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2018) عام 1العدد ( 13مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد

Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 31 (1) 2018

Two Derivative Spectrophotometric Methods for the
Simultaneous Determination of 4-AminoAntipyrine in

Presence of Its Acidic Products

Ruba Fahmi Abbas

Dept. of Chemistry/Collage of Science/ Al-Mustansiryiah University
rubaf1983@uomustansiriyah.edu.iq

Received in:10/October/2017, Accepted in:6/December/2017

Abstract
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.

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Introduction
4-Aminoantipyrine(4AAP) is an antipyretic, analgesic and anti-inflammatory properties
[1]. It is used as a reagent for biochemical reactions producing phenol or peroxides [2], and
used for the estimation of drugs via oxidative coupling reaction forming coloured products [3-
5].
There are many methods for the determination of 4-Aminoantipyrine including
electrochemical method based on fabrication of multi walled carbon nanotube electrodes for
the estimation toxic drugs 4-Aminoantipyrine [6], solid-phase spectrophotometry [7],
electrochemical method by using graphite pencil electrode [8], capillary electrophoresis [9]
and LC/Mass spectrometry [10].
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.

Experimental

Instruments

1. Uv-Visble spectrophotometer (model 1650 PC, SHEMADZU, Japan) with software
program.
2. FTIR spectrophotometer (SHEMADZU, Japan)

Materials
Standard pure powder of 4-Aminoantipyrine (Sigma-Aldrich) (C11H13N3O) (M.wt= 203.24
gm/mol) and hydrochloric acid(BHD) prepared by 2N aqueous solution.

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 700C, 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 70 mg/l).
2. 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).

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2018) عام 1العدد ( 13لمجلد ا مجلة إبن الهيثم للعلوم الصرفة والتطبيقية

Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 31 (1) 2018

Procedure
a. Zero-crossing method
1. 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 1000C for 60 min. the suggested scheme of acidic product might be written as
follows:

NH2

4-Aminoantipyrine

H3C

O
H3C

(2 N) HCl

ref luxed f or 60 min
N

N+

Acidic product

H3C

H3C O

NCl-

The acidic product was subjected to IR spectrophotometry analysis, the comparison of IR-
spectra of 4-Aminoantipyrine with that the acidic product, the characteristic band at 3433.41-
3331.18 corresponding to the NH2 of the amine groups shown in figure(1a), has been
disappeared in the IR- spectra of the acidic products figure (1b) [19].

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Figure (1): IR- spectra of (a) pure 4-Aminoantipyrine and (b) its acidic product

4-Aminoantipyrine and its acidic product UV-spectra are shown in figure (2); direct
estimation of 4-Aminoantipyrine in presence of its acidic products is impossible, zero-
crossing and ratio spectrophotometric methods can be resolved and estimation of 4-
Aminoantipyrine in presence of its acidic products.

Figure (2): Zero order spectra of 4-Aminoantipyrine 30 mg/l(red) and acidic product 30
mg/l (black).

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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 figure (3) and figure (4)
respectively.

Figure (3): Zero-crossing measurements for (10-70 mg/l) 4-Aminoantipyrine (red) and
40 mg/l acidic product(black) a- 1D at valley 255.9nm, b- 2D at valley 234.5nm for

determination of 4-Aminoantipyrine

Figure (4): Zero-crossing measurements for (10-70 mg/l) acidic product(blue) and 40
mg/l 4- Aminoantipyrine(black) a- 1D at peak 243.3nm, b- 2D at peak 227.3nmfor

determination of acidic product.

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).

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Table (1): Validation parameters for the obtained calibration graphs for the
determination of 4AAP and its acidic product using zero-crossing measurements.

Validation
parameters

1D 2D
4AAP
at valley
255.9nm

Acidic product
at peak
243.3 nm

4AAP
at valley
234.5nm

Acidic product
at peak
227.3 nm

Linearity
range
(mg/L)

10-70 10-70 10-70 10-70

equation Y= -0.026x
-0.0041

Y=0.0095x
+0.0012

Y=-0.0037x
-0.018

Y=0.0093x
+0.0135

R2 0.9960 0.9980 0.9970 0.9970
Slope -0. 026 0. 0095 -0.0037 0.0093
Intercept -0.0041 0.0012 -0.018 0.0135
LOD 0.495 1.354 3.478 1.383
LQD 1.5 4.105 1.054 4.193

R2= Coefficient of determination, 4AAP=4-Aminoantipyrine and LOD= limit of detection =
3.3×SDb/S , LQD=10×SDb/S. where, SDb= is the standard deviation of the solvent (n=3) and S= is
the slop of the corresponding linearity graph.

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).

Table (2): The relative error and recovery percentage for estimation of 4AAP and its
acidic product using zero-crossing method

Taken Mixture of
4AAP +acidic product

Found
mg/l
4AAP

1D at
valley
255.9nm

E% Rec% Found
mg/l
4AAP
2D at
valley
234.5nm

E% Rec%

20 mg/l+0 mg/l 19.397 -3.015 96.985 19.781 -1.095 98.905
20 mg/l+20 mg/l 19.718 -1.41 98.590 19.920 -0.400 99.600
40 mg/l+20 mg/l 39.511 -1.222 98.777 39.130 -2.175 97.825
30 mg/l+5 mg/l 30.219 0.730 100.730 30.424 1.413 101.413
30 mg/l+10 mg/l 29.624 -1.253 98.746 29.110 -2.966 97.033
Taken Mixture of
4AAP +acidic product

Found
mg/l
Acidic
product
1D at

E% Rec% Found
mg/l
Acidic
product
2D at

E% Rec%

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peak
243.3nm

peak
227.3nm

0mg/l+30mg/l 29.676 -1.08 98.920 29.522 -1.593 98.406
20mg/l+10mg/l 9.732 -2.68 97.320 9.866 -1.340 98.660
20mg/l+20mg/l 19.995 -0.025 99.975 19.311 -3.445 96.555
10mg/l+10mg/l 10.054 0.54 100.540 10.216 2.160 102.160
30mg/l+10mg/l 10.124 1.24 101.240 10.098 0.980 100.980

4AAP=4-Aminoantipyrine, E% = relative error = = 100 and Rec% =recovery=

E%+100

Ratio spectrophotometric method

In the ratio spectrophotometric method the obtained absorption spectra of the
mixtures of 4-Aminoantipyrine and its acidic product were divided
then by the absorption spectrum of the acidic product standard
solution (as divisor) as shown in figure (5). The first derivative 1DD and
the second derivative 2DD spectra in each case was then obtained,
as shown in figure (6).

Figure (5): Normal ratio spectra (0DD) of 4-Aminoantipyrine (10-70 mg/l) using 50 mg/l
of acidic product as a divisor

Figure (6): Derivative ratio spectra of 4-Aminoantipyrine (10-70 mg/l) using 50 mg/l of
acidic product as a divisor: (a) first derivative ratio spectra (1DD) and (b) second

derivative ratio spectra (2DD)

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In order to optimize the Ratio spectrophotometric method, various concentrations divisor
solution (10, 30, 50, 70 mg/l) were tried of acidic product; the best results were obtained
when a 50 mg/l of acidic product was used as a divisor. Dividing the absorption spectrum of
4-Aminoantipyrine in the range(10-70mg/l) by absorption spectrum of 50 mg/l of acidic
product (as a divisor). The validation parameters of the obtained linear graphs are summarized
in table (3).

Table (3): Validation parameters for the linear graphs for the determination of 4AAP
and its acidic product using 0DD, 1DD and 2DD methods.

Validation
parameters

0DD at
peak
299.9nm

1DD
at peak
290.7 nm

1DD at
valley
310.nm

2DD
at peak
286 nm

2DD at
valley
301 nm

2DD
at peak
316 nm

Linearity
range
(mg/L)

10-70 10-70 10-70 10-70 10-70 10-70

Equation Y=0.314x
+1.338

Y=0.014x
+0.093

Y=-0.014x
-0.078

Y=0.01x
+0.06

Y=-0.02x
-0. 02

Y=0.01x
+0.05

R2 0.9980 0.9980 0.9970 0.9980 0.9970 0.9990
Slope 0.314 0.014 -0.014 0.01 -0.02 0.01
Intercept 1.338 0.093 -0.078 0.006 -0.02 0.05
LOD 0.040 0.919 0.919 1.287 0.643 1.287
LQD 0.124 2.785 2.785 3.9 1.950 3.9

R2= coefficient of determination

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.

Table (4): The relative error and recovery percentage for estimation of 4AAP and its
acidic product using 0DD, 1DD and 2DD methods

parameters 0DD
at 299.9
nm

1DD
at 290.7
nm

1DD
at 310
nm

2DD
at 286
nm

2DD
at 301
nm

2DD
at 316
nm

Taken
mg/l

25 25 25 25 25 25

Found
mg/l

24.92 24.96 24.93 24.96 24.91 24.93

E% -0.32 -0.16 -0.28 -0.16 -0.36 -0.28
Rec% 99.68 99.84 99.72 99.84 99.64 99.73
Taken
mg/l

45 45 45 45 45 45

Found
mg/l

44.95 44.98 45.08 44.93 44.96 45.02

E% -0.11 -0.044 0.111 -0.155 -0.088 0.044
Rec% 99.88 99.95 100.11 99.84 99.91 100.04

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Conclusion

The proposed methods are simple and accurate making them easily for estimation of 4-
aminoantipyrine in presence its acidic product. The purposed methods require neither pH
control nor temperature control and nor solvent extraction. So, the suggested
spectrophotometric methods are appropriate for the estimation 4-aminoantipyrine and its
acidic product in binary mixture.

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