Sayed et al / Physical Chemistry 17(1) (2015) 1-5. Physical Chemistry 17(1) (2015) 1-5
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1 Physical Chemistry 17(1) (2015) 1-5 A comparative study for the quantitative determination of paracetamol in tablets using UV-Visible spectrophotometry and high performance liquid chromatography Murtaza Sayed, M. Ismail, Sanaullah Khan and Hasan M. Khan* Radiation and Environmental Chemistry Laboratory, National Centre of Excellence in Physical Chemistry, University of Peshawar-25120, Pakistan *Corresponding author hmkhan3@gmail.com, hmkhan@yahoo.com A R T I C L E I N F O A B S T R A C T Article type Research article History Received October 2014 Accepted March 2015 August 2015 Issue Keywords Paracetamol HPLC UV-Vis Spectrophotometry Mean recovery Precision This study investigates the comparison of high performance liquid chromatography (HPLC) and UV-Spectrophotometry methods for the quantitative determination of paracetamol in commercially available tablets. HPLC analysis was carried out on reversed phase XDB-C 18 column ( mm, 5 micron) with UV-Visible detection at 230 nm. The mobile phase used was acetonitrile-water (25:75 % v/v) at a flow rate of 1 ml min -1, column temperature of 40 0 C and the determination was completed in less than 5 min. The method developed for HPLC analysis showed excellent linearity in the range of 2-20 µg ml -1 with regression coefficient of and the lower limit of detection was found to be µg ml -1. The lower limit of detection for UV-Visible Spectrophotometer was found to be 4 µg ml -1, while linearity was found in the range 4-10 µg ml -1. After that range deviations were observed from Beer s law. Precision (% R.S.D < 1) and mean recovery was found for UV- visible spectrophotometer in the range of %, while for HPLC recovery was calculated to be in the range of %. The two evaluated methods showed to be adequate to quantify paracetamol in tablets, while HPLC method presented the most reliable results for the analysis of tablets. Paracetamol content in tablets was also tested statistically using test hypothesis NCEPC, University of Peshawar: All rights reserved Cite This Article As: Murtaza Sayed, M. Ismail, Sanaullah Khan and Hasan M. Khan. A comparative study for the quantitative determination of paracetamol in tablets using UV-Visible spectrophotometry and high performance liquid chromatography. Physical Chemistry 17(1) (2015) 1-5 INTRODUCTION Paracetamol (p-hydroxy acetanilide) is a compound with analgesic and antipyretic properties and is commonly used for relief of fever, headaches and other minor aches and pain. The chemical structure of paracetamol is shown in Fig. 1. Already reported work reveals that many analytical methods have been proposed for its quantitative determination in paracetamol tablets, like spectrophotometry (Hoang et al., 2014; Gul et al., 2015), second derivative spectrophotometry (Kokot and Burda, 1997), planer chromatography (Argekar and Sawant, 199), IR spectroscopy (Bouhsain et al., 1997), capillary chromatography (Emre and Özaltın, 2007) and highperformance liquid chromatography (Silvana et al., 2013). This drug is official in Indian pharmacopeia (Riedel and Laufen, 1993), British pharmacopeia (2006), European pharmacopeia (1997) and United States pharmacopeia (2005). Due to the rapid growth, scientific development, and industrialization, various synthetic drugs have been introduced to the market. The increased utilization of these drugs demands the development of fast and economical analytical methods for their successful analysis in raw materials, pharmaceutical tablets and in biological samples. The purpose of the present study was to develop easy and fast analytical methods to quantify paracetamol in raw
2 materials and tablets. The results obtained by these methods were also compared statistically. EXPERIMENTAL Reagents and Chemicals Fig. 1: Structure of paracetamol Paracetamol (acetaminophen), reference standard was obtained from Stanley Pharmacauticals. Methanol and acetonitrile (HPLC Grade) were obtained from Merck (Germany). Grade 1 water was obtained from a Milli-Q ultra pure water purification system. (Millipore, USA). Paracetamol tablets labelled to contain 40 mg/tablet was purchased from local markets. Instruments and analytical conditions Sayed et al / Physical Chemistry 17(1) (2015) 1-5 Method Validation Limit of quantification (LOC) and limit of detection (LOD): For measuring LOC and LOD, a series of diluted standard paracetamol solutions were analysed by both HPLC and UV- visible spectrophotometer. Limit of detection for paracetamol by HPLC method was calculated at the signal to noise ratio of 3, and was found to be µg ml -1. The limit of quantification of paracetamol was calculated at signal to noise ratio of 10 and was found to be µg ml -1. Limits of detection and quantification for UV/Vis spectrophotometry were also calculated and were found to be 4.0 µg ml -1 and 13.3 µg ml -1, respectively. The HPLC analysis was carried out using an Agilent 1200 system, composed of quaternary pump, UV detector and HP Chemstation software. The column used was a symmetry C 18 (250 mm x 4.6 mm i.d., 5 µm particle size) maintained at 40 0 C and UV detection was performed at 230 nm. The mobile phase consisted of acetonitrile and water (25:75 % v/v) at a flow rate of 1 ml min-1 and the injection volume was 4 µl. UV-Spectrophotometric analyses were carried out on Perkin Elmer Lambda 650 UV/Vis spectrophotometer using quartz cuvette having path length of 10 mm. For quantification of paracetamol, scan mode was selected and measurements were obtained by taking water as blank. Preparation of standard and reference solutions Paracetamol standard solution: Approximately 40 mg of paracetamol reference standard was weighed and transferred to 500 ml flask. One ml of 0.5 N NaOH was added to ensure complete solubility and the solution was diluted to volume with distilled water. Subsequent dilutions were made with water to give the concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg ml -1 of paracetamol. Paracetamol sample solution: To analyse the paracetamol content in commercially available tablets, about 10 tablets were accurately weighed, finely powdered and then transferred to 500 ml flask. 0.5 N NaOH (1 ml) was added for solubility and the solution was then diluted to mark with distilled water. The solution was filtered with 0.45 µm membrane filter paper prior to analysis by HPLC and UV- Visible spectrophotometer. Dilutions were then made from stock solution having the concentrations of 2, 4, 6 and 8 µg ml -1. Fig. 2: Plot for standard paracetamol on UV-Visible spectrophotometer showing deviation from Beer s law Linearity: Linearity of HPLC method was evaluated by analysis of working standard solutions of ten different concentrations (n = 3). The regression equation obtained was y = x (r 2 = 0.993, n = 10). The range of linearity was from 2 to 20 µg ml -1. For UV/Vis Spectrophotometry, the linearity was calculated in the range from 4 to 18 µg ml -1. In the range of 4 to 10 µg ml -1, the plot was linear obeying Beer s law with a coefficient of correlation r 2 of Beyond 10 µg ml -1 the data showed Beer s law with negative deviation. By applying Q- test at 90 % confidence interval points beyond 10 µg ml -1 were removed from Fig. 2 and thus a linear plot was obtained as shown in Fig. 3. Fig. 3: Plot showing linear relation between absorbance and concentration by applying Q-test 2
3 Fig. 4: Chromatogram for paracetamol sample solution at 4 µg ml 1 using symmetry C 18 column at 40 0 C and mobile phase composed of water and acetonitrile (75:25 % v/v) at flow rate of 1 ml min -1. Detection wavelength: 230 nm. Accuracy: The accuracy of the method was determined by adding reference standard of paracetamol to the tablet samples at three different concentrations of 4, 6 and 8 µg ml -1. Sample solutions were made in triplicate and were analysed for three consecutive days using HPLC and UV/Vis spectrophotometer. Solutions for the calibration curve were prepared fresh every day. The assay accuracy results from the two methods are summarized in Table 2 showing the recovery percentage of UV and HPLC methods. in the Section The paracetamol contents were determined using the two methods and the obtained results were statistically proved using test hypothesis at 0.01 significance level. RESULTS AND DISCUSSION Different chromatographic conditions were tried by changing the composition of the mobile phase, flow rate and column. Finally, applying mobile phase of water: acetonitrile in a ratio of (75:25 v/v) at a flow rate of 1 ml min -1 showed better results. So using this combination of mobile phase at ph 7.4 and a C 18 column, an adequate peak symmetry (symmetry = 0.5) and short run time of 3 min was achieved as shown in Fig.4 In UV-Visible spectrophotometry, the standard paracetamol sample showed an absorption band at 230 nm (Fig 5). So wavelength of 230 nm was selected for absorption measurement of paracetamol content in the tablets. Fig. 5: UV Spectrum of paracetamol sample solution in water. Concentration 6 µg ml -1 Precision: The intra-day precision was calculated by analysing sample solutions of three different concentrations 4, 6 and 8 µg ml -1 (n = 3) using the UV and HPLC methods. The intermediate precision (inter-day variation in the same concentration) was calculated for three consecutive days (n = 3). Paracetamol content and relative standard deviations (R.S.D) for both UV and HPLC methods were calculated and are summarized in Table 2. Analysis of Paracetamol tablets The samples of paracetamol tablets were analysed by HPLC and UV methods. Before the analysis, the tablets were weighed and finely powdered. The sample solutions for the HPLC and UV analysis were prepared as described earlier 3 Validation A linear relationship was found between paracetamol concentration and response time of both HPLC and UV methods. Table 1 shows regression analysis data for both the methods. HPLC shows the regression coefficient of while UV method shows regression coefficient value of that is little lower than HPLC method. The precision data obtained for the two methods are tabulated in Table 2. Both HPLC and UV-visible spectrophotmetric methods showed R.S.D. values lower than 2% presenting good precision, however HPLC method was more precise than UV method. Accuracy was investigated by means of % recovery experiments (n = 3) using developed methods. Both chromatographic and spectrophotometric methods exhibited recoveries close to 100%, however better recovery was achieved by HPLC-method. The LOD and LOQ for chromatographic method were obtained by considering signal to noise ratio of 3 and 10 and were found to be µg ml -1 and 2.76 µg ml -1, respectively. For the UV-method, LOD and LOQ were found to be 1.25 µg ml -1 and 3.51 µg ml -1, respectively. The HPLC method was found to be a more sensitive method as compared to UV method.
4 Table 1: Overview of the linearity data for paracetamol by the chromatographic and spectrophotometric methods Regression parameters HPLC UV Regression Coefficient (r 2 ) Slope ± Standard Error ± ± Intercept± standard Error ± ± Standard Error of Estimate Concentration Range (µg ml -1 ) 2-10_ 4-10_ Number of Points 10 4 Table 2: Validation Parameters of the elevated methods for Paracetamol determination Validation Parameter HPLC UV Intra-day Precision n=3 (R.S.D %) Inter-day Precision n=3 (R.S.D %) Accuracy n=3 (Recovery, %) LOD (µg ml -1 ) LOQ (µg ml -1 ) Table 2: Quantitative analysis of paracetamol content (Claimed concentration 40 mg/tablet) in commercially available tablets by HPLC and UV methods Factors (n = 5) HPLC UV Mean (%) Amount found (mg) SD R.S.D (%) Analysis of Paracetamol content in tablets The quantitative analysis of paracetamol in tablets was carried out using both HPLC and UV-visble spectrophotometric methods and is presented in Table 3. Both methods showed the nearly same amount of paracetamol content in tablet, however HPLC method showed slightly lower % R.S.D than UV-method. So, both analytical techniques can be employed for quantitative analysis of paracetamol content in the tablets. CONCLUSIONS It can be concluded from the present work that both HPLC and UV methods can be used for the quantitative analysis of paracetamol in tablets and raw material. However, the chromatographic method is showed to be more sensitive, accurate and precise than UV method. The main advantage of the spectrophotometric technique over the chromatographic one is that the UV method is more simple and does not acquire the use of mobile phase and other procedures normally associated with the chromatographic techniques. The results showed that paracetamol content in tablets can be quantitatively determined by using HPLC and UV- spectrophotometric methods and no interference of recipients in the tablets was observed. ACHNOWLEDGMENT The authors are grateful to the Higher Education Commission (HEC) of Pakistan for providing financial support to carry out this research. REFERENCES Argekar, A.P., Sawant, J.G., Simultaneous determination of pyridoxine hydrochloride and doxylamine succinate from tablets by ion pair reversedphase high-performance liquid chromatography (RP- HPLC). Journal of Planer Chromatography 12, Bouhsain, Z., Garrignes, S., de-la Gaurdia, M., Fresenius., PLS-UV spectrophotometric method for the simultaneous determination of paracetamol, acetylsalicylic acid and caffeine in pharmaceutical formulations. Journal of Analytical Chemistry 357, British Pharmacopoeia, Her Majesty s Stationary Office, London,
5 Emre, D., Özaltın,N., Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography. Journal of Chromatography B 847, European Pharmacopoeia, rd Edition, Council of Europe, France, 55. Gul, S., Khanum, K., Mujtaba, N., New validated method for analysis of salymarin in polyherbal formulation (aqueous extract, oral liquid and solid dosage form). Chemistry International 1(3), Hoang, V.D., Ly, D.T.H., Tho, N.H., Nguyen, H.M.T., UV Spectrophotometric Simultaneous Determination of Paracetamol and Ibuprofen in Combined Tablets by Derivative and Wavelet Transforms. The Scientific World Journal, Kokot, Z., Burda, K., Simultaneous determination of salicylic acid and acetylsalicylic acid in aspirin delayed-release tablet formulations by secondderivative UV spectrophotometry. Journal of Pharmaceutical and Biomedical Analysis 18, Riedel, K,D., Laufen, H., High-performance thinlayer chromatographic assay for the routine determination of piroxicam in plasma, urine and tissue. Journal of Chromatography B 276, Silvana, E.V., Teodoro S.K., development and validation of a hplc method for the simultaneous determination of bromhexine, chlorpheniramine, paracetamol, and pseudoephedrine in their combined cold medicine formulations. Journal of Liquid Chromatography & Related Technologies 36(20), United States Pharmacopoeia., The National Formulary, (USP 28/NF 23), The United States Pharmacopoeia Convention, Rockville, MD 16,
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