a novel rp-hplc method for analysis of paracetamol pseudo ephedrine, caffeine and

Journal of Pharmacy Research Vol.4.Issue 4. April 2011

Viswanath Reddy Pyreddy et al. / Journal of Pharmacy Research 2011,4(4),1225-1227

1225-1227

Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

*Corresponding author.Dr. Varaprasad Bobbarala

Chief Scientist,Translational Research Institute of Molecular Sciences, 2-35-72, Sai Narasimha Towers, MVP Colony, Sector-10,Visakhapatnam – 530017, AP, India.Tel.: + 91-9949129539E-mail:[email protected]

INTRODUCTION

A novel RP-HPLC method for analysis of paracetamol, pseudoephedrine, caffeine andchlorpheniramine maleate in pharmaceutical dosage formsViswanath Reddy Pyreddy 1, Useni Reddy Mallu1, Varaprasad Bobbarala2 and Somasekhar Penumajji 3

1Department of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India-515003.2Translational Research Institute of Molecular Sciences (TRIMS), 2-35-72, Sai Narasimha Towers, MVP Colony, Sector-10, Visakhapatnam – 530017, AP, India..3 Vivimed Labs Ltd, Veeranag Towers, Habsiguda, Hyderabad, AP, India

Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011

ABSTRACTOBJECTIVE: To develop a RP-HPLC method for the determination of Caffeine, Paracetamol, Pseudoephedrine hydrochloride and Chlorpheniramine maleate inpharmaceutical products.METHOD: HPLC analysis was carried out by using a C18 (150mm, 4.6mm and 3µm) column with gradient program. Mobile phase composedof sol-A: phosphate buffer (1.0g of KH

2PO

4 in to 1000ml of HPLC water and mixed) and sol-B: acetonitrile. Gradient program was 0-5min, sol-A: 94-94; 5-10min-

sol-A: 94-86; 10-15min- sol-A: 86-54; 15-17min- sol-A: 54-52; 17-20min- sol-A: 52-94 and 20-25min- sol-A: 94-94. Flow for mobile phase elution is 1.0ml per min;column oven temperature is maintained at 40°C and measured the absorbance at 210nm. HPLC water is used as diluent.RESULTS: Paracetamol, Pseudoephedrine HCl,Caffeine and Chlorpheniramine maleate were eluted at 6.5min, 9.7min, 12.0min and 16.2min, respectively. Percent relative standard deviation for five replicatestandard injections area is below 1.5. The method was validated with specificity, precision, linearity, accuracy, ruggedness and robustness. The response was linear overthe concentration range of 10 to 60 µg per mL for each ingredient, with correlation coefficients value is greater than 0.999. Recovery results were between 98 percentto 102 percent. CONCLUSION: The developed RP-HPLC method is single and reproducible, with high resolution and has been successfully applied for the analysisof Caffeine, Paracetamol, Pseudoephedrine hydrochloride and Chlorpheniramine maleate in pharmaceutical drug products.

Key words: Caffeine, Paracetamol, Pseudoephedrine HCl and Chlorpheniramine maleate, RP-HPLC method development and validation.

Caffeine Paracetamol

Pseudoephedrine HCl

Figure-1: Chemical structures ofall active ingredients.

Table-1: List of available combinations.

MATERIALS AND METHODReagents and solutions:High pure (not less than 98.5%) standards (Caffeine, Paracetamol, Pseudoephedrine hydro-chloride and Chlorpheniramine Maleate) were used for analysis. HPLC grade acetonitrile andwater, AR grade Potassium di-hydrogen ortho-phosphate were used for this study.

Apparatus:HPLC system : Waters (Alliance 2695 module-photo diode array detector) and Agilent

(1200series) make systems.Balance : Mettler Toledo analytical balance.pH meter : Lab India Instruments Pvt Ltd.Sonicator : Oscar Ultrasonics Pvt LtdMobile phase: Sol-A : Weighed accurately 1.0g of KH2PO4 in to 1000ml of HPLC water, mixed

and filtered with 0.45µ filter. Sol-B : AcetonitrileDiluent : HPLC water.

Chromatographic Conditions:Column : C18, 150 X 4.6mm, 3µmWave length : 210nmFlow rate : 1.0ml per minInj. volume : 10 µLColumn temp : 40°CRun time : 25minGradient program : (0-5min, sol-A: 94-94; 5-10min- sol-A: 94-86; 10-15min- sol-A: 86-

54; 15-17min- sol-A: 54-52; 17-20min- sol-A: 52-94 and 20-25min- sol-A: 94-94).

Dosage forms Composition

Tablets Paracetamol-125mg, 250mg, 500mg, 650mg and 1000mgSyrup Paracetamol-125mg per 5mLDrops Paracetamol-150mg per 1mLSuppository Paracetamol-80mg and 170mgSuspension Paracetamol-240mg per 5mLInjection Paracetamol-150mg,Infusion Paracetamol-1000mg per 100mLTablets Paracetamol-500mg and Caffeine-25mg

Paracetamol-6500mg and Caffeine-30mgTablets Chlorpheniramine maleate-4mgTablets Caffeine-30mg, Chlorpheniramine maleate-2mg, Paracetamol-500mg and Pseudoephe

drine HCl-60mgCaffeine-20mg, Chlorpheniramine maleate-4mg, Paracetamol-500mg and Pseudoephedrine HCl-60mg

Tablets Caffeine citrate-20mgTablets Caffeine-30mg, Chlorpheniramine maleate-2mg and Paracetamol-650mg

Caffeine-25mg, Chlorpheniramine maleate-4mg and Paracetamol-500mgTablets Acetaminophen-400mg, Caffeine anhydrous-30mg, Chlorpheniramine maleate-4mg

Chlorpheniramine maleate

Chlorpheniramine maleate has protective and therapeutic effects in case of dichlorvos poison-ing in chicks resembling that of atropine. Chemical structures of all ingredients represented infigure-1. List of available dosage forms are listed in table-1. Developed and validated a singleRP-HPLC method with specificity, linearity, accuracy and reproducibility.

Pharmaceutical drug products formulated with individual or combination dosage forms.These dosage forms require qualitative and quantitative analytical methods for the determina-tion of each active ingredient. In this present study, developed a single RP-HPLC method forthe analysis of Caffeine, Paracetamol, Pseudoephedrine hydrochloride and ChlorpheniramineMaleate in pharmaceutical products.

nervousness, excitement, restlessness, irritability, insomnia,diarrhea, nausea and indigestion. Paracetamol or acetami-nophen is used for the treatment of pain and fever reducer (7-9).It is a class of aniline analgesics drugs (10, 11). It has few anti-inflammatory effects in comparison to NSAIDs (12-14). Pseu-doephedrine (15-18) reduces tissue hyperemia, edema and nasalcongestion, colds or allergies. Adverse effects are hyperten-sion, sweating, and anxiety. Pseudoephedrine is useful forsuppressing of cough. Chlorpheniramine maleate (19-21) isused for cough and analgesia, allergy, hay fever, the commoncold, allergic conjunctivitis, vasomotor rhinitis.

Caffeine (1-6) is a xanthine alkaloid (psychoactive stimulant). Caffeine has some legitimatemedical uses in athletic training and in the relief of tension-type headaches. It is a drug that isnaturally produced in the leaves and seeds of many plants. It’s also produced artificially andadded to certain foods. Caffeine is defined as a drug because it stimulates the central nervoussystem, causing increased alertness. Caffeine gives most people a temporary energy boost andelevates mood. The source of caffeine is in tea, coffee, chocolate, many soft drinks and painrelievers. In its natural form, caffeine tastes very bitter but most caffeinated drinks have gonethrough enough processing to camouflage the bitter taste. Overdose of caffeine may cause



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Standard solution preparation:Weighed and transferred accurately about 40mg of each active ingredient working standard into 100ml volumetric flask add 75 ml of diluent and sonicated to dissolve the content and makeup to the volume with diluent. Further dilute 5.0ml of above solution in to 50ml with diluent(40ppm).

Test solution preparation:Prepared a test solution to get 40ppm of known concentration.

System Suitability solution:The tailing factor of all active peaks in standard solution is not more than 2.0. The percentrelative standard deviation for five replicate injections area is not more than 2.0%

Quantification: % of content =Area of test solution x Std. Concentration x average weight x Potency of standard Area of standard solution x sample concentration x Label claim

RESULTS AND DISCUSSION

Method optimization:A single and high resolution RP-HPLC method has been developed for the quantification ofCaffeine, Paracetmol, Pseudoephedrine hydrochloride and Chlorpheniramine Maleate in phar-maceutical formulations. Initial stage of method development, trials were performed with amixture of ammonium acetate buffer and acetonitrile with C18, 250mm column but separationwas not achieved. Finally the separation was achieved with phosphate buffer and acetonitrilewith simple gradient program. Diluent and standard solution chromatograms were representedin figure-2 and 3. Peak shape, resolution (not less than 4.0) and tailing factor (not more than1.5) were within the limit.

Figure-2: Diluent chromatogram

AU

0.00

0.10

0.20

0.30

Minutes0.00 5.00 10.00 15.00 20.00 25.00

Figure-3: Standard solution chromatogram

System suitabilitySystem suitability test parameters were established. Diluent, standard solution (five replicates-each active 40ppm) and test samples were injected in to the chromatographic system andcalculated the percent relative standard deviation for area and retention time. The results foundto be satisfactory and five replicate standard chromatograms represented in figure-4 andtabulated the results in table-2 and 3.

Table-2: System suitability (Area %RSD)

Active Ingredient Name Standard solution AreaInj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD

Paracetamol 1032506 1046366 1044300 1033925 1015106 1034440 1.20Pseudoephedrine HCl 739968 747581 742207 749599 727713 741413 1.16Caffeine 2464124 2417813 2484964 2487315 2460809 2463004 1.13Chlorpheniramine maleate 642030 626598 644278 645360 639925 639638 1.18

Figure-4: System suitability chromatograms

Table-3: System suitability (Retention time %RSD)

Active Ingredient Name Standard solution Retention time (min) Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD

Paracetamol 6.53 6.53 6.50 6.51 6.52 6.52 0.17Pseudoephedrine HCl 9.79 9.76 9.77 9.77 9.78 9.78 0.11Caffeine 12.08 12.08 12.07 12.09 12.08 12.08 0.07Chlorpheniramine maleate 16.12 16.25 16.23 16.24 16.26 16.25 0.06

Method validation:Validation of an analytical method is a necessary step in controlling the quality of quantitativeanalysis. Validation can be defined as the process by which it is established, by laboratorystudies that the analytical parameters of the method should meet the requirements for theintended analytical applications. Thus, with the background knowledge of linearity, accuracy,precision and robustness of the analytical method, it is relatively easy to derive the confidenceand the reliability of the analytical data obtained with it. Validated the developed method asper ICH and FDA (22- 26) guidelines with parameters like specificity, precision, accuracy,linearity and range, ruggedness and robustness etc.

Figure-5: Diluent and standard overlaid chromatograms (zoom).

Precision:Precision was evaluated by carrying out six different sample preparations for all individual andcombination dosage forms. Percentage relative standard deviation (% RSD) was found to beless than 1.5 for within a day and day to day variations, which proves that the developedmethod is precise and reproducible. Results were shown in Table-4.

Table-4: Precision Results.

Active Ingredient Name Sample preparations Average (%)Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6




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Linearity:The linearity of method was evaluated by analyzing six different concentrations of the standardsolution (mixture of all active ingredients). Calibration curve was constructed by plotting peakarea against concentration. The results were shown in table-5. The correlation coefficient valuefound to be within the limit 0.999. All six linearity chromatograms (different concentration)shown in figure-6 and linearity results tabulated in table-5.

Par

acet

amol

- 6

.530

Pse

udoe

phid

rine

HC

l - 9

.846

Caf

fein

e -

12.0

95

Chl

orop

heni

ram

ine

- 16.

264

AU

0.00

0.10

0.20

0.30

0.40

Minutes6.00 8.00 10.00 12 .00 14.00 16.00

Figure-6: Linearity chromatograms

Table-5: Linearity Results.

Active Ingredient Name Linearity solutions areaLevel-1 Level-2 Level-3 Level-4 Level-5 Level-6 Co-relation(10ppm) (20ppm) (30ppm) (40ppm) (50ppm) (60ppm) Coefficient

Paracetamol 220834 501226 775388 1053476 1324473 1615435 0.99996Pseudoephedrine HCl 154748 354840 548999 748824 945629 1153341 0.99996Caffeine 525802 1192511 1834561 2487823 3124217 3804145 0.99997Chlorpheniramine maleate 135283 309801 475354 648731 816912 996747 0.99995

Accuracy:To study the reliability and accuracy of the method, recovery experiments were carried out witha known quantity of the pure drug was added to the placebo sample at the level of 25% to 150%of the test concentration. The contents were determined from the respective chromatograms.The concentration of the drug product in the solution was determined. The mean recoverieswere in range of 98.0-102.0 % which shows that there is no interference from excipients. Table-6 represents the recovery results.

Table-6: Accuracy (recovery) Results.

Active Ingredient Name Spike level Average %25% 50% 75% 100% 125% 150% Recovery


Ruggedness:The ruggedness of the method was determined by carrying out the experiment on differentinstruments like waters HPLC and Agilent HPLC by different operators using differentcolumns of similar types. The percent RSD of six different preparations for all active ingredi-ents assay values with two different instruments, analysts and columns were within the limit.

Table-7: Robustness Results.Parameter System suitability

CONCLUSIONThe proposed single and high resolution RP-HPLC method was validated with linearity,precision, accuracy and specificity. The complete results proved that the developed method tobe convenient and effective for the determination of all active ingredients during the analysis ofthe bulk as well as pharmaceutical dosage forms.

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Source of support: Nil, Conflict of interest: None Declared

Robustness:Robustness of the method was determined by making slight changes in the chromatographic

conditions, such as flow rate and column temperature. It was observed that there were no majorchanges in the chromatograms, which demonstrated that the developed high resolution RP-HPLC method is rugged and robust. The robustness limit for mobile phase, flow rate andtemperature variations were well within the limit. Robustness results were tabulated in table-7.

Standard solution ——- 1.1-1.3 1.2-1.5Flow Rate +0.1mL per min 0.8-1.0 0.8-1.2

-0.1mL per min 0.9-1.1 0.9-1.2Column Oven Temperature +5°C 1.1-1.5 1.0-1.2

-5°C 1.2-1.4 1.1-1.3

Variation Tailing factor Percent (%) RSD

a novel rp-hplc method for analysis of paracetamol pseudo ephedrine, caffeine and

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