CHAPTER-4 A VALIDATED STABILITY-INDICATING...

158

CHAPTER-4

A VALIDATED STABILITY-INDICATING

ANALYTICAL METHOD FOR THE

DETERMINATION OF IMPURITIES IN

FLORFENICOL

159

4.1 Introduction of Florfenicol and survey of analytical Methods

Florfenicol is a synthetic derivative of thiamphenicol. It has a

wide spectrum of antibacterial properities, including efficacy against

Gram-positive and Gram-negative bacteria [1]. The mechanism of

action of Florfenicol similarly to chloramphenicol and thiamphenicol,

relies on slowing down the biosynthesis of bacterial proteins through

reversible binding with an active center of peptydylotransferase on a

subunit 50S of bacterial ribosomes. It is chemically designated as 2,2-

dichloro-N-[(1S,2R)-1-(fluoromethyl)-2-hydroxy-2-[4-(methylsulfonyl)

phenyl]ethyl] acetamide. Florfenicol is a white to almost white,

crystalline powder. It is very soluble in N,N-Dimethylformamide and

soluble in methanol. The molecular formula is C12H14Cl2FNO4S. The

molecular weight of Florfenicol is 358.22.

Fig: 4.1 Chemical structure of Florfenicol

H

Cl

S FO

N

OH Cl

O

H3C

O

2,2-dichloro-N-[(1S,2R)-1-(fluoromethyl)-2-hydroxy-2-[4-

(methylsulfonyl)phenyl]ethyl]acetamide

Molecular formula C12H14Cl2FNO4S

Molecular weight 358.22

160

The different analytical techniques reported so far for the

determination of this drug and its metabolites in biological samples

include capillary electrophoresis [2] and spectrometry [3]. The

determination of Florfenicol in plasma by RP-LC [4], stability of

Florfenicol [5-6], pharmacokinetics studies of Florfenicol in plasma [7-

8] was also reported.

Organic impurities can arise during the manufacturing process

and storage of the drug substances and the criteria for their

acceptance up to certain limits are based on pharmaceutical studies

or known safety data [9]. As per regulatory guidelines, the

pharmaceutical studies using a sample of the isolated impurities can

be considered for safety assessment. It is, therefore, essential to

isolate and characterize unidentified impurities present in the drug

sample. Recently we have developed a process for the synthesis of

Florfenicol in our laboratory. During the development of an analytical

procedure, the LC method was developed for the determination of in-

house synthesized Florfenicol and the impurities arising during its

manufacturing. In the present study, we describe a reverse phase

column liquid chromatography method for the separation and

quantification of process related and degradation impurities of

Florfenicol. The accuracy, precision, limit of detection (LOD), limit of

quantification (LOQ) and robustness of the method were determined in

accordance with ICH guidelines [10]. The target is to develop a suitable

stability-indicating HPLC related substances method for Florfenicol in

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this chapter we describe a stability-indicating LC method for the

determination of Florfenicol and its potential and degradation

impurities and also the method validation.

4.2 Development of a stability-indicating analytical method for

Florfenicol

4.2.1 Materials

Reference standard of Florfenicol and seven impurities namely,

Imp-A, Imp-B, Imp-C, Imp-D, Imp-E , Imp-F and Imp-G (Fig: 4.1)

were synthesized and characterized by use of LC-MS, NMR and IR in

Aurobindo Pharma Ltd., Hyderabad, India. The commercial samples of

Florfenicol are also manufactured by Aurobindo Pharma Ltd. All

reagents used were of analytical reagent grade unless stated

otherwise. Milli Q water, HPLC-grade acetonitrile, HPLC-grade

orthophosphoric acid (OPA) were purchased from Merck (Darmstadt,

Germany).

4.2.2 Equipment

The LC system was equipped with quaternary gradient pumps

with autosampler and auto injector (Alliance, Waters 2695, Milliford,

MA, USA) controlled with Empower software (Waters).

162

Fig: 4.2 Chemical structures of impurities of Florfenicol

Me O2S F

NH2

OH

(1R,2S)-1-[4-(Methylsulfonyl) phenyl]-2-amino-3-fluoro-1-propanol

(Imp-A)

Fig 4.2 (a)

FMe O2S

N

OH

CH3

O

H

(1R,2S)-2-Acetamido-3-fluoro-1-[4-(methyl-sulfonyl)phenyl]-1-

propanol (Imp-B)

Fig: 4.2 (b)

Cl

Me O2S OHO

N

OH ClH

(1R,2R)-2-Dichloroacetamido-1-[4-(methylsulfonyl)phenyl]-1,3-

propanediol (Imp-C)

Fig: 4.2 (c)

Cl

Me O2S FO

N

OHH

(1R,2S)-2-Chloroacetamido-3-fluoro-1-[4-(methylsulfonyl)phenyl]-1-

propanol (Imp-D)

Fig: 4.2 (d)

163

Cl

Me O2S ClO

N

OH ClH

(1R,2S)-2-Dichloroacetamido-3-fluoro-1-[4-(methylsulfonyl)phenyl]-1-

propanol (Imp-E)

Fig: 4.2 (e)

Cl

MeO2S FO

N

OH ClCH3

(1R,2S)-2-(N-Methyl)dichloroacetamido-3-fluoro-1-[(4-methylsulfonyl)-

phenyl]-1-propanol (Imp-F)

Fig: 4.2 (f)

Cl

Me O2S FO

N

OH ClClH

(1R,2S)-2-Trichloroacetamido-3-fluoro-1-[4-(methylsulfonyl)-phenyl]-1-

propanol

(Imp-G)

Fig: 4.2 (g)

4.2.3 Preparation of sample and stock solutions

The stock solutions of Florfenicol (0.5 mg/ml) and spiked with

0.3% of Imp-A of Imp-B, Imp-D, Imp-E, Imp-F and Imp-G with respect

to the Florfenicol analyte concentration. The stock solutions was

further diluted with diluent to obtain a standard solution of 0.0005

164

mg/ml (0.5 µg/ml) for related substances determination and 0.5

mg/ml ( 500 µg/ml) for assay determination.

4.2.4 Generation of stress samples

One lot of Florfenicol drug substance selected for stress

testing. From the ICH stability guideline: “stress testing likely to be

carried out on a single batch of material. Different kinds of stress

conditions (i.e., acid hydrolysis, base hydrolysis, oxidative stress, heat

and humidity) were employed on one lot of Florfenicol drug substance

based on the guidance available from ICH stability guideline (Q1AR2).

The details of the stress conditions are as follows:

a) Acid Degradation: drug in 5.0 M HCl solution was kept at 85°C for

60 mins.

b) Base Degradation: drug in 5.0 M NaOH solution was kept at room

temperature.

c) Oxidative stress: drug in 30% H2O2 solution was kept at 80°C for

120 mins.

d) Thermal Degradation: drug was subjected to dry heat at 105°C for

188 hrs.

e) Phtolytic degradation: drug was subjected to UV at 254 nm (10 K

Lux ) for 188 hrs.

4.2.5 Optimization of chromatographic conditions

The main objective of the chromatographic method was to

seperate Florfenicol from Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F

and Imp-G impurities were coeluted using different stationary phases

165

such as C8, phenyl and cyano as well as different mobile phases

containing buffers like phosphate, sulfate and acetate with different

pH and using organic modifiers like acetonitrile and methanol in the

mobile phase. Apart from the co-elution of impurities, we have also

observed poor peak shapes for Florfenicol, some impurities and

degradants. The chromatographic separation was achieved on a

Hypersil BDS, C18, (250 mm x 4.6 mm), 5 particle size. The gradient

LC method employs solution A and B as mobile phase. The solution A

contains phosphate buffer pH 7.0 and acetonitrile as solution B. The

flow rate of the mobile phase was 1.0 ml/min. The HPLC gradient

program was set as: time% solution B: 0.01/10, 15/20, 30/30,

40/40, 50/60, 60/60, 61/10, 70/10 with a post run time of 10 min.

The detection was monitored at a wavelength of 225 nm. The injection

volume was 20 µl. Standard and test solutions were prepared in pH

3.0 buffer was used as diluent. In the optimized chromatographic

conditions of Florfenicol, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F

and Imp-G were separated with a resolution greater than 3, typical

relative retention times were approximately 0.29, 0.35, 0.59, 0.61,

1.35, 1.36, 1.72 with respect to Florfenicol eluted at 22.45 min.

166

Optimized liquid chromatographic conditions:

Column : Hypersil BDS, C18, (250 mm x

4.6 mm), 5

Mobile phase : The solution A contains

phosphate buffer pH 7.0 and

solution B contains acetonitrile

Pump mode : Gradient

Flow rate : 1.0 ml/min

UV of detection : 225 nm

Injection volume : 20 µl

Run time : 60 min

Retention time : 22.45min

Relative Retention Time (RRT) : Imp-A about 0.29

Imp-B about 0.35

Imp-C about 0.59

Imp-D about 0.61

Imp-E about 1.35

Imp-F about 1.36

Imp-G about 1.72

Diluent : Dissolve 1.36 g of potassium dihydrogen orthophosphate in

1000 ml of water. Adjust pH to 3.0±0.05 with orthophosphoric acid.

No considerable degradation was observed in Florfenicol bulk

samples under stress conditions such as acid hydrolysis, photolytic

and thermal. The degradation of drug substance was observed during

base hydrolysis and oxidative stress condition. Florfenicol was

degraded to Imp-A (7.85%) under acidic conditions (5M HCl/180°C/60

167

min). Florfenicol was degraded to Imp-A (7.80%), Imp-C (1.14%) under

base conditions (5M NaOH/Initial) and it was confirmed by co-

injection with a qualified Imp-A and Imp-C standards. Mild

degradation was observed under oxidative environment (treated with

30% H2O2/85°C/240 min) leads to the formation of some unknown

degradation peaks (3.5%).

Peak purity test results obtained by using a PDA detector

confirmed that the Florfenicol peak is homogenous and pure in all the

analyzed stress samples. The mass balance of Florfenicol in all stress

samples was close to 99.6% (%Assay + %Degradation). This clearly

demonstrates that the developed HPLC method was found to be

specific for Florfenicol in presence of its impurities (Imp-A, Imp-B,

Imp-C, Imp-D, Imp-E, Imp-F and Imp-G) and degradation products.

Figures:

Fig: 4.3 to Fig: 4.7 is the typical HPLC chromatograms showing

the degradation of Florfenicol in various stress conditions and also the

corresponding peak purityplots.

168

Fig: 4.3 Typical HPLC chromatograms of Acid hydrolysis

Fig: 4.3 (a)

Fig: 4.3 (b)

Blank Chromatogram of Acid hydrolysis (5N HCl)

Florfenicol stressed with 5N HCl at 85°C for 60 mins

169

Fig: 4.3 (c) Peak purity plot of Acid hydrolysis

Purity Angle Purity Threshold Purity Flag Peak Purity

0.048

0.249 No Pass

Fig: 4.3 (c)

170

Fig: 4.4 Typical HPLC chromatograms of Base hydrolysis

Fig: 4.4 (a)

Fig: 4.4 (b)

Blank Chromatogram of Base hydrolysis ( 5N NaOH )

Florfenicol stressed with 5N NaOH at room temperature

171

Fig: 4.4 (c) Peak purity plot of Base hydrolysis


0.047

0.247 No Pass

Fig: 4.4 (c)

Fig: 4.5 Typical HPLC chromatograms of Peroxide Degradation

Fig: 4.5 (a)

Fig: 4.5 (b)

Blank Chromatogram of Peroxide Degradation ( 30% H2O2 )

Florfenicol stressed with 30%H2O2 at 80°C for 120 mins

172

Fig: 4.5 (c) Peak purity plot of Peroxide Degradation


0.067

0.272 No Pass

Fig: 4.5 (c)

Fig: 4.6 Typical HPLC chromatograms of Thermal Degradation

Fig: 4.6 (a)

Blank

173

Fig: 4.6 (b)

Fig: 4.6 (c) Peak purity plot of Thermal Degradation


0.063

0.261 No Pass

Fig: 4.6 (c)

Florfenicol stressed at 105°C for 188 hours

174

Fig: 4.7 Typical HPLC chromatograms of Photolytic Degradation

Fig: 4.7 (a)

Fig: 4.7 (b)

Blank

Florfenicol stressed with 12,000 Lux for 188 hours

175

Fig: 4.7(c): Peak purity of Photolytic Degradation


0.061

0.259 No Pass

Fig: 4.7 (c)

Peak purity test performed for the Florfenicol peak using photo

diode array (PDA) detector data confirmed the purity of the peak for

all the stressed samples. This clearly demonstrates that the developed

HPLC method was found to be specific for Florfenicol in presence of

its impurities (Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G

and degradant products.

No degradants were observed after 15 min in the extended run

time of 60 min for all the Florfenicol stressed samples (acid

hydrolysis, base hydrolysis, oxidation stress, heat 120°C, photolysis)

with 90% can in mobile phase.

176

4.2.6 Validation of analytical method and its results:

The developed and optimized HPLC method was taken up to

validation. The analytical method validation was carried out in

accordance with ICH guideline [11].

4.2.6.1 System suitability: A mixture of Florfenicol standard

injections were injected into HPLC system and good resolution was

obtained between the impurities and Florfenicol [Fig: 4.8). The system

suitability results are tabulated (Table: 4.1).

177

Fig: 4.8 Typical Blank, Florfenicol sample and SST

chromatograms

Fig: 4.8 (a)

Fig: 4.8 (b)

Fig: 4.8 (c)

Blank

Florfenicol sample spiked with impurities

Florfenicol Sample

178

Table: 4.1 System suitability results

S.NO Impurity

name USP Plate

count USP

Tailing USP

Resolution

1 Imp-A 4179 1.23 -

2 Imp-B 10231 1.10 3.93

3 Imp-C 29350 1.12 16.68

4 Imp-D 32575 1.13 1.92

5 Florfenicol 56327 1.18 24.53

6 Imp-E 80882 1.10 18.89

7 Imp-F 88309 1.15 0.85

8 Imp-G 173154 1.11 22.69

4.2.6.2 Precision:

The precision of an analytical process experiment the closeness

of agreement between a series of measurements obtained from

multiple sampling of the some homogeneous same under prescribed

conditions.

Precision may be considered at three levels: System precision,

Method precision and Intermediate Precision. Assay method precision

study was evaluated by carrying out six independent assays of

Florfenicol test sample against qualified reference standard and RSD

of six consecutive assays was 0.6% (Table: 4.2 to Table: 4.4).

The results showed insignificant variation in measured

response. Which demonstrated that the assay method was repeatable

with RSD’s below 0.4%.

179

Table: 4.2 System Precision results of the Assay method

Injection ID Florfenicol

1 2194243

2 2174916

3 2189161

4 2184508

5 2192197

6 2191167

Mean 2187699

SD 7082

% RSD

95% Confidence

Interval

0.3

± 7433

Table: 4.3 Method Precision results of the Assay method

Sample ID Assay (% w/w)

1 98.8

2 98.8

3 99.9

4 98.8

5 100.2

6 99.9

Mean 99.4

SD 0.67

% RSD 0.7

95% Confidence Interval

± 0.7

180

Table: 4.4 Intermediate Precision results of the Assay method

Sample ID Assay (% w/w)

1 99.1

2 100.3

3 99.4

4 100.7

5 100.5

6 100.6

Mean 100.1

SD 0.68

% RSD 0.7

95% Confidence

Interval ± 0.7

The precision of the related substance method was checked by

injecting six individual preparations of Florfenicol (0.5 mg/ml) spiked

with 0.3% of Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G

with respect to the Florfenicol analyte concentration. The % RSD of

the area percentage of each impurity (impurities-A, -B, -C, -D, -E, -F

and –G) for six consecutive determinations was respectively as below

(Table: 4.5 to Table: 4.7).

The results showed insignificant variation in measured

response. Which indicated that the related substance method was

repeatable with RSD’s below 1.8%.

181

Table: 4.5 System Precision results of the Related Substance

method

Injection ID Florfenicol

1 64644

2 64842

3 64434

4 64178

5 64439

6 64029

Mean 44290

SD 44423

% RSD 130

95% Confidence

Interval 0.3

Table: 4.6 Method Precision results of the Related Substance

method

Preparation

Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G

1 0.217 0.334 0.461 0.311 0.394 0.341 0.471

2 0.218 0.332 0.462 0.309 0.386 0.344 0.463

3 0.225 0.333 0.464 0.309 0.391 0.347 0.465

4 0.226 0.334 0.469 0.311 0.397 0.343 0.474

5 0.222 0.329 0.463 0.309 0.391 0.346 0.464

6 0.224 0.330 0.466 0.312 0.392 0.346 0.467

Mean 0.222 0.332 0.464 0.310 0.392 0.345 0.467

SD 0.004 0.002 0.003 0.001 0.004 0.002 0.004

%RSD 1.8 0.6 0.6 0.3 1.0 0.6 0.9

95%Confidence

interval ± 0.004 ±0.002 ±0.003 ±0.001 ±0.004 ±0.002 ±0.004

182

In intermediate precision method six preparations individually

using single batch of Florfenicol drug substance spiked with related

substances at specification level (0.3%) as per test method and

injected each solution as per methodology using different column,

system and by another analyst. Results showed insignificant variation

in measured response. Which demonstrated that the related

substance method was repeatable with RSD’s below 1.9%.

Table: 4.7 Intermediate Precision results of the Related substance

method

Preparation Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G

1 0.217 0.341 0.469 0.309 0.373 0.347 0.466

2 0.211 0.341 0.466 0.305 0.378 0.349 0.456

3 0.215 0.346 0.468 0.310 0.377 0.354 0.470

4 0.217 0.344 0.468 0.309 0.389 0.356 0.472

5 0.209 0.337 0.460 0.303 0.388 0.350 0.458

6 0.215 0.339 0.466 0.297 0.372 0.351 0.466

Mean 0.215 0.341 0.466 0.306 0.380 0.351 0.465

SD 0.004 0.003 0.003 0.005. 0.007 0.003 0.006

%RSD 1.9 0.9 0.6 1.6 1.8 0.9 1.3

95%Confidence interval ± 0.004 ±0.003 ±0.003 ±0.005 ±0.007 ±0.003 ±0.006

4.2.6.3 Limit of Detection (LOD) and Limit of Quantification

(LOQ)

LOD and LOQ values of each related substance were predicted

from a separate linearity data at lower concentrations. Each predicted

concentration was verified by preparing the solutions at about

predicted concentration and injecting each solution six times into the

HPLC.

183

4.2.6.4 Limit of Detection (LOD):

The detection limit of an individual analytical procedure is the

lowest amount of analyte is a sample, which can be detected but not

necessarily quantitated as an exact value (Table: 4.8).

Table: 4.8 LOD values of the Florfenicol and its impurities

Injection

ID

Area

Imp-A Imp-B Imp-C Imp-D Florfenicol Imp-E Imp-F Imp-G

1 2820 4918 3541 4858 3144 2615 3920 3307

2 1786 4611 3098 4579 2306 2608 3426 3170

3 2544 4219 3404 4957 2659 2048 2889 2973

4 3132 5132 2663 4304 2905 2376 3041 3656

5 2091 4370 2614 3798 3474 1627 2785 3093

6 2395 3388 2664 3624 3524 2858 2818 2485

Mean 2461 4440 2997 4353 3002 2355 3147 3114

SD 486 616 410 550 475 449 446 387

% RSD 19.7 13.9 13.7 12.6 15.8 19.1 14.2 12.4

Conc.

(µg/mL) 0.113 0.090 0.086 0.084 0.072 0.082 0.077 0.075

Conc. (%

w/w) 0.011 0.018 0.014 0.019 0.013 0.011 0.013 0.018

4.2.6.5 Limit of Quantification (LOQ):

The quantitation limit of (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 quantitative

limit is a parameter of quantitative assays for low levels of compounds

184

in sample matrices, and is used particularly for the determination of

impurities and/ or degradation products (Table: 4.9).

Table: 4.9 LOQ values of the Florfenicol and its impurities

Injection ID

Area

Imp-A Imp-B Imp-C Imp-D Florfenicol Imp-E Imp-F Imp-G

1 4619 8359 6337 8966 5818 6471 6691 6310

2 4489 8470 5986 9014 6006 6230 6093 6536

3 4166 8110 6212 9270 5961 5987 6560 6386

4 4727 8450 6135 8775 6264 5786 6387 6472

5 4175 8646 6031 9487 5948 6288 6558 6106

6 4774 8691 6325 9254 6269 6133 6155 6861

Mean 4492 8454 6171 9128 6044 6149 6407 6445

SD 267 210 147 256 183 240 241 252

% RSD 5.9 2.5 2.4 2.8 3.0 3.9 3.8 3.9

Conc. (µg/mL)

0.227 0.179 0.172 0.169 0.144 0.165 0.154 0.151

Conc. (% w/w)

0.044 0.034 0.028 0.039 0.027 0.030 0.027 0.037

185

4.2.6.6 Linearity

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. The linearity of the assay method was

developed by injecting test sample at 80%, 90%, 100%, 110% and

120% of Florfenicol assay concentration (i.e.100 µg/ml). Each solution

injected twice (n=2) into HPLC and the average area at each

concentration calculated (Table: 4.10).

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

concentration on the X-axis (Fig: 4.9).

186

Table: 4.10 Linearity results of the Assay method

% Concentration Average area

80 1706050

90 1939894

100 2166393

110 2361512

120 2602089

Slope 21874

Intercept -58290

Residual Sum of Squares

12208

Correlation Coefficient 0.9995

Linearity Plot (Concentration Vs Response

Fig: 4.9 Linearity Plot for Assay method

1706050

1916050

2126050

2336050

2546050

80.96 90.86 100.76 110.66 120.56

Are

a

Conc.(µg/mL)

187

Linearity of the related substance method were carried out by

preparing the Florfenicol sample solutions containing Imp- A, B, C, D,

E , F and G from LOQ to 250% (i.e. LOQ 25%, 50%,150%) with respect

to their specifications limit (0.3%). Calibration curve was drawn by

ploting average value of the impurities. (Imp- A, B, C, D, E , F and G)

on the y-axis and concentrations on the X-axis (Fig: 4.10 to 4.17).

188

Linearity results of the Related Substance method

Table: 4.11 Linearity results of the Imp-A

Imp-A

Concentration

(µg/mL) Area Statistical Analysis

0.227 4478 Slope 20120

0.550 9575

0.824 16077 Intercept -669

1.374 27528

1.649 33017 Residual sum of

squares 898

1.924 37772

2.473 47188 Correlation

Coefficient 0.9994

2.748 55545

3.298 65435 Response Factor 2.21

Linearity Plot (Concentration Vs Area)

Fig: 4.10 Linearity Plot for Imp-A

4478

19478

34478

49478

64478

79478

0.227 1.127 2.027 2.927 3.827

Are

a

Conc.(µg/mL)

189

Table: 4.12 Linearity results of the Imp-B

Imp-B

Concentration


0.180 8975 Slope 49241

0.504 24310

0.756 37725 Intercept -99

1.260 62100

1.512 73753 Residual Sum of

Squares 415

1.764 86477


Coefficient 0.9999

2.520 124521

3.024 148654 Response 0.90


Fig: 4.11 Linearity Plot for Imp-B

8975

43975

78975

113975

148975

183975

0.180 1.080 1.980 2.880 3.780

Are

a

Conc.(µg/mL)

190

Table: 4.13 Linearity results of the Imp-C

Imp-C

Concentration


0.172 6691 Slope 43285

0.500 21425

0.749 33058 Intercept -88

1.249 54391


Squares 577

1.749 75611


Coefficient 0.9999

2.498 108358

2.998 128583 Response 1.03


Fig: 4.12 Linearity Plot for Imp-C

6691

36691

66691

96691

126691

156691

0.172 1.022 1.872 2.722 3.572

Are

a

Conc.(µg/mL)

191

Table: 4.14 Linearity results of the Imp-D

Imp-D

Concentration (µg/mL)

Area Statistical Analysis

0.166 9162 Slope 45771

0.507 23428

0.760 35326 Intercept 866

1.266 59613

1.520 70227 Residual Sum of Squares

558 1.773 81942

2.279 104834 Correlation Coefficient

0.9999 2.533 116927

3.039 139386 Response 0.97


Fig: 4.13 Linearity Plot for Imp-D

9162

39162

69162

99162

129162

159162

0.166 1.066 1.966 2.866 3.766

Are

a

Conc.(µg/mL)

192

Table: 4.15 Linearity results of the Florfenicol

Florfenicol

Concentration


0.144 6253 Slope 44463

0.528 23091

0.792 36323 Intercept 81

1.321 59019

1.585 70100

Residual sum of

squares 713 1.849 82184

2.377 105209

2.641 118687

Correlation

coefficient 0.9999 3.170 140187

3.962 176500


Fig: 4.14 Linearity Plot for Florfenicol

6253

39253

72253

105253

138253

171253

0.144 1.094 2.044 2.994 3.944

Are

a

Conc.(µg/mL)

193

Table: 4.16 Linearity results of the Imp-E

Imp-E

Concentration (µg/mL)


0.165 8391 Slope 40988

0.512 20235

0.768 31118 Intercept 889

1.280 53878

1.536 64690 Residual sum of

squares 1194

1.792 75261


Coefficient 0.9997

2.560 107453


3.840 157581

Linearity Plot (Concentration Vs Response)

Fig: 4.15 Linearity Plot of for Imp-E

8391

37391

66391

95391

124391

153391

0.165 1.065 1.965 2.865 3.765

Are

a

Conc.(µg/mL)

194

Table: 4.17 Linearity results of the Imp-F

Imp-F

Concentration (µg/ml)


0.154 8835 Slope 47172

0.493 23432

0.739 34887 Intercept 411

1.232 59155

1.478 70031 Residual sum of squares

1043 1.725 81219

2.218 105102 Correlation Coefficient

0.9998 2.464 115059


3.696 176568

Linearity Plot (Concentration Vs Response)

Fig: 4.16 Linearity Plot of for Imp-F

8835

40835

72835

104835

136835

168835

0.154 1.004 1.854 2.704 3.554

Are

a

Conc.(µg/mL)

195

Table: 4.18 Linearity results of the Imp-G

Imp-G

Concentration


0.151 8863 Slope 34332

0.504 16368

0.756 25112 Intercept 1302

1.261 44784


Squares 1828

1.765 62349


Coefficient 0.9990

2.521 89814


3.782 131349


Fig: 4.17 Linearity Plot of for Imp-G

5230

14230

23230

32230

41230

50230

59230

0.200 0.550 0.900 1.250 1.600 1.950

Are

a

Con. (µg/mL)

196

4.2.6.7 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.

Accuracy of the assay method

Accuracy of the assay method was established by injecting three

preparations of test sample at 80%, 100% and 120% of analyte

concentration (i.e.100 µg/ml). Each solution was injected twice (n=2)

into HPLC and the mean peak area of Florfenicol peak was calculated.

Assay (%w/w) of test solution was determined against three

injections (n=3) of qualified Florfenicol reference standard (Table:

4.19).

The method showed consistent and high absolute recoveries at

all three concentration (80%, 100% and 120%] levels with mean

absolute recovery ranging from 99.3 % to 100.5%. The obtained

absolute recoveries were normally distributed around the mean with

uniform RSD values. The method was found to be accurate with low %

bias (< 1.0).

197

Table: 4.19 Accuracy results of the Assay method

S.NO %

Concentration

Mean recovery

(%) (n=3) %RSD

1 80 100.7 0.6

2 100 100.2 0.3

3 120 100.5 0.3

4.2.6.8 Acuracy/Recovery of the Related substance method

Acuracy of the related substance method established 50%,

100%, 150% the impurities specification limit (0.30%).

Accuracy at 50% impurity specification limit:

Test solution prepared in triplicate (n=3) with impurities (Imp-A,

B, C, D, E, F and G ) at 0.15% level w.r.s analyte concentration (i.e 0.5

mg/m l). Each solution was injected thrice into HPLC. Mean %

recovery of impurities calculated in the sample solution using the area

of impurities standard at 0.3% level with respect to analyte (Table:

4.20).

198

Table: 4.20 Accuracy at 50% level

S.NO Impurity

name Mean

recovery(%) SD %RSD

1 Imp-A 98.2 0.60 0.6

2 Imp-B 97.8 0.40 0.4

3 Imp-C 99.5 0.81 0.8

4 Imp-D 104.6 1.18 1.1

5 Imp-E 101.6 0.75 0.7

6 Imp-F 97.3 1.18 1.2

7 Imp-G 99.2 0.35 0.4


Test solution prepared in triplicate (n=3) with impurities (Imp-A,

B, C, D, E, F and G) at 0.3 % level w.r.s analyte concentration (i.e 0.5

mg/m l). Each solution was injected thrice into HPLC (Table: 4.21).

Table: 4.21 Accuracy at 100% level

S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 106.3 0.70 0.7

2 Imp-B 95.0 0.20 0.2

3 Imp-C 96.1 0.42 0.4

4 Imp-D 99.3 0.58 0.6

5 Imp-E 96.8 0.36 0.4

6 Imp-F 93.2 0.50 0.5

7 Imp-G 103.3 1.65 1.6


Test solution in triplicate (n=3) with impurities (Imp-A, B, C, D,

E, F and G) at 0.9 % level w.r.s analyte concentration (i.e 0.5 mg/m l).

Each solution was injected thrice into HPLC (Table: 4.22).

199

Table: 4.22 Accuracy at 150 % level

S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 99.9 0.42 0.4

2 Imp-B 95.3 0.21 0.2

3 Imp-C 95.7 0.10 0.1

4 Imp-D 98.0 0.06 0.1

5 Imp-E 98.2 0.89 0.9

6 Imp-F 94.4 1.03 1.1

7 Imp-G 96.3 1.45 1.5

The related substance method showed consistent and high obsolute

recoveries of all six impurities at all three different concentrations (50,

100, 150%) levels in drug substance.

4.2.6.9 Solution state stability

The solution state stability of Florfenicol in diluent in the assay

method was carried out by leaving both the test solutions of sample

and reference standard in tightly capped volumetric flasks kept at

room temperature for two days. The same sample solutions were

assayed for every one hour interval up to the study period. The % RSD

of assay of Florfenicol during solution stability experiments was with

in 1.0%.

The solution state stability of Florfenicol related substance

method was carried out by leaving sample solution in tightly capped

volumetric flask at room temperature for two days. Content of Imp A,

B, C, D, E, F and G were checked for every six hours internal up to

the study period. No significant change was observed in the content

200

of all six impurities in drug solution stability experiments up to the

study period. Hence Florfenicol sample solutions are stable for atleast

48 hours in the developed method. In assay method standard and test

solutions injected at each 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h,

11h, 12h. (Table: 4.23).

Table: 4.23 Solution stability results of the Assay method

S.No Time in Hours Assay (% w/w)

1 initial 99.9

2 1 99.8

3 2 99.2

4 3 99.6

5 4 99.5

6 5 99.6

7 6 99.7

8 7 99.2

9 8 99.3

10 9 99.2

11 10 99.4

12 11 99.6

13 12 99.5

% RSD 0.24

In related substances method solution stability studies of

Florfenicol in diluent was done for 15 hrs by injecting sample solution

for every one hour interval up to the study period. The impurity

profiles obtained at different interval were very consistent and

matched with initial value.

201

4.2.7.0 Robustness

The robustness of an analytical procedure is a measure of its

capacity to remain unaffected by small, but deliberate variations in

method parameters and provides an indication of its reliability during

normal usage. To determine the robustness of the developed method

experimental conditions were purposely altered and the resolution

between Imp-C and Imp-D was evaluated. In each of the deliberately

altered chromatographic condition (flow rate 1.3 ml/min and

1.7ml/min, acetonitrile 23% and 27% in the mobile phase, column

temperature 25 C and 35C) the resolution between Imp-B, Imp-C

and Imp-D, Imp-E and Imp-F was greater than 2.0,, illustrating the

robustness of the method.

4.3 Mass balance

The mass balance it 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 [10]. Its establishment hence is a regulatory

requirement. The mass balance is very closely linked to the

development of stability-indicating assay method as it acts as an

approach to establish its validity. The stressed samples of Florfenicol

bulk drug were assayed against the qualified reference standard and

the results of mass balance obtained in each condition is presented

below (Table: 4.24).

202

Table: 4.24 Mass balance of the assay method

Degradation Mechanism

Degradation Condition

% Assay of active

substance)

Mass balance (% Assay+ % impurities+ % degradants)

Remarks

Acid 5M HCl/80°C

/60 min 91.5 99.5

Degraded to Imp-A and some unknown

degradants observed

Base 5M NaOH/

Initial 90.6 99.6

Degraded to Imp-A, Imp-C and some

unknown degradants observed

Peroxide 30% H2O2/

80°C/120 min 96.1 99.5

Some unknown degradants observed

Thermal 105°C/188

Hours 99.5 99.6 No degradation observed

Photolytic 12,000 Lux/ 188 Hours

99.5 99.6 No degradation observed

4.4 Analysis of Florfenicol drug substance stability samples

One manufacturing lot of Florfenicol drug substance was placed

on stability study in chambers maintained at ICH set conditions [11].

The analysis of stability samples were carried up to 12 months period

using the above optimized method. The stability data results obtained

are presented in Table: 4.25 to 4.26. The developed HPLC method

performed satisfactorily for the quantitative evaluation of stability

samples.

203

Table: 4.25 Accelerated stability data (Storage conditions:

40°C/75%RH)

Batch No: KMR(510)128 Packing & storage conditions: Each sample

packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum.

Stability study duration: 6 months Temperature

%Relative humidity

40°C/75%RH

Description

Loss on

drying (%w/w)

Identification

Assay (By HPLC,

%w/w, on

dried basis,)

Specifications

A white to almost

white

crystalline powder

NMT 0.5

IR spectrum should

concordant

with that of standard

NLT 98.0 and NMT 102.0

Initial

A white

crystalline

powder

0.15 Complies 99.5

1M A white

crystalline

powder

0.14 Complies 99.5

2M

A white

crystalline powder

0.13 Complies 99.2

3M

A white

crystalline

powder

0.15 Complies 99.4

6M A white

crystalline

powder

0.14 Complies 99.3

Related substances details on next page.

204

Related

Substances

LOQ

(%w/w)

LOD

(%w/w)

Related Substances (By HPLC, %w/w)

INITIAL 1M 2M 3M 6M

Imp-A 0.044 0.011 ND ND ND ND ND

Imp-B 0.034 0.018 ND ND Below

LOQ

Below

LOQ

Below

LOQ

Imp-C 0.028 0.014 0.08 0.09 0.09 0.09 0.08

Imp-D 0.030 0.011 ND ND ND ND ND

Imp-E 0.027 0.013 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-F 0.032 0.014 ND ND ND ND ND

Imp-G 0.037 0.018 ND ND ND ND ND

Highest

unknown - - ND ND ND ND ND

Total

unknown - - NA NA NA NA NA

Total RS - - 0.08 0.09 0.09 0.09 0.08

ND: Not detected

NA: Not applicable

205

Table: 4.26 Long-term stability data (Storage conditions:

25°C/60%RH)

Batch No: KMR(510)128 Packing & storage conditions: Each sample

packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum

Stability study duration: 12 months Temperature

%Relative humidity

25°C/60%RH

Tests Description

Loss on

drying (%w/w)

Identification

Assay (By HPLC,

%w/w, on

dried basis,)

Specifications

A white to almost

white

crystalline powder

NMT 0.5

IR spectrum should

concordant

with that of standard

NLT 98.0 and NMT 102.0

Initial

A white

crystalline

powder

0.15 Complies 99.5

1M A white

crystalline

powder

0.14 Complies 99.6

2M

A white

crystalline powder

0.13 Complies 99.4

3M

A white

crystalline

powder

0.14 Complies 99.5

6M A white

crystalline

powder

0.13 Complies 99.2

9M

A white

crystalline powder

0.13 Complies 99.3

12M

A white

crystalline

powder

0.12 Complies 99.2

Related substances details on next page.

206

Related Substances

LOQ (%w/w)

LOD

(%w/w)

Related Substances (By HPLC, %w/w)

Initial 1M 2M 3M 6M 9M 12M

Imp-A 0.044 0.011 ND ND ND ND ND ND ND

Imp-B 0.034 0.018 ND Below LOQ

ND Below LOQ

Below LOQ

ND Below LOQ

Imp-C 0.028 0.014 0.07 0.08 0.07 0.06 0.06 0.07 0.08

Imp-D 0.030 0.011 ND ND ND ND ND ND ND

Imp-E 0.027 0.013 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-F 0.032 0.014 ND ND ND ND ND ND ND

Imp-G - - ND ND ND ND ND ND ND

Highest unknown

- - ND ND ND ND ND ND ND

Total unknown

- - NA NA NA NA NA NA NA

Total RS - - 0.08 0.09 0.08 0.08 0.08 0.08 0.09

ND: Not detected NA: Not applicable

207

4.5 Summary and conclusions

Validated stability-indicating HPLC method was developed for

Florfenicol after subjecting the samples to stress testing under ICH

recommendes conditions. The RPLC method was developed for

quantitative and related substance determination of Florfenicol is

rapid precise, accurate and selective. The method was completely

validated showing satisfactory data for all the method validation

parameters tested. The developed method was found to be ‘specific’ to

the drug, as the peaks of the degradation products did not interfere

with the degradation peak. Thus the proposed method can be

employed for assessing the stability of Florfenicol bulk drug samples.

208

Table: 4.27 Summary of Analytical method validation data

Test Parameter

Related Substances method Assay

method

Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G

Precision (RSD) 1.8 0.6 0.6 0.3 1.0 0.6 0.9 0.7

LOD (µg/ml) 0.113 0.090 0.086 0.084 0.082 0.077 0.075 N/A

LOQ (µg/ml) 0.044 0.034 0.028 0.039 0.030 0.027 0.037

N/A

Linearity (corre coefficient

0.9994 0.9999 0.9999 0.9999 0.9997 0.9998 0.9990 0.9995

Accuracy (%) 98.2-106.3 95.0-97.8 95.7-99.5 98.0-104.6 98.2-101.6 93.2-97.3 96.3-103.3 100.2-100.7

Robustness

Resolution b/w Imp-E& Imp-

F>2

Resolution b/w

Imp-E& Imp-F>2

Resolution b/w

Imp-E& Imp-F>2


F>2


F>2

Resolution b/w

Imp-E& Imp-F>2

Resolution b/w Imp-E&

Imp-F>2

Resolution b/w Imp-E&

Imp-F>2

Solution stability Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Mobile phase stability Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

209

4.6 References:

1. Sams, R. A.; in proceedings of the XVIII World Buiatrics

Congress, 1994, Bolonga, 13.

2. Piotr, K.; Lucyna, K.; Aleksandra, C.; Iiona, O.; Alina, P.; Michal

B,; Henry, L.; J. Pharm. Biomed. Anal., 2005, 39, 983.

3. Suxia, Z.; Zhongwei, L.; Xia. G.; Linli. C.; Zhanhui. W.;

Jianzhong. S.; J.Chromatogr. B, 2008, 875, 399.

4. Chue. V.; Larry, S. J.; Guy. S. R.; William, G. H.; J.Chromatogr.

B, 2002, 780, 111.

5. Hayes, J. M.; Eichman, J.; Katz, T.; Gilewicz, R.; J. AOAC,

2003, 86, 22.

6. Marciniec, B.; Stawny, M.; Kachlicki, P.; Jaroszkiewicz, E.;

Michael, N.; Analytical Sciences. 2009, 25, 1255.

7. De Craene, B. A.; Deprez, P.; Haese, E. D.; Nelis, H. J.;

Bossche, W.; De Leenheer, A. P.; Antimicrobial Agents and

Chemotherapy., 1997, 41, 1991.

8. Roy, Y. P. E.; Eric, C. W.; J. Aquatic Animal Health. 2005, 17,

129.

9. ICH, Validation of analytical Procedures: Text and methodology

Q2 (R1), International Conferences on Hormonization, IFPMA,

Geneva, 2005.

10. Steven, B. W.; Pharmaceutical Stress Testing Predicting

Drug Degradation.

11. Validation of Analytical Procedures: Methodology Q2B – ICH

Guidelines.

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