New approach for determination of sulfadiazine in pharmaceutical preparations using 4(4-sulphophenylazo)pyrogallol: Kinetic spectrophotometric method

Article abstract of DOI:10.1016/j.saa.2018.05.012

A new trend describes the development and validation of a simple, sensitive and selective kinetic spectrophotometric methods for the determination of sulfadiazine in pharmaceutical formulations has been conducted. In this paper, sulfadiazine was derivatiz

Full text of DOI:10.1016/j.saa.2018.05.012

Accepted Manuscript
New approach for determination of sulfadiazine in pharmaceutical
preparations using 4(4-sulphophenylazo)pyrogallol: Kinetic
spectrophotometric method
Naser A. Naser, Kasim M. Alasedi, Zainab A. Khan
PII:
DOI:
Reference:
S1386-1425(18)30402-5
doi:10.1016/j.saa.2018.05.012
SAA 16035
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 3 January 2018
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3 May 2018
Please cite this article as: Naser A. Naser, Kasim M. Alasedi, Zainab A. Khan , New
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New approach for determination of sulfadiazine in pharmaceutical preparations
using 4(4-sulphophenylazo)pyrogallol: Kinetic spectrophotometric method
Naser. A. Naser1*, Kasim. M. Alasedi2 and Zainab. A. Khan3
1 Department of Chemistry, Faculty of Science, University of Kufa, P.O. Box(21),
Najaf Governorate, IRAQ.
2 AL-Sheik al-tousi, University College, An-najaf al-ashraf, P.O. Box(9), IRAQ.
3 Department of Chemistry, Faculty of Education for Girls, University of Kufa, P.O.
Box(21), Najaf Governorate, IRAQ.
Naser. A. Naser
Kasim. M. Alasedi
Zainab. A. Khan
E-mail: nasser_an77@yahoo.com
E-mail: dr.kasim@altoosi.edu.iq
E-mail: zainaba.almadani@uokufa.edu.iq
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New approach for determination of sulfadiazine in pharmaceutical preparations
using 4(4-sulphophenylazo)pyrogallol: Kinetic spectrophotometric method
Naser. A. Naser1*, Kasim. M. Alasedi2 and Zainab. A. Khan3
1 Department of Chemistry, Faculty of Science, University of Kufa, P.O. Box(21),
Najaf Governorate, IRAQ.
2 AL-Sheik al-tousi, University College, An-najaf al-ashraf, P.O. Box(9), IRAQ.
3 Department of Chemistry, Faculty of Education for Girls, University of Kufa, P.O.
Box(21), Najaf Governorate, IRAQ.
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Abstract
A new trend describes the development and validation of a simple, sensitive
and selective kinetic spectrophotometric methods for the determination of
sulfadiazine in pharmaceutical formulations has been conducted. In this paper,
sulfadiazine was dervitized as a new organic compound 4(4-sulphophenylazo)
pyrogallol, 4-SPAP, by coupling pyrogallol with diazotized sulfadiazine in medium of
controlled pH. 4-SPAP was characterized by techniques of FT-IR, H-NMR, GC-mass,
TG and DSC thermal analysis methods. Solvatochromic behavior in solvents of
various polarities was also investigated.
The determination of sulfadiazine was accomplished by initial rate and fixed time
methods. These methods were based on the reaction of the compound containing
sulfadiazine, 4-SPAP, with Ca(II) to form colored product with a maximum
absorbance at 520nm. The two methods were adopted for constructing the calibration
curves and examined for their suitability for the quantitation of sulfadiazine in
pharmaceuticals. The limit of detection (LOD)and limit of quantification(LOQ)were
found to be, by initial rate method, 0.35 and1.05 µg.mL-1and that by fixed time method
were found to be 0.69 and2.07µg.mL-1, respectively. The percent relative standard
deviations (%RSD) for the results ranged from 1.04% to 1.76% and 0.85% to 1.42%
for the initial rate and fixed time methods of the proposed kinetic spectrophotometric
method, respectively. The existence of common excipients in the pharmaceutical
formulation did not produce any significant interference. Statistical comparison was
reported as indicated from the F- and t-test data of the proposed methods with that of
reference method showing excellent agreement and indicating no significant
difference in their accuracy and precision.
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Keywords: sulfadiazine; kinetic methods of analysis; pharmaceutical analysis;
solvatochromic.
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1. Introduction
A considerable deal of attention has been simulated for the preparation and
studies of organic reagents in recent years. Numerous of these organic reagents have
drawn much attention as they are sensitive, chromogenic reagents in addition to being
interesting complex-forming agents. Azo compounds with –N=N‒ moiety, are among
the most deep explored classes of organic reagents both from the theoretical and
practical view point. The existence of an azo linkage in aromatic compounds makes
them highly important in dyestuff industry, dosimetry and pharmacy[1-3]. The
medicine of sulpha drugs is the core of a variety of drugs which functions to treat
allergy, coughing and also used for other uses. It should be taken into consideration
that sulfa drugs and sulfites, do not sulfonamide and do not have same effect of
sulfonamides[4,5].
Fig. 1
One of the most important compounds of sulfa antibiotics is sulfadiazine which has
been listed by the world health organization (WHO) as essential medicines of
sulfonamides[6]. It works by stopping the growth of bacteria and other organisms.
Sulfadiazine is used to treat many different types of infections, such as urinary tract
infections, ear infections, meningitis, malaria, toxoplasmosis, and others [7].
Several analytical methods are available to quantify amount of sulfadiazine and other
drugs in pharmaceutical dosage forms. Among these methods, kinetic method has
acquired specific attention in chemical and pharmaceutical analysis due to their
selectivity and elimination of additive interferences, which affect direct
spectrophotometric methods. Furthermore, such methods show high selectivity since
they involve the measurement of the absorbance as a function of reaction time[8,9].
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Such kinetic methods, reported in recent publications[10-22], are employed for the
determination of important molecules used in various fields.
To our knowledge at the date of writing, there is no reference in the analytical
literature reviews demonstrating the kinetic spectrophotometric methods for the
determination of the sulfadiazine. For that reason, our aim is to develop a novel
kinetic spectrophotometric method for sulfadiazine drug determination. In the present
method, sulfadiazine was precipitated through coupling reaction with pyrogallol in
medium of controlled pH, then the amount of sulfadiazine is determined by measuring
the absorbance of solution prepared from the precipitate at 520nm by both initial and
fixed time methods.
2. Experimental
2.1. Apparatus
UV-Visible spectra were measured with matched 1 cm quartz cells by T80 uv-visible
spectrophotometer, PG instruments Ltd., UK. Infrared spectra were recorded using
prestige-21 FT-IR, Shimadzu, Japan. Melting points were determined using SMP30
stuart melting point apparatus, UK and are uncorrected while the pH was monitored
using 340i pH-meter WTW, Germany. Thermal analysis conducted by
thermogravimetry and differential scanning calorimeter were run using Stapt-1000
Linseis. Thermal analysis of the synthesized compound and its starting materials
were performed under a continuously purged nitrogen atmosphere (flow rate
50mL/min). The instrument was calibrated for temperature and enthalpy using
indium. The samples were crimped in non-hermetic aluminum pans
and scanned from 30 to 500 ºC at a heating rate of 10 ºC/min by platinum crucible.
The1H-NMR spectrum was recorded in DMSO using NMR Bruker DPX 400
spectrophotometer operating at 300MHz. TMS was used as internal standard and
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chemical shifts δ recorded in ppm. GC-Mass analysis was accomplished using
GCMS-QP2010, Shimadzu, Japan.
2.2. Chemicals and reagents
All chemicals used were of analytical reagent grade. All solutions were prepared with
deionized water. The solvents were used namely cyclohexane, chloroform, benzene,
ethanol, methanol were purchased from BDH(London, England); diethyl ether, ethyl
acetate, acetic acid were purchased from RDH( Buchs, Switzerland); propanol, 1,4-
dioxane, toluene, n-butanol were purchased from Merck(Darmstadt, Germany);
DMSO, and THF were purchased from GCC(Slidell, USA). Solvents were used as
received. Sulfadiazine, sodium nitrite, pyrogallol and calcium chloride were supplied
from Fluka (Buchs, Switzerland ).
2.3. Synthesis of 4(4-sulphophenylazo)pyrogallol
A 5.44g(0.02mmol) of sulfadiazine was diazotized by adding 5mL of concentrated
HCl and 5mL of NaNO2 which prepared by dissolving amount equivalent to
0.02mmol. The diazotization reaction was accomplished with good stirring at (0-5)ºC
for 30min. The prepared diazotized solution was then added drop-wise to beaker
containing 2.52g(0.02mmol) of pyrogallol. The coupling reaction was maintained at
(0-5)ºC, and pH of the mixture was raised by adding cooled solution of conc. sodium
hydroxide and controlled within range of 5.0-6.0. The resulting solution formed a
colored precipitate. The mixture was diluted with appropriate amount of deionized
water and was allowed to stand in ice bath for 1 hr. with constant stirring, left
overnight then filtered and allowed to dry. The product was purified with absolute
ethanol. Yield of the product was 43%.
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The synthetic route of the product is shown in the following figure:
Fig. 2
2.4 Preparation of solutions for analytical application of 4-SPAP: sulfadiazine
determination
4-SPAP stock solution (0.01M)
This was prepared by dissolving 0.1930g of 4-SPAP in absolute ethanol in 50 mL volumetric
flask. Working standard solutions were freshly prepared by diluting the stock solution with
absolute ethanol to obtain the appropriate concentration.
Calcium(II) solution (0.03M)
Prepared by dissolving 0.1651g of CaCl2 in deionized water and the volume was completed
to 50 mL in a volumetric flask with the same solvent.
Sodium hydroxide solution (1M)
It was prepared by appropriate dilution of the standard concentrated solution (5M) with
deionized water in volumetric flask.
Pharmaceutical preparations
Cream. Ten plastic tube containers of cream as sulfadiazine- containing form (each
contains 1% silver sulfadiazine) were weighed. A weighed amount of the cream
equivalent to 1g of sulfadiazine was dissolved in hot water, cooled and made up to 50
mL with deionized water. The resulting solution was filtered and treated as described
under recommended procedure.
2.5. Recommended procedure for the determination of sulfadiazine
2.5.1 Initial–rate method
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In the first procedure, the initial rates of the reactions were determined by measuring
the slopes of the initials tangents to the absorbance-time curves. An increasing
volume of 4-SPAP working standard solution (0.01M) were transferred into a series
of 10 mL standard flask to cover the range of the calibration graph(2×10-5-3×10-4) at
25ºC. To each flask, 1.4mL of 0.03M Ca(II) was added in the same sequence and
diluted up to the mark with ethanol then the contents of the flask were well mixed. An
increase in absorbance with increase in time was recorded at 520nm compared to a
reagent blank prepared in the same way but containing no 4-SPAP. At selected
concentration of 4-SPAP, the initial rates of the reactions were achieved by
determining the slopes of the initial tangents to the absorbance-time curves. A plot of
calibration graph of log initial rate of the reaction versus the log of the molar
concentration of sulfadiazine-containing product.
2.5.2 Fixed time, absorbance difference (ΔA) method
In this procedure of analysis, two series of 10 mL calibrated flask, increasing volume
of stock solution (0.01M) of 4-SPAP was transferred to cover the range of the
calibration graph (2×10-5-2×10-4) then 0.5mL of 1M sodium hydroxide solution was
added and followed by 1.4 mL of 0.03M Ca(II). The contents were well shaken and
the solution was diluted to the mark with ethanol. The reaction mixture was allowed
to stand for 5 min at 25ºC, and the absorbance of 4-SPAP was measured at 520nm
versus a reference reagent.
2.5.3 Determination of sulfadiazine from dosage form
An accurate weight of plastic tube container equivalent 1% of the silver sulfadiazine
was dissolved in deionized water and the volume was made up to 50mL. The residue
of sample was filtered by using membrane filter. The stock solution was diluted
according to need and analyzed by the recommended procedures.
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3. Results and discussion
3.1 Physical and chemical properties of 4-SPAP
The purity of the synthesized azo compound was examined by melting point and TLC
on silica gel plates using 2:3 methanol:toluene as solvent. The reagent is deep orange
powder, slightly soluble in water. It is soluble in ethanol, methanol, acetone, dioxane
and easily soluble in DMF and DMSO. Its solution with pH less than 5 is
distinguished by yellow color, while its alkaline solution is of brown color.
4-SPAP melts at 167 ºC. FT-IR spectrum of 4-SPAP, (Fig. 3), showed strong bands at
(3421.08 and 3379.28cm-1) which belong to the stretching vibration of OH group,
medium band at 1525cm-1 attribute to stretching of N=N group, strong band at
1334cm-1 to stretching SO2(asym.)[23].
The arylazo dye synthesized in the current study may be found in two tautomeric
forms (Fig. 4). The infrared spectrum of the dye revealed the absence of a carbonyl
band, but an intense hydroxyl band in the region 3379-3421 cm-1, suggesting that this
compound exist predominantly in the 3-hydroxytautomeric form in the solid state.
Fig. 3
Fig. 4
(Fig. 5) shows the 1H-NMR spectrum of reagent with broad band at = 12.3ppm
belonging to the more acidic OH group of pyrogallol moiety. Band at =11.9 ppm(br,
1H, –SO2NH– ). 4-SPAP showed broad band at = 9.5 refer to protons of 2OH
involved in strong intramolecular hydrogen bonding. Further bands; =8.4ppm (s, 3H,
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pyridine-H), group of peaks at and 8.0 ppm belonging to 4H of benzene ring
of sulfadiazine moiety and those absorbed at 6.5 to 7.0 belonging to protons of
pyrogallol and aromatic –CH‒N‒. Singlet broad band at = 3.5ppm refer to water in
the solvent, DMSO, where its band at =2.5ppm[24-26].
Fig. 5
3.2. Thermal analysis of 4-SPAP, Thermogravimetry(TG) and differential scanning
calorimetry(DSC)
The data are extracted from thermal analysis that is related to the variation in the
characteristics of material regarding the effect of temperature especially by combining
the two (TG-DSC) techniques.
Figures 6, 7 and 8 show combined TG-DSC analysis of pyrogallol, sulfadiazine and
the reagent respectively. The TG and DSC of the raw materials and of the product of their
synthesis were markedly different. Unlike inorganic materials, organic compounds show
degradation at low temperatures; therefore, thermal data obtained from thermograms, listed in
table 1, reveal that the initial temperature begins with low thermal stability.
Thermal stability of the compound of interest (the synthesized one) demonstrated from figure
8 which can be used in applied field when it is used without any changes in its structure [27].
TG curve for the synthesized compound presented in figure 8 shows three stages of
decomposition. The first stage is attributed to water loss from the surface and pores of the
powder sample. Large mass changes due to high percentage loss stems in the second stage
from about 200-400ºC which referred to maximum amount of degradation of the organic
reagent. End of decomposition appeared in the third stage which may be attributed to residue
of the compound.
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The DSC curve, shows two peaks; endothermic and exothermic. Broad endothermic peak
attributed dehydration/water loss with melting process did not appear significantly.
Exothermic peak is attributed to decomposition of the prepared reagent.
Fig. 6
Fig. 7
Fig.8
Table (1)
Table (2)
3.3 GC-Mass study
The GC method is an effective method for separating and detectig compounds that
can volatilize and are thermally constant. The difference between the normal GC and
GC-MASS method is that of the detecting carrier gas, where MS is used as a detector
instead of FID or any other detector that generates the molecules and evaporates into a
gas where they are ionised into charged particles. The MS is uesed to identify
constitunt elements of some material or part. It is also used to illustrate chemical
structures of molecules, that is, by depending on the work method of MS to expel
chemical compounds for generating charged particles and measure the mass ratio to
their charge. As a result of thousands of compounds stored in the memory of system,
they are matched with compounds that are analyzed by a certain identical percentage
or by identifying the unknown compound. The reason to prepare the new reagent is
that it is not available in memory of the system. The compound sulfadiazine is
chemically stable when it is transformed into a volatile compound through the highest
peak with retention time 26.611min. As for the pyrogallol, it is thought to decompose
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into different parts through the other peaks as shown in Fig. 9, or it is not found in the
library of the device.
Fig. 9
3.4. Effect of pH on the reagent
The orange solution of 4-SPAP dissolved in ethanol was shown peak in 438nm
assaign to N=N absorption, Fig. 10, which emphesize that the dye was formed where
this band was not found in all reacting molecules.
Fig. 10
Based on the acidity of the medium, the solution of 4-SPAP involves three variable acid-base
-
2-
forms: LH4, LH3 , LH2 , Fig.10. The full protonated neutral species, LH4, yellow colour
424nm exist in acidic medium of pH until 1.0 is reached. The orange neutral form
predominated when pH rangeed between 4.0-6.0 and exhibited maximum absorption at
438nm. In media of pH >7.0, the spectrum of 4-SPAP shows absorption band at 470nm with
isobestic point at 365nm, Fig.11. This behavior explains the presence of an equilibrium
between ionic and neutral forms of 4-SPAP [28, 29].
Fig. 11
Dissociation constant of 4-SPAP
The dissociation constant of 4-SPAP was evaluated by a spectrophotometric
method[30, 31] through the absorbance–pH plot at specific wavelengths which
exhibits S-shaped curve. The values of pKa were 5.10 and 9.7
Fig. 12
Fig. 13
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3.5. Solvent effect
The absorption spectra of the azo compound under study were scanned in different
solvents of varying polarities such as acetic acid, ethanol, methanol, n-propanol, 2-
propanol and butanol which belong to protic solvents and ethyl acetate, acetonitrile,
dioxane, diethyl ether, DMSO, DMF and THF as aprotic solvents.
The behavior of electronic spectra in different solvents is complex. In other words
different phenomena are present in various media. Idealized theories[32,33]
considering the solvent dielectric constant to act as a quantitative measure of solvent
polarity. This view is insufficient as the researches lack to dealing with certain solute-
solvent interactions like hydrogen-bonding and electron pair donor /electron pair
accepter(EPD/EPA) interactions, which has a great effect in those interactions.
According to the already described definition of solvent polarity and from fig. 13
which shows variation of max as a function of solvent dielectric constant, it is clear
that sole physical quantity can't represent such solvent dielectric constant. Thus, the
linear solvation energy relationship(LSER) as multiparameter scale of solvent polarity
as proposed by Kamlet and Taft is considered an effective and aspirational approach
for solvation effects, which entails significant interactions of a solute with its
environment and can make an estimation for the compounds under study to form
hydrogen bonds. Kamlet-Taft solvatochromic equation is of the following form[34].
ν = νo + sπ* + bβ + aα
............ (1)
where π* is an index of the solvent dipolarity/polarizability, β is a measure of the
solvent hydrogen-bonding acceptor (HBA) basicity, α is a measure of the solvent
hydrogen-bonding donor (HBD) acidity and νo is the regression value of the solute
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property in cyclohexane as the reference solvent. The regression coefficients s, b and
a in Eq. 1 measure the relative susceptibilities of the absorption frequencies to the
indicated solvent parameters.
The solvent parameters [35] are presented in table 3. The correlations of the
spectroscopic data were accomplished by means of multiple linear regression
analysis. It was observed that absorption frequencies for azo compound in different
solvents, show agreeable correlation with π*, β and α parameters. The results of the
multiple regressions are given in Table 3, and coefficients νo, s, b and a have
confidence intervals at a level of significance of 95%. The degree of success of eq 1 is
shown in figure 14 by means of a plot of calculated absorption wavenumbers (νcalc)
as a function of the corresponding theoretical values. The negative sign of b
coefficient for the synthesized azo compound in protic solvents denote a
bathochromic shifts with increasing solvent hydrogen bond acceptor basicities. This
indicates stabilization of the electronic excited state relative to the ground state. The
positive sign of s and a coefficients point out a hypsochromic shifts with increasing
solvent dipolarity/polarizability and solvent hydrogen bond donor acidities. This
proposes stabilization of the ground state relative to the electronic excited state.
For aprotic case-solvent, coefficient s of negative sign indicates a bathochromic shifts
with increasing solvent dipolarity/polarizability. Also, this proposes stabilization of
the electronic excited state relative to the ground state. The positive sign of b and a
coefficients refer to a hypsochromic shifts with rising solvent hydrogen bond acceptor
basicities and solvent hydrogen bond donor acidities. This suggests stabilization of the
ground state relative to the electronic excited state.
Table 3
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Table 4
Fig. 14
Fig. 15
3.6. Preliminary investigations between 4-SPAP and Ca(II)
Throughout the preliminary investigation on the reaction between 4-SPAP and Ca(II)
in an alkaline medium, a soluble colored chelate complex was formed. Calcium ion
was selected because it's the only metal ion that reacts with 4-SPAP and gives distinct
color change from more than 30 elements of periodic table at different conditions of
concentration, pH and temperature. The colored product is quantified
spectrophotometrically at 520nm. The absorbance of the colored product measured
versus reagent blank increases with time and then the stability of complex remained
for at least 60 min. This was used as a footing for a suitable kinetic method for the
determination of sulfadiazine drug in pharmaceutical preparations using at least of 20
fold excess amount of Ca(II) in the existence of sodium hydroxide solution.
Therefore, the reaction was remarked as pseudo-first order reactions.
The initial rate and fixed time methods [36, 37] were tested and the most proper
analytical merit was chosen regarding the applicability, sensitivity, the values of the
intercept and correlation coefficient (r).
3.7. Optimization of the experimental conditions
Optimum reaction conditions affecting the reaction of 4-SPAP with calcium(II) was
studied. In subsequent experiments, 1mL of 2×10-4M of 4-SASP was taken in 10mL
final volumes and the absorbance was measured at room temperature(25°C) for a
series of solutions by varying one and fixing the other parameters at λmax versus
reagent blank. All the results recorded in tables and figures are the average of five
measurements.
3.7.1. Selection of reaction medium
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In medium of acidify nature, the product showed pale color and low absorbance was
coherent. Such solutions emphasize weakness of the complex formation; therefore,
they were avoided.
To find a suitable medium for the reaction and to investigate the sensitivity of 4-SPAP
-Ca(II) complex, different aqueous bases and buffer solutions were used, such as
sodium hydroxide, ammonium hydroxide, sodium carbonate, phosphate, glycine and
borate buffers. No significant influence of the buffer type on the absorbance was
observed, and the good results were obtained when sodium hydroxide was used.
In order to determine the optimum concentration of sodium hydroxide, various
volumes of 1M sodium hydroxide solution (0.1-2.0) mL were used. The results
proved that 0.5mLof sodium hydroxide (1M) solution was the optimum for the
determination of 4-SPAP. Larger volumes decreased the absorbance of the colored
species. The above optimum volume was used in all subsequent experiments.
3.7.2. Effect of volume of calcium(II)
The volumes (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.5)mL of metal ion for the maximum
color of the product to attain at 520nm were studied when the reagent concentration is
0.03 M. The absorbance increased with the increase in the volume of Ca(II) and
becomes the greatest at 1.4mL. Thus, 1.4mL was selected as optimum value for the
determination process.
3.7.3. Effect of temperature
The influence of temperature was examined in the range of (0-50)ºC. It was noticed
that the color intensity increased with the increase in temperature up to 30 ºC. After
that it was gradually decreased. Therefore, 25 ºC was selected for the determination
process.
3.8. Quantitation Method(Evaluation of the kinetic methods)
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Initial rate method
Under the above specified optimum conditions, the absorbance time curves for the
reaction of 4-SPAP with Ca(II) were constructed, fig. 15. The initial rates of the
reaction were determined from the slopes tangents of the absorption-time curves
which measured for 19min at intervals of 2min starting from 1min at room
temperature.
The initial rates of 4-SPAP reaction with Ca(II) would follow a pseudo-first order
with respect to 4-SASP concentration, and were found to obey the following equation:
V =∆A/∆t = Kʹ Cn
…………(2)
where V is the reaction rate, A is the absorbance, t is the measuring time, Kʹ is the
pseudo-first order rate constant, C is the molar concentration of 4-SPAP, and n is the
order of the reaction. The logarithmic form of the above equation as follows:
Log V = log ∆A/∆t =log Kʹ + n log C
…………(3)
Regression analysis using the method of least square was done to assess the slope,
intercept and correlation coefficient. The analytical figures of merit and results of
regression analysis are given in table 5. Specific rate constant of the kinetic reaction
of 4-SPAP with Ca(II) (k, at 25ºC) was determined and found to be 11.46min-1 or
0.69s-1. The value of n in the regression equation was 1.0693(≈1), confirming that the
reaction of 4-SPAP with Ca(II) was in first order with respect to the 4-SPAP
concentration. The limit of detection (LOD)and limit of quantification(LOQ)were
found to be 0.35 and1.05 µg.mL-1, respectively. These low values confirmed the
good sensitivity of the initial rate method and consequently its capability to determine
low amounts of 4-SPAP.
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Fig. 16
Table 5
Fig. 17
Fixed time
In this method, the absorbance of varying concentrations retaining 4-SASP was
inspected at a pre-selected fixed time of 1, 3, 5, 7 and 9min. Plots of absorbance
against the concentrations of 4-SPAP were conducted at the fixed periods of time for
the reaction, table 6. The regression equations, correlation coefficients, and the LOD
and LOQ are given in table 6. The lowest limits of detection and quantification were
obtained with fixed times of 1, 3, 5, 7 and 9min. However, the fixed time of 5 min
exhibited broader concentration range for quantification. According to the ICH
guidelines for validation of analytical procedures[38], the limit of detection is not
necessary to be part of the validation. Therefore, depending on wider concentration
range and minimal time of analysis, the fixed time of 5 min is recommended for the
determination of 4-SPAP.
Table 6
3.9. Stoichiometry and the probable structure
The stoichiometry of the reaction of (4-SPAP–Ca(II)) was examined by mole ratio
method[39]. Mole ratio procedure was achieved at 1×10-3M solution of each of the
reagent and metal ion to find the stoichiometry of complex. In this way a sequence of
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solutions were ready in which the concentration of the reagent is kept constant and
that of the metal ion is diverse and the optimum condition was set at maximum
wavelength for the product, the absorbance of the solutions were measured versus
blank and plotted versus the ratio of the moles. The stoichiometric ratio between 4-
SPAP and Ca(II) at 520nm was found to be 1:1.
Based on the obtained molar reactivity, and depending on the nature of 4-SPAP,
the suggested structure of the colored product is shown in fig.18.
Fig. 18
3.10. Validation of the proposed methods
Accuracy and precision. The accuracy and precision for determination of current
kinetic spectrophotometric method were inspected at three concentration levels of 4-
SPAP[40] by examining five replicate samples of each concentration by both the
initial rate and fixed time methods. The percent relative standard deviations (%RSD)
for the results ranged from 1.04% to 1.76% and 0.85% to 1.42% for the initial rate
and fixed time methods of the proposed kinetic spectrophotometric method,
respectively (Table 7) confirming the high reproducibility of the results and the
precision of the method. This good value of precision was advisable for quality
control analysis of sulfadiazine in its pharmaceutical forms.
Table 7
3. 11. Specificity and interference
The specificity of the proposed kinetic spectrophotometric methods(initial rate and
fixed time method) for determination of the studied drug in the presence of frequently
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encountered excipient like; sucrose, glucose, lactose, sodium salicylate, sodium
benzoate, starch and benzoic acid was examined, table 8.
Table 8
The interference was considered acceptable for error lower than ± 2%. It was found
that there was no interferences from these excipients and additives, so the proposed
method can be considered selective one.
3.12. Application of the proposed methods
The initial rate and fixed time methods of the proposed kinetic spectrophotometric
method have been performed on commercial pharmaceutical formulations containing
sulfadiazine.
The method involves weighing appropriate amount of the commercial formulation
and dissolving it in conc. HCl. The solution formed was diazotized by addition of
sodium nitrite at 10 ºC. The diazotized product was coupled with pyrogallol under
controlled condition of pH (less than 7.0) and temperature(5 ºC). The resulting
precipitate was filtered, washed and purified. The precipitate contains sulfadiazine
became ready for analysis within 24 hrs.
The concentration of sulfadiazine was computed from its corresponding regression
equations. The results obtained from the investigations are summarized in table 9,
which indicate that there were no interferences from excipients and the proposed
methods have good selectivity. In these states the proposed methods involved (initial
rate or fixed time) which was statistically compared with official method[41] as
shown in table 10. The determination of t-test and F-test indicated no significant
differences between both the current and the reported methods at 95% confidence
21

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level. That refers to good precision and accuracy in the analysis of sulfadiazine in
pharmaceutical dosage forms.
Table 9
4. Conclusion
Sulfadiazine, for first time, was successfully determined by incorporating it as
an organic solid compound, 4-SPAP, through coupling of pyrogallol with diazotized
sulfadiazine in medium of controlled pH within 5.0-6.0. The procedure of synthesis
which yields 4-SPAP did not prevent the ease of its purification, characterization and
solvatochromic behavior with solvents of different polarities which were studied by
applying Kamelt-Taft equation. The determination process was accomplished using
initial rate and fixed time kinetic spectrophotometric methods. The results showed
that these two methods were simple, accurate and precise with low limit of detection.
References methods for sulfadiazine determination require extraction procedures due
to difficulty of solubility of sulfadizine in aqueous media. The presented method does
not need such tedious treatments. Furthermore, the suggested method of analysis
requires simple apparatus for its performance available in any analytical laboratory. In
accordance to all these features, the proposed method can be regarded valuable and
appropriate for quality control and routine determination of the studied drug in
pharmaceutical dosage forms. Further investigation is required to test other forms of
pharmaceuticals containing sulfadiazine such as tablet and gauze.
Acknowledgments
Financial support from the Faculty of Science-University of Kufa is gratefully
acknowledged.
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Table 1. Effect of temperature on mass changes of analyzed materials.
Step 1
Toffset
ºC
Step 2
Toffset
ºC
Step 3
Toffset
Materials
Loss on
Ignition%
Tonset
ºC
Mass
changes%
Tonset
ºC
Mass
changes/%
Tonset
ºC
Mass
changes%
ºC
Pyrogallol
Sulfadiazine
Reagent
30
25
40
305.631
-69.8284
-28.6071
-2.6528
305.631
423.244
433.581
494.503
-28.4374
-24.0122
-29.7011
-
-
-
-98.2658
-56.6.765
-39.7823
315.128
157.482
494.573
494.762
494.573
494.762
500
500
-3.98835
-7.4284
Table 2. Effect of temperature on DSC of analyzed materials.
1 Effect endo (due to water)
Peak max
2 Effect endo
Peak max
ºC
3 Effect exo
Peak max
ºC
Materials
Tonset
ºC
Tonset
ºC
Toffset
ºC
Tonset
ºC
Toffset
ºC
Toffset
ºC
ºC
Pyrogallol
Sulfadiazine
Reagent
128.6
256.3
60.80
-82.402
-37.167
94.07
143.5
266.4
130.59
215
-
-
-20
-
-
265
-
-
360
275
188.62
74
-3.5
190.41
430
349
194.69
(Tonset – the initial temperature of thermal degradation for each stage, Toffset - the final temperature
at which the degradation process for each stage ends, mass loss (W%) and residue at the end of
decomposition processes)
Table 3. Absorption maxima of azo compound in various solvents and selective Kamlet-Taft
solvent parameters.( : dielectric constant)
A: protic solvents
Solvent
max
ν, cm-1
21929.82
23364.49
23255.81
23041.47
22831.05
22831.05
π*
0.64
0.6
0.54
0.52
0.48
0.47
β
0.45
0.63
0.75
0.9
α
1.12
0.93
0.83
0.84
0.76
0.84
ɛ
6.2
acetic acid 456
Methanol
Ethanol
Propanol
428
430
434
32.6
24.6
20.33
19.92
18
2-propanol 438
Butanol 438
B: aprotic solvents
0.84
0.84
Dioxane
Ether
ethyl acetate
DMF
DMSO
THF
434
420
432
426
436
432
23041.47
23809.52
23148.15
23474.18
22935.78
23148.15
24875.62
0.62
0.27
0.45
0.88
1
0.48
0.47
0.45
0.69
0.76
0.55
0.4
0
0
0
0
0
0
0.19
2.3
4.3
6
36.7
47
0.55
0.66
7.6
37.5
Acetonitrile 402
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Table 4. Regression fits to solvatochromic parameters of Eq 1.
Azo
compound
in
νo
S
B
a
R
F
protic solvent
20399.17 5015.253 -2337.14
22770.57 -1474.66
2275.391
11081.79 2431.977
7.131585
13.95984
0.9145
0.9332
aprotic
solvent
R- Correlation coefficient; F- Fisher's test
Table 5. Analytical information for the initial rate method of the proposed kinetic
spectrophotometric method for determination of 4-SPAP.
Linear range(M)
Leastsquareequation(Log
Correlation coefficient(r)
LOD(M)
LOQ(M)
V=log K ʹ + n log C)a
Intercept (log K)
Slope(n)
2×10-5- 2×10-4
1.6164
0.10693
0.9812
0.09×10-5 (0.35)b 0.27×10-5(1.05)
aV is the reaction rate, K ʹis the conditional rate constant, n is the order of reaction, and C is the molar
concentration of 4-SPAP.
b Figures in parenthesis are the linear range in µg.mL-1.
Table 6. Analytical parameters for the fixed-time method of the proposed kinetic
spectrophotometric method for the determination of 4-SPAP.
Reaction
time(min)
Linear
range(M)
Correlation
coefficient(r)
Slope (b)
Intercept(a)
LOD
(μg.mL-1)
LOQ
(μg.mL-1)
1
3
5
7
9
1×10-5- 1×10-4
1×10-5- 2×10-4
1×10-5- 2×10-4
1×10-5- 2×10-4
1×10-5- 2×10-4
0.9856
0.9745
0.9901
0.9882
0.9868
0.0044
0.0067
0.0147
0.0202
0.0243
0.0021
-0.0001
-0.0022
-0.0025
-0.0037
1.06
0.81
0.69
0.59
0.40
3.18
2.43
2.07
1.77
1.20
29