Methods for the Analysis of Some Chemotherapeutic...
Transcript of Methods for the Analysis of Some Chemotherapeutic...
Chapter – 2
10
Chapter-2
Methods for the Analysis of Some Chemotherapeutic
Agents.
Introduction
The chemical substances, which interact at molecular level altering the body
functions therapeutically, are generally referred to as drugs or chemotherapeutics.
Drugs vary in molecular size and chemical nature. Based on the effect a compound
has on the body, drugs have been variously classified as analgesics, antibiotics,
narcotic analgesic agonists/antagonists, central nervous system (CNS), stimulants/
depressants, tranquilizers, etc. The pharmacological properties of such drugs are
commonly misused for euphonic, homicidal, suicidal and doping purposes. Fatalities
due to the accidental use of excessive dose of drugs are also of very common
occurrence. In this regard, constant updating of the analytical methods for the drug
analysis is very important. The author has given a brief account of the drugs selected
for investigation in the present chapter in the following paragraphs.
Metoclopramide hydrochloride
Metoclopramide hydrochloride (MCP) chemically, 4-amino-5-chloro-N-[2-
(diethylamino) ethyl]-2-methoxy benzamide is an important derivative of benzamide.
It is a potent dopamine receptor antagonist used for its antiemetic and prokinetic
properties. Therefore, it is commonly used to treat nausea and vomiting associated
with conditions including; emetogenic drugs, radiation sickness, malignancy, labor
and infection. It is also used in combination with paracetamol for the relief of
migraine. It is commercially available under version trade names, Reglan (CFL),
Perinorm (IPCA), Segmet (Sigma), Vominorm (Cipla) etc.
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MCP has the following molecular structure:
O
HN
OCH3
N
Cl
H2N
. HCl
MCP is a white crystalline powder with melting point 182-184 °C. Soluble in
water, acetone, alcohol and chloroform. Its aqueous solution is stable for a week at
room temperature (ca. 27 ± 2 °C).
Dapsone
Dapsone (DAP) chemically, 4, 4’-sulfonyl bis (benzene amine), is a potent
anti bacterial (leprostatic). It is most commonly used in combination with rifampicin
and clofazimine as multidrug therapy (MDT) for the treatment of Mycobacterium
leprae infections (leprosy). It is also used to treat dermatitis herpetiformis and other
skin conditions including lichen planus. It has the following structural formula.
S
O
O
H2N NH2
It is soluble in alcohol, methanol, and acetone and dilute hydrochloric acid,
practically insoluble in water. It melts at 175-176 °C.
Cisapride
Cisapride (CPD), chemically 4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)
propyl]-3-methoxy-4-piperidyl]-2, methoxy-benzamide is a parasympathomimetic,
which acts as a serotonin-5-HT4 agonist. It has been used to treat bowel constipation
Chapter – 2
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and it used as an alternative to MCP in the management of diabetic gastroparlsis. It
has the following structure.
O
HN
O N
O
O
F
Cl
H2N
It is freely soluble in methanol and 2-propanol, CPD is commercially known as
Cisawal (Wallace), Cisapro (Zy Alidac), Ciza (Intas) and Unipride (Torrent).
p-Aminobenzoic acid
p-Aminobenzoic acid (PABA), 4-aminobenzoic acid is an important skin
protective agent and it is widely used as a UV filter in sun screen formulations. PABA
is a white crystalline solid freely soluble in ethanol and slightly soluble in water. It
melts at 187-188 °C and it has the following structure.
O
HO
NH2
Literature Survey
The pharmacological importance of these drugs resulted in an increasing need
for analytical methods for their detection and quantification. The important analytical
techniques recommended for the assay of studied drugs include, gas chromatography-
mass-spectrometry, HPLC, HPTLC, electron-capture gas-liquid chromatography,
spectrometry etc., most of the above techniques except spectrophotometry, require an
expensive experimental set-up.
Among the various optical methods, spectrophotometric methods for the
determination of micro quantities of drugs have received considerable attention due to
their simplicity, reliability, rapidity and availability of number of chromogenic
Chapter – 2
13
reagents. The general availability of modern spectrophotometers in most academic
and industrial laboratories, have also contributed to the increased use of the technique.
This is undoubtedly reflected in the great volume of literature on spectrophotometric
analysis of bio-active compounds. In the following paragraphs, a modest attempt is
made to briefly review some of the recently applied spectrophotometric methods for
the determination of metoclopramide hydrochloride [MCP], dapsone [DAP], cisapride
[CPD] and p-.amino benzoic acid [PABA].
Patel et al. [1] have developed two spectrophotometric methods for the
determination of MCP in tablets using 4-dimethyl amino benzaldehyde and alkaline
β-naphthol. The former method results a yellow colour Schiff’s base with a
maximum absorption at 438 nm and the second method include diazo-coupling with
alkaline β-naphthol to form red-dye with a maximum absorption at 553 nm. Beer’s
law range is 10-100 µg mL-1 and 1-10 µg mL
-1 for methods first and second,
respectively.
MCP was spectrophotometrically determined by the treatment of sample
solution with dichlorophenolindophenol [DCPIP] [2] and the resulting bluish violet
radical ion exhibit a maximum absorption at 654 nm. Omran and Ahmed [3] have
proposed benzylacetone [BAC] in alkaline medium as a coupling agent for the
simultaneous spectrophotometric determination of MCP and DAP. The formed azo-
dyes show maximum absorption at 411 nm and at 437 nm for MCP and DAP,
respectively.
A spectrophotometric procedure [4] was developed for the assay of MCP in
pure and dosage forms using 2-naphthol-3, 6-disulphonic acid, the reaction mixture
was heating for about 45s at 100 °C to form an orange coloured complex with λmax
490 nm. Linear range and molar absorptivity values of this method are found to be 1-
25 µg mL-1 and 3.71×10
3 L mol
-1 cm
-1, respectively. In addition 1.10- phenanthroline-
Fe(III) reagent [5] was used for the determination of MCP.
Revanasiddappa and Manju [6] have proposed acetyl acetone as a reagent for
the determination of MCP, DAP, CPD and PABA. The same authors developed a
method [7] for the determination MCP and DAP in pure and in dosage forms by the
Chapter – 2
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diazo-coupling of these drugs with a coupling-agent dibenzoyl methane in an alkaline
medium.
Recently, a derivative spectrophotometric method [8] was developed for the
determination of DAP. Nagaraja et al. [9] have described two spectrophotometric
procedures for the assay of DAP. In the first method, iminodibenzyl (IDB) was used
as a coupling agent in alcoholic medium, and in the second method DAP was made to
react with N-bromosuccinimide and promethazine hydrochloride to give a green
product. Absorbances were measured at 570 nm and at 610 nm in first and second
methods, respectively. Beer’s law range is 0.1-2.5 µg mL-1 and 0.5-5.0 µg mL
-1,
respectively. The same authors have reported [10] resorcinol in sulphuric acid as a
diazo-coupling agent for the determination of dapsone and the formed azo-dye shows
a maximum absorption at 530 nm. Beer’s law range was 0.1-2.5 µg mL-1. In another
method [11], sodium 1,2- naphtoquinone-4-sulphonate in pH 6.98 buffer solution was
used as chromogenic reagent for dapsone determination. The formed pink coloured
species had a molar absorption coefficient value of 3.68 ×104 L mol
-1cm
-1 at 525 nm
and Beer’s law is obeyed from 0.4 to 10 µg mL-1.
Salvador et al. [12] have reported an indirect sequential injection
spectrophotometric method for p-amino benzoic acid determination in sun screen
formulations based on the reaction of it with hypochlorite in acidic medium
and subsequent determination of residual chlorine. Beer’s law is valid from
1 to 20 µg mL-1. Another sequential-injection analysis [13] was described for the
assay of PABA in sun screens by using 8-hydroxy quinoline as a coupling agent
[linear range 2-25 µg mL-1].
In addition to the above recent methods, several other spectrophotometric
methods have also been reported for the determination MCP, DAP, CPD and PABA.
Metoclopramide was spectrophotometrically determined through diazo-
coupling reaction with N- (1-napthyl)-ethylenediamine dihydrochloride (NEDA) [14],
thymol [15], resorcinol [16], 8-anilino-1-naphthalene sulphonic acid [17],
naphtha-2-ol [17] and chromotropic acid [18]. Colorimetrically [19] it was determined
after the reaction with nitrous acid to form a stable yellow nitroso compound with a
maximum absorption at 375 nm. It was determined through ion-pair formation with
Chapter – 2
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thiocyanate and molybdenum(V) or cobalt(II) [20], bromothymol blue [21], and based
on charge-transfer complexes with chloranil or bromanil [22]. Other chromogenic
reactions use sodium vanadate [23], ammonium metavanadate [24], sodium 3,4-
dioxonaphthalene-1-sulphonate [25], ammonium reineckate [26], citric acid –acetic
anhydride [27], catechol [28], Folin-Ciocalteu reagent [29], p-dimethylamino
cinnamaldehyde [30] and 3-methyl benzothiazolin-2-one hydrazone (MBTH) [18]. A
flow-injection spectophotometric method [31] and UV procedures [32, 33] were also
developed for its determination.
A spectrophotometric procedure [34] was developed for the determination of
dapsone [DAP] based on the oxidation of metol by chromium; the resulting
oxidation product was measured at 520 nm. In separate procedures, 3-aminophenol,
1-naphthylamine, salbutamol and clioquinol [35], 8-anilinonaphthalene-1-sulphonic
acid and resorcinol or beta-naphthol [36], 9-chloroacridine [37], NEDA [38], and
cresyl fast violet acetate [39] were some of the reagents proposed for the
spectrophotometric determination of dapsone.
Sastry et al. [40-42] have used MBTH-Fe(III), Fe(II)-1,10-phenanthroline,
chloranilic acid [chromogenic reagents] and Suprachen Violet 3B, Erioglaucine,
Naphthalene Blue, Tropaeolin 000, Wool Fast Blue BL and sulphonaphthalein dyes
[43] for the extractive spectrophotometric determination of cisapride and other basic
drugs. And also, a spectrofluorimetric [44] and derivative spectrophotometric [45]
procedures were reported in literature for the determination of cisapride in
pharmaceutical preparations. A flow-injection spectrophotometric method [46] was
developed for the determination of p-aminobenzoic acid in pharmaceuticals and in
biological fluids. Very few methods have been reported in literature for its
determination. It is appropriate to mention some of the recently reported methods
apart from spectrophotometric methods for the determination of studied drugs, such
as, voltammetric [47, 48], HPLC [49-56] and GC-MS [57].
The survey of chemical literature revealed that no attempt was made use of
citrazinic acid, imipramine hydrochloride and N-bromosuccinimide as the reagents for
the determination of studied drugs (MCP, DAP, CPD and PABA). In this chapter, the
author has presented her findings of the reaction of the above drugs with cited
reagents in three sections 2A, 2B and 2C. Spectrophotometric determination of the
Chapter – 2
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MCP, DAP, CPD and PABA with CZA and MCP and DAP with IPH are presented in
Sections 2A and 2B, respectively. A titrimetric assay of MCP in pure as well as in
dosage forms employing NBS as the oxidimetric titrant, is presented in Section 2C.
Chapter – 2
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Section –2A
Spectrophotometric determination of metoclopramide
hydrochloride, dapsone, cisapride and p - amino benzoic
acid with citrazinic acid
2A.1. Introduction
Citrazinic acid (CZA), chemically 2,6-dihydroxy isonicotinic acid or 1,1-
dihydro-6-hydroxy-2-oxopyridine-4-carboxylic acid. It is yellowish powder with a
greenish tinge. It is insoluble in water and slightly soluble in hot hydrochloric acid.
CZA is freely soluble in alkali hydroxide or carbonate solutions. Alkaline solutions
turn blue on standing. It has the following structure.
N
OHO
HO OH
2,6-dihydroxy isonicotinic acid
Kavlentis has used citrazinic acid as a reagent in the spectrophotometric
determination of uranium(VI) and iron(III) [58], and kinetic spectrophotometric
determination of iron(III), copper(II) and vanadium(V) [59]. Revanasiddappa and
Kiran Kumar [60] have used CZA as a coupling agent with p-amino acetophenone for
the determination of chromium. The same authors have used CZA as a coupling
agent for the determination of nitrite with p-aminoacetophenone [61] and p-
nitroaniline [62]. It has also been found application as the coupling agent in the
indirect spectrophotometric determination of chromium with p-nitroaniline. [63]
2A. 2. EXPERIMENTAL
2A. 2. 1. Apparatus
An Elico model CL-27 digital spectrophotometer with 1cm matched quartz cells was
used for all absorbance measurements.
Chapter – 2
18
2A. 2. 2. Reagents
All chemicals used were of analytical reagent grade.
Citrazinic acid (CZA, 0.1%): Prepared by dissolving 0.1g of the reagent (Fluka,
Switzerland) in 2 mL 4M NaOH and diluting to 100 mL with water.
Sodium nitrite (0.1%): It was prepared by dissolving 0.1g of sodium nitrite in 100
mL distilled water.
Sulphamic acid (2.0%): Freshly prepared by dissolving 2 g of sulphamic acid in 100
mL distilled water. Aqueous solutions of sodium hydroxide (4M) and hydrochloric
acid (1M) were used.
Standard solutions of MCP, DAP, CPD and PABA
The pharmaceutical grade chemotherapeutics were received from different
companies and were used as such. Separate aqueous solutions of MCP (IPCA
Laboratories Ltd., India) and PABA (Franco-Indian Pharma Ltd.) and methanol
solution of DAP (Intas Laboratories Ltd., India) and CPD (Intas Laboratories Ltd.,
India) were prepared. The concentration of these solutions is equal to 1000 µg mL-1.
Solutions of lower concentration were prepared by diluting the standard solutions.
2A.2.3. Standard procedure for the preparation of calibration graph
Different aliquots i,e 0.5 – 6.0 mL [50 µg mL-1 ] of MCP, 0.2 – 2.5 mL [50
µg mL-1] of DAP, 0.5 – 2.5 mL [50 µg mL
-1] of PABA or 0.3 – 1.8 mL [100 µg mL
-1]
of CPD were transferred into a series of 25 mL calibrated flasks. A volume of 1.5 mL
of 0.1 % sodium nitrite was added to each flask, followed by the addition of 1 mL of
1M HCl. After 3 min, 2 mL of 2 % sulphamic acid were added to each flask. Then,
volumes of 2 mL of 0.1 % CZA and 4 mL of sodium hydroxide solution were added
and the contents were diluted to the mark with distilled water and mixed well. After
10 min, the absorbance of the coloured azo-dye was measured at 465 nm for MCP, at
515 nm for DAP, at 500 nm for CPD and at 475 nm for PABA against the
corresponding reagent blank. The amount of drug was computed from the standard
calibration graph [Fig.2A.1] or regression equation.
Chapter – 2
19
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 5 10 15
C on c i n µg mL-1
Absorbance [1 ]
[2]
[3]
[4]
Fig. 2A. 1. Beer’s law curves of [1] MCP [2] DAP [3] PABA and [4] CPD
2A. 2. 4. Recovery of drugs from synthetic mixtures
Two mixtures of each drug (MCP, DAP, CPD and PABA) with several
excipients whose compositions are given in Table 2A.2 were prepared. A portion of a
mixture containing 25 mg of each drug was accurately weighed. Three portions of 20
mL of distilled water were added and the mixture was thouroghly shaken for about 30
min to extract the drug from the powder before filtering the mixture. The residue after
filtration was washed with 20 mL of distilled water. The filtrate and washings were
then combined in a 100 mL calibrated flask and the volume was made up with
distilled water. An aliquot of this solution was treated as described in the Standard
Procedure 2A.2.3. The results are presented in Table 2A.1.
Conc in µµµµg mL-1
Chapter – 2
20
Table 2A. 1. Recovery of drugs from synthetic samples by the proposed method
Drug
Amount
present
(mg)
Excipients (mg) % Recovery
± SD Talc Dextrose Starch
Sodium
alginate Gelatin
Gum
acacia
MCP
100
80
150
200
250
350
150
200
100
75
75
100
50
75
99.8 ± 0.7
100.3 ± 0.5
DAP
50
75
250
200
200
250
180
250
125
100
50
75
100
50
101.3± 0.9
99.9 ± 0.3
CPD
100
120
150
250
200
150
250
150
100
75
75
100
150
100
100.1± 0.8
99.6 ± 0.5
PABA
80
120
300
200
250
300
200
150
120
80
75
50
125
75
99.8 ± 0.4
100.5 ± 0.6
* Average recovery from five experiments.
2A. 2. 5. Procedure for pharmaceutical preparations
An accurately weighed amount of powdered tablets (MCP, DAP or CPD)
equivalent to 25 mg was transferred into a 100 mL calibrated flask; about 50 mL of
water (methanol for DAP and CPD) were added and shaken thoroughly for about 30
min. The volume was made upto the mark with the respective diluents, mixed well
and filtered using a quantitative filter paper. Appropriate aliquots of the drug solution
were taken and the proposed Standard Procedure [2A. 2. 3] was followed for the
analysis of drug content.
For the analysis of an injection solution, the requisite volume was transferred
into a 100 mL calibrated flask and diluted to the mark with distilled water. The
solution was then treated as described above. The same drug samples were also
analyzed by the reference method [6] and the results are given in the Table 2A.2.
Chapter – 2
21
Table 2A. 2. Analysis of studied drugs in pharmaceutical dosage forms
a Average of five determination , b Tabulated value 2.78, c Tabulated value 6.39
Sample
Pharmaceutical
formulation
Amount
taken
(µµµµg mL-1)
Proposed
Amount
found
(µµµµg mL-1 )
Methoda
% Rec ±±±± SD
% C V
Reference
Method [6]
% Rec.±±±± SD
t-valueb
F-valuec
Perinorm
10 mg / tab
4.0
3.99
99.7 ± 0.3
0.08
99.5 ± .0.4
2.29
1.77
8.0 7.99 99.9 ± 0.5 0.06 100.1 ± 0.4 1.11 1.56
12.0 12.0 100.1 ± 0.4 0.03 99.8 ± 0.6 0.85 2.25
Reglan
10 mg / tab
4.0 3.98 99.5 ± 0.8 0.20 99.7 ± 0.6 0.96 1.77
8.0 7.98 99.8 ± 0.2 0.03 100.2 ± 0.2 0.66 1.00
MCP
12.0 11.99 99.9 ± 0.3 0.03 99.9 ± 0.5 0.49 2.77
Emenil
10 mg / tab
4.0
3.98
99.6 ± 0.8
0.20
99.9 ± 0.5
0.99
2.56
8.0 7.96 99.5 ± 0.4 0.05 100.2 ± 0.3 1.89 1.77
12.0 11.97 99.8 ± 0.5 0.04 99.7 ± 0.4 0.23 1.56
Reglan inj
5 mg / mL
4.0 4.00 100.0 ± 0.4 0.10 100.2 ± 0.3 1.62 1.77
8.0 7.98 99.8 ± 0.3 0.04 100.0 ± 0.4 1.15 1.77
12.0 12.0 100.1 ± 0.5 0.12 99.9 ± 0.4 0.99 1.56
Dapsone
25 mg / tab
2.0
1.99
99.6 ± 0.8
0.40
100.1 ± 0.5
1.89
2.56
3.0 2.97 99.0 ± 0.4 0.13 99.6 ± 0.5 1.29 1.56
4.0 3.99 99.8 ± 0.5 0.12 99.1 ± 0.7 0.84 1.96
DAP
Dapsone
100 mg/ tab
2.0
1.99
99.6 ± 0.8
0.40
99.8 ± 0.5
1.90
2.56
3.0 2.98 99.5 ± 0.4 0.13 100.1 ± 0.3 1.75 1.77
4.0 3.98 99.5 ± 0.5 0.12 99.9 ± 0.3 1.65 2.77
Cisawal
10 mg / tab
2.0
1.99
99.6 ± 0.4
0.20
99.8 ± 0.4
2.01
1.56
4.0 3.98 99.5 ± 0.7 0.18 98.9 ± 0.6 0.68 2.25
6.0 5.98 99.8 ± 0.4 0.07 100.1 ± 0.5 0.56 1.56
Ciza
10 mg / tab
2.0
1.99
99.6 ± 0.5
0.25
99.8 ±.0.6
2.28
1.56
CPD 4.0 4.00 100.1± 0.4 0.10 99.1± 0.7 1.09 2.25
6.0 5.98 99.7± 0.4 0.07 99.9 ± 0.4 1.11 1.56
Unipride
10 mg / tab
2.0
2.00
100.1 ± 0.8
0.40
99.9 ± 0.6
1.28
1.77
4.0 3.99 99.8 ± 0.6 0.15 99.5 ± 0.4 1.1 2.25
6.0 5.98 99.6 ± 0.4 0.07 99.9 ± 0.2 0.41 4.00
Chapter – 2
22
2A.3 Results and Discussion
The proposed method is based on the diazo-coupling reaction of the studied
drugs with citrazinic acid in an alkaline medium to give an orange red coloured azo-
dye with a maximum absorption in the range 465 – 515 nm. The absorption spectra
of the azo-dyes are depicted in Fig. 2A. 2.
Two steps are involved in the reaction that produces the coloured dye. In the
first step, these chemotherapeutics are treated with nitrite solution in acidic medium
and they undergo diazotisation to give the diazonium chloride ion. In the second step,
the diazonium ion is coupled with a coupling agent, citrazinic acid, to form an azo-
dye in an alkaline medium. The reaction system is represented in Scheme 1. Here,
MCP is used as the model compound, since the other compounds also behaved
similarly to it (Scheme 2A.1).
+ NO2
_
+ H+
N
++
H2O
N
OHO
HO OH
+
O
NH
OCH3
N
Cl
NH2
O
NH
OCH3
N
Cl
N
[A]
[A]
N
O
NH
OCH3
N
Cl
N
N
O OH
OHHO
Azo-dye
MCP
NaOH
CZA
Scheme. 2A. 1. Reaction pathway of MCP with CZA
2A. 3. 1. Absorption spectra
In order to establish the optimum wavelength for maximum absorption (λmax) of the
formed azo-dye, a known amount of the pure drug solutions [MCP – 10 µg mL-1,
DAP – 4 µg mL-1, PABA – 4 µg mL
-1and CPD – 28 µg mL
-1] were taken and the
Standard Procedure (2.A.2.3) was followed to develop coloured azo-dye of the
Chapter – 2
23
studied drugs. The absorption spectra of the formed dye and its reagent blank were
scanned in the region 400 – 600 nm against the corresponding reagent blank and
distilled water, respectively. The formed coloured azo-dye showed maximum
absorption at 465 nm for MCP, at 515 nm for DAP at 500 nm for CPD and at 475 nm
for PABA, and the reagent blank had negligible absorption at these wavelengths
(Fig 2A.2).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
400 450 500 550 600
Wavelength in nm
Absorbance
[1]
[2]
[3]
[4]
Blank
Fig. 2A. 2. Absorption spectra of [1] MCP, [2] DAP, [3] PABA, [4] CPD and blank with
CZA
2A. 3. 2. Optimization of experimental parameters
The factors influencing the colour development, sensitivity and adherence to
Beer’s law were investigated and were reported below.
2A. 3. 2. 1. Effect of the reagent concentration
The effect of the CZA concentration and its volume on the absorbance of the
coloured dye was studied for the cited drugs at the above-specified wavelengths. The
maximum colour intensity was obtained with 0.5 – 5.0 mL of 0.1 % CZA solution for
all the drugs. A volume of 2 mL of 0.1 % CZA was found to be sufficient in a total
volume of 25 mL of the reaction mixture. Thus, the same volume was employed in
the subsequent studies. The results are presented in the Fig. 2A. 3.
Chapter – 2
24
0.272
0.28
0.288
0.296
0 1 2 3 4 5
Vol.of CZA in mL
Absorb
ance
Fig. 2A. 3. Effect of CZA on the absorbance of the azo-dye
Fig. 2A. 4. Effect of sodium nitrite on the absorbance of the azo-dye
2A. 3. 2. 2 Effect of sodium nitrite
The absorbance values were found to be constant in the volume range 0.5 – 4.0 mL of
0.1 % sodium nitrite under the optimum conditions. An optimum volume of 1.5 mL of
0.1 % NaNO2 was fixed and thus used in further studies and the excess of nitrite could
be removed by the addition of 2 mL of 2 % sulphamic acid otherwise, nitrite strongly
interferes with the method. The results are depicted in Fig.2A.4.
0.36
0.364
0.368
0.372
0.376
0 1 2 3 4 5
Vol.NaNO2in mL
Absorbance
Chapter – 2
25
2A. 3. 2. 3 Effect of acid
The diazotisation reaction was carried out at room temperature (ca. 25 ± 2 °C).
The hydrochloric acid concentration for diazotisation was investigated. A suitable
acidity as evident from the maximum absorbance and stability of the azo-dye formed
was found to be 1 mL of 1 M hydrochloric acid in a 25 mL of reaction mixture.
2A. 3. 2. 4 Effect of alkali
To develop a quantitative method based on this reaction, a study was
conducted to determine the most effective alkalies and optimum alkali concentration
to be used. Sodium hydroxide was found to be the most effective base compared to
sodium carbonate or ammonia. The orange coloured azo-dyes are unstable in
ammonia and do not give maximum colour intensity in sodium carbonate medium.
The orange coloured azo dyes, stable in the optimum concentration of sodium
hydroxide solution leading to a maximum intensity and stability of colour was found
to be 4 mL of 4 M sodium hydroxide for all the studied drugs under optimum
conditions. The results are presented in Fig. 2A.5.
Fig. 2A. 5. Effect of alkali on the absorbance of the azo-dye
0.3
0.34
0.38
0 2 4 6 8
Vol. NaOH, mL
Absorbance
Chapter – 2
26
2A. 3. 2. 5 Effects of the temperature and time
The coloured azo-dyes developed rapidly after the addition of reagents and
attained maximum intensity after about 10 min at room temperature (25 ± 2 °C).
Cooling to 0 – 5° C was not necessary and the formed azo-dyes are stable for a period
of more than 2 h. Beyond this time, the absorbance values are gradually decreasing.
2A. 3. 2. 6 Order of addition of reagents
Reagents were added in the described sequence in order to achieve the
maximum sensitivity of the colour system. Any change in the order of addition of
reagents affects the formation of azo-dye and the sensitivity of the system.
2A. 3. 3 Calibration graphs and Analytical parameters
The calibration graphs for the studied drugs as obtained under optimum
conditions are shown in Fig-2A.1. Good linear relationships were obtained over the
concentration ranges given in Table.2A.3. The corresponding molar absorptivity
values from Beer’s law data and their Sandell’s sensitivity values are presented in
Table.2A.3. The slope, intercept, correlation coefficients, detection limit and
quantitation limit of the method are also given in Table .2A.3.
Chapter – 2
27
Table 2A. 3. Optical characteristics of the studied chemotherapeutics.
Parameter MCP DAP CPD PABA
Beer’s law limit
(µg mL-1)
1.0-12.0 0.4-5.0 1.2-7.2 1.0-5.0
Molar absorptivity
(L mol-1cm
-2). 10
4
1.92 4.52 3.15 2.15
Sandell’s sensitivity
(µg cm-2)
0.0184 0.0054 0.0147 0.0063
Correlation
coefficient [r]
0.9999 0.9999 0.9999 0.9999
Regression equation
[Y*]
Slope [b] 0.0522 0.1606 0.0575 0.1359
Intercept [a] 0.0088 0.0335 0.0292 0.0471
Detection limit [DL]
(µg mL-1)
0.1892 0.0210 0.0845 0.0277
Quantitation limit
[QL] (µg mL-1)
0.5734 0.0638 0 .2562 0.0841
*y = a + bx, where x is the concentration in µg mL-1
Interference studies
In order to evaluate the suitability of the proposed method for the analysis of
pharmaceutical preparations of the studied drugs, the interference of associated
excipients and diluents in dosage forms was investigated. Talc, starch, dextrose,
sodium alginate and gelatin in amounts far in excess of their normal occurrence in
pharmaceutical formulations do not interfere. The results presented in Table 2A.1
shows that there is good agreement between the amounts of drugs taken and found in
the presence of associated excipients and diluents.
Precision
The precision of the proposed method was evaluated by replicate analysis
(within-day and Between-day) of samples containing the studied drugs [MCP, DAP,
CPD and PABA] at three different concentrations. The low values of the coefficient
Chapter – 2
28
of variation (CVs) both at the low and high concentrations reflect the high precision
of the proposed method [Table-2A.4].
Table. 2A. 4. Within-day and between-day studies of chemotherapeutics.
Average value of five determinations carried out over five days.
Applications to the pharmaceuticals
The proposed method was successfully applied to the analysis of MCP, DAP
and CPD in pharmaceutical preparations. The results of an assay of Perinorm,
Regaln, Emenil, Cisawal, Ciza, Unipride and Dapsone tablets and injection solutions
are presented in [Table. 2A.2], compare favorably with the reference method [6]. A
statistical analysis of the results by Student’s t- and F- tests showed no significant
difference in accuracy and precision between the proposed and reference methods
[Table-2A.2].
Sample
Amount
taken
(µµµµg mL-1)
Within day
Amount
found ±±±± SD
(µµµµg mL-1 )
CV%
Amount
taken
(µµµµg mL-1)
Between day
Amount
found ±±±± SD
(µµµµg mL-1 )
CV%
MCP
4.0 3.91 ± 0.05 1.27 4.0 3.90 ± 0.05 1.28
8.0 7.56 ± 0.02 0.25 8.0 7.94 ± 0.04 0.50
12.0 12.05 ± 0.01 0.08 12.0 11.95 ± 0.03 0.25
DAP
2.0
1.99 ± 0.07
3.51
2.0
1.99 ± 0.08
4.02
3.0 2.97 ± 0.05 1.68 3.0 2.91 ± 0.05 1.71
4.0 3.91 ± 0.03 0.76 4.0 4.01 ± 0.03 0.74
CPD
2.0
1.97 ± 0.05
2.53
2.0
1.96 ± 0.08
4.08
4.0 3.89 ± 0.03 0.77 4.0 3.90 ± 0.04 1.02
6.0 5.92 ± 0.02 0.33 6.0 5.97±0.02 0.33
PABA
2.0
1.97 ± 0.06
3.04
2.0
2.01 ± 0.03
2.98
3.0 3.01 ± 0.05 1.66 3.0 2.96 ± 0.06 1.35
4.0 3.98 ± 0.03 0.75 4.0 3.99 ± 0.03 0.75
Chapter – 2
38
Section –2C
Titrimetric method for the determination of metoclopramide
hydrochloride using N-bromosuccinimide
2C. 1. Introduction
Titrimetry is one of the classical methods and finds the application despite and
advent of modern physico-chemical methods. In contrast, e.g, to HPLC, titration is an
absolute method. The titration takes place strictly stoichiometrically and normally
very rapidly. If a titrant with a known titer is used then the content of the sample can
be determined directly. In recent years, titrimetric methods have found wide
applications in the quantitative analysis of therapeutic agents. In the following
paragraphs, a brief review of the oxidimetric titrant N-bromosuccinimide is presented.
N-Bromosuccinimide [NBS] is a chemical reagent first synthesized by
Seliwanow [64]. It is a white solid, melts at 175-178 °C and is soluble in hot water
(1.47 g/100 mL at 25 °C). NBS has the following structural formula.
O
O
NBr
N-bromosuccinimide
It has been used as a valuable reagent for the determination of several
bioactive compounds of therapeutic interest. A brief survey of its analytical utility is
presented in the following paragraphs.
Very recently, NBS was used as a reagent for the spectrophotometric
determination of fluoroquinolone antibiotics [65]. Basavaiah et al. have described
titrimetric and spectrophotometric methods for the assay of salbutanol sulphate [66]
and metoprolol tartrate[67] in pharmaceuticals using NBS.
Chapter – 2
39
Recently, Al-Momani was reported flow injection spectrophotometric
procedure for the determination of meloxicam, piroxicam and tenoxicam [68] and
levofloxacin [69] with NBS.
NBS has been used as a reagent for the spectrofluorimetric determination of
paracetamol [70], a titrimetric determination of nizatindine in capsule [71],
spectrophotometric determination of famotidine [72] and dapsone [73]. A detailed
account of the analytical applications of NBS for the analysis of several organic
compounds is given in the monograph compiled by Mathur and Narang [74]. Up to
2001, the analytical utility of NBS was admirably discussed by Manju [75].
Aforementioned literature survey revealed that no attempt has been made use of NBS
as the oxidimetric titrant for the determination of MCP in pure and in dosage forms.
In this section, an indirect titrimetric assay of MCP with NBS is reported.
2C.2 EXPERIMENTAL
2C. 2. 1. Apparatus
Pre calibrated pipettes, burettes and measuring flasks (Borosil or Corning
make) were used.
2C.2.2 Reagents
All chemicals used were of analytical reagent grade.
N-Bromosuccinimide (NBS) [0.01 M]
0.01 M NBS [Loba Chemie., India] was prepared by dissolving about 1.8 g of
freshly crystal lined powder in 100 mL water with the aid of heat, and diluted to 1
liter with distilled water and standardized iodometrically.
Sodium thiosulphate (0.01M)
Prepared by dissolving 2.48 g of sodium thiosulphate in 1 liter distilled water
and standardized using pure potassium dichromate.
Chapter – 2
40
Others: Potassium iodide [10 %], 5M acetic acid and starch indicator [1%] was
prepared in the usual way.
Standard solution of MCP
Pharmaceutical grade MCP was received from IPCA Laboratories Ltd., India
as gift and was used as received. A stock solution containing 1000 µg mL-1 of drug
was prepared daily by dissolving accurately weighed amount of MCP in distilled
water. Solutions of lower concentration were prepared by diluting the stock solutions
with distilled water.
2C. 2. 3. Determination of drug in pure form
A 10 mL aliquot of pure drug solution containing 1-13 mg of MCP was
accurately measured and transferred into a 100 mL conical flask. The solution was
acidified by adding 5 mL of 5M acetic acid followed by the addition of 0.01 M NBS.
The content was mixed and kept aside for 15 min. Then, 5 mL of 10 % potassium
iodide was added to the flask and the liberated iodine was titrated against sodium
thiosulphate (0.01M) using starch indicator towards the end point. A blank titration
was performed under identical conditions using all the reagents except the drug. The
amount of the drug was calculated from the equation,
(A-B) MwM
Amount of drug found (mg) = N
where, A = Volume of thiosulphate solution used in blank titration.
B = Volume of thiosulphate solution used in sample titration.
Mw = Relative molecular mass of MCP
M = Molarity of thiosulphate
2C. 2. 4. Recovery of drugs from synthetic mixture
The mixtures of MCP with several excipients whose composition are given in
Table 2C.1 were prepared. A portion of a mixture containing 100 mg MCP was
accurately weighed. Three portions of 20 mL of distilled water were added and the
mixture was shaken thoroughly for about 30 min to extract the drug from powder
before filtering the mixture. The residue was then washed with 10 mL of distilled
Chapter – 2
41
water. The filtrate and washings were then combined in a 100 mL calibrated flask and
the volume was made upto the mark with distilled water. An aliquot of this solution
was analysed by following the above Procedure 2C. 2. 3. The results are presented in
Table 2C.1.
Table 2C.1: Analysis of drugs from various excipients
Name of
the
compound
Amount
present
(mg)
Excipients (mg) %
Recovery
± SD
Talc
Dextrose Starch Sodium
alginate
Gelatin Gum
acacia
MCP 75 150 50 150 75 50 75 99.7 ± 0.56
50 200 100 100 50 75 100 99.0 ± 0.78
Average recovery from five experiments.
2C.2.5 Analysis of MCP in pharmaceutical formulations
Tablets: An accurately weighed amount of powdered tablets equivalent to 100 mg
was transferred, extracted and analyzed as described under recovery of drugs from
synthetic mixtures in Section 2C.2.4.
Injections: For analysis of injection solution, an appropriate volume of the sample
containing 100 mg of the drug was transferred into a 100 mL calibrated flask and
diluted to the mark with distilled water. The drug content in the diluted solution was
determined as described above. The samples were also analyzed by the reference
method of British Pharmacopoeia [76] and the results are presented in Table 2C.2
Table 2C.2: Analysis of MCP in pure drug samples.
Formulation % Recovery ± SD
t-value b F-value
c
Reference method Proposed methoda
Perinorm [10 mg/tab] 100.20 ± 0.55 99.90 ± 0.37 2.0 2.20
Reglan [10 mg/tab] 99.80 ± 0.91 100.50 ± 0.50 1.7 3.31
Emenil [10 mg/tab] 100.10 ± 0.68 100.40 ± 0.90 1.4 1.75
Perinorm [5 mg/mL] 99.50 ± 0.75 99.90 ± 0.49 1.09 2.34
Reglan [5 mg/mL] 100.50 ± 0.40 101.0 ± 0.81 1.24 4.10
a Average of five determination , b Tabulated value 2.78, c Tabulated value 6.39
Chapter – 2
42
2C. 3. Results and Discussion
This method is based on the oxidation of MCP by a known excess of NBS and
followed by the determination of unreacted NBS iodometrically. NBS is found to
react quantitatively with MCP in acetic acid medium and the amount of NBS reacted
corresponds to the drug content. Considering the available literature [74] and
stoichiometry, it may be postulated that the MCP undergoes oxidation at suitable
positions. The possible oxidation process based on the literature reports, is as shown
in the following Scheme 2C.1
O
O
N Br
N-bromosuccinimide
+ 3
O
HN
OCH3
N
Cl
H2N
O
BrN
OCH3
N
Cl
H2NBr
Br
Scheme 2C.1. Probable reaction between MCP and NBS
2C. 3. 2. Optimization of experimental variables
In order to establish the optimum conditions for the quantitative estimation of
MCP, the effects of acids and time were studied, and reported in the following
paragraphs.
2C. 3. 2. 1. Effect of acid
At laboratory temperature (ca 25 ± 2 °C), the rate of the reaction was found to
depend on the nature of the acid and its concentration. Different acids such as
hydrochloric acid, sulphuric acid and acetic acid were tried as reaction medium to
obtain reproducible and stoichiometric results. Non-stoichiometric results obtained in
a hydrochloric acid medium as well as sulphuric acid medium Volume of 2-10 mL of
5M acetic acid gave reproducible results and sharp end point in a total volume of
about 25 mL reaction mixture. Thus, a volume of 5 mL of 5M acetic acid was used in
all subsequent work.
Chapter – 2
43
2C. 3. 2. 2. Effect of time
The oxidation reaction was found to be complete and quantitative in 15
minutes and contact times upto 30 min had no effect on the stoichiometry and the
results. Beyond 30 min, a small amount of NBS consumed but without producing any
definite stoichiometry. Hence, it is necessary to terminate the oxidation step at the end
of the 15th min to obtain the reproducible results.
2C. 3. 2. 3. Mole ratio
Under the optimum conditions, three moles of NBS were required for the
complete oxidation of MCP (1:3, drug: NBS), which is conformity, with the scheme
of the oxidation process presented in Scheme 2C.1.
2C. 3. 2. 4. Range of determination
Under the experimental conditions, 10 mL of drug solution containing the
amounts ranging from 1-13 mg of MCP was titrated. It was observed that inaccurate
results (error > 5%) were obtained outside the above limits and do not obtain a sharp
end point beyond the upper limit.
2C. 3. 2. 5. Precision of the method
The precision of the developed method was assessed from the results of
replicate analysis on pure drug at three different concentrations (low, medium and
high). The low values of CVs at both the low and high concentration reflect the high
precision of the method. The results are given in Table 2C.2
2C. 3. 2. 6. Application to pharmaceutical preparations
The applicability of the proposed method for the assay of the different
pharmaceutical formulations containing MCP was examined. The results were
statistically compared with those obtained by the reference method of British
Pharmacopoeia [76].The t-test and F-tests were carried out, which showed that the
proposed method and other reported methods are of comparable accuracy and
precision. The results are given in Table 2C.2
Chapter – 2
44
Conclusions
The methods presented in this chapter have been validated and successfully
used for the determination of selected therapeutics (MCP, DAP, CPD and PABA) in
their pure form and in pharmaceutical formulations, with good accuracy and
precision. Both spectrophotometric methods (2A and 2B) are based on diazo-coupling
reaction utilizing citrazinic acid and imipramine hydrochloride as the coupling agents
shows high sensitivity and simplicity of the methods, and cooling to 0 – 5 °C was not
necessary for diazotization. The formed azo-dyes are stable for a sufficient interval of
time, which makes the methods more practicable.
In section 2C, the developed titrimetric procedure for the assay of MCP using
NBS as the oxidimetric titrant is simple, inexpensive, accurate and more precise than
the other instrumental methods. The methods developed are compared reported
methods and are presented in Table. 2C. 4. The proposed procedures offer the
advantage of simplicity, rapidity and low cost.
Chapter – 2
45
2C. 4. Comparison of the proposed method with other spectrophotometric
methods
Reagent
Range,
µg mL-1
References Remarks
MBTH 2-24 [18] Less sensitive
Catechol 10-40 [28] Less sensitive
Suprachen Violet 3B and 2.5-4 [41] Extractive and time
consuming
Tropaeolin 000 2.5-25 [41]
9-Chloroacridine 20-200 [37] Time consuming and less
sensitive
Ammonium molybdate and
thiocyanate
1-20 [20]
Extractive and less sensitive
Chloranil or bromanil 40-160 [22] Requires heating at 80º C
NaVO3 40-160 [23] Less sensitive and requires
heating
2-naphthol-3,6 disulphonic
acid
1-250 [4] Requires heating at 100º C
and less sensitive
p-dimethylamino
cinnamaldehyde
5-30 [30] Derivative and less sensitive
p-dimethylamino
cinnamaldehyde and
phosphoric acid
160-560 Less sensitive
Bromocresol
green,bromothymol blue and
bromocresol purple
2-10 Less sensitive
8-hydroxy quinoline 2-25 [13] Less sensitive
Citrazinic acid
Imipramine hydrochloride
Titrimetric
1-12 [MCP]
0.4-5[DAP]
1-5 [PABA]
1.2-7.2[CPD]
0 -5.0 [MCP]
0 - 4.0 [DAP]
1 -13 mg
Proposed
methods
Highly sensitive,
inexpensive, rapid and
facile
Chapter – 2
46
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Chapter – 2
29
Section-2B
Diazocoupling method for the determination of
metoclopramide hydrochloride [MCP] and dapsone
[DAP] using imipramine hydrochloride
2B.1. Introduction
Imipramine [IPH] chemically, 10,11-dihydro-N,N-dimethyl-5H-dibenz [b,f]
azepine-5-propanamine hydrochloride. Imipramine hydrochloride crystals from
acetone (m.p -174-175 °C). It acquires a yellow to reddish discolouration under the
influence of light and it has the following structure:
It is freely soluble in water, less soluble in alcohol and sparingly soluble in acetone.
An examination of literature review is revealed that, no researchers used it as a
reagent for the analysis of either metal ions or pharmaceutical samples by
spectrophotometry. In this section, the author has utilized imipramine as the coupling
agent for the determination of MCP and DAP through diazo-coupling reaction.
2B.2. EXPERIMENTAL
2B. 2.1. Apparatus
Instruments used are described in Section 2A.2.1.
2B. 2. 2. Reagents
All chemicals used were of analytical reagent grade.
Imipramine hydrochloride (IPH, 0.5%): It was prepared by dissolving 0.5 g of IPH
(Max Pharma Ltd. India) in 100 mL distilled water.
Sodium nitrite (0.1%), sulphamic acid (2.0 %), 1M and 6 M HCl were used.
Standard solution
Preparation of pure drug samples of MCP and DAP is described in Section 2A.2.2.
N
N
Chapter – 2
30
2B. 2. 3. Preparation of calibration graph
Accurately measured volumes of drug solutions equivalent to 0 -5.0 µg mL-1 and
0 - 4.0 µg mL-1 final solution of MCP and DAP, respectively, were transferred into a
series of 10 mL calibrated flasks. Then, a volume of 1mL of 0.1% sodium nitrite
solution was added to each flask followed by 0.5 mL of 1 M hydrochloric acid. After
3 min, 1 mL of 2% sulphamic acid was added to each flask. A volume of 2 mL of
0.5% IPH solution was added and the contents were diluted to the mark with 6 M
hydrochloric acid and mixed well. After 5 min, the absorbance of the coloured azo
dye was measured at 570 nm for both MCP and DAP against the corresponding
reagent blank. The amount of drug was computed from the standard calibration
graphs [ Fig 2B.1].
0
0.1
0.2
0.3
0.4
0.5
0.6
0 2 4 6
Conc in µg mL-1
Ab
sorb
an
ce
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6
Conc in µg mL-1
Ab
sorb
an
ce
(a) (b)
Fig 2B. 1. Beer’s law curves of (a) MCP and (b) DAP
2B.2.4 Procedure for pharmaceutical preparations
Twenty tablets of MCP and DAP were weighed and finely powdered. An
accurately weighed amount, equivalent to 20 mg of the respective drug was
transferred into a 100 mL calibrated flask and dissolved in 75 mL of distilled water
and the contents were thoroughly shaken for about 30 min and completed to volume
with distilled water (methanol was used for DAP), and then filtered. Appropriate
Chapter – 2
31
aliquots of the drug solution were taken and the above procedure [2B.2.3] was
followed for analyzing the drug content.
To analyze the injection solution, the requisite volume was transferred to a
100 mL standard flask and diluted to the mark with distilled water. The drug content
in the diluted solution was determined as described above. For comparison, the
reference method [6] was employed to determine the drug content in the same
pharmaceutical samples and the results of the analysis are given in Table 2B.1
Chapter – 2
32
Table 2B.1. Results of assay of metoclopramide and dapsone in dosage forms.
a) Average of five determinations; b) Tabulated t - value 2.78; c) Tabulated F value 6.39.
Sa
mp
le
Ph
arm
ace
uti
ca
l
form
ula
tion
Proposed Methoda Reference
Method[ 6 ]
t-valueb
F-valuec Amount
taken
(µµµµg mL-1
)
Amount
found
(µµµµg mL-1
)
% Rec ±±±± SD % Rec.±±±± SD
MC
P
Perinorm
10 mg / tab
1.0
0.99
99.6 ± 0.50
100.2 ± 0.40
2.30
2.10
2.0 1.99 99.5 ± 0.30 99.8 ± 0.40 1.15 1.70
3.0 2.99 99.6 ± 0.10 100.1 ±.0.10 0.80 1.17
Reglan
10 mg / tab
1.0
0.99
99.8 ± 0.30
99.9 ± 0.20
0.23 1.59
2.0 1.99 99.5 ± 0.20 99.9 ± 0.30 0.89 2.25
3.0 3.00 100.0 ± 0.90 100.3 ± 0.70 0.49 1.65
Emenil
10 mg / tab
1.0
1.00
100.0 ± 0.55
99.8 ±. 0.50
1.26
1.21
2.0 1.99 99.4 ± 0.56 99.8 ± 0.49 1.06 1.30
3.0 2.99 99.6 ± 0.58 99.8 ± 0.50 1.47 1.29
Perinorm
5 mg / mL
1.0
0.99
99.5 ± 0.38
99.9 ± 0.19
0.23
3.20
2.0 1.99 99.5 ± 0.44 100.0 ± 0.34 0.24 1.67
3.0 2.99 99.6 ± 0.53 99.8 ± 0.47 1.29 1.27
Reglan inj
5 mg / mL
1.0
0.99
99.7 ± 0.90
99.5 ±.0.50
0.78
1.25
2.0 1.99 99.5 ± 0.53 99.9 ± 0.47 1.29 1.27
3.0 3.00 100.0 ± 0.61 99.9 ± 0.48 1.25 1.61
Dapsone
25 mg / tab
1.0
1.00
100.0±0.55
99.8 ± 0.50
1.26
1.21
2.0 1.99 99.5±0.40 100.2 ± 0.58 2.30 2.10
3.0 2.99 99.6±0.13 100.1 ± 0.12 1.80 1.10
DA
P
Dapsone
100 mg/ tab
1.0
0.99
99.9±0.60
99.8±0.48
1.25
1.61
2.0 1.99 99.9±0.58 99.9±0.51 1.47 1.29
3.0 3.00 100.0±3.0 99.7±0.46 1.75 1.37
Chapter – 2
33
2B.3 Results and Discussion
The proposed method involves the diazo-coupling reaction of MCP or DAP
with IPH in an acidic medium to form a violet coloured azo-dye with a maximum
absorption at 570 nm for both the drugs [Fig 2B.2].
Two steps are involved in the reaction that produces the coloured azo-dye. In
the first step, MCP or DAP are treated with nitrite solution in acidic medium,
undergoes diazotisation to give the diazonium chloride ion. In the second step, the
diazonium chloride couples with a new coupling agent, IPH in an acidic medium to
form an violet coloured azo-dye. IPH has highest electron density at second and
eighth positions, which permits electrophilic substitution by the diazonium ion. The
reaction can be represented as in Scheme 2B.1. Here, MCP is used as the model
compound, since the DAP behaved similarly to it
NO2
_
+ H+
O
NH
OCH3
N
Cl
NH2
+
N
++
H2O
O
NH
OCH3
N
Cl
N
[A]
[A] +
N
R'
R' : CH2CH2CH2N CH3
CH3
N
R'
R' : CH2CH2CH2N CH3
CH3
O
NH
OCH3
N
Cl
NH2
N NHCl
IPH]Azo-dye
MCP
Scheme 2B.1: Proposed diazo-coupling reaction of MCP with IPH
Chapter – 2
34
2B.3.1 Absorption spectra
The general procedure for the determination of MCP and DAP [2B.2.3] was
followed for the formation of azo-dye [MCP- 4 µg mL-1 and DAP- 2.5 µg mL
-1] and
the wavelength of maximum absorption was determined by recording the absorbance
of the azo-dye in the wavelength range 450-650 nm. The absorbance of the reagent
blank against distilled water was also scanned in the same wavelength range.
Absorption spectra [Fig. 2B.2] were obtained by plotting the absorbance values
against wavelength. The azo-dye showed a maximum absorption at 570 nm for both
MCP and DAP and the reagent blank had negligible absobance at this wavelength.
0
0.1
0.2
0.3
0.4
0.5
450 490 530 570 610 650
Wave length in nm
Ab
sorb
an
ce (a)
(b)
blank
Fig .2B. 2: Absorption spectra of (a) MCP and (b) DAP with IPH
2B. 3. 2. Optimization of experimental parameters
Experimental conditions were optimized at 570 nm by studying the influence
of the following parameters with solutions containing a fixed concentration of drugs
2B.3. 2.1 Effect of the reagent
The effect of the concentration of IPH was studied by measuring the
absorbance at 570 nm for solution containing a fixed concentration of drugs and
varying amounts of IPH. A volume of 0.5% solution in a total volume of 10 mL was
found to be sufficient. The results are presented in Fig. 2B.3.
Chapter – 2
35
0
0.1
0.2
0.3
0.4
0 1 2 3
Vol of IPH in mL
Ab
sorb
an
ce
Fig. 2B. 3: Effect of IPH on the absorbance of azo-dye.
2B.3.2.4 Effect of reaction time
The diazotisation was carried out at room temperature (25 ± 2 °C), and 3 min
was the minimum time required for the diazotisation. No cooling (0 – 5 °C) was
required for the diazotisation. A time of 5 min was required for completion of
coupling reaction. The formed azo-dye was stable for more than 3 h.
2B.3.2.5 Effects of acid and nitrite
Different acids such as hydrochloric acid, sulphuric acid, phosphoric acid and
acetic acid were tested for diazotization reaction .HCl medium was the best and 0.5
mL of 1M HCl was used for this purpose. When sulpuric acid was used as a diluent
high blank colour was obtained, with the use of acetic acid very slow colour
development occur and when phosphoric acid was used, the produced dye is less
stable. But, 6 M the formed azo-dye was highly intense and stable in 6 M HCl
medium. Thus, hydrochloric acid found to be necessary for full colour development
and hence 6 M HCl was selected as a diluent for further studies.
Under the optimum conditions, a volume of 1 mL of 0.1 % solution of sodium
nitrite was found to be sufficient in a 10 mL reaction mixture, and the residual sodium
nitrite could be removed by the addition of 1 mL of 2 % sulphamic acid.
Chapter – 2
36
2B. 3 .3. Linearity and Optical characteristics
Under the experimental conditions, standard calibration curves for MCP and
DAP were constructed by plotting absorbance versus concentrations. Linear
relationships were obtained in the concentration range 0.5-5.0 µg mL-1and 0.5-4.0 µg
mL-1 for MCP and DAP, respectively [Fig 2B.1]. The molar absorptivity values from
Beer’s law data and their Sandell’s sensitivity values are presented in Table 2B. 2.
The slope, intercept, correlation coefficient, detection limit and quantitation limit of
the method are also given in Table 2B.2.
Table. 2B. 2. Optical characteristics
Parameter MCP DAP
Linear range
(µg mL-1)
0 -5.0 0 - 4.0
Molar absorptivity
(L mol-1cm
-2)
4.5×104 2.96×10
4
Sandell’s sensitivity (µg cm-2) 0.0078 0.0055
Correlation coefficient [r] 0.9999 0.9999
Regression equation [Y*]
Slope [b] 0.1074 0.1291
Intercept [a] 0.0275 -0.0099
Detection limit [DL] (µg mL-1) 0.0144 0.0132
Quantitation limit [QL] (µg mL-1) 0.0437 0.0402
*y = a + bx, where x is the concentration in µg mL-1
2B.3.3 Recovery Experiments
To assess the selectivity of the method, the interference of those species
accompanying the studied drugs (MCP and DAP) in pharmaceutical formulations was
studied. Samples were prepared by mixing known amount of the drug with various
amounts of common excipients such as talc, starch, dextrose, sodium alginate, gum
acacia and gelatin. The analysis of these laboratory prepared samples was carried out,
and the recovery values were determined. No interference was found from the added
excipients. The results are presented in Table 2B. 3.
Chapter – 2
37
Table 2B.3. Recovery of metoclopramide hydrochloride and dapsone from various
excipients by the proposed method
Nam
e of
the
com
pou
nd
Am
ou
nt
Pre
sen
t (m
g)
Excipients (mg)
Recovery
%
Talc
dextrose Starch Sodium
alginate
Gelatin Gum
acacia
MCP 75 300 400 200 80 50 100 98.7
50 250 350 250 100 75 75 99.0
DAP 75 250 300 250 100 75 100 98.8
50 300 350 200 75 50 75 99.2
* Average recovery from five experiments.
2B. 3. 4. Precision
The precision of the proposed method was determined by analyzing five
replicate samples containing MCP and DAP at three different concentrations.
[Table 2B.4] The intra day precision showed a CV of 1.6 or 0.2% at low
concentration. The inter day precision evaluated over a period of five days showed a
CV of 1.2 or 0.3% at the low concentration. The low values of both the intra and inter
day CVs at the low concentrations reflect the high precision of the proposed method.
The results are given in Table 2B.4.
Table 2B.4. Intra – day and Inter – day precision of the assay of MCP and DAP.
*Average of five determinations carried out over five days.
Sample
in pure
form
Intra - day Inter-day
Analyte
taken
(µg mL-1
)
Analyte
found *
(µg mL-1
) ± SD
CV %
Analyte
taken
(µg mL-1
)
Analyte
found *
(µg mlL-1
) ± SD
CV %
MCP
1
2
3
0.98 ± 0.07
1.96 ± 0.04
2.97 ± 0.04
1.65
0.31
0.22
1
2
3
0.89 ± 0.05
1.94 ± 0. 04
2.95 ± 0.03
1.18
0.60
0.27
DAP
1
2
3
0.99 ± 0.03
1.96 ± 0.03
3.10 ± 0.02
0.63
0.36
0.15
1
2
3
0.98 ± 0.05
1.82 ± 0.02
2.95 ± 0.03
2.40
1.90
0.63