported for the simultaneous

determination of ranitidine hydrochloride and domperidone is not free from limitation [18].

So development of a simple method for the simultaneous estimation of ranitidine

hydrochloride and domperidone is still required.

So the present work was undertaken to develop and validate a economic, rapid

reversed-phase high performance liquid chromatographic method for the quality control

of ranitidine hydrochloride and domperidone in pharmaceutical preparations with lower

solvent consumption along with the short analytical run time that leads to an

environmentally friendly chromatographic procedure that allows the analysis of a large

number of samples in a short period of time.

MATERIAL AND METHODS

Materials

Ranitidine Hydrochloride and domperidone were kind gift from incepta

Pharmaceuticals Limited Dhaka, Bangladesh. Methanol was of HPLC grade and

collected 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.

Instrumentation

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

was used. Ultraviolet detection was achieved with a SPD-20A UV-VIS detector

(Shimadzu, Japan). The drug analysis data were acquired and processed using LC

solution (Version 1.2, Shimadzu, Japan) software running under Windows XP on a

Pentium PC.

Chromatographic conditions

The mobile phase, a mixture of methanol: water (55:45v/v) pumped at a flow rate

of 1.0 ml/min through the column (C18; 5μ, 4.6 X 250 mm, Waters, USA) at 300C. The

mobile phase was degassed prior to use under vacuum by filtration through a 0.2μ nylon

membrane. Concentrations were measured at 280 nm by UV detector at a sensitivity of

0.0001.



Preparation of standard solution

Stock solutions of ranitidine (600 μg/ml) and domperidone (200 μg/ml) were

prepared by dissolving 60 mg ranitidine (as ranitidine hydrochloride) and 20 mg

domperidone in 100 ml mobile phase separately. Several aliquots of standard solutions of

ranitidine and domperidone were taken in different 100ml 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 and

domperidone of five different concentrations (80%, 90%, 100%, 110% and 120% of

target concentration) were prepared in the same way.

Preparation of sample solution

Twenty tablets of marketed brands of ranitidine hydrochloride and domperidone

were weighed separately. Their average weights were determined. Powder of tablets

equivalent to 75 mg of ranitidine hydrochloride and 5 mg of domperidone were weighed

and taken in a 100 ml volumetric flask, dissolved in mobile phase and shaken for about

10 minutes then 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.

Development and validation of HPLC method

Present study was conducted to obtain a new, affordable, cost-effective and

convenient method for HPLC determination of ranitidine hydrochloride and domperidone

in tablet dosage form. The experiment was carried out according to the official

specifications of USP–30, ICH- 1996, Global Quality Guidelines-2002 [3, 19-20]. The

method was validated for the parameters like system suitability, selectivity, linearity,

accuracy, precision, and robustness.

System suitability

In order to assess the system suitability of the method solution containing 100%

target concentration of ranitidine hydrochloride and domperidone were injected in six

replicate and various chromatographic parameters such as retention time, peak area

tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks

were determined. The method was evaluated by analyzing the parameters.

Selectivity



Selectivity was determined in the presence of common excipients used in the

tablet formulation. Sample containing 100% ranitidine hydrochloride and domperidone

were injected first. Then the samples of ranitidine hydrochloride and domperidone along

with two placebo formulations containing common excipients were injected to find out

the selectivity of the method.

Linearity

Linearity of the method was determined by constructing calibration curves.

Standard solutions of ranitidine hydrochloride and domperidone of different

concentrations level (80%, 90%, 100%, 110% and 120%) were used for this purpose.

Each measurement was carried out in six replicates to verify the reproducibility of the

detector response at each concentration level. The peak areas of the chromatograms were

plotted against the concentrations to obtain the calibration curves. The data were then

subjected to regression analysis to calculate calibration equation and correlation

coefficients.

Accuracy

Accuracy was determined by means of spike and recovery method. Both the drugs

at different level were added to placebo formulations. The accuracy was calculated as the

percentage of the analyte recovered by the assay.

Precision

The precision of the method was determined by intra-day (repeatability) and inter-

day (intermediate precision) study. intra-day precision (repeatability) was determined by

performing four repeated analysis of the three standard solutions (90%, 100% and 110%

of target concentration) on the same day and inter-day precision (intermediate precision)

of the method was assessed by carrying out the analysis of standard solutions (90%,

100% and 110% of target concentration) on three different days in the same laboratory.

The relative standard deviation (% RSD) was determined in order to assess the precision

of the method.

Robustness

Robustness of the method was determined by the analysis of the samples under a

variety of conditions making small changes in the buffer pH (2.8 and 3.0), in the



percentage of mobile phase component (water: methanol), in the flow rate (0.9 and 1.1

ml/ min) and in the temperature conditions (30 0C and 28 0C).

RESULTS AND DISCUSSION

Results of system suitability study are shown in Table 1 and 2. All the

chromatograms showed uniform retention time for ranitidine hydrochloride (2.742min

with % RSD 0.050) and for domperidone (5.047min with %RSD 0.102). The mean

theoretical plate count, based on USP tangent calculations , was 2843 for ranitidine

hydrochloride and 5632 for domperidone. %RSD is less than 2% for all the parameters.

TABLE 1: RESULTS OF SYSTEM SUITABILITY TEST OF RANITIDINE

HYDROCHLORIDE

Injection No.

Retention time

Area Theoretical

plates Tailing factor

1 2.740 1121047.00 2822.38 1.505

2 2.742 1120113.00 2841.69 1.497

3 2.743 1120620.00 2864.98 1.478

4 2.740 1121047.00 2822.38 1.505

5 2.742 1120113.00 2841.69 1.497

6 2.743 1120620.00 2864.98 1.478

Average 2.742 1120593.33 2843.017 1.493

SD 0.001 418.208 19.079 0.012

%RSD 0.050 0.037 0.671 0.831

TABLE 2: RESULTS OF SYSTEM SUITABILITY TEST OF DOMPERIDONE

Injection No.

Retention time

Area Theoretical

plates Tailing factor

Capacity factor

Resolution

1 5.04 109870.00 5619.059 1.275 0.835 9.699

2 5.05 110159.00 5641.195 1.279 0.838 9.731

3 5.05 110245.00 5637.248 1.279 0.845 9.744

4 5.04 109870.00 5619.059 1.275 0.835 9.724

5 5.05 110159.00 5641.195 1.279 0.838 9.75

6 5.05 110245.00 5637.248 1.279 0.845 9.78

Average 5.047 110091.33 5632.501 1.278 0.839 9.738

SD 0.005 175.705 10.560 0.002 0.005 0.027

%RSD 0.102 0.160 0.187 0.162 0.547 0.280

Chromatograms shown in figure 2 explain that excipients have no peaks at the

retention time of ranitidine hydrochloride and domperidone. On the other hand



excipients did not change the retention time of ranitidine hydrochloride and

domperidone which proves the selectivity of the method.

Figure 2 Chromatograms of ranitidine hydrochloride and domperidone along with placebo.

A linear relationship between peak areas (average peak area of six replicates)

versus concentrations was observed for ranitidine hydrochloride and domperidone in the

range of 80% to 120% of target concentration. Correlation coefficient was 0.999 both for

ranitidine hydrochloride and domperidone which prove that the method is linear for both

the drugs. Statistical data for linearity are presented in Table 3.

TABLE 3: LINEARITY DATA FOR RANITIDINE HYDROCHLORIDE AND

DOMPERIDONE

Analyte Slope (mean) Intercept ( mean)

Correlation coefficient

Ranitidine hydrochloride

23431 80559 0.999

Domperidone 34286 7727 0.999

Figure 3 and 4 show the result of accuracy study. The measured value was

obtained by recovery test. Spiked amount of both the drug were plotted against the

recovery amount. The correlation coefficient was 0.999 both for ranitidine hydrochloride

and domperidone. In case of ranitidine hydrochloride % recovery was 101.06% and in



case of domperidone % recovery were 99.61%. All the results indicate that the method is

highly accurate.

Figure 3 Accuracy curve of ranitidine hydrochloride

Figure 4 Accuracy curve of domperidone

The measurements for repeatability were done from 9.00 am to 6.00 pm. % RSD

of peak areas was calculated for various run (Table 4). The method is highly precise as %



RSD of peak area was 0.087-0.185 in case of ranitidine hydrochloride and 0.203-0.632 in

case of domperidone.

TABLE 4: REPEATABILITY RESULT OF RANITIDINE HYDROCHLORIDE

AND DOMPERIDONE

Conc. Level

9.00 am 12.00 noon

3.00 pm 6.00 pm Mean SD %RSD

Mean peak area of ranitidine hydrochloride (n= 4)

90% 1008942 1008558 1007102 1008942 1008386 875.205 0.087

100% 1121047 1120620 1120113 1124047 1121457 1768.531 0.158

110% 1233152 1232682 1232124 1237152 1233777 2288.379 0.185

Mean peak area of domperidone (n= 4)

90% 97883 99183 99221 98883 98792 624.802 0.632

100% 109870 110159 110245 109470 109936 349.629 0.318

110% 120757 121175 121270 120857 121015 246.215 0.203

The data of table 5 showed the average results of intermediate precision of

ranitidine hydrochloride and domperidone. Maximum % RSD is 0.274 in case of

ranitidine hydrochloride and 0.945 in case of domperidone.

TABLE 5: INTER-DAY VARIABILITY RESULTS OF RANITIDINE

HYDROCHLORIDE AND DOMPERIDONE

Ranitidine Hydrochloride Domperidone

Days Std conc. Mean area

(n=4)

Std deviation

%RSD Mean area

(n=4) Std

deviation %RSD

90% 1008812 978.546 0.097 99197 694.850 0.700

100% 1122047 768.531 0.068 110347 254.215 0.230 1st

110% 1232987 1288.379 0.104 121307 378.254 0.312

90% 1008499 775.205 0.077 99208 624.800 0.630

100% 1120597 2768.531 0.247 110308 349.600 0.317 2nd

110% 1232703 2548.379 0.207 121099 296.215 0.245

90% 1006960 375.205 0.037 97914 924.802 0.945

100% 1120225 1668.531 0.149 109854 349.629 0.318 3rd

110% 1232451 2148.379 0.174 120699 846.215 0.701

The robustness of a method is its ability to remain unaffected by small changes in

flow rate, amount of methanol, pH and temperature. The results of robustness in the

present method showed no significant changes due to small changes which are



summarized in Table 6. As the changes are not significant we can say that the method is

robust

TABLE 6: RESULTS FOR ROBUSTNESS TEST OF RANITIDINE

HYDROCHLORIDE AND DOMPERIDONE

Parameters Changes % Recovery of ranitidine

% Recovery

of domperidone

0.9 99.4 99.7 Flow rate (ml/min) 1.1 99.5 99.6

28 99.5 99.6 Column temperature

(0C) 30 99.7 99.5

55% 99.6 99.7 methanol variation 52% 99.6 99.6

3.00 99.7 99.7 pH of mobile phase 2.80 99.5 99.6

The proposed method was used to determine the potency of commercially

available tablets. Potency of Ranitidine tablets (150 mg) and domperidone tablet (10 mg)

were determined. Tablet powder was mixed and solution containing both drugs was

prepared. Six replicate determinations were carried out and the results are summarized in

Table 7.

TABLE 7: POTENCY OF RANITIDINE AND DOMPERIDONE IN TABLETS

DETERMINED BY HPLC

Ranitidine Hydrochloride Domperidone Run No.

Label amount

(mg)

Observed amount

(mg) Potency

Label amount

(mg)

Observed amount

(mg) Potency

R-1 150.00 150.21 100.14 10.00 9.98 99.80

R-2 150.00 149.61 99.74 10.00 10.01 100.10

R-3 150.00 149.59 99.73 10.00 9.96 99.60

R-4 150.00 149.47 99.65 10.00 9.97 99.70

R-5 150.00 149.31 99.54 10.00 10.02 100.20

R-6 150.00 149.44 99.63 10.00 9.87 98.70



CONCLUSION

The proposed high-performance liquid chromatographic method was found

accurate, precise and linear. The method is effective for the quality control and identity of

ranitidine hydrochloride and domperidone in pharmaceutical dosage forms. Moreover,

the lower solvent consumption along with the short analytical run time of 10 minutes

leads to an environmentally friendly chromatographic procedure that allows the analysis

of a large number of samples in a short period of time. Therefore, this HPLC method can

be used as a routine sample analysis.

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For Correspondence: S.M. Ashraful Islam Email: [email protected]

A SIMPLE RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF RANITIDINE HYDROCHLORIDE AND DOMPERIDONE IN COMBINE DOSAGE FORM

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