Vous êtes sur la page 1sur 10

See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/279638799

ANALYTICAL METHOD DEVELOPMENT AND


VALIDATION OF THREE COMBINATION DRUGS
PARACETAMOL, CETIRIZINE AND
PSEUDOEPHEDRINE BY RP-HPLC AND ITS
APPLICATION IN ASSAY OF TABLET DOSAGE
FORMS

ARTICLE in WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES · MAY 2015

READS

20

5 AUTHORS, INCLUDING:

Anirbandeep Bose Divakar Goli


Acharya Institutes Acharya & BM Reddy College of Pharmacy, …
46 PUBLICATIONS 279 CITATIONS 49 PUBLICATIONS 176 CITATIONS

SEE PROFILE SEE PROFILE

Sabyasachi Mandal
Himalaya Drug Company
2 PUBLICATIONS 0 CITATIONS

SEE PROFILE

Available from: Sabyasachi Mandal


Retrieved on: 04 February 2016
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 5.210

Volume 4, Issue 06, 1720-1728. Research Article ISSN 2278 – 4357

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF


THREE COMBINATION DRUGS PARACETAMOL, CETIRIZINE AND
PSEUDOEPHEDRINE BY RP-HPLC AND ITS APPLICATION IN
ASSAY OF TABLET DOSAGE FORMS

Shree Narayan Pandit1*, Anirbandeep Bose1, Divakar Goli1, Gurubasavarajswamy 1,


Sabyasachi Mandal1, Sowparnika

1
Department of Quality Assurance, Acharya and B. M Reddy College of Pharmacy.
Bangalore – 560107.

Article Received on ABSTRACT


18 April 2015, A RP-HPLC method was developed for simultaneous estimation of
Revised on 09 May 2015, Paracetamol (PCM), Cetirizine (CTZ) and Pseudoephedrine (PSEDN)
Accepted on 30 May 2015
for bulk drug and marketed tablet formulation using Phenomenex Luna
C18 column (250 mm × 4.6 mm id, 5 µm) and a mobile phase of
*Correspondence for
Acetonitrile: 0.025 mM Potassium Dihydrogen orthophosphate + one
Author
Shree Narayan Pandit drop of Triethylamine in every 100 ml of buffer solution pH 7.5, 50:50
Department of Quality v/v, at flow rate 0.5 ml/min with UV detection at 220 nm. The
Assurance, Acharya and retention time (t R) of Paracetamol, Cetirizine and Pseudoephedrine
B. M Reddy College of
were found to be 5.706, 8.097 and 4.848 respectively. The proposed
Pharmacy. Bangalore–
method was validated for system suitability, specificity, linearity,
560107.
accuracy, precision, LOD, LOQ and robustness. All parameters were
found to be with in the acceptance limit.Linearity for Paracetamol was in the range of 75-600
µg/ml, Cetirizine was in the range of 2.5-20 µg/ml and Pseudoephedrine was in the range of
5-40 µg/ml. LOD and LOQ values were found to be less than 2.2 µg/ml level proving their
sensitiveness towards the developed method.HPLC methods were simple, accurate, precise
and suitable for analysis of marketed tablet formulation containing Paracetamol, Cetirizine
and Pseudoephedrine.

KEYWORDS: RP-HPLC Method, UV Spectroscopy, Paracetamol, Cetirizine and


Pseudoephedrine.

www.wjpps.com Vol 4, Issue 06, 2015. 1720


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

INTRODUCTION
Common cold related to virus is quite common phenomena in our daily life. Due to week
immunity of young age children become more vulnerable to this common cold virus. The
symptom related common cold like headache, running nose, nasal congestion and sneezing
may sometime become troublesome in our common life. The common cold symptoms
usually resolve in seven to ten days but sometimes it can persist for up to three weeks. The
average duration of cough is around 18 days. So to reduce the symptoms sometimes
physician prescribe three or more combination drugs in the form of tablet or syrup. The most
commonly used combination drugs like Paracetamol, cetirizine and pseudoephedrine are
commonly available in market. Paracetamol are used mainly to reduce the headache and fever
related to the common cold. Cetirizine acts as an antihistaminic drug. Pseudoephedrine
reduces nasal congestion and acts as an effective decongestant.Due to different mechanism of
these drugs, the combination dosage form of these three drugs become one of the best options
for general medical practitioner to prescribe for common cold patients. The simultaneous
analysis of these drugs by RP-HPLC in pharmaceutical industry not only reduces the time of
analysis but also it is most cost effective.

So the need of our present study is to provide a simple, sensitive, precise and accurate
validated method of RP-HPLC analysis which will reduce the run cost of routine analysis.

EXPERIMENTAL
Instrumentation
RP-HPLC instrument (Shimadzu, Japan) equipped with a SPD 20A UV-visible detector and
LC-20AT pump, manual Rheodyne injector with 20 µl loop, Phenomenex Luna C18 column
(250 mm×4.6mm i.d., 5µ particle size) and LC solution software.

Chemicals and reagents


Potassium dihydrogen orthophosphate, sodium hydroxide were analytical grade. HPLC grade
acetonitrile and water were from Merck, India. Pure drugs of Paracetamol, Pseudoephedrine
and Cetirizine were provided as gift sample by Micro Labs Ltd, Bangalore.The formulation
of Paracetamol, Pseudoephedrine and Cetirizine received from local pharmacy.

Chromatographic conditions
Chromatographic separation was carried out on Phenomenex Luna C18 column (250 mm x
4.6 mm id, 5µ), mobile phase of Acetonitrile: 0.025 mM Potassium dihydrogen

www.wjpps.com Vol 4, Issue 06, 2015. 1721


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

orthophosphate buffer pH 7.5(50:50 v/v), with flow rate of 0.5 ml/min, detection wavelength
at 220 nm,temperature 25 C and Injection volume was 20 μL.

Preparation of Paracetamol standard stock solution (250 µg/ml)


Weighed accurately 25 mg of Paracetamol and it was transferred into a clean, dry 25 ml
volumetric flask, dissolved with sufficient volume of mobile phase and volume adjusted up to
25 ml with mobile phase to get a concentration of 1000 µg/ml (PCM). 2.5 ml of the above
stock solution was transferred into 10 ml volumetric flask and the volume was made up to 10
ml with mobile phase to get a concentration 250 µg/ml.

Preparation of Cetirizine standard stock solution (10 µg/ml)


Weighed accurately 10 mg of Cetirizine and it was transferred into a clean, dry 10 ml
volumetric flask, dissolved with sufficient volume of mobile phase and volume adjusted up to
10 ml with mobile phase to get a concentration 1000 µg/ml (CTZ). 1.0 ml of the above stock
solution was transferred into 10 ml volumetric flask and the volume was made up to 10 ml
with mobile phase to get a concentration 100 µg/ml. 1.0 ml of above stock solution was
transferred into 10 ml volumetric flask and volume was made up to 10 ml with the mobile
phase to get a concentration 10 µg/ml.

Preparation of Pseudoephedrine standard stock solution (10 µg/ml)


Weighed accurately 10 mg of Pseudoephedrine and it was transferred into a clean, dry 10 ml
volumetric flask, dissolved with sufficient volume of mobile phase and volume adjusted up to
10 ml with mobile phase to get a concentration 1000 µg/ml (PSEDN). 1.0 ml of the above
stock solution was transferred into 10 ml volumetric flask and the volume was made up to 10
ml with mobile phase to get a concentration 100 µg/ml. 1.0 ml of above stock solution was
transferred into 10 ml volumetric flask and volume was made up to 10 ml with the mobile
phase to get a concentration 10 µg/ml.

Preparation of sample stock solution


Twenty tablets were weighed and powdered. Tablet powder equivalent to 25 mg of
Paracetamol was transferred into 25 ml volumetric flask, mixed with mobile phase, sonicated
for 10 min, filtered through a Whatmann’s filter paper and the volume adjusted up to 25 ml
with mobile phase to get a concentration 1000 µg/ml. 2.5 ml from this solution was further
diluted into 10 ml volumetric flask with mobile phase to get a concentration 250 µg/ml.

www.wjpps.com Vol 4, Issue 06, 2015. 1722


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

Figure 1: A typical chromatogram of Pseudoephedrine, Paracetamol and Cetirizine.

Table 1: System suitability study

Parameters Pseudoephedrine Paracetamol Cetirizine


Retention time 4.785 5.706 8.097
Tailing factor 0.877 1.404 1.451
Theoretical Plates 2015.32 2546.27 3810.56
Resolution - 1.540 4.490

Validation of HPLC method


Specificity
This parameter was performed to assess and ensure that the impurities, degraded products and
diluents do not affect the samples analyzed. 20 µl of diluent and sample solutions were
injected into the system and the chromatograms recorded.

Linearity
Thelinearity studies were performed to ensure that the test results are directly proportional to
the concentration of analyte. 20 µl of each standard solution of Pseudoephedrine (5-40
µg/ml), Paracetamol (75-600 g/ml) and Cetirizine (2.5-20 g/ml) was injected into HPLC
system. The peak area v/s concentration were plotted to get a standard calibration curve.

Table 2: Linear regression data for calibration curve

Parameters Pseudoephedrine(g/ml) Paracetamol(g/ml) Cetirizine(g/ml)


Concentration range, μg/ml 5-40 75-600 2.5-20
Slope 22104 64520 108375
Intercept 0.0 0.0 0.0
Correlation coefficient 0.999 0.9963 0.999

www.wjpps.com Vol 4, Issue 06, 2015. 1723


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

Precision
Precision of the assay was determined by intra-day and inter-day precision of the developed
method.Intra-day precision was done by three different analysts in same day whereasInter-
day precision was done in three different days. The results are presented in terms of %
relative standard deviation (% RSD)(Table 3& 4).

Table 3: Intraday precision for PSEDN, PCM and CTZ.


Standard
Concentrations 1 2 3 Mean SD % RSD
(µg/ml)
Pseudoephedrine
10 205505 202444 204289 204079.3 1541.23 0.75
15 277224 278240 280251 278571.7 1540.51 0.55
20 402055 398723 399627 400133 1723.99 0.43
Paracetamol
150 14046964 14032115 14221450 14100176 105288.2 0.746
250 21755412 21856210 22054321 21888648 152071.7 0.694
300 28679776 28269543 28700941 28550087 243188.3 0.851
Cetirizine
5 398820 402677 403328 401608.3 2436.60 0.606
8 1069460 1066796 1065953 1067403 1830.60 0.171
10 1289400 1280202 1269820 127980.3 9795.96 0.765

Table 4: Interday precision for PSEDN, PCM and CTZ.

S. No. Pseudoephedrine Paracetamol Cetirizine


1. 449905 25554624 1413027
2. 450389 25496872 1421026
3. 448986 25532954 1419027
Mean 449760 25528150 1417693
SD 712.65 29174.17 4162.93
%RSD 0.158 0.114 0.293

Accuracy
The accuracy of an analytical method is the closeness of test results obtained by that method
to the true value. The accuracy was performed by addition of known amounts of standard
drug. The accuracy of the method was determined by calculating percentage recovery of
known added amounts of analytes (Table 5, 6 & 7).

www.wjpps.com Vol 4, Issue 06, 2015. 1724


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

Table 5: Recovery studies of Pseudoephedrine

Amount of Amount of Total Total


S.
Sample drug standard drug Peak Area concentration % Recovery concentration
No
taken (µg) added (µg) (µg) found (µg)
1. 15 12 183689 27 100.49 27.13
2. 15 15 199656 30 98.31 29.49
3. 15 18 225846 33 101.09 33.35

Table 6: Recovery studies of Cetirizine

Amount of Amount of Total Total


S.
Sample drug standard drug Peak Area concentration % Recovery concentration
No
taken (µg) added (µg) (µg) found (µg)
1. 5 4 728719 9 99.63 8.96
2. 5 5 806329 10 99.22 9.92
3. 5 6 903351 11 101.05 11.11

Table 7: Recovery studies of Paracetamol

Amount of Amount of Total Total


S.
Sample drug standard drug Peak Area concentration % Recovery concentration
No
taken (µg) added (µg) (µg) found (µg)
1. 250 200 22537211 450 100.58 452.62
2. 250 250 24670937 500 99.05 495.47
3. 250 300 27679466 550 101.07 555.90

Limits of detection and quantification


Limits of detection (LOD) were established at a signal-to-noise ratio (S/N) of 3.3. Limits of
Quantification (LOQ) were established at a signal-to-noise ratio (S/N) of 10. LOD and LOQ
were experimentally verified by six injections of paracetamol, cetirizine and pseudoephedrine
at the LOD and LOQ concentrations. LOD values for PSEDN, PCM and CTZ were found to
be 0.55µg/ml, 0.4 µg/ml and 0.6 µg/ml respectively whereas LOQ values for PSEDN, PCM
and CTZ were found to be 2.0 µg/ml, 1.8 µg/ml and 2.1 µg/ml respectively.

Robustness: This parameter was carried out to check the ability of the system to give
unaffected results for small deliberate changes in system parameters and method parameters.
Robustness was performed by changing the flow rate, wavelength and mobile phase ratio for
standard drugs as well as formulations.

Assay: Formulation used was Tablet and Brand Name – Kold time (500 mg of Paracetamol +
30 mg Of Pseudoephedrine + 10 mg of Cetirizine)

www.wjpps.com Vol 4, Issue 06, 2015. 1725


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

2.5 ml from the sample stock solution of 1000 µg/ml containing PCM, CTZ and PSEDN,
transferred into 10 ml volumetric flask and volume adjusted up to 10 ml with mobile phase to
get 250, 5 and 15 µg/ml concentration respectively.

Fig No.2: Chromatogram of PSEDN, PCM and CTZ sample solution

Table 8: Assay of Pseudoephedrine, Paracetamol and Cetirizine in tablet

Formulation Drug Amount of Amount found % Label


the drugs ( µg/ml) claim found*
( µg/ml)
Pseudoephedrine Paracetamol 250 248.42 98.12
+ Paracetamol + Pseudoephedrine 15 14.94 98.56
Cetirizine Cetirizine 5 4.89 98.00

DISCUSSION
The chromatographic parameters were fixed and HPLC system was studied for the suitability
of drug analysis. The system suitability parameters were given in Table 1. The developed
method was validated to make suitable it for drug analysis. Validation of the HPLC method
was performed for linearity, precision, accuracy, specificity, robustness, LOD and LOQ.

CONCLUSION
HPLC method was developed and validated as per ICH guidelines. It can be concluded that
the method is specific for estimation of PSEDN, PCM and CTZ in pharmaceutical dosage
form. The method has linear response in stated range and is accurate and precise.Statistical
analysis proves that the method is suitable for the analysis of PSEDN, PCM and CTZ as bulk
drug and in pharmaceutical formulation without any interference from the excipients. It may

www.wjpps.com Vol 4, Issue 06, 2015. 1726


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

be extended to study the degradation kinetics of PSEDN, PCM and CTZ and also for its
estimation in plasma and other biological fluids andImpurity profiling for both the drugs.

ACKNOWLEDGEMENT
The authors would like to thank Medriech, Micro Labs, Bangalore andAcharya and B M
reddy college of pharmacy, Bangalore for providing sample of Paracetamol, Cetirizine and
Pseudoephedrine bulk drug.

REFERENCES
1. Willard, Merritt, Dean, Settle. Instrumental methods of analysis. 7 thed. New Delhi: CBS
Publishers and Distributors. 1986; 592-606.
2. Beckett AH, Stenlake JB. Practical pharmaceutical chemistry. 4 thed. New Delhi: CBS
publishers and distributors. 1997; 1.
3. ICH Q2 (R1) Validation of analytical procedures: text and methodology [Online]. 2005
[cited 2014 December]; Available from: URL:
http://www.ich.org/fileadmin/Public_Web_site/ICH_Products/Guidelines/Quality/Q2_R1
_Guideline.Pdf.
4. Nash AR, Wachter AH. Pharmaceutical process validation; 3 rded. New York: Marcel
Dekker Inc. 2005; 129: 512-2.
5. ICH harmonized tripartite guideline (Q2A): Text on validation of analytical procedures
[Online]. 1994 Oct 27 [cited 2014 December]; Available from: URL:
http://www.bioforum.org.il/uploads/editor/Karen/q2_r]_r1_step4.pdf.
6. ICH harmonized tripartite guideline (Q2B): Text on validation of analytical procedures:
Methodology [Online]. 1996 Nov 6 [cited 2014 December]; Available from: URL:
http://www.ics.trieste.it/media/135975/df3559.pdf.
7. Chakraborthy B, Sivasubramanian L, Naidu A. Reverse Phase High Performance Liquid
Chromatographic Method for Simultaneous Determination of Paracetamol, Cetirizine and
Dextromethorphan in Pure and Synthetic Mixture. Res J Pharma, Biol Chem Sci. 2014;
5(3): 183-9.
8. Suryan L, Bhusari VK, Rasal KS, Dhaneshwar SR. Simultaneous Quantitation and
Validation of Paracetamol, Phenylpropanolamine Hydrochloride and Cetirizine
Hydrochloride by RP-HPLC in Bulk Drug and Formulation. Int J Pharm Sci Drug Res.
2011; 3(4): 303-8.

www.wjpps.com Vol 4, Issue 06, 2015. 1727


Pandit et al. World Journal of Pharmacy and Pharmaceutical Sciences

9. Kolhe S, Khose Y, Kale A. Simultaneous estimation of Cetirizine hydrochloride,


Phenylpropanolamine hydrochloride and Paracetamol by RP-HPLC method. Int J of
Pharm Life Sci. 2013; 4(11): 3122-32.
10. Malakar P, Deb AR, Adhikary S, Ahmed S, Maloth R. Simultaneous estimation of
phenylephrine hydrochloride, Paracetamol, Caffeine and Cetirizine dihydrochloride from
tablet dosage form using RP-HPLC. Int J of Biol Pharm Res. 2013; 4(5): 368-76.
11. Deepali G, Pandurang D. RP-HPLC Method for Simultaneous estimation of Nimesulide
and Paracetamol in solid dosage form. Int J Pharm Tech Res. 2010; 2(2): 1330-3.
12. Kallol J, Lopamudra A, Moitra S K, Aninidita B. Analysis of multicomponent drug
formulations Diclofenac and Paracetamol. Asian J Pharm Clin Res. 2011; 4(2): 41-3.
13. Uttam DP, Abhijit VN, Aruna VS, Tirumal AD, Kiran VM. Simultaneous determination
of Aceclofenac, Chlorzoxazone and Paracetamol by HPLC in tablet dose form. Eu J
Chem. 2009; 6(1): 289-94.
14. Pattan SR, Jamdar SG, Godge RK, Dighe NS, Daithankar AV, Nirmal SA. RP-HPLC
method for simultaneous estimation of paracetamol and etoricoxib from bulk and tablets.
J Chem Pharm Res. 2009; 1(1): 329-35.
15. Subramanian G, Shetty R, Agarwal S, Pandey S, Udupa N. Simultaneous reverse phase
HPLC estimation of paracetamol and rofecoxib in tablet. Indian J Pharm Sci. 2005; 67(2):
247-9.

www.wjpps.com Vol 4, Issue 06, 2015. 1728

Vous aimerez peut-être aussi