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Chapter 2 Titrimetric and spectrophotometric assay of atenolol

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SECTION 2.0

DRUG PROFILE AND LITERATURE SURVEY

2.0.1 DRUG PROFILE

Atenolol (ATN), is chemically known as, 2-[4-[(2RS)-2-hydroxy-3-[(1-

methylethyl)amino]propoxy]phenyl]acetamide [1]. Its molecular formula is

C14H22N2O3 and molecular weight is 266.34 g mol-1. The chemical structure of ATN

is as shown below:

O

H2N

NH

OH

O

It is a white powder, soluble in water, acetonitrile, acetic acid, sparingly

soluble in ethanol, acetone, dioxane, dichloromethane and chloroform.

It was first developed by the pharmaceutical company Imperial Chemical

Industries in the late 1970s [2]. ATN is a β1-selective (cardioselective) adrenoreceptor

antagonist drug commonly used for management of hypertension, prevention of heart

diseases as angina pectoris and control of some forms of cardiac arrhythmia. It is also

indicated for prophylaxis of migraine [3].

ATN is officially reported in European Pharmacopoeia (EP) [4], British

Pharmacopoeia (BP) [5], and Indian Pharmacopoeia (IP) [6]. BP and EP both describe

non-aqueous titration with perchloric acid as titrant where the end point is located

potentiometrically. IP describes an UV-spectrophotmetric method for the assay of

ATN.


25

2.0.2 LITERATURE SURVEY OF ANALYTICAL METHODS FOR

ATENOLOL

A literature survey regarding the quantitative analysis of ATN revealed that

several methods based on techniques such as titrimetry, UV/visible spectrophotometry

and chromatography have been developed for the determination of ATN in

pharmaceutical formulations. A brief review of those methods is given below.

2.0.2.1 Titrimetric methods

There are only five reports on the titrimetric determination of ATN. A method

reported by Marmo [7] consisted of dissolving 0.2 g of ATN in 20 mL of 0.05 M

H2SO4 followed by back titration of the residual acid with 0.1 M NaOH using methyl

red-bromocresol green mixed indication for visual location of end point. Basavaiah et

al [8] reported a titrimetric method based on the oxidation of atenolol by a measured

excess of cerium(IV) sulphate in acid medium followed by the determination of the

amount of unreacted oxidant by ferrous ammonium sulphate using ferroin as

indicator. Basavaiah et al [9] reported a titrimetric method involving the oxidation of

ATN by a measured excess of chloramine-T (CAT) in acid medium followed by the

determination of the residual oxidant by iodometric back titration. A method based on

the bromination reaction in which ATN was treated with a known excess of bromate-

bromide mixture in acid medium followed by the determination of unreacted bromine

by iodometry has been reported by the same authors [10]. Three more methods

reported by Basavaiah et al [11] involves the reaction of weakly basic ATN with a

measured excess of acid followed by the titration of residual acid with base by visual,

potentiometric and conductometric end point detection.

2.0.2.2 UV-Visible spectrophotometric methods

There are a few reports on the UV-spectrophotometric determination of ATN

in dosage forms when present in combination with other drugs [12-17].

There are only two reports dealing with the UV-spectrophotometric assay of

ATN in dosage forms when present alone. Huang and Jin [18] have determined ATN

in tablets by measuring the absorbance of the tablet extract in 0.01 M HCl at 224 nm.

The calibration graph was linear over 0.5-25.0 µg mL-1. Measurement of the

absorbance of the tablet extract in anhydrous ethanol at 227 nm enabling the


26

determination of ATN in the concentration range 4.0-24.0 µg mL-1 has been reported

by Wang [19].

To the best of the author’s knowledge, there are twelve reports on the use of

visible spectrophotometry for the determination of ATN in pharmaceuticals. Agrawal

et al [20] reported a method based on the reaction of ATN with hydroxylamine

hydrochloride in NaOH medium followed by the reaction of the resultant hydroxamic

acid derivative with FeCl3 to give a red-violet ferric hydroxamate complex which was

measured at 510 nm and the method is applicable over a concentration range of 50.0-

800.0 µg mL-1. Bashir et al [21] have reported a method in which ATN in basic

medium reacts with sodium nitroprusside to generate a colored complex that absorbs

at 495 nm and Beer’s law was obeyed over the concentration range of 0.5-30.0 µg

mL-1. In a method reported by Korang et al [22] ATN in chloroform treated with

chloranil and propan-2-ol in acetaldehyde and resulting coupled product was

measured at 690 nm. In a method reported by Basavaiah et al [11] ATN reacts with

phenol red and the resulting yellow colored product was measured at 430 nm between

a linear range of 3-30 µg mL-1.

A kinetic method developed by Hiremath et al [23] based on the oxidation of

atenolol by a known excess of permanganate in alkaline medium and unreacted

permanganate was measured at 526 nm by rate-constant, constant-concentration and

constant-time methods between the concentration range 1.06-6.6 mg mL-1

Basavaiah et al [24] have reported a method based on the oxidation of ATN by

a measured excess of CAT followed by determination of the unreacted oxidant by a

charge-transfer complexation reaction involving metol and sulphanilic acid. The

absorbance of the product was measured at 520 nm and the method is applicable over

a concentration range of 2.2-25.0 µg mL-1. Two methods reported by Basavaiah et al

[9] in which the drug is treated with a measured excess of CAT in HCl medium and

subsequent determination of the unreacted oxidant with metanil yellow or indigo

carmine and measuring the absorbance at 530 nm or 610 nm, respectively. Beer’s law

was obeyed over a concentration range of 1.0-20.0 µg mL-1 for metanil yellow

method and 2.2-20.0 µg mL-1 for indigo carmine method. Basavaiah et al [10] devised

one more method by treating ATN with a known excess of bromate-bromide mixture

in HCl medium, and after 10 min, the unreacted oxidant was determined by reacting

with methyl orange and measuring the absorbance at 520 nm and the method is

applicable over a concentration range of 0.5-4.0 µg mL-1.


27

Three methods reported by Basavaiah et al [8] based on the oxidation of ATN

by a measured excess of cerium(IV) sulphate in acid medium followed by the

determination of the amount of unreacted oxidant by using ferroin, methyl orange or

iron(III)-thiocyanate as reagents at 510 nm, 520 nm and 480 nm, respectively. Beer’s

law was obeyed over a concentration range of 2.5-35.0 µg mL-1 for ferroin method,

2.5-60.0 µg mL-1 for methyl orange method and 0.6-8.75 µg mL-1 for iron(III)

thiocyanate method.

Agarwal et al [25] reported a method based on charge transfer complexation

reaction of ATN with choranilic acid and absorbance was measured at 534 nm

between the concentration range 25.0-250.0 µg mL-1. Similarly, Yu et al [26] reported

a charge transfer complexation reaction of ATN with choranilic acid and absorbance

was measured at 530 nm between the concentration range 10.0-280.0 µg mL-1.

2.0.2.3 Other methods

Of the chromatographic techniques, HPLC is perhaps the most frequently

used for the determination of ATN in single as well as combined dosage forms.

Several methods were reported for the quantification of ATN when present

alone [27-31].

HPLC procedure have also been proposed for the determination of ATN when

present in combination with amlodipine [32,33], nifidipine [34-36],

hydrochlorthiazide and amiloride [37,38], nitrendipine [39], hydrochlorthiazide and

chlorthalidone [40] or amiloride and chlorthalidone [41].

Procedures based on several other chromatographic techniques such as liquid

chromatography (LC) [42], ion-pair LC [43], chiral LC [44], LC-mass

spectrophotometry [45], non-supressed ion-chromatography [46], thin layer

chromatography (TLC) [47], HPTLC [48] gas liquid chromatography [49-51] and

ultra performance liquid chromatography (UPLC) [52] are found in the literature for

the assay of ATN in pharmaceuticals, when present in combined dosage forms.

Many other techniques including spectrofluorometry [53-55], atomic

absorption spectrometry [56], nuclear magnetic resonance spectrometry [57],

differential scanning calorimetry-thermogravimetry [58], kinetic mass spectrometry

[59], capillary electrophoresis [60], capillary zone electrophoresis [61-63] and

chemometric-assisted spectrophotometry [64,41] have been applied for the


28

determination of ATN in pharmaceuticals. But, majority of the techniques are devoted

to assay in combined dosage forms.

From the foregoing paragraphs, it is clear that titrimetric methods are indirect,

time consuming [7-11], and require higher acid concentrations [7]. The currently

available UV-spectrophotometric methods [18,19] for assay of ATN in single dosage

forms are not stability indicating. The reported visible spectrophotometric methods

are cumbersome due to heating step [20,21]. In addition, few methods are either less

sensitive or based on the measurement of less stable colored species

[8,11,20,24,25,26].

Other non-spectrophotometric methods require expensive instrumentation

which is less readily accessible to most laboratories in third world countries. There is,

therefore, a need for simple and sensitive methods for the assay of ATN.

Keeping in view the limitations of the reported titrimetric, UV and visible

spectrophotometric methods and the need for alternatives, some more titrimetric and

spectrophotometric methods including a UV- spectrophotometric method which is

stability-indicating were developed. The details are presented in this chapter.


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Section 2.1

DETERMINATION OF ATENOLOL AND ITS PHARMACEUTICAL

PREPARATIONS BY ACID –BASE TITRATION IN NON – AQUEOUS

MEDIUM

2.1.1 INTRODUCTION

Non-aqueous titrations are titrations carried out in the absence of water. These

titrations are based on the principle of Lowry-Bronsted theory and are particularly

useful for the assay of drugs that are very weakly acidic or basic, so weak in fact that

they will not ionize in aqueous conditions. Water, being an amphoteric compound,

acts to suppress the ionization of very weak acids and bases. All the apparatus and

glassware for a non-aqueous titration must be scrupulously dry, as even a drop of

water will ruin the whole assay [65]. The non-aqueous titrations have become of

considerable importance in pharmaceutical analysis and have been accepted by the

majority of modern pharmacopoeias as an official analytical method such as British

pharmacopoeia (2009) [5]. Non-aqueous titration is the most common titrimetric

procedure used in pharmacopoeial assays and serves a double purpose, as it is suitable

for the titration of very weak acids and bases and provides a solvent in which organic

compounds are soluble [66].

A large number of drugs are either weakly acidic (such as barbiturates,

phenytoin or sulfonamides), or weak bases (antihistamines, local anaesthetics,

morphine, etc.). The weak acids are usually titrated with tetrabutylammonium

hydroxide (N(Bu)4OH) or potassium methoxide (CH3OK) in dimethylformamide

(DMF) as solvent. Weak bases are dissolved in glacial acetic acid and titrated with

perchloric acid (HClO4). Acetic acid is a very weak proton acceptor and thus does not

compete effectively with weak bases for protons. Only very strong acids will

protonate acetic acid appreciably according to the following equation:

HA+CH3COOH CH3COOH2+ + A

Perchloric acid is the strongest of the common acids in acetic acid solution and

the titration medium usually used for non-aqueous titration of bases is perchloric acid

in acetic acid. These assays sometimes take some perfecting in terms of being able to

judge precisely the end-point. The end point of this type of titrations is detected either

This work has been published in Der Pharma Lettre, 2012, 4 (5): 1534-1540.


30

visually using indicators or potentiometrically using a potentiometer or pH meter. The

most popular indicator for the non-aqueous titration of bases is crystal violet and other

indicators used include p- naphtholbenzein, quinaldine red and malachite green [67].

In many cases, the color change is complex and the end point can be detected by

measuring the potential of a glass electrode using a potentiometer or pH meter with a

suitable indicator and reference electrodes.

Non-aqueous titration with acetous perchloric acid is used in the

pharmacopoeial assays of many compounds such as adrenaline, aciclovir, adenine,

adenosine, alanine, alcuronium chloride, alfuzosin hydrochloride, alprazolam,

bifonazole, domperidone maleate, gliclazide, loperamide oxide monohydrate,

nikethamide, pefloxacin mesilate and sulpiride, etc., to mention a few [5]. BP and EP

describe non aqueous titrimetry for the quantification of ATN but, require 0.1 M

HClO4 and involve only potentiometric titration. In this section, two non-aqueous

titrimetric procedures for the determination of ATN in tablets have been developed

and validated. The methods are based on the neutralization reaction of the secondary

amino group of ATN with acetous perchloric acid (0.005 M) as titrant in anhydrous

acetic acid medium. The end point was detected both visually using crystal violet as

indicator and potentiometrically using modified glass electrode-SCE electrode

system. The details about the reaction chemistry, method development and validation

are presented in this Section (2.1).

2.1.2 EXPERIMENTAL

2.1.2.1 Instrument

An Elico 120 digital pH meter provided with a combined glass-SCE electrode

system was used for potentiometric titration. The KCl of the salt bridge was replaced

with saturated solution of KCl in glacial acetic acid.

2.1.2.2 Reagents and materials

All chemicals used were of analytical reagent grade. All solutions were made

in glacial acetic acid (S. D. Fine Chem, Mumbai, India) unless mentioned otherwise.

Pharmaceutical grade atenolol (ATN) certified to be 99.89 % pure was gifted by Cipla

India Ltd., Mumbai, India, and was used as received. The following pharmaceutical

preparations were purchased from commercial sources in the local market and

subjected to analysis: Atenex-25 (25 mg ATN per tablet) from Zydas Healthcare, East

Sikkim, India; Atekind-50 (50 mg ATN per tablet) from Mankind Pharma Ltd., New


31

Delhi, India, and Aten-100 (100 mg ATN per tablet) from Zydas Healthcare, East

Sikkim, India.

Perchloric acid (0.005 M): The stock solution of (~0.1 M) perchloric acid (S. D. Fine

Chem, Mumbai, India) was standardized with pure potassium hydrogen phthalate and

crystal violet as indicator [68], and then diluted appropriately with glacial acetic acid

to get a working solution of 0.005 M perchloric acid.

Crystal violet indicator (0.1 %): Prepared by dissolving 50 mg of dye (S. D. Fine

Chem, Mumbai, India) in 50 mL of glacial acetic acid.

Standard ATN solution (1.5 mg mL-1): Stock standard solution containing 1.5 mg

mL-1 drug was prepared by dissolving the required amount of ATN (Cipla India Ltd.,

Mumbai, India) in glacial acetic acid.

2.1.2.3 Assay procedures

Visual titration

An aliquot of the drug solution containing 1.5-15.0 mg of ATN was measured

accurately and transferred into a clean and dry 100 mL titration flask and the total

volume was brought to 10 mL with glacial acetic acid. Two drops of crystal violet

indicator were added and titrated with standard 0.005 M perchloric acid to a blue

colour end point. An indicator blank titration was performed and corrections to the

sample titration were applied. The amount of the drug in the measured aliquot was

calculated from

Amount (mg) = VMwR/n

where V = volume of perchloric acid consumed (mL); Mw = relative molecular mass

of the drug; R = molarity of the perchloric acid and n = number of moles of perchloric

acid reacting with each mole of ATN.

Potentiometric titration

An aliquot of the standard drug solution equivalent to 1.5-15.0 mg of ATN

was measured accurately and transferred into a clean and dry 50 mL beaker and the

solution was diluted to 25 mL by adding glacial acetic acid. The combined glass-SCE

(modified) system was dipped in the solution. The content was stirred magnetically

and the titrant (0.005 M HClO4) was added from a microburette. Near the equivalence

point, titrant was added in 0.1 mL increments. After each addition of titrant, the

solution was stirred magnetically for 30 s and the steady potential (e.m.f) was noted.

The addition of titrant was continued until there was no significant change in potential


32

on further addition of titrant observed. The equivalence point was determined

graphically. The amount of the drug in the measured aliquot was calculated as

described under visual titration.

Procedure for tablets

Twenty tablets each containing 25, 50 or 100 mg of ATN were weighed

accurately and pulverized. An amount of powdered tablet equivalent to 150 mg of

ATN was transferred into a 100 mL calibrated flask and 60 mL of glacial acetic acid

was added. The content was shaken thoroughly for about 15-20 min, diluted to the

mark with glacial acetic acid, mixed well and filtered using a Whatman No. 42 filter

paper. The first 10 mL portion of the filtrate was discarded and a suitable aliquot was

taken and assayed by following the general procedures described for visual and

potentiometric end point detection.

Procedure for selectivity study

A placebo blank containing starch (10 mg), acacia (15 mg), hydroxyl cellulose

(10 mg), sodium citrate (10 mg), talc (20 mg), magnesium stearate (15 mg) and

sodium alginate (10 mg) was made and its solution was prepared as described under

“Procedure for tablets” and then 10 mg of the above mixture subjected to analysis by

the proposed methods.

To 100 mg of the placebo blank described above, 150 mg of ATN was added,

homogenized and the solution of the synthetic mixture was prepared in a 100 mL

calibration flask as described under “Procedure for tablets”. The filtrate was collected

and analyzed by following the procedures of both visual and potentiometric titrations.

2.1.3 RESULTS AND DISCUSSION

2.1.3.1 Chemistry

The methods are based on the principle that substances, which are weakly

basic in aqueous medium, when dissolved in non-aqueous solvents exhibit enhanced

basicity thus allowing their easy determination. In the present titrimetric methods, the

weakly basic property of ATN was enhanced due to the non-leveling effect of glacial

acetic acid and titrated with perchloric acid with visual and potentiometric end point

detection. Crystal violet gave satisfactory end point for the amounts of analyte and

concentrations of titrant employed. A steep rise in the potential was observed at the

equivalence point with potentiometric end point detection (Figure. 2.1.1). With both

methods of equivalence point detection, a reaction stoichiometry of 1:1 (drug:titrant)


33

was obtained which served as the basis for calculation. Using 0.005 M perchloric

acid, 1.5-15.0 mg of ATN was conveniently determined. The relationship between the

drug amount and the titration end point was examined. The linearity between two

parameters is apparent from the correlation coefficients of 0.9989 and 0.9997

obtained by the method of least squares for visual and potentiometric methods,

respectively. From this it is implied that the reaction between ATN and perchloric

acid proceeds stoichiometrically in the ratio 1:1 in the range studied.

(a) (b)

Figure.2.1.1 Potentiometric titration curves for 9 mg ATN Vs 0.005 M HClO4. (a) Normal titration curve and (b) First-derivative curve.

When a strong acid, such as perchloric acid, is dissolved in a weaker acid,

such as acetic acid, the acetic acid is forced to act as a base and accept a proton from

the perchloric acid forming an onium ion [65]. The formed onium ion (CH3COOH2+)

can very readily give up its proton to react with ATN, so basic properties of the drug

is enhanced and hence, titration between ATN and perchloric acid can often be

accurately carried out using acetic acid as solvent. The reactions occurring are as

follows:

HClO4 + CH3COOH CH3COOH2 ClO4

OH2N

NH

OH

OCH3COOH2

OH2N

NH2

OH

O

CH3COOH

0 2 4 6 8 10350

400

450

500

550

600

emf i

n m

V

Volume of 0.005 M HClO4, mL0 2 4 6 8 10

0

50

100

150

200

dE/d

V

Volume of 0.005 M HClO4, mL


34

Overall, the reaction is:

OH2N

NH

OH

OHClO4

OH2N

NH2

OH

OClO4

2.1.3.2 Method validation

The method validation was done according to the present ICH guidelines [69].

Accuracy and precision

The accuracy and precision of the methods was evaluated in terms of

intermediate precision (intra-day and inter-day). Three different amounts of ATN

within the range of study in both methods were analyzed in seven replicates during

the same day (intra-day precision) and five consecutive days (inter-day precision).

The percentage relative standard deviation (RSD %) values were ≤ 1.52 % (intra-day)

and ≤ 2.35 % (inter-day) indicating high precision of the methods. Also, the accuracy

of the methods was evaluated as percentage relative error (RE %) and from the results

shown in Table 2.1.1, it is clear that the accuracy is satisfactory (RE ≤ 1.47%).

Table 2.1.1 Results of intra-day and inter-day accuracy and precision study.

Method

ATN

taken, mg

Intra-day (n=7)

Inter-day (n=5)

ATN founda,

mg RE, %

RSD, %

ATN founda,

mg RE, %

RSD, %

Visual titrimetry

6.00 9.00 12.0

6.07 9.06

12.11

1.17 0.67 0.92

1.52 1.29 0. 90

6.08 9.16

12.14

1.33 1.78 1.17

1.76 2.26 1.52

Potentiometric titrimetry

6.00 9.00 12.0

5.99 8.95

11.91

0.17 0.56 0.75

1.19 0.80 0.49

6.10 9.12

12.23

1.67 1.33 1.92

2.24 1.56 2.35

aMean value of n determinations, RE: relative error, RSD: relative standard deviation.

Selectivity

The selectivity of the proposed methods was determined by placebo blank and

synthetic mixture analyses. In the analysis of placebo blank solution, the titre value in

both the cases was equal to the titre value of blank which revealed no interference. To

assess the role of the inactive ingredients on the assay of ATN, the general procedure

was followed by taking the synthetic mixture extract at three different concentrations


35

of ATN (6, 9 and 12 µg mL-1 in both method A and method B). The percentage

recovery values obtained were in the range 97.65 – 101.3% with RSD < 1.7 % with

clear indication of non-interference by the inactive ingredients in the assay of ATN.

Ruggedness of the methods

Method ruggedness was expressed as the RSD of the same procedure applied

by four different analysts as well as using four different burettes. The inter-analysts

RSD were ≤ 0.72 % whereas the inter-burettes RSD for the same ATN amounts

ranged from 0.38 – 0.75 % suggesting that the developed methods were rugged. The

results are shown in Table 2.1.2.

Table 2.1.2 Results of method ruggedness study.

Method ATN

taken, mg

Ruggedness Inter-analysts

(% RSD) (n=4)

Inter-burettes (% RSD)

(n=4)

Visual end point detection

6.00 9.00 12.0

0.58 0.43 0.72

0.75 0.46 0.38

Potentiometric end point detection

6.00 9.00 12.0

0.32 0.56 0.28

0.46 0.72 0.67

Application to analysis of tablets containing ATN

The described titrimetric procedures were applied to the determination of ATN

in its tablets. The results obtained (Table 2.1.3) were statistically compared with the

official IP method [6]. The official UV-spectrophotometric method involved the

measurement of the absorbance of methanolic ATN tablet solution at 275 nm. The

results obtained by the proposed methods agreed well with those of official method

and with the label claim. The results were also compared statistically by a Student’s t-

test for accuracy and by a variance F-test for precision with those of the official

method at 95 % confidence level as summarized in Table 2.1.3. The results showed

that the calculated t-and F-values did not exceed the tabulated values inferring that

proposed methods are as accurate and precise as the official method.


36

Table 2.1.3 Results of assay of tablets and statistical comparison with the official method.

Recovery studies

Accuracy and the reliability of the methods were further ascertained by

performing recovery experiments. 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 methods. Each test was repeated three times. The results compiled in

Table 2.1.4 show that recoveries were in the range from 99.56 to 101.2 % indicating

that commonly added excipients to tablets did not interfere in the determination.

Table 2.1.4 Results of recovery study via standard addition method.

Visual titrimetry Potentiometric titrimetry

Tablet studied

ATN in tablet

extract, mg

Pure ATN

added, mg

Total ATN

found, mg

Pure ATN recovered*

%

ATN in tablet

extract, mg

Pure ATN

added, mg

Total ATN

found, mg

Pure ATN recovered*

%

Atenex 25

2.98 2.98 2.98

3.0 6.0 9.0

6.01 9.05

12.04

101.0±0.21 101.2±0.21 100.7±0.15

3.03 3.03 3.03

3.0 6.0 9.0

6.04 9.09

11.99

100.3±0.11 101.0±0.15 99.56±0.10

Atekind 50

2.97 2.97 2.97

3.0 6.0 9.0

5.98 8.96

12.00

100.3±0.14 99.83±0.19 100.3±0.16

3.0 3.0 3.0

3.0 6.0 9.0

6.01 9.05

11.98

100.3±0.10 100.8±0.12 99.78±0.09

Aten 100

3.01 3.01 3.01

3.0 6.0 9.0

6.03 9.07

12.00

100.7±0.11 101.0±0.12 99.89±0.11

3.04 3.04 3.04

3.0 6.0 9.0

6.07 9.07

12.15

101.0±0.21 100.5±0.31 101.2±0.21

*Mean value of three determinations.

Brand name

Label claim,

mg/tablet

Found* (Percent of label claim ± SD)

Official method

Proposed methods Visual titrimetry

Potentiometric titrimetry

Atenex 25 25 100.3±0.58 99.3±1.08 t=1.82 F=3.47

101.1±1.05 t=1.49 F=3.28

Atekind 50 50 99.67±0.67 98.89±0.94 t=1.51 F=1.97

100.2±0.78 t=1.17 F=1.36

Aten 100 100 100.6±0.82 100.4±1.11 t=0.32 F=1.83

101.3±1.28 t=1.03 F=2.44

*Average of five determinations. Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95% confidence level is 6.39.


37

Section 2.2

SPECTROPHOTOMETRIC DETERMINATION OF ATENOLOL IN

PHARMACEUTICALS THROUGH CHARGE-TRANSFER COMPLEX

FORMATION REACTION

2.2.1 INTRODUCTION

The molecular interaction between electron donors and acceptors are generally

associated with the formation of intensely colored charge-transfer complex [70] which

absorbs radiation in the visible region. Amines are excellent electron donors and

quinines are good electron acceptors [71-74]. The formation of outer complexes or

electron donor-acceptor (EDA) complexes between quinone and amines is well

known. Since both the donor and the acceptor are often very reactive, chemical

reaction can occur between them. Charge transfer phenomenon was introduced first

by Mulliken [75] and widely discussed by Foster [76] 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 [77].

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 or 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 d - or p -acceptors [78].

Quinones have long been known to react with amines to give coloured

products. The color reaction with p-benzoquinone was first reported by Fouery [79] This work has been published in Acta Poloniae Pharmaceutica-Drug Research, 2012, 69(2): 213-223.


38

and later developed into a quantitative method [80-82]. In addition, a number of

substituted quinones are known to form C-T complexes with amines and amine-

containing substances which have permitted the determination of many

pharmaceutical compounds containing amino group such as mefenamic acid [83],

some psychotropic phenothiazine drugs [84], certain cephalosporins [85], some

pharmaceutical amides [86], some antibacterial drugs [87], some corticosteroid drugs

[88], certain cardiovascular drugs [89], guanidino drugs [90], some pharmaceutical

piperazine derivatives [91], cyproheptadine HCl, methdilazine HCl and promethazine

theoclate [92], nizatidine, haloperidol and droperidol [93], clobetasol propionate,

halobetasol propionate and quinagolide HCl [94], amodiaquine HCl, chloroquine

phosphate and primaquine phosphate [95], mebrophenhydramine HCl and

hydroxyzine HCl [96], astemizole and loratadine [97], cetirizine [98], isoxsuprine

HCl [99], zidovudine [100], amoxycillin [101], clozapine [102], ondansetron HCl

[103], praziquantel [104], ciprofloxacin [105], sulbutamol [106], oxamniquine [107],

metoclopramide [108], disopyramide [109], ranitidine HCl [110], nortriptyline [111],

barbiturates and phenytoin [112], perindopril [113], ganciclovir [114], ketamine HCl

[115], diethylcarbamazine citrate [116], terfenadine [117], loperamide HCl [118],

moclobemide [119], famotidine [120], diclofenac sodium [121] ketorolac

tromethamine [122] and bupropion [123] etc., to mention a few.

Similarly, aromatic nitro compounds also act as electron acceptors. Interaction

between nitrophenols (picric acid, 2,4-dinitrophenol) and compounds containing

amine groups may give rise to the formation of charge transfer complex where a

proton is transferred from nitrophenols to the amine, as well as to charge-transfer

interaction via partial transfer of charge from n-lone pair of the amine to the oxygen-

π* of the nitro-group [124]. Modern understanding of C-T interaction involves the

partial transfer of charge from the highest occupied molecular orbital (HOMO) to the

lowest unoccupied molecular orbital (LUMO) of the C-T complex [70]. Specifically,

the nature of interaction between PA or DNP and ATN is presumed to be of charge-

transfer type (i.e., transfer of electronic charge from the amine donor to nitrophenol

acceptors). Few pharmaceutical compounds [125-129] were quantitatively analyzed

by utilizing nitrophenols as electron acceptors.

From the literature survey of the methods for the assay of atenolol presented in

Section 2.0.2 and from the foregoing paragraphs, it is clear that atenolol has been

assayed by visible spectrophotometry based on charge-transfer complex formation


39

reaction using chloranilic acid as a π-acceptor. The author has studied the reaction

between ATN and DDQ in acetonitrile medium and the reaction between ATN and

nitrophenols in dichloromethane medium based on which three simple and moderately

sensitive methods have been developed for the determination of ATN in tablets. The

details regarding the method development and method validation are presented in this

Section.

2.2.2 EXPERIMENTAL

2.2.2.1 Instrument

A Systronics model 106 digital spectrophotometer (Systronics, Ahmedabad,

Gujarat, India) provided with 1 cm matched quartz cells was used for all absorbance

measurements.


All reagents used were of analytical reagent grade and HPLC grade organic

solvents were used throughout the investigation.

2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ): A 0.1 % (w/v) DDQ solution

was prepared by dissolving 100 mg of the chemical (S.D. Fine Chem Ltd, Mumbai) in

1,4 dioxane and diluted to the mark with same solvent in 100 mL calibrated flask. It

was prepared afresh just before use.

2,6-Dinitrophenol (DNP): A 0.1 % (w/v) solution was prepared by dissolving 100

mg of the chemical (S.D. Fine Chem Ltd, Mumbai) in dichloromethane and made up

to the mark in a 100 mL calibrated flask with the same solvent. This solution was kept

in the dark when not in use.

Picric acid (PA): A 0.05 % (w/v) solution was prepared by dissolving 50 mg of the

chemical (S.D. Fine Chem Ltd, Mumbai) in dichloromethane and made up to the

mark in a 100 mL calibrated flask with the same solvent.

Standard ATN solution: A stock standard solution containing 100 µg mL-1 ATN was

prepared by dissolving 10 mg of pure drug in acetonitrile and diluting to the mark in a

100 mL calibrated flask with the same solvent. This was diluted appropriately with

acetonitrile to get the working concentrations of 60, 40 and 30 µg mL-1 ATN for

methods using DDQ, DNP and PA, respectively.

The pharmaceutical grade ATN and tablets used in this study are those

mentioned in the Section 2.1.2.2.


40


Method A (using DDQ)

Varying aliquots (0.25– 4.0 mL) of a standard ATN solution (60 µg mL-1)

were accurately transferred into a series of 5 mL calibrated flasks using a micro

burette and the total volume in each flask was brought to 4 mL by adding adequate

quantity of acetonitrile. To each flask, 1 mL of 0.1% DDQ solution was added, the

content was mixed well and the absorbance was measured at 590 nm against a reagent

blank similarly prepared without adding ATN solution.

Method B (using DNP)

Different aliquots (0.25–3.0 mL) of standard ATN solution (40 µg mL-1) were

accurately transferred into a series of 5 mL calibration flasks as described above. One

milliliter of 0.1% DNP solution was added to each flask and diluted to volume with

dichloromethane. The content was mixed well and the absorbance was measured at

420 nm against a reagent blank.

Method C (using PA)

Aliquots (0.25–3.0 mL) of a standard ATN (30 µg mL-1) solution were

accurately transferred into a series of 5 mL calibration flasks and the total volume was

brought to 3 mL by adding acetonitrile. To each flask, 1 mL of 0.05% PA solution

was added and the solution made up to volume with dichloromethane. The content

was mixed well and the absorbance was measured at 420 nm against a reagent blank

after 5 min.

Standard graph was prepared by plotting the absorbance versus ATN

concentration, and the concentration of the unknown was read from the calibration

graph or computed from the respective regression equation.


Twenty tablets were weighed and pulverized. An amount of tablet powder

equivalent to 10 mg ATN was extracted with three 30 mL portions of acetonitrile. The

extracts were filtered using Whatmann No 42 filter paper; the filtrate was collected in

a 100 mL calibrated flask and diluted to volume with acetonitrile. A suitable aliquot

of the filtrate (100 µg mL-1 ATN) was diluted to get the working concentrations of 60,

40 and 30 µg mL-1 ATN for analysis by methods A, B and C, respectively, as

described above.


41


A placebo blank was prepared as described under Section 2.1.2.3, and then 10

mg placebo blank extracted in acetonitrile or dichloromethane was analyzed as done

in “Procedure for tablets”.

A synthetic mixture was prepared by adding pure ATN (50 mg) to 30 mg of

the above mentioned placebo blank and the mixture was homogenized. Synthetic

mixture containing 10 mg of ATN was weighed and its solution was prepared as

under “Procedure for tablets”. Two different aliquots were subjected to analysis by

the general procedure and the concentration of ATN was found from the calibration

graph or from the regression equation.


2.2.3.1 Absorption spectra

The reaction of ATN as n-electron donor and the π-acceptors such as DDQ,

DNP and PA results in the formation of C-T complexes. The absorption spectrum of

ATN-DDQ charge-transfer complex exhibited three maxima at 590, 550 and 460 nm

(Figure 2.2.1). These bands can be attributed to the formation of DDQ radical anions

arising from the complete transfer of n-electrons from donor to acceptor moieties in

acetonitrile. The absorption band at 590 nm was selected as analytical wavelength

keeping in view the sensitivity of the reaction product and blank absorbance.

Similarly, the reaction of ATN with DNP or PA results in the formation of an intense

yellow product which exhibits absorption maxima at 420 nm (Figure 2.2.2).

Figure 2.2.1 Absorption spectra of a) ATN-DDQ charge-transfer complex: b) blank.

400 450 500 550 600 6500.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

b

a

Abs

orba

nce

Wavelength, nm

ATN-DDQ C-T complex Blank


42

(a) (b)

Figure 2.2.2 Absorption spectra of a) ATN-DNP C-T complex (method B); b) ATN-PA C-T complex (method C).

2.2.3.2 Chemistry

The chemistry used in method A is based on the reaction of the secondary

aliphatic amine of ATN as n-donor with DDQ as π-acceptor to form charge transfer

complex which subsequently dissociates into radical anions depending on the polarity

of the solvent used [123]. In polar solvents, such as acetonitrile, electron transfer from

the donor to the acceptor moiety takes place with the formation of intensely colored

radical anions (Scheme 2.2.1) [130], according to the following equation:

When an amine is combined 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 phenolate by proton-

transfer, and the latter, to a molecular compound by charge-transfer [131]. Based on

this, the mechanism for method B and method C can be discussed in terms of transfer

of electronic charge from the secondary aliphatic amine of ATN, an electron-rich

molecule (a Lewis-base donor), to the ring of DNP or PA, an electron-deficient

molecule (a Lewis-acid acceptor), and at the same time the proton of the hydroxyl

group of DNP or PA will transfer to the secondary amine of ATN (Scheme 2.2.2).

The explanation for the produced color in method B and method C lies in the

formation of complexes between the pairs of molecules ATN-DNP and ATN-PA, and

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

Abs

orba

nce

Wavelength, nm

ATN-DNP C-T complex Blank

380 400 420 440 460 480 500-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

Wavelength, nm

ATN-PA C-T complex Blank

D + A D A

charge transfer complex

Polar solventD + A

Colored radical anion


43

this complex formation leads to the production of two new molecular orbitals and,

consequently, to a new electronic transition [132].

O

O

Cl

ClNC

NC

O

O

Cl

ClNC

NC

O

O

Cl

ClNC

NC

H2N

O O NHOH CH3

CH3

H2N

OO N

HOHCH3

CH3

Atenolol(ATN) DDQ

CT complexation

H2N

OO N

OHCH3

CH3H

DA complex

DDQ radical anion the measured species

Scheme 2.2.1 The probable pathway for the formation of ATN-DDQ C-T complex.

OHR1

R2

R3

H2N

OO N

HOHCH3

CH3H2N

OO N

HOHCH3

CH3

OHR1

R2

R3

OR1

R2

R3

H2N

OO NH

HOCH3

CH3

Atenolol (ATN) DNP or PA

CT complexthe measured

species

CT complexation

For DNP: R1=R2= NO2 and R3=H For PA: R1=R2=R3= NO2

Scheme 2.2.2 The probable pathway for the formation of ATN-DNP and ATN-PA C-T

complexes.


44

2.2.3.3 Optimization of experimental variables

Many experimental variables which were found to affect the color intensity

and stability of the resulting complexes were optimized to achieve maximum

sensitivity and adherence to Beer’s law.

Effect of reagent concentration

The optimum concentration of the reagent required to achieve maximum

sensitivity for the color developed in each method was ascertained by adding different

amounts of the reagent DDQ, DNP or PA to a fixed concentration of ATN. The

results showed that 1.0 mL each of 0.1 % DDQ, 0.1 % DNP and 0.05 % PA solution

was optimum for the production of maximum and reproducible color intensity

(Figure 2.2.3).

Figure 2.2.3 Effect of volume of reagents on the formation of (ATN-DDQ complex, 24

µg mL-1 ATN), (ATN-DNP complex, 12 µg mL-1 ATN) and (ATN-PA complex, 9 µg mL-1 ATN).

Effect of solvent

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

1,4-dioxane, chloroform, acetonitrile, acetone, t-butanol,2-propanol and

dichloromethane, and the reaction of ATN with DDQ, DNP or PA was followed. In

method A, as shown in Figure 2.2.4, acetonitrile was best suited for preparation of

DDQ solution. The dichloromethane solvent was found to be the ideal solvent for

preparation of both DNP and PA for method B and method C, respectively (Figure

2.2.4). Similarly, the effect of the diluting solvent was studied for all methods and the

results showed that the ideal diluting solvent to achieve maximum sensitivity was

acetonitrile in method A and dichloromethane in method B and method C.

0.5 1.0 1.5 2.0 2.5 3.00.0

0.1

0.2

0.3

0.4

0.5

Abs

orba

nce

Volume of reagent, mL

ATN-DNP CT-Complex DNP blank ATN-DDQ CT-Complex DDQ blank ATN-PA CT-Complex PA blank


45

Figure 2.2.4 Effect of solvents on the formation of (ATN-DDQ complex, 24 µg mL-1

ATN), (ATN-DNP complex, 20 µg mL-1 ATN) and (ATN-PA complex, 9 µg mL-1 ATN).

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 ATN solution at room

temperature. The reaction of ATN with DDQ in method A and DNP in method B was

instantaneous while complete color development was attained after 5 min with PA.

The absorbance of the resulting C-T complexes remained stable for at least 45 min for

method A and for more than 24 hrs for method B and method C.


The proposed methods were validated for linearity, sensitivity, selectivity,

accuracy, precision, robustness, ruggedness and recovery according to the current

ICH guidelines [69].

Linearity and sensitivity

Under the optimum conditions a linear relation was obtained between

absorbance and concentration of ATN (Figure 2.2.5) in the ranges given in Table

2.2.1. The calibration graph in each instance is described by the equation: Y = a + b

X, (where Y = absorbance, a = intercept, b = slope and X = concentration in µg mL-1).

The correlation coefficient, intercept and slope for the calibration data are

summarized in Table 2.2.1. Sensitivity parameters such as apparent molar

absorptivity and Sandell sensitivity values, the limits of detection (LOD) and

1,4-

Dio

xan

Chl

orof

orm

Acet

onitr

ile

Acet

one

t-But

anol

2-Pr

opan

ol

Dic

hlor

omet

hane

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Abs

orba

nce

Solvents

DDQ-blank ATN-DDQ CTcomplex DNP -blank ATN-DNP CTcomplex PA -blank ATN-PA CTcomplex


46

quantification (LOQ) are calculated as per the current ICH guidelines [69] and

compiled in Table 2.2.1. LOD and LOQ were calculated according to the same

guidelines using the following formulae:

SLOD

3.3 &

SLOQ

10

where σ is the standard deviation of six reagent blank determinations and s is the

slope of the calibration curve.

Figure 2.2.5 Calibration curves


In order to determine the accuracy and precision of the proposed methods,

pure drug (ATN) solution at three different concentration levels (within the working

range) were prepared and analyzed in seven replicates during the same day (intra-day

precision) and on five consecutive days (inter-day precision) and the results are

presented in Table 2.2.2. The percentage relative error (RE %) was ≤ 2.85 which

indicate that the accuracy of the methods is satisfactory. Percentage relative standard

0 10 20 30 40 500.0

0.2

0.4

0.6

0.8

1.0 Method A

Abs

orba

nce

Concentration of ATN, µg mL-1

0 5 10 15 20 250.0

0.2

0.4

0.6

0.8

1.0 Method B

Abs

orab

nce


0 2 4 6 8 10 12 14 16 18 200.0

0.2

0.4

0.6

0.8Method C

Abs

orba

nce



47

deviation (RSD %) for intra-day was ≤ 1.31 and for inter-day it was ≤ 2.01 indicating

repeatability and usefulness of the proposed methods in the routine analysis.

Table 2.2.1 Regression and sensitivity parameters.

Parameter Method A Method B Method C max, nm 590 420 420 Beer’s law limits (µg mL-1) 3-48 2-24 1.5-18 Molar absorptivity (L mol-1 cm-1) 5.41×103 1.13×104 1.13×104 Sandell sensitivity* (µg cm-2) 0.0493 0.0236 0.0236 Limit of detection (µg mL-1) 0.26 0.23 0.23 Limit of quantification (µg mL-1) 0.80 0.70 0.69 Regression equation, Y** Intercept, (a)

0.0091

0.0387

-0.0120

Slope, (b) 0.0197 0.0387 0.0446 Correlation coefficient (r) 0.9992 0.9997 0.9999 Standard deviation of intercept (Sa) 0.01078 0.00650 0.00287 Standard deviation of slope (Sb) 0.00040 0.00045 0.00026 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. ** bXaY , where Y is the absorbance, a is the intercept, b is the slope and X is the concentration in µg mL-1.


Selectivity

The selectivity of the proposed methods for the analysis of ATN was

evaluated by placebo blank and synthetic mixture analyses. The procedures were

applied to the analysis of placebo blank and the resulting absorbance readings in all

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

Method ATN taken

(µg mL-1)

Intra-day (n = 7) Inter-day (n = 5) ATN

founda (µg mL-1)

%RSDb %REc ATN founda

(µg mL-1)

%RSDb

%REc

Method A

12.0 11.83 1.31 1.41 12.24 1.56 2.04 24.0 23.81 0.78 0.81 24.42 0.97 1.75 36.0 35.65 1.06 0.98 36.66 1.28 1.84

Method B

8.00 8.19 1.23 2.37 8.23 1.66 2.85 12.0 12.11 1.01 0.90 12.29 2.01 2.42 16.0 16.17 0.71 1.07 16.38 1.85 2.39

Method C

6.00 5.91 0.57 1.47 6.13 1.74 2.18 9.00 9.14 0.25 1.55 9.23 1.36 2.56 12.0 11.79 0.82 1.72 12.29 1.54 2.38

a. Mean value of five determinations; b. Relative standard deviation (%); c. Relative error (%).


48

yielded percent recoveries of 98.3±2.13, 99.1±1.76 and 98.9±1.91 (n=5) for method

A, method B and method C, respectively. The results of this study showed that the

inactive ingredients did not interfere in the assay indicating the high selectivity of the

proposed method and applicability to use for routine determination in pure and in

tablets form.

Robustness and ruggedness

To evaluate the robustness of the methods, two important experimental

variables volume of reagent in all the methods and reaction time in method C, 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 2.2.3 and

indicated that the proposed methods are robust. Method ruggedness was evaluated by

performing the analysis following the recommended procedures by three different

analysts and on three different spectrophotometers by the same analyst. From the

%RSD values presented in Table 2.2.3, one can conclude that the proposed methods

are rugged.

Table 2.2.3 Results of robustness and ruggedness study.


The proposed methods were applied to the determination of ATN in three

brands of tablets and the results are compiled in Table 2.2.4. The results obtained

Method

ATN

taken, µg mL-1

Method robustness Method ruggedness Parameters altered

Reagent volume, mLa

RSD, % (n = 3)

Reaction timeb

RSD, % (n = 3)

Inter-analysts RSD, % (n = 3)

Inter-instruments

RSD, % (n = 3)

A

12.0 0.56 1.24 2.48 24.0 1.24 0.85 2.75 36.0 1.35 0.63 2.16

B

8.00 1.08 1.45 1.67 12.0 1.26 1.38 2.39 16.0 1.48 1.13 1.86

C

6.00 0.79 1.45 0.75 2.75 9.00 0.57 1.03 1.36 2.18 12.0 1.14 0.94 1.12 1.67

aIn all methods, the volume of reagent was 0.8, 1.0 and 1.2 mL. b. The reaction was instantaneous aIn all methods, the volume of reagent was 0.8, 1.0 and 1.2 mL. b. The reaction was instantaneous in methods A and B and the reaction time was 4, 5 and 6 min for method C.


49

were statistically compared with those obtained by the official IP method [6] by

applying the Student’s t-test for accuracy and F-test for precision at 95% confidence

level. As can be seen from the Table 2.2.4, the calculated t- and F- values at 95%

confidence level did not exceed the tabulated values of 2.78 and 6.39, respectively,

for four degrees of freedom. This indicates that there are no significant differences

between the proposed methods and the official method with respect to accuracy and

precision.


Recovery studies

The accuracy and validity of the proposed methods were further ascertained

by performing recovery studies. Pre-analyzed tablet powder was spiked with pure

ATN at three concentration levels (50, 100 and 150 % of that in tablet powder) and

the total was analyzed by the proposed methods. The results of this study are

presented in Table 2.2.5 and indicate that the excipients present in the tablets did not

interfere in the assay.

Tablet Brand name

Label claim

mg/tablet

Found (Percent of label claim ±SD)a

Official method

Proposed methods Method A Method B Method C

Atenex-25

25

100.3±0.58 99.06 ± 1.17 t = 2.12 F= 4.07

99.82± 1.37 t = 0.72 F = 5.58

98.97± 1.32 t = 2.00 F = 5.18

Atekind-50

50

99.67 ± 0.67 98.21± 1.11 t = 2.52 F = 2.74

101.1 ± 1.06 t = 2.55 F= 2.50

98.31 ±1.18 t = 2.24 F= 3.10

Aten-100 100 100.6±0.82 100.9±1.91 t = 0.32 F = 5.43

101.5± 1.06 t = 1.5 F= 1.67

99.32±1.71 t = 1.51 F= 4.35

aMean value of five determinations. Tabulated t-value at the 95% confidence level is 2.78. Tabulated F-value at the 95% confidence level is 6.39.


50


Tablets studied

Method A Method B Method C

ATN in tablets, µg mL-1

Pure ATN

added, µg mL-1

Total found,

µg mL-1

Pure ATN recovered*, Percent±SD


Pure ATN

added, µg mL-1

Total found,

µg mL-1


ATN in tablets

µg mL-1

Pure ATN

added, µg mL-1

Total found,

µg mL-1


Atenex 25

11.9 11.9 11.9

6.00 12.0 18.0

18.1 24.5 30.5

104.4 ±2.77 105.5±0.57 103.2±2.83

7.99 7.99 7.99

4.00 8.00 12.0

12.16 16.43 20.77

104.3±2.21 105.5±2.65 106.5±1.11

5.94 5.94 5.94

3.00 6.00 9.00

9.03 12.21 15.23

103.0±2.92 104.5±2.70 103.2±1.05

Atekind

50

11.8 11.8 11.8

6.00 12.0 18.0

18.0 24.1 30.7

103.5±2.76 102.8±2.84 105.0±1.51

8.09 8.09 8.09

4.00 8.00 12.0

12.40 16.75 21.19

107.8±2.51 108.2±2.76 109.2±1.40

5.90 5.90 5.90

3.00 6.00 9.00

9.04 12.33 15.37

104.5±2.16 107.2±2.07 105.2±1.80

Aten 100

12.1 12.1 12.1

6.00 12.0 18.0

18.4 24.5 30.9

103.9±2.88 103.2±2.15 104.4±0.77

8.12 8.12 8.12

4.00 8.00 12.0

12.49 16.61 20.61

109.3±1.51 106.1±2.75 104.1 ±1.44

5.96 5.96 5.96

3.00 6.00 9.00

9.08 12.50 15.85

104.0±2.37 109.1±1.76 110.0±1.91

* Mean value of three determinations.


51

Section 2.3

SPECTROPHOTOMETRIC DETERMINATION OF ATENOLOL IN

PHARMACEUTICAL FORMULATIONS USING BROMATE-BROMIDE

MIXTURE AS AN ECO-FRIENDLY REAGENT

2.3.1 INTRODUCTION

In acid solution, potassium bromate is a strong oxidant (E0=1.52V) which is

reduced to bromide:

BrO3- + 6H+ +6e Br- + 3H2O

Bromate in the presence of excess bromide yields free bromine.

BrO3- + 5Br- + 6H+ 3Br2 + 3H2O

This reaction occurs in the presence of large excess of bromide ion in acid medium.

Liquid bromine is corrosive to human tissue in a liquid state and its vapors

irritate eyes and throat. Bromine vapors are very toxic with inhalation [133]. Thus the

stable bromate-bromide solution serves for the extemporaneous preparation of a

standard solution of bromine. An acidified mixture of bromate and bromide behaves

as an equivalent solution of bromine. Bromate-bromide mixture is an eco-friendly

green brominating agent [134].

The oxidising action of bromate appears to have been noted by Balard [135],

but the first application of bromate as a titrimetric reagent was due to Kopperchaar

[136] who used it in his well known bromination procedure for the determination of

phenol. Kratscher [137] first recommended the use of bromate as an oxidimetric

reagent. Since then the reagent in combination with bromide has found wide

application in chemical analysis [138-140] including substances of pharmaceutical

importance.

Bromate–bromide mixture in acid medium has been used extensively for

titrimetric and/or spectrophotometric determination of many pharmaceutical

compounds such as atenolol [141], cyproheptadine [142], lamivudine [143],

zidovudine [144], astemizole [145], felodipine [146], stavudine [147], amoxicillin

[148], frusemide [149], simvastatin [150,151], escitalopram oxalate [152],

sumatriptan succinate [153], gatifloxacin [154], citalopram hydrobromide [155],

This work has been published in Journal of Analytical Methods in Chemistry, 2012, doi:10.1155/2012/810156, 12 pages.


52

lansoprazole [156], propranolol HCl [157,158] and captopril [159] etc., to mention a

few.

Many dyes are irreversibly oxidised/destroyed to colorless products by

oxidising agents in acid medium [139] and this observation has been exploited for the

indirect spectrophotometric determination of many oxidisable pharmaceuticals [160-

164]. This bleaching action has successfully been utilized for the indirect

spectrophotometric assay of wide range of pharmaceuticals.

In this section, more sensitive indirect spectrophotometric methods are

described for the determination of ATN using bromate-bromide mixture and two

dyes, meta-cresol purple and erioglaucine as reagents. The methods are based on the

oxidation of ATN by the bromine generated in situ by the action of the acid on the

bromate–bromide mixture followed by the determination of unreacted bromine by

reacting with a fixed amount of either MCP and measuring the absorbance at 540 nm

(method A) and 445 nm (method B) or EGC and measuring the absorbance at 630 nm

(method C).

2.3.2 EXPERIMENTAL

2.3.2.1 Instrument

The instrument used for absorbance measurements was the same as described

in Section 2.2.2.1.


All chemicals and reagents used were of analytical or pharmaceutical grade.

Distilled water was used to prepare the solutions.

Bromate-bromide mixture (40, 80 and 18 µg mL-1): A stock standard bromate-

bromide mixture solution equivalent to 500 µg mL-1 KBrO3 was prepared by

dissolving accurately weighed 50 mg of KBrO3 (S. D. Fine Chem. Ltd., Mumbai,

India) and 0.5 g of KBr (Merck, Mumbai, India) in water and diluted to the mark in a

100 mL calibrated flask. The stock solution was diluted appropriately with water to

get the working concentrations of 40, 80 and 18 µg mL-1 KBrO3 for use in method A,

method B and method C, respectively.

Meta-cresol purple solution (80 and 200 µg mL-1): A 400 µg mL-1 stock solution

was first prepared by dissolving 40 mg of dye (Loba Chemie, Mumbai, India) in 2 mL

of 0.1 M NaOH and diluted to volume with water in a 100 mL calibrated flask. The


53

solution (400 µg mL-1) was diluted further with water to get the working

concentrations of 80 µg mL-1 and 200 µg mL-1 MCP solutions.

Erioglaucine solution (300 µg mL-1): The solution was prepared by dissolving 30

mg of dye (Loba Chemie, Mumbai, India) in water and diluting to the mark with

water in a 100 mL calibrated flask.

Hydrochloric acid (5 M and 1M): The solutions were prepared by appropriate

dilution of concentrated hydrochloric acid (S. D. Fine Chem. Ltd., Mumbai, India. Sp.

gr. 1.18) with water.

Standard ATN solution: A stock standard solution equivalent to 200 µg mL-1 ATN

was prepared by dissolving accurately weighed 50 mg of pure drug with water in a

250 mL calibrated flask. This stock solution was diluted appropriately with water to

get the working concentrations of 40 µg mL-1 for use in methods A and C, and 80 µg

mL-1 for use in method B.

The pharmaceutical grade ATN and tablets used in this study are those

mentioned in the Section 2.1.2.2.


Method A (measuring MCP in acid medium)

Different aliquots (0.25- 5.0 mL) of standard ATN solution (40 µg mL-1) were

accurately transferred into a series of 10 mL calibrated flasks using micro burette and

the total volume was adjusted to 5.0 mL by adding requisite volume of water. To each

flask, 2 mL of 5 M HCl was added followed by 1 mL of bromate-bromide mixture (40

µg mL-1 in KBrO3). The content was mixed well and the flasks were allowed to stand

for 15 min with occasional shaking. Then, 1 mL of 80 µg mL-1 MCP was added to

each flask, diluted to the mark with water, mixed well and the absorbance of each

solution was measured at 540 nm against a reagent blank after 5 min.

Method B (measuring brominated product of MCP)

Varying aliquots (0.25-5.0 mL) of a standard solution (80 µg mL-1ATN) were

accurately measured into a series of 10 mL calibrated flasks and the total volume was

brought to 5 mL by adding water. To each flask were added 2 mL of 5 M HCl and 1

mL of KBrO3-KBr solution (80 µg mL-1 in KBrO3). The content of each flask was

mixed well and kept aside for 10 min with occasional swirling. At last, 1 mL of 200

µg mL-1 MCP solution was added to each flask and diluting up to the mark with


54

water. The absorbance of each solution was measured after 5 min at 445 nm against

water.

Method C (using EGC)

Aliquots (0.25-2.0 mL) of a standard ATN (40 µg mL-1) solution were

accurately transferred into a series of 10 mL calibrated flasks and the total volume

was adjusted to 2.0 mL with water. To each flask, 5 mL of 1 M HCl was added

followed by 1.0 mL of bromate-bromide mixture (18 µg mL-1, in KBrO3). The content

was mixed and the flasks were let stand for 10 min with occasional shaking followed

by addition of 1 mL of 300 µg mL-1 EGC to each flask. The solutions were diluted to

the mark with water, mixed well and the absorbance of each solution was measured at

630 nm after 5 min against a reagent blank.





Tablet extract equivalent to 200 µg mL-1 ATN was prepared as described in

Section 2.1.2.3. A suitable aliquot of the extract (200 µg mL-1 ATN) was diluted to

get the working concentrations of 40 µg mL-1 ATN for the assay by methods A and C,

and 80 µg mL-1 ATN for method B.



mg placebo blank extract was analyzed as done in “Procedure for tablets”.

To the 30 mg of placebo blank of the composition described under Section

2.1.2.3, 20 mg of ATN was added and homogenized, transferred to a 100 mL

calibrated flask and the solution was prepared as described under ‘‘Procedure for

tablets’’, and then subjected to analysis by the procedures described above. The

analysis was used to study the interferences of excipients such as talc, starch, acacia,

methyl cellulose, sodium citrate, magnesium stearate and sodium alginate.


55


2.3.3.1 Absorption spectra

The proposed methods are based on the determination of residual bromine

generated in situ after the reaction between the drug and bromine is judged to be

complete. The red-pink color of unreacted MCP in acid medium absorbed maximally

at 540 nm (method A). The residual bromine was then used to brominate MCP

yielding colored bromo-derivative product with λmax at 445 nm (method B). Similar to

method A, the green color of unreacted EGC in acid medium peaked at 630 nm

(method C). The absorption spectra of all methods are presented in Figure 2.3.1.

Figure 2.3.1 Absorption spectra for the proposed methods (8.0 µg mL-1 MCP for

method A and method B and 30.0 µg mL-1 EGC for method C).

2.3.3.2 Chemistry

Atenolol is reported to undergo oxidation by bromine generated in situ by the

action of the acid on the bromate-bromide mixture [10,165,166]. The proposed

methods are indirect and based on the oxidation of ATN by the bromine followed by

the determination of unreacted bromine by reacting with a fixed amount of either

MCP or EGC and measuring the absorbance at the respective wavelengths. Bromine

generated in situ oxidizes secondary hydroxyl group of ATN to ketone group. The

reaction of unreacted bromine with MCP involved two simultaneous processes i.e.

decrease in the pink color of MCP in acid medium at 540 nm (method A) and increase

in the yellowish-orange color at 445 nm (method B) due to the bromination of the

dye. Similar to method A, unreacted bromine would react with EGC and the decrease

in the absorbance of the green color of EGC in acid medium was measured at 630 nm

(method C). The tentative reaction scheme is given and illustrated in Scheme 2.3.1.

300 350 400 450 500 550 600

0.0

0.2

0.4

0.6

0.8

Abs

orba

nce

Wavelength, nm

Method A Method B

400 450 500 550 600 650 700-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Method C

Abs

orba

nce

Wavelength,nm

Sample Blank


56

BrO3

H2N

OO N

HHOCH3

CH3

Atenolol (ATN)

3Br25Br 6 H+ 3H2O

Known excess of Br2

H2N

OO N

HO

CH3

CH3Unreacted Br2HBr

Unreacted Br2

SO3H

H3C OHO

CH3

4 HBrSO3H

H3C OHO

CH3

Br

Br

Br

Br

red-pink colorMCP in acid medium

measured at 540 nm (method A)

yellowish-orange colorbromo-derivative of MCP

measured at 445 nm (method B)

Unreacted Br2S OOO

N

N

H3C

S OOO

H3C

S OOO

Na +

Na + S OOO

N

N

H3C

S OOO

H3C

S OOO

Na +

Br Br

Br

Br

Br

Br

green colorbromo-derivative of EGC

measured at 630 nm (method C)

6 HBrNa +

Scheme 2.3.1 Tentative reaction scheme for the proposed methods.

2.3.3.3 Basis of the methods

ATN, when added in increasing concentrations to a fixed concentration of in

situ bromine, consumed the latter and there occurred a concomitant fall in bromine

concentration. When a fixed concentration of MCP was added to decreasing

concentrations of bromine, a concomitant increase in the absorbance of MCP resulted

at 540 nm and at the same time decrease in the absorbance resulted at 445 nm.

Similarly, when a fixed concentration of EGC was added to decreasing concentrations

of bromine, a corresponding increase in the absorbance of EGC was observed at 630

nm. These were observed as a proportional increase in the absorbance at 540 nm

(method A) or 630 nm (method C) and decrease at 445 nm (method B) with

increasing the concentration of ATN.


Effect of reagent concentration

Preliminary experiments were performed to fix the upper limits of the MCP

and EGC that could produce a reasonably high absorbance, and these were found to

be 80 and 200 µg mL-1 for MCP in methods A and B, and 300 µg mL-1 for EGC in


57

method C. Bromate concentrations of 4.0 and 1.8 µg mL-1 in the presence of excess

bromide were found optimum to bleach the dye color in method A and method C,

respectively, whereas 8.0 µg mL-1 KBrO3 produced a reasonable maximum

absorbance at 445 nm in method B. Hence, different concentrations of ATN were

reacted with 1.0 mL each of 40, 80 and 18 µg mL-1 bromate in methods A, B and C,

respectively.

Effect of reaction medium

Hydrochloric acid was found to be an ideal medium for the two steps involved

in all the three methods (Figure 2.3.3). In method A, the effect of (1.0-3.0 mL of 5 M

HCl) was studied and the results showed that 2.0 mL of 5 M HCl was optimum for the

oxidation reaction of the drug and bromination reaction of dye. Taking in to account

the maximum absorbance of the measured species and the minimum absorbance of

the blank, 2.0 mL of 5 M HCl was fixed. In method B, 2.0 mL of 5 M HCl was found

optimum and any excess of the acid up to 3.0 mL would not affect the absorbance of

the measured species. In method C, 5.0 mL of 1 M HCl was found optimum to

achieve maximum absorbance for the sample and minimum absorbance for the blank.

Figure 2.3.3 Effect of acid on the colored species.

Reaction time and color stability

The reaction time between ATN and the bromine generated in situ was found

to be 15 min in method A and 10 min in both method B and method C. After

completion the reaction between the drug and the bromine, the residual bromine

would brominate the dyes and this bromination process was found to be complete in 5

min for all three methods. The absorbance of the measured species was constant up to

24 hours.

1.0 1.5 2.0 2.5 3.00.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Blank for method A

Sample for method B

Sample for method A

Abs

orba

nce

Volume of 5 M HCl1 2 3 4 5

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Method C

Abs

orba

nce

Volume of 1 M HCl

Sample Blank


58



A linear relation is found between absorbance and concentration of ATN

within the Beer’s law range given in Table 2.3.1. The calibration graphs (Figure

2.3.2) are described by the equation: Y = a + b X (where Y = absorbance, a =

intercept, b = slope and X = concentration in µg mL-1) obtained by the method of least

squares. The apparent molar absorptivity (ε), Sandell’s sensitivity, limits of detection

(LOD) and quantification (LOQ) are also given in the Table 2.3.1. Limits of detection

(LOD) and quantification (LOQ) were calculated from the following equations [69]:

SLOD

3.3 &

SLOQ

10

where σ is the standard deviation of “n” reagent blank determinations and S is the

slope of the calibration curve.

Figure 2.3.2 Calibration curves.

0 5 10 15 200.0

0.2

0.4

0.6

0.8

1.0Method A

Abs

orba

nce


0 10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8 Method B

Abs

orba

nce


0 2 4 6 80.0

0.2

0.4

0.6

0.8

1.0 Method C

Abs

orba

nce



59

Table 2.3.1 Regression and sensitivity parameters


In order to study the accuracy and precision of the proposed methods, three

concentrations of pure ATN within the linearity range were analyzed, each

determination being repeated seven times (intra-day precision) on the same day and

one time each for five days (inter-day precision). The percentage relative standard

deviation (% RSD) was ≤ 2.09 % (intra-day) and ≤ 2.63 % (inter-day). In addition, the

accuracy of the proposed method was measured by calculating the percentage relative

error (% RE), which was varied between 0.46 % and 3.54 %. The results of this study

indicate the high accuracy and precision of the proposed methods (Table 2.3.2).


To evaluate the robustness of the methods, two important experimental

variables, viz., the amount of acid and reaction time, were slightly varied, and the

capacity of the methods was found to remain unaffected by small deliberate

variations. The results of this study are presented in Table 2.3.3 and indicate that the

proposed methods are robust. Method ruggedness is expressed as % RSD of the same

procedure applied by three analysts and using three different spectrophotometers by

the same analyst. The inter-analysts’ and inter-instruments’ RSD values were ≤ 3.42%

indicating ruggedness of the proposed methods. The results of this study are presented

in Table 2.3.3.

Parameter Method A Method B Method C max, nm 540 445 630 Beer’s law limits (µg mL-1) 1-20 2-40 1-8 Molar absorptivity (L mol-1cm-1) 1.20×104 4.51×103 3.46×104 Sandell sensitivity* (µg cm2) 0.0223 0.0591 0.0077 Limit of detection (µg mL-1) 0.12 0.56 0.05 Limit of quantification (µg mL-1) 0.36 1.69 0.14 Regression equation, Y** Intercept, (a)

0.0038

0.7755

0.0217

Slope, (b) 0.0443 -0.0154 0.1229 Correlation coefficient (r) 0.9996 -0.9973 0.9992 Standard deviation of intercept (Sa) 0.00664 0.08471 0.01436 Standard deviation of slope (Sb) 0.00059 0.00378 0.00292 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. ** bXaY , where Y is the absorbance, a is the intercept, b is the slope and X is the concentration in µg mL-1.


60



Selectivity

In the present methods, a study of some potential interference was performed

by selecting the excipients often used in pharmaceutical formulations or as possible

co-active substances. Selectivity was evaluated by both placebo blank and synthetic

mixture analyses. The placebo blank, consisting the composition as mentioned under

Method ATN taken

(µg mL-1)

Intra-day (n = 7) Inter-day (n = 5) ATN

founda (µg mL-1)


(µg mL-1)

%RSDb

%REc

Method A

4.00 4.14 1.49 1.71 4.09 1.86 2.25 8.00 8.12 0.75 1.56 8.16 1.34 2.00 12.0 4.00 0.67 1.04 12.31 1.28 2.58

Method B

8.00 8.22 1.74 2.69 8.19 2.14 2.38 16.0 16.25 1.06 1.58 16.44 2.08 2.75 24.0 24.63 0.56 2.62 24.85 1.72 3.54

Method C

2.00 1.99 1.64 0.46 2.05 2.14 2.50 4.00 4.09 2.09 2.44 4.14 2.56 3.50 6.00 6.07 1.47 1.09 6.16 2.63 2.67

a Mean value of five determinations; b Relative standard deviation (%); c Relative error (%).

Method

ATN taken, µg mL-1

Method robustness Method ruggedness Parameters altered Volume of acid, mLa

RSD, % (n = 3)

Reaction timeb

RSD, % (n = 3)

Inter-analysts’ RSD, % (n = 3)

Inter-instruments’

RSD, % (n = 3)

A

4.00 1.26 1.46 1.34 2.64 8.00 0.72 1.72 0.85 3.18 12.0 0.64 1.28 1.03 3.03

B

8.00 0.85 1.39 1.42 2.86 16.0 0.52 0.92 1.17 2.47 24.0 1.18 1.15 1.33 3.26

C

2.00 1.26 1.26 1.06 3.42 4.00 0.96 1.39 0.88 2.78 6.00 1.08 0.76 1.24 2.37

aIn methods A and B, the volume of 5 M HCl was 1.8, 2.0 and 2.2 mL whereas in method C, the aIn methods A and B, the volume of 5 M HCl was 1.8, 2.0 and 2.2 mL whereas in method C, the volume of 1 M HCl was 4.8, 5.0 and 5.2 mL b. The reaction time in methods A was 14, 15 and 16 min whereas in methods B and C, the same was 9, 10 and 11 min.


61

“Procedure for selectivity study” was prepared and analyzed as described under the

recommended procedures. The resulting absorbance readings for the methods were

same as the reagent blank, inferring no interference from the placebo. The selectivity

of the methods was further confirmed by carrying out recovery study from synthetic

mixture. The percent recoveries of ATN were 102.1±1.35, 101.9±1.18 and101.4±1.63

for method A, method B and method C, respectively. This confirms the selectivity of

the proposed methods in the presence of the commonly employed tablet excipients.



different brands of tablets, namely, Atenex-25, Atekind-50 and Aten-100. The results

presented in Table 2.3.4 showed that there was a close agreement between the results

obtained by the proposed methods and the label claim. The results were also

compared with those of the official IP method [6] statistically by a Student's t- test for

accuracy and variance ratio F- test for precision at 95 % confidence level. The

calculated t- and F-values indicate that there is no significant difference between the

proposed methods and the reference method with respect to accuracy and precision.


Tablet Brand name

Label claim

mg/tablet


Official method

Proposed methods Method A Method B Method C

Atenex-25b

25

100.3±0.58

100.9 ± 1.06 t = 1.11 F= 3.34

99.65 ± 0.96 t = 1.2 F= 2.74

101.0 ± 1.12 t = 1.28 F= 3.33

Atekind-50c

50

99.67 ± 0.67

101.0 ± 1.09 t = 2.32 F= 1.48

100.6 ± 1.36 t = 1.42 F= 4.12

99.81 ±1.42 t = 0.20 F= 4.49

Aten-100d

100

100.6±0.82

100.6 ±1.11 t = 0.03 F= 1.83

101.1 ± 1.37 t = 0.69 F= 2.79

99.72 ±1.69 t = 1.05 F= 4.25

aMean value of five determinations. b,dMarketed by Zydas Healthcare, East Sikkim, India, c Marketed by Mankind Pharma Ltd., New Delhi, India,

Tabulated t-value at the 95% confidence level is 2.78. Tabulated F-value at the 95% confidence level is 6.39.


62

Recovery studies

To further establish the accuracy of the methods, a standard addition technique was followed. A fixed amount of drug from pre-analyzed

tablet powder was taken and pure drug at three different concentrations (50, 100 and 150 % of that in tablet powder) was added. The total

concentration was found by the proposed methods. The determination with each concentration was repeated three times and the percent recovery

of the added standard was calculated. Results of this study presented in Table 2.3.5 reveal that the accuracy of methods was unaffected by the

various excipients present in the formulations.


Tablets studied

Method A Method B Method C ATN in tablets, µg mL-1

Pure ATN added,

µg mL-1

Total found,

µg mL-1



Pure ATN added,

µg mL-1

Total found,

µg mL-1


ATN in tablets

µg mL-1

Pure ATN added, µg mL-1

Total found,

µg mL-1


Atenex 25

4.08 4.08 4.08

2.0 4.0 6.0

6.06 8.14 10.23

99.50±2.29 98.75±2.79 101.2±1.22

7.97 7.97 7.97

4.00 8.00 12.0

12.01 15.93 20.10

101.0±2.74 99.50±2.35 101.1±0.98

2.02 2.02 2.02

1.0 2.0 3.0

3.05 4.09 5.11

103.0±1.76 103.5±1.98 103.0±1.52

Atekind 50

3.99 3.99 3.99

2.0 4.0 6.0

6.10 8.16 10.29

103.5±1.91 103.8±1.04 104.0±2.51

8.05 8.05 8.05

4.00 8.00 12.0

12.16 16.15 20.44

102.8±2.15 101.2±2.76 103.2±2.40

1.99 1.99 1.99

1.0 2.0 3.0

3.00 4.02 5.03

101.0±0.97 101.5±1.58 101.3±1.79

Aten 100

4.02 4.02 4.02

2.0 4.0 6.0

6.07 8.03 10.15

104.0±2.31 103.5±1.43 104.5±2.77

8.09 8.09 8.09

4.00 8.00 12.0

12.18 16.35 20.34

102.2±1.56 103.2±2.45 102.1±1.47

1.99 1.99 1.99

1.0 2.0 3.0

3.02 4.05 5.07

103.0±2.13 103.0±2.06 102.7±1.91


63

Section 2.4

APPLICATION OF BROMATE-BROMIDE MIXTURE AS AN OXIDISING

AGENT FOR THE SPECTROPHOTOMETRIC DETERMINATION OF

ATENOLOL IN PHARMACEUTICALS

2.4.1 INTRODUCTION

The chemistry and applicability of bromate-bromide mixture has been

reviewed in Section 2.3.1.

The bromate-bromide reagent has been used in the assay of many

pharmaceutical compounds based on oxidation and/or bromination of the drug by

bromine generated in situ after the drug solution is treated with a known excess of

bromate-bromide mixture in acid medium followed by determination of the residual

bromine iodometrically [10,142-148].

Literature survey presented in Section 2.0.2 reveals that bromate-bromide

reagent has not been used with potassium iodide and starch reagents. Based on this

two simple, sensitive and economic spectrophotometric methods have been developed

for the determination of ATN in bulk drug and pharmaceuticals.

2.4.2 EXPERIMENTAL

2.4.2.1 Instrument

The instrument used for absorbance measurements was the same as described

in Section 2.2.2.1.


All reagents and chemicals used were of analytical or pharmaceutical grade

and distilled water was used to prepare the solutions.

Bromate-bromide mixture: A standard stock solution of KBrO3–KBr equivalent to

300 µg mL-1 KBrO3 was prepared by dissolving accurately weighed 30 mg of KBrO3

(S. D. Fine Chem. Ltd., Mumbai, India) and 0.3 g of KBr (Merck, Mumbai, India) in

water and diluted to the mark in a 100 mL calibrated flask. The stock solution was

diluted with water to get bromate–bromide mixture solutions containing 30.0 µg mL-1

in KBrO3 for use in method A and 15 µg mL-1 in KBrO3 for method B.

This work has been published in Chemical Industry & Chemical Engineering Quarterly, 2012, 18 (1): 43-52.


64

Potassium iodide: A 2% potassium iodide (Merck, Mumbai, India) solution was

prepared by dissolving 2 g potassium iodide with water in a 100 mL calibrated flask.

This solution was prepared a fresh daily.

Starch solution: One gram of starch (LOBA Chemie Ltd., Mumbai, India) was made

in to paste with water and slowly poured with constant stirring into 100 mL boiling

water, boiled for 5 min, cooled and used. This solution was prepared freshly every

day.

Hydrochloric acid: Concentrated acid (Merck, Mumbai, India, Sp. gr. 1.18) was

diluted appropriately with water to get 3 M HCl for use in both methods.

Sodium acetate: A 3 M aqueous solution of sodium acetate was prepared by

dissolving suitable quantity of sodium acetate trihydrate crystals (Merck, Mumbai,

India) in water for use in method A.

Standard ATN solution: A stock standard solution equivalent to 100 µg mL-1 ATN

was prepared by dissolving accurately weighed 25 mg of pure drug with water in a

250 mL calibrated flask. This stock solution was diluted with water to get the working

concentrations of 20 and 15 µg mL-1 for method A and method B, respectively.

Tablets used in this study are those mentioned in the Section 2.1.2.2.

2.4.2.3. Assay procedures

Method A (based on the measurement of tri-iodide ion)

Varying aliquots (0.25–4.5 mL) of standard ATN solution (20 µg mL-1) were

accurately transferred into a series of 10 mL calibrated flasks and the total volume

was adjusted to 4.5 mL with water. One mL of 3 M HCl was added to each flask

followed by the addition of 1 mL bromate–bromide mixture solution (30 µg mL-1 in

KBrO3). The content was mixed well and let stand for 15 min with occasional

shaking. Then, 1.0 mL of 3 M sodium acetate solution was added to each flask

followed by 1 mL of 2 % potassium iodide. The volume was brought up to the mark

with water and the absorbance of the resulting triiodide ion was measured at 360 nm

after 5 min against the water.

Method B (based on the measurement of starch-iodine complex)

Into a series of 10 mL calibrated flasks, different aliquots (0.2, 0.5, 1.0, 2.0,

3.0 and 4.0 mL) of standard ATN (15 µg mL-1) solution were transferred using a

micro burette. The total volume in each flask was brought to 4 mL by adding required

quantity of water. The solution was acidified by adding 1.0 mL of 3 M HCl, and 1.0


65

mL of bromate–bromide (15 µg mL-1 in KBrO3) solution was then added to each

flask. The flasks were kept aside for 15 min with periodic shaking; 1.0 mL of 2%

potassium iodide was added and the content was mixed well. After 5 min, 1 mL of 1%

starch solution was added to each flask and the volume was made up to the mark with

water and mixed well. The absorbance of the resulting blue chromogen was measured

at 570 nm against water blank after 5 min.





Tablet extract equivalent to 100 µg mL-1 ATN was prepared as described in

Section 2.1.2.3. A suitable aliquot of the extract (100 µg mL-1 ATN) was diluted

appropriately with water to get 20.0 and 15.0 µg mL-1 ATN for the assay by methods

A and B, respectively.




A synthetic mixture was prepared by adding 10 mg of ATN to the 20 mg of

the placebo blank, homogenized and the solution was prepared as done under

“Procedure for tablets”. The filtrate was collected in a 100 mL flask. The synthetic

mixture solution (100 µg mL-1 in ATN) was appropriately diluted with water to get

20.0 and 15.0 µg mL-1 ATN solutions, and appropriate aliquots were subjected to

analysis by method A and method B, separately.


The proposed methods use the bromine generated in situ by the action of the

acid on bromate-bromide mixture which can be considered as a green

brominating/oxidizing agent and are based on the oxidation reaction of ATN with a

known excess of bromate-bromide mixture in acid medium. The unreacted bromine is

made oxidizes iodide and liberated iodine which will form tri-iodide ion (I3 -) in the

presence of excess iodide. The amount of iodine liberated, by the reaction of

unreacted bromine with potassium iodide, was either measured directly at 360 nm

(method A) or reacted with starch and resulting blue colored chromogen of starch-


66

iodine complex is measured at 570 nm (method B) (Figure 2.4.1). The reaction

pathway of the proposed methods is illustrated in Scheme 2.4.1.

BrO3-

H2N

OO N

HHOCH3

CH3

Atenolol(ATN)

3Br25Br- 6 H+ 3H2O

Known excess of Br2

H2N

OO N

HOCH3

CH3Unreacted Br2HBr

Unreacted Br2 + Excess of KI

I3- measured at 360 nm

(method A)

blue complex measured at 570 nm(method B)

I3- + Starch

Scheme 2.4.1 Reaction pathway of the proposed methods

Figure 2.4.1 Absorption spectra of tri-iodide ion (method A) and starch-iodine

complex (method B).


Effect of acid concentration

The effect of acid concentration on the measured species was investigated by

following the assay procedures. The effect of 1 mL of HCl of different concentrations

(1.0, 2.0, 3.0, 4.0, 5.0 and 10.0 M) was studied by measuring the absorbance of the

colored product using a fixed concentration of ATN (6.0 µg mL-1 in method A and 2.0

µg mL-1 ATN in method B). From Figure 2.4.2, it is clear that the absorbance of the

colored product remained constant with 1.0 mL of 3 to 10 M HCl. Therefore, 1.0 mL

of 3.0 M HCl was selected as the optimum for both methods.

300 350 400 450 500 550 600 650 700 7500.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

Wavelength, nm

Method A Method B


67

Figure 2.4.2 Effect of acid concentration on the formation of colored products.

Reaction time and color stability

The effect of time on the reaction between ATN and bromate-bromide mixture

in the presence of HCl was studied by keeping all other reaction conditions

unchanged. The absorbance of the colored species was measured after different

reaction times (5.0-45.0 min) and the results showed that the reaction time was

complete within 15 min in both the methods. The stability of yellow tri-iodide ion in

method A was stable up to 45 min where as the absorbance of the blue colored starch-

iodine complex chromogen in method B remained stable for at least 1 hr.

Effect of sodium acetate

The liberation of iodine did not stop even after 30 min under the specified acidic

conditions in method A, but on adding sodium acetate the reaction ceased

immediately. The amount of sodium acetate required was optimized and 1mL of 3 M

sodium acetate in a total volume of 10 mL was found optimum.


The proposed methods were validated in according to the current ICH guidelines

[69].


The standard calibration curves (Figure 2.4.3) under the optimum

experimental conditions were constructed by plotting the absorbance versus

concentration. A linear correlation was found between absorbance at λmax and

concentration of ATN in the concentration ranges given in Table 2.4.1. The graphs

are described by the regression equation: Y = a + bX (where Y = absorbance; a =

0 2 4 6 8 10

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

Abs

orba

nce

Molarity of HCl

Method A Method B


68

intercept; b = slope and X = concentration in µg mL-1). The regression parameters

such as slope (b), intercept (a) and correlation coefficient (r) are presented in Table

2.4.1. The molar absorptivity (ε), Sandell’s sensitivity, limits of detection (LOD) and

quantitation (LOQ) of both methods are also given in Table 2.4.1. The high values of

ε, low values of Sandell’s sensitivity, LOD and LOQ values indicate the high

sensitivity of the proposed methods.

Figure 2.4.3 Calibration curves


To compute the accuracy and precision of the proposed methods, the assay

procedures described above were repeated seven times within the day to determine the

repeatability (intra-day precision) and five times on different days to determine the

intermediate precision (inter-day precision). These assays were performed for three

concentration levels of ATN and the results of this study are summarized in Table

2.4.2. Accuracy was evaluated as percentage relative error (% RE) between the

measured mean concentrations and taken concentrations of ATN and the results are

also presented in Table 2.4.2. The % RE values were ≤ 1.57 and demonstrate the high

accuracy of the proposed methods. The percentage relative standard deviation

(%RSD) values were ≤ 2.25 (intra-day) and ≤ 2.74 (inter-day) indicating high

precision of the methods.

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0 Method A

Abs

orba

nce


0 1 2 3 4 5 6

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8 Method B

Abs

orba

nce



69

Table 2.4.1 Regression and sensitivity parameters

Parameter Method A Method B max, nm 360 570 Beer’s law limits (µg mL-1) 0.5-9.0 0.3-6.0 Molar absorptivity (L mol-1cm-1) 2.36×104 2.89×104 Sandell sensitivity* (µg cm-2) 0.0113 0.0092 Limit of detection (µg mL-1) 0.10 0.08 Limit of quantification (µg mL-1) 0.32 0.25 Regression equation, Y** Intercept, (a) 0.9111 0.7485 Slope, (b) -0.0835 -0.1035 Correlation coefficient (r) -0.9991 -0.9992 Standard deviation of intercept (Sa) 0.08487 0.08826 Standard deviation of slope (Sb) 0.01688 0.02829 *Limit of determination as the weight in µg per mL of solution, which corresponds to an absorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. ** bXaY , where Y is the absorbance, a is the intercept, b is the slope and X is the concentration in µg mL-1.



The robustness of the methods was evaluated by making small incremental

changes in the volume of acid and reaction time, and the effect of the changes on the

absorbance of the measured species was studied. The changes had negligible

influence on the results as revealed by small intermediate precision values expressed

as % RSD (≤ 2.26). Method ruggedness was expressed as the % RSD of the same

procedure applied by four different analysts as well as using three different cuvettes.

The inter-analysts RSD were within 1.63 whereas inter- cuvettes % RSD for the same

ATN concentrations ranged from 1.92-2.77 suggesting that the developed methods

were rugged. The results of this study are shown in Table 2.4.3.

Method ATN taken

( µg mL-1)

Intra-day (n = 7) Inter-day (n = 5) ATN founda ( µg mL-1)


( µg mL-1) %RSDb %REc

Method

A

2.0 2.02 2.25 1.06 2.01 2.74 1.28 4.0 4.05 0.92 1.18 4.03 1.26 1.57 6.0 6.02 0.50 0.28 6.01 1.04 1.03

Method

B

1.5 1.49 1.85 0.58 1.48 2.14 1.37 3.0 3.01 1.34 0.47 2.98 2.06 1.15 4.5 4.46 0.29 0.81 4.48 1.42 1.08

a Mean value of five determinations; b Relative standard deviation (%); c Relative error (%).


70


Selectivity

The proposed methods were tested for selectivity by placebo blank and

synthetic mixture analyses. A convenient aliquot of placebo blank was extracted with

water and was subjected to analysis following the recommended procedures. In both

the methods, there was no interference by the inactive ingredients as shown by the

near absorbance of the respective reagent blanks. A separate test was performed by

applying the proposed methods to the determination of ATN in a synthetic mixture.

The synthetic mixture solution was prepared as described under “Procedure for

tablets” and a suitable aliquot was subjected to analysis by method A and method B,

separately. The percentage recovery of ATN was 101.02 ± 1.21 for method A and

102.0 ± 1.67 for method B. This confirms the selectivity of the proposed methods in

the presence of commonly employed tablet excipients.



representative tablets Atenex-25, Atekind-50 and Aten-100 purchased from local

stores. The results presented in Table 2.4.4 showed that the methods are successful to

the determination of ATN in tablets without any detectable interference from the

excipients. The obtained results were statistically compared with the official IP

method [6]. The results obtained by the proposed methods agreed well with those of

the official method. When the results were statistically compared with those of the

reference method by applying the Student’s t-test for accuracy and F-test for

precision, the calculated Student’s t- value and F-value at 95% confidence level did

Method

ATN

taken, µg mL-1

Method robustness Method ruggedness Parameters altered Volume of acid, mLa

RSD, % (n = 3)

Reaction timeb

RSD, % (n = 3)

Inter-analysts RSD, % (n = 3)

Inter-cuvettes RSD, % (n = 3)

A

2.0 0.78 1.68 0.85 2.06 4.0 1.18 2.03 1.24 2.12 6.0 0.84 1.85 1.26 2.28

B

1.5 1.26 2.26 1.28 2.36 3.0 0.94 1.78 1.63 2.77 4.5 1.52 1.34 1.09 1.92

In methods A and B, the volume of 3 M HCl was 0.8, 1.0 and 1.2 mL b. The reaction time a was 13, 15 and 17 min in methods A and B


71

not exceed the tabulated values of 2.78 and 6.39, respectively. Hence, no significant

difference exists between the proposed methods and the official method with respect

to accuracy and precision.

Table 2.4.4 Results of assay of tablets and statistical comparison with the official

method.

Recovery studies

To assess the accuracy of the methods, recovery experiments were performed

by applying the standard-addition technique. To a fixed and known amount of ATN in

tablet powder (pre-analyzed), pure ATN was added at three concentration levels (50,

100 and 125 % of the level present in the tablet) and the total was measured by the

proposed methods. The determination with each concentration was repeated three

times. In all the cases, the recovery percentage values ranged between 99.0 and 104.5

with relative standard deviation in the range 0.31-3.29 %. The results of this study

presented in Table 2.4.5 indicated that the various excipients present in the

formulations did not interfere in the assay.

Tablet Brand name

Label claim

mg/tablet


Reference method

Proposed methods Method A Method B

Atenex-25

25

100.3±0.58

101.1 ± 0.94 t =1.62 F=2.63

100.6 ± 0.63 t = 0.78 F=1.18

Atekind-50

50

99.67 ± 0.67

100.8 ± 1.07 t = 2.00 F= 2.55

100.3± 0.89 t = 1.12 F= 1.76

Aten-100

100

100.6±0.82

101.2 ± 1.15 t = 0.95 F= 1.97

101.4 ±1.33 t = 0.64 F= 2.63



72


*Mean value of three determinations.

Tablets studied

Method A Method B ATN in tablets, µg mL-1

Pure ATN

added, µg mL-1

Total found,

µg mL-1



Pure ATN

added, µg mL-1

Total found,

µg mL-1


Atenex 25

4.04 4.04 4.04

2.0 4.0 5.0

6.13 8.18 9.21

104.5 ±1.36 103.5±0.56 103.4±0.53

2.01 2.01 2.01

1.00 2.00 3.00

3.02 4.05 5.08

101.0±1.90 102.0±0.84 102.3±0.49

Atekind 50

4.03 4.03 4.03

2.0 4.0 5.0

6.06 8.08 9.07

101.5±2.37 101.3±0.98 100.8±0.31

2.00 2.00 2.00

1.00 2.00 3.00

2.99 3.98 5.02

99.00±3.29 99.00±0.85 100.7±0.50

Aten 100

4.05 4.05 4.05

2.0 4.0 5.0

6.12 8.16 9.14

103.5±2.36 102.8±1.49 101.8±0.54

2.03 2.03 2.03

1.00 2.00 3.00

3.06 4.07 5.06

103.0±3.28 102.0±0.84 101.0±0.86


73

Section 2.5

UV- SPECTROPHOTOMETRIC ASSAY OF ATENOLOL IN

PHARMACEUTICALS AND STUDY OF ITS FORCED DEGRADATION

2.5.1 INTRODUCTION

As a wide variety of pharmaceutical substances absorb radiation in the near-

ultraviolet (200–380 nm) region of the electromagnetic spectrum, the UV-

spectrophotometric technique is widely employed in pharmaceutical analysis. UV-

spectrophotometry [167-170], because of its simplicity, reproducibility and speed and

minimum solvent/reagent system required and less analysis time, is widely used for

the assay of many therapeutic compounds used as medications. UV-

spectrophotometry is perhaps the most widely used spectrophotometric technique for

the quantitative analysis of chemical substances as pure materials and as components

of dosage forms. It has found increasing usefulness as a means of assaying

pharmaceutical substances described in the pharmacopeias [171]. There are a lot of

pharmaceutical compounds such as quetiapine fumarate [172], oxcarbazepine [173],

lomefloxacin [174], ascorbic acid [175], doxazosin [176], simvastatin [177],

rosiglitazone maleate [178], ranitidine HCl [179], piroxicam [180], ketoconazole

[181], and famotidine [182], etc., to mention a few, which have been successfully

assayed by UV-spectrophotometry.

In spite of many-fold advantages, only two methods have been reported for the

determination of ATN in pharmaceuticals when present alone by UV-

spectrophotometry and none of them is stability-indicating.

Stability testing and stress testing (forced degradation studies) are critical

components of drug development strategy. The studies help us to understand the

mechanism of a drug’s decomposition, which further helps in obtaining information

on physical and chemical factors that result in instability [183]. Stress testing is

defined as the stability testing of drug substances and drug products under conditions

exceeding those used for accelerated testing. The International Conference on

Harmonization (ICH) guidelines entitled “stability testing of new drug substances and

products” requires that stress testing be carried out to elucidate the inherent stability

This work has been communicated to Malaysian Journal of Pharmaceutical Sciences.


74

characteristics of the active substance [184]. The required tests include susceptibility

to alkaline, acidic, oxidative and UV degradation.

In the present Section 2.5, a simple, inexpensive, accurate, reproducible, and

stability-indicating UV-spectrophotometric method for ATN has been described. The

method is based on the measurement of absorbance of ATN solution in 0.1 M NaOH

at 273 nm. The method development and complete validation procedure followed by

stress studies as per the current ICH guidelines [69] are presented in the following

paragraphs

2.5.2 EXPERIMENTAL

2.5.2.1 Instrument

Shimadzu Pharmaspec 1700 UV/Visible spectrophotometer was used for

absorbance measurements.


All chemicals used were of analytical reagent grade. Doubly-distilled water

was used to prepare solutions wherever required. Hydrogen peroxide (H2O2),

hydrochloric acid and sodium hydroxide were purchased from Merck (Mumbai,

India).

A 5 M sodium hydroxide (Merck, Mumbai, India) solution was prepared by

dissolving required amount of the pellets in water. This solution was diluted to 0.1 M

and standardized and used as solvent to dissolve drug. Hydrochloric acid (5 M) was

prepared by appropriate dilution of concentrated acid (Merck, Mumbai, India; sp. gr.

1.18) with water. A 5% solution of H2O2 (LOBA Chemie Pvt. Ltd., Mumbai, India,

30% w/v) was prepared by diluting required volume of the commercially available

reagent with water.

Standard ATN solution: A stock standard solution of 250 µg mL-1 ATN was

prepared by dissolving 25 mg of pure ATN in 0.1 M NaOH and diluted to 100 mL

with the same solvent in a calibrated flask.

Tablets used in this study are those mentioned in the Section 2.1.2.2.


Construction of calibration curve

Aliquots of 0.2, 0.5, 1.0, 2.0, 3.0…….8.0 mL of 250 µg mL-1 ATN standard

solution were accurately transferred into a series of 10 mL calibrated flask and made


75

up to the mark with 0.1 M NaOH. The absorbance of the resulting solution was

measured at 273 nm against 0.1 M NaOH blank. Calibration curve was prepared by

plotting the absorbance versus concentration of drug. The concentration of the

unknown was read from the calibration curve or computed from the regression

equation derived using the Beer’s law data.


Twenty tablets were weighed accurately and ground into a fine powder. An

accurately weighed amount of the powdered tablet equivalent to 25 mg of ATN was

transferred to a 100 mL calibrated flask and shaken with 60 mL of 0.1 M NaOH for

about 20 min, then made up to the mark with the same solvent, mixed and filtered

using a Whatman No. 42 filter paper. The first 10 mL portion of the filtrate was

discarded, and a convenient aliquot, say 5 or 6 mL was taken for assay following the

procedure described above.




To the 50 mg placebo blank of the described composition, 25 mg of ATN was

added and homogenized, transferred to a 100 mL calibrated flask and solution

prepared as described under ‘Procedure for tablets’. The synthetic mixture solution

(250 µg mL-1 in ATN) was then subjected to analysis.

Preparation of acid and base induced-degradation products

Five mL of 500 µg mL-1 ATN solution in 0.1 M NaOH was taken (in

triplicate) in 25 mL calibrated flasks, 5.0 mL each of 5.0 M HCl (acid hydrolysis) and

5.0 M NaOH (alkaline hydrolysis) were added separately to each flask. The flasks

were kept on a water bath for 2.0 h at 80 °C, and then cooled to room temperature.

The flasks were neutralized with 5.0 mL of 5.0 M NaOH (for acid hydrolysis) and 5.0

mL of 5.0 M HCl (for alkaline hydrolysis) followed by diluting to the mark with 0.1

M NaOH. The absorption spectrum for each flask was recorded from 250-335 nm

versus the solvent blank.

Procedure for oxidative degradation

To 5.0 mL of 500 µg mL-1 ATN solution in 0.1 M NaOH taken in a 25 mL

calibrated flask, 5 mL of 5 % hydrogen peroxide was added. The flask was kept on a

water bath at 80 °C for 2.0 h. The flask was cooled to room temperature, made up to


76

the mark with the 0.1 M NaOH and the absorption spectrum was run from 250-335

nm against the solvent blank.

Procedure for dry heat and photo-degradation

The powdered sample (0.5 g) of ATN was taken on a Petri dish and kept in the

oven at 105 °C for 24 h, the sample cooled to room temperature and used to prepare

40 µg mL-1 ATN in 0.1 M NaOH. Also, another powdered sample (0.5 g) of ATN was

taken on a Petri dish, exposed to UV radiation in a UV chamber of 1200 lux-hr for 48

h and used to prepare 40 µg mL-1 ATN in 0.1 M NaOH. The absorption spectrum of

each solution was run from 250-335 nm against the corresponding solvent.


2.5.3.1 Absorption spectrum

The absorption spectrum of 40 µg mL-1 ATN solution in 0.1M NaOH was

recorded between 251.70-347.85 nm and showed an absorption maximum at 273 nm,

and at this wavelength 0.1 M NaOH had insignificant absorbance. Therefore, 273 nm

was used as analytical wavelength (λmax). Figure 2.5.1 represents the absorption

spectra of ATN in 0.1 M NaOH along with 0.1 M NaOH blank.

Figure 2.5.1 Absorption spectra of ATN in 0.1 M NaOH and blank. 2.5.3.2 Degradation studies of ATN

Forced degradation studies provide an indication of the stability-indicating

property of the drug. The study was carried out after subjecting ATN to dry heat

treatment, UV-degradation, acid and alkali hydrolysis; and oxidation. The UV-spectra

of ATN samples which were subjected to dry heat treatment and UV-degradation


77

(Figure 2.5.2) were similar to that of the standard ATN sample (Figure 2.5.1) and it

showed that ATN did not undergo degradation under these conditions. ATN subjected

to acid and alkali hydrolysis also did not undergo degradation, since the absorbance

values obtained under these stressed conditions (Figure 2.5.3) were similar to those of

standard ATN sample (Figure 2.5.1). The absorption spectrum (Figure 2.5.4)

obtained under oxidation condition with H2O2 shows complete degradation of ATN.

The overall degradation summary was compiled in Table 2.5.1.

(a) (b)

Figure 2.5.2 UV-spectra of ATN after subjected to a) thermal degradation and b) photo degradation.

(a) (b) Figure 2.5.3 UV-spectra of ATN after subjected to a) acid hydrolysis and b) base

hydrolysis.


78

Figure 2.5.4 UV-spectrum of ATN after subjected to peroxide degradation.

Table 2.5.1 Forced degradation summary


The proposed method was validated for linearity, sensitivity, precision,

accuracy, robustness, ruggedness, specificity, interference and recovery.


Linear correlation was obtained between the absorbance and concentration of

ATN in the range of 5.0 - 200.0 µg mL-1. The calibration graph (Figure 2.5.5) is

described by the equation:

Y = a + b X

(where Y= absorbance, a= intercept, b= slope and X= concentration in µg mL-1)

obtained by the method of least squares. Correlation coefficient, intercept and slope

for the calibration data are summarized in Table 2.5.2. Sensitivity parameters such as

Degradation condition % Assay* Observation Control sample 99.89 Not applicable Acid hydrolysis (5M HCl , 80°C, 2 hours)

99.81 No degradation observed

Base hydrolysis (5 M NaOH , 80°C, 2 hours)


Oxidation (5 % H2O2, 80°C, 2 hours)

- Extensively degraded

Thermal (105°C, 3 hours) 98.92 No degradation observed Photolytic (1.2 million lux hours)


* Percentage against standard ATN.


79

apparent molar absorptivity and Sandell sensitivity values, the limits of detection and

quantification calculated as per the current ICH guidelines [69] are compiled in the

same table. The limits of detection (LOD) and quantification (LOQ) were calculated

using the formulae:

sLOD

3.3 &

sLOQ

10

where σ is the standard deviation of five reagent blank absorbance measurements and s is the slope of the calibration curve.

Figure 2.5.5 Calibration curve.

Table 2.5.2 Sensitivity and regression parameters

Parameter Proposed method max, nm 273 Linear range, µg mL-1 5.0 – 200.0 Molar absorptivity(ε), L mol-1cm-1 1.34×103

Sandell sensitivity*, µg cm-2 0.20 Limit of detection (LOD), µg mL-1 0.24 Limit of quantification (LOQ), µg mL-1 0.73 Regression equation, Y** Intercept (a) 0.0093 Slope (b) 0.0049 Regression coefficient (r) 0.9995 Standard deviation of intercept (Sa) 0.00586 Standard deviation of slope (Sb) 5.0 × 10-5 *Limit of determination as the weight in µg per mL of 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.

0 50 100 150 2000.0

0.2

0.4

0.6

0.8

1.0

Abs

orba

nce



80


Within-day accuracy and precision of the proposed method were evaluated by

replicate analysis (n=7) of calibration standards at three different concentration levels

on the same day. Between-day accuracy and precision were determined by assaying

the calibration standards at the same concentration levels on five consecutive days.

Percentage relative standard deviations (% RSD) as precision and percentage relative

error (% RE) as accuracy of the proposed method were calculated. These results of

accuracy and precision showed that the proposed methods have good repeatability and

reproducibility (Table 2.5.3).



Method robustness was checked by determination of ATN at three different

concentrations by measuring the absorbance at 272, 273 and 274 nm whereas the

method ruggedness was performed 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. 2.5.4.


ATN taken,

µg mL-1

Intra-day accuracy and precision (n=7)

Inter-day accuracy and precision (n=5)

ATN found, µg mL-1 %RE %RSD ATN found,

µg mL-1 %RE %RSD 50.00 100.0 150.0

49.70 99.37 149.6

0.59 0.63 0.27

0.18 0.28 0.33

50.38 100.53 151.22

0.76 0.53 0.81

0.85 1.03 0.96

%RE: Percentage relative error, %RSD: Percentage relative standard deviation.

ATN 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)

50.00 2.58 1.25 1.75 100.0 3.12 0.74 1.24 150.0 2.64 0.85 1.07

*Wavelengths used were 272, 273 and 274 nm.


81

Selectivity

The proposed method was tested for selectivity by placebo blank and synthetic

mixture analyses. The placebo blank solution showed nearly the same absorbance as

that 0.1 M NaOH blank indicating no interference from the inactive ingredients.

A separate experiment was performed with the synthetic mixture. The analysis

of synthetic mixture solution yielded percent recoveries which ranged from 100.47 –

101.78 with standard deviation of 0.65 – 1.19. The results of this study showed that

the inactive ingredients did not interfere in the assay. These results further

demonstrate the accuracy as well as the precision of the proposed method.


In order to evaluate the analytical applicability of the proposed method to the

quantification of ATN in commercial tablets, the results obtained by the proposed

method were compared to those of the official method [4] by applying Student’s t-test

for accuracy and F-test for precision. The official method described the non-aqueous

potentiometric method in acetic acid medium. The results (Table 2.5.5) 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 official method with respect to accuracy and

precision.


Tablet Brand name

Label claim

mg/tablet


Official method

Proposed method

Atenex-25b

25

100.3±0.58

99.32 ± 0.73 t = 2.35 F=1.58

Atekind-50c

50

99.67 ± 0.67

100.9 ± 0.77 t = 2.69 F= 1.32

Aten-100d

100

100.6±0.82

98.72 ± 1.01 t = 3.23 F= 1.52



82

Recovery studies

The accuracy and validity of the proposed method were further ascertained by

performing recovery studies. Pre-analyzed tablet powder was spiked with pure ATN

at three concentration levels (50, 100 and 150 % of that in tablet powder) and the total

was found by the proposed method. The added ATN recovery percentage values

ranged from 99.39-102.50 % with standard deviation of 0.32 – 0.85 (Table 2.5.6)

indicating that the recovery was good, and that the co-formulated substance did not

interfere in the determination.


Tablets studied


Pure ATN added,

µg mL-1

Total found,

µg mL-1


Atenex

25

49.60 49.60 49.60

25.0 50.0 75.0

75.00 100.1 126.5

101.4 ±0.49 100.8±0.32 102.4±0.50

Atekind

50

50.45 50.45 50.45

25.0 50.0 75.0

75.86 101.6 126.9

101.6±0.85 102.3±0.33 101.9±0.50

Aten 100

49.36 49.36 49.36

25.0 50.0 75.0

74.21 100.0 125.9

99.40±0.49 101.4±0.33 102.1±0.77

*Mean value of n determinations.


83

SECTION 2.6

SUMMARY AND CONCLUSIONS –Assessment of methods

Two titrimetric, eight visible spectrophotometric and one UV-

spectrophotometric methods were developed and validated for the assay of ATN in

pharmaceuticals. The performance characteristics of the methods developed and those

of the existing methods are compiled in Table 2.6.1. The EP [4] and BP [5] methods

which are based on the potentiometric titration of drug with 0.1 M HClO4 is

applicable for macroscale assays and unsuitable for semimicro analysis. Even though

few titrimetric methods were previously reported, they are time consuming as they are

indirect titrations. To fill this void, two methods using 5 mM HClO4 as titrant were

developed. Here, the end points was detected either visually or potentiometrically.

The methods are applicable over 1.5 – 15.0 mg of ATN. The methods were applied

successfully to the determination of ATN in tablets. Compared to all reported

methods for ATN, the proposed methods have two additional advantages of simplicity

of operations and low-cost per analysis. These advantageous features advocate their

use in quality control laboratories for routine use.

Many of the previously reported spectrophotometric methods suffered from

one or other disadvantage like poor sensitivity, heating or extraction step, use of

expensive chemical and/or complicated experimental setup as can be seen from Table

2.6.1. The proposed spectrophotometric methods are simple, sensitive and use non

hazardous eco-friendly reagents. As an overall review, BB: MCP method with ε value

3.46 × 104 L mol-1 cm-1 is more sensitive among the proposed methods.

The previously reported UV-spectrophotometric methods [12-19] are confined

to the assay and are not stability-indicating. The present method employs 0.1 M

NaOH as the solvent and thereby reducing cost of analysis. The method offers an

additional advantage of wide linear dynamic range (5.0-200.0 µg mL-1). From the

stress study it clearly demonstrated that ATN undergoes extensive degradation under

oxidative stress condition and stable to acidic, thermal, photolytic, and alkaline

conditions. This study is a typical example of development of a stability indicating

assay, established following the recommendations of ICH-guidelines.


84

Table 2.6.1 Comparison of the proposed and the existing titrimetric and visible spectrophotometric methods. I Titrimetry

Sl. No.

Reagent Titration conditions Range, mg

Remarks Ref.

1. 0.05 M Sulphuric acid

Residual acid was back titrated by 0.1 M NaOH using methyl red-bromocresol green mixed indicator

NA Back titration, time consuming, requires higher acid medium

7

2. Cerium(IV) sulphate in acid medium

Unreacted oxidant was determined by ferrous ammonium sulphate using ferroin as indicator

4.0-12.0 Back titration, time consuming

8

3. Chloramine-T in acid medium

Residual oxidant was determibed by iodometric back titration

3.0-20.0 Back titration, time consuming, require

regular standardization.

9

4. Bromate-bromide mixture in acid medium

Determination of unreacted bromine by reaction with excess iodide and the liberated iodine is titrated to the starch end point using sodium thiosulphate

3.0-20.0 Back titration, time consuming.

10

5. 0.01 M Hydrochloric acid

Residual acid was then back titrated with 0.01 M sodium hydroxide using bromothymol blue-phenol red mixed indicator.

2.0-20.0 Back titration, time consuming.

11


85

II Spectrophotometry

6 0.1 M Perchloric acid Increased basisity of drug due to non leveling effct of acetic acid was determined using crystal violet as indicator.

NA Macro-scale analysis. 4,5

7. 0.005 M Perchloric acid

Increased basisity of drug due to non leveling effct of acetic acid was determined using crystal violet as indicator

1.5-15.0 Rapid, simple, no need of regular

standardization and applicable to semi

micro scale analysis.

Present method

Sl. No.

Reagent/s used Methodology λmax (nm)

Linear range, µg mL-1 and

ε, L mol-1cm-1

Remarks Ref.

1. Hydroxylamine hydrochloride- iron(III)

Ferric hydroxamate complex measured

510 50-800 (ε = 5.3×102)

Less sensitive, heating required

20

2. Sodium nitroprusside Complex of ammonia and nitroprusside measured

495 0.5-30 (ε = 3.01×105)

Heating required 21

3. Chloranil - propan-2-ol in acetaldehyde

Coupled product measured

690 NA Use of organic solvents, expensive reagents.

22

4. Phenol red The change in the color of phenol red measured

430 3.0-30 (ε = 3.47×103)

Less sensitive 11


86

5. Potassium permanganate- in alkaline medium

Unreacted KMnO4 measured Rate-constant method Fixed-concentration method Fixed-time method

526 1.06-6.6

(NA)

Time –consuming, involve judicial control of many experimental variables

23

6. Chloramine-T-metol-sulphanilic acid

Unreacted chloramine-T measured

520 2.5-25 (ε = 3.24×103)

Less sensitive 24

7. Chloramine-T: a) Metanil yellow

b) Indigo carmine

Unreacted chloramine-T measured

530

610

1-12

(ε = 1.19×104) 2.5-20

(ε = 6.65×103)

-

9

8. Bromate-bromide mixture- methyl orange

Unreacted bromine measured

520 0.5-4.0 (ε = 4.13×104)

- 10

9. Ce(IV) sulphate a) Ferroin b)Methyl orange c)Iron(III) thiocyanate

Unreacted oxidant measured

510 520 480

2.5-35.0 (3.5x103) 2.5-60.0 (1.0x103) 0.6-8.75 (1.1x104)

Less sensitive 8

10. Chloranilic acid Charge transfer complex measured

534 25-250

Less sensitive, use of organic solvents

25

11 Chloranilic acid Charge transfer complex measured

530 10.0-280.0 (NA)

Less sensitive, use of organic solvents

26

12 a) DDQ b)2,4-Dinitrophenol

Radical anion was measured Charge transfer

590

420

3.0-48.0 (ε =5.41×103)

2.0-24.0

Rapid, simple, sensitive selective and no heating

step. Use a single reagent

Present methods


87

c) Picric acid complex measured 420

(ε =1.13×104) 1.5-18.0

(ε =1.13×104)

and a single step reaction

13. Bromate-bromide mixture: a) MCP

b) MCP

c) EGC

Ureacted MCP in acid measured Bromo-derivative of MCP measured Ureacted EGC in acid measured

540

445

630

1.0 -20.0

(ε =1.20×104) 2.0-40.0

(ε =4.51×103) 1.0-8.0

(ε =3.46×104)

Simple, sensitive and no heating step. No use of

organic solvent. Use of an eco-friendly reagent

Present methods

14. Bromate-bromide mixture: Iodine Starch-iodine

Tri-iodide ion measured Starch-iodine complex measured

360

570

0.5-9.0 (ε =2.36×104)

0.3 -6.0 (ε =2.89×104)

Simple, sensitive and no heating step. No use of

organic solvent. Use of a green oxidizing reagent

Present methods

MCP: Meta-cresol purple, EGC: Erioglaucine, NR: Not reported, NA: Not available.


88

In the analysis of placebo blank and synthetic mixture the participation of inactive

ingredients in the reaction either with the reagent or chromogenic agent is almost

aught. This was confirmed by comparing the consumption of titrant or absorbance

values obtained in the presence and absence drug in the test solution. This confirms

the factor that the proposed methods are more selective. The accuracy (RE, %) and

precision (RSD, %) of all the methods were evaluated. With RE (%) and RSD (%)

values less than 3.54, methods are considered to be fairly accurate and precise. Upon

comparing the results of the proposed methods with those of the reference method

using Student’s t-test and F-test, all calculated values were found below the tabulated

t- and F- value. The results of the method robustness and ruggedness, both express in

terms of RSD (%), were less than 3.42 % for all the proposed methods. The reaction

scheme proposed for all the methods are tentative since the chromogenic products

were not isolated and characterized. Such inference is based either on reaction

stoichiometry or literature knowledge.


89

REFERENCES

1. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 14th

Edn., Merck & Co., Inc., Whitehouse Station, New Jersey, USA, 2006, p. 742.

2. A.M. Barrett, J. Carter, R. Hull, D.J. Le Count, C.J. Squire, US Patent 3,663,607;

May 16, 1972; Assigned to Imperial Chemical Industries Ltd., England.

3. M.R. Alan, D.S. Fred, J.T. Stewart, “Conquering headache”, 4th Edn., Decker

DTC, Hamilton, London, 2003, p. 76.

4. European Pharmacopoeia, Vol. II, European Department for the Quality of

Medicines, Council of Europe, Stranbourg, France, 2005, p. 1032.

5. British Pharmacopoeia, Vol. II, Her Majesty’s Stationary Office, London, 2009.

6. Indian Pharmacopeia, 4th Edn., Ministry of Health and Family Welfare,

Government of India, New Delhi, 1996, p. 74.

7. E. Marmo, Drugs Exptt. Clin. Res., 1980, 6, 639.

8. K. Basavaiah, U. Chandrashekar, P. Nagegowda, Bulg. Chem. Comm., 2003, 35,

174.

9. K. Basavaiah, U. Chandrashekar, P. Nagegowda, Indian J. Chem. Technol., 2004,

11, 769.

10. K. Basavaiah, U. Chandrashekar, P. Nagegowda, J. Serb. Chem. Soc., 2006, 71,

553.

11. K. Basavaiah, U. Chandrashekar, B.C. Somashekar, V. Ramakrishna, Proc. Nat.

Acad. Sci. India., 2005, 75(A), 233.

12. C.V.N. Prasad, C. Parihar, K. Sunil, P. Parimoo, J. Pharm. Biomed. Anal., 1998,

17, 877.

13. C.V.N. Prasad, C. Parihar, T.R. Chowdhary, S. Purohit, P. Parimoo, Pharm.

Pharmacol. Comm., 1998, 4, 325.

14. D. Bonazzi, R. Gotti, V. Andrisano, V. Cavrini, Farmaco, 1996, 51, 733.

15. S. Singh, R. Jain, Indian Drugs, 34, 1997, 678.

16. D. Satyanarayana, K. Kannan, R. Manavalan, Chem. Anal. (Warsaw)., 2006, 51,

771.

17. W. Wehner, Die Pharmazie, 2000, 55, 543.

18. J. Huang, J. Jin, Zhongguo Yiyao Gongye Zazhi, 1989, 20, 19.

19. S. Wang, Yaowu Fenxi Zazhi, 1990, 10, 110.

20. Y.K. Agarwal, K. Raman, S. Rajput, S.K. Menon, Anal. Lett., 1992, 25, 1503.

21. N. Bashir, S.W.H. Shah, M. Bangesh, Riazullah, J. Sci. Ind. Res., 2011, 70, 51.


90

22. M.A. Korang, M.H. Abdel-Hay, S.M. Galal, M.A. Elsayed, J. Pharm. Belg., 1984,

40, 178.

23. G.C. Hiremath, R.M. Mulla, S.T. Nandibewoor, Chem. Anal. (Warsaw)., 2005,

50, 449.

24. K. Basavaiah, U. Chandrashekar, P. Nagegowda, Acta Cienc. Indica, Chem.,

2004, XXX C, 075.

25. S.P. Agarwal, V. Singhal, A. Prakash, Indian J Pharm. Sci., 1998, 60, 53.

26. L.L. Yu, J.C. Liu, H.K. Li, Yaowu Fenxi Zazhi, 2010, 30, 538.

27. L. Gung, Yaowu Fenxi Zazhi, 1989, 9, 175.

28. R. Mehvar, J. Chromatogr. Biomed. Appl., 1989, 85, 402.

29. D. Radulovic, L.J. Zivanovic, G. Velimirovic and D. Stevanovic, Anal. Lett.,

1991, 24, 1813.

30. Z. Pawalk and B.J. Clark, J. Pharm. Biomed. Anal., 1992, 10, 329.

31. S.V. Grram and H.P. Tipnis, Indian Drugs, 1993, 30, 195.

32. S.S. Zarapkar, S.S. Kolte and S.H. Rane, Indian Drugs, 1997, 34, 350.

33. A.P. Argekar and S.G. Powar, J. Pharm. Biomed. Anal., 2000, 21, 1137.

34. S.V. Erram and H.P. Tipnis , Indian Drugs, 1992, 29, 436.

35. R. Jain and C.L. Jain, Microchem. J., 1991, 44, 187.

36. H.L. Rau, A.R. Aroor and P.G. Rao, Indian Drugs, 1991, 28, 283.

37. R.T. Sane, V.R. Bhate, V.G. Nayak and K.D. Ladge, Indian Drugs, 1991, 28, 322.

38. M.S. Bhatia, S.G. Karkhedikar and S.G. Chaturvedi, Indian Drugs, 1997, 34, 576.

39. A.P. Argekar and J.G. Sawant, J. Liq. Chromatogr. Rel. Technol., 1999, 22, 1571.

40. S.I. Sasa, I.M. Jalal and H.S. Khalil, J. Liq. Chromatogr., 1988, 11, 1673.

41. A. El-Sindy, S, Emara and A. Mostafa, IL Farmaco, 2005, 60, 269.

42. A. Holbrook, A.M. Krstulovic, J.H. Miller and J. Rysaluk, Pharmeuropa, 1991, 3,

218.

43. S.T. Sasa , J. Liq. Chromatogr., 1988, 11, 929.

44. A.K. Singh, E.R.M. Kedor-Hackmann and M.I.R.M. Santora, J. AOAC Int., 2001,

84, 1724.

45. M.E. Abdel-Hamid, Farmaco, 2000, 55, 136.

46. R. Ghanem, M.A. Bello, M. Callejon and A. Guiraum, J. Pharm. Biomed. Anal.,

1996, 15, 383.

47. H.Y. Aboul-Enein, M.I. El-Awady and C.M. Heard, Pharmazie, 2002, 57, 169.


91

48. I. Kaliappan, B.S.M. Pabbisetty and S.N. Karunanidhi, Indian Drugs, 2000, 37,

497.

49. M. Veronico, G. Ragno and C. Vetuschi, Spectrosc. Lett., 1995, 28, 407.

50. G.S. Sadan and A.B. Ghogare, Indian Drugs, 1990, 28, 147.

51. G.R. Rao, A.B. Avadhanutu, R. Siridhar, A.R.R. Pantalu and G.K. Kokate, East

Pharm., 1990, 33, 113.

52. G.S. Sadana, A.B. Ghogare, Indian Drugs, 1990, 28, 142.

53. J.A. Murillo-Pulgarin, A. Alanon Molina and P. Fernandezlopoz, Anal. Chim.

Acta, 1998, 370, 9.

54. M. Gajewska, G. Glass and J. Kostelecki, Acta Pol. Pharm., 1992, 49, 1.

55. H.C. Zhao, N.H. Yan and J.X. Guo, Fenxi Shiyanshi, 1994, 13, 36.

56. M.A. El-Ries, Anal. Lett., 1995, 28, 1629.

57. M.A. Iorio, A. Mazzeo Farina and A. Doldo, J. Pharm. Biomed. Anal., 1987, 5, 1.

58. G. Pyramides, J.W. Robinson and S.W. Zito, J. Pharm. Biomed. Anal., 1995, 13,

103.

59. W.A. Tao, F.C. Gazzo and R.G. Cooks, Anal. Chem., 2001, 73, 1692.

60. F.M. Han and Y. Chen, Fenxi Kexme x-uebao, 1998, 14, 135.

61. A. Saffati and B.J. Clark, J. Pharm. Biomed. Anal., 1996, 14, 1547.

62. A. Saffati and B.J. Clark, Anal. Proc. (London), 1993, 30, 481.

63. B.Y. Sun, A.J. Huang, Y.L. Sun and Z.P. Sun, Chin. Chem. Lett., 1997, 8, 1001.

64. M.C.F. Ferraro, P.M. Castellano and T.S. Kaufmann, J. Pharm. Sci., 1981, 18, 60.

65. D. Cairns. “Essentials of pharmaceutical chemistry”, 3rd Edn., Pharmaceutical

Press, Cornwall, UK, 2008.

66. D.G. Watson. “Pharmaceutical analysis: a textbook for pharmacy students and

pharmaceutical chemists”, 2nd Edn., Elsevier/Churchill Livingstone, UK, 2005.

67. K.G. Bothara. “A hand book of inorganic pharmaceutical chemistry”, 9th Edn.,

Pragati Books Pvt. Ltd., 2008.

68. R.N.J. Kucharsky, L. Safarik, “Titrations in non-aqueous solvents”, Elsevier

publishing company, Netherlands. 1965.

69. “International Conference on Harmonization of Technical Requirements for

Registration of Pharmaceuticals for Human Use”, ICH Harmonisation Tripartite

Guideline. Validation of Analytical Procedures: Text and Methodology Q2 (R 1),

Complementary Guideline on Methodology dated 06 November 1996,

incorporated in November 2005, London.


92

70. R.S. Mulliken and W.B. Pearson, “Molecular Complexes”, Wiley Interscience,

NewYork, 1969.

71. R.Foster, “Molecular Complexes” Vol II , Elok Science, London, 1974, 251.

72. S. Patil (Edn.), “The Chemistry of Quinonoid Compounds”, Part-I, John-Wiley

and Sons, London, 1974.

73. R.H. Thomson, “Naturally Occurring Quinones”, Academic Press, London,

1971.

74. J. Yarwood (Edn.), “Spectorscopy and Structure of Molecular Complexes”,

Plenum., Press, London, 1973.

75. R.S. Mulliken, J. Am. Chem. Soc., 1950, 72, 600.

76. R. Foster. Charge transfer complexes, Academic Press, London, 1969.

77. P. Shahdousti, M. Aghamohammadi, N. Alizadeh, Spectrochim. Acta Part A.,

2008, 69, 1195.

78. J.O. Onah, J. E. Odeiani. J. Pharm. Biomed. Anal., 2002, 29, 639.

79. M. Fouery, J. Pharm. Chim., 1934, 20, 116.

80. J.W. Carett and J.P. Heotis, J. Assoc. Off. Agri. Chem., 1955, 41, 323.

81. H.F. Beckmann and L. Feldman, J. Agri. Food. Chem., 1960, 8, 227.

82. M.F. Loucks and L. Nauer Jr., J. Assoc. Off. Anal. Chem., 1967, 50, 268.

83. A. Raza. J. Anal. Chem., 2008, 63, 244.

84. K. Basavaiah. IL Farmaco, 2004, 59, 315.

85. G.A. Saleh, H.F. Askal, M.F. Radwan, M.A. Omar, Talanta, 2001, 54, 1205.

86. G.A. Saleh, H.F. Askal. J. Pharm. Biomed. Anal., 1991, 9, 219.

87. C.S. Xuan, Z.Y. Wang, J.L. Song. Anal. Lett., 1998, 31, 1185.

88. A.A. El Kheir, S.F. Belal, M.M. Ayad, S.M. El Adl, Anal. Lett., 1986, 19, 1019.

89. M.A. Elsayed, M. Barary, M. Abdel-salam, S. Mohamed, Anal. Lett., 1989, 22,

1665.

90. A.M. Wahbi, M.M. Bedair, S.M. Galal, A.A. Gazy, J. Pharm. Biomed. Anal.,

1993, 11, 639.

91. F.M. Abdel-Gawad, J. Pharm. Biomed. Anal., 1997, 15, 1679.

92. K. Basavaiah, V.S. Charan, Turk. J. Chem., 2002, 26, 653.

93. H.F. Askal, Talanta, 1997, 44, 1749.

94. A.A. Mostafa, L.I. Bebawy, H.H. Refaat, J. Pharm. Biomed. Anal., 2002, 27, 889.

95. A.M. Al-brashy, Anal. Lett., 1993, 26, 2595.

96. K. Basavaiah, V.S. Charan, Indian J. Pharm. Sci., 2003, 65, 660.


93

97. K. Basavaiah, V.S. Charan, Sci. Asia., 2002, 28, 359.

98. A.F.M. El-Walily, M.A. Korany, A. El-Gindy, M.F. Bedair, J. Pharm. Biomed.

Anal., 1998, 17, 435.

99. K. Basavaiah, K. Tharpa, K.B. Vinay, Croat. Chem. Acta., 2010, 83, 415.

100. K. Basavaiah, U.R. Anil kumar. E-J.Chem., 2007, 4, 173.

101. K. Basavaiah, K. Tharpa, N. Rajendraprasad, S.G. Hiriyanna, K.B. Vinay, Indian

J. Chem. Technol., 2009, 16, 265.

102. C.S.P. Sastry, T.V. Rekha, A. Satyanarayana, Mikrochim. Acta., 1998, 128, 201.

103. A. Raza, A.S. Ijaz, Atta-ur-Rehmana, U. Rasheed, J. Chin. Chem. Soc., 2007, 54,

223.

104. A.S. Amin, I.S. Ahmed, Mikrochim. Acta., 2001, 137, 35.

105. F.M. Abdel-Gawad, Y.M. Issa, H.M. Fahmy, H.M. Hussein, Mikrochim. Acta

1998, 130, 35.

106. G.G. Mohamed, S.M. Khalil, M.A. Zayed, M.A. El-Shall, J. Pharm. Biomed.

Anal., 2002, 28, 1127.

107. N.A. El Ragehy, M.F. El Tarras, F.I. Khattab, A.K.S. Ahmad, Spectrosc. Lett.,

1991, 24, 81.

108. A.E. El-Gendy, Spectrosc. Lett., 1992, 25, 1297.

109. C.S.P. Sastry, J. Murali, P.Y. Nalidu, Indian Drugs, 1998, 35, 364.

110. M. Walash, M. Sharaf-El Din, M.E.S. Metwalli, M. RedaShabana, Arch. Pharm.

Res., 2004, 27, 720.

111. F.M. Abou-Attia. IL Farmaco, 2000, 55, 659.

112. G.A. Saleh, Talanta, 1998, 46, 111.

113. H.E. Abdellatef, J. Pharm. Biomed. Anal., 1998, 17, 1267.

114. A.A. Gouda, Talanta, 2009, 80, 151.

115. G.B. Okide, U.E. Odoh, Indian J. Pharm. Sci., 1998, 60, 368.

116. M.U. Adikwu, K.C. Ofokansi, A.A. Attama, Chem. Pharm. Bull., 1999, 47, 463.

117. E. Khaled, Talanta, 2008, 75, 1167.

118. Z.A. El-Sherif, A.O. Mohamed, M.I. Walash, F.M. Tarras, J. Pharm. Biomed.

Anal., 2000, 22, 13.

119. M.U. Adikwu, K.C. Ofokansi, J. Pharm. Biomed. Anal., 1997, 16, 529.

120. B.V. Kamath, K. Shivram, S. Vangani, Anal. Lett., 1992, 25, 2239.

121. B.V. Kamath, K. Shivram, G.P. Oza, S. Vangani, Anal. Lett., 1993, 26, 665.

122. B.V. Kamath, K. Shivram, S. Vangani, Anal. Lett., 1994, 27, 103.


94

123. K. Basavaiah, S.A.M. Abdulrahman, Thai J. Pharm. Sci., 2010, 34, 134.

124. M. Hasani, M. Irandoust, M. Shamsipur, Spectrochim. Acta A., 2006, 63, 377.

125. F.A. El-Yazbi, A.A. Gazy, H. Mahgoub, M.A. El-Sayed, R.M. Youssef, J. Pharm.

Biomed. Anal., 2003, 31, 1027.

126. M.H. Abdel-Hay, S.M. Sabry, M.H. Barary, T.S. Belal, Anal. Lett., 2004, 37,247.

127. N. Rajendraprasad, K. Basavaiah, K.B. Vinay, J. Serb. Chem. Soc., 2011, 76,

1551.

128. A.M.A. Sameer, K. Basavaiah, Int. J. Anal. Chem. 2011,

doi:10.1155/2011/619310, Vol. 2011, Article ID 619310, 9 pages.

129. M.S. Raghu, K. Basavaiah, Journal of the Association of Arab Universities for

Basic and Applied Sciences, 2012, 12, 33.

130. M.E. Abdel-Hamid, M. Abdel-Salam, M.S. Mahrous, M.M. Abdel-Khalek,

Talanta, 1985, 32, 1002.

131. G. Saito, Y. Matsunaga, Bull. Chem. Soc. Jpn., 1971, 44, 3328.

132. W. Kemp, Organic Spectroscopy, 3rd edn., Replika press, India, 2006, p. 274.

133. http://www.lenntech.com/periodic/elements/br.htm

134. P. Anastas, J.C. Warner, “Green Chemistry: Theory and Practice”, Oxford

University Press, New York, 1998, p. 30.

135. Balard, Ann. Chim. et. Phys., 1826, 32, 337.

136. W.F. Koppeschaar, Z. Anal. Chem., 1876, 15, 233.

137. Kratschmer , Z. Anal. Chem., 1885, 24, 546.

138. M.R.F. Ashworth, “Titrimetric OrganicAnalysis”, Part I, Interscience Publishers,

London, pp. 118.

139. I.M. Kolthoff and R. Belcher, “Volumetric Analysis”, Vol. III, Interscience

Publishers, Inc, New York, 1957, pp. 529.

140. T. Higuchi and E. Brochmann-Hausen (edn.), “Pharmaceutical Analysis”, CBS

Publishers and Distributors, New Delhi (Indian edition, 1997).

141. K. Basavaiah, U. Chandrashekar, P. Nagegowda. J. Serb. Chem. Soc., 2006, 71,

553.

142. K. Basavaiah. Ind. J. Chem. Technol., 2006, 13, 360.

143. K. Basavaiah, B. C. Somashekar. J. Sci. Ind. Res., 2006, 65, 349.

144. U.R. Anilkumar, K. Basavaiah. Bulgarian Chem. Commun., 2007, 39, 53.

145. P. Nagegowda, K. Basavaiah. J. Braz. Chem. Soc., 2005, 16, 821.


95

146. K. Basavaiah, U. Chandrashekar, P. Nagegowda. J. Serb. Chem. Soc., 2005, 70,

969.

147. K. Basavaiah, V. Ramakrishna, B. C. Somashekar, U. R. Anilkumar. An. Acad.

Bras. Cienc., 2008,80, 253.

148. V. Ramakrishna, K. Basavaiah. Ind. J. Chem. Technol., 2005, 12,543.

149. K. Basavaiah, U. Chandrashekar, P. Nagegowda. Ind. J. Chem. Technol., 2005,

12, 149.

150. K. Tharpa, K. Basavaiah. J. Anal. Chem., 2009,64, 1193.

151. K. Basavaiah, K. Tharpa. Chem. Ind. Chem. Eng. Q., 2008, 14, 205.

152. B.G. Chaudhari, H.R. Parmar. Int. J. Pharm. Qual. Assur., 2010, 2, 9.

153. K.V.V. Satyanarayana, P.N. Rao. E-J. Chem., 2011, 8, 269.

154. K. Basavaiah, U.R. Anilkumar, K. Tharpa. Chem. Ind. Chem. Eng. Q., 2008,

14,185.

155. B. Narayana, K. Veena. J. Mex. Chem. Soc, 2010, 54, 98.

156. K. Basavaiah, V. Ramakrishna, U.R. Anilkumar, B.C. Somashekar. Ecl. Quím.,

2007, 32, 57

157. A.M. El-Didamony. Drug Test. Anal., 2010, 2,122.

158. K. Basavaiah, U. Chandrashekar, H.C. Prameela, P. Nagegowda, Acta Cienc.

Indica, Chem., 2003, XXIX, 25.

159. A.M. El-Didamony, E.A.H. Erfan. Spectrochim. Acta Part A., 2010, 75, 1138.

160. C.S.P. Sastry, S.G. Rao, J.S.V.L.M. Rao and P.Y. Naidu, Anal. Lett., 1997, 30,

2377.

161. A.S. Amin and G.H. Ragab, Anal, Sci., 2003, 19, 247.

162. C.S.N. Sharma, C. Kamala Sastry and C.S.P. Sastry, Acta Cieneia Indica Chem.,

2002, 28, 221.

163. C.S.P. Sastry and J.S.V.L.M. Rao, East. Pharm., 1996, 39, 117.

164. C.S.P. Sastry, V.A.N. Sarma, U.V. Prasad and C.S.R. Lakshmi, Indian J. Pharm,

Sci, 1997, 59, 161.

165. K. Basavaiah, U.R. Anilkumar, J. Mex. Chem. Soc., 2007, 51, 106.

166. K. Basavaiah, K. Tharpa, J. Mex. Chem. Soc., 2008, 52, 193.

167. J.H. Miyawa, S.G. Schulman In: L. Ohannesian, A. J. Streeter, Editors,

“Handbook of pharmaceuticals analysis”, Marcel Dekker, Inc., New York, 2002

(Chapter 5).


96

168. K. Basavaiah, N. Rajendraprasad, P.J. Ramesh, K.B. Vinay. Thai J. Pharm. Sci.

2010, 34, 146.

169. K. Basavaiah, N. Rajendraprasad, M.X. Cijo, K.B. Vinay, P.J. Ramesh. J. Sci. Ind.

Res. 2011, 70, 346.

170. G.C. Gomes, H.R.N. Salgado. Acta Farm. Bonaerense, 2005, 24, 406.

171. R.N. Saha, C. Sajeev, P.R. Jadhav, S.P. Patil, N. Srinivasan. J. Pharm. Biomed.

Anal., 2002, 28,741.

172. C. Sajeev, P.R. Jadhav, D. RaviShankar, R.N. Saha. Anal. Chem. Acta., 2002, 463,

207.

173. M.S. Arayne, N. Sultana, F. Hussain, S.A. Ali. J. Anal. Chem., 2007, 62, 536.

174. M.I. Walash, A. El-Brashy, N. El-Enany, M.E. Kamel. Pharm. Chem. J., 2009,

43, 697.

175. E.M.A. Orsine, J.L.S. Martins. Anal. Lett., 1993, 26,1933.

176. E.R.M. Hackmann, E.A.S. Gianotto, M.I.R.M. Santoro. Anal. Lett., 1993, 26,259.

177. E.R.M. Hackmann, M.M.F. Nery, M.I.R.M. Santoro. Anal. Lett., 1994, 27,363.

178. O.Z. Devi, K. Basavaiah, P.J. Ramesh, K.B. Vinay, Farmacia, 2011, 59, 647.

179. B.R. Matthews, Drug Dev. Ind. Pharm. 1999, 25, 831.

180. ICH-Q1A (R2), Stability testing of new drug substances and products,

International Conference on Harmonisation, Geneva, February, 2003.

181. D.A. Skoog, D.M. West, “Principles of Instrumental Analysis”, Sounders College

Publications, USA.1980.

182. B.G. Nagavi, “Laboratory Handbook of Instrumental Drug Analysis Advanced

Pharmaceutical Analysis”, Vallabh Prakashan, India, 2009.

183. Thomas, “Ultraviolet and visible spectroscopy”, John willey and sons ltd. U.K.,

2nd ed., 1996, 131.

184. D. Harvey, “Modern Analytical Chemistry”- 1st Edn. James M. Smith Publishers,

2000.

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