31 International Journal of Chromatographic Science 2013, 3(2): 31-34

Original Article

QUANTITATIVE DETERMINATION OF PARACETAMOL AND CAFFEINE

FROM FORMULATED TABLETS BY REVERSED PHASE-HPLC

SEPARATION TECHNIQUE

R. Chandra1, K. Dutt Sharma

Department of Pharmaceutical Chemistry, Dolphin P.G. Institute of Biomedical and Natural Sciences, Dehradun-248001,

Uttarakhand, INDIA

*Corresponding author. Email: rajvimalchandra@gmail.com

Received 10 July 2013; accepted 20 August 2013

Abstract

This RP-HPLC method has been described for the determination binary formulated tablets of paracetamol and caffeine.

However, none have been cross validated using formulated products. The present study depicts an accurate, stable, rapid,

simple, precise and reducible reversed-phase high performance liquid chromatographic (RP-HPLC) separation method.

The present isocratic method was carried out on C18-column with mobile phase methanol and water in the ratio of 40:60 (by

volume) at the flow rate 1.0 mL/minute. The detection was carried out at λmax=243 nm. The retention time of paracetamol

and caffeine were 3.03 and 4.23 minutes, respectively. Under the optimal condition, the linearity were found for

paracetamol R2=0.999 and for caffeine R

2=0.994. The limit of detection and limit of quantification for paracetamol were

computed 0.04 and 0.12 µg/mL and for caffeine 0.05 and 0.15 µg/mL, respectively. The recoveries of paracetamol and

caffeine were in the range of 99.62-99.45 % and 104.48-100.56 %. The proposed method was successfully validated to

determine paracetamol and caffeine from its formulated tablets.

© 2013 Universal Research Publications. All rights reserved

Key words: Development, Paracetamol, Caffeine, RP-HPLC, Validation

1. INTRODUCTION Paracetamol is used as antipyretic, analgesic and anti-

inflammatory. Chemically, it is 4-Hydroxy Acetanilide.

This is available in combination form in different

formulations. The antipyretic, analgesic and anti-

inflammatory effect of paracetamol is due to inhibiting

prostaglandin synthesis cyclooxygenase-1(COX-1) and

cyclooxygenase-2 (COX-2) [1-3]. It is prevent to fever,

headaches, pain of arthritis, aches, colds, flu and period

pain. Paracetamol in combination form with caffeine used

in migraine attack, child birth and avoid postpartum

hemorrhage [4]. Caffeine is the most wanted drug of the

world [5]. The chemical name of caffeine is 1, 3, 7-

trimethylxanthine. Caffeine is used as a stimulant, due to

acts on the central nervous system [6]. Paracetamol in

formulated combination form with caffeine and other drugs

has been validated various methods with spectrophotometry

and high performance liquid chromatography [7-14]. The

main aim of this study was to develop a new RP-HPLC

method for determination of paracetamol and caffeine from

formulated tablets, in accordance with International

conference harmonized guidelines [15].

2. EXPERIMENTAL

2.1 Chemicals and Reagents

Paracetamol and caffeine reference standard (label claim

99.7%pure) was purchased from Ranbaxy Pharmaceutical

Ltd. Tablets of paracetamol and caffeine with 250 mg and

100 mg purchased from Lupin Pharmaceutical Ltd. HPLC

grade methanol and water were purchased from Merck

India Limited and 0.45µm nylon membrane filter was

supplied by Pall life Sciences, Mumbai.

2.2 Instrumentation

The instrument, a reversed phase-high performance liquid

chromatograph was used for method development. The

chromatograph consisting, a binary pump, column

thermostat and a UV-detector. The eluent was analyzed

using a C18 column at wavelength (λmax) 243 nm with

mobile phase methanol and water in the ratio of 40:60 (by

volume). Injection volume was used 20 µL. The mobile

phase was filtered through 0.45µm nylon filter membrane

followed by sonicate for five min prior to use.

2.3 Preparation of Stock Solution

Stock solution containing 250 µg/mL of paracetamol and

100 µg/mL of caffeine were prepared separately in mobile

phase. The stock solutions were filtered through a 0.45 µm

nylon membrane followed by sonicate for five minutes.

Serial dilutions were prepared by appropriate dilution of the

stock solutions with mobile phase.

2.4 Methods Validation

The methods were validated according to ICH guidelines

Available online at http://www.urpjournals.com

International Journal of Chromatographic Science

Universal Research Publications. All rights reserved

32 International Journal of Chromatographic Science 2013, 3(2): 31-34

with respect to linearity, specificity, precision, and system

suitability test, limit of detection (LOD) and limit of

quantification (LOQ) [15].

2.5 Linearity

For the measuring linearity were prepared serial dilutions

of 5, 10, 20, 25, 50 and 100 µg/mL from the stock solution

of paracetamol and caffeine. A total volume of 10 mL was

maintained with mobile phase methanol and water. These

different serial dilutions were filtered through a 0.45µm

nylon membrane and sonicate. The each solution of 20 µL

was injected into the column in thrice. The calibration

curves were obtained by plotting peak areas versus known

concentrations in µg/mL.

2.6 Specificity

Specificity was determined with exepient of formulated

tablets. An equivalent weight was taken and solution

prepared similarly to the sample solution. The prepared

solution was determined as per the described method. After

determination was not reported any interference with

exepient at the retention time of the peaks of paracetamol

and caffeine. Therefore, it is concluded that the method is

specific.

2.7 Accuracy

The accuracy of the method was determined by recovery

method. The recovery was checked at the five theoretical

concentrations levels 10, 20, 40, 50 and 100 µg. The

chromatograms were recorded and the percentage recovery

was calculated.

2.8 System Suitability test

The Reproducibility of sample was checked of the system

to measurement of peak area and was carried out using

three replicates of same concentration of standard and

sample, respectively.

2.9 Limit of Detection (LOD) and limit of

Quantification (LOQ)

It is a lowest response of the instrument at lowest

concentration, so the detectable signal value called as limit

of detection and quantifiable noise value called as limit of

quantification. It was measured by signal to noise ratio. To

determine the limit of detection (LOD) and limit of

quantification (LOQ), to prepare three replications [16-19]

of low concentrations serial dilutions of mixed standard of

paracetamol and caffeine from the standard stock solution.

3. Results and Discussion

3.1 Selection of mobile phase

It was a basic need of liquid chromatography. To select the

mobile phase various composition of mobile phases were

checked with water and methanol in different ratio 25:75,

35:65 and 45:55 (by volume) on C18

column at wave length

243 nm and methanol and water 20:80, 30:70 and 40:60 (by

volume) on C18

column at wave length 243 nm. The mobile

phase methanol and water in the ratio of 40:60 (by volume)

was selected. At this mobile phase was obtained suitable

retention time and peak area of both drugs with better

resolution.

3.2 Chromatographic Conditions Under optimal experimental conditions, a mobile phase

methanol and water in the ratio of 40:60 (by volume), and a

flow rate of 1.0 mL/min, paracetamol and caffeine depicted

a well defined chromatographic separation (resolution

factor more than 1.423±0.07) within a run time of 6 min.

The retention times of paracetamol and caffeine 3.03

minutes±0.36 and 4.23 minutes±0.39, respectively. Figure

1 depicts chromatograms of paracetamol and caffeine in

binary mixture.

Figure 1. Chromatogram of binary mixture

3.3 System suitability test To establish the chromatographic conditions were

performed system suitability test (SST) during the

development and optimization of the method. The test was

performed by injecting the standard mixture in triplicate

and the various parameters retention time, tailing factor,

resolution factor and theoretical plates were computed as

reported by USP [20] and International conference

harmonized guidelines. System suitability parameters were

shown in table 1.

3.4 Linearity

Linearity was determined by the regression analysis. The

calibration curves were plotted between known

concentrations and average peak areas. The method was

linear with a correlation co-efficient (R2) 0.999 and caffeine

having correlation co-efficient (R2) 0.994. The result was

shown in table 2.

3.5 Limits of Detection (LOD) and Limit of

Quantification (LOQ)

The limit of detection (LOD) and limit of quantification

(LOQ) were calculated using the signal-to-noise-ratio of 3

and 10. The limit of detection (LOD) and limit of

quantification (LOQ) values were found for paracetamol

0.04 and 0.12 µg/mL and for caffeine 0.05 and 0.15 µg/mL,

respectively.

3.6 Specificity

The specificity of the method was determined by checking

the interference with the components from placebo. No

interference was observed for any of the components like

excipients of both drugs (figure 1).

3.7 Accuracy

The accuracy of the method was computed by

determination of recovery for five concentrations. The

amount of paracetamol and caffeine recovered and then

percentage of drug content calculated. The mean recovery

33 International Journal of Chromatographic Science 2013, 3(2): 31-34

Tab. 1. Statistical Summary of validation system suitability parameters.

Parameters Paracetamol Caffiene

Mean SE Mean SE

Retention time 3.03 minutes ± 0.36 4.23 minutes ± 0.39

Tailing factor 0.82 ± 0.06 0.81 ± 0.07

Resolution factor 1.42 ± 0.07 1.33 ± 0.06

Theoretical plates 475.34 ± 0.44 2012 ± 0.05

SE= Standard Error (±)

Tab. 2. Statistical summary of Linearity, limit of detection and limit of quantification.

Parameters Paracetamol Caffeine

Number of samples per curve 6 6

Correlation range (µg/mL) 10-100 10-100

Regression equation Y=4629x+34584 Y=860.4x-1056

Regression coefficient R2=0.999 R

2=0.994

Limit of detection (µg/mL) 0.04 0.05

Limit of quantification(µg/mL) 0.12 0.15

Tab. 3. Recovery experiment for paracetamol and caffeine

Paracetamol Caffeine

Added in µg Found Recovery % Added in µg Found Recovery %

10 9.96 99.62 10 10.44 104.48

20 19.14 95.72 20 20.93 104.68

40 39.9 99.96 40 38.21 95.53

50 51.4 102.89 50 49.83 99.67

100 99.45 99.45 100 100.56 100.56

Mean=99.53

SD=2.55

CV%=2.56

Mean=100.98

SD=3.79

CV%=3.75

SD=standard deviation

CV%=coefficient variation percentage

of paracetamol and caffeine were found 99.53 % and

100.98 % with less than 10 % coefficient variation (table 3)

[7]. Hence, the results were statistically significant. The

recovery results showed that the method was very accurate

for quantitative determination of paracetamol and caffeine

from formulated tablets.

4. Conclusion

The RP-HPLC validation method was developed with

isocratic mode. Selected experimental methods were

providing high resolution and repeatability of peaks. For

the repeatability of the peaks and retention time the

required temperature was 200C [21].This validated method

more reliable to simultaneous determination of paracetamol

and caffeine from its binary formulated tablets. There was

no interference from the excipients used in the tablet

formulations and hence the methods are suitable for

analysis of formulated tablets. The results of validation

show that the described HPLC reverse phase separation

methods are simple, linear, precise, accurate and selective.

Hence the above method can be recommended for

simultaneous determination of paracetamol and caffeine.

References

1. Kumble, RM; Singh, SG; Stability-indicating RP-

HPLC method for analysis of paracetamol and

tramadol in a pharmaceutical dosage form. E Journal

of Chemistry. 9(3): 1347-1356 (2012).

2. Satoskar, RS; Bhandarkar, SD; Ainapare, SS;

Pharmacology and pharmacotherapeutics. 16th ed.

Popular prakashan; Mumbai: 1999. p. 164.

3. The Indian pharmacopoeia. Published by the Indian

pharmacopoeia commission, Ghaziabad, Vol-III, pp.

1516 (2007).

4. www.healthcaremagic.com

5. Alvi, SN; Hammami, M M; Validated HPLC Method for Determination of Caffeine Level in Human Plasma using Synthetic Plasma: Application to Bioavailability Studies. Journal of

Chromatographic Science. 49 (4): 292-296 (2011). 6. Nehlig, A; Daval, JL; Debry, G; Caffeine and the

central nervous system: mechanisms of action,

biochemical, metabolic and psychostimulant effects.

Brain Research Reviews. 17(2): 139–70 (1992).

7. Suzen, S; Akay, C; Tartilmis, S; Erdol, RS; Onal, A;

Cevheroglu, S; Quantitation of acetaminophen in

pharmaceutical formulations using high-performance

liquid chromatography. J. Fac. Pharm. Ankara. 27(2):

93-100 (1998).

8. Mulla, T;, Rao, JR; Yadav, SS; Bharekar, VV; Rajput,

MP; Development and Validation of HPLC Method

For Simultaneous Quantitation of Paracetamol And

Dexketoprofen Trometamol in Bulk Drug and

Formulation. International Journal of Comprehensive

Pharmacy. 2(7): 1-4 (2011).

9. Altun, ML; HPLC method for the analysis of

paracetamol, caffeine and dipyrone. Turk J Chem. 33:

521-528 (2002).

10. ‎Tsvetkova, B; Pencheva, I; Zlatkov, A; Peikov, P;

34 International Journal of Chromatographic Science 2013, 3(2): 31-34

Simultaneous high-performance liquid chromato-

-graphy determination of paracetamol and ascorbic

acid in tablet dosage forms.African Journal of

Pharmacy and Pharmacology. 6(17): 1332-1336

(2012).

11. Pattan, SR; Jamdar, SG; Godge, RK; Dighe, NS;

Dathankar, AV; Nirmal, SA; Pai, MG; RP-HPLC

Method for simultaneous estimation of Paracetamol

and Etoricoxib from Bulk and Tablets. Journal of

chemical and pharmaceutical research. 1(1): 329-335

(2009).

12. Ashraful, SM; Abuzar, SM; Paul, PK; Validation of

UV-Spectrophotometric and RP-HPLC methods for

the simultaneous analysis of Paracetamol and

Aceclofenac in marketed tablets. International journal

of pharmacy and Life Science. 2(12): 1267-1275

(2011).

13. El-Din, MS; Zeid, ME; Zeid, AM; Development and

Validation of RP-HPLC Method for simultaneous

Determination of Ascorbic acid and Salicylamide in

their binary mixtures: Application to combined tablets.

J Chromat Separation Techniques. 3(5): 1-7 (2012).

14. Dimal, AS; Neel, JP; Sunil, LB; Usman, KC; Kashyap,

KB; Stability Indicating LC-Method for Estimation of

Paracetamol and Lornoxicam in Combined Dosage

Form. J Sci Pharm. 79:113-122 (2011).

15. International conference on harmonization. Q1A (R-2)

stability testing of new drug substances and products

international conference on harmonization. IFPMA,

Geneva (2003).

16. Guide for Validation of Analytical and Bioanalytical

Methods. Resolution R.E.n. 899. Brazilian Sanitary

Surviellance Agency, Brasflia,DF; (2003).

17. Q2B Validation of Analytical Procedures:

Methodology. International Conference on

Harmonization. Washington, DC (1996).

18. US Pharmacopeia 27th

US pharmacopeial Convention.

Rockville, MD, pp: 2256, (2004).

19. Rolim, A; Maciel, CPM; Validation Assay for Total

Flavonoids, as Rutin Equivalents, from Trichilia

catigua Adr. Juss (Meliaceae) and Ptychopetalum

olacoides Bentham (Olacaceae) Commercial Extract. J

AOAC International. 88: 1015-1019 (2005).

20. The united States Pharmacopoeia 30th

and the National

formulary 25th

. Rockville, MD, USA (2007).

21. Upadhyay, HC; Verma, RK; Srivastava, SK;

Quantitative Determination of Bioactive 4-Hydroxy-a-

Tetralone, Tetralone-4-O-b-D-Glucopyranoside and

Ellagic Acid in Ammannia baccifera (Linn.) by

Reversed-Phase High-Performance Liquid

Chromatography. Journal of Chromatographic Science.

51:21–25 (2013).

Source of support: Nil; Conflict of interest: The authors declare no conflict of interest.