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DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD USED FOR SIMULTANEOUS DETERMINATION OF PARACETAMOL, CAFFEINE AND ORPHENADRINE CITRATE BY HPLC, IN PHARMACEUTICAL FORMULATION.

Byline: Ammara Nazir, Yasra Naseer, Sahar Raza and Roohi Shahid

ABSTRACT: New, rapid, simple, and precise reversed-phase high-performance liquid chromatographic method was validated and developed for the simultaneous determination of caffeine, Paracetamol and Orphenadrine citrate. Good resolution and quantization were attained on reversed-phase. The mixture containing KOH buffer: Acetonitrile = (60: 40 v/v) was chosen as Mobile Phase. The flow rate balanced was 1 mL/min, UV quantization was set at 220 nm. Satisfactory validation results were ascertained in terms of, percentage recovery (99.1) for Paracetamol, (98.2) for Caffeine and (98.9) for Orphenadrine Citrate precision and Specificity. This method was proved to be specific and accurate for the determination of mentioned drugs in pharmaceutical formulations along with their degradation products.

Key words: Orphenadrine citrate; Paracetamol; caffeine; Reversed-phase HPLC; Acetonitrile; Potassium Hydroxide

INTRODUCTION

Caffeine is a naturally occurring substance belongs to the methyl xanthine family and commonly found in seeds, leaves, and fruits worldwide. The systematic name of Caffeine is 1, 3, 5 Trimethylxanthine and the chemical formula is C8H10N4O2 (Fig: 1) [1]. For cardiovascular, respiratory and the central nervous system caffeine is used as a drug. It is also used with aspirin, decrease the cerebral eye blood flow and also used for headaches treatment [2]. The density of caffeine is 1.2/cm, melting point is 237 degC and boiling point is 178 degC (sublimes) [3,4,5]. Caffeine enhances heartbeat, blood pressure and causes an irregular heart rhythm. Paracetamol or 4-hydroxyacetanilide N-(4-hydroxyphenyl) acetamide is a para-aminophenol derivative (Fig: 2). It has antipyretic and analgesic properties and do not show any anti-inflammatory activities [6,7]. Paracetamol is a white crystalline powder with 151.16256 g/mole molecular weight and 169-171Co melting point.

Paracetamol reduces the temperature of patients suffering from fever and it is a mild painkiller. In different formulations many of these products are available for the relief of cold and influenza. Allergic skin reactions and gastrointestinal problems are often caused by paracetamol. Nephropathy can be caused by Paracetamol with drugs combinations containing phenacetin [8].

Orphenadrine citrate also known as dimethyl [2-(2-methylbenzhydrylox ethyl] amine dihydrogen citrate (Fig: 3). Molecular formula of orphenadrine citrate is C18H23NO.C6H6O. Orphenadrine is an anti-muscarinic, anticholinergic, centrally acting skeletal muscle relaxant [9]. Orphenadrine is a white crystalline powder and its molecular weight is 461.5 gm [10,11,12]. It is use to relieve pain due to spasm of voluntary muscle [13]. Orphenadrine citrate is used as an alternative to quinine nocturnal leg cramps [14].

MATERIALS AND METHODS

Materials

The Standards of Paracetamol, Caffeine and Orphenadrine Citrate were used of Analytical grade and obtained from Medipak Company Lahore. All reagents and chemicals such as Acetonitrile, methanol, HCl and KOH of HPLC grade were got from Merck, Karachi Pakistan. The tablets named Medigesic were obtained from SP, Pakistan. The tablets were commercially available and labeled to contain 450 mg Paracetamol, 30 mg Caffeine and 50 mg Orphenadrine Citrate.

Method Development

A RP-HPLC method was improved for the simultaneous determination of Caffeine, Paracetamol and Orphenadrine citrate. Equipments used for the development of method were HPLC system LC 100 UV visible spectrophotometer and Column for strongly acidic molecules Purespher RP-18 endcapped, 5.0 u, 100 Ao 4.6 x 250 mm. The development of Analytical method has been done step by step. Various types of solvents were used for checking solubility of Caffeine, Paracetamol and Orphenadrine citrate. From different compositions the Mobile phase selected was Potassium hydroxide buffer: Acetonitrile = (60: 40). The mobile phase was filtered with the 0.45 um Nylon membrane after sonication of 10 mins. The UV detection was checked at 220 nm. The flow rate was 1min/ml at ambient temperature.

Injection Volume was 20 uL.

Preparation of 0.1 M Buffer

In 1000 ml volumetric flask 4.0 g Potassium Hydroxide was added. The distilled water was used to dissolve it and volume was up to the mark with distilled water. pH was maintained to 3.8+ 0.1 with Hydrochloric Acid.

Preparation of Mobile Phase

In 1000 ml volumetric flask 600 ml of Potassium Hydroxide buffer was transferred and then 400 ml Acetonitrile to prepare a mobile phase.

Standard Solution of Caffeine

In 100 ml measuring flask 30 mg of caffeine was transferred, added 80 ml mobile phase, sonicated to dissolve and again mobile phase was added to made volume up to the mark. 10 ml of this standard solution was taken into another 100 ml measuring flask and mobile phase was used to make up the volume. It contains 30 ug/ml.

Standard Solution of Paracetamol

In 100 ml measuring flask 450 mg of Paracetamol was transferred, added 80 ml mobile phase, sonicated to dissolve and again mobile phase was added to made volume up to the mark.10 ml of this standard solution was taken into another 100 ml measuring flask the mobile phase was used to make up the volume. It contains 450 ug/ml.

Standard Solution of Orphenadrine Citrate.

In 100 ml measuring flask 50 mg of Orphenadrine citrate was transferred, added 80 ml mobile phase, sonicated to dissolve and again mobile phase was added to made volume up to the mark. 10 ml of this standard solution was taken into another 100 ml measuring flask the mobile phase was used to make up the volume. It contains 50 ug/ml.

Preparation of Sample

An accurately weigh amount of powdered drug equivalent to 450 mg of Paracetamol, 50 mg of Orphenadrine citrate and 30 mg of Caffeine was transferred in 100 ml volumetric flask and added 80 ml of mobile phase, sonicated to dissolve and again mobile phase was added to made volume up to the mark. From this 10 ml of sample solution was transferred into another 100 ml volumetric flask and up to mark with mobile phase and mixed well. The concentration of the above solution was Caffeine = 30 mg/ml, Paracetamol = 450 mg/ml and Orphenadrine citrate = 50 mg/ml

Preparation of Mixture of Standards

30 mg of Caffeine, 450 mg of Paracetamol and 50 mg of Orphenadrine citrate was added in 100 ml volumetric flask and 80 ml of mobile phase was added, sonicated to dissolve and volume was up to the mark with mobile phase. From this solution 10 ml of sample was transferred into another 100 ml volumetric flask and up to mark with mobile phase and mixed well. This solution contains 30 mg/ml of Caffeine, 450 mg/ml of Paracetamol and 50mg/ml of Orphenadrine citrate.

RESULTS AND DISCUSSION

A simple rapid and precise reverse phase HPLC method with UV detection was developed and validated for the simultaneous determination of Caffeine, Paracetamol and Codeine Phosphate in pharmaceutical formulations. Accuracy, precision and specificity were found from this validated method. Accuracy of the developed HPLC technique was assessed by ascertaining %age recovery and Relative standard deviation for distinctive dilution (80 %. 100 % and 120 %) of Paracetamol, Caffeine and Orphenadrine citrate. The accuracy of method was observed by standard addition method and the study was proceed in triplicate, at each level of concentration such as 80 %. 100 % and 120 %. The %age recovery and standard deviation of the %age recovery were calculated and results are mentioned in Table # 2.

To prove the sufficiency of the method, a laboratory mixture of Paracetamol, Caffeine and Orphenadrine citrate was prepared. This mixture was Prepared from the stock solutions that contains equal ratio of quantities in the dosage form. For quantitative determination of the mixture, a series of six replicas at 100 % concentration were prepared and twelve solutions for each concentration (twelve Replicas) were injected. There %age recovery, theoretical plates, tailing factor and resolution results are mentioned in shown # 1.

The specificity of the method, Paracetamol RS (single APIs 100 %), Caffeine RS (single APIs 100 %), and Orphenadrine citrate RS (single APIs 100 %) solutions were prepared. All of the APIs were mixed together. solutions of APIs, their mixture, placebo and sample solutions were run and results obtained. The chromatogram of mixture of standards was obtained which matched with others as shown in Fig 4.

DISCUSSION

Accuracy of the developed HPLC technique was assessed by ascertaining %age recovery and Relative standard deviation for distinctive dilution (80 %. 100 % and 120 %) of Paracetamol, Caffeine and Orphenadrine citrate. The normal Recoveries for all dilutions were inside indicated limit.

The R.S.D qualities were under 2 %, which showed that developed method was accurate and suitable for expected utilization. No interference was found from placebo at the retention time of Paracetamol, Caffeine and Orphenadrine citrate.

CONCLUSION

A reverse phase high performance liquid charomatographic method was developed for simultaneously determined the Paracetamol, Caffeine and Orphenadrine citrate in pharmaceutical formulations. The results proved that the developed method was simple, accurate and reproducible. The HPLC method was developed and validated by analytical parameters. The validation results shows good precision, accuracy, and specificity.

The simplicity of mobile phase, short run time, less expensive chemicals, isocratic mode of elution, good resolution and simple method of standard and sample solutions preparation was many advantages of the developed RP-HPLC method. The method accurately determined the amounts of all APIs in the presence of impurities and excipients.

Table No. 1: Results of precision of Caffeine, Paracetamol and Orphenadrine citrate

Drugs###Peak Area###%age Recovery###% RSD###T.F.

Caffeine###113262###100.06###48630###1.46

Paracetamol###634212323###100.12###0.286###1.63

Orphenadrine###634033###644860###6831###2824

Table No. 2: Results of % age recovery of Caffeine, Paracetamol and Orphenadrine citrate

Drugs###Theoretical###Weight of###Amount###Concentration###Peak Area###%age###% RSD###T. F.###Theoretica

###Contents (%)###Placebo###Added per 100###injected into###of Sample###Recovery###l Plates

###mL###HPLC

###80.2%###340 mg###24.04 mg###24 ug/mL###622177###80.63###0.66###2.44###22312

###100.2%###380 mg###30.06 mg###30 ug/mL###662105###100.38###0.61###3.64###23085

Caffeine

###120.2%###420 mg###36.06 mg###36 ug/mL###696775###120.07###0.68###4.47###23174

###80.2%###340 mg###360.9 mg###360 ug/mL###10566354.7###80.63###0.64###1.44###25281

Paracetamol###100.2%###380 mg###450.9 mg###450 ug/mL###12066315.5###100.38###0.51###1.78###06210

###120.2%###420 mg###540.9 mg###540 ug/mL###14066456.0###120.07###0.23###1.47###49843

###80.2%###340 mg###40.1 mg###40 ug/mL###100576###80.63###0.76###2.11###22321

Orphenadrine###100.2%###380 mg###50.1 mg###50 ug/ml###120588###100.38###0.71###1.80###22578

citrate

###120.2%###420 mg###60.1 mg###60 ug/mL###140572###120.07###0.68###6842###22432

REFERENCES

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[8]. Frank, E. Paracetamol - a curriculum resource, (1st Ed) pp. 1-24, Royal Society of Chemistry, Burlington house, Piccard illy, London, UK (2002)

[9]. Karim, A., Jumaa, F. A. A., Abdul, R., Hikmet, A. A. Formulation and Clinical Evaluation of Orphenadrine citrate as a Plain Tablet. J.Pharm.Sci., 15: 1-7(2006)

[10]. George, H., Cocolas. (1991). Wilson and Gisvoldorganic medicinal and pharmaceutical chemistry, (9th Ed) pp. 483, Lippincott Company, USA.

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[13]. Elenbass, J. K. (1980). Centrally acting oral skeletal muscle relaxants. J. Hosp. pharm., 37: 1313 - 1323. [14]. Latta, D., Turner, E. (1989). An alternative to quinine nocturnal legs cramps. Curr. Ther. Res., 45: 833 - 837.
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