Research & Reviews in Pharmacy and Pharmaceutical Sciences
Menue-ISSN:2320-1215 p-ISSN: 2322-0112
e-ISSN:2320-1215 p-ISSN: 2322-0112
S Dhruvi*, B Ankita, and P Bhagirath
Sat Kaival College of Pharmacy, Sarsa-388365, Ta. Dist. Anand, Gujarat, India.
Received date: 13/02/2014 Accepted date: 17/03/2014 Revised date: 12/03/2014
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A simple, rapid and precise Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was developed for simultaneous estimation of paracetamol and pamabrom in bulk and combined pharmaceutical dosage form by using BDS hypersil C18 column. The sample was analysed using phosphate buffer (pH-4): methanol (65:35v/v) as a mobile phase at a flow rate of 1.0 ml/min and detected at 270 nm. The retention time for paracetamol and pamabrom was found to be 4.04 min and 7.36 min respectively. The linearity of developed method was achieved in the range of 32.5-97.5μg/ml for paracetamol and 2.5-7.5μg/ml for pamabrom. The method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and robustness as per International Conference Harmonization (ICH) guidelines.
Development, Validation, paracetamol (PCM), pamabrom (PAM)
Paracetamol (N- 4-hydroxyphenyl acetamide) is an Analgesic-antipyretic drugs with poor Anti-inflammatory action and belongs to para-aminophenol derivative categories of NSAIDs. The main mechanism of action of paracetamol is considered to be the inhibition of cyclooxygenase (COX). Recent findings suggest that it is highly selective for COX-2 because of its selectivity for COX-2 it does not significantly inhibit the production of the pro-clotting thromboxanes [1].
Pamabrom is Diuretic drug that increases the rate of urine flow; however, clinically useful diuretics also increase the rate of excretion of Na+ (natriuresis) and of an accompanying anion, usually Cl- [2].
Paracetamol and pamabrom tablet is used for premenstrual dysphoric disorder, back pain, premenstrual syndrome [3].
Analytical method development is defined as development, revision and application of validated, standardized and official methods of analysis [4]. Method validation is the process of documenting or proving that selected method provides analytical data for the intended use. Method is validated by using parameters like accuracy, precision, linearity, limit of detection, limit of quantitation, system suitability, selectivity and specificity [5,6].
Paracetamol is an official in Indian Pharmacopoeia, British Pharmacopoeia and United Pharmacopoeia. Pamabrom is an official in United Pharmacopoeia. Very few analytical methods have been reported for estimation of paracetamol and pamabrom in combined pharmaceutical dosage form.
Combined formulation of paracetamol and pamabrom is approved by Central Drugs Standard Control Organization (CDSCO) dated on 07/12/2011 [7].
Figure 1: Chemical structure of paracetamol [6]
Figure 2: Chemical structure of pamabrom[9].
HPLC grade water, methanol and acetonitrile were used for preparing mobile phase. Potassium di hydrogen phosphate (AR grade) and ortho phosphoric acid (AR grade) were used for preparing the buffer. Pure sample of paracetamol and pamabrom were gifted by Astron chemicals, Ahmadabad and Ami Life sciences Pvt Ltd, Vadodara. MENSODOLTM TAB (paracetamol-325mg, pamabrom-25mg) was gifted from Anthus Pharmaceuticals, Chennai.
The HPLC system was SPD-20AT, Shimadzu consisting of pump LC-20AT, detector SPD-20AT, column BDS hypersil C18, 250mm × 4.6mm, 5μ(particle size), Thermo scientific, injector rheodyne injector (20 μl capacity) and syringe hamilton (25 μl). Data were processed using chromatographic software Spinchrom.
A freshly prepared mixture of phosphate buffer (pH-4): methanol (65:35v/v) used as the mobile phase. Mobile phase was filtered through a 0.45μm membrane filter and sonicated before use. The flow rate of the mobile phase was maintained at 1.0 ml/min.
Mobile phase was prepared by mixing 350 ml of methanol and 650 ml of phosphate buffer (pH: 4.0), filtered through 0.45μm whatman filter and sonicated for 10 min.
Reference standard of PCM 65 mg and PAM 50 mg were transferred to 10 ml volumetric flask separately and dissolved in methanol. Flasks were shaken for 20 min and the volume was made up to the mark with mobile phase to obtain standard stock solution of PCM (650μg/ml) and PAM (500μg/ml). Stock solutions were filtered through a 0.45μm whatman filter. 1ml PAM solution was withdrawn to 10 ml volumetric flask and diluted up to mark with mobile phase to get working standard solution of PAM (50μg/ml). The working standard solutions of PCM and PAM were prepared from suitable aliquots of stock solutions.
Twenty tablets were weighed, their average weight was determined and finally powdered. An accurately weighed tablet powder equivalent to 650 mg of PCM and 50 mg PAM was then transferred to 10 ml volumetric flask containing 5 ml methanol and sonicated for 20 min. The solution was filtered through whatman filter paper and the volume was adjusted up to the mark with mobile phase. This solution is expected to contain 650μg/ml PCM and 50μg/ml PAM. This solution (0.5 ml) was transferred in to a 10 ml volumetric flask and the volume was adjusted up to mark with mobile phase to get a concentration of PCM (32.5μg/ml) and PAM (2.5μg/ml).
The standard solution of PCM (32.5μg/ml) and PAM (2.5μg/ml) were scanned in the UV region of 200-400 nm using methanol as a blank and the overlain spectra was recorded. 270nm analytical wavelength was selected for estimation of PCM and PAM. (Figure 3).
Figure 3: Selection of detection wavelength (270 nm).
The pure drug solutions of PCM (32.5μg/ml) and PAM (2.5μg/ml) were injected individually into HPLC system and allow to run in different mobile phases like methanol, water: methanol, water: acetonitrile, phosphate buffer: methanol and phosphate buffer: acetonitrile were tried in order to find the optimum conditions for the separation of PCM and PAM. It was found that mobile phase containing phosphate buffer (pH-4): methanol (65:35v/v) at a flow rate of 1.0 ml/min with detection wavelength 270 nm gave satisfactory results with sharp, well defined and resolved peaks with minimum tailing as compared to other mobile phases. Under these conditions the retention times were typically 4.04 min for PCM and 7.367 min for PAM (Figure 4) and optimized chromatographic conditions described in (Table 1).
Figure 4: Chromatogram of standard solution for PCM (tR 4.04 min) and PAM (tR 7.367 min).
Table 1: Optimized chromatographic conditions for simultaneous estimation of PCM and PAM
Validation of the optimized RP-HPLC method was carried out with respect to the following parameters.
Linearity responses for PCM and PAM were assessed in the concentration range 32.5-97.5μg/ml and 2.5-7.5μg/ml of standard solutions, respectively.
The sensitivity measurement of PAM and PCM by the use of proposed method was estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). The LOD and LOQ were calculated using following equations.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where,
σ = the standard deviation of the response
S = slope of the calibration curve.
The precision of the method was verified by repeatability, interday and intraday precision. Repeatability study was performed by analysis of three different concentrations of the drug in six replicates on the same day. Intraday precision was determined by analysing sample solutions at different time intervals on the same day and on different day for interday precision.
To the pre-analysed sample, a known amount of standard solution of pure drug (PCM and PAM) were spiked at three different levels. These study was carried out at 80%, 100% and 120% level.
Robustness was performed by deliberately changing the chromatographic conditions. The important parameter to be studied was the resolution factor between two peaks. The robustness was checked by changing following parameters one by one:
Change in the ratio of mobile phase by ± 0.2 ml [phosphate buffer (pH 4): methanol (67:33v/v) and phosphate buffer (pH 4): methanol (63:37v/v)]
Change in flow rate by ± 0.2 ml/minute (0.8 ml/min. and 1.2 ml/min.) After each change, sample solution was injected and % assay with system suitability parameters were checked.
Change in pH of mobile phase by ± 0.2 pH [phosphate buffer (pH 4.2): methanol (65:35v/v) and phosphate buffer (pH 3.8): methanol (65:35v/v)].
To check System suitability, number of theoretical plates, resolution, retention time and tailing factor were determined [17].
Twenty tablets were weighed, their average weight was determined and finally powdered. An accurately weighed tablet powder equivalent to 650 mg of PCM and 50 mg PAM was then transferred to 10 ml volumetric flask containing 5 ml methanol and sonicated for 20 min. The solution was filtered through 0.45μm filter and the volume was adjusted up to mark with methanol. From the above solution 1 ml was taken into a 10 ml volumetric flask and the volume was adjusted up to mark with methanol to get a final concentration of PCM (65μg/ml) and PAM (5μg/ml). 20 μl of the test solution was injected and chromatogram was recorded for the same and the amount of the drug was calculated [18].
The results of method development and validation studies on simultaneous estimation of PCM and PAM in the current study involving phosphate buffer (pH-4) and methanol (65:35v/v) as the mobile phase for RP-HPLC are given below.
PCM and PAM were completely separated on C18 column by RP-HPLC using the iso-cratic elution of phosphate buffer and methanol as mobile phase. When the methanol percentage was reduced starting from 80% by a decrement of every 5%, broadening, fronting and tailing of peaks were observed. As a result of decrease in the percentage of methanol and using phosphate buffer (pH-4) a sharp pointed and well separated peak was observed. As methanol concentration gradually decreases the peak broadening, fronting and tailing were remarkably reduced. Eventually proper resolution was achieved at flow rate of 1ml/min and using phosphate buffer (pH-4): methanol (65:35 v/v) as the mobile phase for RP-HPLC. (Figure 4) (Table 1)
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.[6] The drug response was linear (R2 = 0.9995 for PCM and 0.9995 for PAM) over the concentration range between 32.5-97.5μg/ml for PCM and 2.5-7.5μg/ml for PAM. The result is shown in (Table 2).
Table 2: Linearity data of PCM and PAM by proposed method.
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy [6]. The LOD and LOQ were calculated by respective equations. The LOD values were found to be 2.26 and 6.86μg/ml for PCM and PAM respectively. The LOQ values were found to be and 0.16 and 0.55μg/ml for PCM and PAM respectively. (Table 2)
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility [6]. The results of the repeatability, intra-day and inter-day precision experiments are shown respectively as given in (Table 3) and (Table 4). The developed method was found to be precise as the RSD values for repeatability of intra-day and interday precision studies were < 2 %.
Table 3: Repeatability study of PCM and PAM.
Table 4: Intra-day and inter-day study of PCM & PAM.
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.[6] As shown in (Table 5) and (Table 6), good recoveries of the PCM and PAM in the range from 99 to 101 % were obtained at various added concentrations.
Table 5: Determination of accuracy for PCM.
Table 6: Determination of accuracy for PAM
The standard deviation of the peak areas was calculated for each parameter and the % RSD was found to be less than 2 %. Result shows low values of % RSD as shown in (Table 7) and signify the robustness of the method.
Table 7: Robustness study for PCM and PAM.
System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.[6] The column efficiency, resolution and peak asymmetry were calculated for the standard solutions and the results are mention in (Table 8) [17].
Table 8: System suitability parameters.
When dosage form was analysed, PCM and PAM gave sharp and well defined peaks at retention time 4.04 min. and 7.36 min. respectively, when scanned at 270 nm. Assay result of marketed formulations is shown in (Table 9) [18].
Table 9: Quantitative determination in pharmaceutical formulation.
Development and validation of RP-HPLC method was found to be simple, accurate, precise and economical. These methods can be applied for routine quantitative analysis of paracetamol and pamabraom in bulk and pharmaceutical dosage forms.
Authors are grateful to Ami life sciences Pvt Ltd and Astron pharmaceuticals, Gujarat, India for providing gratis sample. We wish to express our gratitude to Anthus Pharmaceuticals, Chennai, India for providing marketed formulation. Our heartily thanks Sat Kaival College of Pharmacy, Gujarat, India for the perfect logistic support and guidance they have extended to us.