CHAPTER-5 A VALIDATED STABILITY-INDICATING ANALYTICAL ...shodhganga.inflibnet.ac.in/bitstream/10603/4517/14/14_chapter 5.pdf · d) Thermal Degradation: drug was subjected to dry heat

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CHAPTER-5

A VALIDATED STABILITY-INDICATING

ANALYTICAL METHOD FOR THE

DETERMINATION OF IMPURITIES IN

CEFTAZIDIME PENTAHYDRATE

211

5.1 Introduction on Ceftazidime Pentahydrate and survey of

analytical methods

Ceftazidime is a third generation semi-synthetic cephalosporin

antibiotic having broad spectrum of activity with enhanced activity

against pseudomonas aeruginosa [1]. It is chemically designated as

(6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2-[(1-carboxy-1-

(methylethoxy)imino]acetyl]amino]-8-Oxo-3-[(1-pyridinio)methyl-5-

thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylatepentahydrate.

Ceftazidime is a hygroscopic, optically active, powder. It is freely

soluble in ethanol and water and practically in soluble in acetonitrile.

The empirical formula is C22H22N6O7S2.5H20. The molecular weight of

Ceftazidime is 636.6.

Fig: 5.1 Chemical structure of Ceftazidime Pentahydrate

HH

S

N

H2N

CC

N

N

O

O N

HS

N

COO

O

H3C

H3C COOH

5H2O

(6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2-[(1-

carboxymethylethoxy)imino]acetyl]amino]-8-Oxo-3-[(1-

pyridinio)methyl-5-

thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylatepentahydrate

Molecular formula: C22H22N6O7S2.5H20

Molecular weight: 636.6.

A literature survey revealed that several methods have been

reported for the determination of Ceftazidime [2-9]. Organic impurities

can arise during the manufacturing process and storage of the drug

212

substances and the criteria for their acceptance up to certain limits

are based on pharmaceutical studies or known safety data [10]. As per

regulatory guidelines, the pharmaceutical studies using a sample of

the isolated impurities can be considered for safety assessment [11]. It

is, therefore, essential to isolate and characterize unidentified

impurities present in the drug sample [12]. Recently we have

developed a process for the synthesis of Ceftazidime in our laboratory.

During the development of an analytical procedure, the LC method

was developed for the determination of in-house synthesized

Ceftazidime 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 Ceftazidime. The

accuracy, precision, limit of detection (LOD), limit of quantification

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

with ICH guidelines. The target is to develop a suitable stability-

indicating HPLC related substances method for Ceftazidime in this

chapter we describe a stability-indicating LC method for the

determination of Ceftazidime and its potential and degradation

impurities and also the method validation.

213

5.2 Development of a stability-indicating analytical method for

Ceftazidime Pentahydrate

5.2.1 Materials

Reference standard of Ceftazidime and ten impurities namely,

Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G, Imp-H, Imp-I and

Imp-J (Fig: 5.2) were synthesized and characterized by use of LC-MS,

NMR and IR in Aurobindo Pharma Ltd., Hyderabad, India. The

commercial samples of Ceftazidime 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).

5.2.2 Equipment

The LC system was equipped with quaternary gradient pumps

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

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

Fig: 5.2 Chemical structures of impurities of Ceftazidime

Pentahydrate

S

NHN CHO

O

N

H2N

O

H3CCO2H

H3C

2-[[[(1Z)-1-(2-Aminothiazol-4-yl)-2-[(oxoethyl) amino]-2-oxoethylidene]

amino] oxy]-2-methylpropanoic acid (Imp-A) Fig: 5.2 (a)

214

H2N

N

S

ON

H H

COO

. HI

(6R,7R)-7-Amino-3-(1-pyridiniummethyl)-3-cephem-4-carboxylate

Monohydroiodide (Imp-B)

Fig: 5.2 (b)

N

O C COOH

CH3

CH3

N

S

C N

N

S

ONO

H H

COO

O

H2N

H

(6R,7R)-7-[(Z)-2-(2- Aminothiazol-4-yl)-2-(2-carboxyprop-2-

oxyimino)acetamido]-3-(1- pyridiniummethyl)-3-cephem-4-carboxylate

sulfoxide (Imp-C)

Fig: 5.2 (c)

*

N

S

C N

N

S

ON

H COO

N

O C COOH

CH3

CH3

O

H H

H2N

H

4RS,6R,7R)-7-[(Z)-2-(2-Aminothaizol-4-yl)-2-[(2-carboxyprop-2-

oxyimino) acetamido]-3-(1-pyridiniummethyl]-2-cephem-4-carboxylate

(Imp-D)

Fig: 5.2 (d)

215

H

N

S

C N

N

S

ON

N

O

H H

COO

OCHOOC

CH3

CH3

H2N

(6R,7R)-7-[(E)- 2-(2-Aminothaizol-4-yl)-2-[(2-carboxyprop-2-oxyimino)

acetamido]-3-(1-pyridiniummethyl]-2-cephem-4-carboxylate (Imp-E)

Fig: 5.2 (e)

N

O C COOH

CH3

CH3

N

SOHCHN

C N

N

S

ONO

H H

COO

H

(6R,7R)-7-[(Z)-2-(2-N-Formylaminothiazol-4-yl)-2-(2-carboxy -prop-2-

oxyimino) acetamido]-3-(1-pyridiniummethyl)-3-cephem-4-carboxylate

(Imp-F)

Fig: 5.2 (f)

Pyridine

(Imp-G)

Fig: 5.2 (g)

216

N

O C COOH

CH3

CH3

N

S

C N

N

S

OO

H H

CH2

OO

H2N

H

6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxiimino)

acetamido]-3-hydroxymethyl-3-cephem-4-carboxylic acid lactone

(Imp-H)

Fig: 5.2 (h)

N

O C COOC(CH3)3

CH3

CH3

N

S

C N

N

S

ONO

H H

COO

H2N

H

(6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-tert-butoxycarbonylprop-2-

oxyimino)acetamido]-3-(1-pyridiniummethyl)-3-cephem -4-carboxylate

(Imp-I)

Fig: 5.2 (i)

H

N

SPh3CHN

C N

N

S

ONO

H H

COO

N

O C COOC(CH3)3

CH3

CH3

(6R,7R)-7-[(Z)-2-(2-tert-Butoxycarbonylprop-2-oxyimino)-2-(2-trityl-

aminothiazol-4- yl)acetamido]-3-[1-pyridiniummethyl)-3-cephem-4-

caboxylate [Imp-J]

Fig: 5.2 (j)

217

5.2.3 Preparation of Mobilephase:

Dissolve 2.3 g of disodium hydrogen orthophosphate anhydrous

and 1.75 g of potassium dihydrogen orthophosphate in 1000 ml of

water. Observe pH should be 7.0±0.05, otherwise adjust pH to 7.0 ±

0.05 with dilute orthophosphoric acid solution or dilute sodium

hydroxide solution.

5.2.4 Preparation of sample and stock solutions

The stock solutions of Ceftazidime (1.25 mg/ml) and spiked with

0.5% of Imp-A, Imp-B, Imp-D, Imp-E, Imp-H, Imp-I and 0.1% of Imp-

C, Imp-F, Imp-G with respect to the Ceftazidime analyte

concentration. The stock solutions were further diluted with diluent to

obtain a standard solution of 0.0125 mg/ml (12.5 µg/ml) for related

substances determination and 1.0 mg/ml (1000 µg/ml) for assay

determination.

5.2.5 Generation of stress samples

One lot of Ceftazidime drug substance selected for stress testing.

From the ICH stability guideline: Stress testing likely to be carried out

on a single batch of material [12]. Different kinds of stress conditions

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

and light) were employed on one lot of Ceftazidime drug substance

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

are as follows:

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

for 120 mins.

218

b) Base Degradation: drug in 1 M NaOH solution was kept at 85°C

for 30 mins.

c) Oxidative Degradation: drug in 30% H2O2 solution was kept at

85°C for 240 mins.

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

for 120 hrs.

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

K Lux ) for 48 hrs.

5.2.6 Optimization of chromatographic conditions

The main objective of the chromatographic method was to

seperate Ceftazidime from Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-

F, Imp-G, Imp-H, Imp-I and Imp-J impurities were coeluted using

different stationary phases 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 ceftazidime,

some impurities and degradants. The chromatographic separation was

achieved on a YMC Pack ODS-A (250 x 4.6 mm), 5µ 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: 0.01/05,

15/25, 25/70, 40/70, 42/05, 50/05 with a post run time of 10 min.

219

The column temperature was maintained at 30°C and the detection

was monitored at a wavelength of 255 nm. The injection volume was

10µl. Standard and test solutions were prepared in mixture of

phosphate buffer and acetonitrile in the ratio of 95:5 v/v was used as

diluent. In the optimized chromatographic conditions of Ceftazidime,

Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G, Imp-H, Imp-I and

Imp-J were separated with a resolution greater than 2, typical relative

retention times were approximately 0.45, 0.50, 0.64, 0.88, 1.00, 1.34,

1.40, 1.88, 1.99, 3.47, 4.75 with respect to Ceftazidime eluted at

5.919.

No considerable degradation was observed in ceftazidime bulk

samples under stress conditions such as acid hydrolysis, base

htdrolysis, oxidative environment, thermal and photolytic conditions.

The degradation of drug substance was observed during base

hydrolysis and oxidative stress condition. Ceftazidime was degraded to

Imp-B (5.3%), Imp-D (2.1%), Imp-F (10.2%) under Base conditions (1M

NaOH/85°C/90 min) and it was confirmed by co-injection with a

qualified Imp-B, Imp-D and Imp-F standards. Ceftazidime was

degraded to Imp-C (7.46%) under oxidative environment (treated with

3% H2O2 initial ) and it was confirmed by co-injection with a qualified

Imp-C. Ceftazidime was degraded to Imp-E (1.1%) under photolytic

environment (10 K Lux/168 hours) and it was confirmed by co-

injection with a qualified Imp-E.

Peak purity test results obtained by using a PDA detector

confirmed that the Ceftazidime peak is homogenous and pure in all

220

the analyzed stress samples. The mass balance of Ceftazidime in all

stress samples was close to 99.9% (%Assay + %Degradation). This

clearly demonstrates that the developed HPLC method was found to

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

Imp-C, Imp-D, Imp-E, Imp-F, Imp-G, Imp-H, Imp-I and Imp-J ) and

degradation products.

Although liquid chromatographic method was available in

Pharma Europa for ceftazidime pentahydrate. In Pharma Europa

method the Imp-C (ceftazidime sulfoxide) and Imp-A (ATIBAA glycine

analog- Ph. Eur. Imp-G) were not separate and peak shapes also very

broad in nature. But in house method Ph .Eur. impurities and in

house impurities are very well separated and peak shapes also very

sharp.

221

Fig: 5.3 Typical HPLC chromatograms of Pharma europa

method and In house method

Fig: 5.3 (a) Spiked chromatogram as per Pharma europa

method

Fig: 5.3 (b) Spiked chromatogram as per In house method

No degradants were observed after 15 min in the extended run

time of 40 min for all the Ceftazidime stressed samples (acid

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

with 90% can in mobile phase.

222

Optimized liquid chromatographic conditions

Column : YMC Pack ODS-A, 250 x 4.6 mm,

5µ particle size

Mobile phase : The solution A contains

phosphate buffer pH 7.0 and

Solution B contains acetonitrile

Gradient program : Time / % B: 0/05, 15/25, 25/70,

40/70, 42/05 and 50/05

Flow rate : 1.0 ml/min

Column temperature : 30°C

UV detection : 255 nm

Injection volume : 10 µl

Run time : 40 min

Retention time : 5.919

Relative Retention Time (RRT) : Impurity-A about 0.45

Impurity-B about 0.50

Impurity-C about 0.64

Impurity-D about 0.88

Impurity-E about 1.34

Impurity-F about 1.40

Impurity-G about 1.88

Impurity-H about 1.99

Impurity-I about 3.47

Impurity-I about 4.75

Diluent : mixture of phosphate buffer and

acetonitrile in the ratio of 95:5 v/v.

223

Figures:

Fig: 5.4 to Fig: 5.8 is the typical HPLC chromatograms showing

the degradation of Ceftazidime in various stress conditions and also

the corresponding peak purity plots.

Fig: 5.4 Typical HPLC chromatograms of Acid hydrolysis

Fig: 5.4 (a)

Fig: 5.4 (b)

Blank Chromatogram of Acid hydrolysis (1N HCl)

Ceftazidime stressed with 5M HCl at 85C for 120 mins

224

Fig: 5.4 (c) Peak purity plot of Acid hydrolysis

Purity

Angle

Purity

Threshold Purity Flag Peak Purity

0.065

0.253 No Pass

Fig: 5.4 (c)

225

Fig: 5.5 Typical HPLC chromatograms of Base hydrolysis

Fig: 5.5 (a)

Fig: 5.5 (b)

Blank Chromatogram of Base hydrolysis ( 1N NaOH )

Ceftazidime stressed with 1N NaOH at room temperature

226

Fig: 5.5 (c) Peak purity plot of Base hydrolysis

Purity Angle Purity Threshold Purity Flag Peak Purity

0.064

0.255 No Pass

Fig: 5.5 (c)

227

Fig: 5.6 Typical HPLC chromatograms of Peroxide Degradation

Fig: 5.6 (a)

Fig: 5.6 (b)

Blank Chromatogram of Peroxide Degradation ( 3% H2O2 )

Ceftazidime stressed with 30%H2O2 at room temperature

228

Fig: 5.6 (c) Peak purity plot of Peroxide Degradation


0.073

0.259 No Pass

Fig: 5.6 (c)

229

Fig: 5.7 Typical HPLC chromatograms of Thermal Degradation

Fig: 5.7 (a)

Fig: 5.7 (b)

Ceftazidime stressed at 105°C for 168 hours

230

Fig: 5.7 (c) Peak purity plot of Thermal Degradation


0.080

0.263 No Pass

Fig: 5.7 (c)

231

Fig: 5.8 Typical HPLC chromatograms of Photolytic Degradation

Fig: 5.8 (a)

Fig: 5.8 (b)

Ceftazidime stressed with 10K Lux for 168 hours

232

Fig: 5.8 (c) Peak purity of Photolytic Degradation


0.087

0.268 No Pass

Fig: 5.8 (c)

233

5.2.7 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 is

accordance with ICH guideline [8].

5.2.7.1 System suitability : A mixture of Ceftazidime standard

injections were injected into HPLC system and good resolution was

obtained between impurities and Ceftazidime [Fig : 5.9 (c)].The system

suitability results were given below table (Table: 5.1).

Fig: 5.9 Typical Blank, Ceftazidime sample and SST Chromatograms

Fig: 5.9 (a)

234

Fig: 5.9 (b)

Fig: 5.9 (c)

235

Table: 5.1 System suitability results

Compound (n=3)

USP Platecount (N)

USPTaling factor (T)

USP Resolution(Rs)

Imp-A 2548 1.18 -

Imp-B 12723 1.25 2.23

Imp-C 9600 1.09 6.22

Imp-D 20591 0.94 8.95

Imp-E 25934 1.05 4.82

Imp-F 81125 0.97 15.46

Imp-G* 66416 1.12 2.87

Imp-H 65105 0.84 21.68

Imp-I 751144 1.08 61.37

Imp-J 810803 1.07 67.03

*Separate content method was available for Imp-G.

5.2.7.2 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

Ceftazidime test sample against qualified reference standard and RSD

of six consecutive assays was 0.2% (Table: 5.2 to Table: 5.4).

The results showed insignificant variation in measured

response. Which demonstrated that the assay method was repeatable

with RSD’s below 0.1%.

236

Table: 5.2 System Precision results of the Assay method

Injection ID Area

1 18562078

2 18595314

3 18575275

4 18532557

5 18579886

6 18559486

Mean 18567433

SD 21457

% RSD 0.1

95% Confidence

Interval ± 0.22521

Table: 5.3 Method Precision results of the Assay method

Injection ID Assay (% w/w)

1 98.8

2 99.2

3 98.7

4 99.0

5 98.6

6 98.5

Mean 98.8

SD 0.26

% RSD 0.3

95% Confidence Interval

± 0.3

237

Table: 5.4 Intermediate Precision results of the Assay method

Injection ID Assay (% w/w)

1 99.0

2 98.5

3 98.6

4 99.1

5 99.1

6 98.9

Mean 98.9

SD 0.26

% RSD 0.3

95%

ConfidenceInterval ± 0.3

The precision of the related substance method was checked by

injecting six individual preparations of Ceftazidime (1.25 mg/ml)

spiked with 0.1% of Imp-A, Imp-C, Imp-F, Imp-H and 0.5% of Imp-B,

Imp-D, Imp-E, Imp-I, Imp-J with respect to the Ceftazidime analyte

concentration. The % RSD of the area percentage of each impurity (

impurities- A, -B, -C, -D, -E, -F, -H, -I and -J ) for six consecutive

determinations was respectively as below (Table: 5.5 to Table: 5.7).

The results showed insignificant variation in measured

response. Which demonstrated that the related substance method was

repeatable with RSD’s below 1.7%.

238

Table: 5.5 System Precision of the analytical method

Injection Area Statistical Analysis

1 261511 Mean 261836

2 261099

3 261530 SD 713

4 262976 % RSD 0.3

5 262438 95% Confidence

Interval ± 748

6 261462

Table: 5.6 Method Precision results of the analytical method

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

1 0.184 0.516 0.168 0.604 0.552

2 0.186 0.511 0.170 0.601 0.546

3 0.186 0.515 0.169 0.605 0.549

4 0.188 0.516 0.167 0.588 0.545

5 0.187 0.513 0.168 0.586 0.542

6 0.189 0.512 0.171 0.583 0.543

Mean 0.187 0.514 0.169 0.595 0.546

SD 0.002 0.002 0.001 0.010 0.004

%RSD 1.1 0.4 0.6 1.7 0.7

95% Confidence

interval

±0.002 ±0.002 ±0.001 ±0.001 ±0.004

Preparation Imp-F Imp-H Imp-I Imp-J

1 0.143 0.114 0.550 0.483

2 0.143 0.113 0.543 0.478

3 0.144 0.113 0.543 0.484

4 0.142 0.113 0.542 0.485

5 0.143 0.116 0.539 0.485

6 0.141 0.115 0.540 0.491

Mean 0.143 0.114 0.543 0.484

SD 0.001 0.001 0.004 0.004

%RSD 0.7 0.9 0.7 0.8

95%

Confidence interval

±0.001 ±0.001 ±0.004 ±0.004

239

Table: 5.7 Intermediate Precision results of the analytical method

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

1 0.186 0.518 0.167 0.585 0.523

2 0.186 0.492 0.168 0.584 0.528

3 0.184 0.521 0.168 0.581 0.529

4 0.187 0.507 0.167 0.595 0.526

5 0.188 0.507 0.167 0.572 0.522

6 0.189 0.505 0.167 0.589 0.526

Mean 0.187 0.508 0.167 0.584 0.526

SD 0.002 0.010 0.001 0.008 0.003

%RSD 1.1 2.0 0.6 1.4 0.6

95%

Confidence interval

±0.002 ±0.010 ±0.001 ±0.008 ±0.003

Preparation Imp-F Imp-H Imp-I Imp-J

1 0.149 0.123 0.530 0.515

2 0.150 0.125 0.535 0.463

3 0.149 0.120 0.530 0.517

4 0.149 0.122 0.536 0.452

5 0.149 0.122 0.532 0.484

6 0.148 0.121 0.536 0.508

Mean 0.149 0.122 0.533 0.490

SD 0.001 0.002 0.003 0.028

%RSD 0.7 1.6 0.6 5.7

95%

Confidence

interval

±0.001

±0.002

±0.003 ±0.029

5.2.7.3 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: 5.8).

240

Table: 5.8 LOD values of the Ceftazidime and its impurities.

Injection

ID

Area

Imp-A Imp-B Imp-C Imp-D Ceftazidime

1 3458 4684 3061 2797 3677

2 3592 3759 2485 2622 3788

3 3816 5031 4360 2687 2520

4 3370 3393 3798 2507 4022

5 2780 3264 3389 2539 3445

6 3772 3787 3231 1852 3202

Mean 3465 3986 3387 2501 3442

SD 377 713 642 335 533

% RSD 10.9 17.9 19.0 13.4 15.5

Conc.

(µg/mL) 0.401 0.142 0.142 0.134 0.137

Conc. (% w/w)

0.027 0.012 0.014 0.011 0.011

Injection

ID

Area

Imp-E Imp-F Imp-H Imp-I Imp-J

1 2235 2051 1856 2507 3461

2 1489 2360 2041 3197 3638

3 2277 2381 2011 2115 2537

4 2058 2562 1994 2647 3473

5 2202 2767 1711 2775 3196

6 2065 2992 1296 2443 3007

Mean 2054 2519 1818 2614 3219

SD 291 332 284 362 402

% RSD 14.2 13.2 15.6 13.8 12.5

Conc.

(µg/mL) 0.140 0.130 0.131 0.130 0.400

Conc. (%

w/w) 0.012 0.012 0.009 0.012 0.017

241

5.2.7.4 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 quantitative

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

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

impurities and/ or degradation products. (Table: 5.9).

242

Table: 5.9 LOQ values of the Ceftazidime and its impurities

Injection

ID

Area

Imp-A Imp-B Imp-C Imp-D Ceftazidi

me

1 8944 6685 6151 4852 6960

2 8367 7264 6213 5544 6888

3 8741 6210 5945 4660 6463

4 8956 7547 5521 4892 7607

5 8611 7584 6053 4567 6942

6 8991 6652 6742 5152 7022

Mean 8768 6990 6104 4945 6980

SD 246 557 397 357 367

% RSD 2.8 8.0 6.5 7.2 5.3

Conc. (µg/mL)

1.002 0.284 0.284 0.267 0.274

Conc. (%

w/w) 0.067 0.022 0.026 0.021 0.023

Injection

ID

Area

Imp-E Imp-F Imp-H Imp-I Imp-J

1 3878 5197 4250 5587 6342

2 3938 4212 3914 5046 6327

3 4069 4828 4281 4988 6022

4 3928 4888 3634 4909 5263

5 3658 4671 4361 5440 5418

6 3931 4657 4270 4729 5564

Mean 3900 4742 4118 5117 5823

SD 135 325 284 329 471

% RSD 3.5 6.9 6.9 6.4 8.1

Conc.

(µg/mL) 0.280 0.260 0.262 0.260 0.801

Conc. (%

w/w) 0.023 0.023 0.020 0.023 0.031

243

5.2.7.5 Linearity

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. The linearity of the assay method was

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

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

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

concentration calculated (Table: 5.10). Calibration curve drawn by

plotting average area on the Y-axis and concentration on the X-axis

(Fig: 5.10).

244

Table: 5.10 Linearity results of the Assay method

%

Concentration Average area

80 16696833

90 19072546

100 21053265

110 23241940

120 25335028

Slope 21079

Intercept 58267

Residual Sum of

Squares 33995

Correlation Coefficient 0.9999

Linearity Plot (Concentration Vs Response)

Fig: 5.10 Linearity Plot for Assay method

16696833

18816833

20936833

23056833

25176833

790.000 890.000 990.000 1090.000 1190.000

Are

a

Conc.(µg/mL)

245

Linearity of Related substance method

Linearity experiment were carried out by preparing the

Ceftazidime sample solutions containing Imp- A, B, C, D, E, F, H, I

and J from LOQ to 150% (i.e. LOQ 25%, 50%, 70, 100, 120 and 150%)

with respect to their specifications limit (0.5%). Calibration curve was

drawn by ploting average value of the impurities. (Imp-A, B, C, D, E,

F, H, I and J ) on the y-axis and concentrations on the X-axis (Fig:

5.11 to Fig: 5.20).

246

Linearity results of the related substance method.

Table: 5.11 Linearity results of Imp-A

Imp-A

Concentration (µg/mL)

Area Statistical Analysis

0.254 3889 Slope 18211

0.324 5325 Intercept -601

0.648 10959 Residual Sum of

Squares 304

0.907 16458 Correlation

Coefficient 0.99971

1.296 22806

1.555 27885 Response factor 1.28

1.943 34621

Linearity Plot (Concentration Vs Area)

Fig: 5.11 Linearity Plot for Imp-A

247

Table: 5.12 Linearity results of Imp-B

Imp-B

Concentration

(µg/mL) Area Statistical Analysis

0.250 5554 Slope 24996

1.567 38326 Intercept -949


Squares 1143


Coefficient 0.9999

6.269 154135


9.404 234953


Fig: 5.12 Linearity Plot for Imp-B

5554

40554

75554

110554

145554

180554

215554

0.250 1.750 3.250 4.750 6.250 7.750 9.250

Are

a

Con.(µg/mL)

248

Table: 5.13 Linearity results of Imp-C

Imp-C

Concentration


0.250 3892 Slope 18181

0.324 5323 Intercept -546


Squares 339


Coefficient 0.9996

1.296 22807


1.943 34611


Fig: 5.13 Linearity Plot for Imp-C

3892

10892

17892

24892

31892

0.250 0.550 0.850 1.150 1.450 1.750

Are

a

Con.(µg/mL)

249

Table: 5.14 Linearity results of Imp-D

Imp-D

Concentration


0.262 4506 Slope 18309

1.577 32563 Intercept 1480


Squares 2003


Coefficient 0.9995

6.309 117807


9.464 175773


Fig: 5.14 Linearity Plot for Imp-D

4506

44506

84506

124506

164506

0.262 2.062 3.862 5.662 7.462 9.262

Are

a

Con.(µg/mL)

250

Table: 5.15 Linearity results of Ceftazidime

Ceftazidime

Concentration (µg/mL)

Area Statistical Analysis

0.265 6000 Slope 23346

1.547 34714 Intercept -798

3.094 71842

4.332 99914 Residual Sum of Squares

659 6.188 142860


Coefficient 0.9999

9.283 216083


Fig: 5.15 Linearity Plot for Ceftazidime

6000

56000

106000

156000

206000

0.265 2.065 3.865 5.665 7.465 9.265

Are

a

Con.(µg/mL)

251

Table: 5.16 Linearity results of Imp-E

Imp-E

Concentration


0.270 3459 Slope 13111

1.585 20724 Intercept 53


Squares 186


Coefficient 0.9999

6.341 83010


9.511 124682


Fig: 5.16 Linearity Plot for Imp-E

3459

23459

43459

63459

83459

103459

123459

0.270 2.070 3.870 5.670 7.470 9.270

Are

a

Con.(µg/mL)

252

Table: 5.17 Linearity results of Imp-F

Imp-F

Concentration


0.258 4252 Slope 16354

0.326 5542 Intercept 156


Squares 313

0.914 14595 Correlation Coefficient

0.9996 1.306 21781


1.959 32153


Fig: 5.17 Linearity Plot for Imp-F

4252

9252

14252

19252

24252

29252

0.258 0.658 1.058 1.458 1.858

Are

a

Con.(µg/mL)

253

Table: 5.18 Linearity results of Imp-H

Imp-H

Concentration


0.250 4121 Slope 15916

0.324 5083 Intercept -154


Squares 329

0.908 14131 Correlation Coefficient

0.9995 1.297 19955


1.945 31073


Fig: 5.18 Linearity Plot for Imp-H

4121

9121

14121

19121

24121

29121

0.250 0.650 1.050 1.450 1.850

Are

a

Con.(µg/mL)

254

Table: 5.19 Linearity results of Imp-I

Imp-I

Concentration


0.256 4484 Slope 17131

1.551 28177 Intercept 722


Squares 832


Coefficient 0.9999

6.203 107561


9.305 160565


Fig: 5.19 Linearity Plot for Imp-I

4484

34484

64484

94484

124484

154484

0.256 2.056 3.856 5.656 7.456 9.256

Are

a

Con.(µg/mL)

255

Table: 5.20 Linearity results of Imp-J

Imp-J

Concentration


0.837 8019 Slope 14602

1.572 29501 Intercept 18


Squares 6602


Coefficient 0.9916

6.286 84719


9.429 145429

Linearity Plot (Concentration Vs Response)

Fig: 5.20 Linearity Plot for Imp-J

8019

33019

58019

83019

108019

133019

0.837 2.837 4.837 6.837 8.837

Are

a

Con.(µg/mL)

256

5.2.7.6 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 developed by injecting three

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

concentration (i.e.1250 µg/ml). Each solution was injected twice (n=2)

into HPLC and the mean peak area of Ceftazidime peak was

calculated.

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

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

5.21). The method was showed consistent and high absolute

recoveries at all three concentration (80%, 100% and 120% ] levels

with mean absolute recovery ranging from 99.9 % to 100.1%. The

obtained obsolute recoveries were normally distributed around the

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

with low % bias (< 1.0).

257

Table: 5.21 Accuracy results of the Assay method

S.NO %

Concentration

Mean recovery

(%)

(n=3)

%RSD

1 80 100.0 0.5

2 100 100.1 0.2

3 120 99.9 0.2

5.2.7.7 Accuracy/Recovery of the Related substance method

Accuracy of the related substance method established 50%,

100%, 150% the impurities specification limit (0.5%).

Accuracy at 50% Impurity specification limit:

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

E, F, H, I and J) at 0.25% (Imp-A, B, D, E, I, J) and 0.05% (C, F, H)

level w.r.s analyte concentration (i.e 1.25 mg/ml). Each solution was

injected thrice into HPLC (Table: 5.22).

258

Table: 5.22 Accuracy at 50% level

S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 104.4 0.22 0.2

2 Imp-B 104.1 0.23 0.2

3 Imp-C 103.4 1.21 1.2

4 Imp-D 103.1 0.23 0.2

5 Imp-E 103.3 0.46 0.4

6 Imp-F 103.8 1.95 1.9

7 Imp-H 102.0 1.95 1.9

8 Imp-I 103.6 0.06 0.1

9 Imp-J 104.2 0.61 0.6


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

E, F, H, I and J) at 0.5% [Imp-A, B, D, E, I, J ] and 0.1% [C, F, H] level

w.r.s analyte concentration (i.e 1.25 mg/ml). Each solution was



S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 105.2 0.50 0.5

2 Imp-B 105.1 0.50 0.5

3 Imp-C 100.7 2.94 2.9

4 Imp-D 104.2 0.53 0.5

5 Imp-E 104.3 0.23 0.2

6 Imp-F 101.6 1.10 1.1

7 Imp-H 103.9 1.95 1.9

8 Imp-I 103.9 0.59 0.6

9 Imp-J 101.0 0.87 0.9

259


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

E, F, H and I) at 0.75% [Imp-A, B, D, E, I, J] and 0.15% [C, F, H] level

w.r.s analyte concentration (i.e 1.25 mg/m l). Each solution was


The related substance method showed consistent and high

absolute recoveries of all six impurities at all three different

concentrations (50, 100, 150%) levels in drug substance.


S.NO Impurity

name Mean

recovery(%) SD %RSD

1 Imp-A 103.6 0.71 0.7

2 Imp-B 103.9 0.72 0.7

3 Imp-C 102.5 0.40 0.4

4 Imp-D 102.2 0.26 0.3

5 Imp-E 103.1 0.83 0.8

6 Imp-F 101.5 1.35 1.3

7 Imp-H 100.6 0.65 0.6

8 Imp-I 101.7 0.44 0.4

9 Imp-J 100.0 2.04 2.0

260

5.2.7.8 Solution state stability

The solution state stability of Ceftazidime 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 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

Ceftazidime during solution stability experiments was with in 1.0%.

The solution state stability of Ceftazidime 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, H, I and J were checked for every six hours internal up

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

of all ten impurities drug solution stability experiments up to the

study period. Hence Ceftazidime sample solutions are stable for

atleast 48hours in the developed method.

261

5.2.7.8 (a) Assay method

Standard and test solution injected at each 0h, 1h, 2h, 3h, 4h,

5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h. (Table: 5.25).

Table: 5.25 Solution stability results of the Assay method

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

1 initial 99.1

2 1 98.7

3 2 99.0

4 3 99.3

5 4 99.1

6 5 98.9

7 6 99.2

8 7 99.1

9 8 98.8

10 9 99.1

11 10 98.9

12 11 98.8

13 12 99.0

% RSD 0.17

The solution state stability of Ceftazidime 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, H, I and J were checked for every six hours internal up

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

of all ten impurities drug solution stability experiments up to the

study period. Hence Ceftazidime sample solutions are stable for

atleast 48 hours in the developed method.

262

5.2.7.9 Robustness

To determine the robustness of the developed method

experimental conditions were purposely altered and the resolution

between Imp-E and Imp-F was evaluated. In each of the deliberately

altered chromatographic condition (flow rate 0.9 ml/min and 1.1

ml/min, acetonitrile in the mobile phase, column oven temperature

25°C and 35°C) the resolution between Imp-C and Imp-D was greater

than 2.0., illustrating the robustness of the method.

5.3 Mass balance

The mass balance is a process of adding both 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 Ceftazidime

bulk drug were assayed against the qualified reference standard and

the results of mass balance obtained were very close to 99.8%. The

results of mass balance obtained in each condition is presented below

(Table: 5.26).

263

Table: 5.26 Mass balance of the assay method

Degradation

Mechanism

Degradation

Condition

% Assay of

active substance

Mass balance

(% Assay+ %

impurities+ %

degradants)

Remarks

Acid

1M

HCl/85°C/

30 min

90.1 99.8

Some unknown

degradants

observed

Base 1M NaOH/

Initial 80.3 99.7

Degraded to Imp-B, Imp-D

and Imp-F

Peroxide 3% H2O2/

Initial 89.6 99.9

Degraded to

Imp-C

Thermal 105°C/168

Hours 99.1 99.8

No degradation

observed

Photolytic 10K Lux/168

Hours 98.9 99.8

Degraded to Imp-E

5.4 Analysis of Ceftazidime drug substance stability samples

USP states that stability analysis for Ceftazidime should be

performed by validated stability-indicating test method [12]. One

manufacturing lot of Ceftazidime was placed on stability study in

chambers maintained at ICH defined conditions. 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: 5.27 and 5.28. The developed HPLC method was performed

satisfactorily for the quantitative evaluation of stability samples.

264

Table: 5.27 Accelerated stability data (Storage conditions:

40°C/75%RH)

Batch No: VRR(428)72 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

Tests Description

Water

content (%w/w,

by KF)

Identification

Assay (By HPLC,

%w/w, on

anhydrous basis)

Specifications

A white to

almost

white crystalline

powder

Between

13.0 and 15.0

IR spectrum

should

concordant with that of

standard

NLT 95.0 and

NMT 102.0

Initial

A white

crystalline powder

14.20 Complies 99.5

1M

A white

crystalline

powder

13.97 Complies 99.8

2M A white

crystalline

powder

14.24 Complies 99.7

3M

A white

crystalline powder

14.09 Complies 99.7

6M

A white

crystalline

powder

14.15 Complies 99.8

Related substances details on next page.

265

Related Substances

LOQ (%w/w)

LOD (%w/w)

Related Substances (By HPLC, %w/w)

Initial 1M 2M 3M 6M

Imp-A 0.024 0.011 Below LOQ

Below LOQ

ND ND ND

Imp-B 0.022 0.012 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-C 0.026 0.014 Below

LOQ 0.03 0.03 0.05 0.03

Imp-D 0.021 0.011 ND ND ND ND ND

Imp-E 0.023 0.012 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-F 0.023 0.012 ND ND ND ND ND

Imp-H 0.020 0.009 ND ND ND ND ND

Imp-I 0.023 0.012 ND ND ND ND ND

Imp-J 0.031 0.017 ND ND ND ND ND

Highest

unknown - - ND ND ND ND ND

Total unknown

- - NA NA NA NA NA

Total RS - - NA 0.03 0.03 0.05 0.03

ND: Not detected

NA: Not applicable

266

Table: 5.28 Long-term stability data (Storage conditions:

25°C/60%RH)

Batch No: VRR(428)72 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

Water content

(%w/w, by KF)

Identification Assay

(By HPLC, %w/w, on anhydrous basis)

Specifications

A white to almost white

crystalline powder

Between 13.0 and 15.0

IR spectrum should

concordant with that of

standard

NLT 95.0 and NMT 102.0

Initial A white

crystalline powder

14.20 Complies 99.5

1M A white

crystalline powder

13.97 Complies 99.8

2M A white

crystalline powder

14.09 Complies 99.7

3M A white

crystalline powder

14.12 Complies 99.7

6M A white

crystalline powder

14.16 Complies 99.8

9M A white

crystalline powder

14.20 Complies 99.7

12M A white

crystalline powder

14.19 Complies 99.8

Related substances details on next page.

267

Related Substances

LOQ

(%w/w) LOD

(%w/w)

Related Substances (By HPLC, %w/w)

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

Imp-A 0.024 0.011 Below LOQ

Below LOQ

ND ND ND ND ND

Imp-B 0.022 0.012 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

ND ND

Imp-C 0.026 0.014 Below LOQ

0.04 0.03 0.05 0.03 0.08 0.09

Imp-D 0.021 0.011 ND ND ND ND ND ND ND

Imp-E 0.023 0.012 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-F 0.023 0.012 ND ND ND ND ND ND ND

Imp-H 0.020 0.009 ND ND ND ND ND ND ND

Imp-I 0.023 0.012 ND ND ND ND ND ND ND

Imp-J 0.031 0.017 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 - - NA 0.03 0.03 0.05 0.03 0.08 0.09

ND: Not detected NA: Not applicable

268

5.5 Summary and conclusions

Validated stability-indicating HPLC method was developed for

Ceftazidime after subjecting the samples to stress testing under ICH

recommendes conditions. The RPLC method developed for quantitative

and related subsatance determination of Ceftazidime is rapid specific,

accurate, linear, and precise. The method was completely validated

showing satisfactory data for all the method validation parameters

tested. The developed method was found ‘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 Ceftazidime bulk drug samples.

269

Table: 5.29 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-H

Imp-I Imp-J

Precision

(RSD) 0.3 0.4 0.6 1.7 0.7 0.7 0.9 0.7 0.8 0.3

LOD

(µg/ml) 0.132 0.142 0.142 0.134 0.140 0.130 0.131 0.130 0.404 N/A

LOQ

(µg/ml) 0.282 0.284 0.284 0.267 0.280 0.260 0.262 0.260 0.801

N/A

Linearity

(corre coefficient 0.9999 0.9999 0.9996 0.9995 0.9999 0.9996 0.9995 0.9999 0.9916 0.9999

Accuracy (%) 101.5-102.4 103.9-105.1 100.7-103.4 102.0-104.2 103.1-104.3 101.5-103.8 100.6-103.9 101.7-103.9 100.0-104.2 99.9-100.1

Robustness

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-E&

Imp-EF>2

Resolution

b/w Imp-

E& Imp-

EF>2

Resolution

b/w Imp-

E& Imp-

EF>2

Resolution

b/w Imp-

E& Imp-

EF>2

Resolution

b/w

Montelukast

Imp-D>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

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

Stable up to

15hr

Stable up to

15hr

270

5.6 References:

1. Sean, C. S.; Martindale-The complete drug reference,

35th edition, 2007, Vol. 2, p. 209.

2. Hiremath, B.; Mruthyunjayaswamy, B. H. M.; Acta Pharm.,

2008, 58, 275.

3. Moreno, A. H.; Salgado, H. R.; JAOAC. Int., 2008, 91, 739.

4. Schmidt, C. A.; Carazzo, M.; Laporta, LV.; Bittencourt, C. F.;

Santos, M. R.; Friedrich, M.; JAOAC Int., 2008, 91, 59.

5. Jiang, E.; HU, C.; Se. Pu., 2008, 26, 75.

6. Doadrio, T. H.; Rumelin, A. K.; Orenga, U. F.; Arzneim.

Forsch., 2004, 54, 320.

7. Porra, R.; Farina, A.; Cotichini, V.; Lecce, R.; J. Pharm and

Biomedical Analysis., 1998, 18, 241.

8. Hanes, S. D.; Herring, V. L.; Wood, G.C.; J. Chromatogr B:

Biomedical Sci Appl., 1998, 719, 245.

9. Myers, C. M.; Blumer, J. L.; Antimicrob Agents Chemother.,

1983, 24, 343.

10. Impurities in New Drug Substances Q3A (R2), ICH Harmonised

Tripartite Guidelines, 25 October 2006.

11. Good Manufacturing Practice Guide to Active Pharmaceutical

Ingredients Q7A, ICH Harmonised Tripartitie Guidelines,

November 2005.

12. Validation of Analytical Procedures: Text and Methodology Q2

(R1), ICH Harmonised Tripatite Guidelines , November 2005.

Documents

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