DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF PARACETAMOL AND PAMABROM IN BULK DRUG AND IN ITS SYNTHETIC MIXTURE

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About Authors:
Nimish Talaviya1*, Nirav Chandegara2, Rakesh Radadiya3, Chetana Ribadiya4, Chandani Joshi5, Dhara Kanjaria6
1,5Department of Q.A., Smt. R. D. Gardi B. Pharmacy College, Rajkot
2Department of Pharmacology, K. B. Institute of Technology, Gandhinagar
3Department of Q.A., Mts. V.B. Manvar College of Pharmacy, Dumiyani
4Department of Q.A., Smt. R. B. Patel Mahila Pharmacy College, Atkot
6Department of Q.A., Shree H. N. Shukla Institute of Pharmaceutical Education & Research, Rajkot
*nimish.talaviya@gmail.com

Abstract
This paper presents a RP-HPLC method for the simultaneous estimation of Paracetamol and Pamabrom in its synthetic mixture. The process was carried out on C18 (250 x 4.6mm, 5µ) (Spincotech Pvt. Ltd.) column using Phosphate buffer pH 4.5: Acetonitrile (75:25 %v/v) where pH was maintained using 0.1% orthophosphoric acid as a mobile phase at a flow rate of 0.8ml/min. Detection wavelength was fixed at isobestic point 268 nm. Linearity was observed in the concentration range of 10-50 μg/ml for Paracetamol (r2=0.9986) and 15-75 μg/ml for Pamabrom (r2=0.9971). The retention time of Paracetamol and Pamabrom was found to be 2.665 and 6.907 minutes respectively with resolution 13.70. The developed method is simple, precise, specific, robust, rapid, reproducible, and sensitive and it can be used for estimation ofParacetamol and Pamabrom in its synthetic mixture.

REFERENCE ID: PHARMATUTOR-ART-1943

INTRODUCTION
Paracetamol (PCM)is chemically 4-hydroxyacetanilide (Figure 1-A) used as analgesic and antipyretic. [1]Paracetamol acts primarily in the CNS, increasing the pain threshold by inhibiting both isoforms of cyclooxygenase, COX-1, COX-2 and COX-3 enzymes involved in prostaglandin (PG) synthesis. [2]

Pamabrom (PAM) is chemically 8-Bromo-3,7-dihydro-1,3-dimethyl-1-H-purine-2;6-dione compound with 2-amino-2-methyl-1-propanol (1:1) (Figure 1-B) is a diuretic drug. [3]Pamabrom is xanthine diuretics reduce salt and water reabsorption in the proximal tubule and increase renal blood flow, yet their diuretic effect is modest.[4]

The therapeutic importance of these two compounds justifies establishing analytical methods for its determination in bulk and laboratory mixture.

The chemical structures of Paracetamol and Pamabromare shown in Figure 1 (A), (B). [1, 3]


Figure-1: Chemical structure of (A) Paracetamol and (B) Pamabrom

Paracetamol is official in IP, BP and USP and is estimated by UV-Visible Spectrophotometric method as per IP, USP and BP. [3, 5, 6]In BP a redox titration for PCM is given for drug substance.[6]Pamabrom is official in US Pharmacopoeia. [3]It is assayed by Liquid chromatography as per USP. Literature review also reveals HPLC, UV spectrophotometric and HPTLC method for the estimation of PCM with other drugs. [7, 8, 9]Literature survey does not reveal any simple spectrophotometric method of Paracetamol and Pamabrom in synthetic mixture or Pharmaceutical dosage form.  So the objective of this work was to develop simple, precise and rapid spectrophotometric methods for combined synthetic mixture containing Paracetamol, Pamabrom and excipients.

MATERIALS AND METHODS

Instrumentation
Isocratic HPLC system with LC-20AD pump, Hamilton injector with injection volume 20μL, Phenomenex C18(Luna 5micron, 250×4.6mm,i.d) column, Double beam spectrophotometer of Thermo Electron Corporation (HeλIOS α), Digital pH meter (Janki Impex Pvt Ltd) etc.

Material and reagent
Reference standard of Paracetamol(gift sample from AUM Research Laboratory, Ahmedabad)and Pamabrom (gift sample from SUVEN Life Science, Hyderabad).HPLC grade methanol from Finar chemicals, Ahmadabad. All other chemicals and reagents used were of AR grade. Mili Q water was use for this study.

Preparation of Standard Solution:
100 mg of Paracetamoland 100mg of Pamabromwas weighedand transferred to a 100ml volumetric flask. About 30ml of diluent was addedand sonicated to dissolve, made up the volume with diluents to get finalconcentration of 1000μg/ml for Paracetamol and 1000μg/ml for Pamabrom. From that 10 ml of PCM and 20 ml of PAM stock solution was withdraw and volume was made upto 100 ml with diluent (Acetonitrile: Phosphate buffer (pH 4.5) (25:75 v/v) which give final concentration of 100 μg/ml and 200 μg/ml respectively.

Selection of analytical wavelength:
The standard solutions of Paracetamol (10 µg/ml) and Pamabrom (10 µg/ml) in methanol were scanned separately in the UV region of 200 to 400 nm and the overlain spectra were recorded as shown in figure 2.

Preparation of calibration curve:
1.0, 2.0, 3.0, 4.0 and 5.0 ml of working standard solution of Paracetamol (100 µg/ml) and 0.75, 1.5, 2.25, 3.0 and 3.75ml of working standard solution of Pamabrom (200 µg/ml) was pipette out and mixed in 10 ml volumetric flask which gives 10-50 µg/ml of PCM and 15-75 µg/ml of PAM. The chromatogram was recorded under the finalized chromatographic conditions as described above after getting a stable baseline. Peak areas were recorded for all the peaks. Calibration curves of Paracetamol and Pamabrom were constructed by plotting the peak area versus concentration (µg/ml) respectively.

VALIDATION PARAMETERS
Parameters such as Linearity and range, Accuracy, Precision, LOD and LOQ were taken up as tests for analytical method validation shown in table 6.

Linearity andRange:
Linearity of the proposed method was verified by analyzing five combined different concentrations in the range of 10-50 μg/ml for Paracetamol and 15-75 μg/ml for Pamabrom. Each concentration was made three times. The calibrationcurve of Peak area versus respective concentration was plotted and regression line equation for Paracetamol and Pamabrom was constructed as shown in figure 6.

Precision
Precision of the method was determined in the terms of Repeatability, Intraday and Interday precision.  Repeatability (% RSD) was assessed by analyzing test drug solution within the calibration range, six times on the same day. Intraday variation (% RSD) was determined by analysis of this solution three times on the same day. Interday precision (%RSD) was determined by analysis of this solution on three different days.

Limit of detection (LOD) and limit of quantitation (LOQ)
They were calculated as 3.3 σ/S and 10 σ/S respectively. Where σ is the standard deviation of the response (y-intercept) and S, is the mean of the slope of calibration plot.

Accuracy:
Recovery studies were done so as to check the accuracy of the method. Known amounts of standard solutions of PCM and PAM were added to pre-quantified sample solutions of PCM and PAM and peak area were determined. Concentration of the drug in the sample was calculated using the equations.  The analysis was done in a set of 3 replicates at 3 levels (80%, 100% and 120%) as shown in table 3.

Application of Proposed Method to synthetic mixture:
The synthetic mixture of Paracetamol and Pamabrom was prepared in ratio of 13:1. Accurately weighed 325 mg of Paracetamol and 25 mg of Pamabrom were transferred to 100 ml volumetric flask, and 70 ml of methanol was added. Excipients, such as microcrystalline cellulose, povidone, stearic acid, sucroseand corn starch which were used in tablet formulation, were added in this mixture and mechanically stirred for 20 minutes. This solution was filtered through the whatmann filter paper no. 41 and filtrate was collected. Residues were washed with methanol. The filtrate and washing were combined and volume of solution was made up to 100 ml with methanol. 1 ml of this solution was diluted up to 10 ml with diluent. 1.2 ml of this solution was further diluted up to 10 ml with diluent. Chromatogram of the resulting solution was recorded and percentage of label claim was calculated and mentioned in table 4.

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