Method development and validation of paracetamol drug by RP-HPLC

Abstract

A simple and reproducible method was developed for paracetamol by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Paracetamol was separated on C18 column [4.6x250mm, particle size 5µm], using ortho phosphoric acid buf-fer with pH of 3.5 at the UV detection of 207nm. Isocratic elution of acetonitrile (ACN) and water was used as a mobile phase with various ratios and flow rates, eventually 25:75 v/v ACN and water was being set with the flow rate of 1mL/min. The statistical valida-tion parameters such as linearity, accuracy, precision, inter-day and intra-day variation were checked, further the limit of detection and limit of quantification of paracetamol concentrations were found to be 120ng/mL and 360ng/mL. Recovery and assay studies of paracetamol were within 99 to 102% indicating that the pro-posed method can be adoptable for quality control analysis of pa-racetamol. [PUBLICATION ABSTRACT]

Headnote

Abstract

A simple and reproducible method was developed for paracetamol by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Paracetamol was separated on C18 column [4.6x250mm, particle size 5µm], using ortho phosphoric acid buf-fer with pH of 3.5 at the UV detection of 207nm. Isocratic elution of acetonitrile (ACN) and water was used as a mobile phase with various ratios and flow rates, eventually 25:75 v/v ACN and water was being set with the flow rate of 1mL/min. The statistical valida-tion parameters such as linearity, accuracy, precision, inter-day and intra-day variation were checked, further the limit of detection and limit of quantification of paracetamol concentrations were found to be 120ng/mL and 360ng/mL. Recovery and assay studies of paracetamol were within 99 to 102% indicating that the pro-posed method can be adoptable for quality control analysis of pa-racetamol.

Key words: RP-HPLC, Validation, paracetamol, acetonitrile

© 2013 Deccan College of Medical Sciences. All rights reserved.

Article history:

Received 08 December 2012

Accepted 18 February 2013

Early online 25 February 2013

Print 28 February 2013

Paracetamol is an acetanilide derivative chemically 4-hydroxy acetanilide having analgesic, antipyretic and weak antiinflam-matory action1,2 and also administered in the man-agement of more severe pains in advanced can-cers3. In literature several analytical techniques like colorimetric4, spectroflurimetric5 methods have been reported on assay of paracetamol in combi-nation with other drugs. However pure drug analy-sis was studied by spectrometry6 and scanty litera-ture is available on paracetamol pure drug analysis with HPLC. Several HPLC methods using a variety of columns and detection techniques have been reported on paracetamol in combination with other drugs7,8,9 and physical and chemical stability stu-dies10. In the present study, novel method was de-veloped with acetonitrile (ACN) as solvent in HPLC; it is a simple method to study, detect and separate the paracetamol from mixture of com-pounds and can be adopted for regular quality as-sessment in pharmaceutical industry and scientific laboratories.

Materials and Methods

All reagents used were of analytical-reagent grade. Water purification systems, reverse osmosis and ultra pure water (Nanopure Human Corporation, Korea), sonicator (Digital citizen ultra sonic clean-er) for degassing of HPLC grade ACN and ortho phosphoric acid 88% (S.D. FineChem Limited, Mumbai, India) and pure paracetamol drug.

Chromatographic system

The RP-HPLC system composed of Agilent 1200 series instant pilot software: ChemStation Plus, EZChrom Elite, and certified for pharmaceutical QA/QC. It constitutes micro vacuum degasser G1379B, binary pump G1312B, diode array detec-tor SL G1315C, thermostatted column compart-ment G1316B with C18 column [4.6x250mm, pore size 5µm], high performance autosampler G1367C, thermostat for high performance auto-sampler G1330B. It is most flexible configuration for the maximum in gradient and low flow rate ac-curacy and precision, high-speed, multi-wavelength and full spectral UV-visible detection for peak purity analysis and spectral confirmation.

The chromatographic and integrated data were recorded in computer system.

Chromatographic conditions

Two solvents were used, solvent-A containing ACN filtered through 0.2µm filter paper and solvent-B containing ultra pure water filtered through 0.25µm PTFE (Poly Tetra Fluoro Thylene) filter [pH ad-justed to 3.5 with 88% ortho phosphoric acid], de-gassed with sonication. Various flow rates and sol-vent compositions were provided at room tempera-ture [28°C] to develop a method. The detection was performed at 207nm using diode array detector SL G1315C.

Method

Solution 10mg/mL of pure paracetamol was pre-pared in the mobile phase. The solution was ade-quately diluted to 50µg/mL and it was taken to de-velop a method by applying various solvent ratios and flow rates as shown in table 1 and chromato-grams in figure 1.

Validation of the method

Validation of the optimized HPLC method was car-ried out with the following parameters.

Linearity

Paracetamol standard stock solution of 10mg/mL was used for preparation of subsequent aliquots; aliquots of 100, 50, 25, 12.5 and 6.25 µg/mL con-centrations were prepared by serial dilution. The solution of 200 µL was loaded in autosamplar tray and 20 µL was being injected into column. All measurements were repeated three times for each concentration. The calibration curves of the area under curve versus concentration were recorded.

Accuracy

Paracetamol standard stock solution of 10mg/mL was used to prepare 10, 35, 55 µg/mL concentra-tions and injected for the accuracy studies. The area under curve obtained was checked and ana-lyzed for the recovery percentage.

Precision

The precision of method was checked and verified by repeatability, inter-day and intra-day precision. Repeatability was checked by injecting 80g/mL concentration of paracetamol for 6 times on the same day and for intraday precision one concen-tration was injected and analyzed at different time intervals on the same day. Similarly, for the inter-day precision another concentration was analyzed on different days.

Limit of detection (LOD) and limit of quantification (LOQ)

The LOD and LOQ of paracetamol were separately determined on the basis of signal (S) and noise (N) ratio, LOD and LOQ concentrations of paracetamol were confirmed and recorded by the S/N ratio where 3 and 9 were shown.

Robustness of the method

To determine the robustness of the developed me-thod, minute changes were made in the flow rate, percentage of ACN and the pH of the mobile phase and is studied for the deviations from opti-mized method.

System suitability parameters

To perform the system suitability tests the standard solution was freshly prepared and injected under the condition of optimized method to study the fol-lowing parameters.

Analysis of the marketed drug

Ten tablets of paracetamol (Crocin) were weighed and crushed into fine powder. The average weight for one tablet was calculated and weighed. The weighed fine powder of paracetamol tablet was dissolved in 100 mL of mobile phase in volumetric flask by shaking it for 30 minutes. Then, the solu-tion was kept for ultrasonication for 20 min and filtered through 0.2 µm filter paper. Further dilu-tions were made for the analysis of the drug con-tent in the tablet.

Results and discussion

The developed and validated method of paraceta-mol was aimed to establish chromatographic con-ditions, capable of qualitative and quantitative de-termination of paracetamol in pharmaceutical preparations. Paracetamol was completely sepa-rated on C18 column by RP-HPLC using the iso-cratic elution of ACN and water as mobile phase. When the ACN percentage was reduced starting from 80% by a decrement of every 5%, broaden-ing, fronting and tailing of peaks were observed (Fig 1). As a result of higher concentrations of ACN in mobile phase and decrease in the percentage of ACN the peak was sharp pointed and well sepa-rated. The unusual peaks could be the result of improper dissolution of paracetamol in higher con-centration of ACN, therefore the chromatographic column was not able to resolve the paracetamol properly and even there was decrease in the re-covery percentage of paracetamol (Table 1). As ACN concentration gradually decreases the peak broadening, fronting and tailing were remarkably reduced. It is evident that the flow rate of mobile phase in chromatography plays an important role in resolving the paracetamol, as the flow rate in-creases from 0.75 mL/min to 1.50 mL/min the re-tention time also decreased with fluctuation in pa-racetamol recovery (Table 1), eventually proper resolution was achieved at flow rate of 1mL/min and retention time of 3.6 minutes.

Linearity

The method gave a linear response to paracetamol drug within the concentration range of 6.25 - 100 µg/mL with r2= 0.999 as shown in figure 2.

Accuracy

The paracetamol was recovered in the range of 98.8 to 102.0 % for various concentrations as shown in the table 2.

Precision

The repeatability, intra-day and inter-day precision results are shown in the table 3. The RSD values were below 3%, indicating a good precision. The t-test value for inter-day precision was less than 0.1%, indicating the significant precision.

LOD and LOQ

The LOD and LOQ concentrations of paracetamol were found to be 120ng/mL and 360ng/mL.

Robustness of the method

The robustness of the method gave the mean, standard deviation (SD) and RSD within the limits. Results are shown in table 4.

Analysis of marketed drug

The labeled amount of paracetamol in two different tablet strips of 250 and 500 mg/tablet recovered 99.56% and 99.75% respectively. The RSD value is below 2%. The retention time was found to be 3.6 min and the results are shown in table 5.

System suitability studies

The system suitability parameters such as reten-tion time, capacity factor, theoretical plate number, peak purity and resolution factor of optimized me-thod were associated with confined values as shown in the table 6.

Conclusion

The optimized reverse phase HPLC method for paracetamol is linear, accurate, precise, robust, simple, rapid and selective. It can be adopted ap-parently for routine quality control analysis of raw materials, formulations and testing.

Acknowledgments

Authors would like to thank Mr. Srinivasa Rao, Managing Director, Onan Biotech, Hyderabad for laboratory facility; and Praveen Kumar for technical assistance.

Conflict of interest: None

References

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