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CHAPTER 8
SPECTROPHOTOMETRIC ASSAY OF
CHLOROQUINE PHOSPHATE
356
SECTION 8.0
DRUG PROFILE AND LITERATURE SURVEY
8.0.1.0 DRUG PROFILE
Chloroquine phosphate (CQP) is chemically known as 7-chloro-[4-(4-
diethylamino-1-methylbutyl amino]-quinoline diphosphate. Its molecular formula is
C18H26ClN3·2H3PO4, with a molecular weight of 515.87. The structural formula is:
NCl
NHN CH3
CH3
2 H3PO4.
Physically, CQP is a white crystalline powder soluble in water; sparingly
soluble in chloroform and acetonitrile.
It is a antimalarial drug and found effective against erythrocytic forms of
Plasmodium vivax, P. ovale and P. malariae. It also used in the treatment of amebiasis,
rheumatoid arthritis, discoid lupus erythematosus and photosensitive diseases [1].
INH is officially reported in British Pharmacopoeia (BP) [2] and United State
Pharmacopeia (USP) [3]. BP describes non-aqueous titration with perchloric acid as
titrant where the end point is located potentiometrically. USP describes a UV-
spectrophotometric method, where the absorbance of CQP in HCl medium is measured
at 343 nm.
357
8.0.2.0 LITERATURE SURVEY - ANALYTICAL FRAMEWORK
8.0.2.1Titrimetric methods
The literature survey reveals that only four titrimetric procedures have been
reported for the assay of CQP in dosage forms. An acid-base titrimetric method was
developed, where the drug was titrated with 0.1 M NaOH in MeCN: H2O medium
using thymol blue as indicator [4]. A non-aqueous titrimetric method involoving
conductometric titration of drug with HClO4 has also been reported [5]. Gravimetric
estimation of CQP was also carried out using NaBPh4 and picric acid [6].
8.0.2.2 Spectrophotometric methods
A number of visible spectrophotometric methods [7-22] have been reported for
the assay of CQP in pharmaceuticals. Nagaraj et al., [7] reported a colorimetric method
for CQP in tablets and in urine. The method involved the extraction of the drug with
chloroform, the chloroform extract with bromocresol purple (BCP) at pH 5.4, re-
extraction of the aqueous layer with chloroform followed by absorbance measurement
at 420 nm. The method was applicable over a concentration range of 2.5-7.5 µg ml-1
.
The drug has also been determined spectrophotometrically based on ion-pair complex
formation with bromothymol blue (BTB) followed by extraction into dichloromethane
and measurement at 410 nm [8]. BTB has also been used for another based on
measurement of chloroform extactable ion-pair complex at 410 nm [9].The method has
been applied to pure form, pharmaceuticals as well as urine. Onyegbule et al., [10] have
reported a method based on the formation of nitrobenzene-soluble ion-associate
complex formed by the interaction of drug with cobalt thiocyante and absorbance
measurement at 625 nm. Beer’s law is obeyed over a concentration range of 2 -60 µg
ml-1
. Based on the similar reaction, another method was developed by Khalil et al., [11].
The method involved the ion-pair formation between drug and Mo(V)SCN followed by
extraction with methylene chloride. Tetrabromophnolphthalein has also been used as a
chromogenic agent for ion-pair reaction followed by extraction with dichloromethane
[12]. Another ion-association reaction using methyl orange has also been developed for
the assay of CQP in pharmaceuticals [13]
358
Ramana et al., [14] developed a method for the determination of CQP in tablets
and injections. The method involves the reaction between drug and CoCl2-KSCN
followed by the extraction of the blue complex with iso-BuCOMe and measurement of
absorbance at 625 nm. CQP on reaction with ammonium molybdate and SnCl2 formed
molybdenum blue complex which was extracted with iso-BuOH and measured at 720
nm [15].
Zayed et al., [16] developed two simple methods for the quantification of CQP
in pure form and in dosage forms. The methods were based on charge-transfer (CT)
reaction between drug and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) π-acceptor
or iodine σ-acceptor followed by measurement of the colored complex at 462 or 287
nm. Based on similar reaction another method has also been developed by reacting base
form the drug with chloranilic acid [17]. Chloranil has also been used for the charge-
transfer reaction [18].
A method was developed by Mohamed [19] based on oxidation with KBrO3
followed by measurement of tri-iodide ion at 343 nm. A similar method with bromate-
bromide mixture in acid medium where the yellow colored tri-iodide ion is measured at
350 nm [20] has also been reported. N-bromosuccinimide has also been used for the
determination of CQP, where the yellow colored product was measured at 410 nm.
Beer’s law was obeyed over a concentration range of 2.5-7.5 µg ml-1
[21].
The reaction between drug and ammonium molybdate yielded a colored product
peaking at 465 nm [22] and served as the basis for assay.
A derivative UV-spectrophotometric method was developed by Singh et al.,
[23], which involvedmeasurement of absorbance of CQP solution in 0.1 M H2SO4 at
343 nm.
8.0.2.3 Other techniques
Several chromatographic techniques have been employed for the determination
of CQP either in biological fluids or in pharmaceuticals and include HPLC [24-51], gas
chromatography [52-54] and HPTLC [55].
359
Several other techniques have been employed for the determination of CQP in
pharmaceuticals and include capillary electrophoresis [56, 57], fluorimetry [58-60] and
polarography [61].
From the literature survey presented above, it is clear that sixteen visible and
one UV-spectrophotmetric methods are available for the assay of CQP in
pharmaceuticals. The reported spectrophotometric methods suffer from one or the other
limitations such as poor sensitivity and narrow linear dynamic range. The extractive
spectrophotometric methods [7-13] suffer from such disadvantages as rigid pH control
tedious and time consuming extraction step and judicious control of all experimental
variables. Reported UV-spectrophotometric method is not stability-indicating in nature.
Keeping in view the drawbacks of the reported methods, the author has
attempted to develop two visible and one stability-indicating UV-spectrophotometric
spectrophotometric methods giving due consideration to various parameters involved in
the validation and assay of CQP both in pure form as well as in pharmaceuticals. The
details are presented in Section 8.1 and 8.2 and a separate section (Section 8.3) has
been devoted to assess the performance characteristics of the proposed methods in
comparison with the reported methods.
360
SECTION 8.1
SIMPLE AND SELECTIVE SPECTROPHOTOMETRIC DETERMINATION
OF CHLOROQUINE IN TABLETS USING TWO NITROPHENOLS AS
CHROMOGENIC AGENTS
8.1.1.0 INTRODUCTION
Charge-transfer (C-T) complexes, also called electron-donor-acceptor (EDA)
complexes, may be formed when one interactant can perform as the electron donor and
the other as the electron acceptor. The appearance of a new electronic absorption band,
not attributable to either the donor or the acceptor, often, is taken as evidence for
charge-transfer complexing [62]. Charge transfer phenomenon was introduced first by
Mulliken [63, 64] and widely discussed by Foster [65] to define a new type of adduct to
explain the behavior of certain classes of molecules which do not conform to classical
patterns of ionic, covalent, and coordination of hydrogen bonding components. While
such adducts largely retain some of the properties of the components, some changes are
apparent, e.g., its solubility, the diamagnetic and paramagnetic susceptibility. The
charge-transfer complexation arises from the partial transfer of an electron from a
donating molecule having sufficient low ionization potential to an accepting one having
sufficient high electron affinity and as a result, formation of intensely colored charge-
transfer complexes which absorb radiation in the visible region [66] occurs. The source
molecule from which the charge is transferred is called the electron donor (D) and the
receiving molecule is called the electron acceptor (A).
D + A → DA
Compounds with unshared pairs of electrons may interact with other compounds
through the donation of such electrons in a manner different from the traditional dative
bond formation. Those interactions giving rise to intermolecular forces may be
sufficiently strong to show features that do not exactly fit the definition of the classical
dipole–dipole, dipole-induced dipole and/or van der Waals interactions. Depending
upon the orbital that accepts these electrons, these acceptors may be described as δ - or
π -acceptors [67].
361
Amines are excellent electron donors because of their low ionization potentials
and can strongly interact with electron acceptors [68, 69]. Charge transfer complexation
reactions have been extensively utilized for the determination of many pharmaceutical
compounds containing amino group such as some psychotropic phenothiazine drugs
[70], some pharmaceutical amides [71], some antibacterial drugs [72], ganciclovir [73],
diethylcarbamazine citrate [74], terfenadine [75], loperamide HCl [76], moclobemide
[77], famotidine [78], diclofenac sodium [79], hydroxyzine hydrochloride [80],
mycophenolate mofetil [81], bupropion hydrochloride [82], atenolol [83], etc., to
mention a few.
From the literature survey presented in Section 8.0.2, it is clear that there is no
report dealing with the determination of CQP in pharmaceutical formulations, based on
its reaction with nitrophenols such as 2,4-dinitrophenol (DNP) or 2, 4, 6-trinitrophenol
(picric acid; PA) The reagents under study have numerous applications as analytical
reagents and they have been used for the spectrophotometric determination of many
drugs in pharmaceutical formulations [84-87]. In this Section (8.1), the author has used
PA and DNP as chromogenic agents to develop two spectrophotometric methods for the
determination of CQP in pure drug and in its formulations. Since CQP is a diphosphate
salt, transfer of non-bonding electrons is restricted. Hence it was found necessary to
convert CQP to base form and thus CQP was treated with base and the free base form
(CRQ) was extracted into chloroform. The methods involve the charge-transfer(C-T)
complex formation reaction of the base form of the CQP with DNP (method A) and PA
(method B) in chloroform to form intensely colored radical anions measurable at 420
nm in method A and at 430 nm in method B. The details about the reaction chemistry,
method development and validation as well as applications of all the methods are
presented in this Section (8.1).
8.1.2.0 EXPERIMENTAL
8.1.2.1 Apparatus
The instrument used for absorbance measurements was the same as described in
Section 2.1.2.1.
362
8.1.2.1 Reagents
All chemicals used were of analytical reagent grade and distilled water was used
throughout the study.
Pharmaceutical grade CQP (certified to be 99.95% pure) was procured from
Cipla India Ltd., Mumbai, India, and used as received. Cadiquin 200 mg (Zydus Cadila
Healthcare Ltd., Bangalore) tablets were purchased from local market and chloroform
(spectroscopic grade) was purchased from Merck, Mumbai, India.
Dinitrophenol (0.1%): Prepared by dissolving 0.1 g of dinitrophenol (S.D. Fine Chem
Ltd, Mumbai, India) in 100 ml of chloroform and used for the assay in method A.
Picric acid (0.025%): Prepared by dissolving 0.025 g of picric acid (S.D. Fine Chem
Ltd, Mumbai, India) in 100 ml of chloroform and used for the assay in method B.
Sodium hydroxide (1.0 M): Accurately weighed 4 g of the pure NaOH (Merck,
Mumbai, India) was dissolved in water; the solution was made up to 100 ml with water.
Preparation of CQP base (CRQ) solution
Into a 125 ml separating funnel, an accurately weighed 32.5 mg of pure CQP was
transferred and dissolved in about 30 ml of water and the solution rendered alkaline by
adding 5 ml of 1 M NaOH and the content was shaken for 5 min. The free base (CRQ)
formed was extracted with three 20.0 ml portions of chloroform, the extract was passed
over anhydrous sodium sulphate and collected in a 100 ml volumetric flask. The volume
was made up to mark with chloroform and the resulting solution (200 µg ml-1
CRQ) was
further diluted with chloroform to get a working concentration of 100 µg ml-1
CRQ for
method A and 50µg ml-1
CRQ for method B.
8.1.3.0 ASSAY PROCEDURES
8.1.3.1 Method A (using DNP)
Different aliquots (0.1, 0.25, 0.5………3.5 ml) of standard CRQ solution (100
µg ml-1
) were accurately transferred into a series of 5 ml calibration flasks using a micro
363
burette .One ml of 0.1% DNP solution was added to each flask and diluted to volume
with chloroform. The content was mixed well and the absorbance was measured at 420
nm against a reagent blank
8.1.3.2 Method B (using PA)
Aliquots (0.1, 0.25, 0.5……..3.0 ml) of a standard CRQ (50 µg ml-1
) solution
were accurately transferred into a series of 5 ml calibration flasks .To each flask, 1 ml
of 0.025% PA solution was added and the solution made up to volume with chloroform.
The content was mixed well and the absorbance was measured at 430 nm against a
reagent blank.
Standard graph was prepared by plotting the absorbance versus drug
concentration, and the concentration of the unknown was read from the calibration
graph or computed from the respective regression equation
8.1.3.3 Procedure for tablets
Twenty tablets were weighed and pulverized. The amount of tablet powder
equivalent to 32.5 mg of CQP was transferred into a 100 ml volumetric flask containing
30 ml of water. The content was shaken well for 20 min. The resulting solution was
filtered through Whatmann No 42 filter paper and the filtrate was collected in to a 125
ml separating funnel. The salt was converted to free base as described earlier, CRQ
solutions of concentrations 100 and 50 µg ml-1
for method A and method B,
respectively, were prepared as described under the general procedure for pure drug and
a suitable aliquot was used for assay by applying procedures described earlier.
8.1.3.4 Placebo blank synthetic mixture analyses
A placebo blank containing lactose (20mg), starch (40 mg), acacia (35 mg),
sodium citrate (35 mg), hydroxyl cellulose (35 mg), magnesium stearate (35 mg), talc
(40 mg) and sodium alginate (35 mg) was prepared by mixing all the components into a
homogeneous mixture. A 20 mg of the placebo blank was accurately weighed and its
solution was prepared as described under ‘tablets’, and then subjected to analysis by
following the general procedures.
364
To 30 mg of the placebo blank, 32.5 mg of CQP was added and homogenized,
transferred to 100 ml volumetric flask and the solution was prepared as described under
“Procedure for tablets”. A convenient aliquot was diluted and then subjected to analysis
by the procedures described above.
5.1.4.0 RESULTS AND DISCUSSION
Absorption spectra
The reaction of chloroquine base (CRQ) as n-electron donor and the π-acceptors
DNP and PA, result in the formation of yellow C-T complexes having absorption
maxima at 420 and 430 nm, respectively (Figure 8.1.1). The respective blanks had
negligible absorbance at this wavelength.
360 380 400 420 440 460 480 500 520
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorb
ance
Wavelength, nm
CRQ-DNP C-T complex
Blank
340 360 380 400 420 440 460 480 500 520
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Ab
so
rba
nce
Wavelength, nm
CRQ-PA C-T complex
Blank
(a) (b)
Figure.8.1.1. Absorption spectra: (a) CRQ-DNP (b) CRQ–PA charge transfer
complexes.
Reaction pathway
Charge-transfer complex is a complex formed between an electron-donor and an
electron-acceptor and is characterized by electronic transition(s) to an excited state in
which there is a partial transfer of electronic charge from the donor to the acceptor
moiety. As a result, the excitation energy of this resonance occurs very frequently in the
visible region of the electro-magnetic spectrum [65]. This produces the usually intense
colors characteristic for these complexes. Therefore, CRQ, a nitrogenous base a n-
365
donor, was made to react with DNP and PA and produce a coloured charge transfer
complexes in dichloromethane.
DNP and PA were earlier used for the determination of some amine derivatives
through formation of intense yellow coloured complexes [83,84, 88,89]. When an
amine is reacted with a polynitrophenol, one type of force field produces an acid-base
interaction, and the other, an electron donor-acceptor interaction. The former interaction
leads to the formation of true phenolate by proton-transfer, and the latter, to a true
molecular compound by charge-transfer [85]. The explanation for the produced color in
both methods lies in the formation of complexes between the pairs of molecules CRQ-
DNP and CRQ-PA, and this complex formation leads to the production of two new
molecular orbitals and, consequently, to a new electronic transition [90].
Because CRQ has two tertiary amino groups and one secondary amino group in
its molecular structure with the availability of non-bonding electron donors, it reacts
with dinitrophenol and picric acid in chloroform to yield a yellow coloured C-T
complex peaking at 420 and 430 nm (Figure 5.1.1). The interaction between CRQ (D),
an n-donor and nitrophenols (A), π-acceptors, is a charge transfer complexation reaction
followed by the formation of radical ions [91] according to the Scheme 8.1.1.
D•• + A → [D
••→ A] → D
•+ + A
• −
[Donor + Acceptor → Complex → Radical ions]
366
NCl
NH
CH3
N
CH3
CH3
R1R3
R2
OH NCl
NH
CH3
N
CH3
CH3
R1R3
R2
OH
NCl
NH
CH3
N
CH3
CH3
R1R3
R2
O
+.
.For DNP: R1=R2= NO2 and R3=H
For PA: R1=R2=R3= NO2
Radical anion measured species
Scheme 8.1.1 Possible reaction pathway for the formation of C-T complex between
drug (CRQ) and DNP or PA.
8.1.4.1 Optimization of reaction conditions
Choice of solvent
Several organic solvents such as chloroform, dichloromethane,
1,2-dichloroethane were tried for the extraction of base form of the chloroquine. Only
chloroform favored the extraction of the drug to its base form. In order to select a
suitable solvent for preparation of the reagent solutions used in the study, the reagents
were prepared separately in different solvents such as chloroform, acetonitrile, acetone,
2-propanol and dichloromethane, and the reaction of CRQ with DNP or PA was
followed. The chloroform solvent was found to be the ideal solvent for preparation of
both DNP and PA for method A and method B, respectively. Similarly, the effect of the
367
diluting solvent was studied for all methods and the results showed that the ideal
diluting solvent to achieve maximum sensitivity was chloroform in both methods.
Effect of reagent concentration
The optimum concentration of the reagent required to achieve maximum
sensitivity of the developed color species in each method was ascertained by adding
different amounts of the reagent DNP or PA to a fixed concentration of CRQ. The
results showed that 1.0 ml of 0.1% DNP or 0.025% PA solution was optimum for the
production of maximum and reproducible color intensity (Figure.8.1.2).
0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Ab
so
rban
ce
Volume of dye, ml
Blank
CRQ-DNP
Blank
CRQ-PA
Figure 8.1.2 Effect of reagent concentration on the formation of (CRQ-DNP complex,
40 µg ml-1
CRQ) and (CRQ -PA complex, 20 µg ml-1
CRQ)
Effect of reaction time and stability of the C-T complexes
The optimum reaction times were determined by measuring the absorbance of
the complex formed upon the addition of reagent solution to CRQ solution at room
temperature. The reaction in both methods was instantaneous. The absorbance of the
resulting C-T complexes remained stable for at least more than 45-90 min in both the
methods.
Composition of the C-T complexes
The composition of the C-T complex was established by Job’s method of
continuous variations [92] using equimolar concentrations of the drug and reagents
368
(6.25 x 10-4
M in method A, 8.99 x 10-4
M in method B). Five solutions containing CRQ
and the reagent (DNP or PA) in various molar ratios with a total volume of 5 ml in both
the methods were prepared. The absorbance of solutions was subsequently measured at
420 and 430 nm. The CRQ contains one secondary and two tertiary amino groups, the
secondary amine being more basic in nature than tertiary amine and is vulnerable for
charge transfer reaction. The steric hindrance around tertiary amines suppresses their
basicity. The graphs of the results obtained (Figure 8.1.3) gave a maximum at a molar
ratio of Xmax = 0.5 in both the methods which indicated the formation of a 1:1 C-T
complex between CRQ and reagent (DNP or PA).
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Absorb
ance
Mole ratio
VCRQ
+(VCRQ
+ VDNP
)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.1
0.2
0.3
0.4
Ab
so
rba
nce
Mole ratio
VCRQ
+(VCRQ
+VPA
)
(a) (b)
Figure 8.1.3 Job’s continuous variation plot a) CRQ+DNP and b) CRQ+PA
8.1.4.2 Method validation
Linearity and sensitivity
Under the optimized experimental conditions for CRQ determination, the
standard calibration curves for CRQ with DNP and PA were constructed by plotting
absorbance versus concentration (Figure 8.1.4). The linear regression equations were
obtained by the method of least squares and the Beer's law range, molar absorptivity,
Sandell’s sensitivity, correlation coefficient, standard deviation of intercept (Sa),
369
standard deviation of slope (Sb), limits of detection and quantification for both methods
are calculated according to ICH guidelines [93] and are summarized in Table 8.1.1.
Figure 8.1.4 Calibration curves
Accuracy and precision
In order to determine the accuracy and precision of the proposed methods, pure
drug (CRQ) solution at three different concentration levels (within the working range)
Table 8.1.1 Sensitivity and regression parameters
Parameter Method A Method B
λmax, nm
Color stability, min
420
45
430
90
Linear range, µg ml-1 2-70 1-30
Molar absorptivity(ε), l mol-1
cm-1
4.7× 10 3 1.1× 10
4
Sandell sensitivity*, µg cm
-2 0.0673 0.0313
Limit of detection (LOD), µg ml-1
4.01 0.62
Limit of quantification (LOQ), µg ml-1
1.32 0.20
Regression equation, Y**
Intercept (a) 0.0085 0.0147
Slope (b) 0.0142 0.0313
Standard deviation of a (Sa) 0.0869 0.0787
Standard deviation of b (Sb) 0.0047 0.0022
Regression coefficient (r) 0.9994 0.9995 *Limit of determination as the weight in µg ml-1 of solution, which corresponds to an
absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1.0 cm2 and l = 1.0 cm.
bXaY +=** , where Y is the absorbance and X concentration in µg ml-1
370
were prepared and analyzed during the same day (intra-day precision) and on five
consecutive days(inter-day precision) and the results are presented in Table 8.1.2.
Table 8.1.2 Results of intra-day and inter-day accuracy and precision study
Method
CRQ
taken
µg ml-1
Intra-day accuracy and
precision
(n=5)
Inter-day accuracy and
precision
(n=5)
CRQ
found
µg ml-1
%RE %RSD
CRQ
found
µg ml-1
%RE %RSD
A
20.0
40.0
60.0
19.50
40.75
59.44
2.45
1.89
0.91
1.92
1.50
1.37
20.45
40.50
60.92
2.28
1.26
1.54
2.26
1.64
1.34
B
10.0
20.0
30.0
9.88
20.17
29.60
1.18
0.86
1.31
1.25
0.96
1.14
9.83
20.21
29.52
1.69
1.08
1.57
1.73
1.21
1.25
Selectivity
The selectivity of the proposed methods for the analysis of CRQ was evaluated
by placebo blank and synthetic mixture analyses. The recommended procedures were
applied to the analysis of placebo blank and the resulting absorbance readings in both
methods were same as that of the reagent blank, confirming no interference from the
placebo. The analysis of synthetic mixture solution prepared as described earlier yielded
percent recoveries of 99.8±1.13 and 99.1±1.06 (n=5) for method A, and method B,
respectively. The results of this study showed that the inactive ingredients did not
interfere in the assay indicating the high selectivity of the proposed methods and its
utility for routine determination in pure drug and in tablets form
Robustness and ruggedness
To evaluate the robustness of the methods, a important experimental variable
volume of reagent in both the methods were altered incrementally and the effect of this
change on the absorbance of the C-T complexes was studied. The results of this study
are presented in Table 8.1.3 and indicated that the proposed methods are robust. Method
ruggedness was evaluated by performing the analysis following the recommended
371
procedures by three different analysts and on three different cuvettes by the same
analyst. From the %RSD values presented in Table 8.1.3, one can conclude that the
proposed methods are rugged.
Table 8.1.3 Results of robustness and ruggedness expressed as intermediate
precision (%RSD)
Method
CRQ
taken,
µg ml-1
Robustnessa
(%RSD)
Ruggedness
Inter-analysts
(%RSD), (n=4)
Inter-cuvettes
(%RSD), (n=4)
A
20.0
40.0
60.0
1.26
1.21
1.17
1.56
0.84
1.72
2.53
3.08
2.68
B
10.0
20.0
30.0
1.34
1.28
1.23
0.76
1.26
1.01
2.98
2.62
3.12 DNP,PA volumes used were 0.8, 1.0 and 1.2 ml
Application to tablets
The proposed methods were applied to the determination of CRQ in tablets and
the results are compiled in Table 8.1.4. The results obtained were statistically compared
with those obtained by the reference method [2], by applying the Student’s t-test for
accuracy and F-test for precision at 95% confidence level. The reference method
involved the potentiometric titration of the drug with perchloric acid. As can be seen
from the Table 8.1.4, the calculated t- and F- values at 95% confidence level did not
exceed the tabulated values for four degrees of freedom. This indicates that there are no
significant differences between the proposed methods and the reference method with
respect to accuracy and precision.
372
Recovery studies
To further ascertain the accuracy of the proposed methods, a standard addition
technique was followed. A fixed amount of drug from pre-analyzed tablet powder/syrup
was taken and pure drug at three different levels (50, 100 and 150 % of that in tablet
powder/syrup content) was added. The total was found by the proposed methods. The
determination at each level was repeated three times and the percent recovery of the
added standard was calculated. Results of this study presented in Table 8.1.5 reveal that
the accuracy of methods was unaffected by the various excipients present in the
formulations.
Table 8.1.4 Results of analysis of tablets by the proposed methods and
statistical comparison of the results with the reference method
Tablet
brand
nameb
Label claim
Founda (Percent of label claim ±SD)
Reference
method
Proposed methods
Method A Method B
Cadiquin 200 mg
98.56±1.36
98.04±1.86
t=2.44
F=1.16
99.38±2.51
t= 2.11
F= 1.87
Mean value of five determinations,
The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are
2.77 and 6.39, respectively.
Table 8.1.5 Results of recovery study by standard addition method
Method A Method B
Tablet
Studied
CRQ
in
tablet
µg ml-1
Pure
CRQ
added
µg ml-1
Total
found
µg
ml-1
Pure CRQ
recovereda
Percent ±
SD
CRQ
in
tablet
µg ml-1
Pure
CRQ
added
µg ml-1
Total
found
µg
ml-1
Pure CRQ
recovereda
Percent ±
SD
Cadiquin
9.80 5.0 14.67 99.16 ± 1.78 19.87
19.87
19.87
10.0
20.0
30.0
30.11
39.23
49.52
100.8±0.84
98.40±2.58
99.31±1.03 9.80 10.0 20.09 101.5 ± 1.31
9.80 15.0 24.46 98.69± 1.24
373
SECTION 8.2
DEVELOPMENT AND VALIDATION OF A UV-SPECTROPHOTOMETRIC
METHOD FOR THE DETERMINATION OF CHLOROQUINE AND ITS
STABILITY STUDIES
8.2.1.0 INTRODUCTION
A smart profile and utilization of UV-spectrometry in different assay have been
presented in Section 3.4. From the literature survey presented Section 8.0.2.0 it is
evident that, a UV-spectrophotometric method was found in the literature which
involves measurement of absorbance of CQP solution in 0.1 M H2SO4 at 343 nm.
In the literature, no stability-indicating UV-spectrophotometric methods have
ever been reported for the assay of CQP. In the present Section 8.2, a simple,
inexpensive, accurate, reproducible, and stability-indicating UV- spectrophotometric
method for CQP is described. The methods are based on the measurement of
absorbance of CQP solution in 0.1 M HCl at 342 nm. Besides, the method was used to
study the degradation of the drug under stress conditions as per the ICH guidelines [94].
8.2.2.0 EXPERIMENTAL
8.2.2.1 Apparatus
The instrument is the same that was described in Section 3.4.2.1.
8.2.2.2 Reagents
All chemicals used were of analytical reagent grade. Doubly-distilled water was
used to prepare solutions wherever required. Pure drug and tablets used were the same
as described in Section 8.1.2.
Hydrochloric acid (5 M), hydrogen peroxide (5% v/v), sodium hydroxide solution
(5 M) were prepared as described under Section 4.2.2.2.
374
Standard drug solution
A stock standard solution of 100 µg ml-1
CQP was prepared by dissolving 20 mg
of pure CQP in 0.1 M HCl and diluted to 100 ml with the same solvent in a calibrated
flask.
8.2.3.0 ASSAY PROCEDURES
8.2.3.1 Preparation of calibration curve
Into a series of 10 ml calibration flasks, aliquots of standard drug solution (0.25
to 2.5 ml of 100 µg ml-1
) equivalent to 2.5-25.0 µg ml-1
CQP were accurately
transferred and the volume was made up to the mark with 0.1 M HCl. The absorbance
of each solution was then measured at 342 nm against 0.1 M HCl as the blank.
A calibration curve was prepared by plotting the absorbance versus
concentration of drug. The concentration of the unknown was read from the respective
calibration curve or computed from the regression equation derived using the Beer’s
law data.
8.2.3.2 Procedure for tablets
Twenty cadiquin tablets containing CQP (200 mg/tablet) were weighed and
pulverized. The amount of tablet powder containing 10 mg CQP was transferred into a
100 ml volumetric flask. The content was shaken well with about 60 ml of 0.1 M HCl
for 20 min and the extract was diluted to the mark with the same solvent. It was filtered
using Whatman No 42 filter paper. First 10 ml portion of the filtrate was discarded and
a subsequent portion was subjected to analysis following the general procedure
described earlier.
8.2.3.3 Placebo blank and synthetic mixture analyses
Thirty mg of the placebo blank prepared in Section 8.1.3.3 was taken and its
solution prepared as described under ‘Procedure for tablets’ and then analyzed using
the procedures described above.
375
To 20 mg of the placebo blank, 10 mg of CQP was added and homogenized,
transferred to 50 ml volumetric flask and the solution was prepared as described under
“Procedure for tablets”. A convenient aliquot was diluted and then subjected to analysis
by the procedures described above.
8.2.3.4 Forced degradation studies
Ten µg ml-1
CQP ( 2.5 ml of 100 µg ml-1
CQP) was taken (in triplicate) in a 25
mL volumetric flask and mixed with 5 ml of 5 M HCl (acid hydrolysis) or 5 M NaOH
(alkaline hydrolysis) or 5% H2O2 (oxidative degradation) and boiled for 2 h at 80 °C in
a hot water bath. The solution was cooled to room temperature and diluted to the mark
with 0.1 M HCl after neutralization with 5.0 ml of 5 M NaOH (for acid hydrolysis) and
5 ml of 5 M HCl (for alkaline hydrolysis). In thermal degradation, solid drug was kept
in Petri dish in oven at 100 °C for 24 h. After cooling to room temperature, 10 µg ml-1
CQP solution in 0.1 M HCl was prepared and absorbance measured. For UV
degradation study, the stock solutions of the drug (100 µg ml-1
) were exposed to UV
radiation of wavelength 254 nm and of 1.2K flux intensity for 48 h in a UV chamber.
The solutions after dilution with 0.1 M HCl was assayed as described above.
8.2.4.0 RESULTS AND DISCUSSION
Spectral characteristics
The absorption spectrum of 10 µg ml-1
CQP solution in 0.1 M HCl recorded
between 200 and 400 nm showed an absorption maximum at 342 nm, and at this
wavelength 0.1 M HCl had insignificant absorbance. Therefore, 342 nm was used as
analytical wavelength (λmax). Figure 8.2.1 represents the absorption spectrum of CQP in
0.1 M HCl along with blank.
376
Figure 8.1.1 Absorption spectrum of CQP (10 µg ml-1
) in 0.1 M HCl
8.2.4.1 Forced degradation studies
The CQP was subjected to acid, neutral, base and hydrogen peroxide induced
degradation in solution state, and photo and thermal degradation in solid state. The
study was performed by measuring the absorbance of CQP solution only after
subjecting to forced degradation. The results of this are presented in Table 8.2.1. The
results revealed that, the drug was stable under the acid hydrolysis, photo and thermal
degradation. There was significant change in the absorbance due in base hydrolysis and
slight degradation under oxidative degradation. The absorption spectra (Figure. 8.2.2)
was recorded for this degraded CQP and there was almost completely diminished signal
was observed. This confirms that CQP is susceptible to oxidative degradation.
Table 8.2.1 Results of degradation study
Degradation condition % Degradation
No degradation ( control) No degradation
Acid hydrolysis (5 M HCl , 80°C, 2 hours) No degradation
Base hydrolysis (5 M NaOH , 80°C, 2 hours) 51
Oxidation (5% H2O2 , 80°C, 2 hours) 16
Thermal (100°C, 24 hours) No degradation
Photolytic (1.2 K flux, 48 hours) No degradation
377
(a)
(b)
(c)
378
(d)
(e)
Figure 8.2.2 Absorption spectra of 10 µg ml-1
CQP after
(a) acid hydrolysis, (b) base hydrolysis, (c) photo degradation (d) thermal
degradation and (e) peroxide degradation.
8.2.1.2 Method validation
Linearity and sensitivity
A linear correlation was found between absorbance at λmax and concentration of
CQP (Figure 8.2.3). The slope (b), intercept (a) and correlation coefficient (r) were
evaluated by using the method of least squares. Optical characteristics such as Beer’s
law limits, molar absorptivity and Sandell sensitivity values of the method was
379
calculated. The limits of detection (LOD) and quantitation (LOQ) were also calculated
according to ICH guidelines [93], and all these data are presented in Table 8.2.2.
Figure 8.2.3 Calibration curve
Table 8.2.2 Sensitivity and regression parameters
Parameter Value
λmax, nm 342
Linear range, µg ml-1
2.5 – 25.0
Molar absorptivity(ε), l mol-1
cm-1
8.88 × 103
Sandell sensitivity*, µg/cm
2 0.0401
Limit of detection (LOD), µg ml-1
0.39
Limit of quantification (LOQ), µg ml-1
1.18
Regression equation**
Intercept (a) 0.0043
Slope (b) 0.0238
Sa 0.0987
Sb 0.0035
Regression coefficient (r) 0.9998 *Limit of determination as the weight in µg ml
-1of solution, which corresponds to an absorbance of
A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, where Y is
the absorbance, X is concentration in µg ml-1, a is intercept and b is slope
Accuracy and precision
Accuracy was evaluated as percentage relative error between the measured
concentrations and the concentrations taken for CQP (Bias %). The results obtained are
compiled in Table 8.2.3 and they show that both accuracy and precision are good.
Precision of the method was calculated in terms of intermediate precision (intra-day and
inter-day). Three different concentrations of CQP were analysed in seven replicates
380
during the same day (intra-day precision) and for five consecutive days (inter-day
precision). RSD (%) values of the intra-day studies showed that the precision was good.
Robustness and ruggedness
Method robustness was tested by measuring the absorbance at 341, 342 and 343
nm whereas the method ruggedness was tested by comparing the RSD values of the
results obtained by four different analysts, and also with three different cuvettes by a
single analyst. The intermediate precision, expressed as percent RSD, which is a
measure of robustness and ruggedness was within the acceptable limits as shown in the
Table 8.2.4.
Table 8.2.3 Results of intra-day and inter-day accuracy and precision study
CQP
taken,
µg ml-1
Intra-day accuracy and
precision
(n=7)
Inter-day accuracy and
precision
(n=5)
CQP
found,
µg ml-1
%RE %RSD
CQP
found,
µg ml-1
%RE %RSD
10.0
15.0
20.0
09.95
15.08
19.73
0.46
0.58
1.34
1.93
1.54
1.37
9.82
15.13
19.70
0.72
0.89
1.47
2.48
1.76
1.58 RE: Relative error and RSD: Relative standard deviation.
Table 8.2.4 Results of robustness and ruggedness expressed as intermediate
precision(%RSD)
CQP
taken,
µg ml-1
Method robustness Method ruggedness
Parameter altered
Wavelength*, nm,
%RSD (n = 3)
Inter-analysts’
%RSD
(n = 4)
Inter-cuvettes’
%RSD
(n = 3)
10.0 1.78 0.97 1.18
15.0 2.16 1.12 1.39
20.0 2.63 1.03 1.07 *Wavelengths used were 341, 342 and 343 nm.
381
Selectivity
The proposed method was tested for selectivity by placebo blank and synthetic
mixture analyses. The placebo blank solution was subjected to analysis according to the
recommended procedure and found that there was no interference from the inactive
ingredients, indicating a high selectivity for determining CQP in its tablets.
When the synthetic mixture solution was subjected to analyses at 10, 15 and 20
µg ml-1
CQP concentration levels, the percent recoveries were 98.48, 97.36 and 102.7
respectively, with % RSD being less than 2.5% implying that the assay procedure is free
from matrix interference.
Application to tablets
In order to evaluate the analytical applicability of the proposed method to the
quantification of CQP in commercial tablets, the results obtained by the proposed
method were compared to those of the reference method [2] by applying Student’s t-test
for accuracy and F-test for precision. The results (Table 8.2.5) showed that the
Student’s t- and F-values at 95 % confidence level did not exceed the tabulated values,
which confirmed that there is a good agreement between the results obtained by the
proposed method and the reference method with respect to accuracy and precision.
Table 8.2.5 Results of analysis of tablets by the proposed methods and
statistical comparison of the results with the reference method
Tablet
brand
nameb
Label claim
Founda (Percent of label claim ±SD)
Reference
method
Proposed methods
Cadiquin 200 mg
98.56±1.36
99.34±1.28
t=1.68
F=2.47
Mean value of five determinations,
The value of t and F (tabulated) at 95 % confidence level and for four degrees of freedom are
2.77 and 6.39, respectively.
382
Recovery study
To a fixed amount of drug in formulation (pre-analysed): pure drug at three
different levels was added, and the total was found by the proposed method. Each test
was repeated three times. The results compiled in Table 8.2.6 show that recoveries were
in the range from 98.51 to 102.5% indicating that commonly added excipients to tablets
did not interfere in the determination
Table 8.2.6 Results of recovery study via standard
addition method
Tablet
studied
CQP
in
tablet,
mg
Pure
CQP
added,
mg
Total
found,
mg
Pure CQP
recovered*
Percent ±
SD
Cadiquin-
200
9.85 5.00 14.58 98.24±1.28
9.85 10.0 19.96 100.6±1.32
9.85 15.0 24.49 98.58±0.87
*Mean value of three determinations
383
SECTION 8.3
CONCLUSION ON CHAPTER 8 –Assessment of methods
To sum up, two visible and one UV-spectrophotometric methods were
developed and validated for the determination of chloroquine phosphate in bulk drug
and in tablets. The UV-spectrophotometric method was additionally used to evaluate the
behavior of the drug towards several stress conditions such as acid and base hydrolysis,
peroxide oxidation, light and heat which are first of their kind for CQP in
pharmaceuticals.
Visible spectrophotometry is one of the most widely used methods of analysis in
pharmaceutical labs because many substances can be selectively converted to a colored
derivative. In addition, the instrumentation is readily available and generally fairly easy
to operate. Considering these advantages and based on various reaction chemistries,
many reports employing visible spectrophotometry are found in the literature for the
assay of CQP in pharmaceuticals. However, most of the reported methods suffer from
one or other disadvantage such as poor sensitivity, poor selectivity, tedious and time
consuming liquid-liquid extraction step, strict pH control and narrow linear range, etc.,
as indicated in Table 8.3.1. In this Chapter, the author has made an attempt to develop
and validate two visible spectrophotometric methods for the determination of CQP
using different reagents and is based on charge transfer reaction. Also, the methods
based on charge-transfer complexation reaction the very simple ones since they are
based on one step reaction i.e., mixing of drug solution in its base form and the dyes and
the formed colored complexes were directly measured at the respective wavelength.
Besides being simple in sample pretreatment, the methods are accurate, precise,
applicable over wide linear dynamic ranges and they can be applied for the analysis of
CQP in tablets.
384
Table 8.3.1 Comparison of performance characteristics of the proposed methods with the existing methods
Sl.
No. Reagent/s used Methodology
λλλλmax (nm)
Linear range
(µg ml-1
)
(ε = l mol-1
cm-1
)
Remarks Ref
1 Bromocresol purple
Measurement of chloroform
extractable ion-pair complex
420
1.25-8.75
Time consuming and tedious extraction step
7
2 Bromothymol blue Measurement of dichloromethane extractable ion-pair complex
410
1-12
Time consuming and tedious extraction step 8
3
Cobalt-thiocyanate Measurement of nitrobenzene
extractable ion-pair complex
625
2-60
(1.18 x 104)
Requires close pH control, time consuming,
requires extraction
10
4 Mo(V)-SCN Measurement of nitrobenzene
extractable ion-pair complex
610
2-42 Time consuming, requires close pH control
11
5 Tetrabromophnolphthalein Measurement of dichloromethane
extractable ion-pair complex
530
5-25
Requires close pH control, time consuming,
requires extraction
12
6 Methyl orange Measurement of ion-associate
complex 510
2-16
-
13
7 CoCl2-KSCN Measurement of iso-BuCOMe
extractable redox complex
625 - Time consuming and tedious extraction step 14
8 Ammonium molybdate Measurement of redox complex 720 1.0-15
- 15
9 2,3-dichloro-5,6-dicyano-p-
benzoquinone
Iodine
Measurement of charge-transfer
complex
462
287
5-53
(6.1 x 103)
1-40
(9.92 x 103)
Moderately sensitive
Measurement at lower analytical wavelength
16
10 Chloranilic acid Measurement of charge-transfer
complex
520
335
0.8-8 -
17
385
For the first time, the author developed stability-indicating UV-spectrophotometric method and validated according to ICH
guidelines. All the proposed methods are applicable over wide linear dynamic ranges and found to be unaffected by the inactive
ingredients added to tablets as shown by the results of tablet analysis apart from placebo blank and synthetic mixture analyses.
The proposed methods in this chapter (8) have certain disadvantages. The two spectrophotometric methods using DNP and PA,
as reagents involve use of organic solvents. However, the use has been scaled down to the barest minimum and easier compare to the
reported extraction ion-pair reactions. The C-T methods entail apparatus to be completely free from water and spectrophotometric
cells to be dried with acetone before measurement, otherwise the accuracy and precision of the methods will be affected. The
measured reaction product was not isolated for characterization and the reaction scheme was proposed purely based on literature
knowledge.
11 KBrO3
Measurement of tri-iodide ion 343
0.5-5
Longer reaction time 19
12 KBro3-KBr Measurement of tri-iodide ion 350
3-45
Time consuming, moderately sensitive 20
13 N-bromosuccinimide Measurement of tri-iodide ion 410 2.5-7.5
Unstable oxidant, time consuming 21
14 a) dinitrophenol
b) picric acid
Measurement of radical anion in
chloroform
420
430
2-70.0 (4.7× 10
3)
1.0-30.0
(1.1× 10 4)
No heating or extraction step and eco-friendly chemicals used, inexpensive
instrumental setup employed.
Present work
15 0.1 M HCl Measurement of absorbance in HCl
medium 342
2.5-25
(8.88× 10 3)
Simple, stability-indicating Present work
386
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