Development and validation of stability indicating HPTLC method for simultaneous estimation of Domperidone maleate and Naproxen sodium in pharmaceutical formulations.

 

Lata P. Kothapalli*, Rahul R Shahane, Rabindra K. Nanda, Asha B. Thomas

Department of Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune 18, Maharashtra, India

*Corresponding Author E-mail: lata_pk@yahoo.co.in

 

A high-performance thin-layer chromatographic method was developed and validated for simultaneous determination of Domperidone maleate (DOM) and Naproxen Sodium (NAP) in combined dosage formulation. The chromatography was performed on pre-coated silica gel 60 F 254 plates using Toluene: Dichloromethane: Ethyl acetate: GAA (5:3:2:0.1 v/v/v/v) as mobile phase. Densitometric evaluation of the chromatograph was performed at 272 nm. The two drugs were satisfactorily resolved with Rf values 0.12 ± 0.02 and 0.70 ± 0.03 for DOM and NAP, respectively. The accuracy and reliability of the method was assessed by studying linearity for DOM (10–60 ng/band) and for NAP (250-1500 ng/band), presicion and accuracy and specificity, in accordance with ICH guidelines. The method determined the two drugs simultaneously from dosage forms without any interference of the tablet excipients. DOM and NAP were also subjected to acid, base, oxidation, heat and photo-degradation studies. The degradation products obtained were well resolved from the pure drugs when subjected to acid and alkali degradation and oxidative stress. The developed method could effectively separate the drugs from its degradation products and hence can be used as a stability-indicating assay.

 

 

 

INTRODUCTION:

Domperidone maleate (DOM0 is a specific blocker of dopamine receptors. It speeds gastrointestinal peristalsis, causes prolactin release, and is used as antiemetic and is a tool in the study of dopaminergic mechanisms. Chemically it is 5-chloro-1-(1-[3-(2-oxo-2,3-dihydro-1-H-benzo[d]imidazole-1-yl)propyl]piperidine-4-yl)-1-H-benzo[d]-imidazole-2-(3H)-one.(Fig.1)^[1]

 

Fig. 1: Chemical structure of Domperidone Maleate

 

Naproxen sodium (NAP) belongs to class of aryl acetic acid derivatives used as nonsteroidal anti inflammatory drugs (NSAIDs). Naproxen has analgesic and antipyretic properties. Chemically it is (+)-(S)-2-(6-methoxynapthalene-2-yl) propanoic acid. ( Fig 2) ^[2]

 

Fig. 2: Chemical structure of Naproxen Sodium

 

Literature survey revealed HPTLC,^[3] HPLC^[4,5,6] and Spectrophotometry,^[7,8]  methods for analysis of Domperidone maleate and Naproxen Sodium in combination. The present work describes a simple, precise, rapid, selective, economic, stability indicating high-performance thin-layer chromatography procedure for determination of Domperidone Maleate and Naproxen Sodium in pharmaceutical formulation.

 

Materials and methods

Pure drugs, Domperidone maleate and Naproxen Sodium were obtained as gift samples from Wockhardt Aurangabad. Combined dosage formulation Naxdom 250 tablets of Sun Pharmaceutical Industries Ltd contain 10 mg of Domperidone maleate and 250 mg of Naproxen sodium, which were purchased from local market. All other chemicals and reagents used during analysis were of HPLC and AR grade.

 

Preparation of standard stock solution

Accurately about 10 mg of Domperidone maleate (DOM) and 250.0 mg of Naproxen Sodium (NAP) were weighed and transferred to 100.0 ml volumetric flask, added 40 ml of methanol and ultrasonicated  for 10 minutes, volume was then made up to the mark with methanol (100 µg/ml DOM and 2500 µg/ml NAP). From the above solution, 1.0 ml of solution was diluted to 10.0 ml with methanol (10µg/ml DOM and 250µg/ml NAP).   

 

Selection of mobile phase

Aliquots of standard stock solutions were applied on Merck HPTLC plates in the form of band (band size: 6 mm) and the plates were run in different solvent systems in an attempt to achieve the desired Rf value. With a compact band, several trials were made by using different solvent systems containing non-polar solvents and relatively polar like toluene: methanol, Toluene: Dichloromethane: Ammonia, Methanol: Ethyl acetate: Glacial acetic acid (GAA)etc., in different proportions were taken in order to determine the best conditions for the effective separation of Domperidone and Naproxen. Among the different mobile phase combinations tested Toluene: Dichloromethane: Ethyl Acetate: GAA (5:3:2:0.1 v/v/v/v) gave resolved, compact bands which showed symmetrical peaks and desired Rf value. The Rf values were 0.12 ± 0.02 for DOM and 0.70 ± 0.03 for NAP, respectively.

 

Selection of analytical wavelength for densitometry evaluation

Standard stock solution (4 µl) of both the drugs were individually applied on the plate with the help of CAMAG LINOMAT-V automatic sample applicator, the plate was chromatographed in twin-trough glass chamber saturated with mobile phase for 15 minutes. After chromatographic development, the plate was removed and air dried. The bands on the TLC plate were scanned over the wavelength range of 200-700 nm. From the overlain spectra (Fig. 3), it was observed that Domperidone Maleate and Naproxen Sodium both exhibited absorbance at about 272 nm which was selected as the analytical wavelength for further analysis.

        

Fig. 3: Overlay spectrum of Domperidone and Naproxen

 

Instrumentation and optimized chromatographic conditions:

HPTLC studies were done using 10 cm × 10 cm aluminium backed plates coated with 0.2 mm layers of silica gel 60 F254 (E. Merck Germany). Samples were applied on the plates as 6 mm bands, 5 mm apart, under the continuous flow of nitrogen using Camag Linomat V sample applicator fitted with a 100 micro liter syringe (Hamilton, Bonaduz, Switzerland). A constant application rate of 150 nL s-1 was used. Linear ascending development of the plates to a distance of 80 mm was performed with Toluene: Dichloromethane: Ethyl acetate: glacial acetic acid (5:3:2: 0.1 v/v/v/v) as mobile phase in a twin-trough glass chamber previously saturated with mobile phase vapor for 15 min at room temperature (25°C). After chromatographic development, the plate was air dried and scanned at 272nm by means of a Camag TLC scanner III, controlled by WINCAT’s software version 4, in reflectance-absorbance mode using the deuterium lamp. The slit dimensions were 5 mm × 0.45 mm and the scanning speed was 20 mm s-1. Concentrations of the compounds chromatographed were determined from the intensity of the diffused light and peak areas were used for evaluation.

 

Fig. 4: Chromatogram showing standard Domperidone (Peak 1) and Naproxen (Peak 2)

 

Calibration plots for Domperidone Maleate and Naproxen Sodium

From standard stock solution, 1.0 ml each of DOM and NAP was taken in 10 ml volumetric flask and volume made to 10.0 ml with methanol (Concentration: 10 µg/ml of DOM and 250 µg/ml of NAP). The diluted solution was applied on the TLC plate in increasing range with the help of micro syringe using LINOMAT-V automatic sample applicator. The plate was then developed and scanned under the above mentioned chromatographic conditions. Peak area was recorded for each drug concentration and the calibration curves of the concentration Vs peak area were constructed for both the drugs.       

 

Preparation of sample solution of marketed formulation

A combined oral dosage formulation containing Domperidone maleate (10 mg) and Naproxen Sodium (250mg) is available in the market with a brand name Naxdom 250. Twenty tablets were weighed and crushed to fine powder and sample equivalent to accurately about 10 mg of DOM and 250 mg of NAP was transferred to 100.0 ml volumetric flask and 40 ml methanol was added and ultrasonicated for 15 min. Volume was then made up to the mark with methanol. Further appropriate dilution were done in the concentration ratio of 1:25 (10 ng/band: 250 ng/band of DOM and NAP, respectively), applied on HPTLC plates and analysed under the optimized chromatographic conditions. Amount of drug present in sample was calculated by comparing the mean peak area of sample band with that of the standard.

 

Method validation

The method was validated in compliance with ICH guidelines ^[9].

Accuracy

An accurately weighed quantity of a sample equivalent to about 10 mg Domperidone and 250 mg Naproxen was transferred individually in nine different 100.0 ml volumetric flasks, added 8.0 / 200.0 mg, 10./250.0 mg and 12.0/300 mg of standard sample of DOM/NAP to the sample for 80 %, 100 % and 120 % level of recovery, respectively. Further 1 ml was diluted to 10 ml with methanol. Appropriate dilutions were prepared in triplicate and analyzed. Accuracy was determined and expressed as percent recovery.

 

Precision

To ascertain repeatability and reproducibility of the method precision studies were performed. Sample solution was prepared and analyzed in similar manner as described under analysis of the marketed formulation. Intra-day precision was determined by analyzing a sample solution at three different time intervals on the same day and inter-day precision was determined by analyzing a sample solution on three consecutive days.

 

Robustness

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. By introducing small changes in the mobile phase composition, mobile phase volume, duration of chamber saturation with mobile phase, time from spotting to development (15 min, 40 min and 1.2hrs) and time from development to scanning (15min, 40 min and 1.2 hrs), the effects on Rf value and peak area of drugs were examined. The composition of mobile phase was changed slightly (± 0.1 ml for component). TLC plates with standard and sample bands were run with mobile phases of composition, Toluene: Dichloromethane: ethyl acetate (4.9:2.9:1.9.v/v/v and 5.1:3.1:2.1. v/v/v) while content of GAA was maintained constant. Mobile phase volume and duration of chamber saturation were varied at 10.0 ml ±1.0 ml (9, 10 and 11 ml) and 15 min ± 20% (12, 15 and 18 min), respectively.

 

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ were separately determined based on the standard deviation of response of the calibration curve. The standard deviation of y-intercept and slope of the calibration curves were used to calculate the LOD and LOQ.

 

Forced degradation studies

In forced degradation studies, the individual drugs and sample were exposed to following stress conditions like acidic (1M HCl), alkaline (1M NaOH), and oxidation (3 % H₂O₂). Standard drugs and sample were taken in the flasks and were refluxed in a water bath at 80°C for 3hr. For heat and photo degradation sample was kept at 60°C and in UV light (254 nm) for 24 hr, respectively. After the respective time intervals all the flasks were removed and allowed to cool. The samples were then analyzed in similar manner as described under analysis of DOM and NAP in marketed formulation.

 

RESULT AND DISCUSSION:

Linearity

Peak areas were found to have good linear relationship with the concentration than the peak heights. Domperidone maleate and Naproxen sodium were found to give linear detector response in the concentration range of 10-60 ng/band and 250-1500 ng/band, respectively. The coefficients of correlation for DOM and NAP calibration curves were found to be 0.9992 and 0.999, respectively (Fig. 5 and 6).

 

Fig. 5: Standard calibration curve for Domperidone maleate

 

Fig. 6: Standard calibration curve for Naproxen sodium

 

Analysis of marketed formulation

Analysis of marketed formulation containing Domperidone Maleate and Naproxen sodium was carried out and results are expressed as percentage amount of the label claim. There was no interference from the excipients. The DOM and NAP content was found to be close to 100 % and the results are summarized in Table no. 1. The low SD value indicated the suitability of this method for routine analysis.

 

Accuracy studies

To ascertain the accuracy of proposed method, recovery studies were carried out by standard addition method and the results are expressed as percent recovery. The mean percentage recovery for each compound was calculated at each concentration level and reported with its standard deviation. The percentage recovery at three levels (80 %, 100 % and 120 %) for both the drugs was found to be satisfactory (Table no. 2) Indicating the accuracy of developed method.

 

 

Table no. I: Results of analysis of marketed formulation

Brand Name	Label Claim (mg)		Amount  Estimated* (mg)		% Label Claim*, SD
	DOM	NAP	DOM	NAP	DOM	NAP
Naxdom 250	10	250	9.96	249.92	99.65± 0.31	99.96± 0.98

*Mean of six determinations

 

 

Table no.2 :  Results of recovery studies

% Level of Recovery	Amount of drug added (mg)		Percent recovery*
	DOM	NAP	DOM	NAP
80 100 120	8.0 10.0 12.0	200.0 250.0 300.0	101.22 99.53 100.73	101.29 99.31 101.30

*Mean of three determinations.

 

 

Table no. 3: Results of precision studies

Drug	% Label claim*	S. D.	R.S.D
Intra-day precision
DOM	10.04	0.164	0.162
NAP	248.96	1.155	1.161
Inter day precision
DOM	10.09	0.242	0.240
NAP	248.85	1.169	1.175

*Mean of three determinations

 

Table no. 4: Results of robustness studies

*Mean of three determinations

 

 

 

Precision

Precision was evaluated by carrying out independent sample preparation of a single lot of formulation 3 times on same day and on 3 different days. Standard deviation and percentage relative standard deviation (% RSD) was found to be less than 2% for intraday and inter day precision (Table no. 3) indicating the repeatability and reproducibility of the developed method.

 

 

Robustness

The effect of change in mobile phase composition (± 0.1 ml), chamber saturation period (± 20 %), time from application to development (0, 15, 40, 1.2 hr), time from development to scanning (0, 10, 30, 1 hr) on the Rf value of drugs was studied. The method was found to be unaffected by small changes in method parameters with % RSD for Rf values under varied method parameters less than 2 %. The developed method is considered to be robust (Table no. 4).

 

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD values for DOM and NAP was found to be 8 ng/band and 61 ng/band respectively, and LOQ values for DOM and NAP was found to be 25 ng/band and 187 ng/band respectively. The low LOD and LOQ values indicate that the method is sensitive.

 

Forced degradation studies

Intentional degradation of DOM and NAP was tried under different stress conditions such as acid hydrolysis, alkaline hydrolysis, oxidation, heat and exposure to UV radiations. DOM and NAP was found to degrade in acid, alkaline and oxidized conditions. The percent assay of active substance and the Rf values of degradation products are given in Table no 5. Densitogram of acid, alkaline and H₂O₂ treated samples are shown in Fig. 7, 8 and 9 respectively.

 

 

Fig. 7: Chromatogram of 1M HCl treated sample

          

Fig. 8: Chromatogram of 1M NaOH treated sample

 

Fig. 9: Chromatogram of H₂O ₂ (3%) treated tablet sample

 

 

Table no. 5: Results of forced degradation study

Stress condition	Percent assay of active substance		Rf value of degraded product
	DOM	NAP
Acid (1M HCl)	70.82	74.63	0.06, 0.10, 0.28, 0.66, 0.75
Alkali (1M NaOH)	80.5	77.59	0.09, 0.64, 0.70
Oxide (3.0 % H2O2)	75.54	72.74	0.09, 0.20, 0.43, 0.59, 0.78.
Neutral Hydrolysis	98.17	99.36	-
Heat (60 ºC)	99.43	99.58	-
UV-Exposure (254nm)	95.96	97.00	-

 

CONCLUSION:

Introducing HPTLC in pharmaceutical analysis represents a major step in terms of quality assurance. The proposed HPTLC method gives well resolved peaks for Domperidone maleate and Naproxen sodium. Based on the results obtained it is concluded that the method is sensitive, accurate, precise and reproducible, where Domperidone maleate and Naproxen sodium can be determined in bulk and in pharmaceutical formulation without interference from the excipients. The proposed HPTLC method was also able to selectively quantitate Domperidone maleate and Naproxen sodium in presence of the degradation products obtained in forced degradation study. Hence, the method can be employed as a stability indicating one. ICH guidelines were followed throughout method validation and the suggested method can be applied for routine quality control analysis of pharmaceutical formulation containing these drugs.

 

ACKNOWLEDGEMENT:

The authors are thankful to Principal, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune for providing necessary facilities. Authors are also thankful to Wockhardt Pvt. Ltd. Aurangabad Maharashtra, for providing gift sample of pure drugs.

 

REFERENCES:

1.           Indian Pharmacopoeia, Govt. of India Ministry of health and family welfare. Vol.-II. Gaziabad: Published by the Controller of Publication; (2007). p. 1246

2.           British Pharmacopoeia 2008 Volume I. London: Stationery Office; 2007. Naproxen Sodium; p. 40-41.

3.          Pawar  SM,  Patil BS,  Patil RY. Validated HPTLC method for simultaneous quantitation of domperidone maleate and naproxen sodium in bulk drug and formulation. Eurasian J Anal Chem, 5(3): (2010); 284-292.

4.          Shozan M, Ahsanul H, Safiqul I, Ashraful I. Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Domperidone and Naproxen in Tablet Dosage Form. Journal of Applied Pharmaceutical Science, 1(7): (2011); 145-148.

5.          Pawar SM, Fegade DJ, Choudhary YR. Validated RP-HPLC Method for Simultaneous Quantitation of Domperidone Maleate and Naproxen Sodium in Bulk Drug and Formulation. Der Pharmacia Lettre, 2(5): (2010); 229-236.

6.          Shekhar CE, Kumar RS, Sankar RM, Prasanti P. Simultaneous Estimation of Naproxen Sodium and Domperidone Maleate in Bulk and Pharmaceutical Dosage Form by Modified RP-HPLC Method. International Journal of Pharmaceutical and Chemical Sciences, 1(4): (2012); 615-1623.

7.          Lofty HM, Amer SM, Zaazaa HE, Mostafa NS. Spectrophotometric Methods for Quantitative Determination of Binary Mixture of Naproxen Sodium and Domperidone Maleate. Austin Journal of Analytical and Pharmaceutical Chemistry, 2(3): (2015); 1044.

8.          Singh S, Sharma S, Yadav A, Gautam H. Simultaneous estimation of naproxen and Domperidone using uv spectrophotometry in Tablet dosage form. Bulletin of Pharmaceutical Research; 3(2): (2013); 66-70.

9.          Guidelines prepared within the international conference on harmonization of technical requirements for the registration of pharmaceuticals for human use. Validation of analytical procedures: Text and Methodology Q2(R1), (2005); 1-13.

 

 

 

Received on 13.07.2016         Modified on 15.07.2016

Accepted on 28.07.2015         © AJRC All right reserved