chapter 2 titrimetric, spectrophotometric...

CHAPTER 2

TITRIMETRIC, SPECTROPHOTOMETRIC

AND CHROMATOGRAPHIC ASSAY OF

OFLOXACIN

18

SECTION 2.0

DRUG PROFILE AND LITERATURE SURVEY

2.0.1 DRUG PROFILE

Ofloxacin (OFX) is chemically known as (RS)-7-fluoro-2-methyl-6-(4-

methylpiperazine-1-yl)-10-oxo-4-oxa-1-azatricyclo[7.3.1.0]trideca-5(13),6,8,11-tetraene-

11-carboxylic acid. Its empirical formula is C18H20FN3O4, with a molecular weight of

361.4. The structural formula is:

O

NN

NCH

3

F

O

OH

O

CH3

OFX is a pale yellow or bright yellow crystalline powder. It is slightly soluble in

water, methanol and methylene chloride, soluble in glacial acetic acid.

OFX is a new synthetic second generation fluoroquinolone antibiotic with a broad

spectrum of activity against gram-positive and gram-negative bacteria [1]. OFX is widely

used in the treatment of respiratory tract and urinary tract infections [2]. OFX possesses

two relevant ionizable functional groups: a basic piperazinyl group and a carboxylic

group. The carboxylic group and the carbonyl groups are required for antimicrobial

activity. OFX is official in the United States Pharmacopoeia (USP) [3] and the British

Pharmacopoeia (BP) [4], which recommends non-aqueous titrimetry with acetous

perchloric acid towards potentiometric end point [3, 4]. BP also recommends a High

performance liquid chromatography (HPLC) [3] technique for the assay of OFX in bulk

and dosage forms.

19

2.0.2.0 LITERATURE SURVEY - ANALYTICAL FRAMEWORK

2.0.2.1 Titrimetric methods

Other than official methods [3, 4] no titimetric methods were found in the

literature for the determination of OFX in pharmaceuticals. The official methods involve

the titration of the drug with 0.1 M perchloric acid towards potentiometric end point.

Only one visual titrimetric procedure was found in the literature for the assay of OFX in

bulk drug as well as in its formulations [5]. The method was based on the bromination of

the drug in HCl medium followed by the determination of unreacted NBS iodometrically.

The method was applicable over an amount of 1-8 mg OFX.

2.0.2.2 Spectrophotometric methods

Several visible spectrophotometric methods [5-12] employing different reactions

have been reported for the assay of OFX either alone or with some other therapeutic

agents. Vinay et al.,[5] developed two methods based on bromination of the drug,

followed by the determination of unreacted NBS by reacting with fixed amount of indigo

carmine or metanil yellow. The Sastry et al., [6] have reported a method based on

coupling reaction using Ce(IV). The method involved the measurement of absorbance of

oxidative coupled product formed between Ce(IV) and 3-methyl-2benzothiazoline

hydrazone. Absorbance of the colored product was measured at 630 nm and applicable

over a concentration range of 1-10 µg ml-1

. OFX was determined by complex formation

reaction using Fe(III) nitrate nonahydrate and absorbance of the amber colored

chromogen was measured at 370 nm [7]. By using the similar reaction, Mathur et al, [8]

developed a method using FeCl3 in HCl medium. The method was found to be less

sensitive and applicable over a concentration range of 20-160 µg ml-1

. A method based

on the measurement of 2:1 complex formed between OFX and iron(III) in H2SO4

medium, followed by the measurement of absorbance of the complex at 420 nm [9]. The

method was applicable to pharmaceutical formulations as well as in spiked human urine


. Issa et al., [10] have developed three

methods based on the formation of chloroform extractable ion-pair complexes between

OFX and bromophenol blue (BPB), bromothymol blue (BTB) and bromocresol purple

(BCP). The methods were applicable over a concentration of 5-25, 2-15 and 2-20 µg ml-1

20

for BPB, BTB and BCP methods. Another two extractive spectrophotometric methods

were developed by Sastry et al., [11]. The methods were based on formation of

chloroform soluble ion-pair complex between Tropaelin 000 (TP 000) and Supracene

violet 3B (SV 3B). Absorbance of the ion-pair complexes were measured at 485 and 575

nm for TP 000 and SV 3B methods. Patel et al., [12] have developed a method based on

the formation of pink colored chromogen having maximum absorbance at 552.5 nm, by

the reaction between OFX and citric acid-acetic anhydride. The method was applicable


.

Vinay et al., [9] developed two UV-spectrophotometric methods in

pharmaceutical formulations and in spiked human urine. The methods were based on the

the measurement of the absorbance of the drug in 0.1 M HCl at 293 nm and in 0.1 M

NaOH at 287 nm. Another UV-spectrophotometric method [14] has been reported by for

the assay of OFX in pharmaceuticals and spiked human urine.

2.0.2.3 Chromatographic techniques

High performance Liquid Chromatography (HPLC), despite its versatility, speed,

sensitivity and specificity, has sparsely been employed for the assay of OFX. The

technique has been applied for the assay of OFX in pharmaceuticals [14-18]. A method

was found in the literature using octadecyl silane chemically bonded to porous silica

particles (Waters Spherisorb, 5 µ ODS 1, 4.6 x 150 mm) column with a 0.05 M phosphate

buffer-acetonitrile (65:35, v/v) as mobile phase and the detection was made at 210 nm.

The method was rectilinear over a concentration range of 24-120 µg ml-1

[14]. Kalta et

al, [15] developed a method using acetonitrile: potassium dihydrogen phosphate buffer

(80:20) with 0.5% v/v of triethylamine and the pH was adjusted to 2.5 by adding

orthophosphoric acid. Phenomenex C18 column with 0.24% sodium lauryl sulphate with

a mobile phase consisting of acetonitrile: acetic acid (pH-4.0) (58:40:02) and detection

made at 295 nm. The method was less sensitive and found rectilinear over a range of 160-

240 µg ml-1

[16]. A method was developed by Dharuman et al., [17] using Kromasil C8

(5µ, 15 cm × 4.6 mm id) column with mobile phase consisting of 0.5%v/v triethylamine

buffer of pH 3.0 and acetonitrile in the ratio of 73: 27 and detection was made at 303

nm.C-18 column (RP-18, 5µ) coupled with a guard column of same material, in isocratic

mode with mobile phase mixture of acetonitrile: water: tri ethylamine (25:75:1) followed

21

by UV-detection at 300 nm [18].

HPLC method was also used for the assay of OFX in blood serum [19-21], blood

plasma [22, 23], waste waters [24, 25] and urine [26, 27]. Ligand exchange HPLC has

recently been applied for the direct determination of OFX enantiomers in human urine

[28]. A stability indicating high-performance thin layer chromatographic [29] assay has

also been described for OFX.

2.0.2.4 Other techniques

Various other techniques have been reported for the determination of OFX in pure

drug, pharmaceutical dosage forms and in biological fluids, and they include

spectrofluorimetry [30-34], potentiometry and conductometry [35], polarography and

voltammetry [36], differential pulse polarography [37], ion-selective electrode based

potentiometry [38], adsorptive stripping voltammetry [39], voltammetry, linear sweep

voltammetry and electrochemical impedance spectroscopy [40], chemiluminescence

spectrometry [41-45] and capillary electrophoresis [46-48].

Many of the above techniques are deficient on simplicity, cost-effectiveness and

easy accessibility. The reported HPLC methods for OFX in dosage forms [14-18] either

require an internal standard [14, 17] or are applicable for combined dosage forms [14-18].

Assays of OFX in urine [26-28] are generally complex and lengthy, mostly because of

the sample preparation, like solid-phase extraction or liquid-liquid extraction.

Titrimetry and visible spectrophotometry may serve as useful alternatives to many

of the aforesaid sophisticated techniques because of their cost-effectiveness, ease of

operation, sensitivity, remarkable accuracy and precision and wide applicability. The

reported titrimetric method have disadvantages of narrow dynamic range, use of unstable

reagent, use of hazardous chemical etc. The reported spectrophotometric methods, though

sensitive, suffer from one or the other disadvantage such as use of boiling [12] or

extraction step [10, 11], strict pH control [7, 10, 11 ], use of organic solvent/expensive

chemical [6] and measurement of less sensitive species [8].

Realizing the importance of titrimetry in pharmaceutical analysis, and

inadequacies of the currently available visible spectrophotometric methods, the author

has made an attempt to develop new titrimetric and visible spectrophotometric methods

for the determination of OFX. The methods use cerium(IV) as the oxidimetric reagent,

22

and 0-dianisidine and p-tolidine as auxiliary reagents. In addition, extraction-free

spectrophotometric procedures using anionic dyes as ion-pair reagents have also been

developed. Using a different column and mobile phase, a new and sensitive HPLC

method with a wide linear dynamic range was also developed. The details concerning the

method development and method validation are presented in the following sections of

this chapter.

23

SECTION 2.1

TITRIMETRIC ASSAY OF OFLOXACIN IN PHARMACEUTICALS USING

CERIUM(IV) AS AN OXIDIMETRIC REAGENT

2.1.1.0 INTRODUCTION

Cerium(IV) sulfate is a chemical compound which is frequently used as an

oxidising agent in titration. The orange-colored cerium(IV) ion is reduced to the colorless

cerium(III) ion. The potential for reduction is high, about +1.44V. It is a very safe

oxidizer, one of the most nontoxic ones [49].

Ce4+ + e-Ce3+

It can be used only in acid solution, best in 0.5 M or higher concentrations.

Solutions of cerium(IV) sulphate (Ce(IV)SO4) in dilute H2SO4 are stable even at boiling

temperature [50].

Ce(IV) has been used for the titrimetric analysis of some pharmaceuticals [51-53],

ascorbic acid [54] etc. It has also been used for the spectrophotometric assay of

simvastatin [55], olanzapine [56, 57], lansoprazole [58], ciprofloxacin [59], propranolol

[60], atenolol [61], methylthiouracil [62], antiamoebics and anthelmintics [63] and

diuretics [64], methdilazine[65], ganciclovir [66] and carbamazepine [67].

Although, Ce(IV) has been extensively used for the titrimetric and

spectrophotometric assay of several pharmaceuticals, there is no report found for the

assay of OFX using cerium(IV). In this section, titrimetric assays of OFX are described

based on the oxidation of drug by Ce(IV). In direct titrimetry, the acidified solution of

OFX is titrated directly with Ce(IV) using ferroin as indicator, and indirect titrimetry

involves the addition of known excess of Ce(IV) to an acidified solution of OFX followed

by the determination of unreacted oxidant by back titration with ammonium ferrous

sulphate (FAS) using the same ferroin indicator. The details of the method development

and validation are presented in this Section 2.1.

24

2.1.2.0 EXPERIMENTAL

2.1.2.1 Reagents and Standards

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

distilled water was used throughout the experiment.

Cerium(IV) sulphate (0.025 M): The solution was prepared by dissolving 5.05 g of the

chemical (LOBA Chemie, Mumbai,India, assay 99.9%) in 14 ml of conc. H2SO4 and

diluting to volume in a 500 ml calibrated flask with water, mixed well, filtered using

glass wool, standardized [68] and used in both the methods.

FAS (0.025 M): The solution of FAS (0.05 M) was prepared by dissolving 4.9 g of the

chemical (LOBA Chemie, Mumbai, India, assay 99-101%) in water containing 5 ml of

2M H2SO4 and diluted to volume in a 500 ml calibrated flask with water and used in

indirect titrimetry.

Sulphuric acid (2 M): Prepared by appropriate dilution of the conc. H2SO4 (S. D. Fine

Chem, Mumbai, India, sp. gr. 1.84) with water.

Ferroin indicator: Prepared by dissolving 0.742 g of 1,10-phenanthroline monohydrate

in 50 ml of 0.025 M ferrous sulphate solution (0.348 g of ferrous sulphate heptahydrate in

50 ml water).

Preparation of Standard OFX Solution

Pure OFX (Pharmaceutical grade) sample was kindly provided by Cipla India Ltd,

Mumbai, India, and was used as received. Standard OFX solution (1.5 mg ml-1

) was

prepared by dissolving calculated quantity of pure drug in 0.1 M H2SO4.

Two brands of tablets containing OFX, Zenflox-400 (Mankind Pharma Pvt Ltd.,

New Delhi, India) and Ofloxin-400 (J. B. Chemicals and Pharmaceuticals Ltd, Mumbai,

India), used in the investigation were purchased from local commercial sources.

2.1.3.0 ASSAY PROCEDURES

2.1.3.1 Direct titrimetry

A 10 ml aliquot of pure drug solution containing 1.5-15 mg of OFX was

accurately measured and transferred into a 100 ml titration flask. Five ml of 2 M H2SO4

was added, and titrated with 0.025 M Ce(IV)SO4 using 1 drops of ferroin as indicator

until the sky blue color appeared.

25

2.1.3.2 Indirect titrimetry

A 10 ml aliquot of standard solution containing 1.5-15 mg of OFX was measured

accurately and taken in a 100 ml titration flask, and five ml of 2 M H2SO4 was added to

the flask. By means of a pipette, 10 ml of 0.025 M Ce(IV) was added, content mixed and

the flask kept aside for 15 min. Then, the unreacted Ce(IV) was titrated against 0.025 M

FAS using ferroin as indicator to a first appearance of orange red color. A blank titration

was performed with 10 ml water in place of OFX solution.

The amount of OFX in the aliquot was computed from the formula:

Amount (mg) = VMwS/n

where V = ml of Ce(IV) reacted

Mw = relative molecular mass of drug

S = strength of Ce(IV), moles l-1

n = number of moles of Ce(IV) reacting with per mole of OFX.

2.1.3.3 Procedure for tablets

Ten tablets were accurately weighed and powdered. A portion equivalent to 150

mg OFX was accurately weighed and transferred into a 100 ml calibrated flask, 60 ml of

0.1 M H2SO4 was added to the flask and the content was shaken thoroughly for 15-20

min to extract the drug into the liquid phase; the volume was finally diluted to the mark

with the same acid, mixed well and filtered using a Whatman No. 42 filter paper. The

filtrate equivalent to 1.5 mg ml-1

OFX was assayed by both the methods.

2.1.3.4 Placebo blank and synthetic mixture analysis

A placebo blank of commonly employed tablet excipients, consisting of talc (50

mg), starch (50 mg), acacia (20 mg), methyl cellulose (30 mg), sodium citrate (20 mg),

magnesium stearate (20 mg) and sodium alginate (2 mg) was made and its solution was

prepared by taking about 30 mg as described under Section 2.1.3.3.

To 50 mg placebo blank of the composition described above, 50 mg and 100 mg

of OFX was added separately and homogenized, transferred to two separate 50 ml

calibrated flask and the solution was prepared as described under Section 2.1.3.3.

2.1.4.0 RESULTS AND DISCUSSION

From the preliminary experiments, OFX was found to undergo oxidation with

Ce(IV). Based on this observation, two titrimetric methods were developed. In direct

26

titrimetry, OFX was titrated directly with Ce(IV) using ferroin as indicator, and indirect

titrimetry involved back titration of the unreacted Ce(IV) with FAS using the same

indicator.

2.1.4.1 Optimization of variables

The experimental variables which provided accurate and precise results were optimized.

In both the methods, the reactions were carried out in H2SO4 medium, since in

this medium the reactions were found to be stoichiometric. In direct titrimetry,

reproducible and regular stoichiometry was obtained when 0.33-1.67 M H2SO4

concentration was maintained. Hence, 5 ml of 2 M H2SO4 solution in a total volume of 15

ml (0.67 M overall) was found to be the most suitable concentration for a quantitative

reaction between OFX and Ce(IV). In indirect titrimetry, the reaction stoichiometry was

unaffected when 0.2-1.0 M H2SO4 was maintained. Hence, 5 ml of 2 M H2SO4 in a total

volume of 25 ml (0.4 M overall) was used. At the laboratory temperature (30 ± 2o C), the

time required for complete oxidation was found to be 15 min and the contact time up to

60 min, a small amount of Ce(IV) is consumed but without producing a definite

stoichiometry. Hence, the reaction time of 15 min was fixed in method B. In both the

methods, the reaction stoichiometry of OFX: Ce(IV) was calculated to be 1:5 in the range

of 1.5-15 mg in both the methods.

2.1.4.2 Method validation

The proposed methods were validated for precision, accuracy, robustness

and ruggedness and specificity, according to the International Conference on

Harmonization (ICH) [69] guidelines.

Quantitative data

Over the range investigated 1.5-15 mg, a fixed stoichiometry of 1:5 (OFX:

Ce(IV)) in both the methods was obtained which served as the basis for calculations.

Precision and accuracy

Intra-day precision and accuracy of the proposed methods were evaluated by

replicate analysis (n=7) of standards at three different levels on the same day. Inter-day

precision and accuracy were determined by assaying the standards at the same amount

levels on five consecutive days. Precision and accuracy were based on the calculated

27

relative standard deviation (RSD, %) and relative error (RE, %) of the found amount

compared to the taken one, respectively (Table 2.1.1).

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

Method

OFX

taken,

mg

Intra-day accuracy and

precision

(n=7)

Inter-day accuracy and

precision

(n=7)

OFX found %RE %RSD OFX

found %RE %RSD

Direct

titrimetry

6.00 5.92 1.32 2.40 6.09 1.50 2.51

9.00 8.86 1.56 1.20 9.18 2.00 1.67

12.0 12.10 0.83 2.15 12.20 1.67 1.97

Indirect

titrimetry

6.00 6.19 3.17 2.40 5.80 3.26 2.57

9.00 8.74 2.89 1.46 9.27 2.95 1.73

12.0 12.18 1.50 1.20 12.28 2.32 1.54

RE: Relative error and RSD: Relative standard deviation.

Robustness and ruggedness

Method robustness was tested by making small incremental change in H2SO4

concentration in both the methods, and in method B, even reaction time was slightly

altered. To check the ruggedness, analysis was performed by four different analysts; and

on three different burettes by the same analyst. The robustness and the ruggedness were

checked at three different drug levels. 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. 2.1.2.

Selectivity

The proposed methods were tested for selectivity by placebo blank and synthetic

mixture analyses. A convenient aliquot of the placebo blank solution was subjected to

analysis according to the recommended procedures. In both the cases, there was no

interference by the inactive ingredients as shown by near zero ml consumption of Ce(IV).

A separate experiment was performed with the synthetic mixture. The analysis of

synthetic mixture solution yielded percent recoveries which ranged of 95.71- 104.3

with standard deviation of 1.17 – 1.36 in both the cases. The results of this study are

presented in Table 2.1.3 indicating that the inactive ingredients did not interfere in the

assay. These results further demonstrate the accuracy as well as the precision of the

proposed methods.

28

Table 2.1.3 Results of recovery of the drug from synthetic mixture

*Mean value of five determinations

Application to tablets

In order to evaluate the analytical applicability of the proposed methods to the

quantification of OFX in commercial tablets, the results obtained by the proposed

methods were compared to those of the reference method [4] by applying Student’s t-test

for accuracy and F-test for precision. The reference method involved the titration of OFX

in anhydrous acetic acid with acetous perchloric acid to potentiometric end point

detection. The results (Table 2.1.4) show that the Student’s t- and F-values at 95 %

confidence level are less than the theoretical values, which confirmed that there is a good

agreement between the results obtained by the proposed methods and the reference

method with respect to accuracy and precision.

Table 2.1.2 Results of robustness and ruggedness expressed as intermediate

precision (%RSD)

Method

OFX

taken,

mg

Method robustness

Method ruggedness Parameter altered

H2SO4 , ml*

% RSD,

(n = 3)

Reaction

time**

,

min

Inter-

analysts’

% RSD,

(n = 4)

Inter-

burettes’

% RSD,

(n = 3)

Direct

titrimetry

6.00 1.19 - 0.87 1.21

9.00 1.07 - 0.82 1.14

12.0 1.02 - 0.71 1.31

Indirect

titrimetry

6.00 1.16 1.09 1.03 1.25

9.00 1.07 1.12 0.96 1.29

12.00 0.89 0.98 0.83 1.35 * H2SO4 volumes used were 4.8, 5.0 and 5.2 ml of 2 M in both the methods. **Reaction times altered were 13, 15 and 17 min in method B.

Method OFX in synthetic

mixture taken, mg

OFX recovered*

(Percent ± SD)

Direct titrimetry

6.00 103.5 ± 1.23

9.00 102.9 ± 1.17

12.00 104.3 ± 1.21

Indirect titrimetry

6.00 95.97 ± 1.29

9.00 96.78 ±1.24

12.00 95.71 ± 1.36

29

Recovery studies

The accuracy and validity of the proposed methods were further ascertained by

performing recovery studies. Pre-analysed tablet powder was spiked with pure OFX at

three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was

found by the proposed methods. In both the cases, the added OFX recovery percentage

values ranged of 94.58 – 101.0 % with standard deviation of 1.13 – 1.32 (Table 2.1.5)

indicating that the recovery was good, and that the co formulated substance did not

interfere in the determination.

Table 2.1.4 Results of analysis of tablets by the proposed methods and statistical

comparison with reference method

Tablet brand

name

Label

claim,

mg/tablet

Found* (Percent of label claim ± SD)

(n=5)

Reference

method

Direct titrimetry Indirect titrimetry

Zenflox** 400

98.12 ± 1.18

96.58 ± 1.24 t = 2.01

F = 1.10

96.50 ± 1.38 t = 2.00

F = 1.37

Ofloxin***

400

99.14 ± 1.07

98.42 ± 1.11

t = 1.04

F = 1.08

97.13 ± 1.36 t = 2.57

F = 1.62 *Mean value of five determinations. ** Mankind Pharma Pvt Ltd., New Delhi, India;*** J. B. Chemicals and Pharmaceuticals Ltd,

Mumbai, India.

The value of t (tabulated) at 95 % confidence level and for four degrees of freedom is 2.77.

The value of F (tabulated) at 95 % confidence level and for four degrees of freedom is 6.39.

Table 2.1.5 Results of recovery study by standard addition method

Method Tablet

studied

OFX in

tablet,

mg

Pure

OFX

added,

mg

Total

found, mg

Pure OFX

recovered*

Percent ± SD

Direct

titrimetry

Ofloxin-

400

5.91 3.00 8.94 101.0 ± 1.32

5.91 6.00 11.81 98.33 ± 1.23

5.91 9.00 14.42 94.58 ± 1.15

Indirect

titrimetry

Ofloxin-

400

3.89 2.00 5.81 96.15 ± 1.21

3.89 4.00 7.77 97.08 ± 1.13

3.89 6.00 9.85 99.38 ± 1.28 *Mean value of three measurements.

30

SECTION 2.2

SPECTROPHOTOMETRIC DETERMINATION OF OFLOXACIN IN

PHARMACEUTICALS BY REDOX REACTION USING CERIUM(IV)


The chemistry and applicability of Ce(IV) has been reviewed in Section 2.1.1.0.

In this Section, 2.2, two spectrophotometric methods (method A and method B) are

described based on the oxidation of OFX by a measured excess of cerium(IV) in H2SO4

medium. The unreacted oxidant was determined by reacting with either p-toluidine (p-

TD) and measuring the absorbance at 525 nm (method A) or o-dianisidine (o-DA) and

measuring the absorbance at 470 nm (method B). The proposed methods were validated

for linearity, sensitivity, selectivity, robustness and ruggedness besides intra-day and

inter-day precision and accuracy.


2.2.2.1 Apparatus

All absorption measurements were made using a Systronics model 106 digital

spectrophotometer (Systronica Ltd, Ahmedabad, India) with 1 cm path length quartz

cells.

2.2.2.2 Reagents and standards

All chemicals used were of analytical reagent grade. Distilled water was used

throughout the investigation.

Cerium(IV) (6000 and 300 µg ml-1

): An approximately 0.025 M cerium (IV) sulphate was

prepared by dissolving 5.1 g of chemical (LOBA Chemie, Mumbai,India, 99.9 % pure) in

0.5 M H2SO4 with the aid of heat, and filtered using Whatmen No 42 filter paper, and

diluted to 500 ml in a calibrated flask with the same acid and standardized against standard

0.025 M ferrous ammonium sulphate in the presence of ferroin as indicator [64]. The

solution was then diluted appropriately with 0.5 M H2SO4 to get working concentrations of

6000 µg ml-1

and 300 µg ml-1

Ce(IV) for use in method A and method B, respectively.

Acetic acid (1:1 and 3:2 v/v): In two different 100 ml calibrated flasks 50 ml and 60 ml of

glacial acetic acid (S.D. Fine Chem Ltd, Mumbai, India, purity 100%) were diluted upto

the mark with water to get 1:1 and 3:2 acetic acid, respectively.

31

p-Toluidine, (p-TD) (1 %): Prepared by dissolving 1 g of the chemical (Merk, Mumbai,

India, 99 % assay) in 1:1 acetic acid and diluted to 100 ml with the same solvent in a

calibrated flask.

o-Dianisidine, o-DA (0.01 %): The solution was prepared by dissolving accurately

weighed 10 mg of the chemical (Loba chemie, Mumbai, India, 99 % assay) in 100 ml of

3:2 acetic acid.

Sulphuric acid (10 M, 5 M and 0.1 M): Prepared by diluting the concentrated acid

(Merck, Mumbai, India, Sp. gr. 1.84, 98%) appropriately with water to get 10 M, 5 M and

0.1 M acid solutions.

Preparation of standard OFX solutions (300 and 20 µg ml-1

)

A stock standard solution equivalent to 600 µg ml-1

was prepared by dissolving 60

mg of pure OFX (99.8% pure) in 0.1 M H2SO4 and diluting to the mark in a 100 ml

calibrated flask with the same acid. The stock solution was diluted stepwise with the same

acid to obtain a working concentration of 300 and 20 µg ml-1

for method A and method B,

respectively.

Two brands of tablets, Zenflox-400 (Mankind Pharma Pvt Ltd., New Delhi, India)

and Ofloxin-400 (J. B. Chemicals and Pharmaceuticals Ltd, Mumbai, India), used in the

investigation were purchased from local market and used in the investigation.


2.2.3.1 Method A (using p-TD)

Into a series of 10.0 ml calibrated flasks, 0.0, 1.0, 2.0, 3.0 and 4.0 ml of 300 µg ml-1

standard OFX solution were placed by means of a microburette, and the total volume was

adjusted to 4 ml by adding requisite quantity of 0.1 M H2SO4. To each flask were added 2

ml of 5 M H2SO4 and 1 ml of 6000 µg ml-1

cerium(IV) sulphate, the last being measured

accurately by means of a microbuttete. The content was mixed and let stand for 5 min with

occasional shaking. Finally, 1 ml of 1% p-TD solution was added and diluted to the mark

with 1 M H2SO4, mixed and absorbance of each solution measured at 525 nm against a

water blank.

2.2.3.2 Method B (using 0-DA)

Different aliquots containing 0.0, 0.5, 1.0, 1.5 and 2.0 ml of 20 µg ml-1

of drug

solution were accurately measured and transferred into a series of 10 ml calibrated flasks

32

and the total volume was brought to 2 ml with 0.1 M H2SO4. To each flask were added 5

ml of 10 M H2SO4 and 1 ml of 300 µg ml-1

cerium(IV) sulphate, the last being measured

accurately. The content was mixed and kept aside for 20 min before adding 1 ml of 0.01 %

o-DA to each flask. The volume was brought to the mark with 10 M H2SO4 immediately,

mixed and absorbance measured at 470 nm against water.

In either method, a calibration graph was prepared by plotting the measured

absorbance vs concentration (µg ml-1

) of OFX, and the concentration of the unknown was

read from the calibration graph or computed from the regression equation derived using the

absorbance-concentration data.


Twenty tablets were weighed and pulversized. A quantity of tablet powder

containing 60 mg of OFX was transferred into a 100 ml calibrated flask. The content was

shaken with about 70 ml of 0.1 M H2SO4 for 20 min. The mixture was diluted to the mark

with the same acid. It was filtered using Whatman No 42 filter paper. First 10 ml portion

of the filtrate was discarded and the resulting solution (600 µg ml-1

) was diluted to 300

µg ml-1

and used in method A. The tablet extract was diluted to get 20 µg ml-1

OFX and

subjected to analysis by following the procedure described under method B.

2.2.3.4 Placebo blank and synthetic mixtures analysis

Twenty mg of the placebo blank was extracted and its solution was prepared as described

under tablets; and a convenient aliquot was subjected to analysis.

A synthetic mixture was prepared by adding pure OFX (100 mg) to the above

mentioned placebo blank and the mixture was homogenized. Synthetic mixture

containing 60 mg of OFX was weighed and its solution was prepared as described under

section 2.2.3.3. Three different aliquots were subjected to analysis by the general

procedure described in method A after diluting to 300 µg ml-1

with 0.1 M H2SO4. The

stock solution was diluted to get 20 µg ml-1

and used for the assay in method B. The

concentration of OFX was found from the calibration graph or from the regression

equation.


Ce(IV) has the ability to rapidly oxidize OFX and to form colored products with

two amines viz. p-toluidine and o-dianisidine [70, 71], as these reactions were

33

successfully employed to develop two indirect procedures for the determination of OFX

in pharmaceuticals. The methods are based on the oxidation of OFX in H2SO4 medium

by a measured excess of Ce(IV) followed by the determination of the residual oxidant by

reacting with p-toluidine or o-dianisidine and measuring the absorbance of the resultant

colored oxidation product at 525 or 470 nm. The possible reaction scheme is shown in

Scheme 2.2.1. The measured absorbance was found to decrease linearly with increasing

concentration of OFX, thus forming the basis for the quantification of drug.

OFX + Ce(IV)H+

Oxidation product

of OFXUnreacted Ce(IV)

+

Unreacted Ce(IV)

NH2

H2N

O

O+

NH2+

H2N

O

O

+

Unreacted Ce(IV)

+

NH2

CH3

p-TD

o-DAOxidation product of o-DA

( measured at 470 nm)

CH2

NH2+

Oxidation product of p-TD

(measured at 525 nm) Method A

H+

Method B

H+

Scheme 2.2.1 Possible reaction scheme for the reaction between Ce(IV) and p-TD and o-

DA in H2SO4 medium.

When a fixed concentration of Ce(IV)SO4 is reacted with increasing concentrations

of OFX, there occurred a concomitant decrease in the concentration of Ce(IV). When

treated with a fixed quantity of p-TD or o-DA, this resulted in decreasing concentrations of

the resultant colored product showing a proportional decrease in absorbance at the

respective analytical wavelength. The decrease in absorbance of the resultant color

compared to the reagent blank was found to be proportional to the concentration of OFX,

and this formed the basis of determination.

34


Various experimental variables involved in the oxidation of OFX by Ce(IV) and the

latter’s determination with p-TD or o-DA were optimized. Preliminary experiments with p-

TD or o-DA were performed to determine the upper Beer’s law limits for Ce(IV) and these

were found to be 600 µg ml-1

and 30 µg ml-1

with p-TD and o-DA, respectively, under the

optimized experimental conditions. Hence, different concentrations of OFX were reacted

with 1 ml of 6000 µg ml-1

or 300 µg ml-1

Ce(IV) in acid medium before determining the

unreacted oxidant with either p-TD or o-DA. This enabled to evaluate the concentration

ranges over which OFX could be determined by either method.

For both steps involved in the assay, i.e., the oxidation of OFX by Ce(IV) and the

latter’s determination of with p-TD or o-DA, H2SO4 medium was selected after preliminary

experiments. In method A, 2 ml of 5 M H2SO4 in a total volume of 6 ml (overall acidity

~1.7 M) and in method B, 5 ml of 10 M H2SO4 in a total volume of 7 ml (overall ~7 M)

was adequate for the oxidation in 5 min and any delay upto 20 min had no effect in method

A whereas in method B, 10 min was required for complete oxidation of OFX. At lower

acid concentrations oxidation reaction was found to be incomplete. In method A, the

purple color formed (within 5 min) on adding p-TD and peaking at 525 nm was stable upto

90 min; where as in method B, 10 min standing time was necessary for the formation of red

colored product (λmax 470 nm) with a stability period of 20 min. The colored products

formed with p-TD and o-DA required higher H2SO4 concentration for stability, and hence,

1 M and 10 M H2SO4 were used as diluent after adding p-TD and o-DA to the residual

Ce(IV) respectively.

For each system, two blanks were prepared. The first blank, which contained all

reactants except OFX, gave maximum absorbance (intercept of the calibration graph). The

second blank was prepared in the absence of OFX and Ce(IV)SO4 to assess the

contribution of other reactants to the signal (absorbance) of the system. Since the second

blank had insignificant absorbance in both methods, measurements were made against

water.


The proposed methods were validated for linearity, sensitivity, precision, accuracy,

robustness, ruggedness, selectivity and recovery.

35

Linearity and sensitivity

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

OFX in the ranges given in Table 2.2.1. The graphs are described by the regression

equation:

Y = a + bX

(where Y = absorbance of 1-cm layer of solution; a = intercept; b = slope and X =

concentration in µg ml-1

). Here, Beer’s law is obeyed in the inverse manner. Regression

analysis of the Beer’s law data using the method of least squares was made to evaluate

the slope (b), intercept (a) and correlation coefficient (r) for each system and the values

are presented in Table 2.2.1. A plot of log absorbance and log concentration, yielded

straight lines with slopes equal to 1.00 and 1.01 for method A and method B,

respectively, further establishing the linear relationship between the two variables. The

optical characteristics such as Beer’s law limits, molar absorptivity and Sandell

sensitivity values of both the methods are also given in Table 2.2.1. The limits of

detection (LOD) and quantitation (LOQ) calculated according to ICH guidelines [69]

using the formulae: LOD = 3.3 S/b and LOQ = 10 S/b, (where S is the standard deviation

of blank absorbance values, and b is the slope of the calibration plot) are also presented in

Table 2.2.1. The high values of ε and low values of Sandell sensitivity and LOD indicate

the high sensitivity of the proposed methods.

Accuracy and precision

The assays described under “general procedures” 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) of the

methods. These assays were performed for three levels of analyte. The results of this

study are summarized in Table 2.2.2. The percentage relative standard deviation (%RSD)

values were ≤ 7.69% (intra-day) and ≤ 4.75% (inter-day) indicating good precision of the

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

measured mean concentrations and taken concentrations for OFX. Bias {bias % =

[(Concentration found - known concentration) x 100 / known concentration]} was

calculated at each concentration and these results are also presented in Table 2.2.2.

36

Percent relative error (%RE) values of ≤ 4% demonstrates the good accuracy of the

proposed methods.

Table 2.2.1 Sensitivity and regression parameters

Parameter Method A Method B

λmax, nm 525 470

Linear range, µg ml-1

0.0 – 120 0.0 – 4.0

Molar absorptivity(ε), l mol-1

cm-1

2.43 × 103 5.99 × 10

4

Sandell sensitivity*, µg cm

-2 0.1485 0.006

Limit of detection (LOD), µg ml-1

0.63 0.02

Limit of quantification (LOQ), µg ml-1

1.92 0.07

Regression equation, Y**

Intercept (a) 0.6260 0.6446

Slope (b) -0.0051 -0.1579

Standard deviation of a (Sa) 0.3654 0.3436

Standard deviation of b (Sb) 0.0033 0.0973

Variance (Sa2) 0.1339 0.1181

Regression coefficient (r) -0.9973 -0.9995 *Limit of determination as the weight in µg per ml of solution, which corresponds to an

absorbance of A = 0.001measured 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.


% RE: Percentage relative error; %RSD: Percentage relative standard deviation


The robustness of the methods was evaluated by making small incremental

changes in the volume of reagents and contact time after adding the reagents, and the

effect of the changes was studied on the absorbance of the coloured systems. The changes

had negligible influence on the results as revealed by small intermediate precision values

Method

OFX

taken,

µµµµg ml-1


precision

(n=7)


precision

(n=5)

OFX found,

µµµµg ml-1

%RE %RSD

OFX

found,

µµµµg ml-1

%RE %RSD

A

30.0

45.0

60.0

31.05

46.22

61.09

3.50

2.70

1.82

7.69

4.00

1.85

30.77

46.37

59.96

2.58

3.04

3.75

3.28

4.75

3.46

B

1.0

2.0

3.0

1.02

2.08

3.11

2.00

4.00

3.67

2.58

2.88

1.33

1.04

2.06

3.09

4.00

3.14

2.86

4.58

3.94

3.08

37

expressed as %RSD (≤ 3.62%). Method ruggedness was demonstrated having the

analysis done by four analysts, and also by a single analyst performing analysis on four

different instruments in the same laboratory. Intermediate precision values (%RSD) in

both instances were in the range 0.86 – 4.64% indicating acceptable ruggedness. The

results are presented in Table 2.2.3.

Table 2.2.3 Results of robustness and ruggedness expressed as intermediate

precision (% RSD).

Method OFX

taken*

Robustness Ruggedness

Parameter altered Inter-analysts,

(%RSD)

(n=4)

Inter-

instruments,

(%RSD)

(n=4)

Volume of

Reagent*

(%RSD)

Reaction

time**

A

30.0

60.0

90.0

2.67

3.42

1.83

3.58

2.16

2.78

1.62

1.35

0.96

3.58

4.64

2.85

B

1.0

2.0

3.0

2.45

3.62

2.76

1.64

2.37

1.85

1.26

0.86

1.35

2.72

3.14

4.35 *Volumes of reagents added were 1 ± 0.2 ml. ** In method A, reaction times studied were 5±1

min and in method B reaction times were 10±1 min.

Selectivity

To determine the selectivity of the methods, the placebo solution was subjected to

analysis according to the recommended procedures. The absorbance readings for placebo

blank were nearly zero for both the methods, inferring no interference from the placebo.

The analysis of synthetic mixture solution yielded percent recoveries in the range

98.65 - 102.6 % with standard deviation of 1.10 - 1.24. The results of this study indicate

that the inactive ingredients did not interfere in the assay. This showed a high selectivity

of the methods.


The described procedures were successfully applied to the determination of OFX

in its pharmaceutical formulations. The results obtained (Table 2.2.4) were statistically

compared with the BP method [4]. The method involved the titration of OFX in

anhydrous acetic acid with acetous perchloric acid to a potentiometric end point. The

results obtained by the proposed methods agreed well with those of reference 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 reference method at 95%

38

confidence level as summarized in Table 2.2.4. 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 reference method.

Recovery studies

To further assess the accuracy of the proposed methods, recovery experiment was

performed by applying the standard-addition technique. The recovery was assessed by

determining the agreement between the measured standard concentration and added

known concentration to the sample. The test was done by spiking the pre-analysed tablet

powder with pure OFX at three different levels (50, 100 and 150 % of the content present

in the tablet powder (taken) and the total was found by the proposed methods. Each test

was repeated three times. From this test the percentage recovery values were found in the

range of 97.58 – 104.8 with standard deviation values from 0.33 – 1.50%. Closeness of

the results to 100% showed the fairly good accuracy of the method. These results are

shown in Table 2.2.5.

Table 2.2.4 Results of analysis of tablets by the proposed methods and statistical

comparison of the results with the reference method.

Brand

name Nominal

amount,

(mg/tablet)


Reference

method

Proposed methods

Method A Method B

Zenflox** 400

103.5±1.28

101.6±2.14 t = 1.76

F = 2.79

102.1±2.65 t = 1.13

F = 1.13

Ofloxin***

400

101.3±1.08

100.8±2.58 t = 0.43

F = 5.71

99.67±2.06 t = 1.64

F = 3.64 *Average of five determinations. ** Mankind Pharma Pvt Ltd., New Delhi, India;*** J. B. Chemicals and Pharmaceuticals Ltd,

Mumbai, India.

Tabulated t value at the 95% confidence level is 2.77. Tabulated F value at the 95% confidence

level is 6.39.

39

Table 2.2.5 Results of recovery study using standard addition method

Method A Method B

OFX in

tablet

extract,

µµµµg ml-1

Pure

OFX

added,

µµµµg ml-1

Total

OFX

found,

µµµµg ml-1

Pure OFX

recovered

(Percent±SD*)

OFX in

tablet

extract,

µµµµg ml-1

Pure

OFX

added,

µµµµg ml-1

Total

OFX

found,

µµµµg ml-1

Pure OFX

recovered

(Percent±SD*)

30.48

30.48

30.48

15.0

30.0

45.0

45.28

60.51

75.32

98.65±0.86

100.1±1.06

99.65±0.56

1.02

1.02

1.02

0.5

1.0

1.5

1.51

2.04

2.59

97.58±0.77

102.3±0.89

104.5±1.50

30.24

30.24

30.24

15.0

30.0

45.0

45.44

60.42

76.59

101.3±0.89

100.6±0.33

103.0±0.65

1.00

1.00

1.00

0.5

1.0

1.5

1.52

2.02

2.57

104.0±1.25

101.6±1.08

104.8±1.11 * Mean value of three determinations

40

SECTION 2.3

SIMPLE AND SENSITIVE SPECTROPHOTOMETRIC ASSAY OF

OFLOXACIN IN PHARMACEUTICALS BASED ON ION-PAIR REACTION


Ion-association is a chemical reaction whereby ions of opposite electrical charge

come together in solution to form a distinct chemical entity. Ions of opposite charge are

naturally attracted to each other by the electrostatic force. This is described by Coulomb's

law.

F is the force of attraction, q1 and q2 are the magnitudes of the electrical charges,

ε is the dielectric constant of the medium and r is the distance between the ions [72].

Sulphonphthalein dyes are commonly used as anionic dyes to form ion-pair complex with

the nitrogenous compound present in positively charged protonated forms.

Ion-pair complexation reaction has been applied for the spectrophotometric

determination of many organic compounds [73-76] to mention a few. In response to the

problem resulting from extraction of the ion pair few articles were published for the

analysis of pharmaceutical compounds namely β-blockers [77], acebutalol hydrochloride

[78], benzalkonium chloride [79], quetiapine fumarate [80], tramadol hydrochloride

[81], pheniramine maleate [82], dothiepin hydrochloride [83], domperidone [84] and

rizatriptan benzoate [85] etc, through ion pair formation without extraction.

In the literature survey present in section 2.0.2.2, two spectrophotometric assay of

OFX based on ion-pair complex formation reaction through extraction step [10, 11]. The

methods were based on the formation of chloroform extractable ion-pair complexes

between OFX and bromophenol blue (BPB), bromothymol blue (BTB) and bromocresol

purple (BCP) [10]. The methods were based on formation of chloroform soluble ion-pair

complex between Tropaelin 000 and Supracene violet 3B [11]. The ion-pairs formed at

specific pH were extracted into chloroform before measurement. These procedures

41

involve strict pH control besides the cumbersome extraction step. Many a time,

incomplete separation may lead to erratic results.

In this section 2.3, the author explored the utilization of simple ion-pair

extraction free technique for the determination of OFX in pure form and in tablets. The

present methods employed bromothymol blue (BTB) in method A and bromophenol blue

(BPB) in method B as chromogenic agents and the resulting ion pairs are measured

directly in dichloromethane. The methods were demonstrated to be more sensitive than

the reported extraction methods [8, 9] for OFX and offer the advantages over the existing

spectrophotometric methods in terms of simplicity, sensitivity and speed.


2.3.2.1 Apparatus

The instrument is the same that was described in Section 2.2.2.1.

2.3.2.2 Reagents

All chemicals used were of analytical reagent grade and distilled water was used

throughout the study.

BTB and BPB: The solutions of 0.03 % BTB and 0.05 % BPB (both from Loba Chemie,

Mumbai, India) were prepared in dichloromethane (Merck, Mumbai, India).

Preparation of Standard OFX Solution

A stock standard solution containing 250 µg ml-1

OFX was prepared by

dissolving 25 mg of pure OFX in dichloromethane (Merck, Mumbai, India). From this

stock solution prepared 25 and 20 µg ml-1

OFX and used for the assay in Method A and

Method B, respectively.

Tablets used were the same as described in section 2.1.2.0.


2.3.3.1 Method A (using BTB)

Aliquots of 0.25, 0.5……….4.0 ml OFX standard solution in dichloromethane

(25 µg ml-1

) were measured accurately and transferred into a series of 5 ml calibrated

flask. To each flask, 1 ml of 0.03 % BTB was added, diluted to the mark with

dichloromethane and mixed well. The absorbance of the resulting yellow color

chromogen was measured at 410 nm against the reagent blank.

42

2.3.3.2 Method B (using BPB)

Aliquots of 0.25, 0.5……….4.0 ml OFX standard solution in dichloromethane

(20 µg ml-1

) were measured accurately and transferred into a series of 5 ml calibrated

flask. To each flask, 1 ml of 0.05 % BPB was added, diluted to the mark with

dichloromethane and mixed well. The absorbance of the resulting yellow color

chromogen was measured at 410 nm against reagent blank.

In both the methods, a standard graph was prepared by plotting the increasing

absorbance values versus concentration of OFX. The concentration of the unknown was

read from the standard graph or computed from the respective regression equation

derived using the Beer’s law data.


Ten tablets were accurately weighed and powdered. A portion equivalent to 25

mg OFX was accurately weighed and transferred into a 50 ml calibrated flask, 30 ml of

dichloromethane was added to the flask and the content was shaken thoroughly for 15-20

min to extract the drug into the liquid phase; the volume was finally diluted to the mark

with dichloromethane, mixed well and filtered using a Whatman No. 42 filter paper. An

aliquot of the filtrate (500 µg ml-1

in OFX) was further diluted to get required

concentrations and analysed for OFX following the procedures described for the

calibration curve.

2.3.3.4 Placebo blank and synthetic mixture analyses

Placebo blank solution was prepared as described under section 2.3.3.3 by taking

20 mg of placebo; and then analysed using the procedures described above.

Ten mg of placebo blank of the composition described above, 10 mg of OFX was

added and homogenized, transferred to 50 ml calibrated flask and the solution was

prepared as described under section 2.3.3.3. After dilution to 20 µg ml-1

OFX, 2.5 ml in

each case was then subjected to analysis by the procedures described above.

2.3.3.6 Procedure for stoichiometric relationship

Job’s method of continuous variations of equimolar solutions was employed

6.805 × 10-5

M each of OFX and BTB (method A) and 5.444 × 10-5

M each of OFX and

BPB (method B) solutions in dichloromethane were prepared separately. A series of

solutions was prepared in which the total volume of OFX and the dye was kept at 5 ml.

43

The drug and reagent were mixed in various complementary proportions (0:5, 1:4,

2:3,………..5:0, inclusive) and completed as directed under the recommended

procedures. The absorbance of the resultant ion-pair complex was measured at 410 nm.


OFX is an amino compound containing a piperazine moiety. Hence, attempts

were made to determine it by applying ion pair technique in which basic cationic nitrogen

in OFX forms ion pair with an anionic dye, where a highly yellow colored ion-pair

complex is formed.

Absorption spectra

OFX in dichloromethane does not absorb in the visible region and the new broad

absorptions in the visible region after the addition of the dyes to the fixed concentrations

of the drug indicates the formation of ion pair complex between the drug and dye. Figure

2.3.1 shows the absorption spectra of the ion pair complex, OFX-dye (BTB and BPB) in

dichloromethane. The absorption maximum of the ion pair in dichloromethane is at 410

nm where the absorbance of the reagent blank is insignificant (0.02) in both the methods.

0

0.1

0.2

0.3

0.4

0.5

0.6

360 380 400 420 440 460 480 500 520

Wavelength, nm

Ab

so

rba

nc

e

BTB

BTB (BLANK)

BPB

BPB (BLANK)

Figure 2.3.1 Absorption spectra of ion-pair complexes [OFX (10 µg ml-1

): BTB (0.03%)

and OFX (8 µg ml-1

): BPB (0.05%)] against respective reagent blank


Optimum reaction conditions for quantitative determination of ion-pair complexes

were established via various preliminary experiments such as choice of organic solvent,

concentration of the dye and reaction time.

44

Choice of organic solvent

A number of organic solvents such as dichloromethane, acetone, methanol,

dioxane and carbon tetrachloride were examined since OFX is soluble in these solvents.

Among these solvents, dichloromethane was preferred as the most suitable solvent

because in this medium, the reagent blank gave negligible blank absorbance and the ion-

pair complex formed was found to exhibit higher sensitivity and stability. In other

solvents, the reagent blank yielded high absorbance value.

Effect of dye concentration

The influence of the concentration of BTB and BPB on the intensity of the color

developed at the selected wavelength and constant drug concentration was studied using

different amounts (0.25-3.0 ml) of 0.03% of BTB in method A and (0.25-2.0 ml) of

0.05% BPB in method B. As shown in Figure 2.3.2 the constant absorbance readings

were obtained between (0.5-3.0 ml) of 0.03% of BTB in method A and (0.5-2.0 ml) of

0.05% BPB in method B. Hence, 1 ml of each 0.03% BTB and 0.05% BPB was used for

method A and method B, respectively.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2 2.5 3 3.5

volume of dyes

Ab

so

rba

nc

e

BTB (0.03%)

BPB (0.05%)

Figure 2.3.2 Effect of dye concentration (10 µg ml-1

OFX in method A & 8 µg ml-1

OFX

in method B)

Effect of reaction time

The optimum reaction time for the development of color at ambient temperature

(25±2oC) was studied and it was found that complete color development was

instantaneous in both the methods. The formed color was stable for at least 60 min in

both the cases (Figure 2.3.3).

45

Figure 2.3.3 Effect of time (10 µg ml-1

OFX in method A & 8 µg ml-1

OFX in method B)

Stoichiometric ratio

Job’s method of continuous variations was applied to establish the reaction

stoichiometry. In both the cases, the plot reached a maximum value at a mole fraction of

0.5 which indicated the formation of 1:1 (OFX:Dye) complex (Fig. 2.3.4) between

positive protonated OFX+ and BTB

- or BPB

- anion as shown in Fig. 2.3.5. In OFX, the

nitrogen atom bonded to electron donating methyl group in the piperazine ring is the most

vulnerable one for protonation [86].

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

so

rba

nce

[OFX]/[OFX]+[DYE]

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Absorb

ance

[OFX]/[OFX]+[DYE]

(a) (b)

Figure 2.3.4 Job’s Continuous - variations plots (a) OFX + BTB and (b) OFX + BPB

46

O

NN

NCH3

F

O

OH

O

CH3

O

NN

NCH

3

F

O

OH

O

CH3

(a)

Br

HO

Br

O

C

BrOH

BrO

SO2

BrHO

Br

O

C

BrO

Br

SO3H

BrHO

Br

O

C

BrO

Br

SO3-

+ H+

+

BrHO

Br

O

C

BrO

Br

SO3-

+ H+

+

BrHO

Br

O

C

BrO

Br

SO3-

Bromophenol blue(lactoid ring)

(quinoid ring)

1:1 complex OFX:BPB

OFX

H

(b)

Figure 2.3.5 Possible reaction schemes (a) OFX: BTB and (b) OFX: BPB

Conditional stability constants (Kf) of the ion-pair complexes

The conditional stability constants (Kf) of the ion-pair complexes for OFX were

calculated from the continuous variation data using the following equation [87]:

Kf =

A/Am

[1-A/Am]n+2 CM(n)n

where A and Am are the observed maximum absorbance and the absorbance value when

all the drug present is associated, respectively. CM is the mole concentration of drug at the

maximum absorbance and n is the stoichiometry with which dye ion associates with drug.

47

The log Kf ± CL (Confidence limit at 95 %, n =3) values for OFX-BTB and OFX-BPB

ion-pair associates were 7.339 ± 0.559 and 6.757 ± 0.671, respectively.


The proposed methods have been validated for linearity, sensitivity, precision,

accuracy, selectivity and recovery.


Under optimum conditions a linear relation was obtained between absorbance and

concentration of OFX in the range of 1.25-20.0 µg ml-1

(method A) and 1.0-16.0 µg ml-1

(method B) (Figure 2.3.6). Correlation coefficient, intercept and slope for the calibration

data are summarized in Table 2.3.1. Sensitivity parameters such as apparent molar

absorptivity and sandell sensitivity values, the limits of detection and quantification are

calculated as per the current ICH guidelines [69] are compiled in Table 2.3.1 speak of the

excellent sensitivity of the proposed method. The limits of detection (LOD) and

quantification (LOQ) were calculated according to the same guidelines.

Table 2.3.1 Sensitivity and Regression Parameters

Parameter Method A Method B

λmax, nm 410 410

Linear range, µg ml-1

1.25-20.0

1.0-16.0

Molar absorptivity(ε), l moL-1

cm-1

1.74 x 104

2.18 x 104

Sandell sensitivity*, µg cm

-2 0.021 0.017

Limit of detection (LOD), µg ml-1

0.14 0.08

Limit of quantification (LOQ), µg ml-1

0.43 0.23

Regression equation, Y**

Intercept (a) 0.002 0.0006

Slope (b) 0.046 0.061

Standard deviation of a (Sa) 0.126 0.013

Standard deviation of b (Sb) 0.007 0.001

Regression coefficient (r) 0.999 0.999 *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, b is slope.

48

0

0.1

0.2

0.3

0.40.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25

Concentration of drug (µg mL-1)

Ab

so

rba

nc

e

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16

Concentration of drug (µg mL-1)

Ab

so

rba

nc

e

Method A Method B

Figure 2.3.6 Calibration curves


In order to evaluate the precision of the proposed methods, solutions containing

three different concentrations of the OFX were prepared and analyzed in seven replicates.

The results obtained from this investigation are summarized in Table 2.3.2. The low

values of the relative standard deviation (% R.S.D) and percentage relative error (% R.E)

indicate the high precision and the good accuracy of the proposed methods. The assay

procedure was repeated seven times, and percentage relative standard deviation (%

R.S.D) values were obtained within the same day to evaluate repeatability (intra-day

precision) and over five different days to evaluate intermediate precision (inter-day

precision).


Method OFX

taken

( µg ml-1

)


precision

(n= 7)


precision

(n= 7)

OFX

found

( µg ml-1

)

%RE %RSD

OFX

found

( µg ml-1

)

%RE %RSD

A

10.0 9.78 2.21 1.02 9.70 3.00 1.18

15.0 14.81 1.27 1.14 14.61 2.61 1.29

20.0 19.42 2.89 1.22 19.38 3.10 1.31

B

4.0 3.92 2.00 1.17 3.88 3.00 1.24

8.0 7.83 2.12 1.04 7.78 2.75 1.13

12.0 11.73 2.25 1.21 11.7 2.50 1.29 RE. Relative error and RSD. Relative standard deviation.

49


Method robustness was evaluated by making small incremental change in dyes

concentrations (n = 3). The %RSD with the altered dyes concentration was ≤ 1.32 %. The

capacity remains unaffected by small deliberate variations. In order to demonstrate the

ruggedness of the methods, determinations at three different concentrations of the drug

were carried out by four different analysts, and also with three different instruments by a

single analyst. The inter-analysts RSD were ≤ 0.91 %, whereas the inter-instrumental

variation expressed as RSD were ≤ 1.08 %. These low values of intermediate precision

demonstrate the robustness and ruggedness of the proposed methods (Table 2.3.3).

Selectivity

The proposed methods were tested for selectivity by placebo blank and synthetic

mixture analyses. The placebo blank solution prepared as described earlier was subjected

to analysis according to the recommended procedures. The resulting absorbance readings

for both the methods were same as the reagent blank, inferring no interference from

placebo. The analysis of synthetic mixture solution prepared above yielded percent

recoveries which ranged from 98.45 to103.6 with standard deviation of 0.91–1.13 in all

the cases. The results of this study indicated that the inactive ingredients did not interfere

in the assay. These results further demonstrate the accuracy as well as the precision of the

proposed methods.

Table 2.3.3 Results of robustness and ruggedness expressed as intermediate precision

(%RSD)

Method

OFX

taken,

µg

ml-1

Method robustness

Method ruggedness Parameter altered

BTB, ml*

%RSD

(n = 3)

BPB,

ml*

%RSD

(n = 3)

Inter-analysts’

%RSD

(n = 4)

Inter-

instruments’

%RSD

(n = 3)

A 4.0 1.21 1.18 0.83 1.35

8.0 1.34 1.11 0.98 1.21

12.0 1.27 1.23 0.92 1.27

B

4.0 1.12 1.41 0.89 1.52

8.0 1.18 1.32 0.96 1.47

12.0 1.23 1.29 0.83 1.41 * Dyes (BTB and BPB) volumes used were 0.8, 1.0 and 1.2 ml

50


In order to evaluate the analytical applicability of the proposed methods to the

quantification of OFX in commercial tablets, the results obtained by the proposed

methods were compared to those of the reference method [4] by applying Student’s t-test

for accuracy and F-test for precision. The reference method involved the titration of OFX

in anhydrous acetic acid with acetous perchloric acid to a potentiometric end point

detection. The results (Table 2.3.4) show that the Student’s t- and F-values at 95 %

confidence level are less than the tabulated values, which confirmed that there is a good

agreement between the results obtained by the proposed methods and the reference

method with respect to accuracy and precision.

Table 2.3.4 Results of analysis of tablets by the proposed methods and statistical comparison with the reference method

Tablet Brand

name

Label

claim,

mg/tablet


Reference

method

Method A Method B

Zenflox**

400

99.92 ± 1.11

98.12 ± 1.19 t=2.47

F= 1.15

98.87 ± 1.15

t=1.47

F= 1.07

Ofloxin***

400

100.3 ± 1.06

98.95 ± 1.12

t=1.96

F=1.12

102.1 ± 1.17

t=2.55

F=1.21 *Mean value of five determinations. ** Mankind Pharma Pvt Ltd., New Delhi, India;*** J. B. Chemicals and Pharmaceuticals Ltd, Mumbai,India. The value of t (tabulated) at 95 % confidence level and for four degrees of freedom is 2.77. The value of F (tabulated) at 95 % confidence level and for four degrees of freedom is 6.39.

Recovery studies

The accuracy and validity of the proposed methods were further ascertained by

performing recovery studies. Pre-analysed tablet powder was spiked with pure OFX at

three concentration levels (50, 100 and 150 % of that in tablet powder) and the total was

found by the proposed methods. In all cases, the added OFX recovery percentage values

ranged of 98.60-102.9 % with standard deviation of 1.07-1.21 (Table 2.3.5) indicating

that the recovery was good, and that the co formulated substance did not interfere in the

determination.

51

Table 2.3.5 Results of recovery studies by standard addition method

Method

Tablet

studied

OFX in

tablet,

µg ml-1

Pure

OFX

added,

µg ml-1

Total

found,

µg ml-1

Pure OFX

recovered*

Percent ± SD

A Ofloxin-400

5.0 2.5 7.48 99.20 ± 1.14

5.0 5.0 9.93 98.60 ± 1.07

5.0 7.5 12.45 99.33 ± 1.11

B

Ofloxin-400

4.0 2.0 6.02 101.0 ± 1.17

4.0 4.0 8.12 102.9 ± 1.09 4.0 6.0 9.97 99.50 ± 1.21

*Mean value of three measurements.

52

SECTION 2.4

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION

OF OFLOXACIN IN PHARMACEUTICALS AND HUMAN URINE


Chromatography, literally "color writing", was first employed by Russian scientist

Michael Tsvet in 1900 for the separation of plant pigments. Chromatographic technique

was developed subsequently as a result of the work of Archer John Porter Martin and

Richard Laurence Millington Synge during the 1940s and 1950s, and their work

encouraged the rapid development of several chromatographic techniques including

HPLC [88].

The most characteristic feature of the development in the methodology of

pharmaceutical and biomedical analysis during the past 25 years is that HPLC became

undoubtedly the most important analytical method for identification and quantification of

drugs, either in their active pharmaceutical ingredient or in their formulations [89]. HPLC

offers enhanced detection sensitivity, improved accuracy, and reproducibility of drug

analysis in the course of drug research, development and quality control testing of

marketed drug products.

The literature survey on the HPLC methods for the assay of OFX presented in

Section 2.0.2.3 reveals that the currently available HPLC methods for OFX in

pharmaceutical [12-16] or urine [24, 25] are associated with one or other disadvantages.

Hence the aim of the present work was to develop and validate a simple and rapid HPLC

method for determination of OFX in its tablets and human urine without involving any

unwelcome steps like use of the internal standard, solid-phase or liquid-liquid extraction

step etc. The proposed method is simpler than all the reported methods for urine, and is

more sensitive than the reported HPLC methods for OFX in its dosage forms. The details

of method development and method validation are presented in this section, 2.4.


2.4.2.1 Apparatus

Chromatographic analysis was carried out using Alliance Waters HPLC system

equipped with Alliances 2657 series low pressure quaternary pump, a programmable

53

variable wavelength UV-visible detector, Waters 2996 photodiode array detector and

auto sampler. Data were collected and processed using Waters Empower 2.0 software.

2.4.2.2 Reagents

All the reagents used were of analytical reagent grade and all solutions were

prepared in bi-distilled water.

Phosphate buffer (pH 7.0): The solution was prepared by dissolving 0.35 g of

potassium dihydrogen phosphate in 100 ml of water and the pH was brought to 7.0 by

adding 4% orthophosphoric acid using a pH meter.

Preparation of standard solutions

A 400 µg ml-1

stock standard solution of OFX was prepared by dissolving 40 mg

of the pure drug in mobile phase solution [potassium dihydrogen phosphate buffer (pH

7.0) and methanol (35:65, v/v)]. Working solutions were prepared by diluting the stock

solution with the mobile phase. The same tablets mentioned in the previous section were

also used in this work.


2.4.3.1 Chromatographic conditions

Chromatographic assay was performed using an Intersil C8-3 (5 µm, 3.9 × 150

mm i.d.,) column. The mobile phase was composed of potassium dihydrogen phosphate

buffer (pH 7.0) and methanol (35:65, v/v). The flow rate was maintained at 1.0 ml min-1

.

The column effluent was monitored on UV detector set at 254 nm.

2.4.3.2 Procedure for preparation of calibration curve

Working solutions equivalent to 0.05 - 60.0 µg ml-1

OFX were prepared by

diluting appropriate aliquots of the stock solution. Aliquots of 10 µL were injected

(triplicate) and eluted with the mobile phase under the reported chromatographic

conditions. The average peak area versus the concentration of OFX in µg ml-1

was

plotted. Alternatively, corresponding regression equation was derived using the mean

peak area-concentration data and the concentration of the unknown was computed from

the regression equation.

54


Twenty tablets were accurately weighed, finely pulverized and mixed using a

mortar and pestle. An amount of tablet powder equivalent to 40 mg of OFX was weighed

and transferred into a 100 ml calibrated flask, 50 ml of mobile phase was added and was

sonicated for 20 min in an ultrasonic bath to complete dissolution of the OFX, the content

was then diluted to the mark with the mobile phase, mixed well and filtered using a

Whatman No. 42 filter paper. Aliquots of this solution were successively diluted with the

mobile phase and then subjected to analysis as per the general procedure described for the

calibration curve.

2.4.3.4 Placebo blank analysis

Employing 20 mg of the placebo prepared as described in section 2.1, its solution

was prepared as described under section 2.4.3.3 and then subjected to analysis.

2.4.3.5 Procedure for synthetic mixture analysis

To assess the role of the inactive ingredients on the assay of OFX, a synthetic

mixture was separately prepared by adding 20 mg of OFX to about 20 mg placebo

mentioned above. The drug was extracted and solution prepared as described under the

section 2.4.3.3. The solution after appropriate dilution was analyzed following the

recommended procedure.

2.4.3.6 Procedure for analysis of spiked human urine

Urine sample was collected from a healthy male aged about 34 years. Urine

samples, 3 ml each were spiked with varying amounts of OFX (10, 20 and 40 µg ml-1

)

and 2 ml of methanol (urine protein precipitating agent). The content was vortexed for 30

s and the precipitated protein was separated out by centrifugation for 20 min at 4000 rpm.

The centrifugate was mixed with the mobile phase and the pH was adjusted to 7 (by

adding 4% orthophosphoric acid/1% KOH). The volume of the final solution was brought

to 10 ml by adding the mobile phase. Aliquots of 20 µL were injected (triplicate) and

eluted with the mobile phase under the reported chromatographic conditions. The

concentration of OFX was found using regression equation and the percentage recovery

of OFX was calculated.

55


Method development and optimization

The proposed method permits the quantitation of OFX in commercial tablets and

in human urine. In order to obtain good linearity, sensitivity and selectivity, the method

was optimized and validated in accordance with the current ICH guidelines [64].

A well defined symmetrical peak and good results were obtained upon measuring

the response of eluent under the optimized conditions after thorough experimental trials

that could be summarized as follows:

Choice of column

Two different columns were used for performance investigations, including

hypersil BDS C8 (250 mm x 4.0 mm i.d, 5.0 µm particle size) thermo column, Intersil C8-

3 (5 µm, 3.9 × 150 mm i.d.,) column and chromatopack (250 mm x 4.6 mm i.d., 5 µm

particle size) column. The experimental studies revealed that the Intersil C8-3 column

was more suitable since it gave better sensitivity.

Choice of wavelength

Spectroscopic analysis of the compound showed that OFX has maximum UV

absorbance at 254 nm. Therefore, the chromatographic detection was performed at 254

nm.

Mobile phase composition

Chromatographic conditions were optimized by changing the mobile phase

composition and buffers used in the mobile phase. Different experiments were carried out

to optimize the mobile phase. Several modifications in the mobile phase composition

were performed in order to study the possibilities of changing the selectivity of the

chromatographic system. These modifications included the change of the type and ratio

of the organic modifier, the pH, the strength of the phosphate buffer, and the flow rate.

Precise and accurate results with maximum number of theoretical plates and good peak

were obtained when the mobile phase of potassium dihydrogen phosphate buffer (pH 7.0)

and methanol (35:65, v/v) was used.

Type of organic modifier

Methanol was replaced by acetonitrile but it did not give a good peak. Methanol

was the organic modifier of choice giving nice, elegant and highly sensitive peak.

56

Ratio of organic modifier

The effect of ratio of organic modifier on the selectivity and retention time of the

test solute was investigated using mobile phases containing 25-75% methanol. At 65%

methanol well defined peak and the highest number of theoretical plates were observed.

Effect of pH and ionic strength of buffer

The effect of pH of the mobile phase on the selectivity and retention time of the

test solute was investigated using mobile phases of pH ranging from 4.0 – 9.0. The results

revealed that pH 7.0 was most appropriate and giving well defined peak and the highest

number of theoretical plates. At lower and higher pH, non-symmetrical peak and smaller

number of theoretical plates were observed. Therefore, pH 7.0 was fixed as optimum.

The same trend was observed after making alteration in the ionic strength of the buffer

and ~0.4% phosphate buffer was used as working buffer throughout the investigation.

The effect of flow rate

The effect of flow rate on the symmetry, sensitivity and retention time of the peak

was studied and a flow rate of 1 ml min-1

was optimal for better symmetry and reasonable

retention time.



Stock standard solution of OFX (400 µg ml-1

) was appropriately diluted with the

mobile phase to obtain solutions in the concentration range 0.05 - 60 µg ml-1

OFX.

Twenty microlitre of each solution was injected in triplicate onto the column under the

operating chromatographic conditions described above. The least squares method was

used to calculate the slope, intercept and the correlation coefficient (r) of the regression

line. The relation between mean peak area Y (n=3) and concentration, X expressed by the

equation Y = 17718.5476 X + 2820.7403 (r2 = 0.9999), was linear. A plot of log peak

area Vs log concentration was a straight line with the slope of 0.9240 and this coupled

with a high value of the correlation coefficient (r-value >0.999) indicated excellent

linearity between mean peak area and concentration in the range 0.05 - 60.0 µg ml-1

OFX. Related statistical data are presented in Table 2.4.1.

The limit of quantification (LOQ) was determined by establishing the lowest

concentration that can be measured according to ICH recommendations [69], below

57

which the calibration graph is non linear and was found to be 50 ng ml-1

. The limit of

detection (LOD) was determined by establishing the minimum level at which the analyte

can be reliably detected and it was found to be 17 ng ml-1

.

Selectivity

A chromatogram obtained for placebo solution is shown in Figure 2.4.1 and did

not show any interference from the above substances as shown by the retention time

1.86±0.02.

The peak area value resulting from 40 µg ml-1

OFX in synthetic mixture solution

had nearly the same as that obtained for pure OFX solutions of identical concentration.

This unequivocally demonstrated the non-interference of the inactive ingredients in the

assay of OFX. Further, the slope of the calibration plots prepared from the synthetic

mixture solution was about the same as that prepared from pure drug solution. Method

selectivity was checked by comparing the chromatograms obtained for placebo blank

(Figure 2.4.1), pure OFX solution (Figure 2.4.2), synthetic mixture and tablet solution

(Figure 2.4.3). An examination of the chromatograms of the above solutions revealed the

absence of peaks due to additives present in tablet preparations and urine (Figure 2.4.4).

Table 2.4.1 Regression and Sensitivity parameters

Parameters Value

Linearity range, µg ml-1

0.050 – 60.0

Regression (Y* = a + bX)

Slope (b) 17718.55

Intercept (a) -2820.74 Standard deviation of intercept (Sa) 4804.85

Standard deviation of Slope (Sb) 99.54

Correlation co-efficient (r) 0.9999 Limit of detection (LOD, ng ml

-1) 17.0

Limit of quantification (LOQ, ng ml-1) 50.0

Variance (Sa2) 2.31 × 10

7

ntSa

/±

5041.24

ntSb

/±

104.44

*Y = a+bX, where Y is the area and X concentration in µg ml-1.

ntSa

/± =confidence limit for intercept, ntSb

/± =confidence limit for slope.

58

Figure 2.4.1 Typical chromatogram obtained for placebo blank.

Figure 2.4.2 Typical chromatogram obtained for pure OFX solution (20 µg ml

-1).

Figure 2.4.3 Typical chromatogram obtained for OFX from tablet extract (20 µg ml

-1).

59

Figure 2.4.4 Typical chromatogram obtained for OFX from spiked human urine

(20 µg ml-1

)


Method precision was evaluated from the results of seven independent

determinations of OFX at three different concentrations, 20.0, 40.0 and 60.0 µg ml-1

on

the same day. The inter-day and intra-day relative standard deviation (RSD) values for

peak area and retention time for the selected concentration of OFX were less than 1%.

The method accuracy, expressed as relative error (%) was determined by calculating the

percent deviation found between concentrations of OFX injected and concentrations

found from the peak area. This study was performed by taking the same three

concentrations of OFX used for precision estimation. The intra-day and inter-day

accuracy (expressed as %RE) was less than 2% and the values are compiled in Table

2.4.2.


OFX

injected,

µg ml-1

Intra-day accuracy and precision Inter-day accuracy and precision

OFX

found* %RE %RSD

** %RSD

***

OFX

found* %RE %RSD

** %RSD

***

20.0 19.70 1.50 0.23 0.10 20.18 0.90 0.58 0.12

40.0 40.32 0.80 0.19 0.07 40.65 1.63 0.44 0.13

60.0 59.93 0.12 0.08 0.10 60.6 0.93 0.48 0.09 *Mean value of seven determinations. **Based on peak area. ***Based on retention time

60

Robustness

To determine the robustness of the method small deliberate changes in the

chromatographic conditions like detection wavelength and column temperature were

made, and the results were compared with those of the optimized chromatographic

conditions. The results indicated that changing the detection wavelength (±1 nm) had

some effect on the chromatographic behavior of OFX. However, the alteration in the

column temperature (±1 °C) had no significant effect. The results of this study expressed

as %RSD are summarized in Table 2.4.3.


The developed and validated method was successfully applied to the assay of

OFX commercial tablets. The results shown in Table 2.4.4 are in good agreement with

those obtained with the BP method [4]. The method involved the titration of OFX in

anhydrous acetic acid with acetous perchloric acid to a potentiometric end point.

Table 2.4.3 Results of robustness study (OFX concentration, 40 µg ml-1

, n = 3)

Chromatogra

phic

condition

Modification

(n = 3)

Peak area precision (n=3) Retention time

precision (n=3)

Mean area ±

SD %RSD

Mean RT ±

SD,

(min)

%RSD

Wavelength

(nm)

253 651854 ±

26142 4.01 1.82±0.01 0.49 254

255

Column

temperature

(°C)

24 711936 ±

1565.9

0.22 2.12±0.03 1.42 25

26

Table 2.4.4 Results of analysis of tablets by the proposed method and statistical

comparison with reference method

Tablet

brand

name**

Nominal

amount, mg


Reference method Proposed method

OF-400 400 99.04±1.58 98.56±0.64

t = 0.68

F = 6.09

Zenflox 400 400 101.3±1.26

102.5±0.85

t = 1.79 F = 2.19

*Mean value of five determinations

61

Application to spiked human urine sample

The developed and validated method was successfully applied to determine OFX

in spiked urine sample with satisfactory recovery (Table 2.4.5). Figure 2.4.4 shows the

OFX peak obtained from spiked human urine. The recovery of OFX from urine sample

was measured under the procedure as described above. The recovery for OFX in spiked

human urine analysis was calculated at three concentrations (10.0, 20.0 and 40.0 µg ml-1

).

The recovery for a OFX-spiked human urine samples was in the range of 97.15 –

100.2%.

Recovery studies

To further assess the accuracy and reliability of the method, recovery studies via

standard addition method was performed. To the pre-analyzed tablet powder, pure OFX

was added at three levels and the total was found by the proposed method. Each test was

triplicated. When the test was performed on two different brands of tablets, the percent

recovery of pure OFX was in the range of 94.66 – 103.1 with standard deviation values of

0.36 – 0.84. The results indicated that the method is very accurate and that common

excipients found in tablet preparations did not interfere. The results are compiled in Table

2.4.6.

Table 2.4.5 Results of recovery of OFX in spiked urine sample

Spiked OFX

concentration

(µg ml-1

)

Found ± SD*

% Recovery

10.0

20.0 40.0

10.02±0.04

19.50±0.14 38.90±0.02

100.2

97.84 97.15

*Mean value of five determinations.

62

Table 2.4.6 Results of recovery study by standard addition method

Tablet studied

OFX in

tablet, µg

ml-1

Pure OFX

added,

µg ml-1

Total found,

µg ml-1

Pure OFX

recovered*,

Percent ± SD

OF-400

Zenflox 400

19.71 10.0 28.47 94.66±0.76

19.71 20.0 39.37 98.32±0.36

19.71 30.0 48.38 95.58±0.84

20.50 10.0 30.66 101.6±0.42

20.50 20.0 41.12 103.1±0.74

20.50 30.0 51.31 102.7±0.65 *Mean value of three determinations

63

SECTION 2.5

CONCLUSIONS ON CHAPTER 2-Assessment of the Methods

A comparison of performance characteristics of the proposed titrimetric and

spectrophotometric methods with those of the existing methods is presented in Table

2.6.1.

Table 2.5.1 Comparison of performance characteristics of the proposed methods with the

existing methods A. Titrimetry

Sl.

No.

Reagent Titration conditions

Range,

mg

Remarks

Ref.

No.

1. Anhydrous

acetic acid & perchloric

acid

Non-aqueous potentiometric

titration of OFX in anhydrous acetic acid with perchloric acid

- Fairly large quantities of

0.300 g of OFX for each titration.

3

2. Acetic

anhydride and

perchloric

acid

0.1 g of OFX in 275 ml acetic

anhydride titrated against perchloric acid to a

potentiometric end point

detection.

- Employ large amount of

acetic acid and acetic anhydride

4

3 NBS Unreacted NBS titrated

iodometrically

1-8 narrow liner range, use

of hazardous and unstablen reagent

5

4. Ce(IV)

a. Direct titration of

OFX with Ce(IV)SO4

using ferroin as

indicator

b. Oxidation of OFX by

Ce(IV)SO4,and back

titration of residual

Ce(IV)SO4 with iron

(II) ammonium

sulphate in H2SO4

medium

1.5-15.0

Uses stable reagent,

wide range of

determination and

used aqueous medium

Present

method

64

B. Spectrophotometry

Sl.

No. Reagent/s used* Methodology

λλλλmax

(nm)

Linear range

(µg ml-1

)

(ε = l mol-1

cm-1

)

Remarks Ref

1

NBS-

indigocarmine

NBS-metanil

yellow

Increase in absorbance

of blue color measured

Increase in absorbance

of pink color measured

610

530

0.5-5.0

(ε= 5.53 x 104)

0.3-3.0

(ε= 9.24x104)

Use of unstable

and hazardous reagent

5

2.

Ce(IV)-MBTH

Oxidative coupling

reaction product

measured

640

1-10

Uses expensive

reagent, less stable species

measured.

6

3.

Iron(III) nitrate

Ambered colored

complex was measured

370

0-62.5 (A1% = 207)

Buffers used.

7

4.

Iron(III)

chloride/HCl

Yellow complex measured

410 20-160

Less sensitive

8

5

Iron(III)-

ammonium sulphate in acid

medium

Complex formed

measured 420

10-120

(ε=2.51 x 103)

Less sensitive 9

6.

a) Bromophenol blue

Chloroform extractable

yellow colored 1:1ion-

pair complex was measured

410 5-25

(ε= 1.04 x 104) Require close pH

control and involve extraction

step

10 b) Bromothymol

blue 415

2-15 (ε= 2.01 x 10

4)

c)Bromocresol

purple 410

2-20 (ε= 1.04 x 10

4)

7.

a) Tropaeolin 000

(TP 000)

Red chloroform extractable ion-pair

complex measured

485

2.5-30 (ε= 8.24 x 10

3)

Require close pH

control and involve

extraction step

11

b) Supracene Violet 3B (SV

3B)

Chloroform extractable

ion-pair complex

measured

575

2.5-25 (ε= 1.09 x 10

4)

8.

Citric acid-acetic

anhydride Pink colored

chromogen measured 552

5-55 (ε= 6.04 x 10

3)

Boiling for 20 min required.

12

65

9.

a) Ce(IV)-p-TD

Coloured product

formed between Ce(IV)-

p-TD measured

525 0.0 - 120

(ε= 2.4 x 103)

Very simple,

sensitive and

precise. No

heating /extraction

involved. Free

from critical

experimental

variables. stable coloured species

measured

This

work

b) Ce(IV)-o-DA

Coloured product formed between Ce(IV)-

o-DA measured

470 0.0 - 4.0

(ε= 5.99 x 104)

10.

a) Bromothymol

blue

b) Bromophenol

blue

In both the methods,

resulting yellow colored

ion-pair complexes were

measured.

410

a) 1.25-20 µg ml-1

(ε=1.74x 104)

b) 1.0-16 µg ml-1

(ε =2.18 x 104)

Highly sensitive

with wide linear

dynamic ranges,

no heating or

extraction step, no

pH-adjustment,

single step

reaction

This

work

C. High performance liquid chromatography

Sl.

No.

Chromatographic conditions Detector Linear

range,

µg ml-1

Remarks Ref. No.

1. A column containing octadecyl silane

chemically bonded to porous silica

particles (Waters Spherisorb, 5 microm ODS 1, 4.6 x 150 mm) with

0.05 M phosphate buffer-acetonitrile

(65:35, v/v), and the pH is adjusted to

2.7 with orthophosphoric acid as mobile phase

UV detection at

210 nm

24-120

Less

sensitive,

used propylparabe

n (POP) as

the internal

standard

14

2. C18 column containing mobile phase used was a combination of

acetonitrile:0.25M potassium

dihydrogen phosphate buffer (80:20)

with 0.5% v/v of triethylamine and the pH was adjusted to 2.5 by adding

orthophosphoric acid.

UV detection at 320 nm

5 -25

Less sensitive,

narrow linear

range

15

3. Phenomenex C18 column with 0.24%

sodium lauryl sulphate: acetonitrile:

acetic acid (pH-4.0) (58:40:02) as the

mobile phase.

UV detection at

295 nm

160 - 240

Less

sensitive,

narrow linear

range

16

66

4. Kromasil C8, 5µ, 15 cm × 4.6 mm id

column with mobile phase consisting of 0.5%v/v Triethylamine buffer of

pH 3.0 and Acetonitrile in the ratio of

73: 27

PDA detection at

303 nm

10-50

Less

sensitive, narrow linear

range

15

5. C-18 column (RP-18, 5µ) coupled

with a guard column of same material, in isocratic mode with

mobile phase mixture of Acetonitrile:

Water: Tri ethylamine (25:75:1)

UV detection at

300 nm

NA NA

16

6. Intersil C8-3 (5 µm, 3.9 × 150

mm i.d.,) column at ambient

temperature with mobile phase

consisting of potassium

dihydrogen phosphate buffer

(pH 7.0) and methanol (35:65,

v/v)

UV-detection

at 254 nm

0.05 - 60 Highly

sensitive,

wide linear

dynamic

range and

uses no

internal

standard

This

work

Two titrimetric, four spectrophotometric and one HPLC methods were developed

and optimized for the determination of ofloxacin. The titrimetric and spectrophotometric

procedures are based on well established and characterized redox and complexation

reactions. For redox reaction Ce(IV)SO4 has been used as an oxidizing agent in both titrimetry

and spectrophotometry.

A close examination of the performance characteristics of the existing titrimetric

and spectrophotometric methods summarized in Table 2.6.1 reveals that most of the

reported methods are suffer from one or the other disadvantage. Only two titrimetric

methods have been reported for the assay of OFX. Both the methods used perchloric acid

as titrant [3, 4]. In USP method, a fairly large quantity of OFX is required for each

titration and used non-aqueous medium in both the methods [3, 4]. The proposed

titrimetric methods employed Ce(IV)SO4 which is a very stable reagent and the methods

are simple and applicable over a wide range of determination (1.5-15 mg).

The author used Ce(IV)SO4 and ion-pair complex forming reagents like

bromothymol blue and bromophenol blue for the spectrophotometric assay of OFX in

pharmaceuticals. The Ce(IV)SO4 has earlier been used for the assay of OFX based on the

oxidative coupling reaction with MBTH [5]; however MBTH is a very expensive reagent

and the reaction product is less stable. Ion-pair complex formation reactions have also

67

been applied for the assay of OFX using same dyes bromothymol blue and bromophenol

blue. However, the reported methods suffer from certain disadvantages like extraction of

ion-pair complex in chloroform and close pH control. In contrast the proposed methods

are found to be free from such unwelcome experimental steps. Among the proposed

methods, the method employing Ce(IV)SO4 – o-DA is the most sensitive method with ε

value of 5.99 × 104 l mol

-1 cm

-1 and the methods using Ce (IV)SO4 – p-TD quantifies

OFX concentration over a widest linear dynamic range (0.0-120 µg ml-1). The use of

strong oxidizing agent like Ce(IV)SO4 is certainly a non-selective reagent, yet the

reaction is found to be selective under the optimized experimental conditions and offers

quantification of OFX in wide range.

The specificity of the proposed HPLC method is higher than the titrimetric and

spectrophotometric methods proposed so far. Some of the remarkable advantage of the

proposed HPLC method are- it does not use an internal standard, short retention time and

run time and flow rate (1.0 ml min-1

), makes the method attractive since these features

help in saving cost and time of analysis.

The developed methods have been validated according to the current ICH guidelines.

All the methods were optimized for maximum sensitivity, demonstrated being robust and

rugged, and fairly accurate and precise. The accuracy and precision of all the methods are

evaluated in terms of % RE and % RSD, respectively. All the titrimetric and

spectrophotometric methods are 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 each less

than 4.64 % for all the proposed methods.

68

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