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Chapter-7

7.1. Introduction

Levofloxacin is described chemically as (-)-(S)-9-fluoro-2,3-dihydro-3-

methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-

benzoxazine-6-carboxylic acid(Figure 7.1). The drug is an antibiotic of the

3rd-generation fluoroquinolone family inhibits the bacterial enzymes DNA

gyrase and topoisomerase IV [1]. The empirical formula is C18H20FN3O4 and

the molecular weight is 361.368. Levofloxacin is a off-white to yellow

crystalline powder.

(-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-

pyrido [1, 2, 3-de]-1,4-benzoxazine-6-carboxylic acid (Mol. Wt.:361.37)

Figure 7.1: Chemical structure of Levofloxacin

7.2. Literature Survey

Levofloxacin is a synthetic chemotherapeutic antibiotic of the

fluoroquinolone drug class [2-3] and is used to treat severe or life-

threatening bacterial infections or bacterial infections that have failed to

respond to other antibiotic classes [4-5] It is sold under various brand

names, such as Levaquin and Tavanic, the most common. In form of

ophthalmic solutions it is known as Oftaquix, Quixin and Iquix.Levofloxacin

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

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

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is a chiral fluorinated carboxyquinolone. Investigation of ofloxacin, an older

drug that is the racemic mixture, found that the l form [the (–)-(S)

enantiomer] is more active. This specific component is levofloxacin[6-7].

Stress testing is a part of developmental strategy under the ICH

requirements and is carried out under more severe conditions than

accelerated conditions. These studies serve to give information on drug’s

inherent stability and help in the validation of analytical methods to be used

in stability studies.

Validation of a Levofloxacin HPLC assay in plasma and dialysate for

pharmacokinetic studies was published using fluorescence detection [8]. In

one publication an hplc assay and a microbiological assay to determine

levofloxacin in soft tissue, bone, bile and serum was described [9]. Analysis of

Levofloxacin in pharmaceutical preparations by high performance thin layer

chromatography was also described [10]. The high performance liquid

chromatography tandem mass spectrometry method (HPLC/MS/MS) has

been used to determine Levofloxacin in human plasma [11]. So far several

articles were published for determination of Levofloxacin in metabolites and

in biological fluids [12, 13].

As on date, no validated stability-indicating HPLC method for quality

control testing of Levofloxacin in bulk drugs or drug products was published

in any of the journals neither by the innovator nor by any other

manufacturer. Attempts were made to develop a stability-indicating HPLC

method for the related substance determination and quantitative estimation

of Levofloxacin. This chapter mainly deals with the forced degradation of

http://en.wikipedia.org/wiki/Chirality_(chemistry)

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

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

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

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Levofloxacin under stress conditions like water hydrolysis, acid hydrolysis,

base hydrolysis, oxidation, heat and light. This chapter also deals with the

validation of the developed method for the accurate quantification of

impurities and assay of Levofloxacin in bulk samples.

7.3. Development and optimization of HPLC method

7.3.1. Samples, Chemicals and Reagents

Samples of Levofloxacin and its three process impurities (Figure 7.1 to

Figure 7.4) were received from Bulk Actives, Unit-II of Dr. Reddy’s

Laboratories, Hyderabad, India. HPLC grade Methanol and Acetonitrile was

purchased from Rankem, Mumbai, India. Ortho-phosphoric acid was

purchased from Qualigens Fine Chemicals, Mumbai, India. Sodium

dihydrogen ortho phosphate dihydrate was purchased from Qualigens Fine

Chemicals, Mumbai, India. Triethylamine was purchased from Loba Chemie

Mumbai, India. High pure water was prepared by using Millipore Milli Q plus

purification system.

7.3.2. Equipment

The LC method development, validation and forced degradation studies

were done using Agilent 1200 series HPLC system with diode array detector.

The data were collected and the peak purity of the Levofloxacin peak was

checked using chemistation software. The photolytic degradation was carried

out using Binder KBS240 photolytic chamber.

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(-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-piperazinyl-7-oxo-7H-pyrido[1,2,3-

de]-1,4-benzoxazine-6-carboxylic acid (Mol. Wt.: 347.34)

Figure 7.2: Chemical structure of impurity-1

Ethyl(-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-

oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylate(Mol. Wt.: 389.42)


(-)-(S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de]-1,4-

benzoxazine-6-carboxylic acid (Mol. Wt.: 281.21)


7.3.3. Sample preparation

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A working solution of 300 µg mL-1 of Levofloxacin was prepared for the

determination of assay and related substances analysis. Separate stock

solutions of impurities (impurity-1, impurity-2 and impurity-3) at 300 µg mL-

1 were also prepared in diluent.

7.3.4. Specificity of the test method and generation of stress samples

Specificity is the ability of the method to assess unequivocally the

analyte in presence of components, which may be expected to present.

Typically, these might include impurities, degradants, matrix, etc. [14]. The

specificity of the developed LC method for Levofloxacin was carried out in the

presence of its impurities.

One lot of Levofloxacin drug substance was chosen for stress study

experiment. From the ICH Stability guideline: “stress testing is likely to be

carried out on a single batch of material [15]. Various kinds of stress

conditions (i.e., heat, humidity, acid, and base, water, oxidative and light)

were employed on one lot of Levofloxacin drug substance based on the

guidance available from ICH stability guideline (Q1AR2). The details of the

stress conditions applied are as follows:

a) Acid hydrolysis: drug substance in 0.5 N HCl solution was exposed at 70

°C

for 7 days.

b) Base hydrolysis: drug substance in 0.5 N NaOH solution was exposed at

70 °C for 7 days.

c) Oxidative stress: drug substance in 0.01% v/v H2O2 solution was

exposed at room temperature for 12 hours.

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d) Water hydrolysis: drug solution in water at 70 °C for 7 days.

e) Thermal stress: bulk drug was subjected to dry heat at 100 °C for 5

days.

f) Photolytic degradation: bulk drug was subjected to ICH Q1B conditions.

Stress testing of the drug substance can help to identify the likely

degradation products, which can in turn help to establish the degradation

pathways and the intrinsic stability of the molecule.

Specificity is the ability of the method to measure the analyte response

in the presence of its potential impurities. All stress degradation studies were

performed at an initial drug concentration of 300 µg mL-1. Acid hydrolysis

was performed in 0.5 N HCl at 70°C for 7 days. The study in basic solution

was carried out in 0.5 N NaOH at 70°C for 7 days .For study in neutral

solution, the drug dissolved in water and was kept at 70°C for 7 days.

Oxidation studies were carried out at ambient temperature in 0.01%

hydrogen peroxide for 12 hours. Photo degradation studies were carried out

according to Option 2 of Q1B in ICH guidelines [16].The drug sample was

exposed to light for a overall illumination of 1.2 million lux hours and an

integrated near ultraviolet energy of 200 W h m2 . The drug sample was

exposed to dry heat at 100 °C for 5 days. Samples were withdrawn at

appropriate times and subjected to LC analysis after suitable dilution (300 µg

mL-1) to evaluate the ability of the proposed method to separate Levofloxacin

from its degradation products.

Photodiode array detector was employed to check and to ensure the

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homogeneity and purity of Levofloxacin peak in all the stressed sample

solutions. Assessment of mass balance in the degraded samples was carried

out to confirm the amount of impurities detected in stressed samples

matches with the amount present before the stress was applied. Quantitative

determination of Levofloxacin was carried out in all the stressed samples

against qualified working standard and the mass balance (% assay + % sum

of all impurities + % sum of all degradation products) was tabulated in table

7.14.

7.3.5. Method development

The main target of the chromatographic method is to get the separation

of impurity-1, impurity-2, impurity-3 and the degradation products generated

during stress studies from the analyte peak. Impurities were co-eluted by

using different stationary phases like C8, Cyno, XTerra and Phenyl and

different mobile phases containing buffers like phosphate, sulphate and

acetate with different pH (4- 10) and using organic modifiers like acetonitrile,

methanol and ethanol in the mobile phase. Apart from the co-elution of

impurities, poor peak shapes for some impurities and degradation products

were also noticed.

Sodium dihydrogen orthophosphate buffer with pH 6.0 and methanol

at 1.0 mL min-1 flow was chosen for initial trail with a 250 mm length X 4.6

mm ID column and 5 µm particle size C18 stationary phase. When impurity

spiked sample was injected the resolution between impurities and analyte

was poor.

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To get the good resolution of impurities from analyte, in the phosphate

buffer Triethyl amine added from 0.1% to 0.5% (v/v) then pH adjusted to 6.0

at each level of Triethyl amine and injected impurity spiked sample solution,

from Figure 7.5 at 0.5% Triethyl amine level the resolution was good among

impurities and analyte.

At low concentrations of Triethyl amine the resolution between

oxidative drgradant, impurity-1 and also the resolution between impurity-2,

impurity-3 was poor although they were well separated from analyte. At 0.5

%( v/v) level of Triethyl amine at pH 6.0 all the impurities and degradation

products were well separated amongst as well as from anlayte

.

Figure 7.5: Effect of Triethyl amine in mobile phase on the resolution

between ■Levofloxacin; Impurity-1; oxidative degradant; Impurity-2;

Impurity-3.

The effect of buffer pH (Figure 7.6) was also studied under the above

168

conditions and it was found that at higher and lower pH the tailing of the

Levofloxacin peak was more and also resolution was poor between impurities

and degradants and also from the analyte.

Figure 7.6: Effect of mobile phase pH on the resolution between ■

Levofloxacin; Impurity-1; oxidative degradant; Impurity-2;

Impurity-3.

The effect of Buffer concentration on the retention of Levofloxacin and

its impurities was also studied (Represented in Figure 7.7). At low

concentration of buffer the retention time of analyte as well as impurities was

very high to decrease the retention time of analyte and impurities buffer

concentration was increased to 25 mM without changing other conditions.

169

Figure 7.7: Effect of Buffer concentration on the retention of ■

Levofloxacin; Impurity-1; Impurity-2; Impurity-3.

At these chromatographic conditions all the impurities and degradants

were well separated amongst and also from Levofloxacin. The effect of solvent

B was also studied, when Acetonitrile used instead of Methanol the resolution

between imp-1, oxidative degradant and resolution between imp-2, imp-3

was very poor when 100% Methanol used as solvent B all the impurities were

well separated.

The results clearly indicated that on ACE C18 column 250 mm

length X 4.6 mm ID with 5 µm particle size and solvent A as 0.5% Triethyl

amine in Sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0),

solvent B as Methanol with a gradient programme: Time(t)/ % solvent B:

0/30, 20/50, 25/80, 30/80 with a post run time of 5 minutes at detection

wavelength 294 nm was successful in separation of drug from its impurities

and degradation products.

Under the above conditions, results were as follows, retention time of

Levofloxacin was around 9.7 min, with a tailing factor of 1.1, number of

theoretical plates (N) for the Levofloxacin peak was 72659 and % RSD for 5

replicate injections was 0.1% the typical retention times imp-1,imp-2,imp-3

were about 3.4,12.3,13.5 min respectively (Figure 7.22). Peak purity of

stressed samples of Levofloxacin was checked by using a photodiode array

detector of Agilent 1200 series, the purity factor is within the threshold limit

in all the stress samples, demonstrating the homogeneity of analyte peak.

Accelerated and long term stability study results as per ICH Q1A (R2) for

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Levofloxacin were generated for 12 months by using the developed LC method

and the results were well within the limits, this further confirms the stability

indicating of the developed LC method.

Optimized chromatographic conditions

Column : ACE C18, 250mm x 4.6mm, 5m particle size

Mobile phase A : pH6.0 Buffer*

Mobile phase B : Methanol

HPLC program : Gradient

Gradient Programme : T/%B: 0/30, 20/50, 25/80, 30/80

Post run time : 5 minutes

Flow rate : 1.0 mL/min

Column temperature : 40 ± 2 °C

Wavelength of detection : 294 nm

Injection volume : 20L for related substances

10L for assay determination

Diluent : Water :Acetonitrile (60 :40)

Run time : 35 min

Retention time : Levofloxacin about 9.7 min.

Relative Retention Time : Impurity-1 about 0.35

Impurity-2 about 1.27

Impurity-3 about 1.39

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pH6.0 Buffer*: 0.5% Triethyl amine in Sodium dihydrogen orthophosphate

dihydrate (25 mM; pH 6.0)

Figure 7.8 to Figure 7.19 is the typical HPLC chromatograms showing

the degradation of Levofloxacin in various stress conditions and also the

corresponding peak purity plots.

X-axis: Retention time in min and Y-axis: Peak response in mAU

Figure 7.8: Typical HPLC chromatograms of acid hydrolysis

Figure 7.9: Peak purity plot of acid hydrolysis

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Figure 7.10: Typical HPLC chromatograms of alkali hydrolysis

Figure 7.11: Peak purity plot of alkali hydrolysis


Figure 7.12: Typical HPLC chromatograms of oxidative degradation

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Figure 7.13: Peak purity plot of oxidative degradation


Figure 7.14: Typical HPLC chromatograms of thermal degradation

174

Figure 7.15: Peak purity plot of thermal degradation


Figure 7.16: Typical HPLC chromatograms of water hydrolysis

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Figure 7.17: Peak purity plot of water hydrolysis


Figure 7.18: Typical HPLC chromatograms of photolytic degradation

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Figure 7.19: Peak purity plot of photolytic degradation

7.4. Comments on the stress degradation of Levofloxacin

No considerable degradation observed when the Levofloxacin sample

was subjected to acid, base, water, thermal and photolytic stress.

Considerable degradation was observed during oxidative degradation (Figure

7.12) at 0.50 RRT which was identified by LCMS/MS. However, the

developed method is able to well resolve all the degradants from the analyte

peak (i.e. Levofloxacin) generated from oxidative degradation and the

Levofloxacin peak was observed to be pure and homogeneous when checked

under DAD and also assay of levofloxacin was unaffected by impurities and

degradation products, thus establishes the stability-indicating power of the

developed method.

7.4.1. Identification of major degradation product (at 0.50 RRT) formed

in oxidative stress condition

LCMS/MS analysis was carried out for the oxidative stress sample of

Levofloxacin using Agilent 6410 QQQ mass spectrometer with suitable

volatile buffer ammonium acetate(10 mM, pH=6.0) as mobile phase. The

degradation product formed at 0.50 RRT shows the mass of 377 which is 16

higher mass than Levofloxacin mass 361.The fragmentation for the degradant

was also carried out for degradation product and Levofloxacin using product

ion scan by LCMS/MS with optimum collision energy of 25.

The fragmentation pattern (Figure 7.20) clearly indicates that formed

degradant was N-Oxide of Levofloxacin which was supported by chemical

properties of Levofloxacin. The fragment 361.20 formed from the cleavage of

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CO-OH bond in acid group and the fragment 317.2 results from the cleavage of

N-Oxide ( mass of 16) followed by loss of carbonyl group( mass of 28).Due to

steric hindrances and localization of lone pair on nitrogen the N-Oxide will

form in piperazine ring at N-Methyl position. So the probable structure as

shown in Figure 7.21. The N-Oxide was formed due to oxidation so this

impurity was reduced by adding antioxidant during purification of

Levofloxacin.

Figure 7.20 Fragmentation mass spectrum of 0.50 RRT degradation

product formed in oxidative degradation of Levofloxacin.

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Figure 7.21 Structure of 0.50 RRT degradation product (M.Wt:377.37)

formed in oxidative degradation of Levofloxacin

7.5. Validation of Analytical method and its results

The developed and optimized HPLC method was taken up for

validation. The analytical method validation was carried out in accordance

with ICH guidelines [17].

7.5.1. System Suitability Test (SST)

A mixture of Levofloxacin standard, impurity-1, impurity-2 and impurity-3,

were injected into HPLC system and good resolution was obtained between

impurities and Moxifloxacin. A typical blank, pure Moxifloxacin and spiked

HPLC chromatograms were presented below (Figure 7.22). These results are

tabulated in table 7.1.

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Figure 7.22: System Suitability Test chromatogram

Table 7.1: System Suitability Test results

Compound (n=3) USP Resolution (RS)

USP Tailing factor (T)

No. of theoretical plates (N) / USP

Levofloxacin - 1.2 72659

Impurity-1 9.7 1.1 42186

Impurity-2 5.4 1.3 29360

Impurity-3 7.6 1.3 19360

n = Number of determinations

7.5.2. Precision

The precision of an analytical procedure expresses the closeness of

agreement between a series of measurements obtained from multiple

sampling of the same homogenous sample under the prescribed conditions

[18]. Assay method precision study was evaluated by carrying out six

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independent assays of Levofloxacin test sample against qualified reference

standard and RSD of six consecutive assays was 0.2% (Table 7.2).

Table 7.2: Precision results of the assay method

Preparation Assay (% w/w)

1 99.2

2 100.4

3 99.7

4 99.6

5 99.3

6 99.9

%RSD 0.4

The precision of the related substance method was checked by

injecting six individual preparations of Levofloxacin (0.3 mg/ml) spiked with

0.10% of Impurity-1, Impurity-2 and Impurity-3 with respect to Levofloxacin

analyte concentration. The % RSD of area of Impurity-1, Impurity-2 and

Impurity-3 for six consecutive determinations was 4.3%, 3.7% and 6.0%.

(Table 7.3).

Table 7.3: Precision results of the RS method

Preparation Peak area of

Impurity-1

Peak area of

Impurity-2

Peak area of

Impurity-3

1 65.217 75.217 33.217

2 60.665 70.401 36.53

3 61.213 71.298 31.304

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4 66.209 73.447 32.665

5 59.454 75.673 31.007

6 62.409 77.433 32.675

%RSD 4.3 3.7 6.0

7.5.3. Limit of quantification (LOQ) and limit of detection (LOD)

LOQ and LOD established for Impurity-1, Impurity-2 and Impurity-3

based on signal to noise ratio method [19,20].

Limit of quantification (LOQ)

The quantitation limit (LOQ) of an analytical procedure is the

lowest amount of analyte in a sample, which can be quantitatively

determined with suitable precision and accuracy. The quantitation limit is a

parameter of quantitative assays for low levels of compounds in sample

matrices, and is used particularly for the determination of impurities. A

series of diluted solutions of impurities at low concentrations were prepared

and injected the LOQ concentrations and their signal to noise ratios were

tabulated in table 7.4.

Table 7.4: LOQ values of the impurities

S.No. Impurity Name Concentration Signal to Noise ratio

1 Impurity-1 90 ng/mL 10.5



Limit of detection (LOD)

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The detection limit of an individual analytical procedure is the lowest

amount of analyte in a sample, which can be detected but not necessarily

quantitated as an exact value. A series of diluted solutions of impurities at

low concentrations were prepared and injected the LOD concentrations and

their signal to noise ratios was tabulated in table 7.5.

Table 7.5: LOD values of the impurities

S.No Impurity Name Concentration Signal to Noise ratio




7.5.4 Linearity

7.5.4.1. Linearity of the assay method

The linearity of an analytical procedure is its ability to obtain test

results, which are directly proportional to the concentration of analyte in the

test sample [21]. The linearity of the assay method was established by

injecting test sample at 50%, 75%, 100%, 125% and 150% of Levofloxacin

assay concentration (i.e.300 µg/ml). Each solution was injected twice (n=2)

into HPLC and calculated the average area at each concentration (Table 7.6).

Calibration curve was drawn by plotting average area on the Y-axis and

concentration on the X-axis (Figure 7.23).

Table 7.6: Linearity results of the assay method

Concentration (%) Mean peak area

50 38938.048

183

75 58307.071

100 79876.095

125 99202.119

150 118660.143

Correlation coefficient 0.9998

Figure 7.23: Linearity graph of assay method

7.5.4.2. Linearity of the related substances method

Linearity experiments were carried out by preparing the Levofloxacin

sample solutions containing impurity-1, impurity-2 and impurity-3, from

LOQ to 200% (i.e. LOQ, 50%, 75%, 100%, 150% and 200%) with respect to

their specification limit (0.10% w/w) Please refer Table 7.7 below.

Calibration curve was drawn by plotting mean area of the individual

impurity (Impurity-1, Impurity-2 and Impurity-3) on the Y-axis and

concentration on the X-axis (Figure 7.24 and Figure 7.26).

y = 801.3x - 1139R² = 0.999

20000

40000

60000

80000

100000

120000

140000

40 70 100 130 160

P

e

a

k

A

r

e

a

Concentration in %

Linearity curve for Assay

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Table 7.7: Linearity results of the RS method

Figure 7.24: Linearity graph of Impurity-1

y = 0.657x + 1.877R² = 0.990

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

P

e

a

k

A

r

e

a

Concentration %

Linearity curve for Impurity-1

S.No Concentration

(in %)

Impurity-1

(Mean peak area)

Impurity-2

(Mean peak area)

Impurity-3

(Mean peak area)

1 LOQ 4.807 3.115 2.708

2 50 32.667 34.996 54.477

3 75 47.734 57.453 75.661

4 100 65.345 73.264 108.908

5 150 108.407 109.665 162.725

6 200 130.454 151.768 220.556

Slope 0.66 0.76 1.09

Intercept 1.88 -0.15 -0.67

Correlation coefficient 0.995 0.998 0.999

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7.5.5. Accuracy / Recovery

The accuracy of an analytical procedure expresses the closeness of

agreement between the value, which is accepted either as a conventional

true value or an accepted reference value and the value found [22].

7.5.5.1. Accuracy of the assay method

Accuracy of the assay method was established by injecting test sample

at 50%, 100% and 150% of analyte concentration (i.e., 0.3mg/mL). Each

y = 0.755x - 0.150R² = 0.996

0

20

40

60

80

100

120

140

160

180

0.00 50.00 100.00 150.00 200.00 250.00

P

e

a

k

A

r

e

a

Concentration %


y = 1.093x - 0.673R² = 0.998

0

50

100

150

200

250

0 50 100 150 200 250

P

e

a

k

A

r

e

a

Concentration %


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solution was injected twice into HPLC and the average peak area of

Levofloxacin peak was calculated. % Recovery of assay method was carried

out in triplicate at each concentration level (Table 7.8).

Table 7.8: Recovery of the assay method

S.No. Concentration (%) % Mean recovery (n=3)

1 50 100.7

2 100 99.5

3 150 99.7

n= number of Determinations

7.5.5.2. Accuracy of the RS method

Accuracy of the related substances method established at 50%, 100%

and 150% of the impurities specification limit (0.10% w/w).

Accuracy at 50% impurity specification level

Prepared test solution in triplicate (n=3) with impurities Impurity-1

and Impurity-2 at 0.05% level w.r.t. analyte concentration (i.e. 0.3mg/mL).

Injected each solution once into HPLC. Calculated % mean recovery of

impurities in the test solution using the peak area obtained in impurities

solution injected without test solution (Table 7.9).

Table 7.9: Recovery at 50% level

S.No. Impurity Name % Mean recovery (n=3)

1 Impurity-1 92.6

2 Impurity-2 96.8

3 Impurity-3 93.4

187


Prepared test solution in triplicate (n=3) with impurities Impurity-1,

Impurity-2 and Impurity-3 at 0.10% level w.r.t. analyte concentration (i.e.

0.3mg/mL). Injected each solution once into HPLC. Calculated the %mean

recovery of impurities in the test solution using the area obtained in

impurities solution injected without test solution (Table 7.10).


S.No. Impurity Name % Mean recovery (n=3)

1 Impurity-1 101.5

2 Impurity-2 96.7

3 Impurity-3 103.4


Prepared test solution in triplicate (n=3) with impurities Impurity-1,

Impurity-2 and Impurity-3 at 0.15% level w.r.t. analyte concentration

(i.e.0.3mg/mL). Injected each solution once into HPLC. Calculated the

%mean recovery of impurities in the test solution using the area obtained in

impurities solution injected without test solution (Table 7.11).


S.No Impurity Name % Mean recovery (n=3)

1 Impurity-1 95.7

2 Impurity-2 94.2

3 Impurity-3 97.5

188

7.5.6. Solution and mobile phase stability

Leaving both the test solutions of sample and reference standard in

tightly capped volumetric flasks at room temperature for two days carried

out the solution stability of Levofloxacin in the assay method. The same

sample solutions were assayed at six hours interval up to the study period.

The RSD of assay of Levofloxacin during solution stability experiments was

within 1.0%.

No significant change was observed in the content of Impurity-1,

Impurity-2 and Impurity-3 during solution stability and mobile phase

stability experiments up to the study period. The data obtained in both the

above experiments proves that sample solutions and mobile phase used

during assay and related substance determination were stable up to 48 h.

Assay method

Injected standard and test solution each at 0 h, 6 h, 12 h, 18 h, 24 h,

30 h, 36 h, 42 h and 48 h. Table 7.12 summarizes assay content obtained at

different interval.

Table 7.12: Solution stability results of the assay method

S.No. Interval Assay (%w/w)

1 0 h 99.1

2 6 h 98.5

189

3 12 h 99.5

4 18 h 100.4

5 24 h 99.4

6 30 h 98.8

7 36 h 99.3

8 42 h 98.6

9 48 h 98.3

%RSD 0.7

Related substances method

Solution and mobile phase stability was established for 48 h by

injecting test solution at an interval of 6 h. The impurity profiles obtained at

different interval were very consistent and matched with initial value.

7.5.7. Robustness

In all the varied chromatographic conditions (flow rate, composition of

organic modifier and column temperature) the resolution between critical

pair i.e. Impurity-2 and Levofloxacin was greater than 3, illustrating the

robustness of the developed method. The results obtained were captured in

the below Table 7.13.

Table 7.13: Results of the robustness study

S.No. Parameter Variation Resolution between

Levofloxacin and

Impurity-2

Impurity-2 and

Impurity-3

190

1 Temperature (± 5°C

of set temperature)

a) At 35°C

b) At 45°C

5.5

4.8

3.1

2.9

2 Flow rate (± 20% of

set flow)

a) At 1.2

mL/min

b) At 0.8

mL/min

4.8

5.2

2.6

2.8

3

pH of the buffer(±

0.5 of pH-6.0)

a) At 6.5

b) At 5.5

5.1

4.5

3.2

2.9

7.6. Mass balance

The mass balance is a process of adding together the assay value and

the levels of degradation products to see how closely these add up to 100%

of the initial value, with due consideration of the margin of analytical error

[23]. Its establishment hence is a regulatory requirement. The mass balance

is very closely linked to the development of stability-indicating assay method

as its acts as an approach to establish its validity.

Mass balance is also important in understanding alternate

degradation pathways. The stressed samples of Levofloxacin bulk drug were

assayed against the qualified reference standard and the results of mass

balance obtained were very close to 100%. The results of mass balance

obtained in each condition are presented below (Table 7.14).

Table 7.14: Summary of forced degradation results

191

S.No Stress condition Duration

% Assay of

active

substance

Mass balance

(% Assay+ %

impurities+ %

degradants)

Remarks

1 Acid hydrolysis

(0.5 N HCl at70 °C) 7 days 96.7 99.4

Slight degradation

observed

2

Alkali hydrolysis

(0.5 N NaOH at

70°C)

7 days 99.1 99.5 No degradation

was observed

3 Oxidation

(0.01% H2O2 at RT) 12 hours 87.3 99.3

Major degradation

was observed

4 Water hydrolysis at

70 °C 7 days 99.5 99.8

Mild degradation

was observed

5 Thermal Stress

(at 100 °C) 5 days 99.2 99.6

No degradation

observed

6 Photolytic stress

(ICH Q1B) 11 days 99.3 99.7

No degradation

observed

7.7. Quality control monitoring of Levofloxacin in three production

batches

Using the developed HPLC method Levofloxacin samples of three

production batches was analyzed and results (Table 7.15) indicates that the

method was able to determine the Levofloxacin without interference of other

impurities as well as its related substances. Hence the developed was suitable

for determination of Levofloxacin as well its impurities in bulk samples.

192

Table 7.15: Results of quality monitoring of Levofloxacin in three production batches

Batch No: Description

Water

Content by KF

Specific optical rotation

(°)

Related substances by HPLC Any

Unknown

impurity

Total impurit

ies

Assay on

Anhydrous

basis Imp-1 Imp-2

Imp-3

LP001E08

Yellowish white

crystalline powder

2.5 -79 ND ND 0.03 0.01 0.05 99.7

LP001G09

Yellowish white

crystalline

powder

2.2 -78 ND ND 0.04 0.01 0.06 100.3

LP001H08

Yellowish white

crystalline powder

2.6 -78 ND ND 0.03 0.02 0.05 99.8

ND-Not detected

7.8. Analysis of Levofloxacin stability samples

USP states that stability testing for both drug substance and drug

product should be performed by validated stability-indicating test method

[24]. One manufacturing lot of Levofloxacin monohydrate was placed for

stability study in chambers maintained at ICH set conditions. The analysis

of stability samples was carried up to 12 months period using the above

stability-indicating method. The stability data results obtained are presented

in Table 7.16 and 7.17. The developed HPLC method performed satisfactorily

for the quantitative evaluation of stability samples.

193

Table 7.16: Accelerated stability data (storage conditions 40 °C / 75%

RH)

Accelerated Stability data

Product: Levofloxacin Batch No:LP001F07

Packing conditions: Each sample placed in poly bag which is placed in triple laminated bag

Stability duration:6 months

Storage condition

Period Description

Water

Content by KF

Specific

optical rotation(°)

Related substances by

HPLC Any

Unknown impurity

Total

impurities

Assay on

Anhydrous basis

Imp-1 Imp-2

Imp-3

Temperature 40°C±2°C,

Relative humidity 75±5%

Initial Yellowish white

crystalline

powder

2.3 -78 ND ND 0.02 0.01 0.03 99.5

1 st

month

Yellowish white

crystalline powder

2.5 -78 ND ND 0.01 ND 0.02 99.9

2 nd

month

Yellowish white

crystalline powder

2.6 -79 ND ND 0.01 ND 0.02 99.8

3 rd

month

Yellowish white crystalline

powder 2.4 -79 ND ND 0.02 ND 0.02 99.9

6 th

month


powder 2.4 -79 ND ND 0.02 ND 0.02 99.7

Long term Stability data

Product: Levofloxacin Batch No:LP001F07

Packing conditions: Each sample placed in poly bag which is placed in triple laminated bag

Stability duration:12 months

Storage condition

Period Description Water

Content by KF

Specific optical

rotation(°)

Related substances by

HPLC Any

Unknown impurity

Total impuritie

s

Assay on Anhydrou

s basis Imp

-1

Imp

-2

Imp

-3

Temperature

30°C±2°C, Relative humidity

60±5%

Initial

Yellowish white

crystalline powder

2.3 -78 ND ND 0.02 0.01 0.03 99.5

1 st

month

Yellowish white

crystalline powder

2.4 -79 ND ND 0.01 0.01 0.02 99.5

2 nd month


powder 2.4 -78 ND ND 0.02 ND 0.02 99.9

3 rd month


powder 2.3 -78 ND ND 0.02 ND 0.02 99.4

6 th

month


powder 2.5 -78 ND ND 0.01 ND 0.01 99.7

9 th

month

Yellowish white

crystalline 2.6 -79 ND ND 0.02 ND 0.02 99.3

194

Table 7.17: Long-term stability data (storage conditions: 30 °C / 60% RH)

7.9. Summary

A simple specific validated stability indicating RP-HPLC method

developed for the determination of related substances and assay of

Levofloxacin drug substance for the first time. The developed method was

precise, accurate and selective. The method was fully validated showing

satisfactory data for all the method validation parameters tested. The

developed method was stability-indicating and can be conveniently used by

quality control department to determine the related substances and assay in

regular Levofloxacin production samples and also stability samples.

powder

12 th

month


powder

2.4 -79 ND ND 0.02 ND 0.02 99.7

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