A Friendly Environment Approach for determination of paracetamol

This paper involves a determination of paracetamol using less environment harmful reagent; the hydroxy analog of the pharmaceutical naproxen replaces the chemical reagent in which it is used as a coupling agent for the diazotized p-aminophenol (the hydrolysis product of paracetamol).The paper offers a determination of an analgesic paracetamol in the presence of high content of another analgesic . Paracetamol azo-dye formed shows the higher absorption peak at 500 nm. Absorbanceconcentration relation is liner over the range from 10 to 650 μg/20ml, (i.e. 0.5-32.5 ppm) with a good sensitivity (molar absorptivity 1.2x10 l.mol.cm) ; good precision (RSD better than ±0.275%) and high accuracy (relative error less than + 0.77%), Sandell's sensitivity index is 0.0124μg.cm, the calculated limit of detection (LOD) is 0.0030 μg/ml and the evaluated limit of quantitation (LOQ) is 0.0101 μg/ml. The application of the method exhibits a successful results for determination of paracetamol in dosage forms and it's already applied for the determination of paracetamol in presence of naproxen .


Introduction
Nowadays, the most important development concept in analytical chemistry is the modified version of an environmentally friendly approach [1], some of these concepts are the selection of the safer compounds [2].
A green, and economic method for determination of paracetamol in pharmaceutical preparations established, the method uv spectrophotomtry, which based on dissolving of sample in water, estimating the quantity of paracetamol at two wavelengths , then replacing the concentration in the equation of multi component system [3].
There were other procedures that can be classified under friendly environment these are phenylephrine via oxidative coupling [4], sulphacetamide as a diazotized agent coupled to paracetamol [5] and anthranilic acid as a coupling agent for diazotized paracetamol which is form a colored azo dye measured at 421 nm. [6] The same principle for determination of paracetamol but with more harmful reagents has been reported .The hemolysis compound [7] nitroaniline used for determination of paracetamol [8] , the irritant [9] thymol was used as a coupling agent for determination of paracetamol in alkaline medium to produce a colored product measured at 600nm. [10] and irritant [11] hexacyanoferrate was used for determination of paracetamol as an oxidant in the presence of ammonia followed by coupling reaction with phenol to form a colored product measured at 630nm. [12] ,in addition to that the skin inflammatory compound [13] phloroacetophenone was used as a coupling compound to determine the diazotized p-aminophenol (hydrolyzed paracetamol) in basic medium [14]. Dichlorodicyano benzoquinone which produces the toxic CN in water [15] was used for determination of paracetamol via charge transfer reaction [16].
The aim of this work is the determination of a diazotized p-aminophenol (hydrolyzed paracetamol) using modified naproxen (mNap) as a safer compound than chemical reagents .

Apparatus
All spectral and absorbance measurement were carried out on double-beam Jasco V-630 spectrophotometer with 1.0 cm matched quartz cells. pH measurements were performed using HANNA 301 pH meter, BEL balance was used for weight measurements, reflux was utilized by electrothermal heater and stirring was utilized by Wisd stirrer.

The chemicals
All chemical reagents used were of an analytical grade -Hydrolyzed paracetamol solution (HPAR) (100 µg/ml) : A10 ml ethanol was used to dissolve accurately weighed quantity of powder 0.025 g PAR (SDI) , then 100-150 ml distilled water was added, (shaking was used to increase the solubility) ,filtered in to 250.0 ml in volumetric flask , and the procedure for hydrolysis of paracetamol had been followed [17].
-Paracetamol solution (1000 µg/ml): 0.25 g of paracetamol was dissolved in 10 ml ethanol then the solution was completed to 250.0 ml in a volumetric flask with distilled water.
-Modified Naproxen ( hydroxy naproxen) synthesized and identified in previous research [18] was used in this article , Modified Naproxen (mNap) (8 x 10 -4 M): 0.0184 g of mNap (SDI) was dissolved in 2 ml ethanol and the volume was completed to 100 ml with distilled water in a volumetric flask. The solution was kept in dark bottle and is stay stable for at least one month.
-Sodium Nitrite (NaNO2) 1%:, to prepare the solution, 1.0 g of pure sodium nitrite was dissolved in distilled water and the volume was completed to 100 ml in a volumetric flask.
-Sodium hydroxide solution (1M): to prepare this solution an appropriate dilution of the concentrated solution (Fluka) was diluted with distilled water to 1000 ml in a volumetric flask and then transferred to a plastic bottle.
-Paracetamol Tablet solution (1000 µg.ml -1 ): 10 tablets (each one contains 500 mg PAR) was weighted and finely powdered. Quantity of powder exactly equivalent to 0.25 g PAR was dissolved in 10.0 ml ethanol, a dilution by 100-150 ml distilled water was done then it was filtered and completed to 250.0 ml in volumetric flask, similar procedure for hydrolysis of paracetamol had been followed.
-Paracetamol injection solution(1000 µg.ml -1 ): The contain of 3 injections were mixed , a 2.5 ml equivalent to 250 mg paracetamol was diluted to 250.0 ml with distilled water in a volumetric flask , 150.0 ml of the last solution was prepared by following the preparation of HPAR in Tablet.
-Paracetamol syrup solution(1000 µg/ml):A 10.41ml of antipyrol syrup (each 5 ml contain 120 mg PAR ) was diluted to 250 ml with distilled water in a volumetric flask ; 150 ml of the last solution was prepared by following the preparation of HPAR in Tablet.

Procedure and calibration graph:
To increasing volume (0.1-6.5) ml of 100µg.ml -1 standard paracetamol solution, the following reagents has been added in the following orders : 1.0 ml of H3PO4 (1M), 1.0 ml of 1% NaNO2, 0.5 ml of 3% sulphamic acid, 3 ml of (8 x 10 -4 M) mNap and 2 ml of NaOH(1M) has been finally added , then they were diluted to 20 mL, The absorbance has been measured at 500 nm against blank. The relation between absorbance and concentration shows a good linearity over the range of the concentration between 10 to 650 µg of Paracetamol in 20 ml (0.5-32.5 ppm) with good sensitivity in which that the molar absorptivity is 1.2 x 10 4 l.mol -1 .cm -1 and sensitivity index of Sandell is 0.0124 µg/cm 2 .

The study of the optimum reaction conditions The selection of acid and its quantity
The effect of different quantities (0.5-2.5 ml) of many acids (hydrochloric, sulphuric, nitric, phosphoric, and acetic acid) (1M) on absorption intensity has been studied Table (1) shows the results.  Table (1) makes it clear that , the maximum absorptions intensity of the colored product results when 1.0 ml of H3PO4 was used to justify the reaction medium.

Effect of nitrite quantity with time
Between 0.1 ml to 1.2 ml of NaNO2 (1%) has been checked with a standing time from 0 to 5 min, Table (2).  Table (2) exhibits that the reaction needs two minutes to reach optimum and 1.0 ml of 1% NaNO2 is the best quantity required for the completion.

Effect of sulphamic acid quantity with time
Between 0.3-1.5 ml of 3% of sulphamic acid was used, and absorbance of the solutions was measured at different standing time.  Table (3) indicate that five minutes as a standing time was preferred and 0.5 ml of 3% of sulphamic acid was selected .

The Effect of coupling agent quantity
The effect of (1-5) ml of (810 -4 M) coupling agent has been studied against 10-150 µg of Paracetamol/20 ml ; the determination coefficient of a measured absorbance has been evaluated. Table (4) shows that 3 ml and 4 ml of coupling agent solution gives the higher determination coefficient but 3 ml exhibits the best sensitivity.

Selection of base and its quantity
A higher conjugation and a bigger chromophor area are the results of removing the acidic hydrogen from conjugated product; for this reason, four types of bases or basic salts at different volumes (1)(2)(3)(4)(5) ml of each have been tested. See Table (5). Table (5) shows that 2 ml of sodium hydroxide (1M) is the best one and it was used from the beginning.

The Effect of surfactant
In order to study the effect of surfactants on absorption intensity, 2ml of SDS, CPC, and CTAB surfactants with different orders of additions were followed as its shown in Table (6).  II. Para +H+S+N+F+D+B III. Para +H+N+S+F+D+B IV. Para +H+N+F+S+D+B V. Para +H+N+F+D+S+B VI. Para +H+N+F+D+B+S

The Stability of reaction
The stability of the colored product against time has been followed using three different concentrations of Paracetamol, Table (7) indicates that the colored product keeps stable for at least 60 minutes. Under the optimum reaction conditions, the absorption spectrum of the colored product against blank (Fig.2) shows that wavelength of maximum absorption intensity is 500 nm.this wavelength has been used in subsequent investigations.

Accuracy and Precision
Paracetamol is determined at three different concentrations for checking the accuracy and precision, Table (8) indicates that the reaction is accurate (the relative error from -2.11 to 0.77 ) and precise (the relative standard deviation is from ±0.068 to ±0.275) .

Effect of organic solvents
The characteristic of the colored product is more detectable using acetic acid and water than that using other solvents which they were turbid or completely separated into two layers. Water is still being a choice.

Figure (3): Effect of Organic Solvents
A: Acetic acid B: Water Effect of foreign compounds to realize the application of this method, the interferences of foreign compounds have been studied by following the recommended procedure of 100 µg of paracetamol in the presence of 100, 300, 500, 1000 µg of expected foreign compounds using the recommended procedure.   (10) indicates that there was no interfering effect caused by the studied foreign compounds at different fold excess of these compounds.

Application of the method
The method has been applied to the determination of paracetamol in the dosage forms. The results are listed in Table (10) indicating a good applicability of the method.

Evaluation of analytical data
T-test has been used for evaluation of the method statistically ; The present method and British Pharmacopeia one [19] have been applied at the same time for t-test calculation [20] ; the value compared with statistical Tables for four degrees of freedom at 95% validation level; Table (11) shows that there is an identical ability to application.

Conclusion
A Friendly environment precise with good sensitivity method has been used for the determination of paracetamol in their pharmaceutical preparations (tablet, syrup, and injection) excluding the toxic reagents, don't need organic solvent; and don't need separation steps or adjustment of pH.