CHAPTER 2

NEW METHODS FOR THE SPECTROPHOTOMETRIC

DETERMINATION OF SILVER SULFADIAZINE AND
DICLOFENAC DIETHYLAMINE

SECTION I:
VISIBLE SPECTROPHOTOMETRIC METHODS FOR THE DETERMINATION
OF SILVER SULFADIAZINE IN BULK AND DOSAGE FORMS

SECTION II:
VALIDATED SPECTROPHOTOMETRIC METHODS FOR THE
DETERMINATION OF DICLOFENAC DIETHYLAMINE

f/jrs'/sr/M ■jr/jt/jrs<ars,m-ar.’

ABSTRACT
The present chapter describes new spectrophotometric methods for the determination of \
I
\ two drugs namely, silver sulfadiazine and diclofenac diethylamine. For the determination of silver *
I i
I sulfadiazine, />-dimethylaminobenzaldehyde and 1,2-napthoquinone-4-sulfonic acid sodium are f

| used as chromogenic reagents. Diclofenac diethylamine is determined by using two new coloring
I
| reagents namely, ferric ammonium sulfate with 1,10-phenanthroline and p-chloranilic acid.
I Developed spectrophotometric methods are simple, sensitive and selective that can be %
i
{ successfully applied for the analysis of studied drugs in pure and dosage forms. *
**
4, 4
\
4 4
4 4
4
4
1 4
i 4
'4
4
f 4
4
« 4
4
i f,
i 4
i* 4
i 4
i
l 4
s
,
l 4
4.
* Silver sulfadiazine Diclofenac diethylamine 4
i 4
i
>•••< r.-jr-jr. *r/jr- r/.ff, sr/ir- A'-Jt
4
waTsM'*’/-*' ■&'*»

23
CHAPTER 2 SECTION I

VISIBLE SPECTROPHOTOMETRIC METHODS FOR THE

DETERMINATION OF SILVER SULFADIAZINE IN BULK AND
DOSAGE FORMS

2.1.1. DRUG PROFILE

Silver sulfadiazine chemically, silver-[(4-aminophenyl)sulfonyl](pyrimidin-2-

yl)azanide belongs to the class of drugs known as sulfa antibiotics. This medication is
mainly used to prevent the growth of bacteria that may taint an open wound. It also helps
in reducing the spreading of bacteria to the surrounding skin or to the blood where it
causes serious blood infection (sepsis) [1-4]. Currently, silver sulfadiazine is used
unanimously in hospitals to prevent serious bum infections. It kills bacteria [5-7], herpes
virus [8] as well as the protozoal parasite Plasmodium berghei (malaria) [9]. Silver
sulfadiazine also kills various yeasts, including several Aspergillus varieties, Mucor
pusillus, Rhizopus nigricans and 50 different clinical isolates of Candida albicans [10].
Silver sulfadiazine is a topical sulfonamide/silver antibacterial drag [11] which has been
effectively used for the topical remedy of contaminated bums, including chemical bums.
It affects a broad spectrum of organisms and fights against bacteria as well as yeast on the
infected areas of burned skin. So, it is considered as an antimicrobial agent. Furthermore,
it prevents the growth of a wide array of bacteria as well as yeast on the infected bums.

2.1.2. LITERATURE SURVEY- ANALYTICAL FRAME WORK

A limited number of analytical methods have been described in the literature for
the quantitative determination of silver sulfadiazine which include high performance
liquid chromatography (HPLC) [12-14], titrimetry [15], atomic absorption spectrometry
[16], oscillographic titration [17] and spectrophotometric [18] methods. Brief reviews of
those methods are given below.
Ding et al., [12] proposed new HPLC method to determine the content of silver
sulfadiazine in chitosan silver sulfadiazine mucilages. The method was carried out using

24
an Agilent Eclipse XDB-C18 (250 mm x 4.6 mm, 5 pm) column with the detection
wavelength at 254 nm. The flow rate of the mobile phase [acetonitrile-1% phosphoric
acid (8:92)] was 1 mL’min'1.
Nataly and Myriam [13] reported HPLC method for the quantification of silver
sulfadiazine in a cream formulation, which was based on the official method mentioned
in United States Pharmacopoeia (USP 30) and it can be applied in quality control with
good reliability.
Li et al., [14] reported HPLC method for the determination of silver sulfadiazine
in gel. The column used was Nova-pak C18 (5.0 mm x 300 mm, 4 mm) with 30 °C
column temperature. The mobile phase was 0.1 % phosphoric acid-acetonitrile (92:8).
The flow rate was 1.0 mL min'1. The detector wavelength was 265 nm.
Abramovic et al., [15] proposed titrimetric method for the determination of silver
sulfadiazine as Ag in dermazin cream using potassium iodide solution.
Zhenxing and Zhujun [16] developed atomic absorption spectrometric method for
the determination of silver sulfadiazine. Method was based on the standard curve method
where recovery rate of the sample was 99.89 % with RSD of 0.21 %.
Kun-hong et al, [17] developed oscillographic titration method for the
determination of silver sulfadiazine. Method was studied by dissolving the sample into
nitric acid solution. Sodium tetraphenylborate (TPB) was added to precipitate silver and
excessive TPB was titrated with the solution of tetraethylammonium chloride in the
media of NaOH-NaAc (pH 12).
Usman and Mohamed [18] described spectrophotometric method for the
determination of silver sulfadiazine. The methods included determination of silver in
silver sulfadiazine pharmaceutical creams and ointments. Organic residues in the
products were destroyed with sulfuric acid and nitric acid mixture. After evaporation of
acids the silver was reduced with formaldehyde in alkaline solution, pH 11-12.5, to the
metallic state; which was then re-oxidized at pH 3.5 with Fe(III) in the presence of
femozine. Resulting absorbance was measured at 562 nm.

25
2.1.3. AIM AND SCOPE OF THE PRESENT WORK

As per discussion in the literature review, most of the developed methods include
determination of amount of silver present in the silver sulfadiazine but not the
determination of bulk drug. There are no reports in literature on visible
spectrophotometric determination of silver sulfadiazine in pure and dosage forms. Thus,
the aim of the present work is to develop and validate new, simple spectrophotometric
methods for the determination of silver sulfadiazine. The present investigation deals with
the development of simple spectrophotometric methods for the determination of silver
sulfadiazine by utilizing p-dimethyl aminobenzaldehyde and l,2-napthoquinone-4-
sulfonic acid sodium. The proposed methods are sensitive, accurate and precise that gave
good results when applied for the formulations containing silver sulfadiazine.

2.1.4. EXPERIMENTAL

2.1.4.1. Apparatus

A UV-visible spectrophotometer (SHIMADZU, Model No: UV 2550) with 1 cm

quartz cells was used for the absorbance measurements.

2.1.4.2. Reagents and Solutions

All solutions were prepared with double distilled water. Chemicals used were of
analytical reagent grade. Solutions of 1 % /?-dimethylaminobenzaldehyde (PDAB) and
0.05 % l,2-napthoquinone-4-sulfonic acid sodium (NQS) were prepared in water.
Standard drug solution (1000 pg mL"1) was prepared by dissolving silver sulfadiazine

(SSD) in cone. H2SO4 and diluted with distilled water. The stock solution was diluted
approximately to get working concentration.

2.1.4.3. Assay Procedures

2.1.43.1. Determination ofSSD using PDAB (Method A)

Aliquots containing 5.00-30.00 pg mL'1 of SSD were transferred into a series of

10 mL volumetric flasks. To each flask, 1 mL of PDAB (1 %) solution was added and

made up to the mark with ethanol. The absorbance of yellow colored chromogen was

26
measured at 452 nm (Figure 2.1.1) against the reagent blank. The amount of SSD present
in the sample was computed from its calibration curve.

2.1.4.3.2. Determination of SSD using NQS (Method B)

Aliquots containing 5.00-25.00 pg mL'1 of SSD were transferred into a series of

10 mL volumetric flasks. To each flask, 1 mL of NQS (0.05 %) solution and 0.5 mL of

NaOH (0.1 M) were added. The solution was kept aside for 15 minutes and made up to
the mark with distilled water. The absorbance of reddish orange colored chromogen was
measured at 479 nm (Figure 2.1.2) against the reagent blank. The amount of SSD present
in sample solution was computed from its calibration curve.

2.1.4.3.3. Assay offormulations

SSD ointments (Silver sulfadiazine and Silverdine) were dissolved in cone.

H2SO4 [19] and stirred vigorously for 20 min at 50 °C. After cooling to room temperature
the solution was diluted with 100 mL distilled water. A convenient aliquot was then
subjected to the analysis using proposed methods.

2.1.5. RESULTS AND DISCUSSION

2.I.5.I. Chemistry of Colored Species

In the present work PDAB and NQS is used as chromogenic reagents for the
determination of SSD.
Certain amines condense with various aldehydes in strongly acidic medium to
give colored product. Among many, PDAB, vanillin, formaldehyde, benzaldehyde,
salicylaldehyde, paraldehyde, aminobenzaldehyde, m- and p-nitrobenzaldehyde, etc. are
widely used aldehydes as an analytical reagent for the determination of drugs containing
amine group [20]. The reaction of PDAB with aromatic amines to give Schiff base is
equally well documented [21-25]. In the present method the amino group in benzene
moiety of SSD undergoes condensation with PDAB in acid medium to form yellow
Schiff base with maximum absorbance at 452 nm. The probable reaction mechanism is
given in Scheme 2.1.1.

27
 NQS is a chromogenic reagent which reacts with amino group in alkaline
condition to form colored species which are successfully measured by using
spectrophotometry. In the present investigation, amino group in the drug act as a
nucleophile due to the presence of lone pair of electron of the nitrogen atom. This free
amino group of SSD molecule reacts with NQS to give red colored product (Scheme
2.1.2) which is estimated spectrophotometrieally at 479 nm.

2.1.5.2. Optimization of Reaction Conditions

The optimum conditions for the developed methods are fixed based on the study
of the effects of various parameters such as concentration of the drug solution,
concentration of the reagents such as NQS and PDAB and choice of the base. Controlled
experiment is carried out by measuring absorbance at 452 and 479 nm for method A and
B respectively.
To find suitable reagent concentration for the reaction, reagent (PDAB) in
different concentrations (0.5-3.0 %) is used in method A. After the number of preliminary
experiments, it is found that 1 mL of 1% PDAB is optimal for the formation of color with
maximum intensity. In order to optimize the reagent concentration, NQS in different
concentrations (0.025-1.5 %) are used in method B. It is found that 1 mL of 0.05 % NQS
is optimum for the formation of stable colored species. Reaction involved in method B is
mainly based on nucleophilic substitution reaction. Alkaline medium is necessary to
activate the nucleophilic substitution reaction. Best results are obtained in case of NaOH
as base and 0.5 mL of 0.1 M NaOH is found to be optimum for SSD-NQS reaction.

2.1.5.3. Analytical Data and Method Validation

2.15.3.1. Linearity and sensitivity

The linearity is evaluated by linear regression analysis of Beer’s law data using
least - square regression method (Figures 2.1.3 & 2.1.4), which is used to calculate the
correlation coefficient (r), intercept (a) and slope (b) of the regression line. The
sensitivity parameters such as molar absorptivity, Sandell’s sensitivity, detection limit
and quantification limit of proposed methods are calculated and obtained results are
summarized in Table 2.1.1.

28
2.1.5.3.2. Accuracy and precision

The accuracy and precision of the methods are established by analyzing the pure
drug solution at three different levels (with in working limit). The results of relative error
(RE %) which is the measure of accuracy and relative standard deviation (RSD %),
a measure of precision are presented in Tables 2.1.2A & 2.I.2B. RE and RSD are less
than 3 % which reveals the high accuracy and precision of the methods.

2.1.5.3.3. Selectivity

In pharmaceutical analysis, it is important to analyze the magnitude of selectivity

towards the some excipients which often associated with pharmaceutical preparation. The
effect of the presence of some common excipients (starch, lactose, citric acid and sodium
carbonate) have been studied and optimized. The results suggest the lack of interference
from drug formulation.
The selectivity of the proposed methods is tested by preparing the placebo blank
(solution containing all the tablet excepients except SSD). A solution of analytical
placebo is prepared according to the sample preparation procedure and subjected to the
analysis using the procedure described earlier. The absorbance measured is nearly same
as that of the reagent blank which indicates that the change in absorbance with respect to
the reagent blank is caused only by the analyte. Interference by common additives is
analyzed by preparing the test solution containing following components: SSD (10 mg),
starch (40 mg), lactose (30 mg), citric acid (35 mg) and sodium carbonate (45 mg). The
entire mixture is transferred to 100 mL calibrated flask, the contents shaken for 20 min;
volume diluted to the mark with distilled water, mixed and filtered. The filtrate after
suitable dilution is analyzed by proposed method. Good recovery result obtained by the
analysis confirms the selectivity of the proposed methods in the presence of excipients.

2.1.5.3.4. Robustness

Robustness is a measure of the performance of a method when small, deliberate

changes are made to the experimental conditions. Robustness of the method is
determined by making slight deliberate changes in reaction time and reaction condition. It
is observed that there are no marked changes in the experimental results, which
demonstrated that the developed spectrophotometric methods are robust.

29
2.1.6. APPLICATIONS

The proposed methods are successfully applied to determine SSD in

pharmaceutical formulation. The content of the formulation is calculated by applying
suitable dilution factor. The results for the formulation are compared statistically with
those of the tabulated value at 95 % confidence level. The calculated student’s t-test did
not exceed the tabulated value, indicating that there is no significant difference between
the proposed methods and the tabulated value with respect to accuracy and precision.
Table 2.1.3 gives the results of the determination from which it is clear that there is close
agreement between the results obtained by the proposed methods and label claim.

2.1.7. CONCLUSIONS
❖ In this study simple, rapid and sensitive visible spectrophotometric methods are
developed and validated for the determination of SSD in bulk and dosage forms.
❖ The procedure requires shorter reaction time, inexpensive reagents and gives
stable colored species.
❖ There is no influence from common additives and excipients. The result
demonstrated that the procedure is accurate, precise and reproducible.
❖ The methods thus can be successfully applied for the determination of SSD in
pure and dosage forms.

30
Table 2.1.1: Spectral and statistical data for the determination of SSD

Parameters SSD-PDAB SSD-NQS

Xmax (nm) 452 479
Beer’s Law Limits (pg mL'1) 5.00 - 30.00 5.00 - 25.00

Molar Absorptivity (L mol'1 cm'1) 0.19 x 104 0.46 x 104

Sandell’s Sensitivity (pg cm'2) 1.87 x 10'2 3.4 x 10'2

Limit of Detection* (pg mL'1) 0.8887 0.6263

Limit of Quantification * (pg mL'1) 2.6931 1.8981

Regression Equation** Y=a+b X Y=a+bX

Slope (b) 0.0518 0.0285

Intercept (a) -0.0290 0.0004

Correlation Coefficient (r) 0.9980 0.9509

* Calculated according to ICH guidelines

** Y is the absorbance and X is the concentration in pg mL'1

31
Table 2.I.2A: Evaluation of accuracy and precision (SSD-PDAB)

Amount taken Amount found* RE SD RSD

(l»g mL1) (jig mL'1) (%) (pg mL'1) (%)

15.00 14.85 1.00 0.01 0.06

20.00 19.90 0.50 0.01 0.05

25.00 24.88 0.48 0.47 1.92

* Mean value of five determinations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 2.I.2B: Evaluation of accuracy and precision (SSD-NQS)

Amount taken Amount found* RE SD RSD

(M mL'1) ( pg mL'1) (%) (fig mL'1) (%)

5.00 5.03 -0.60 0.06 1.19

20.00 19.97 0.15 0.08 0.40

25.00 24.84 0.64 0.33 1.32

* Mean value of five determinations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 2.1.3: Result of assay of formulations by the proposed method

Brand name Labeled amount Found* ± SD using Found* ± SD using

PDAB NQS
(mg)

14 ±0.18 15.12 ±0.01

Silver sulfadiazine 15
t = 0.38 t = 1.52

200.60 ± 0.07 200.14 ±0.04

Silverdine 200
t = 0.34 t = 0.18
*Mean value of five determinations

Tabulated t value at 95% confidence level is 2.77

32
 wavelength (nm) Wavelength (nm)

Figure 2.1.1: Absorption spectrum Figure 2.1.2: Absorption spectrum

for SSD-PDAB for SSD-NQS

Concentration (jig mL'1)

Figure 2.1.3: Calibration curve Figure 2.1.4: Calibration curve

for SSD-PDAB for SSD-NQS

33
 Yellow colored product

Scheme 2.1.1: Reaction of SSD with PDAB

SSD NQS

NaOH

Orange colored product

Scheme 2.1.2: Reaction of SSD with NQS

34
CHAPTER 2 SECTION II

VALIDATED SPECTROPHOTOMETRIC METHODS FOR THE

DETERMINATION OF DICLOFENAC DIETHYLAMINE

2.11.1. DRUG PROFILE

Diclofenac diethylamine chemically, A-ethylethanamine 2-[(2,6-dichloro phenyl)

amino jbenzeneacetate is a powerful non-steroidal anti-inflammatory drug (NSAIDs)
[26]. It has been used extensively in Europe since 1985 to relieve the symptoms of the
knee inflammation as well as other painful inflammatory tendon, ligament, muscle, and
joint conditions [27]. Diclofenac diethylamine works by blocking the production of some
of the body chemicals that cause inflammation, pain, stiffness, tenderness and swelling
[28], Diclofenac diethylamine is applied as a gel and usually only a small amount is
absorbed into the body. The drug undergoes substantial hepatic first-pass metabolism and
thus only about 50 % of the administered dose reaches systemic circulation [29].
Therefore, the chances of medicine interactions and side-effects occurring are small. The
drug, diclofenac diethylamine also possesses the ideal characteristics, such as poor
bioavailability (40 to 60 %), short biological half-life (2 to 3 hrs), smaller dose (25 to 50
mg), etc., to be formulated into a transdermal patch [30]. Transdermal patches offer
added advantages, such as maintenance of constant and prolonged drug level, reduced
frequency of dosing, minimization of inter and intra patient variability, self­
administration and easy termination of medication, leading to patient compliance [31].
Diclofenac diethylamine is a prostaglandin synthetase enzyme inhibitor [32] which is
mainly used to relieve acute pain. It is generally used in addition to other non-medication
measures (such as getting enough rest) to relieve discomforts. Diclofenac diethylamine
may take a few weeks to improve inflammation but can start to relieve pain after the first
dose.

2.11.2. LITERATURE SURVEY- ANALYTICAL FRAME WORK

A literature survey revealed that only few analytical methods are widely known
for the determination of diclofenac diethylamine in pharmaceutical formulations.

35
Reported methods include, high performance liquid chromatography (HPLC) [33-36],
reversed phase high performance liquid chromatographic (RP-HPLC) [37-38], thermal
analysis [39] and spectrophotometric methods [40-42]. Brief reviews of those methods
are given below.
Wang et al, [33] developed HPLC method for the determination of relevant
substances of diclofenac diethylamine in emulgel. An Ultimate Cl8 column was used
with the mobile phase of 0.2 % acetic acid (pH = 3.0) - methanol (34:66). The detection
wavelength was 254 nm. Blank adjuvant didn't interfere with the detection of impurities.
The relevant substances were separated from diclofenac diethylamine and the detection
limit was 0.02 mg mL'1.
Silva et al., [34] proposed new analytical method for the quantitation of
diclofenac diethylamine in human skin by HPLC, in accordance with Regulation
899/2003 of the National Sanitary Surveillance Agency (ANVISA). The HPLC column
was a reversed-phase Shimpack Cl8, with a 5 mm particle bed at flowing rate 1.2 mL
min'1. Analytes were measured by a UV detector set at 280 nm.
Shah et al., [35] reported simple and precise HPLC method for the determination
of diclofenac diethylammonium in gels. The drug was chromatographed on a reverse
phase C18 column. The eluants were monitored at a wavelength of 282 nm utilizing a
mixture of 0.01M disodium hydrogen orthophosphate-MeCN (50:50).
Mulgund et al, [36] proposed simple, specific, accurate and stability-indicating
reversed phase high performance liquid chromatographic method for the simultaneous
determination of mephenesin and diclofenac diethylamine, using a Spheri-5-RP-18
column and a mobile phase composed of methanol: water (70:30, v/v), pH 3.0 adjusted
with o-phosphoric acid. The retention times of mephenesin and diclofenac diethylamine
were found to be 3.9 min and 14.5 min respectively.
Wei-ling [37] proposed RP-HPLC method for the analysis of related substances in
diclofenac diethylamine. The test used an Ultimate Cl8 column (250 mm x 4.6 mm,
5 mm). The mobile phase consisted of methanol-4 % acetic acid solution (65:35) at the
flow rate of 1 mL min'1 and the detection wavelength was set at 254 nm.
Asghar et al., [38] described new specific, precise, accurate, and robust
RP-HPLC method for the simultaneous determination of lidocaine and diclofenac

36
diethylamine in a pharmaceutical gel formulation. The stationary phase was Princeton
SPHER 100 C18 column (250mm x 4.6mm, 5m). The mobile phase was
acetonitrile:potassium dihydrogen phosphate (0.01M):butane sulfonic acid sodium salt
(45:55:0.1 %) and adjust to pH 6.8 ± 0.05 with triethylamine. Detection was carried out
at 261 nm using Jasco UV 2075.
Fini et al, [39] reported thermal (DSC, TGA and HSM) methods for the analysis
of diclofenac salts. A variety of alkyl, hydroxyalkyl and alkyl hydroxyalkyl linear amines
of drug were prepared and analyzed. All the salts demonstrated thermal instability at
temperature above the melting point, showing a dramatic loss of weight. In each case the
TGA profile indicates that it corresponds to the base content inside the salt which is
associated with a broad endotherm in the DSC thermogram that follows or overlaps with
that of the melting endotherm.
Chaudhary et al, [40] developed simultaneous spectrophotometric assay of
diclofenac diethylamine and curcumin in transdermal gels using methanol as the solvent,
in which diclofenac diethylamine was measured at 275 nm.
Bucci et al, [41] proposed spectrophotometric method for the analysis of
diclofenac diethylamine. Drug was analyzed by measuring the absorbance of the sample
at 276 nm using a multi-wavelength computational program.
Vaidya and Parab [42] reported visible spectrophotometric method which
involved the determination of diclofenac diethylamine using 1 % potassium ferricyanide
in the presence of 0.5 % NaOH which produced an orange chromogen with maximum
absorbance at 450 nm and the absorbance obeyed Beer's law in the concentration range of
2.00 to 12.00 mg mL'1. The chromogen was stable for more than 30 minutes.

2.II.3. AIM AND SCOPE OF THE PRESENT WORK

Few analytical methods for quantitative determination of diclofenac diethylamine

in formulations are reported in the literature. Among this only one method describes the
visible spectrophotometric determination of diclofenac diethylamine in pharmaceutical
formulation. The aim of the work is to develop and validate appropriate analytical
method by using visible spectrophotometry for the estimation of diclofenac diethylamine
in bulk and pharmaceutical formulation. In the present investigation, two new

37
chromogenic reagents namely ferric ammonium sulfate with 1,10-phenanthroline and p-
chloranilie acid are used for the determination of diclofenac diethylamine.

2.H.4. EXPERIMENTAL

2.11.4.1. Apparatus

A UV-visible spectrophotometer (SHEVLADZU, Model No: UV 2550) with 1 cm

quartz cells was used for the absorbance measurements.

2.11.4.2. Reagents and Solutions

All solutions were prepared with double distilled water. Chemicals used were
of analytical reagent grade. Solutions of ferric ammonium sulfate (FAS) (0.2 %),
1,10-phenanthroline (0.4 %) were prepared with distilled water. A solution of chloranilic
acid (CAA) (1 %) was prepared in 1,4-dioxane. A stock solution of 1000 pg mL'1
diclofenac diethylamine (DDEA) was prepared by dissolving 0.1 g of pure drug in 100
mL ethanol. The stock solution was diluted approximately to get working concentrations.

2.11.4.3. Assay Procedures

2.11.4.3.1. Determination ofDDEA using FAS (Method A)

Aliquots containing 2.00-14.00 jig mL'1 of DDEA were transferred into a series
of 10 mL volumetric flasks. To each flask, 1 mL of FAS solution and 1 mL of
1,10-phenanthroline solution were added. The solutions were mixed and kept for 20 min
on a water bath at 70 ± 1 °C. Resulting solutions were cooled to room temperature and
then diluted to 10 mL with 0.1 M H2SO4. The absorbance of orange red colored complex
was measured at 510 nm against the blank by UV-visible spectrophotometer. The amount
of DDEA present in the sample was computed from its calibration curve.

2.11.4.3.2. Determination ofDDEA using CAA (Method B)

Aliquots containing 20.00-160.00 pg mL'1 of DDEA were transferred into a series

of 10 mL volumetric flasks. To each flask, 2 mL of CAA was added. The solution was
kept aside for few minutes and the diluted to 10 mL with 1,4-dioxane. The absorbance of

38
the colored species was measured at 526 nm against the blank. The amount of DDEA
present in sample solution was computed from its calibration curve.

2.11.43.3. Assay offormulation

A gel containing DDEA (Voveran® emulgel® 11.60 mg) was dissolved in 100 mL
ethanol. The above solution was filtered using Whatmann No. 1 filter paper. The stock
solution was diluted appropriately to get final concentration of 116 pg mL'1.
A convenient aliquot was then subjected to the analysis using the proposed methods.

2.II.5. RESULTS AND DISCUSSION

2.II.5.1. Chemistry of Colored Species

In the present work FAS with 1,10-phenanthroline and CAA are used as a
coloring reagents for the spectrophotometric determination of DDEA.
Fe(III) salts play a prominent role in the spectrophotometric determination of
pharmaceutical drugs [43], In the present method, Fe(III) in the form of FAS is selected
as a analytical reagent for the determination of DDEA. Method is based on the reducing
properties of the drug, it reduces Fe(III) to Fe(II) and this amount is corresponds to the
drug concentration. The amount of Fe(II) can be determined by using
1,10-phenanthroline through the formation of colored ferroin complex which is measured
at 510 nm (Figure 2.II.1), Thus, the colored complex resulting from DDEA is due to the
fact that, each of two nitrogen atom of 1,10-phenanthroline has an unshared pair of
electrons that can be shared with Fe(II) ion which is formed by the reaction of DDEA
with Fe(III) (Scheme 2.II.1),
CAA has been used for the spectrophotometric determination of drugs containing
n-electron donors such as nitrogen and oxygen [44]. In this method the amino group in
DDEA serves as n-electron donors and is responsible for the formation of charge transfer
complexes with CAA which act as a rc-electron acceptor (Scheme 2.II.2). CAA in
1,4-dioxane forms purple colored complex which exhibits absorption maxima at 526 nm
(Figure 2.II.2). The complex formed is stable for 5 hrs.

39
2.II.5.2. Optimization of Reaction Conditions

The optimum conditions for the development of this method are established by
varying the parameters, one at a time and keeping the others fixed and observing the
effect produced on the absorbance of the colored species.

2.11.5.2.1. Effect of reagent concentration

The absorbance of the reaction increased with increasing the reagent

concentration. It is found that 1 mL of 0.2 % FAS and 2 mL of 1 % CAA is optimal for
the formation of colored product.

2.11.5.2.2. Effect of temperature

The reaction between the drug and Fe(III) salts in the presence of 1,10-
phenanthroline is found to be slow at room temperature and required a longer time for
completion. Hence, reaction is accelerated by carrying out the reactions at higher
temperature. It is found that the maximum absorbance is obtained after heating the
reaction mixture at 60 °C for 20 min.

2.11.5.2.3. Effect of organic solvent

Different solvents like chloroform, 1,4-dioxane, acetonitrile and ethanol have

been tried in order to obtain maximum sensitivity. Among these solvents, 1,4-dioxane is
found to be the best solvent for the formation of stable CAA-DDEA complex.

2.11.5.3. Analytical Data and Method Validation

2.II.5.3.1. Linearity and sensitivity

The calibration graph for the proposed methods is constructed by plotting the
absorbance versus concentration (Figures 2.II.3 & 2.II.4). The validity of molar
absorptivity and Sandell's sensitivity indicates the high sensitivity of the methods. The
high value of correlation coefficient (r) obtained for the proposed methods indicate the
excellent linearity for the determination of DDEA. Statistical analysis is carried out as per
the ICH guidelines [45], Obtained results are summarized in Table 2.II. 1.

40
2.II. 5.3.2. Accuracy and precision

Accuracy is calculated as the percentage recoveries of DDEA in their pure form

and it is evaluated by standard addition technique at three different concentration levels.
Percentage recoveries of both the methods are found to be acceptable. Precision of the
methods are assessed as percentage of relative standard deviation (% RSD) at different
concentration levels. Reproducibility of the methods is evaluated by the analysis of the
drug at three different concentrations for five replicates. RE and RSD are less than 3 %
which reveals the high accuracy and precision of the methods. Detailed results are given
in Tables 2.II.2A & 2.II.2B,

2.II. 5.3.3. Selectivity

The selectivity of the methods are investigated by observing that no interference

by excipients are encountered which are often associated with pharmaceutical
preparation. The effect of the presence of some common excipients (starch, lactose,
ascorbic acid, citric acid and sodium carbonate) have been studied and optimized. The
selectivity of the proposed methods is tested by preparing the placebo blank (solution
containing all the tablet excepients except DDEA). A solution of analytical placebo is
prepared according to the sample preparation procedure and subjected to the analysis
using the procedure described earlier. The absorbance measured is nearly same as that of
the reagent blank which indicates that the change in absorbance with respect to the
reagent blank is caused only by the analyte. Non interference by common additives is
analyzed by preparing the test solution containing following components: DDEA
(15 mg), starch (30 mg), lactose (30 mg), sodium carbonate (35 mg) and citric acid
(20 mg). The entire mixture is transferred to 100 mL calibrated flask, the contents shaken
for 20 min; volume diluted to the mark with distilled water, mixed and filtered. The
filtrate after suitable dilution is analyzed by proposed method. The recovery result
obtained by this measurement is 97.83 ± 0.16 and 98.16 ± 0.19 for both the methods. The
result confirms the selectivity of the proposed methods in the presence of excipients.

2.II. 5.3.4. Robustness

Robustness is examined by evaluating the influence of small variation in the

method variables on its analytical performance. In these experiments, one parameter is

41
changed whereas the others are kept unchanged and the recovery percentage is calculated
each time. It is found that small variation in the method variables does not significantly
affect the procedures.

2.11.6. APPLICATIONS

The proposed methods can be successfully applied for the determination DDEA
in commercial gel (Voveran® Emulgel®). DDEA content in gel (with the declared
amounts of 11.60 mg w/w) has been analyzed by the above described visible
spectrophotometric methods and the results obtained are listed in Table 2.II.3.
No significant differences are found between the calculated and theoretical values of t
tests at 95 % confidence level proving good accuracy in the analysis of the investigated
drug in their dosage form.

2.11.7. CONCLUSIONS

❖ The proposed methods are found to be simple, selective and sensitive.

❖ The results of statistical parameters and recovery studies clearly indicated the
reproducibility and accuracy of the proposed methods.
❖ Analysis of the authentic sample containing drug shows no interference from the
common excipients.
❖ Thus, it can be extended for routine analysis of DDEA in pharmaceutical
industries and research laboratories.

42
Table 2.II.1: Spectral and statistical data for the determination of DDEA

Parameters DDEA-FAS DDEA-CAA

A-max (nm) 510 526

Beer’s Law Limits (pg mL'1) 2.00 - 8.00 20.00 - 120.00

Molar Absorptivity (L mol'1 cm'1) 0.98 x 104 1.62 x 104

Sandell’s Sensitivity (pg cm'2) 3.77 x 10'2 2.27 x 10'2

Limit of Detection * (pg mL1) 0.1976 1.2617

Limit of Quantification * (pg mL'1) 0.5988 2.9411

Regression Equation** Y = a + bX Y = a + bX

Slope (b) 0.0167 0.0034

Intercept (a) 0.0392 -0.0140

Correlation Coefficient (r) 0.9854 0.9933

* Calculated according to ICH guidelines

** Y is the absorbance and X is the concentration in pg mL'1

43
Table 2.II.2A: Evaluation of accuracy and precision (DDEA-FAS)

Amount taken Amount found* RE SD RSD

(fig mL*1) (pg mL1) (%) (fig mL1) (%)

2.00 1.95 2.50 0.27 1.30

4.00 3.92 2.00 0.52 0.13

6.00 5.95 0.83 0.86 0.14

* Mean value of five determinations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 2.II.2B: Evaluation of accuracy and precision (DDEA-CAA)

Amount taken Amount found* RE SD RSD

(fig mL*1) (fig mL*1) (%) (fig mL*1) (%)

20.00 19.94 0.30 0.04 0.20

40.00 39.91 0.22 0.40 1.00

60.00 59.90 0.16 0.70 1.32

* Mean value of five determinations

RE - Relative Error; SD - Standard Deviation; RSD - Relative Standard Deviation

Table 2.II.3: Result of assay of formulation by the proposed method

Brand name Labeled amount Found* ± SD using Found* ± SD

(mg) FAS using CAA

Voveran® emulgel® 11.63 ±0.73 11.46 ± 1.00

11.60
t = 0.52 t = 0.38
*Mean value of five determinations

Tabulated t value at 95 % confidence level is 2.77

44
 fM
H
00
ri ri
VO

Absorbance
Absorbance
<N
tH
O O
bo
CT)
oNJ o4*

(N
oo

oo
VO
oo

8o
D
O
C
oo
o
fS

00
oo
I

Wavelength (nm)
Wavelength (nm)

Figure 2.11,1; Absorption spectrum of Figure 2.II.2: Absorption spectrum of

DDEA-FAS DDEA-CAA
ob>
o
m cn

din
O NJ
NJ
Absorbance

Absorbance
o
h*
U1

o
co
O

o
CM

0.05 - J
0 4*—i—i—i—i—i—i—i—i
0 2 4 6 8 10 12 14 16 0 40 80 120 160 200

Concentration (ng ml/1) Concentration (jig ml/1)

Figure 2.II.3: Calibration curve for Figure 2.11.4: Calibration curve for
DDEA-FAS DDEA-CAA

45
Scheme 2.II.1: Reaction of DDEA with Fe(III) followed by the reaction of Fe(II)
with 1,10-phenanthroline

46
47
REFERENCES
[1] Moyer, C.A., Brentantol, G.H.W. & Margof, W., Arch Szrrg, 90 (1965) 812.
[2] Pegg, S.P., Ramsey, K. & Meldrum, L., ScandJPlaslReconsir Svrg, 13 (1999) 95.
[3] Fox, C.L., Arch. Szrrg, 96 (1968) 184.
[4] Modak, S.M., Sampath, L. & Fox, C.L., JBurn Care Res, 9 (1988) 359.
[5] Grier, N., (1983) “Silver and Its Compounds; Sterilization and Preservation”, Lea
& Febiger, Philadelphia.
[6] Carr, H.S, Wlodkowski, T.J. & Rosenkranz, H.S., Antimicrob Agents Chemother, 4
(1973)585.
[7] Chang, T.W. & Weinstein, L., Antimicrob Agents Chemother, 7 (1975) 538.
[8] Chang, T.W. & Weinstein, L., Antimicrob Agents Chemother, 8 (1975) 677.
[9] Wysor, M„ Chemother, 21 (1975) 302.
[10] Wlodkowski, T. & Rosenkranz, H., Lancet, 29 (1973) 739.
[11] Fisher, N.M., Marsh, E. & Lazova., J Am Acad Dermatol, 49 (2003) 730.
[12] Ding, J., Zhang, H. & Huarong L., Zhongguo Yaoshi, 16 (2013) 1491.
[13] Nataly, T.C. & Myriam, E.T., Revista Colombiana de Ciencias Quimico-
Farmaceuticas, 37 (2008) 191.
[14] Li, Y., Huang, Z., Peng, W. & Chen, Y., Zhongguo Yiyuan Yaoxue Zazhi, 26
(2006) 1311.
[15] Abramovic, B.F., Gaal, F.F. & Marinkovic, M.M., Acta Pharmaceutica
Jugoslavia, 39 (1989) 129.
[16] Zhenxing, F. & Zhujun, C., Huaxi Yaoxue Zazhi, 27 (2012) 217.
[17] Kun-hong, L.M., Jing-yu, L. & Wei-lin, L., Fenxi Kexue Xuebao, 19 (2003) 97.
[18] Usman, Z.H. & Mohamed, J.B., Anal Lett, 31 (1998) 147.
[19] Tsipouras, N„ Rix, C.J. & Brady, P.H., Clin Chem, 41 (1995) 87.
[20] Sushma, K., Somsubhra, G. & David, B., IntJMedPharm Res, 1 (2013) 433.
[21] Anil, K.T., Gurupadayya, B.M. & Reddy, M.B., Indian J Chem Technol, 19
(2012) 56.
[22] Siddappa, K., Mallikaijun, M., Reddy, P.T. & Tambe, M., Ecletica Quimica, 33
(2008)41.
[23] Tehseen, A., Asrar, A.K. & Bushra, M„ Anal Lett, 38 (2005) 1899.

48
[24] Prakash, S.S., Kapse, G.K. & Appala, R.S., Asian JChem, 14 (2002) 545.
[25] Shiniehi, N., Rie, N., Mie, K., Kenichiro, N. & Shuzo, A., Chem Pharm Bull, 30
(1982)2467.
[26] John, V.A, Rheumatol Rehabil, 2 (1979) 22.
[27] Fritz, U.N., Morris, S.G., Gail, S.S., Jiun-min, L., Markus, U., Helmut, H.A.,
Francois, E., J Rheumatol, 32 (2005) 2384.
[28] Kriwet, K. & Muller-Goymann, C.C., EurJPharm Biopharm, 66 (1993) 234.
[29] Haroutinaian, S., Drennan, D.A. & Lipman, A.G., Pain Med, 11 (2010) 535.
[30] Nauman, R.K., Gul, M.K., Abdur, R.K., Abdul, W., Muhammad, J.A.,
Muhammad, A. & Abid, H., Afr J Pharm Pharmaco, 6 (2012) 434.
[31] Bucher, U. & Sanger, A. Diclofenac Emulgel in the Treatment of Localized
Rheumatic Disorders. Presented at XVIth Int Congr Rheumatol (IL:AR) Sydney,
Australia, 1985.
[32] Hema, C., Kanchan, K., Saima, A., Saurabh, A., Vikash, K., Sushila, R. &
Permender. R., JLiq Chromatogr Relat Tech, 35 (2013) 174.
[33] Wang, Y., Zhang, H., Zhou, Y., Tingyan, L. & Ding, J., Zhongnan Yaoxue, 9
(2011)909.
[34] Silva, J.A., Bedor, D.C.G, Sousa, C.E.M., Santana, D.P. & Egito, E.T., Revista de
Ciencias Farmaceuticas Basica e Aplicada, 31 (2010) 41.
[35] Shah, Y., Joshi, S., Jindal, K.C. & Khanna, S., Drug Dev Ind Pharm, 20 (1994)
1303.
[36] Mulgund, S.V., Phoujdar, M.S., Londhe, S.V., Mallade, P.S., Kulkami, T.S.,
Deshpande, A.S. & Jain, K.S., Indian J Pharm Sci, 71 (2009) 35.
[37] Wei-ling, L., Yaowu Fenxi Zazhi, 33 (2013) 1546.
[38] Asghar, S., Sheikh, S. & Showkat, P., Int J Res Pharm Sci, 2 (2012) 78.
[39] Fini, A., Fazio, G., Benetti, L. & Ghedini, V., Thermochim Acta, 464 (2007) 65.
[40] Chaudhary, H., Kanchan, K., Vikash, K., Sushila, R. & Permender, R., Anal Chem
Lett, 1 (2011)224.
[41] Bucci, R., Magri, A.D. & Magri, A.L., Fresenius J Anal Chem, 362 (1998) 577.
[42] Vaidya, R. & Rajesh, P.S., Indian Drugs, 32 (1995) 194.

49
[43] Vinay, K.B., Revanasiddappa, H.D., Devi, O.Z. & Basavaiah, K., Chem Ind Chem
Eng Q, 16(2010) 1.
[44] Zachary, N.A., Rizk, M., Ibrahim, F. & Walash, M., Talanta, 33 (1986) 111.
[45] International Conference on Hormonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use, ICH Topic Q2 (Rl), Validation of
Analytical Procedures (2005).

50