INTRODUCTION:

Paracetamol (acetaminophen) is one of the most popular over-the-counter analgesic and antipyretic drugs. Paracetamol is available in different dosage forms; such as tablet, capsules, drops, elixirs, suspensions and suppositories etc. Dosage forms of paracetamol and its combinations with other drugs have been listed in various pharmacopoeias ^1,2. The combination of paracetamol with dipyrone is used as an antipyretic, analgesic and anti-inflammatory drug. Numerous methods have been reported for the analysis of paracetamol alone and its combinations with other drugs in pharmaceuticals or in biological fluids. Paracetamol has been determined in combination with other drugs using titrimetry ^3,4, voltammetry⁵, fluorimetry⁶, colorimetry⁶, UV-spectrophotometry⁷, high-performance liquid chromatography (HPLC)⁸. The determination of paracetamol in tablets by spectrophotometry was reported in the literature. A suitable HPLC method to determine paracetamol with simple composition of mobile phase was not located in the literature survey. The objective of this study was to develop and validate a specific, accurate, precise and reproducible quality control method for paracetamol with effective simple composition of mobile phase.

MATERIAL AND METHODS:

Chemicals:

Paracetamol (acetaminophen) was used from USP reference standard received from S. D. Fine-Chem Limited, Mumbai, M.H., India. Chromatographic grade double-distilled water, HPLC-grade methanol (Riedel-de Haen-34860 were used.

Apparatus:

The method development was performed with a HPLC-LC type system consisting of a dual plunger, pulse compensating operation solvent delivery system; isocratic, constant pressure, gradient and external input solvent delivery mode (make Younglin Instrument, Korea; Model no. Acme 9000 HPLC). Dual wavelength UV/Vis absorbance detector; wavelength range 190-900nm and Basic Marathan type 816 version 1.4, march 1994 autosampler using a 20 μl sample loop with SP 930 D gradient pump. The detector was set at 245 nm and peak areas were integrated automatically by computer using the Autochro-2000 software program. Separation was carried out at room temperature using a Varian, Microsorb-MV 100-5 C18 column (250 x 4.6 mm; North America). All the calculations concerning the quantitative analysis were performed with external standardization by the measurement of peak areas.

Stock and Standard Solutions:

Paracetamol (100.00 mg) was accurately weighed in a 100 ml volumetric flask and dissolved in the mobile phase and filled up to volume with the mobile phase.

Standard Working Solution:

Standard working solutions were prepared individually in mobile phase for paracetamol. Aliquots from working solution was combined and diluted with mobile phase to yield a solution with final concentrations of 5 μg/ml, 10 μg/ml and 15 μg/ml. Studies on the stability of the analytes in standard working solution showed that there were no decomposition products in the chromatogram or difference in areas during analytical procedure, even after storage for four days at 4⁰C.

PROCEDURE:

Chromatographic Conditions:

HPLC analysis was performed by isocratic elution with a flow rate of 0.5 ml/min. The mobile phase composition was methanol-water (30:70) (v/v). All solvents were filtered through a 0.25 μm millipore filter before use and degassed in an ultrasonic bath. Volumes of 10 μl prepared solutions and samples were injected into the column using auto sampler. Quantification was effected by measuring at 245 nm as established from the three-dimensional chromatogram. The chromatographic run time was 10 min and the column void volume was 1.735 min. Throughout the study, the suitability of the chromatographic system was monitored by overlapping figures.

Figure 1: Chromatogram of 245nm and 0.5 Flow Rate at Room Temperature (5 μg/ml)

Figure 2: Chromatogram of 245nm and 0.5 Flow Rate at Room Temperature (10 μg/ml)

Calibration:

Standard solutions containing paracetamol between 5-15 μg/ml was prepared in the mobile phase. Triplicate 20 μl injections were made for each standard solution to see the reproducibility of the detector response at each concentration level. The peak area of each was plotted against the concentration to obtain the calibration graph. The three concentrations of each sample were subjected to regression analysis to calculate the calibration equation and correlation coefficients.

RESULTS AND DISCUSSION:

Method Development:

The mobile phase was chosen after several trials with methanol, isopropyl alcohol, acetonitrile, water and buffer solutions in various proportions and at different pH values. A mobile phase consisting of methanol-water (30:70) (v/v) was selected to achieve maximum sensitivity with minimum composition and quantity of organic mobile phase.

Table 1: Calibration Result (Figure 6)

Sr. No.	Name	Area	Height	Amount	Response factor
1.	5 μg/ml	680.250	50.375	5.000	0.007350
2.	10 μg/ml	1320.304	99.183	10.000	0.007574
3.	15 μg/ml	2002.388	137.858	15.000	0.007491

Table 2: Precision of the development method at LOQ level (n = 6)

Compound	λ	Peak Area (mean) n = 6	Amount	SD %
Paracetamol (5 μg/ml)	245 nm	680.250	5.000	1.18
Paracetamol (10 μg/ml)	245 nm	1320.304	10.000	1.83
Paracetamol (15 μg/ml)	245 nm	2002.388	15.000	2.01
Paracetamol (100 μg/ml)	245 nm	9446.444	100.000	5.61
Unknown (about 25 μg/ml)	245 nm	3.27.488	23.468	2.36

Flow rates between 0.5 and 1.0 ml/min were studied. A flow rate of 0.5 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed-phase C18 column, the retention times for paracetamol was observed to be 8.1 min. Total time of analysis was less than 9 min. The maximum absorption of paracetamol detected at 245 nm and this wavelength was chosen for the analysis. The chromatogram at 245 nm showed a complete resolution of all peaks with selected composition of mobile phase (Figure 1, 2, 3 and 4)

Figure 1, 2, 3 and 4 Chromatogram of the paracetamol by the RP-HPLC method

Linearity:

Table 1 presents the equation of the regression line, correlation coefficient (r²), standard deviation (SD), values of coefficient of determinant for compound. Excellent linearity was obtained for compounds between the peak areas and concentrations of 5- 15 μg/ml with correlation coefficient r² = 0.99980 for paracetamol.

Equation of regression line:

Amount=a*Area+b (Y=a*Area+b); a: 0.00750 b: 0

Standard Deviation: 0.10039

Correlation Coefficient: 0.99980

Coefficient of Determinant: 0.99960

Figure 3: Chromatogram of 245nm and 0.5 Flow Rate at Room Temperature (15 μg/ml)

Figure 4: Chromatogram of 245nm and 0.5 Flow Rate at Room Temperature (Unknown sample of the drug paracetamol)

Figure 5: Overlap graph of 5, 10, 15 μg/ml drug samples at 245nm and 0.5 Flow Rate at Room Temperature

Figure 6: Calibration report of 5, 10, 15 μg/ml drug samples at 245nm and 0.5 Flow Rate at Room Temperature

Limits of Detection and Quantification:

Limits of detection (LOD) were established at a signal-to-noise ratio (S/N) of 3. Limits of quanti cation (LOQ) were established at a signal-to-noise ratio (S/N) of 6. LOD and LOQ were experimentally veried by four injections of paracetamol at the LOD and LOQ concentrations. The LOD was calculated to be 0.816 μg/ml and the LOQ was calculated to be 1.226 μg/ml for paracetamol.

Suitability of the Method:

The chromatographic parameters such as resolution, selectivity and peak asymmetry were satisfactory for compounds (Figure 5) overlap 5, 10, 15

Table 3: Accuracy of the Developed Method (n= 6)

Compound	Spiked concentration (μg/ml)	Measured concentration (μg/ml)	SD %	% deviation
Paracetamol (100 μg/ml)	100	107.862	5.61	7.862
Unknown (about 25 μg/ml)	25	23.468	2.36	6.128

Precision:

The precision of the method (within-day variations of replicate determinations) was checked by injecting paracetamol 6 times at the LOQ level. The precision of the method, expressed as the SD % at the LOQ level was for each samples of paracetamol showed in Table 2.

Accuracy:

A standard working solution containing paracetamol drug concentrations of 100 μg/ml and 25 μg/ml respectively was prepared. The prepared sample of standards was injected 6 times as a test sample. From the respective area counts, the concentrations of the each paracetamol samples calculated using the detector responses. The accuracy, defined in terms of % deviation of the calculated concentrations from the actual concentrations, is listed in Table 3.

% Deviation = (Spiked Concentration - Mean Measured Concentration) / Spiked concentration X 100

Compound