Two Derivative Spectrophotometric Methods for the Simultaneous Determination of 4-AminoAntipyrine in Presence of Its Acidic Products

Simple, economic and sensitive mathematical spectrophotometric methods were developed for the estimation 4-aminoantipyrine in presence of its acidic product. The estimation of binary mixture 4-aminoantipyrine and its acidic product was achieved by first derivative and second derivative spectrophotometric methods by applying zero-crossing at (valley 255.9nm and 234.5nm) for 4-aminoantipyrine and (peak 243.3 nm and 227.3nm) for acidic product. The value of coefficient of determination for the liner graphs were not less than 0.996 and the recovery percentage were found to be in the range from 96.555 to 102.160. Normal ratio spectrophotometric method 0DD was used 50 mg/l acidic product as a divisor and then measured at 299.9 nm with correlation coefficient 0.998 and limit of detection 0.04098. ratio derivative methods 1DD and 2DD; are based on measuring the first derivative and second derivative for normal ratio spectrum at (peak 290.7 nm and valley 310 nm) for 1DD and (peak 286, valley 301 and peak 316nm) for 2DD the correlation coefficient for linearity graph not less than 0.997 and the recovery percentage were found to be in the range from 99.64 to 100.11. Keyword: 4-Aminoantipyrine; zero-crossing; spectrophotometric; ratio derivative; 1DD and 2DD.

On the other hand, spectrophotometric methods such as zero-crossing [11][12] and ratio derivative [13][14] methods are still favorite studies because of the simplicity, accuracy and availability of the instrumentation [15][16].Therefore, the aim of this study is to develop rapid, simple and inexpensive spectrophotometric methods for the analysis of 4-Aminoantipyrine in the presence of its acidic product without chemical process separation.Furthermore, the zero-crossing and ratio derivative spectrophotometric methods could be used for the estimation of 4-Aminoantipyrine without any interference from its acidic product.

a.
Preparation of acidic product for IR-spectral 0.01 gm of pure 4-Aminoantipyrine was refluxed with 100 ml 2N HCl for 60 min.after cooling, the solution was evaporated to dryness by using oven at 70 0 C, the residue was extracted with ethanol, filtered and then dried in air.The separated acidic product was subjected to IR spectral analysis [17][18].

b. Preparation of standard solutions
1.
4-Aminoantipyrine stock standard solution 100mg/l was prepared by dissolving 0.01 gm of pure 4-Aminoantipyrine with distilled water and then complete to the 100ml with the same solvent.After then different volumes of 4-Aminoantipyrine stock standard solution (100mg/l) ranging from (2.5 ml to 17.5 ml) were transferred into a series of 25 ml volumetric flasks and completed to the mark with distilled water to obtain a range of concentrations from (10 to  4-Aminoantipyrine acidic product stock standard solution was prepared by refluxing 0.01 gm of pure 4-Aminoantipyrine with 100ml 2N HCl for 60 min.after cooling, were transferred into a series of (2.5, 5, 7.5, 10, 12.5, 15, 17.5 ml) of this solution to 25 ml volumetric flasks and complete to the mark with distilled water to obtain the acidic product solutions in the concentration range of (10 to 70 mg/l).
The first derivative 1D and second derivative 2D of the uv spectrum of pure 4-Aminoantipyrine standard solutions were measured.Two calibration graphs were obtained by plotting the valley of 1D spectra at 255.9nm and the valley of 2D spectra at 234.5nm (corresponding to zero-crossing of its acidic product) of 1D and 2D spectra against the corresponding concentrations.In the same way, the 1Dand 2D of 4-Aminoantipyrine acidic product standard solution were recorded.Two calibration graphs were obtained by plotting the values of 1D spectra at 243.3 nm and peak of 2D spectra at 227.3nm (corresponding to zero-crossing of pure 4-Aminoantipyrine) of 1D and 2D spectra against the corresponding concentrations.

2.
To series of binary mixtures solutions of pure 4-Aminantipyrine standard and its acidic product were prepared; the first series was prepared by using a concentration of 20 mg/l of acidic product with different concentrations of pure 4-aminoantipyrine, while the second series of mixture was prepared by using a concentration 20 mg/l of pure 4-aminoantipyrine with difference concentrations of the acidic product.

b. Ratio spectra method
Standard solutions of 4-Aminoantipyrine ranging from 10 to 70 mg/l were scanned in the range (from 200 to 400nm), and then divided by a spectrum of standard solution (50 mg/l) of its acidic product (as a divisor).The value of peak 299.9nm of the resultant spectra were plotted against the corresponding concentrations to obtain a calibration graph.The first derivative of ratio spectra(1DD) at peak 291 nm and valley 310 nm was selected, the value of the resultant at these pair of wavelength was measured and plotted against the concentrations to obtain two calibration graphs for 1DD method.The second derivative of ratio spectra (2DD) at peak 286nm, valley 299.9 nm and peak 316nm were selected, three calibration graphs were constructed for 2DD method to the corresponding 4-Aminoantipyrine concentration and the regression validation parameters were calculated.

Results and discussion
The derivative spectrophotometric methods for the estimation of organic and pharmaceuticals compounds in presence of its acidic degradation products without chemical separation are always of interest.In this study, the acidic product of 4-Aminoantipyrine was carried out by dissolving the pure organic compound 4-Aminoantipyrine in 2M HCl and reflux at 100 0 C for 60 min.the suggested scheme of acidic product might be written as follows: The acidic product was subjected to IR spectrophotometry analysis, the comparison of IRspectra of 4-Aminoantipyrine with that the acidic product, the characteristic band at 3433.41-3331.18corresponding to the NH2 of the amine groups shown in (Figure1a) , has been disappeared in the IR-spectra of the acidic products (Figure1b) [19].

Zero-crossing method
Zero-crossing method used individual determination of 4-Aminoantipyrine and its acidic product in binary mixture at the selected wavelengths as shown in (Figure3) and (Figure4) respectively.Linearity graphs for 1D and 2D at zero-crossing measurements of standard solutions of (10-70mg/l) for each of 4-Aminoantipyrine and its acidic product were obtained.linear equation, limit of detections and other validation parameters are listed in Table 1.1D and 2D methods used for the estimation 4-Aminoantipyrine in presence of its acidic product by using zero-order measurements at valley 255.9nm and 234.5nm respectively.In the same way, 1D and 2D methods were used for estimation of the acidic product of 4-Aminoantipyrine in presence of 4-Aminoantipyrine by using zero-crossing measurements at peak values at 243.3 nm and 227.3 nm respectively.The relative error and recovery percents for the estimation of 4AAP and in presence of its acidic product in synthetic mixtures were calculated for triplicate measurements for the suggested zero-crossing spectrophotometric method.The values of recovery percentage were found in the range between 96.555 and 102.160 indicating that the recommended zero-crossing method is accurate as shown in Table 2.The relative error and recovery percentage were calculated and presented in Table 4.The obtained values correspond to triplicate analysis of 4-Amionantipyrine solution in the concentration range 25 -45 mg.l-1 using the ratio derivative method.The values of recovery percentage were found to be in the range between 99.64 and 100.11, indicating that the ratio derivative method is reliable and accurate.

Figure ( 1 )
Figure (1): IR-spectra of (a) pure 4-Aminoantipyrine and (b) its acidic product4-Aminoantipyrine and its acidic product UV-spectra are shown in (Figure2); direct estimation of 4-Aminoantipyrine in presence of its acidic products is impossible, zerocrossing and ratio spectrophotometric methods can be resolved and estimation of 4-Aminoantipyrine in presence of its acidic products.