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Chapter – III Levofloxacin Introduction

169

LEVOFLOXACIN- AN ANTIBACTERIAL AGENT

3.01 Drug Profile

Levofloxacin chemically known as (-)-(S)-9fluoro-2,3-dihydro-3-methyl-10-

(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]1,4benzoxazine-6-carboxylic acid

hemihydrate. It is the L-isomer of the racemate, ofloxacin, a quinolone antimicrobial

agent. Levofloxacin is a broad-spectrum antibiotic that is active against both Gram-

positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase [221-

222], a type II topoisomerase, and topoisomerase IV, which is an enzyme necessary to

separate replicated DNA, thereby inhibiting cell division.

Physical Properties:

The Chemical formula : C18H20FN3O4 • ½ H2O

Molecular weight : 370.38grams/mole

Apperance : Light yellowish-white to yellowwhite

crystal

Solubility : Water and methanol.

Structure:

N

O

F

O

OH

O

H

N

N

http://en.wikipedia.org/wiki/Broad-spectrum_antibiotic

http://en.wikipedia.org/wiki/Gram-positive

http://en.wikipedia.org/wiki/Gram-positive

http://en.wikipedia.org/wiki/Gram-negative

http://en.wikipedia.org/wiki/DNA_gyrase

http://en.wikipedia.org/wiki/Topoisomerase


170

Formulations: Levofloxacin is available by prescription in tablet form, injection

solution (Oral 250mg/10ml) as well as used in prescription eye and ear drops.

1) Levoflox -Protec (Cipla) :250 mg, 500mg Tablets

2) Fynal –Mankind (Discover): 250mg, 500mg, 750mg Tables; 100ml Injection

3) Leon -Dr.Reddy’s : 250mg, 500mg, 750mg Tablets

Literature Survey:

Some Spectrophotometric methods have been reported in the literature for the

analysis of Levofloxacin such as ion-ion pair complex formation reactions with some

acid-dyes [223-225], oxidative coupling reactions [226-227] and complexation

formation reactions [228-230] derivative spectrophotometry of their Cu(II)

complexes[231], charge-transfer complexation with n-acceptors [232-237] and ternary

complex formation with eosin and palladium have been strongly reported. Other

methods include titrimetry [237-239] atomic absorption spectrometry [240] florimetry

[241-243] luminescence [244-245] conductometry [246] voltametry and polarography

[247-248] flow injection analysis [249] HPTLC [250-252] and capillary

electrophoresis [253-254]. Several workers [255-263] developed chromatographic

methods for simultaneous quantification of levofloxacin in combination with other

drugs present in human plasma and in chicken. Baietto et al [255] developed a HPLC

method for the determination of levofloxacin in combination with linezolid,

rifampicin, and moxifloxacin .A UPLC method is developed by Park et al [256] for

the determination of levofloxacin in blood plasma in humans. Zhou et al [257] have

developed method for the determination of levofloxacin in human plasma and its

applications to bioequivalence studies.


171

Different authors have developed HPLC methods to determine levofloxacin in

combination with rifampicin [258], with moxifloxac [259] in plasma and amniotic

fluid. Siewart.S [260] developed a HPLC method for the assay of LEF in plasma and

dialysate for pharmacokinetics. Ji et al [261] developed a hydrophilic interaction LC-

MS for the determination of LEF in human plasma, [262] combination with

mexifloxacin, with cefepime, garenoxacinand moxifloxacin [263], Djabarouti, S [264]

developed a HPLC method to determine levofloxacin present in plasma, bone tissues

and bronchoalveolar lavage. Liu, P.Y et al [265] developed a HPLC method to

determine levofloxacin present in chicken.

Hurtado et al [266] developed a HPLC and UV Spectrophotometric method

for the determination of levofloxacin present in formulations. Patel and co-workers

[267] developed column high performance liquid chromatographic and derivative

spectrophotometric methods for the determination of levofloxacin in combination

with ornidazole. A rapid liquid chromatography-tandem mass spectrometry method

for the determination of broad mixtures of pharmaceuticals in surface water is

developed by Conley and co-workers [268]. Kothekar et al [269] also developed

HPLC method to determine levofloxacin combination with ambroxol HCl. Santoro,

M.I.R.M et al [270] reported a HPLC method for the assay of levofloxacin in

combination with gatifloxacin, lomefloxacin and pefloxacin.

Chapter-III Levofloxacin Part-A: HPLC-Method

172

DETERMINATION OF LEVOFLOXACIN BY REVERSE PHASE-

HPLC

3.02 Introduction

A simple method of separating an individual component from a mixture and to

determine its purity is chromatography. The same technique is also used in assay of a

component. Several workers worked on estimation of levofloxacin in combination with

other drugs, in biological fluids and formulations [258-270]. The reported methods are

too cost, time consuming and not applicable in low concentrations, hence the author has

attempted in developing a new HPLC method for quantification of Levofloxacin in

pharmaceutical formulations. The main advantage of the method is less expensive and

time required for the quantification is reduced.

3.03 Experimental

Shimadzu LC- 20AT Prominance liquid chromatographic system is used for

the analysis. Shimadzu LC- 20AT system equipped with binary gradient pump, UV-

VIS SPD 20A detector, Column Oven and controlled by spinchrom software.

Chromatographic conditions:

Column : C18 (250mm, 4.6mm-ID; 5µm particle

size)

Detector : UV-VIS SPD 20A Prominance

Wavelength (λmax) : 295nm

Flow Rate : 1.0ml/min.

Injection Volume : 20 μl


173

Temperature : 30oC

Run Time : 20 min.

Retention Time : 3.918min.

3.04Method Development

(i)Materials and Reagents

All reagents and chemicals used are of HPLC grade. Acetonitril, methanol,

and phosphoric acid 85% are purchased from Merck, India and HPLC grade water is

used to prepare the mobile phase. Stock solutions of levofloxacin and sample

solutions are prepared in the mobile phase. All solutions are filtered through 0.45 µm

membrane filter and degassed using a sonicator. Gift samples of levofloxacin and

working reference standard from Chandra Lab Hyderabad are used. The formulations

are purchased locally from market.

(ii) Preparation of Solutions

Buffer solution (0.1% o-phosphoric acid, Merck; v/v): About 1.0ml of o-phosphoric

acid is transferred into 1000ml volumetric flask made up to the mark with HPLC

grade distilled water and degassed by sonication.

Mobile phase(Buffer, Methanol and Acetonitrile;70:12:18 v/v): Mobile phase is

prepared by mixing accurately measured volumes of 350ml of buffer,60ml of

methanol and 90ml of acetonitrile in a 1000ml beaker, stirred well, filtered through

0.45μ or finer porosity membrane filter, sonicated and used for the analysis.


174

Drug Standard Solutions

Stock solution: About 12.5mg of Levofloxacin is accurately weighed, transferred into

a cleaned and dried 100ml volumetric flask, dissolved in atetonitrile and made up to

the mark with mobile phase. The solution is sonicated, filtered through 0.45μ or finer

porosity membrane and used for the analysis.

Working standard solution (12.5μg/ml): Working standard solution is prepared daily

by accurately measuring 5ml of the stock solution into 50ml volumetric flask and

made up to the mark with mobile phase.

Preparation of test solutions: Tablets of 250mg/500mg are powdered and mixed

thoroughly; an amount of the powder equivalent to 12.5mg of the drug is accurately

weighed and transferred into a 100ml volumetric flask dissolved in methanol, shaken

well and filtered. The filtrate is evaporated to dryness carefully and the residue is

dissolved in 10ml of acetonitrile and sonicated and made up to 100ml with the mobile

phase

(iii) Procedure:

Chromatographic conditions are fixed for the Shimadzu HPLC system. The

mobile phase is allowed to pump into the column C18 (250mm, 4.6mm-ID; 5µm

particle size) at a flow rate of 1.0 ml/min., keeping the column in thermostat at a

constant temperature of 30oC about 30min. The response of the system is recorded

against time at maximum wavelength 295nm. After the elution of the mobile phase

through the column, it is found that the base line of the chromatogram is almost

parallel to x-axis. Working standard solution of levofloxacin (20μl) is injected into the

system, the mobile phase is allowed to pass through the column for 20minutes and the


175

chromatogram is recorded. Typical chromatograms for the standard and

pharmaceutical formulation are represented in Fig.3.01 and Fig.3.02; P: 181.

(iv)Optimization of the proposed method:

The developed method is optimized and optimum conditions are established

by varying the parameters such as concentration of the standard, type of column,

temperature of the column, flow rate, injection volume, composition of the mobile

phase (polarity), pH of the buffer one at a time, keeping the others fixed and

observing the effect on the retention time, tailing factor and other system suitability

parameters.

3.05. Method Validation:

Validation of analytical methods is important in the analysis of pharmaceutical

formulations. The ability to control the quality is dependent upon the ability of the

analytical methods, as applied under well-defined conditions and at an established

level of sensitivity, to give a reliable demonstration of all deviation from target

criteria. Analytical method validation is now required by regulatory authorities for

marketing authorizations and guidelines have been published. It is important to isolate

analytical method validation from the selection and development of the method. The

most widely applied validation characteristics are given below

3.05(i) System Suitability and System Precision:

Working standard solution (15.5μg/ml) of levofloxacin is injected six times

into the HPLC system and the chromatograms are recorded. The system suitability

parameters and system precision are evaluated based on the area of the peaks and


176

found to be within the limits. The results are incorporated in Table-3.01(a), P: 183.

The % RSD for peak areas of six replicate injections is determined and found to be

1.089. The results are summarized in Table-3.01(b), P: 183.

3.05(ii) Linearity of detector response:

Linearity between the response of the detector and the different concentrations

of the standard drug solutions is established by plotting a graph to concentration

versus average area of two peaks. The regression parameters are calculated. A series

of six working standard solutions are prepared in the concentration range of about

25.0μg/ml to 150μg/ml corresponding to 25% to 150% of target concentration. Each

solution is injected into the system and recorded the chromatogram under the test

conditions. A graph is plotted to concentration in µg/ml on X-axis versus response on

Y-axis Fig.3.03 (a), P: 182. The detector response is found to be linear with a squared

correlation coefficient of 0.9998. The results are summarized in Table-3.02, P: 183.

3.05(iii) Precision of test method:

Commercial formulations of levofloxacin tablets of 250mg and 500mg are

successfully analyzed. The precision of the test method is evaluated by taking six

replicate measurements. The average percent of assay of levofloxacin in tablets is found

to be 100.53% and 99.99% and %RSD is found to be 0.818 and 0.564 respectively. The

results are summarized in Table-3.03, P: 184.

3.05(iv) Accuracy of the method:

A study of levofloxacin from spiked placebo is conducted. Different

concentration solutions of levofloxacin formulations are prepared equivalent to about


177

75%, 100%, and 125% of the target concentration. Sample solutions are prepared in

triplicate for each spike level and assayed as per test method. The % recovery is found

to be 100.52(±0.828), 100.38(±0.891) and 100.08(±1.047). The results are

summarized in Table-3.04, P: 184.

3.05(v) Linearity of the test method:

A series of six different concentration solutions of the drug are prepared as per the

test method and for each concentration the chromatogram is recorded. The amount of

drug recovered is estimated. A linear plot is drawn to average amount of levofloxacin

added (mg) versus average of levofloxacin recovered (mg) in accuracy (Fig.3.03 (b), P:

182). The results of the recovery experiments by the developed method are summarized

in Table-3.05, P: 185.

3.05 (vi)Limit of Detection and Limit of Quantitation

Limit of detection and Limit of quantitation (LOD and LOQ) are calculated based

on the standard deviation of the response and the slope of the calibration curve. The

results of LOD and LOQ are summarized in Table-3.02, P: 183.

3.05(v) Ruggedness

Ruggedness is the degree of reproducibility of results obtained by the analysis of

the same sample under a variety of normal test conditions different, laboratories,

instruments, reagents, assay temperatures, small variations in mobile phase, different

days etc

(a) Intra Day & Inter Day variability: This study is conducted on day-1 and day-2

using same columns on the same HPLC system by assaying six different sample


178

preparations of Levofloxacin formulations under similar conditions. The system

suitability parameters are evaluated as per the standard method and found to be within

limits. The average % assay was found to be 99.98 and 100.21 with a relative standard

deviation of 0.695% and 0.576% respectively. The results are summarized in Table-

3.06(a), P: 185. Comparison of the results obtained on two days shows that the assay

method is rugged for day to day variability.

(b) System to System variability: System to system variability study is conducted on

two HPLC systems by using the same column by assaying six separately prepared

sample solutions of levofloxacin formulations under similar conditions. The system

suitability parameters are evaluated as per the test method on both the systems and

found to be within limits. The average % assay for the two systems is found to be

100.22and 100.27 with a relative standard deviation of 0.454% and 0.587%

respectively. The results are summarized in Table-3.06(b), P: 186. Comparison of the

results obtained on two systems shows that the assay method is rugged for system to

system variability.

(c) Column to Column variability: This study is conducted using two columns on the

same HPLC system by assaying six separately prepared sample solutions of

levofloxacin formulations under similar conditions. The system suitability parameters

are evaluated as per the test method on both the systems and found to be within limits.

The average % assay for the two systems was found to be 100.24and 100.25 with a

relative standard deviation of 0.562% and 0.547% respectively. The results are

summarized in Table-3.06(c), P: 186. Comparison of the results obtained on two

columns shows that the assay method is rugged for column to column variability.


179

3.05(vi) Robustness

Robustness is a measure of the capacity of an analytical method to remain

unaffected by small but deliberate variations in method parameters such as per cent

organic content in the mobile phase, pH of the mobile phase, buffer concentration,

temperature, injection volume and column temperature. These parameters may be

evaluated one factor at a time or simultaneously as part of a factorial experiment. It

also provides some indication of the reliability of an analytical method during normal

usage

Six separately prepared sample solutions are injected into the HPLC system

with flow rates of 0.9ml/min. and 1.1ml/min, at two different wavelengths 290nm and

300nm, keeping the column at 28oC and 32

oC, using mobile phase of different

composition and maintaining buffer solution at different pH. The instrument response

is recorded against time in each case. The system suitability parameters are evaluated

as per the test method and found to be within limits and are shown in Table-3.07, P:

187.

3.06 Results and Discussion:

Reverse phase high performance liquid chromatography method is chosen for

the analysis of levofloxacin in pure and pharmaceutical formulations. The optimized

chromatographic conditions are adjusted before running the system. The

chromatograms are recorded by injecting standard and test solutions of the drug in to

the column. The system suitable parameters are evaluated and found within the limits.

The developed method is simple, rapid, precise, accurate and linear. The results are

given in Table-3.01(a) and Table-3.01(b); P: 183. A plot is drawn between


180

concentration and the instrument response and found to be linear with good

correlation coefficient(r=0.9996) (Table-3.03, P: 184; Fig.3.03 (a), P: 182). Precision

and accuracy of the developed method are expressed in %RSD and % of recovery of

the active pharmaceutical ingredient. Low %RSD values 0.813 and 0.668 and high

%recovery 100.45%, 99.77% and 100.74%corresponding to 75% ,100% and 125%

spike levels indicate that the method is highly precise and accurate (Table-3.04, P:

184). Pharmaceutical formulations are analyzed by the developed method by

estimating the amount of drug recovered by standard addition method.

A graph is drawn between the amount of drug added and the amount of drug

recovered, the plot is linear and regression equation is given by y=1.010x+0.0727with

r2 =

0.9997(Table-3.05; P: 185). A study is conducted between two analysts, two

different systems, and two columns and compared the results. The system suitability

parameters are evaluated as per the test method on both the systems, columns and for

analysts and found to be within limits. The average % assay and relative standard

deviation are within the limits (Table-3.06(a)-Table-3.06(c), P: 185-186). The

change in system suitability parameters are evaluated by studying the effect of change

chromatographic parameters and found to be acceptable (Table-3.07, P: 187).

3.07 Conclusion: The developed method for the determination of Levofloxacin is

optimized and validated using the test method and found to be linear, precise,

accurate, rugged and robust. The developed RP-HPLC method is cheap, economical

and completed within short time. The developed method can be successfully applied

for the routine analysis of levofloxacin in bulk pharmaceutical formulations.


181

Fig.3.01Chromatogram of Levofloxacin (Standard)

Fig.3.02 Chromatogram of Levofloxacin (Formulation)


182

Linearity of detector responce to the concentration of the Levofloxacin

y = 6796.7x + 4253.8

R2 = 0.9996

0.0E+00

3.0E+05

6.0E+05

9.0E+05

1.2E+06

1.5E+06

0 50 100 150

Concentration of Levofloxacinin µg/ml

Dete

cto

r R

esp

on

ce

Fig.3.03 (a) Linearity of the detector response

Linearity of the test Methody = 1.0099x - 0.0727

R2 = 0.9997

0.0

5.0

10.0

15.0

20.0

0.0 5.0 10.0 15.0 20.0

Weight of Drug added in µg/ml

Weig

ht

of

dru

g r

eco

vered

in

µg

/ml

Fig. 3.03(b) Linearity of the test Method


183

Table-3.01(a): System Suitability

System suitability parameters Observed value Acceptance criteria

USP tailing factor 1.752 NMT2.0

Number of theoretical plates 3764 NLT2000

RSD(six replicate measurements) 1.089 NMT 2.0%

Table-3.01(b): System Precision

Injection ID Average Area Statistical parameters Value of the parameters

1 689881 Mean 690857

2 691864

3 687963 SD 1736

4 690795

5 691846 %RSD 0.251

6 692793

Table-3.02: Linearity of detector response

Injection

ID

Concentration

μg/ml

Average

Area Regression Parameter

1 3.13 172470 Slope 55013

2 6.25 344941 Intercept 2507

3 9.38 517411 Correlation Coefficient 0.9996

4 12.50 689881 LOD µg/ml 0.341

5 15.63 862351 LOQ µg/ml 1.137

6 18.75 1011000


184

Table-3.03: Precision of the test method

Percent of Assay Labeled Amount

Sample No. Labeled Amount 250mg/Tablet Labeled Amount 500mg/tablet

Amount Found %Recovery Amount Found %Recovery

1 253.30 101.32 498.40 99.68

2 254.00 101.60 499.35 99.87

3 249.65 99.86 497.75 99.55

4 249.45 99.78 503.60 100.72

5 252.00 100.80 503.35 100.67

6 249.58 99.83 497.25 99.45

Mean 251.33 100.53 499.95 99.99

SD 2.044 0.818 2.820 0.564

%RSD 0.813 0.813 0.564 0.564

Table-3.04: Accuracy of the method

Spike

level

Sample

ID

Amount

Added(μg/ml)

Amount

Found(μg/ml)

% of

Recovery Statistical Analysis

75% 1 9.38 9.51 101.37 Mean 100.52

2 9.38 9.35 99.72 SDV 0.828

3 9.38 9.42 100.45 %RSD 0.824

100% 1 12.50 12.68 101.40 Mean 100.38

2 12.50 12.50 99.96 SDV 0.891

3 12.50 12.47 99.77 %RSD 0.888

125% 1 15.63 15.45 98.88 Mean 100.08

2 15.63 15.73 100.64 SDV 1.047

3 15.63 15.75 100.74 %RSD 1.047


185

Table-3.05: Linearity of test method

Spike

level (%)

Drug

added(mg)

Drug

Recovered(mg) Regression parameters

25 3.13 3.00 Slope 1.010

50 6.25 6.22

75 9.38 9.59 Intercept 0.0727

100 12.50 12.53

125 15.63 15.66 Correlation Coefficient 0.9997

150 18.75 18.84

Table -3.06(a) Intra Day and Inter Day precision

Percent of assay of Levofloxacin formulation

Sample No. Day-1 Day-2

1 98.86 100.9

2 99.92 100.8

3 99.82 99.75

4 100.5 99.85

5 100.9 99.53

6 99.85 100.4

Mean 99.98 100.21

SD 0.695 0.577

%RSD 0.695 0.576

Comparison of the precision with method precision

F=1.384,t=1.955 F=2.011,t=1.381

Comparison of precision between two analysts

F=1.451 t=0.572


186

Table -3.06(b) System to System Variation


Sample No. Systemt-1 System-2

1 99.99 101.02

2 100.6 100.94

3 99.94 99.84

4 100.35 99.73

5 99.62 100.32

6 100.84 99.78

Mean 100.22 100.27

SD 0.455 0.588

%RSD 0.454 0.587


F=3.226,t=1.649 F=1.933,t=1.076


F=1.671 t=0.190

Table -3.06(c) Column to Column Variation


Sample No. Column-1 Column-2

1 99.88 100.36

2 99.84 100.69

3 100.88 99.94

4 99.93 99.85

5 101.04 100.75

6 99.85 99.94

Mean 100.24 100.25

SD 0.563 0.402

%RSD 0.562 0.401


F=2.108,t=1.275 F=4.142,T=1.676


F=1.961 t=0.043


187

Table 3.07: Study of Robustness

Variable Variation USP Plate Count USP Tailing %RSD

Standard -------- 3764 1.752 0.000

Flow Rate 0.9ml/min. 3491 1.639 1.100

1.1 ml/min. 3871 1.674 0.790

Wavelength 300nm 3175 1.352 0.921

290 nm 3769 1.726 0.721

Temperature 28oC 3879 1.598 1.040

32oC 3582 1.526 0.974

%Mobile

Phase

70%:10%:20% 3498 1.498 0.737

70%:15%:15% 3762 1.521 1.694

Buffer pH 2.8 3751 1.427 1.436

3.2 3462 1.519 1.489

Acceptance Criteria USP Tailing Factor not more than 2.0, Theoretical Plates

not less than 2000

Chapter – III Levofloxacin Part-B: Visible Spectrophotometry

188

DETERMINATION OF LEVOFLOXACIN BY VISIBLE

SPECTROPHOTOMETRY

3.08 Introduction

The functional groups that are present in levofloxacin are not fully exploit

hence the author has made some attempts to develop new spectrophotometric methods

for the determination of levofloxacin in pure and formulations. The developed

methods have better sensitivity, selectivity, precision and accuracy.

3.09(i) Experimental

UV-Visible Spectrophotometer: ElicoSL159 model, 2nm high resolution, double

beam, 1cm length quartz coated optics and wavelength range190-1100nm instrument

is used for all the spectral measurements.

3.09(ii) Preparation of solutions

3.09(ii) (a) Standard Solution of Levofloxacin

The stock solution of Levofloxacin is freshly prepared by transferring

accurately weighed 100mg of Levofloxacin into 100ml volumetric flask, dissolving,

and then made up to the mark with double distilled water. Working standard solution

200μg/ml and 250μg/ml are prepared and used in the reactions M2(a), M3, M18 and

M1(a), M1(d), M5(c), M12, M14, M17 respectively.

3.09(ii) (b) Preparation of reagents

Analytical grade chemicals and reagents are used in the preparation of

solutions and all the solutions are prepared in double distilled water.


189

Method M1(a) and M1(d)

Aqueous solutions of ARS (0.2%, 5.49 x 10-3

M), BTB (0.1%, 1.60 x 10-3

M),

TPooo (0.2%, 5.71 x 10-3

M) and HCl solution (0.1M) are prepared in the same way

as described under NTT in Chapter – II, P: 81-82.

Method M2(a)

MB solution (Fluka; 0.2%, w/v 6.25x10-3

M): Prepared by dissolving 200mg of MB in

100ml of distilled water.

Buffer solution pH 9.8 NH4OH – NH4Cl: 7.0gms of NH4Cl and 6.8ml of ammonia

solution are dissolved in 100ml of distilled water and pH is adjusted to 9.8.

Method – M3

Fe (III) solution (Wilson labs; 0.054%, 3.33x10-3

M): Prepared by dissolving 54mg

of anhydrous Ferric chloride in 100ml of distilled water

O-PHEN solution (E.Merck, 0.2%, 1.10x10-2

M): Prepared by dissolving 200mg of

o-phenanthroline in 100ml of distilled water with warming.

O-phosphoric acid solution (Qualigens, 2.0x10-2

M): Prepared by mixing 1.27ml of

o-phosphoric acid with 100ml of distilled water. 10.0ml of this stock solution is

further diluted to 100ml with distilled water


190

Method M5(c)

Aqueous solutions of MBTH (0.2%, 8.56 x 10-3

M) and Ce (IV) (1%, 9.35 x 10-

3M), NaIO4 solution (0.2%, 9.35 x 10

-3M), AcOH solution (Qualigens; 2.3 M) are

prepared in the same way as described under NTT in Chapter – II P: 83.

Method M12

NaIO4 solution (Loba; 0.855%, 4.00x10-2


NaIO4 in 100ml of 0.3M HCl.

PHH (Loba; 4.0%, 6.90x10-2

M): Prepared by dissolving 4.0g of Phenyl Hydrazine

Hydrochloride (PHH) in 100ml of distilled water and filtered.

K3Fe(CN)6 solution (Sd-fine;.2.0%,6.00x10-2

M): Prepared by dissolving 2.0g of

K3Fe(CN)6 in 100ml distilled water.

NaOH solution (BDH, 0.4%, 0.1M): Prepared by dissolving 400mgs of NaOH to

100ml distilled water and standardized.

Method M14

CTC solution (E.Merck; 2.86x 10-1

M): Prepared by dissolving 7.25g of cobaltous

nitrate and 3.8g of ammonium thiocyanate in 100ml of distilled water.

Buffer solution (pH 2.0): Prepared by mixing 306ml of trisodium citrate (0.1M) with

694ml of HCI (0.1M) and the pH of the solution were adjusted to 2.0.


191

Method M17

Aqueous solutions of PA (0.1%, 4.36 x 10-3

M) and Buffer pH – 9.8 are

prepared in the same way as described under NTT in Chapter – II, P: 84.

Method M18

4-AP Solution (BDH; 0.5%, 2.45 x 10-2

M): 500mg of 4-AP is accurately weighed

and dissolve din 100ml of methanol

3.09(iii) Procedures proposed for the assay of Levofloxacin

After a systematic and detailed study of the various parameters involved, the

following procedures are proposed for the assay of levofloxacin in bulk and

pharmaceutical formulations.

Method – M1(a) and M1(d)

Into a series of 125ml separating funnels containing aliquots of standard LEF

solution 8.3-50.0μg/ml and 16.7-100.0μg/ml, for methods M1(a) and M1(d) respectively,

6.0ml of 0.1M HCI solution and 1.0ml of 0.2%ARSdye solution for M1(a) and 2.0ml

0.2%TP ooo dye solution for M1(d) are added successively. The total volume of aqueous

phase in each separating funnel is adjusted to 15ml with distilled water. To each

separating funnel 10ml of chloroform is added and the contents are shaken for 2min. The

two phases are allowed to separate and the absorbance of the separated chloroform layer

is measured at max (415nm for ARS; and 515nm for TPooo) against a similar reagent

blank. The amount of levofloxacin is deduced from the calibration curve ARS and TPooo

(Fig.3.13 – Fig.3.14, P: 207).


192

Method M2(a)

Aliquots of standard drug solution (LEF) 13.3-80.0μg/ml and 1.0ml of pH 9.8

buffer solution are placed separately in a series of 125ml separating funnels. 0.5ml of

MB is added and the total volume of aqueous phase in each funnel is adjusted to

10.0ml with distilled water. Then 10.0mlof chloroform is added in each separating

funnel and the contents are shaken for 2min and allowed to separate. The organic

layer is collected through cotton plug and the absorbance is measured immediately at

650nm against a reagent blank. All the colored species are stable for 2 hours. The

amount of drug in a sample solution is obtained from the Beer’s Lambert plot

(Fig.3.15, P: 207).

Method – M3

Into a series of 25ml calibrated tubes aliquots of standard Levofloxacin

solution (20.0-120.0g/ml) are transferred and then 3.0ml (3.33 x 10-3

M) of Fe (III),

2.0ml of (1.01 x 10-2

M) o-phenanthroline are added successively. The total volume in

each tube is brought to 10.0ml with distilled water. The tubes are kept on a boiling

water bath for 30min. The tubes are removed and cooled to room temperature. 2.0ml

of (2.16 x 10-2

M) o-phosphoric acid is added and volume in each tube is made up to

the mark with distilled water. The absorbance of the colored complex solution is

measured after 5 min at 510nm against a reagent blank. The content of the drug is

computed from the appropriate calibration graph (Fig.3.16, P: 208)


193

Method – M5(c)

Aliquots of standard LEF solution (10.0-60.0g/ml) are transferred into a series of

25ml calibrated tubes. Then 1.0ml (9.35 x 10-3

M) of NaIO4 solution,1.0ml of acetic

acid solution are and the total volume is adjusted to 10.0 ml and kept in a water bath

for 45min. The solutions are cooled suddenly. After that 1.0ml (8.56 x 10-3

M) of

MBTH solution is added and kept aside for 10min. The volume is made up to the

mark with distilled water. The absorbance is measured at 610nm against a similar

reagent blank. The amount of LEF is computed from its calibration graph. (Fig.3.17,

P: 208).

Method-M12

Different aliquots of standard LEF solution (10.0-60.0g/ml) are transferred

into a series of 25ml-calibrated tubes. Then 1.0ml of NaIO4 solution is added to each

tube and the volume made up to 5ml with distilled water. After keeping the tubes in a

hot water bath for 5min., 1.5ml of PHH solution and 1.0ml of K3Fe(CN)6 solutions

are added successively and shaken well. The tubes are kept in ice water for

5min.Later 5.0ml of HCl is added and the solution in each tube is made up to 25ml

with ethanol. The absorbance is measured after 15min. at 430nm against reagent

blank. The amount of LEF is computed from its calibration graph (Fig.3.18, P: 208).

Method - M14

Into a series of 125ml separating funnels, aliquots of standard Levofloxacin

solution (5.0-30.0g/ml) are taken and 2.0ml of buffer (pH 2.0) and 5.0ml (2.5x10-

1M) of CTC solutions are added. The total volume of aqueous phase in each


194

separating funnel is adjusted to 15.0ml with distilled water. To each separating funnel,

10.0ml of nitrobenzene is added and the contents are shaken for 2 min. The two

phases are allowed to separate and the absorbance of the separated nitrobenzene layer

is measured at 660nm against a similar reagent blank. The amount of LEF is

computed from its calibration graph (Fig. 3.19, P: 208).

Method M17

Into a series of 50 ml separating funnels containing aliquots of drug (8.3 –

50.0μg/ml) solutions, 2 ml of buffer of pH 9.8 and 2.0 ml of 0.1% picric acid

solutions are added successively. The total volume of aqueous phase in each

separating funnel is adjusted to 15 ml with distilled water. To each separating funnel

10 ml of chloroform is added and the contents are shaken for 2 min. The two phases

are allowed to separate and the absorbance of the separated chloroform layer is

measured at 415nm against a reagent blank prepared under similar conditions. The

amount of drug is deduced from the calibration graph (Fig.3.20, P: 209).

Method: M18

Delivered aliquots of standard LEF solution (10.0 – 60.0g/ml) into a series of

10ml calibrated tubes, then 3.0ml (5.83 x 10-3

M) of 4-AP is added to each tube and

kept aside for 15min. Later the solution in each tube is made up to 10ml with methanol.

The absorbance is measured at 440nm against the reagent blank. The amount of LEF is

computed from its calibration graph. (Fig.3.21, P: 209).


195

3.09 (iii) Optimum Conditions

Optimum conditions for the maximum color development and maximum

stability of the proposed methods (M1(a), M1(d), M2(a), M3, M5(c), M12, M14,

M17 and M18 are established by varying the parameters one at a time, keeping the

others fixed and observing the effect produced on the absorbance of the colored

species. The optimum conditions thus established are incorporated in recommended

procedures.

Method - M1(a) and M1(d)

The Procedure to establish optimum conditions for these methods is as same as

described in Chapter II of NTT Table 2.08(a), P: 97.

Method – M2(a)

The author performed controlled in pediments by measuring absorbance at

max 655nm of a series of solutions varying one and fixing the other parameter in

order to fix the optimum conditions. Various parameters such as type of acid for

buffer, concentration of acid, concentration of dye or choice of organic solvent, ratio

of organic phase to aqueous phase, shaking time, temperature, intensity and stability

of the colored species in organic phase are investigated and the results are recorded in

Table 3.08 (a), P: 200.

Method – M3

In order to establish optimum conditions necessary for rapid and quantitative

formation of the colored complex with maximum stability and sensitivity, the author


196

has performed control experiments by varying one and fixing the other parameters,

such as effect of pH of the buffer solution, volume of buffer solution, volume of Fe

(III) and o-phenanthroline solution, temperature, time of heating, order of addition of

reagents and nature of solvents for final dilution. The optimum conditions are

incorporated in Table 3.08(b), P: 201.

Method –M5(c)

The optimum conditions established for method M5(c) are found to be same as

described in Chapter II of NTT Table 2.08(d) and Table 2.08(e) P:100-101.

Method M12

In developing this method, the effect of various parameters likes strength and

volume of NaIO4, time and temperature required for oxidation, volume and strength

of reagents such as PHH and K3Fe(CN)6], volume of HCl, solvent for final dilution in

developing color of maximum stability and intensity are studied. The results are

incorporated in Table 3.08(c), P: 202.

Method - M14

The optimum conditions incorporated in the recommended procedure are

ascertained by performing systematic investigations. The volume of cobalt

thiocyanate, volume of buffer solution, solvent for the extraction of colored species,

time required for maximum color development and stability period of the colored

species are studied and the results are incorporated in Table 3.08(d), P: 203.


197

Method – M17

The optimum conditions established for methods M17 are found to be same as

described in Chapter II of NTT Table 3.08(i), P: 105.

Method: M18

The optimum conditions are established basing on the study of the effects of

various parameters such as volume of (2.54 x 10-2

M) 4-AP solution, volume of

solvents solution used initially and subsequently for final dilution and the stability of

colored species after final dilution, measuring absorbance at 430nm. The optimum

conditions developed and actual conditions chosen for the procedure are recorded in

Table 3.08(e), P: 204.

3.10 Validation of the methods

3.10 (i) Linearity

Concentration of the drug is plotted against absorbance and is found to be

passing linearly through the origin. Slope, intercept and correlation coefficient are

evaluated and found within the limits and are presented in Table 3.10(a) – Table

3.10(b), P: 212.

3.10 (ii) Precision

The precision of the developed method is ascertained from the absorbance

values obtained by actual determination of six replicates of a fixed amount of LEF in

total solution. The percent relative standard deviation and percent range of error (at


198

0.05confidence limits) are calculated for the proposed methods and represented in

Table 3.11(a) – Table 3.11(b), P: 213.

3.10(iii) Accuracy

To determine the accuracy three different concentrations of bulk samples of

LEF within the Beer’s law limits are taken and analyzed by the proposed method.

The results are recorded in Table 3.12(a) – Table 3.12(i), P: 213-217.

3.10(iv) Limit of Detection and Limit of Quantification

The limits of detection and quantification are calculated from the standard

deviation of intercept and slope of the absorbance measurements obtained for a series

of solutions and are presented in Table 3.10(a) – Table 3.10(b), P: 212.

3.11 Assay of pharmaceutical formulations

An accurately weighed portion of powdered tablets equivalent to 100mg of

LEF is dissolved in 20ml of methanol (MeOH), shaken well and filtered, the filtrate

is diluted to 100ml with MeOH to get 1mg/ml of drug in formulations and then

25.0ml, 20.0ml and 10.0ml of this solution are further diluted to 100ml to obtain

working standard solutions of 250μg/ml, 200μg/ml and 100μg/ml respectively.

Commercial formulations containing Levofloxacin are successfully analyzed by the

proposed methods. The values obtained by the proposed and reference methods for

the formulations are compared statistically with F-test and t-test and found to be not

different significantly. Present of recoveries are determined by adding standard drug

to preanalysed formulations. The results of the recovery experiments by the

proposed methods are also listed in Table 3.13(a) – Table 3.13(e), P: 218-222.


199

3.12 Results and Discussions

3.12 (i) Spectral Characteristics

In order to ascertain the optimum wavelength of maximum absorption (max) of the

colored species, specified amounts of the drug are taken and color is developed separately

by following the above mentioned procedures. The absorption spectra are scanned on a

spectrophotometer in the wavelength region of 370 to 900nm against similar reagent blank.

The results are graphically represented in Fig.3.04 – Fig.3.12, P: 205-207.

3.12(ii) Optical Characteristics

Absorbences at maximum wavelength of a set of six standard solutions

containing different amounts of levofloxacin and specified of amounts of reagents are

recorded against the corresponding reagent blanks. In order to test whether the

colored species formed in above methods adhere to Beer’s law, a plot of concentration

against absorbance is drawn and it is found to be linear. Beer’s law limits, molar

absorptivity, Sandell’s sensitivity and optimum photometric range in each method are

calculated. The values are presented in Table 3.09 (a) – Table 3.09(b), P: 212. The

optimum photometric range of the proposed methods is determined from the Ringbom

plots Fig.3.22-Fig.3.30, P: 209-211.


200

Table 3.08(a)

Optimum conditions established for Method M2(a) for LEF Parameter Optimum range Conditions in

procedure

Remarks

max (nm)

650 - 660 650 --

Effect of buffer on color development 9.0 - 10.0

pH-9.8

Variations of the pH less than 6.0 and greater than11.0 resulted in low

absorbance values

Volume of buffer required for maximum

intensity of color (ml)

0.5 - 1.5

1.0

Optimum volume of 1.0ml of buffer is sufficient for maximum color

development

Effect of volume of dye MB

1.0 - 5.0

1.0

1.0ml of MB dye is necessary for covering the broad range of beer’s law

limits

Choice of organic solvent for extraction of

colored complex

Chloroform

Chloroform

Water immiscible solvents tested for the extraction of the colored

complex into organic phase include chlorobenzene, dichloro methane,

CCl4, C6H6 and butanol. CHCl3 is preferred for its selective extraction of

the colored drug-dye complex from the aqueous phase.

Effect of the ratio of organic to aqueous

phase on extraction

1:1

1:1

The extraction of the colored species in to Chloroform layer is in

complete when the ratio of chloroform to aqueous phase is more than the

specified ratio in each case

Effect of shaking time (min)

1 – 5

2

Constant absorbance values are obtained for the shaking period of 1-5

min.

Effect of temperature on the colored species

Laboratory -

Temperature

(285)

Laboratory-

Temperature

(285)

At low temperature (<200C) and at high temperature (>35

0C) the

extraction of the colored species is found to be improper and the stability

of the colored species is found to be very less.

Stability of the colored species

Immediate to

60 min

10 min

The colored species after separation from organic phase is stable for 60

min, after wards the absorbance gradually decreases.


201

Table 3.08(b)

Optimum conditions established for Method M3 for LEF

Parameter Optimum range Conditions in

procedure

Remarks

max (nm) 490-510 510

Effect of volume of 3.33x10-3

M of Fe(III)

solution on color development

1.0-4.0ml

3.0ml

Variation of volume below and above of this range gave erratic results.


M of

O-PHEN on color development 1.5 –3.0ml 2.0ml

Variation of volume below and above of this range gave erratic results.

Effect of temperature on colored species

90-990C

Boiling Water

Bath.

It is found that boiling water bath is necessary for uniform temperature

and maximum color development. Below this temperature the intensity

of the colored complex is weak.

Effect of heating time

25-40min.

30min.

Below 25min. the colored complex is not completely formed.


M of o-

phosphoric acid.

1.0-3.0ml

2.0ml

To complex excess of Fe (III) ions, a minimum of 1.0 ml of o-

phosphoric acid is required.

Effect of order of addition of reagents on

color development

LEF, Fe (III)

solution, o-Phen

before heating

and phosphoric

acid after heating.

LEF, Fe (III)

solution and o-Phen

before heating and

phosphoric acid after

heating.

Interchanging the order of LEF, Fe (III) solution and o- Phen has no

effect on absorbance of the colored species.

Nature of solvent for final dilution.

Distilled Water

Distilled Water

Other water miscible solvents, like acetonitrile, methanol, ethanol,

acetone and 1, 4-dioxan did not enhance the intensity of the final colored

product.

Stability of the colored species after final

dilution.

5-60 min

5 min

The absorbance of the colored product decreased slowly beyond 60 min.


202

Table 3.08(c)

Optimum conditions established in Method M12 for LEF

Parameter

Optimum range

Conditions in

procedure

Remarks

max (nm) 510-550

430

Volume of (4.0×10-2

M) NaIO4 solution 0.5-2.0ml

1.0ml

Beyond the upper and lower limits low absorbance is observed either in

lower or upper Beer’s law limits.

Time and temperature.

25-35min. at

Laboratory

temperature

30min. at

Laboratory

temperature

30min. is necessary to complete oxidation.

Volume of (2.76×10-1

M) PHH solution. 0.5-2.5ml 1.5ml

1.5ml of PHH solution is found necessary for color development.

Volume of Hexacyano ferrate (III)

(3.02x10-3

M) solution 0.5-2.5ml 2.0ml

Addition of <0.5ml results in erratic values especially in upper region of

Beer’s law limits.

Time and temperature prior to the addition

of Conc.HCl.

5min.

0-50C

5min. in ice bath. Minimum cooling for 5min. in ice bath has been found to be necessary.

Addition of Conc.HCl.

4-6ml

5ml

4.0ml of Conc. HCl is found necessary for maximum color

development.

Solvent for final dilution Ethanol Ethanol Ethanol is found to be the best solvent for final dilution.

Stability period after final dilution. Immediate-

30min. 15min.


203

Table 3.08(d)

Optimum conditions established in Method M14for LEF

Parameter Optimum range Conditions in

procedure

Remarks

max (nm) 610 - 630 660

Effect of buffer pH on color.

pH - 2.0

Variation of pH of the buffer beyond the upper and lower limits resulted

in low absorbance values.

Volume of buffer required for maximum

intensity of color.

1.5 - 2.5ml

2.0ml

2.0 ml of buffer of pH 2.0 is found to be necessary in aqueous phase for

maximum color development in nitrobenzene layer.

Effect of volume of (2.86 x 10-1

M) of CTC

solution required for complex formation.

4.0 – 6.0ml

5.0 ml

5.0 ml of CTC is found to be necessary for complex formation and to

cover broad range of Beer’s law limits. No added advantage is observed

with more than 5.0ml.

Solvent for extraction of complex.

Nitrobenzene

Nitrobenzene

Among the various solvents tried for extraction (dichloromethane,

chloroform, chlorobenzene and nitrobenzene), nitrobenzene is found to

be better due to higher sensitivity.

Effect of the ratio of organic to aqueous

phase on extraction.

1:1,1:2,2:3

2:3

A ratio of 2:3 organic to aqueous phases is required for the efficient

extraction of the complex.

Stability period.

5 - 60 min

5 min

The intensity the colored species begins to decrease slowly after 60 min.


204

Table 3.08(e)

Optimum conditions established in method M18 for LEF

Parameter

Optimum

range

Conditions in

procedure

Remarks

max (nm)

400 - 470

440

Effect of volume (2.45 x 10-2

M) of 4-AP

in MeOH and waiting time. 2 - 4ml, 15min 3.0ml ,15min

3ml of 4-AP and 15min waiting time are preferred for covering

broad range in Beer’s law limits.

Solvent for final dilution. Methanol Methanol MeOH has been found to be suitable for final dilution to give

better absorbance values.

Stability period after final dilution. Immediate -

40min 10min

After 40min the absorbance of colored species diminish slowly

with time.


205

Method M1(a):ARS+CHCl3

[LEF]=9.00x10-5

M,[ARS]=3.66x10-4

M

[HCl]=4.00x10-2

M

0.000

0.030

0.060

0.090

0.120

0.150

0.180

375 400 425 450 475 500

Wavelength nm

Ab

so

rba

nc

e

BlankTest

Fig. 3.04 Absorption Spectra of LEF

with ARS M1(a)

Method M1(d):TPooo+CHCl3

[LEF]=9.00x10-5M,

[TPooo]=3.81x10-4M,[HCl]=4.00x10-2M

0.000

0.100

0.200

0.300

475 525 575Wavelength nm

Ab

so

rba

nc

e

Ox.DyeTestAq. Dye


with TPooo M1(c)

MethodM2(a):MB+CHCl3

[LEF]=7.20x10-5

M

[MB]=4.17x10-4

M,Buffer of pH=9.8

0.000

0.100

0.200

0.300

550 600 650 700 750Wavelength nm

Ab

so

rba

nc

e

Blank

Test

Fig. 3.06Absorption Spectra of LEF

with MB M2(a)

Method M3:Fe(III)+O-PHEN

[LEF]=1.08x10-4

M, [Fe(III)]=3.99x10-4

M

[O-PHEN]=8.07x10-4

M

[O-PHOS]=1.73x10-3

M

0.000

0.030

0.060

0.090

0.120

0.150

0.180

400 450 500 550 600Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with Fe(III)+O-PHEN M3


206

Method M5(c):MBTH+NaIO4+AcOH

[LEF]=5.40x10-5M,[MBTH]=3.42x10-4M

[NaIO4]=3.74x10-4M,[AcOH]=1.28x10-1M

0.000

0.050

0.100

0.150

0.200

500 550 600 650 700Wavelength nm

Ab

so

rba

nc

e

BlankTest


with MBTH+NaIO4+AcOH M5(a)

MethodM12:PHH+NaIO4+[Fe(CN)6]

[LEF]=5.40x10-5M, [PHH]=1.66x10-3

[Fe(CN)6]-3=2.42x10-4M,[HCl]=2.40M

0.000

0.050

0.100

0.150

0.200

375 400 425 450 475

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with PHH+NaIO4+[Fe(CN)6]-3

M12

Method

M14:CTC+Buffer+Nitrobenzene

[LEF]=5.40x10-5M,[CTC]=5.71x10-2M

Buffer of pH=2.0

0.000

0.100

0.200

0.300

550 600 650 700 750Wavelength nm

Ab

so

rba

nc

e

Blnk

Test


with CTC +Buffer+Nitrobenzene M14

Method M17:PA+CHCl3

[LEF]=4.50x10-5

M,[PA]=5.82x10-4

M

[HCl]=4.00x10-2

M

0.000

0.100

0.200

0.300

375 400 425 450 475

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with PA M17


207

Method M18:4- AP

[LEF]=1.08x10-4

M

[4-AP]=2.45x10-2

M

0.000

0.100

0.200

0.300

0.400

380 400 420 440 460 480 500

Wavelength nm

Ab

so

rba

nc

e

Blank

Test


with 4-AP M18

Method M1(a):ARS+CHCl3

[LEF]=2.25x10-5M - 1.35x10-4M

[ARS]=3.66x10-4M

[HCl]=4.0x10-2M

0.000

0.200

0.400

0.600

0 10 20 30 40 50 60

Weight of the Drug in μg/ml

Ab

so

rba

nc

e

Fig.3.13 Beer’s Law Plot of LEF with

ARS M1(a)


[LEF]=4.50x10-5M - 2.70x10-4M

[TPooo]=3.81x10-4M, [HCl]=4.0x10-2M

0.000

0.250

0.500

0.750

0 25 50 75 100


Ab

so

rba

nc

e


TPooo M1(d)

Method M2(a):MB+Buffer+CHCl3

[LEF]=3.6x10-5M - 2.16x10-4M

[MB]=4.17x10-4M,Buffer of pH=9.8

0.000

0.300

0.600

0.900

0 20 40 60 80 100


Ab

so

rba

nc

e


MB M2(a)


208


[LEF]=5.40x10-5M - 3.24x10-4M

[Fe(III)]=3.99x10-4M,

[O-PHEN]=8.07x10-4M

[O-PHOS]=1.73x10-3M

0.000

0.200

0.400

0.600

0 40 80 120


Ab

so

rba

nc

e


Fe(III)+O-PHEN M3


[LEF]=2.70x10-5M - 1.62x10-4M

[MBTH]=3.42x10-4M,

[NaIO4]=3.74x10-4M

[AcOH]=1.28x10-1M

0.000

0.250

0.500

0.750

0 20 40 60

Weight of the Drug in μg/mlA

bs

orb

an

ce


MBTH+NaIO4+AcOH M5(c)

MethodM12:PHH+NaIO4+[Fe(CN)6]-3

[LEF]=2.70x10-5M - 1.62x10-4M

[PHH]=1.66x10-2M,[NaIO4]=1.60X10-3M

[[Fe(CN)6]-3]=2.42X10-4M

0.000

0.200

0.400

0.600

0.800

0 20 40 60


Ab

so

rba

nc

e


PHH+NaIO4+[Fe(CN)6]-3

M12

MethodM14:CTC+Buffer+NB

[LEF]=1.35x10-5M - 8.10x10-5M

[CTC]=5.71x10-2M,Buffer of pH=2.0

0.000

0.100

0.200

0.300

0.400

0.500

0 10 20 30


Ab

so

rba

nc

e


CTC+Buffer+Nitrobenzene M14


209

Method M17:PA+CHCl3

[LEF]=2.25x10-5M - 1.35x10-4M

[PA]=5.82x10-4M,[HCl]=4.0x10-2M

0.000

0.300

0.600

0.900

0 20 40 60


Ab

so

rba

nc

e


PA M17

Method M18 4-AP

[LEF]=2.25x10-5

M - 1.35x10-4

M

[4-AP]=2.45x10-2

M

0.000

0.200

0.400

0.600

0 25 50 75


Ab

so

rba

nc

e

Fig.3.21 Beer’s Law Plot of LEF with 4-

AP M18

Method M1(a) :ARS+CHCl30

30

60

90

0.50 1.00 1.50 2.00

LOG(Concentration in μg/ml)

%T

ran

sm

ita

nc

e

Fig.3.22 Ringbom Plot of LEF with ARS

M1(a)


30

60

90

1.00 1.25 1.50 1.75 2.00


%T

ran

sm

ita

nc

e

Fig.3.23 Ringbom Plot of LEF with

TPooo M1(d)


210

Method M2(a):MB+CHCl3

0

20

40

60

80

1.00 1.50 2.00


%T

ran

sm

ita

nc

e

Fig.3.24 Ringbom Plot of LEF with MB

M2(a)


30

60

90

1.00 1.50 2.00 2.50


%T

ran

sm

ita

nc

e


Fe(III)+O-PHEN M3


0

30

60

90

0.40 0.80 1.20 1.60


%T

ran

sm

ita

nc

e


MBTH+NaIO4+AcOH M5(c)

Method M12:PHH+NaIO4+[Fe(CN)6]-3

0

20

40

60

80

0.50 1.00 1.50 2.00


%T

ran

sm

ita

nc

e


PHH+NaIO4+[Fe(CN)6]-3

M12


211

Method M14:CTC+Buffer+NB20

40

60

80

100

0.50 0.75 1.00 1.25 1.50


%T

ran

sm

ita

nc

e


CTC+Buffer+Nitrobenzene M14

Method M17:PA+CHCl30

20

40

60

80

0.50 1.00 1.50 2.00


%T

ran

sm

ita

nc

e

Fig.3.29 Ringbom Plot of LEF with PA

M17

Method M18:4-AP

25

50

75

100

0.50 1.00 1.50 2.00 2.50

Concentration of the Drug in μg/ml

%T

ran

sm

ita

nc

e

Fig.3.30 Ringbom Plot of LEF with 4-AP

M18


212

Table-3.09(a): Optical Characteristics of the proposed methods for LEF

Name of the Parameter M1(a) M1(d) M2(a) M3 M5(c)

Maximum Wavelength λmax 415 nm 515 nm 650 nm 510 nm 610 nm

Beer's Law Limits µg/ml 8.3-50.0 16.7-100.0 13.3-80.0 20.0-120.0 10.0-60.0

Optimum Photometric Range µg/ml 16.7-41.7 33.3-83.3 26.7-66.7 40.0-100.0 20.0-40.0

Sandell's Sensitivity µg/cm2 / 0.001 Abs 8.59E-02 1.52E-01 9.46E-02 2.60E-01 9.62E-02

Molar Absorptivity lt/mole/cm 3.85E+03 2.36E+03 3.64E+03 1.62E+03 3.55E+03

Table-3.09(b): Optical Characteristics of the proposed methods for LEF

Name of the Parameter M12 M14 M17 M18

Maximum Wavelength λmax 430 nm 660 nm 415nm 440

Beer's Law Limits µg/ml 10.0-60.0 5.0-30.0 8.3-50.0 10.0-60.0

Optimum Photometric Range µg/ml 20.0-50.0 10.0-25.0 17.7-41.7 10.0-50.0

Sandell's Sensitivity µg/cm2 / 0.001 Abs 8.33E-02 7.46E-02 6.27E-02 0.1031

Molar Absorptivity lt/mole/cm 3.91E+03 5.01E+03 5.93E+03 3.21E+03

Table-3.10(a): Linear least square regression analysis

Name of the Parameter M1(a) M1(d) M2(a) M3 M5(c)

Slope (b) 1.04E-02 6.38E-03 9.82E-03 4.38E-03 9.57E-03

Intercept(a) 2.27E-03 6.00E-04 -5.33E-04 -1.05E-02 6.00E-03

Standard Deviation on Slope(Sb) 1.45E-04 2.95E-05 1.35E-04 4.94E-05 8.72E-05

Standard Deviation on Intercept(Sa) 4.69E-03 1.91E-03 7.02E-03 3.85E-03 3.40E-03

Correlation coefficient (r ) 0.9994 0.9999 0.9994 0.9999 0.9998

Limit of Detection (LOD) µg/ml 1.362 0.906 2.150 2.646 1.066

Limit of Quantification (LOQ) µg/ml 4.525 3.006 7.158 8.802 3.552

Table-3.10(b): Linear least square regression analysis

Name of the Parameter M12 M14 M17 M18

Slope (b) 1.06E-02 1.35E-02 1.60E-02 8.65E-03

Intercept(a) 1.60E-03 -4.00E-04 6.33E-03 2.27E-03

Standard Deviation on Slope(Sb) 1.73E-04 4.47E-05 2.00E-04 1.21E-04

Standard Deviation on Intercept(Sa) 6.74E-03 8.69E-04 6.48E-03 4.69E-03

Correlation coefficient (r ) 0.9991 0.9999 0.9995 0.9994

Limit of Detection (LOD) µg/ml 1.922 0.197 1.214 1.627

Limit of Quantification (LOQ) µg/ml 6.394 0.643 4.053 5.423


213

Table 3.11(a) Precision of the proposed methods

Statistical Parameter Value M1(a) M1(d) M2(a) M3 M5(c)

Concentration (μg/ml) 33.33 66.67 53.33 80.00 40.00

Mean( of six replicates) (μg/ml) 33.26 66.56 53.25 79.81 40.05

Standard Deviation (s) 0.252 0.280 0.304 0.533 0.191

%Relative Standard Deviation(%RSD) 0.763 0.436 0.553 0.676 0.484

0.05 level confidence limit µg/ml 0.415 0.474 0.495 0.884 0.326

Table 3.11(b) Precision of the proposed methods

Statistical Parameter Value M12 M14 M17 M18

Concentration (μg/ml) 40.00 20.00 33.33 40.00

Mean( of six replicates) (μg/ml) 39.95 19.95 33.32 39.93

Standard Deviation (s) 0.213 0.208 0.185 0.251

%Relative Standard Deviation(%RSD) 0.525 0.985 0.552 0.629

0.05 level confidence limit µg/ml 0.343 0.327 0.304 0.413

Table-3.12(a): Accuracy of the proposed method M1 (a)

Sample

ID

Amount

Taken µg/ml

Amount

Found µg/ml

Percent of

Recovery

Statistical analysis

1 25.00 25.10 100.41 Mean 100.16

2 25.00 25.07 100.29 SD 0.340

3 25.00 24.94 99.77 %RSD 0.340

1 33.33 33.58 100.75 Mean 100.53

2 33.33 33.72 101.17 SD 0.774

3 33.33 33.22 99.67 %RSD 0.770

1 41.66 42.07 100.98 Mean 100.06

2 41.66 41.23 98.96 SD 1.020

3 41.66 41.76 100.23 %RSD 1.019


214

Table-3.12(b): Accuracy of the proposed method M1(d)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 50.00 49.21 98.42 Mean 99.922

2 50.00 49.74 99.48 SD 1.773

3 50.00 50.94 101.87 %RSD 1.774

1 66.67 66.94 100.40 Mean 99.830

2 66.67 65.99 98.98 SD 0.751

3 66.67 66.74 100.10 %RSD 0.752

1 83.34 84.21 101.05 Mean 100.251

2 83.34 82.96 99.55 SD 0.754

3 83.34 83.47 100.16 %RSD 0.752

Table-3.12(c): Accuracy of the proposed methodM2(a)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 40.00 40.95 102.38 Mean 101.448

2 40.00 39.92 99.81 SD 1.426

3 40.00 40.86 102.16 %RSD 1.406

1 53.33 53.99 101.24 Mean 100.444

2 53.33 52.96 99.31 SD 1.011

3 53.33 53.75 100.79 %RSD 1.006

1 66.66 67.05 100.58 Mean 99.986

2 66.66 66.94 100.42 SD 0.892

3 66.66 65.97 98.96 %RSD 0.892


215

Table-3.12(d): Accuracy of the proposed Method M3

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 60.00 61.22 102.03 Mean 100.139

2 60.00 59.72 99.53 SD 1.676

3 60.00 59.31 98.85 %RSD 1.674

1 80.00 81.42 101.78 Mean 100.629

2 80.00 79.16 98.95 SD 1.486

3 80.00 80.93 101.16 %RSD 1.477

1 100.00 101.31 101.31 Mean 100.067

2 100.00 98.96 98.96 SD 1.181

3 100.00 99.93 99.93 %RSD 1.180

Table-3.12(e): Accuracy of the proposed method M5(c)

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 30.00 30.71 102.37 Mean 100.956

2 30.00 30.41 101.37 SD 1.655

3 30.00 29.74 99.13 %RSD 1.640

1 40.00 40.77 101.93 Mean 100.500

2 40.00 40.32 100.80 SD 1.596

3 40.00 39.51 98.78 %RSD 1.588

1 50.00 50.73 101.46 Mean 99.793

2 50.00 49.21 98.42 SD 1.541

3 50.00 49.75 99.50 %RSD 1.544


216

Table-3.12(f): Accuracy of the proposed method M12

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 30.00 30.07 100.23 Mean 99.820

2 30.00 29.95 99.83 SD 0.413

3 30.00 29.82 99.40 %RSD 0.414

1 40.00 40.74 101.85 Mean 100.534

2 40.00 39.85 99.63 SD 1.167

3 40.00 40.05 100.13 %RSD 1.161

1 50.00 50.21 100.42 Mean 99.587

2 50.00 49.66 99.32 SD 0.737

3 50.00 49.51 99.02 %RSD 0.740

Table-3.12(g): Accuracy of the proposed method M14

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 15.00 15.30 102.00 Mean 101.333

2 15.00 14.98 99.87 SD 1.272

3 15.00 15.32 102.13 %RSD 1.255

1 20.00 19.94 99.70 Mean 100.083

2 20.00 20.08 100.40 SD 0.355

3 20.00 20.03 100.15 %RSD 0.354

1 25.00 25.08 100.30 Mean 99.833

2 25.00 24.95 99.80 SD 0.451

3 25.00 24.85 99.40 %RSD 0.452


217

Table-3.12(h): Accuracy of the proposed method M17

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 25.00 24.85 99.41 Mean 99.837

2 25.00 24.98 99.93 SD 0.389

3 25.00 25.04 100.17 %RSD 0.389

1 33.33 33.14 99.43 Mean 100.350

2 33.33 33.52 100.57 SD 0.832

3 33.33 33.68 101.05 %RSD 0.829

1 41.66 41.17 98.82 Mean 100.026

2 41.66 41.98 100.76 SD 1.055

3 41.66 41.87 100.50 %RSD 1.054

Table-3.12(i): Accuracy of the proposed method M18

Sample

ID

Amount taken

µg/ml

Amount found

µg/ml

Percent of

Recovery


1 30.00 29.75 99.17 Mean 100.233

2 30.00 30.31 101.03 SD 0.961

3 30.00 30.15 100.50 %RSD 0.959

1 40.00 39.85 99.63 Mean 100.817

2 40.00 40.62 101.55 SD 1.041

3 40.00 40.51 101.28 %RSD 1.033

1 50.00 50.46 100.92 Mean 99.827

2 50.00 49.77 99.54 SD 0.982

3 50.00 49.51 99.02 %RSD 0.984


218

Table3.13 (a): Assay of Formulations of Levofloxacin; Reference Method [235]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery

Ref.Method Proposed methods**

M1(a) M1(d) M1(a) M1(d)

Levoflox 250 Mean 250.69 252.14 %REC 100.42 100.28 100.86

SD ±2.131 ±2.312 %RSD ±0.86 ±0.851 ±0.916

F-test 1.026 1.147

t-test 0.561 1.601

Leon 250 Mean 250.05 250.95 %REC 100.08 100.02 100.038

SD ±2.074 ±2.841 %RSD ±0.5 ±0.829 ±1.132

F-test 1.083 1.731

t-test 0.042 0.578


SD ±3.814 ±3.869 %RSD ±0.86 ±0.763 ±0.284

F-test 1.282 1.245

t-test 0.151 0.474

Leon 500 Mean 500.51 503.26 %REC 100.08 100.10 100.65

SD ±2.984 ±2.96 %RSD ±0.5 ±0.596 ±0.589

F-test 2.094 2.116

t-test 0.296 1.900

*Average of six determinations are considered, AVG=Average, SD=Standard

deviation, F=F-test value, t=t-test value; Theoretical values at 0.05 level of

confidence limit F=5.19, t=1.833.

**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and

500.0mg formulations (Average of six determinations)


219

Table3.13 (b): Assay of Formulations of Levofloxacin, Reference Method [235]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M2(a) M3 M2(a) M3


SD ±1.984 ±2.136 %RSD ±0.86 ±0.794 ±0.849

F-test 1.184 1.021

t-test 0.305 1.061

Leon 250 Mean 24.94 248.94 %REC 100.08 99.58 99.58

SD ±1.975 ±2.136 %RSD ±0.5 ±0.793 ±0.858

F-test 0.1.195 1.022

t-test 0.929 0.859


SD ±3.853 ±2.537 %RSD ±0.86 ±0.0.767 ±0.505

F-test 1.255 2.896

t-test 0.992 1.609

Leon 500 Mean 495.82 500.78 %REC 100.08 99.16 100.16

SD ±3.741 ±2.946 %RSD ±0.5 ±0.754 ±0.588

F-test 1.332 2.147

t-test 1.933 0.458

*Average of six determinations are considered, AVG=Average, SD=Standard deviation,

F=F-test value, t=t-test value; Theoretical values at 0.05 level of confidence limit F=5.19,

t=1.833.

**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and

500.0mg formulations (Average of six determinations)


220

Table 3.13 (c): Assay of Formulations of Levofloxacin, Reference Method [235]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M5(c) M12 M5(c) M12


SD ±1.683 ±1.924 %RSD ±0.86 ±0.671 ±0.773

F-test 1.645 1.259

t-test 0.843 1.174

Leon 250 Mean 248.57 251.02 %REC 100.08 99.43 100.41

SD ±2.617 ±2.154 %RSD ±0.5 ±1.052 ±0.858

F-test 1.469 1.004

t-test 0.945 0.821


SD ±2.859 ±4.142 %RSD ±0.86 ±0.480 ±0.833

F-test 2.281 1.087

t-test 1.337 1.261

Leon 500 Mean 499.43 501.53 %REC 100.08 99.89 100.31

SD ±3.754 ±1.994 %RSD ±0.5 ±0.751 ±0.397

F-test 1.323 0.4.689

t-test 0.265 0.265



t=1.833.

**%REC=% of Recovery, %RSD=%of Relative standard deviation; 250.0 and 500.0mg

formulations (Average of six determinations)


221

Table 3.13 (d): Assay of Formulations of Levofloxacin, Reference Method [235]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed methods*

Percent of Recovery


M14 M17 M14 M17


SD ±1.376 ±2.142 %RSD ±0.86 ±0.548 ±0.862

F-test 2.462 1.016

t-test 1.353 1.163

Leon 250 Mean 248.95 251.24 %REC 100.08 99.58 100.50

SD ±2.164 ±1.794 %RSD ±0.5 ±0.869 ±0.714

F-test 1.005 1.448

t-test 0.839 1.196


SD ±3.868 ±2.794 %RSD ±0.86 ±0.776 ±0.557

F-test 1.246 2.388

t-test 0899 0.848

Leon 500 Mean 502.11 498.79 %REC 100.08 100.42 99.76

SD ±2.415 ±2.995 %RSD ±0.5 ±0.481 ±0.601

F-test 3.196 2.078

t-test 1.512 0.699



t=1.833.




222

Table 3.13 (e): Assay of Formulations of Levofloxacin, Reference Method [235]

Sample

Amount

Taken

(mg/tablet)

Amount found in

proposed method*

Percent of Recovery

Ref.Method Proposed method**

M18 M18

Levoflox 250 Mean 249.57 %REC 100.42 99.83

SD ±1.859 %RSD ±0.86 ±0.745

F-test 1.348

t-test 0.400

Leon 250 Mean 251.31 %REC 100.08 100.52

SD ±2.125 %RSD ±0.5 ±0.845

F-test 1.032

t-test 1.066

Levoflox 500 Mean 499.57 %REC 100.42 99.91

SD ±3.513 %RSD ±0.86 ±703

F-test 1.511

t-test 0.212

Leon 500 Mean 500.68 %REC 100.08 100.14

SD ±3.152 %RSD ±0.5 ±0.629

F-test 1.876

t-test 0.373



t=1.833.




223

3.13 Nature of the colored species

The important functional groups that are responsible for the color development

in Levofloxacin are substituted quinoline, substituted pyrazine, carboxylic acid and

carbonyl (keto) group. Based on the reviews concerning the reagents used for the

development of color by exploiting appropriate functional moieties in Levofloxacin,

an attempt has been made to indicate the nature of colored species in each of the

proposed methods.

Tertiary nitrogen atom of pyrazine is responsible for the development of ion-

ion association complex formation with acid dyes such as Alizarin Red-S (ARS) and

Tropaeolin ooo (TPooo) in method M1(a) and method M1(d) respectively; Another

extractive method is developed with basic dye MB which forms ion-ion association

complex with carboxylic acid functional group of the drug in method M2(a). Ortho-

position of substituted quinoline is favorable for electrophlic substitution reactions in

oxidative coupling reaction in method M5(c). The tertiary nitrogen in pyrazine is also

responsible for ion-ion association complex with PA in basic buffer medium method

M17. 4 – amino phenazone (4- AP) gave a colored hydrazone with keto groups of the

drug M18 . Fe (III) converts into a Fe (II) due to oxidation of the drug and reduced

form of iron is detected by the usual reagent o-phenanthroline methodM3. The

colored species formed is the coordination complex of the drug (electron donor) and

the central atom of cobalt thiocyanite, which is extractable into nitrobenzene from

aqueous solution method M14.

Methods - M1(a) and M1(d)

Tertiary nitrogen atom of substituted pyrazine ring is protonated in acidic

medium. The protonated drug molecule is associated with anion of the acidic dyes


224

such as ARS and TP ooo and behaves as a single unit being held together by

electrostatic force of attraction which is extractable into chloroform from aqueous

phase. The scheme of the colored product is given below.

N

O

F

O

OH

O

HN

NH N

O

F

O

OH

O

HNH

N

Protonation

Levofloxacin Cation of the Drug

Anion of ARSN

O

F

O OH

O

H

HN N

O

O

-O3S

HON

O

F O

OHO

H

NH

N

Cation of the DrugColored Product

Anion of TPooo

N

O

F O

OHO

H

NH

N

OH

N

N

O3S

N

O

F O

OHO

H

NH

N

Cation of the Drug

Colored Product

Scheme 3.01

Method - M2(a)

Levofloxacin possesses carboxylic acid in substituted quinoline ring which is

responsible for color formation in ion association complex with basic dye methylene

blue which is extractable into chloroform. The carboxylate anion of LEF is expected to


225

attract the oppositely charged part of methylene blue and behaves as a single unit being

held together by electrostatic attraction. The scheme of the reaction is given below

N

O

F

O

OH

O

HN

N

N

O

F

O

O

O

H

N

N + HBasic Buffer

LevofloxacinAnion of Levofloxacin

N

O

F O

OO

H

N

N

NS

CH

3

N

N+

CH

3

CH3CH3

Cation of the Dye

+ N

O

F

O

O

O

HN

N

N

SCH3

NN+

CH3

CH3

CH3

Anion of Levofloxacin Colored Product

Scheme 3.02

Method M3

Levofloxcin is oxidized in the presence of oxidizing agent Fe (III) which is

reduced into Fe (II).The reduced form of iron can be easily converted into colored

product by using o-phenanthroline. The reduction product is tris complex of Fe (II),

well known as ferroin.

Fe(III) + Levofloxacin Fe(II) + Levofloxacin

Reduction

Oxidation

Unreacted Fe(III)

Unreacted Fe(III) + O-Phosphoric Acid Fe(III)-O-Phosphoric Acid Complex

+


226

N

N

3 + Fe(II)

NN

N

N N

NFe(II)

Colored ComplexO-PHEN

Scheme 3.03

Method –M5(c)

Under the reaction conditions, MBTH on oxidation in the presence of oxidant

loses two electrons and one proton forming and electrophilic intermediate, which is the

active coupling species that reacts with the coupler levofloxacin by electrophillic attack

on ortho position of the most nucleophilic site on cyclic ring substituted quinoline.

N

S

CH3

N-NH2

MBTH

N

S

CH3

N NH Ce(IV)

Electrophilic intermediate

-2e , H

/ NaIO4+AcOH

N

S

CH3

N


NHN

S

CH3

N NH


N

O

F

O

OH

O

HN

N

N

S

CH3

N NH


N

O

F

O

OH

O

HN

NN

S

CH3

NNH

Colored Product

Scheme 3.04


227

Method M12

In this method aldehyde formed from epoxide portion of levofloxacin through

oxidation, is first converted into its phenyl hydrazone, then a red color is developed

under the action of the oxidizing agent [(hexacyanoferrate (III)] in acid medium. The

colored species formation may be represented as under scheme.

N

O

F

O

HO

O

HN

N

H2O2

N

O

F

O

HO

O

HN

NOHO

N

O

FO

HO O

H

N

N

OH

Levofloxacin

Epoxide

N

NH2

H

.HCl

+

H

CO

HN

N C

H

N

NH2

H

.HCl

[Fe(CN)6]-3

N NCl

I

II

I + II

HN

N C

H

NNFormazine Dye

Scheme 3.05

Method – M14

Formation of the colored complex is obtained when LEF is treated with CTC

due to the presence of tertiary amine group. The colored species formed is the

coordination complex of the drug (electron donor) and the central atom of cobalt

thiocyanite, which is extractable into nitrobenzene from aqueous solution.


228

CNS

NCS

SCN

Co

SCN

N

O

F

O

OH

O

H

N

N

N

O

F

O

HO

O

H

N

N

Scheme 3.06

Method M17

In basic medium picric acid immediately loses the acidic proton, to produce a

stable anionic species. Levofloxacin possesses a tertiary nitrogen atom in substituted

Pyrazine ring and it immediately accepts the proton thus the drug molecule behaves as

a cation. The cation ion of the drug and the negative ion of the dye form a stable ion-

ion associated complex which is extractable into chloroform solvent. The scheme of

the colored product is represented in scheme

OH

NO2

NO2

O2N

O

NO2

NO2

O2N + HBasic Buffer

Stable Anion of Picric AcidPicric Acid

N

O

F

O

OH

O

HN

N + H NH

O

F

O

OH

O

HN

NProtonation

Levofloxacin Protonated Cation


229

NH

O

FO

OHO

H

N

N O

NO2

NO2

O2N+

NH

O

F

O

OH

O

HN

N

O

NO2

NO2

O2N

Ion-Ion Associated ComplexCation of the Drug Anion of the reagent

Scheme 3.07

Method: M18

4 – Amino phenazone (4- AP) gave a colored hydrazone with keto group of

the drug Levofloxacin due to condensation. The colored species formation may be

represented as under scheme.

CH3

CH3H2N

O

C6H5

-H2O

Condensation

4-AP

N

O

FO

HO O

H

N

N

+

CH3H3C

O

C6H5

N

FO

HO O

H

N

N

N

Scheme 3.08

3.14 Conclusions

The proposed methods exploit the various functional groups that are present in

Levofloxacin. These methods are simple, sensitive and reliable and can be used for

routine determination of LEF in bulk and pharmaceutical formulations. Statistical


230

analysis of the results shows that the proposed procedures have good precision and

accuracy. Results of the analysis of pharmaceutical formulations reveal that the

proposed methods are suitable for their analysis with virtually no interference of the

usual additives present in pharmaceutical formulations. The descending order of

sensitivity (εmax) and maximum wavelength (λmax) among the proposed methods are

shown below.

The descending order of sensitivity (εmax) among the proposed methods is shown

below.

M17›M14›M12›M1(a) ›M2(a) ›M5(c) ›M18›M1(d) ›M3

The descending order maximum wavelength (λmax) among the proposed methods is

shown below.

M14›M2(a)› M5(c) ›M1(d) ›M3›M12›M18›M1(a)›M17

Chapter – III Levofloxacin Part-C: Derivative Spectrophotomerty

231

ESTIMATION OF LEVOFLOXACIN BY DERIVATIVE

SPECTROPHOTOMETRY

3.15Introduction

The derivatization of spectra can lead to more accurate determination of the

wavelengths of broad peak maxima, of peaks which appear only as shoulders, as

well as the isolation of small peaks from interfering large background absorption.

The influence of an impurity on the absorption spectrum of a substance can be

eliminated by considering derivative curves. The main advantage of Derivative

spectrophotometry is background correction, multicomponent analysis with better

selectivity which is not possible in normal spectrophotometry.

For quantitative purpose, peak heights are usually measured for the long-

wave peak satellite of the second derivative curves or the short – wave peak satellite

of the same curves. Single or multi-components can be analyzed in the presence of

broad, interfering background matrix absorption. Such measurements are of

particular importance in clinical, biological, biochemical and food laboratories. An

extensive literature survey reveals that a few derivative spectrophotometric methods

are reported to determine the amount of Levofloxacin. The objective of the present

work is to develop simple, rapid and economical Second Order Derivative

Spectrophotometric methods for determination of levofloxacin in bulk and

formulations.

3.16(i) Experimental

UV-Visible Spectrophotometer: ElicoSL159 model, 2nm high resolution, double

beam, 1cm length quartz coated optics; Wavelength range190-1100nm; High


232

stability, linearity, and precision instrument is used for all the spectral

measurements.

3.16 (ii) Preparation of stock, working standard and reagent solutions

Stock solution of levofloxacin is freshly prepared by transferring accurately

weighed 100mg of levofloxacin into 100ml volumetric flask and dissolved in triply

distilled water and made up to the mark. Then working standard solutions 250μg/ml

for methods M2(a), M5(a) and M15; 200μg/ml for method M3 are prepared by

transferring 25.0ml and 20.0ml of the stock solution into two 100ml standard flask

and made up to the mark with triple distilled water.

Preparation of reagents

Method M2(a)

MB solution (Fluka; 0.2%, w/v 6.25x10-3


MB in 100ml of distilled water.

NH4OH – NH4Cl Buffer solution: 7.0gms of NH4Cl and 6.8ml of liquid ammonia

solution are mixed and diluted to 100ml with distilled water and pH is adjusted to

9.8

Method – M3

Fe (III) solution (Wilson labs; 0.054%, 3.33x10-3

M): Prepared by dissolving

54mg of anhydrous Ferric chloride in 100ml of distilled water.


233

O-PHEN solution (E.Merck, 0.2%, 1.10x10-2

M): Prepared by dissolving 200mg

of o-phenanthroline in 100ml of distilled water with warming

O-phosphoric acid solution (Qualigens, 2.0x10-2

M): Prepared by mixing 1.27ml

of o-phosphoric acid with 100ml of distilled water. 10.0ml of this stock solution is

further diluted to 100ml with distilled water.

Method M5(a)

MBTH Solution (Fluka; 0.2%, 8.56 x 10-3

M): 200mg of 3-Methyl-2-

BenzaThiazolinone Hydrazone (MBTH) is accurately weighed, transferred into

100ml volumetric flask, made up to the mark with distilled water

Ce(IV)solution(Merck); 1%, 9.35 x 10-3

M): 1% Ce(IV) solution is prepared by

dissolving accurately weighed 1.0g of potassium dichromate in 100ml of distilled

water

Method – M15

p-CA solution (Sd-fine; 0.1%, 4.785x10-3

M: Prepared by dissolving 100mg of p-

chloranilic acid initially in 20ml of isopropyl alcohol followed by dilution to 100ml

with chloroform.

3.16 (iii) Procedures of the proposed methods

Method M2(a)

Aliquots of standard drug solution of levofloxacin 6.7 – 106.7 μg/ml are

added into a series of 25ml tubes. 1.0ml buffer solution of pH 9.8 is placed


234

separately, and then 0.5ml of MB is added. The total volume of aqueous phase in

each funnel is adjusted to 10.0ml with distilled water. Then 10.0mlof chloroform is

added in each separating funnel and the contents are shaken for 2min and allowed to

separate. The organic layer is collected through cotton plug into another tube. The

zero order, first derivative and second derivative spectra are recorded using the

prepared solutions against reagent blank (Fig.3.31, Fig.3.35, Fig.3.39; P: 238-240).

The second derivative amplittiud values are plotted against concentration to

construct the calibration curves (Fig.3.43; P : 241).

Method – M3

Aliquots (10.0-160.0g/ml) of standard LEF solution are transferred into a

series of 25ml calibrated tubes and then solutions of 3.0ml (3.33 x 10-3

M) of Fe

(III), 2.0ml of (1.01 x 10-2

M) o-phenanthroline are added successively. The total

volume in each tube is brought to 10.0ml with distilled water. The tubes are kept on

a boiling water bath for 30min. The tubes are removed and cooled to room

temperature. 2.0ml of (2.16 x 10-2

M) o-phosphoric acid is added and volume in each

tube is made up to the mark with distilled water. The absorption spectra, first

derivative and second derivative spectra are recorded using the prepared solutions

against reagent blank (Fig.3.32, Fig.3.36, Fig.3.40; P: 238-240). The values of the

second derivative amplitude are obtained using five different concentrations

(Fig.3.44; P: 241).

Method – M5(a)

Aliquots of standard LEF solution (2.5 – 40.0g/ml) are transferred into a

series of 25ml calibrated tubes. Then 0.5ml (8.56 x 10-3

M) of MBTH solution is


235

added and kept aside for 5min. After that 2.0ml (1.58 x 10-2

M) of ceric ammonium

sulphate is added and kept aside for 10min. The volume is made up to the mark

with distilled water. Zero order, first and second derivative spectra are recorded

against reagent blank (Fig.3.33, Fig.3.37, Fig.3.41; P: 238-240). Calibration plot is

constructed by plotting the values of second derivative amplitude against

concentration of the drug (Fig.3.45; P: 241).

Method – M15

Into a series of 10ml calibrated tubes containing aliquots of standard LEF

solution (6.25 – 100.0g/ml), 2.0ml of chloranilic acid (4.785 x 10-3

M) is added and

kept aside for 30 min at lab temperature. The volume in each tube is made up to the

mark with chloroform. The spectra of the extracted organic layer are recorded

against reagent blank (Fig.3.34, Fig.3.38, Fig.42; P: 238-240). The values of the

second derivative amplitude are obtained for five different concentrations, the

values of are plotted against concentration to construct the calibration curve

(Fig.3.46; P: 241).

3.17 Method Development

The zero-order (0D) spectra of levofloxacin are showed in Fig.3.31 –Fig.

3.34; P: 238.The zero-crossing points are assigned from the 1D and

2D spectra of

the proposed methods are shown Fig3.35- Fig.3.42; P: 239-240. The 1D zero-

crossing point of levofloxacin for the proposed methods are found at 640nm for

method M2(a), 517nm for method M3, , 640nm for method M5(a), and 525 nm

for method M15 (Fig.3.35-Fig.3.38; P: 239 ) respectively; and 2D zero-crossing

point of LEF for the proposed methods are found to be 600nm and 680nm for


236

method M2a, 470nm, 530nm for method M3; 610nm and 670 nm for method

M5(a) and 485nm and 567 nm for method M15 (Fig3.39-Fig3.42; P:240)

respectively. The selection of the optimal wavelength is based on the fact that the

absolute value of the total derivative spectrum at the selected wavelength has the

best linear response to the analyte concentration. Second derivative amplitudes are

measured at wavelength 650nm, 510nm, 640nm and 540nm for the methods M2(a),

M3, M5(a) and M15respectively.

3.18. Method Validation

Linearity and Range: The calibration curves are constructed by plotting the 2D

value against concentration at the zero-crossing wavelengths. The linearity of the

calibration curves and the adherence of the method to Beer’s law are validated by

the high value of the correlation coefficient and the value of intercept on ordinate

which is close to zero. The calibration plots are linear in the range of 6.7 –

106.7μg/ml (M2a); 10.0-160.0 μg/ml for (M3), 2.0-16.0 μg/ml (M5a) and 6.2 –

100.0 μg/ml (M15) (Fig.3.43 – Fig 3.46; P: 241). The 2D spectra of levofloxacin

showed better resolution and linearity hence chosen for the determination of

levofloxacin. The correlation coefficient of the standard curves (n = 5) for all the

methods is greater than 0.999.

Precision: One of the most common statistical terms employed to express precision

is the standard deviation. The precision of each proposed method is determined for

five replicates of a constant amount of LEF in total sample solution. The standard

deviation and percent relative standard deviation are calculated for the proposed

methods and are presented in Table 3.15, P: 242.


237

Accuracy: To determine the accuracy of each proposed method, different amounts

of samples (75%, 100%, and 125%) of levolfoxacin within the linearity limits are

taken and analyzed by the proposed methods. The results (percent error) are

recorded in Table 3.16(a)-Table 3.16(d), P: 242-244.

LOD and LOQ: The limit of detection and limit of quantitation are calculated and

the results are presented in Table 3.14, P: 242.

3.19Results and Discussion

Spectrophotometric parameters including derivative order, wavelength and

Δλ values should be optimized to obtain sensitivity and reproducibility. In this study

second-derivative technique (2D) traced with Δλ=2.0 nm is used. The plots drawn

between second derivative amplitude and concentration of the drug are linear

(Fig.3.43-Fig.3.46, P: 241). Calculated parameters such as linearity limits, slope

intercept, correlation coefficient, LOD and LOQ are presented in Table: 3.14, P:

242. Correlation coefficient values are found to be more than 0.999. This indicates

that there is good correlation between the second derivative amplitude and the

concentration of the drug in the developed method. Low percent of relative standard

deviation values 0.437, 0.171, 0.240 and 0.292 for the methods M2(a), M5(a), M7

and M15 indicate that these methods are precise ( Table: 3.15, P: 242). Mean

percent of recovery for the four methods is found in between 99.43% to 100.57% in

three concentration levels within the linearity limits reveals that the methods are

highly accurate


238

Zero Order Spectra of LEF with

MB,CHCl3

0.000

0.250

0.500

0.750

1.000

550 650 750

Wavelength nm

Ab

so

rba

nc

e4.17μg/ml8.33μg/ml16.67μg/ml33.33μg/ml66.67μg/ml

Fig.3.31 Absorption Spectra of LEF with

MB M2(a)


Fe(III),O-PHEN

0.000

0.250

0.500

0.750

400 450 500 550 600 650

Wavelength nm

Ab

so

rba

nc

e

10μg/ml20μg/ml40μg/ml80μg/ml160μg/ml

Fig.3.32 Absorption Spectra of LEF

with MB M3


MBTH,Ce(IV)

0.000

0.200

0.400

0.600

0.800

550 650 750

Wavelength nm

Ab

so

rba

nc

e

2.5μg/ml5.0μg/ml10.0μg/ml20.0μg/ml40.0μg/ml

Fig.3.33 Absorption Spectra of LEF with

MBTH+ Ce(IV) M5(a)

Zero Order Spectra of LEF

with p-CA,CHCl3

0.000

0.250

0.500

0.750

450 500 550 600 650

Wavelength nm

Ab

so

rba

nc

e

6.25μg/ml12.5μg/ml25.0μg/ml50.0μg/ml100μg/ml

Fig.3.34 Absorption Spectra of LEF

with p-CA M15


239

First Derivative Spectra of LEF

with MB, CHCl3

-1.5E-02

-1.0E-02

-5.0E-03

0.0E+00

5.0E-03

1.0E-02

1.5E-02

500 600 700 800

Wavelength nm

ΔA

/Δλ


Fig.3.35 First Derivative Spectra of

LEF with MB M2(a)

First Derivative Spectra of LEF with

Fe(III),O-PHEN

-1.E-02

-8.E-03

-6.E-03

-4.E-03

-2.E-03

0.E+00

2.E-03

4.E-03

6.E-03

8.E-03

375 425 475 525 575 625

Wavelength nm

ΔA

/Δλ



LEF with MB M3

First Derivative Spectra of LEF with

MBTH,Ce(IV)

-1.E-02

-9.E-03

-6.E-03

-3.E-03

0.E+00

3.E-03

6.E-03

9.E-03

500 600 700 800

Wavelength nm

ΔA

/Δλ



LEF with MBTH+ Ce(IV) M5(a)

First Derivative Spectra of LEF

with p-CA,CHCl3

-2.E-02

-1.E-02

-5.E-03

0.E+00

5.E-03

1.E-02

2.E-02

425 475 525 575 625

Wavelength nm

ΔA

/Δλ



LEF with p-CA M15


240

Second Derivative Spectra of LEF

with MB,CHCl3

-6.0E-04

-4.0E-04

-2.0E-04

0.0E+00

2.0E-04

4.0E-04

500 600 700 800

Wavelength nm

Δ2A

/Δ2λ


Fig.3.39 Second Derivative Spectra of

LEF with MB M2(a)

Second DerivativeSpectra of LEF

with Fe(III),O-PHEN

-4.0E-04

-3.0E-04

-2.0E-04

-1.0E-04

0.0E+00

1.0E-04

2.0E-04

3.0E-04

375 425 475 525 575 625

Wavelength nm

Δ2A

/Δ2λ



LEF with Fe(III)+O-PHEN M3


with MBTH,Ce(IV)

-6.E-03

-4.E-03

-2.E-03

0.E+00

2.E-03

4.E-03

500 600 700 800

Wavelength nm

Δ2A

/Δ2λ



LEF with MBTH+Ce(IV) M5(a)


with p-CA,CHCl3

-4.E-03

-3.E-03

-2.E-03

-1.E-03

0.E+00

1.E-03

2.E-03

3.E-03

425 475 525 575 625

Wavelength nm

Δ2A

/Δ2λ



LEF with p-CA M15


241

Plot of Second Derivative

Amplitudes aginst Concentration

(MB,CHCl3)

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

5.0E-04

0 40 80 120

Concentration in μg/ml

Am

plitu

de

Fig.3.43 Calibration plot of LEF with

MB Method M2(a)

Plot of Second Derivtive

Amplitudes against Concentration

(Fe(III),O-PHEN)

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

0 50 100 150 200


Am

plitu

de

Fig.3.44 Calibration plot of LEF with

Fe(III)+O-PHEN Method M3

Plot of Second Derivative

Amplitudes aginst Concentrtion

(MBTH,Ce(IV)

0.0E+00

2.0E-03

4.0E-03

6.0E-03

0 10 20 30 40


Am

plitu

de

Fig.3.45Calibration plot of LEF with

MBTH +Ce(III) Method M5(a)

Plot of the Second Derivative

Amplitudes against Concentration

(p-CA,CHCl3)

0.0E+00

1.0E-03

2.0E-03

3.0E-03

0 30 60 90 120


Am

plitu

de

Fig.3.46 Calibration plot of LEF with p-

CA Method M15


242

Table 3.14 Regression parameters for the proposed methods

S.No Name of the Parameter M2(a) M3 M5(a) M15

1 Linearity Limits µg/ml 6.7-106.7 10.0-160.0 2.5-40.0 6.25-100.0

2 Slope (b) 3.65E-06 2.16E-06 1.28E-04 3.00E-05

3 Intercept(a) 2.08E-06 -7.33E-07 7.17E-05 -2.03E-05

4 Correlation Coefficient ( r ) 0.9999 0.9998 0.9997 0.9998

5 Limit of Detection (LOD) µg/ml 0.345 4.665 1.471 1.554

6 Limit of Quantification (LOQ) µg/ml 1.149 15.551 4.904 5.180

Table 3.15 Precision of the proposed methods

S.No Name of the Parameter M2(a) M3 M5(a) M15

1 Amount Taken (µg/ml) 33.33 80.00 50.00 20.00

2 Mean (n=5)( µg/ml) 33.29 79.97 49.96 19.99

3 Standard Deviation (S) 0.145 0.137 0.05 0.146

4 %Relative Standard Deviation 0.437 0.171 0.240 0.292

5 0.05 level confidence limit 0.253 0.238 0.084 0.254

6 %Recovery 99.87 99.96 99.93 99.99

Table-3.16(a): Accuracy of the method M2(a)

Concentration-1 Concentration-2 Concentration-3

Taken Found %Recovery Taken Found %Recovery Taken Found %Recovery

24.99 25.32 101.29 33.33 33.65 100.96 41.66 42.06 100.95

24.99 24.87 99.49 33.33 33.78 101.35 41.66 41.45 99.49

24.99 24.97 99.89 33.33 33.11 99.33 41.66 41.26 99.03

24.99 25.31 101.25 33.33 33.12 99.36 41.66 42.18 101.24

24.99 25.21 100.85 33.33 33.77 101.32 41.66 41.39 99.34

24.99 25.32 101.29 33.33 33.65 100.96 41.66 42.06 100.95

Mean 25.13 100.55 33.486 100.46 41.66 100.01

SD 0.205 0.342 0.420

%RSD 0.815 1.022 1.010


243

Table-3.16(b): Accuracy of the method M3



60 59.63 99.38 80 80.56 100.70 100 98.96 98.96

60 60.42 100.70 80 79.63 99.53 100 99.79 99.79

60 60.13 100.22 80 79.16 98.95 100 101.85 101.85

60 59.75 99.58 80 80.47 100.58 100 101.45 101.45

60 59.44 99.07 80 80.84 101.05 100 100.75 100.75

60 59.63 99.38 80 80.56 100.70 100 98.96 98.96

Mean 59.87 99.79 80.13 100.16 100.56 100.56

SD 0.396 0.706 1.19

%RSD 0.662 0.881 1.18

Table-3.16(c): Accuracy of the method M5(a)



15 14.93 99.53 20 19.64 98.20 25 25.45 101.80

15 15.22 101.47 20 19.52 97.60 25 24.87 99.48

15 15.11 100.73 20 20.32 101.60 25 24.98 99.92

15 14.97 99.80 20 20.21 101.05 25 24.81 99.24

15 14.89 99.26 20 19.74 98.70 25 25.61 102.44

15 14.93 99.53 20 19.64 98.20 25 25.45 101.80

Mean 15.02 100.16 19.88 99.43 25.14 100.57

SD 0.137 0.356 0.362

%RSD 0.915 1.794 1.439


244

Table-3.16(d): Accuracy of the method M15



37.5 37.88 101.01 50 49.84 99.68 62.5 62.96 100.73

37.5 37.54 100.11 50 49.61 99.22 62.5 62.41 99.85

37.5 37.23 99.28 50 50.71 101.42 62.5 62.11 99.37

37.5 37.89 101.04 50 50.41 100.82 62.5 62.86 100.57

37.5 37.91 101.09 50 50.53 101.06 62.5 62.32 99.71

37.5 37.88 101.01 50 49.84 99.68 62.5 62.96 100.73

Mean 37.69 100.50 50.22 100.44 62.53 100.05

SD 0.299 0.471 0.363

%RSD 0.794 0.938 0.581

3.20 Conclusion

The developed methods are simple, rapid, precise, reliable, sensitive,

reproducible and economical. These methods could be readily applied to routine

quality control analysis of levofloxacin by ordinary laboratories.

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