Viswanath Reddy Pyreddy et al.

/ Journal of Pharmacy Research 2011,4(4),1225-1227

Research Article Available online through
ISSN: 0974-6943
www.jpronline.info
A novel RP-HPLC method for analysis of paracetamol, pseudoephedrine, caffeine and
chlorpheniramine maleate in pharmaceutical dosage forms
Viswanath Reddy Pyreddy 1, Useni Reddy Mallu 1, Varaprasad Bobbarala2 and Somasekhar Penumajji 3
1
Department of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India-515003.
2
Translational 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

ABSTRACT
OBJECTIVE: To develop a RP-HPLC method for the determination of Caffeine, Paracetamol, Pseudoephedrine hydrochloride and Chlorpheniramine maleate in
pharmaceutical products.METHOD: HPLC analysis was carried out by using a C18 (150mm, 4.6mm and 3µm) column with gradient program. Mobile phase composed
of sol-A: phosphate buffer (1.0g of KH2PO4 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 replicate
standard injections area is below 1.5. The method was validated with specificity, precision, linearity, accuracy, ruggedness and robustness. The response was linear over
the 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 percent
to 102 percent. CONCLUSION: The developed RP-HPLC method is single and reproducible, with high resolution and has been successfully applied for the analysis
of 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.

INTRODUCTION
Pharmaceutical drug products formulated with individual or combination dosage forms. Table-1: List of available combinations.
These dosage forms require qualitative and quantitative analytical methods for the determina- Dosage forms Composition
tion of each active ingredient. In this present study, developed a single RP-HPLC method for
the analysis of Caffeine, Paracetamol, Pseudoephedrine hydrochloride and Chlorpheniramine Tablets Paracetamol-125mg, 250mg, 500mg, 650mg and 1000mg
Maleate in pharmaceutical products. Syrup Paracetamol-125mg per 5mL
Drops Paracetamol-150mg per 1mL
Suppository Paracetamol-80mg and 170mg
Caffeine (1-6) is a xanthine alkaloid (psychoactive stimulant). Caffeine has some legitimate Suspension Paracetamol-240mg per 5mL
medical uses in athletic training and in the relief of tension-type headaches. It is a drug that is Injection Paracetamol-150mg,
naturally produced in the leaves and seeds of many plants. It’s also produced artificially and Infusion Paracetamol-1000mg per 100mL
Tablets Paracetamol-500mg and Caffeine-25mg
added to certain foods. Caffeine is defined as a drug because it stimulates the central nervous Paracetamol-6500mg and Caffeine-30mg
system, causing increased alertness. Caffeine gives most people a temporary energy boost and Tablets Chlorpheniramine maleate-4mg
elevates mood. The source of caffeine is in tea, coffee, chocolate, many soft drinks and pain Tablets Caffeine-30mg, Chlorpheniramine maleate-2mg, Paracetamol-500mg and Pseudoephe
relievers. In its natural form, caffeine tastes very bitter but most caffeinated drinks have gone drine HCl-60mg
Caffeine-20mg, Chlorpheniramine maleate-4mg, Paracetamol-500mg and Pseudoephe
through enough processing to camouflage the bitter taste. Overdose of caffeine may cause drine HCl-60mg
Tablets Caffeine citrate-20mg
Tablets Caffeine-30mg, Chlorpheniramine maleate-2mg and Paracetamol-650mg
Caffeine-25mg, Chlorpheniramine maleate-4mg and Paracetamol-500mg
Tablets Acetaminophen-400mg, Caffeine anhydrous-30mg, Chlorpheniramine maleate-4mg

MATERIALS AND METHOD

Reagents 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 and
Caffeine Paracetamol Pseudoephedrine HCl water, AR grade Potassium di-hydrogen ortho-phosphate were used for this study.
nervousness, excitement, restlessness, irritability, insomnia, Apparatus:
diarrhea, nausea and indigestion. Paracetamol or acetami- HPLC system : Waters (Alliance 2695 module-photo diode array detector) and Agilent
nophen is used for the treatment of pain and fever reducer (7-9). (1200series) make systems.
It is a class of aniline analgesics drugs (10, 11). It has few anti- Balance : Mettler Toledo analytical balance.
inflammatory effects in comparison to NSAIDs (12-14). Pseu- pH meter : Lab India Instruments Pvt Ltd.
doephedrine (15-18) reduces tissue hyperemia, edema and nasal Sonicator : Oscar Ultrasonics Pvt Ltd
congestion, colds or allergies. Adverse effects are hyperten-
Mobile phase:
sion, sweating, and anxiety. Pseudoephedrine is useful for
Sol-A : Weighed accurately 1.0g of KH2PO4 in to 1000ml of HPLC water, mixed
suppressing of cough. Chlorpheniramine maleate (19-21) is Chlorpheniramine maleate
and filtered with 0.45µ filter.
used for cough and analgesia, allergy, hay fever, the common Figure-1: Chemical structures of
cold, allergic conjunctivitis, vasomotor rhinitis. all active ingredients. Sol-B : Acetonitrile
Chlorpheniramine maleate has protective and therapeutic effects in case of dichlorvos poison- Diluent : HPLC water.
ing in chicks resembling that of atropine. Chemical structures of all ingredients represented in
figure-1. List of available dosage forms are listed in table-1. Developed and validated a single Chromatographic Conditions:
RP-HPLC method with specificity, linearity, accuracy and reproducibility. Column : C18, 150 X 4.6mm, 3µm
Wave length : 210nm
Flow rate : 1.0ml per min
*Corresponding author. Inj. volume : 10 µL
Dr. Varaprasad Bobbarala Column temp : 40°C
Chief Scientist, Run time : 25min
Translational Research Institute of Molecular Sciences, Gradient program : (0-5min, sol-A: 94-94; 5-10min- sol-A: 94-86; 10-15min- sol-A: 86-
2-35-72, Sai Narasimha Towers, MVP Colony, Sector-10, 54; 15-17min- sol-A: 54-52; 17-20min- sol-A: 52-94 and 20-25min-
Visakhapatnam – 530017, AP, India.
Tel.: + 91-9949129539 sol-A: 94-94).
E-mail:[email protected]
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 Viswanath Reddy Pyreddy et al. / Journal of Pharmacy Research 2011,4(4),1225-1227
Standard solution preparation: Table-3: System suitability (Retention time %RSD)
Weighed and transferred accurately about 40mg of each active ingredient working standard in Active Ingredient Name Standard solution Retention time (min)
to 100ml volumetric flask add 75 ml of diluent and sonicated to dissolve the content and make Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD
up to the volume with diluent. Further dilute 5.0ml of above solution in to 50ml with diluent Paracetamol 6.53 6.53 6.50 6.51 6.52 6.52 0.17
(40ppm). Pseudoephedrine HCl 9.79 9.76 9.77 9.77 9.78 9.78 0.11
Caffeine 12.08 12.08 12.07 12.09 12.08 12.08 0.07
Chlorpheniramine maleate 16.12 16.25 16.23 16.24 16.26 16.25 0.06
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 percent
relative 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 of
Caffeine, Paracetmol, Pseudoephedrine hydrochloride and Chlorpheniramine Maleate in phar-
maceutical formulations. Initial stage of method development, trials were performed with a
mixture of ammonium acetate buffer and acetonitrile with C18, 250mm column but separation
was not achieved. Finally the separation was achieved with phosphate buffer and acetonitrile
with simple gradient program. Diluent and standard solution chromatograms were represented
in figure-2 and 3. Peak shape, resolution (not less than 4.0) and tailing factor (not more than
1.5) were within the limit.

0.30

0.20
AU

Figure-4: System suitability chromatograms

0.10 Method validation:

Validation of an analytical method is a necessary step in controlling the quality of quantitative
analysis. Validation can be defined as the process by which it is established, by laboratory
0.00 studies that the analytical parameters of the method should meet the requirements for the
0.00 5.00 10.00 15.00 20.00 25.00 intended analytical applications. Thus, with the background knowledge of linearity, accuracy,
Minutes precision and robustness of the analytical method, it is relatively easy to derive the confidence
and the reliability of the analytical data obtained with it. Validated the developed method as
Figure-2: Diluent chromatogram per ICH and FDA (22- 26) guidelines with parameters like specificity, precision, accuracy,
linearity and range, ruggedness and robustness etc.

Figure-3: Standard solution chromatogram

Figure-5: Diluent and standard overlaid chromatograms (zoom).
System suitability
System suitability test parameters were established. Diluent, standard solution (five replicates- Precision:
each active 40ppm) and test samples were injected in to the chromatographic system and Precision was evaluated by carrying out six different sample preparations for all individual and
calculated the percent relative standard deviation for area and retention time. The results found combination dosage forms. Percentage relative standard deviation (% RSD) was found to be
to be satisfactory and five replicate standard chromatograms represented in figure-4 and less than 1.5 for within a day and day to day variations, which proves that the developed
tabulated the results in table-2 and 3. method is precise and reproducible. Results were shown in Table-4.

Table-2: System suitability (Area %RSD) Table-4: Precision Results.

Active Ingredient Name Standard solution Area Active Ingredient Name Sample preparations Average (%)
Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD Prep-1 Prep-2 Prep-3 Prep-4 Prep-5 Prep-6
Paracetamol 1032506 1046366 1044300 1033925 1015106 1034440 1.20 Paracetamol 99.81 99.36 98.85 99.3 99.36 98.7 99.23
Pseudoephedrine HCl 739968 747581 742207 749599 727713 741413 1.16 Pseudoephedrine HCl 100.16 99.00 100.1 99.62 100.1 99.09 99.68
Caffeine 2464124 2417813 2484964 2487315 2460809 2463004 1.13 Caffeine 99.75 99.74 99.81 99.78 99.81 99.18 99.68
Chlorpheniramine maleate 642030 626598 644278 645360 639925 639638 1.18 Chlorpheniramine maleate 99.41 99.92 99.24 99.74 100.33 99.78 99.74

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 Viswanath Reddy Pyreddy et al. / Journal of Pharmacy Research 2011,4(4),1225-1227
Linearity: conditions, such as flow rate and column temperature. It was observed that there were no major
The linearity of method was evaluated by analyzing six different concentrations of the standard changes in the chromatograms, which demonstrated that the developed high resolution RP-
solution (mixture of all active ingredients). Calibration curve was constructed by plotting peak HPLC method is rugged and robust. The robustness limit for mobile phase, flow rate and
area against concentration. The results were shown in table-5. The correlation coefficient value temperature variations were well within the limit. Robustness results were tabulated in table-
found to be within the limit 0.999. All six linearity chromatograms (different concentration) 7.
shown in figure-6 and linearity results tabulated in table-5.
CONCLUSION
0.40 The proposed single and high resolution RP-HPLC method was validated with linearity,
Pseudoephidrine HCl - 9.846

Chloropheniramine - 16.264
precision, accuracy and specificity. The complete results proved that the developed method to
0.30 be convenient and effective for the determination of all active ingredients during the analysis of

Caffeine - 12.095
Paracetamol - 6.530

the bulk as well as pharmaceutical dosage forms.

AU

0.20
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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1225-1227