Spectrophotometric Assay of Paracetamol in Pharmaceutical Preparations

Accepted Received 9/1/2007  22/8/2006  لا صخلم تل ةيفيط ةقيرط ثحبلا نمضتي ريدق لوماتيسا رابلا نم رغصلا يف هيهانتم تايمك . دمتعت ةتوزأ ىمع ةقيرطلا ا راب هتمعافمب كلذو لوماتيسا رابمل يضماحلا لمحتلا نم جتانلا لونيف ونيما كيرومكورديهلا ضماح دوجوب تيرتنلا نويا عم نا رتقا مث  يدعاق طسو يف جتانلا موينوزايادلا حمم عم نا رتقلاا فشاك  يفوتيساورومف نون ةغبص نيوكتل ةيوزآ ءاملا يف ةبئاذو ةرقتسم ةيلاقترب  و مت ل صاصتملاا ةدش سايق ةغبصل  ةجتانلا  يجوملا لوطلا دنع  472  رتيمونان  تناكو  دودح  نوناق زيكرتلا ىدم يف ريب  نم  10  ىلإ 180  ما رغوركيام ـلا نم لوماتيسا راب / 25  لم ةميق تناكو  ةيرلاوملا ةيصاصتملاا  يه  2.16  10  4  رتل . م لو 1 . مس 1 يبسنلا أطخلاو ،  حوا رت  نيب -


ABSTRACT
A spectrophotometric method for the assay of micro amounts of paracetamol has been developed.The method is based on the reaction of p-aminophenol which results from the acid hydrolysis of paracetamol, with nitrite ion to form the corresponding diazonium salt followed by coupling reaction in an alkaline medium with phloroacetophenone to form a stable and soluble orange azo dye.The intensity of absorbance for the resulting azo dye is measured at 472 nm and Beer's law is obeyed in the concentration range of 10-180 g of paracetamol in a final volume of 25 ml, with a molar absorptivity of 2.1610 4 l.mol -1 .cm - , a relative error of -0.64 to +1.64% and a relative standard deviation of 0.4 to 1.24 %, depending on the concentration level of paracetamol.The method has been successfully applied to the assay of paracetamol in various pharmaceutical preparations.
Paracetamol can be determined by nitration and subsequent reaction with acetone as nucleophilic reagent (9) or by complexation with Co(III) and Cu(II) ions (10).Another method based on reaction of hydrolysed paracetamol (p-aminophenol) at ambient temperature with sodium sulphide in the presence of Ce(IV) or Fe (III) to produce a methylene blue-like dye (11).Also, a charge transfer complex formation has been used for the determination using 2,3-dichloro-5,6-dicyano-1,4benzoquinone (12).Oxidation reduction reaction can be used in the determination of paracetamol using Fe (III) in the presence of 2,2`bipyridyl (13).Many chromatographic methods have been published.These included high performance liquid chromatography, gas chromatography and liquid chromatography (14)(15)(16).
The present method involves the diazotisation of hydrolysed paracetamol, followed by coupling with phloroacetophenone to form a highly coloured dye that has been applied successfully to the assay of paracetamol in pharmaceutical preparations.

Experimental Instruments
All spectrophotometric measurements are performed on Shimadzu UV-Visible Recording Spectrophotometer UV-160 using 1-cm silica cells, pH meter type Philips PW 9420 is used for pH reading.

Reagents
All chemicals used in this investigation are of analyticalreagent grade, and paracetamol standard material is provided from general establishment for medical appliance and drugs / SDI -Samaraa / Iraq.Solutions Paracetamol solution , 1000 g.ml -1 .This solution is prepared by dissolving 0.25 g of paracetamol in 10 ml of ethanol and diluted to 250 ml in a volumetric flask with distilled water.Phloroacetophenone , 0.1% (w/v).This solution is prepared fresh daily by dissolving 0.1 g of phloroacetophenone (Fluka) in 100 ml distilled water.Sodium nitrite solution, 1% (w/v).This solution is prepared by dissolving 1 g of sodium nitrite (BDH) in 100 ml distilled water.Sulphamic acid solution, 3% (w/v).This solution is prepared by dissolving 3 g of sulphamic acid (Fluka) in 100 ml distilled water.
Hydrochloric acid solution, 1N.This solution is prepared by diluting 8.5 ml of concentrated acid (11.8 N) to 100 ml with distilled water.Solution of hydrolyzed paracetamol, , 100g.ml - .This solution is prepared by transferring 150 ml of 1000 g.ml -1 paracetamol into 250-ml round-bottomed flask provided with condenser,25ml of hydrochloric acid(11.8N) is added then refluxed for 1 hour, after that the cold solution is neutralised with 20% sodium carbonate and diluted to 250 -ml with distilled water in a volumetric flask ( 17).To prepare 100g.ml - paracetamol, 16.6 ml of the above solution is diluted to 100 ml in a volumetric flask using distilled water.Paracetamol tablets solution, 100g.ml - .10 tablets (each one contains 500 mg paracetamol) are weighed and finely powdered.An accurately weighed amount of powder equivalent to 0.25g paracetamol is dissolved in 10 ml ethanol, then 100-150 ml distilled water is added, shaking to increase the solubility, filtered into 250 ml calibrated flask, then the solution is completed to the volume with a distilled water, and proceed as mentioned above in preparation of hydrolysed paracetamol solution Paracetamol syrup solution, 10g.ml - .A 10.4 ml of syrup (each 5ml contains 120 mg paracetamol) is transferred into a 250 ml calibrated flask and the total volume is diluted with distilled water, and proceed as mentioned in preparation of hydrolysed paracetamol solution.Paracetamol suppositories solution, 100g.ml - .Weigh and mix well 4 suppositories (each suppository contain 250 mg paracetamol).An accurate weighed amount of mixture equivalent to 0.250 g paracetamol is dissolved in boiling distilled water, filtered, and the residues are washed with 10 ml ethanol and boiling distilled water and the volume is completed to 250 ml in a calibrated flask with distilled water, and proceed as mentioned in preparation of hydrolysed paracetamol solution.

Procedure and calibration graph
To a series of 25 -ml calibrated flasks, transfer 0.1 -2.2 ml of hydrolysed paracetamol solution (equivalent to 100 g.ml-1 paracetamol) , then 0.5 ml of 1N hydrochloric acid and 0.5 ml of 1% (w/v) sodium nitrite solution are added and the mixture is allowed to stand for 5 minutes and then 0.2 ml of 3% (w/v) sulphamic acid solution is added with occasional shaking for 2 minutes.After that a 2 ml of 0.1% (w/v) phloroacetophenone solution and 2 ml of 1N sodium hydroxide are added .The volumes are completed to the mark with distilled water and the absorbance is read at 472 nm against the reagent blank after 10minutes standing time.A linear calibration graph is obtained over the concentration range of 10 -180 g paracetamol / 25 ml and a concentration above 180 g / 25 ml gives a negative deviation (Fig. 1).The molar absorptivity has been found to be 2.1610 4 l.mol -1 .cm - .

Results and Discussion
During the investigation, hydrolysed paracetamol solution equivalent to 100 g.ml -1 paracetamol, is taken and the final volumes are brought to 25 ml with distilled water.

Effect of diazotisation acid
Different amounts and types of acids have been used in diazotisation of paracetamol, the results show that the solution of 1N hydrochloric acid gives the best results when added in a volume of 0.5 ml (Table 1).

Effect of sodium nitrite amount and time
The maximum absorbance reading is obtained by adding 0.2 ml of 1% sodium nitrite with 5 minutes reaction time (Table2).

Effect of sulphamic acid amount and time
The excess of nitrite can be removed by the addition of sulphamic acid solution (18).The effect of sulphamic acid amount and time has been studied.(Table 3)

B
The results in Table 3 indicate that 0.2 ml of sulphamic acid solution (3%, w/v) with 2 minute standing time for the reaction, give the most suitable effect on the intensity of the dye.

Effect of phloroacetophenone amount
The effect of different amounts of phloroacetophenone solution (0.1%) on the intensity of absorbance at different amounts (5-200 g) of paracetamol/25ml has been studied.A 2 ml of phloroacetophenone solution (0.1%) in a total volume of 25 ml give the higher sensitivity and higher value of correlation coefficient (r), therefore it has been selected for subsequent experiments (Table 4).

Effect of base
Previous experiments have shown that the coloured azo dye formed in alkaline medium, therefore different amounts and types of strong and weak bases have been studied (Table 5).The results indicate that the strong bases give high intensity and high colour contrast and the formed orange azo dye is stable while sodium bicarbonate gives turbid solution after 30 minutes.A volume of 2 ml of 1N sodium hydroxide (final reaction mixture pH=11.7)has been selected for the subsequent experiments.

Table 5. Effect of bases on absorbance and colour contrast
Where S = The dye, B = Blank ** Turbid solution after 30 minutes

Effect of time
The coloured azo dye developed rapidly after addition of base and the stability period (at least one hour) is sufficient to allow several measurements to be performed and the results are given in Table 6.

Final absorption spectrum
The absorption spectrum of the orange azo dye formed from coupling of diazotised p-aminophenol with phloroacetophenone in alkaline medium shows a maximum absorption at 472 nm.The reagent blank has very nill absorption(0.023)at this wavelength (Fig. 2).

Nature of the dye
The stoichiometry of the formed azo dye between diazotised pamino-phenol and phloroacetophenone is investigated by applying the continuous-variations method and mole-ratio method.The results indicate that the azo-dye has formed in the ratio of 1:1 diazotised paminophenol to phloroacetophenone, and the azo dye may have the following suggested structure:

Accuracy and precision
To check the accuracy and precision of the calibration graph, three different concentrations of paracetamol are determined.The results shown in Table 7 indicate that the method is satisfactory.

Analytical application
The proposed method is applied to determine paracetamol in different pharmaceutical preparations.On applying proposed procedure, good recovery is obtained as shown in table 8

Comparison of the methods
A comparison between the present method and British pharmacopeia standard method (1) for the determination of paracetamol in tablets drug, is based on the t-test to show the ability of using the present method in the determination of investigated drug (Table 9).The results in Table 9 indicate that the calculated experimental tvalue is less than its value in the statistic table at confidence level (95%) and for four degrees of freedom (2.776).These result indicated that there is no significant difference between the present method and the standard method.
According to the difficulties of availability of some reagents used in standard method for determination of paracetamol in syrup and suppositories ,so we used standard addition method in order to prove that the proposed method can be applied to determination of paracetamol in syrup and suppositories without interferences (Table 10) The results in Table 10 indicated that the proposed method can be used in determination of paracetamol in syrup and suppositories with satisfactory results.
Table 11 shows the comparison between some of analytical variables obtained from the present method with that of another Literature spectrophotometric method.
Table11.The comparison of the methods.

Application of the method
The results indicate that the proposed method has a good sensitivity compared with the above literature method which is more sensitive.

Fig. 2 .
Fig. 2. Absorption spectra of 100g paracetamol / 25ml treated according to the recommended procedure and measured against (A) reagent blank, (B) distilled water and (C) reagent blank measured against distilled water

Table 1 . Effect of diazotisation acids / ml of acid used Absorbance and colour contrast 1N Acid solution used
 max S - max B Where S = The dye , B = Blank

Table 7 . Accuracy and precision
.

Table 10 . The results of standard addition method
*Average for three determinations