chapter-7 development and validation of a rp-hplc...

Chapter-7 Page 212

CHAPTER-7

Development and Validation of a RP-HPLC

method for related compund-C in Ziprasidone

hydrochloride monohydrate

Chapter-7

This chapter describes about the

method for related compound

of literature, materials and methods, results and

conclusion were covered.

benzisothiazol-3-yl)-1-piperazinyl]ethyl]

the treatment of schizophrenia

bipolar disorder. Its intramusc

schizophrenic patients for whom treatment with just

monohydrate is appropriate

Ziprasidone hydrochloride

UPLC methods are observed in publicat

method is developed for Ziprasidone

compound-C.

Figure- 7.1: Structure of Zipr

Chemical name

CAS Registry Number

7.1. INTRODUCTION

This chapter describes about the Development and validation of a RP

ated compound-C in Ziprasidone hydrochloride monohydrate

of literature, materials and methods, results and discussion , summary and

covered. Ziprasidone hydrochloride monohydrate

piperazinyl]ethyl]-6-chloro-1,3-dihydro-2H-indol

schizophrenia, and acute mania and mixed states

. Its intramuscular injection form is approved for acute agitation in

schizophrenic patients for whom treatment with just Ziprasidone

is appropriate. Analytical HPLC method has been developed for

ydrochloride monohydrate and impurities. Many HPLC, LC

LC methods are observed in publications during method development.

od is developed for Ziprasidone hydrochloride monohydrate(1

Structure of Ziprasidone hydrochloride monohydrate

: 5-[2-[4-(1,2-benzisothiazol-3-yl)-1-

piperazinyl]ethyl]-6-chloro-1,3-dihydro

one

: 146939-27-7

13

alidation of a RP-HPLC

monohydrate, survey

discussion , summary and

monohydrate 5-[2-[4-(1,2-

indol-2-one is

associated with

ular injection form is approved for acute agitation in

iprasidone hydrochloride

been developed for

rities. Many HPLC, LC-MS and

ions during method development. An HPLC

(1-11) and related

monohydrate.

dihydro-2H-indol-2-

Chapter-7 Page 214

Molecular formula : C21H21 CLN4OS

Molecular weight : 412.936

Therapeutic category : Schizophrenia and acute mania

Table- 7.1: Ziprasidone hydrochloride monohydrate impurities details:

S. No Impurity structure Chemical name Molecular

weight

Related compound-C

1

Methyl (-)-(R)-(o-

chlorophenyl)-

6,7-dihyrothieno

[3,2-c]

pyridine-5 (4H)-

acetate,

hydrogen sulfate

871.85

C42H40Cl2N8O5S2

Related compound-D

2

(3-

(benzo[d]isothiazol-

3-yl)-5-(2-(4-

(benzo[d]

isothiazol-3-

yl)piperazil-1-yl)

ethyl)-6-

chloroindolin-2-one)

545.11

C28H24 ClN5OS2

Chapter-7 Page 215

ZPH Acetone

3. HN

O

Cl

N

N

SN

5-(2-(4-

(benzo[d]isothiazol-

3-yl)piperazin-1-

yl)ethyl)-6-chloro-3-

(prop-1-en-2-

yl)indolin-2-one

453

C24H25ClN4OS

7.2. REVIEW OF LITERATURE

A simple and reliable head space gas chromatographic method has been

developed for the determination of residual methyl chloride, ethyl chloride and

isopropyl chloride in Ziprasidone hydrochloride monohydrate. The proposed

method is based on flame ionization detection technique with DB-624 as stationary

phase. Linearity of detector response was established up to 13.5μg/g and the

detection limit was 0.8μg/g for methyl chloride, ethyl chloride and 0.9μg/g for

isopropyl chloride respectively. No interference of organic solvents used in the

synthesis was observed. Performance of the method was assessed by evaluating the

recovery, repeatability, reproducibility, linearity and limits of detection and

quantification. The proposed method has a potential for application to drug

substances which may contains traces of alkyl chloride. Results prove that the

validated method was suitable for determining the residual methyl chloride, ethyl

chloride and isopropyl chloride in Ziprasidone hydrochloride monohydrate drug

substance. To widen the scope of this method, interference of 17 commonly

employed solvents in the synthesis has been studied for any possible interference

with methyl chloride, ethyl chloride and isopropyl chloride. The potentiality of

method has been studied for various drug substances containing possible alkyl

chlorides residue present in their drug matrix.

Chapter-7 Page 216

Ultra Performance Liquid Chromatography (UPLC) was employed to develop

a rapid and robust method for the analysis of Ziprasidone hydrochloride

monohydrate, both as a drug substance and in the final dosage forms. The

application of this method in stability analyses was verified. Tests were carried out

according to ICH/FDA guidelines, European Pharmacopeia, and United States

Pharmacopeia rules, which take into account factors such as specificity, linearity,

accuracy, and precision. Separation was performed on an acquity UPLC BEH phenyl

1.7μm column with a simple mobile phase, consisting of acetonitrile and water

adjusted to pH 2.0 with ortho-phosphoric acid. Using this mobile phase and gradient

elution, the separation was completed with in 5 min. This method is very sensitive,

and allows performing simultaneous identification, assay, and determination of

impurities and related substances in one injection.

A reverse phase HPLC method is described for the determination of

Ziprasidone hydrochloride monohydrate in bulk and pharmaceutical dosage forms.

Chromatography was carried out on an ODS C18 column using a mixture of

methanol and phosphate buffer (55:45 v/v) as the mobile phase at a flow rate of

1mL/min. Detection was carried out at 314nm. The retention time of the drug was

4.522 min. The method produced linear responses in the concentration range of 0.5-

30 μg /mL of Ziprasidone hydrochloride monohydrate. The method was found to be

applicable for determination of the drug in capsules.

Ziprasidone is known as a novel "a typical" or "second-generation"

antipsychotic drug. A sensitive and reproducible method was developed and

validated for determination of Ziprasidone hydrochloride monohydrate and its

major impurities, which are significantly different in polarity. The separation is

performed on a Waters spherisorb octadecylsilyl column (5.0 μm , 250 x 4.6 mm

I.D.) using a gradient with mobile phase A [buffer:acetonitrile] [80:20, v/v] and

mobile phase B [buffer:acetonitrile] [10:90, v/v] at a working temperature of 25°C.

The buffer was 0.05 M KH2PO4 solution with an addition of 10 mL triethylamine

/Lsolution, adjusted to pH 2.5 with ortho phosphoric acid. The flow rate was 1.5

mL/min, and the elute was monitored at 250 nm using a diode array detector.

Chapter-7 Page 217

Optimization of the experimental conditions was performed using partial least

squares regression, for which four factors were selected for optimization: buffer

concentration, buffer pH, triethylamine concentration, and temperature. The

proposed validated method is convenient and reliable for the assay and purity

control in both raw materials and dosage forms.

7.3. OBJECTIVE

The main objective of this research work is to develop method for separation

of related compound-C and Ziprasidone Acetone impurity in Ziprasidone

hydrochloride monohydrate . After review of so many literature is reveals that the

reported methods are not reported for the separation of these two impurities in any

where.

7.4. MATERIALS AND METHODS

7.4.1. Reagents & Chemicals.

a. Water : Merck

b. Acetonitrile HPLC grade : Merck

c. Mono basic potassium phosphate : Merck

d. Potassium hydroxide : Merck

e. Methanol : Merck

f. Hydrochloric acid : Merck

7.4.2. Drug substances:

Ziprasidone hydrochloride monohydrate and related compound-C and related

compound-D samples were received from M/S Aurobindo Laboratories,

Hyderabad(A.P),India.

Chapter-7 Page 218

7.4.3. Instrument details:

The High performance Liquid Chromatography using Waters HPLC instrument

having quaternary pumps including auto injector. This HPLC connected with PDA

detector, make Waters. All the components are controlled with Empower2 software.

7.4.4. Method development:

Development trials were performed with all neutral buffer salts and different make

HPLC columns but finally the chromatographic conditions were optimized with the

potassium phosphate salt, acetonitrile and methanol with simple gradient program.

7.4.4.1. Wave length Selection:

The UV spectrums were generated for Ziprasidone hydrochloride monohydrate and

related compound-C using with photo diode array detector (PDA). Ziprasidone

hydrochloride monohydrate and its impurities were found to have varying

absorption maxima over a range of wave length. But it was found that at about

229nm, Ziprasidone hydrochloride monohydrate and its impurities were found to

have optimum UV absorption. Therefore, 229nm was selected for the study and

quantification of Ziprasidone hydrochloride monohydrate and it’s related

impurities.

Figure- 7.2: UV Spectra of (a) Ziprasidone hydrochloride monohydrate, (b)

related compound-C (c) related compound-D.

Chapter-7 Page 219

7.4.4.2. Selection of mobile phase and stationary phase:

Ziprasidone hydrochloride monohydrate and related compound-C were

found that different functional groups, shows different affinities with mobile phases

and stationary phase. A different column with different selectivity provides good

separation for method development. Two parameters were chosen to get required

resolutions and separations and symmetrical peaks for Ziprasidone hydrochloride

monohydrate and impurities. i.e., Selection of the mobile phase and column.

7.4.4.3. Selection of Mobile phase:

Ziprasidone hydrochloride monohydrate acetone, related compound-C were

co-eluted using with different mobile phases. Ziprasidone hydrochloride

monohydrate and the impurities of Ziprasidone hydrochloride monohydrate were

having wide range of polarities and the separation of these impurities mainly

depends on the column stationary phase. An gradient method was mobile phase of

buffer is 0.02M ammonium dihydrogen phosphate in water pH adjusted to 9.85 and

acetonitrile and methanol was suitable for the separation of Ziprasidone

hydrochloride monohydrate and its related substances. Mobile phase was degassed

and filtered through 0.22 µm millipore filter paper.

7.4.4.4. Selection of stationary phase:

Separation was achieved with Zorbax RX C8 150 x 4.6 mm I.D.,5.0µm column.

Different stationary phases were studies for the separation of Ziprasidone

hydrochloride monohydrate such as C18 and C8 using the mobile phase specified.

The experimentation was started using Waters Symmetry C18, 250 x 4.6mm,I.D.,

5.0µm, column.

Trail-1:

The complete experiment details are as follows.

Column : Waters symmetry C18, 250 x 4.6mm I.D.,5.0µm

Mobile Phase-A

:

0.25% phosphoric acid mixed 2.50 g of ortho

phosphoric acid in 1000 ml of water. Mixed

well, filtered and degassed

Chapter-7 Page 220

Mobile phase-B

Sample Preparation :

:

Mixed acetonitrile and water in the ratio of 95 and 5

and sonicate to degassed

0.5mg/mL

Flow rate : 0.9 mL/ min

Oven temperature

Injection volume

:

:

30oC

20µL

Elution

Gradient program

:

Gradient

Time in min Mobilephase- A

(%)

Mobilephase-

B (%)

0 85 15

20 70 30

25 65 35

30 60 40

45 60 40

46 85 15

50 85 15

55 85 15

Figure-7.3: Blend Chromatogram by using waters Symmetry C18, 250 x 4.6mm

I.D., 5.0µm column and trail-1 method conditions.

Chapter-7 Page 221

Observation: Related compound-C is not stable in trail-1method conditions, hence

waters symmetry C18, 250 x 4.6mm,I.D., 5.0µm, column and trail-1 method

conditions not suitable for the separation of related compound-C.

Trail-2:


Column

Buffer

:

:

Zorbax RX C8 150 x 4.6 mm I.D., 5.0µm column

Dissolved 6.8 g/L of monobasic potassium phosphate in

water and adjust with 5N potassium hydroxide to a pH

of 6.0

Mobile Phase : Acetonitrile: methanol : buffer ( 11:1:8)

Wavelength : 229 nm

Flow rate

Diluent

Elution

:

:

:

1.0 mL/min

Methanol, water and hydrochloric acid (20:5:0.01)

Isocratic

Sample preparation

Injection volume

Run time

:

:

:

0.5mg/mL

20µL

60min

Figure-7.4: Blend Chromatogram by using Zorbax RX C8 150 X 4.6 mmI.D.,

5.0µm column and trail-2 method conditions.

Chapter-7 Page 222

Observation: Related compound-C and ZPH Acetone are coeluting. Hence, the

isocratic elution and trail-2 method conditions is not suitable for the separation of

related compound-C.

Trail-3:


Column

Buffer(Mobile phase-A)

:

:

Zorbax RX C8 150 x 4.6 mm I.D., 5.0μm column

Dissolve 6.8 g/L of monobasic potassium phosphate in

water and adjust with 5N potassium hydroxide to a pH of

6.0

Mobile phase-B : Buffer and acetonitrile in the ratio of 75:25 v/v

Sample preparation : 5 mg in 25 mL of diluent

Wavelength : 229 nm

Flow rate : 1.0 mL/ min

Oven temperature : 40oC

Diluent

Elution

Injection volume

Runtime

Gradient program

:

:

:

:

100% acetonotrile

Gradient

20μL

60 min

Time in min Mobile phase-

A(%)

Mobile phase B

(%)

0 65 35

5 50 50

10 50 50

23 20 80

35 20 80

45 50 50

60 65 35

Chapter-7 Page 223

Figure-7.5: Blend Chromatogram by using Zorbax RX C8 150 X 4.6 mm I.D.,

5.0µm column and trail-3 method conditions.

Conclusion: Method needs to modify for getting to reduce blank peaks and baseline

noise. Based on the above study on stationary phase, it was concluded related

compound-C in Ziprasidone hydrochloride monohydrate were well separated from

each other in Zorbax RX C8 150 X 4.6 mm I.D., 5.0µm column.

7.4.5. Optimized method:

Based on the above study, the below mentioned HPLC parameters was chosen for

the separation and quantification of related compound-C and Ziprasidone

hydrochloride monohydrate.

Column : Zorbax RX C8 150 x 4.6 mm I.D., 5.0μm column

Buffer preparation

: dDissolved 6.8 g/L of mono basic potassium phosphate in

water adjust with 5N of potassium hydroxide to a pH 6.0

Mobile phase-A

Mobile phase-B

Sample preparation

Auto sampler

:

:

:

A degassed mixture of buffer: methanol: acetonitrile

in the ratio of 40:15:45 (v/v/v)

Acetonitrile (100%)

45 mg in 100 mL of diluent

5oC

Wavelength : 229 nm

Chapter-7 Page 224

Flow rate : 1.0 mL/Min

Oven temperature

Injection volume

Run time

:

:

:

35oC

20μL

65min

Diluent : Methanol: water : hydrochloric acid (80:20:0.04)

Elution

Gradient programme

:

:

Gradient

Time in min Mobile phase-A

(%)

Mobile phase-B

(%)

0 100 0

20 100 0

35 60 40

50 60 40

55 100 0

65 100 0

a) Reference stock solution: Weighed about each 18 mg of Ziprasidone

hydrochloride monohydrate working standard and related compound-D into

100 ml volumetric flask, dissolved and diluted to volume with diluent and

mixed well (0.18mg/mL).

b) Reference Solution (0.2%): Diluted 0.5 mL of reference stock solution to 100

mL with diluent (0.0009 mg/mL).

c) Preparation of Sample solution: Accurately weighed 45 mg of sample into

100 mL volumetric flask, dissolved and diluted to volume with diluent.

Procedure:

Injected all above solutions once and reference solution six times and calculated the

system suitability parameters i.e. the theoretical plates, Tailing factor, %RSD for

reference solution.

System suitability criteria:

% RSD for six replicate injections of reference solution should be not more than

10.0.

Chapter-7 Page 225

The tailing factor for Ziprasidone hydrochloride monohydrate peak reference

solution should be not more than 1.5.

The number of theoretical plates for Ziprasidone hydrochloride monohydrate peak

reference solution should be not less than 3000.

Table -7.2: Specification:

S. No Name of the impurity Specification

01 Related compound-C Not more than 0.20%

Calculation: calculate the impurity using below formula:

Related compound- C area in test solution 1

Related compound C = --------------------------------------------------------- X ------ X 0.2

Avg. area of Ziprasidone peak in reference solution RRF

Total impurities: % known impurities + % other unknown impurities calculated by

area normalization.

RRF for related compound-C: 0.42

RRT for related compound-C: 2.70

Figure- 7.6: A typical HPLC Chromatogram of diluent.

Chapter-7 Page 226

Figure- 7.7: A typical HPLC Chromatogram of reference solution (0.2%).

Figure- 7.8: A typical HPLC Chromatogram of Ziprasidone hydrochloride

monohydrate as such sample.

Figure- 7.9: A typical HPLC Chromatogram of Ziprasidone hydrochloride

monohydrate and all impurities blend solution.

Chapter-7 Page 227

7.5. RESULTS AND DISCUSSION

7.5.1. Method validation:

Analytical method validation was performed as per ICH and USFDA guidelines with

specificity, precision, accuracy, linearity, limit of detection, limit of quantification,

ruggedness and robustness.

7.5.1.1. Related substances by HPLC:

7.5.1.2. System suitability:

a) Reference stock solution: Weighed about each 18 mg of Ziprasidone

hydrochloride monohydrate working standard and related compound-D into

100 ml volumetric flask, dissolved and diluted to volume with diluent and

mixed well (0.18mg/mL).

b) Reference solution (0.2%): Diluted 0.5mL of reference stock solution to 100

mL with diluent (0.0009 mg/mL).

c) Preparation of sample solution: Transferred 45mg of sample into 100mL

volumetric flask, dissolved and diluted to volume with diluent.

Injected all above solutions once and calculated the system suitability

parameters i.e. the resolution between adjacent peaks, Tailing factor and

tangent for Ziprasidone hydrochloride monohydrate.

Conclusion: Under optimized Chromatographic conditions, related compound-D

and Ziprasidone hydrochloride monohydrate, were separated well, retention times

being about 20.08 and 5.70 min, respectively. The system suitability results are

given in table-7.3.

Table- 7.3: System suitability results:

S. No Name Retention

time(min)

Relative retention

time(min)

% RSD Theoretical

plates(N)

Tailing

factor( T)

01 Ziprasidone

reference

solution

5.70 1.00 0.5 6050 1.10

02 Related

compound-

D

20.08 3.52 2.4 ------ -----

Chapter-7 Page 228

7.5.1.3. Specificity:

a) Thermal degradation: Accurately weighed 1 gm of Ziprasidone hydrochloride

monohydrate sample is taken and kept under thermal condition i.e., at 105°C

for 7days and sample collected after 48hours and sample analysed related

compound -C by HPLC and checked for % degradation and determine the peak

purity of main peak.

Observation: Ziprasidone hydrochloride monohydrate sample is stable

under thermal condition.

b) Photo degradation: Weighed 1 gm of sample is taken and kept in UV chamber

i.e., at 254 nm for 48 hours and sample collected after 48 hours and sample

analyzed.

Observation: A Ziprasidone hydrochloride monohydrate sample is stable

under photo condition.

c) Acid hydrolysis: Sample was dissolved in 0.1N HCl at room temperature and

collected the sample after 30minutes. The 30 minutes sample was analyzed for

related compound -C by HPLC and checked for % degradation and determine

the peak purity of main peak.

Observation: Ziprasidone hydrochloride monohydrate sample is stable under

acid hydrolysis.

d) Base hydrolysis: Sample was dissolved in 0.1N NaOH at room temperature

and collected the sample after 1hr. The 1hr sample was analyzed for related

compound -C by HPLC and checked for % degradation and determine the peak

purity of main peak. As 1 hour sample was found at RRT 5.2 impurity only

observed as 0.06% and all other individual unspecified impurities eluting after

Ziprasidone hydrochloride monohydrate are detected less than disregard limit.

Hence, sample was dissolved in 0.1N NaOH and reflux at 70oC. Collected the

sample after 12 hours and analyzed for related compound-C by HPLC.

Chapter-7 Page 229

Calculated the % degradation of related compound-C, unspecified impurity and

determine the peak purity of Ziprasidone hydrochloride monohydrate peak.

Observation: Ziprasidone hydrochloride monohydrate was degraded under

base hydrolysis.

e) Oxidation degradation: Sample was dissolved in 3% H2O2 in dark at room

temperature up to 24 hours and samples were analyzed for related

compound-C by HPLC and checked for % degradation and determine the peak


Note: Initial sample was dissolved in formic acid and found Ziprasidone

hydrochloride monohydrate main peak is completely degraded. Hence,

experiments were conducted without co-solvent for Water, base, acid and

oxidation.

Observation: Ziprasidone hydrochloride monohydrate was degraded to

under peroxide solution.

f) Water hydrolysis: Sample was dissolved in water and refluxed at 70°C

temperature for 12 hours. 12th hour, sample was analyzed for related

compound-C by HPLC and checked for % degradation and determine the peak


Observation: Ziprasidone hydrochloride monohydrate was not degraded to

under water hydrolysis.

Conclusion:

Related compound-C found to be not degraded under the all stress study conditions.

Un specified impurity eluting after Ziprasidone hydrochloride monohydrate peak:

Found to be degraded unspecified impurities eluting after Ziprasidone

hydrochloride monohydrate peak in base degradation.

The un specified impurity was found to be degraded drastically from not

detected to 1.02% at about RRT 6.07 minutes and 0.90% at about RRT 5.00 minutes

under stressed condition of base hydrolysis (0.1N NaOH) at 70oC temperature.

Chapter-7 Page 230

Ziprasidone hydrochloride monohydrate:

However, Ziprasidone hydrochloride monohydrate peak found drastically degraded

under stressed condition of 0.1N HCl and 0.1N NaOH and not degraded under the

stress condition of UV, Visible, heat, water hydrolysis and oxidation. The degradation

results are given in below table - 7.4.

Table- 7.4: Ziprasidone hydrochloride monohydrate degradation data :

Stressed condition Time (hrs) % Purity

Thermal degradation 7 x 24hrs 99.87

Photo degradation 7 x 24hrs 99.85

Acid hydrolysis 0.5hrs 97.75

Base hydrolysis 12hrs 53.13

Oxidation degradation 24hrs 90.0

Water hydrolysis 12 hrs 99.90

Figure- 7.10: A typical HPLC Chromatogram of thermal degradation sample.

Chapter-7 Page 231

Figure- 7.11: A typical HPLC Chromatogram of photo degradation sample.

Figure- 7.12: A typical HPLC Chromatogram of acid degradation sample.

Figure-7.13: A typical HPLC Chromatogram of base degradation sample.

Chapter-7 Page 232

Figure- 7.14: A typical HPLC Chromatogram of oxidation degradation sample.

Figure- 7.15: A typical HPLC Chromatogram of water hydrolysis degradation

sample.

7.5.1.4. Limit of Detection and Limit of Quantification:

a) LOQ solution-1 preparation (0.05%): Transferred 5.0µL of related compound-

C stock solutions into 10mL volumetric flask, dissolved and diluted to volume

with diluent.

b) LOQ solution-2 preparation: Transferred 5.0µL of related compound-C stock

solutions into 10mL volumetric flask, dissolved and diluted to volume with

diluent.

Chapter-7 Page 233

c) LOD solution-1 preparation: Transferred 3.3mL of above LOQ solution-2

stock solutions into 10mL volumetric flask, dissolved and diluted to volume

with diluent.

Injected all above solutions and calculated the Limit of detection and Limit of

quantification for each impurity.

Conclusion:

The LOD for related compound-C was found 0.013% respectively. The LOQ for

related compound-C was found to be 0.05 % respectively. The results are

summarized in the table - 7.5.

Table- 7.5: Limit of detection and Limit of Quantification data:

Concentration Related compound-C (%)

LOD 0.013

LOQ 0.05

7.5.1.5. Precision and accuracy at Limit of Quantification level:

a) Related compound-C stock Solution preparation: Transferred 45mg of

related compound-C into 100mL volumetric flask, containing 20mL of diluent

dissolved and diluted to volume with diluent.

Prepared six times the solution as mentioned above and inject all the above

solutions each preparation once, calculated the % RSD for six preparations for

impurity.

Accuracy:

b) Sample + related compound-C stock solution preparation: Accurately

weighed 45mg of sample into 100mL volumetric flask, dissolved in 50mL of

diluent and added 50µL of related compound-C dissolved and diluted to

volume with diluent.

c) Sample solution preparation: Weighed 45mg of sample into 100mL


Chapter-7 4

Prepared three times the solution as mentioned above and inject each

preparation once and calculated the % recovery for related compound-C at

Limit of Quantification level.

Conclusion:

The repeatability and recovery at the LOQ concentrations for related compound-C

were 3.7% and 105.7% respectively. The results are summarized in the table- 7.6.

Table- 7.6: Precision and accuracy at Limit of Quantification level data:

S. No Impurity % RSD (n=6) % Recovery (n=3)

1 Related compound-C 3.7 105.7

7.5.1.6. Linearity:

a) Linearity solution-1 (0.049%): Transferred 5µL related compound-C stock

solution into 10mL volumetric flask, containing 5mL of diluent dissolved and

diluted to volume with diluent.

b) Linearity solution-2 (0.099%): Transferred 10µL of related compound-C

stock solution into 10mL volumetric flask, containing 5mL of diluent dissolved

and diluted to volume with diluent.

c) Linearity solution-3(0.148%): Transferred 15µL of related compound-C

stock solution in to 10mL volumetric flask, containing 5mL of diluent

dissolved and diluted to volume with diluent.

d) Linearity solution-4 (0.198%): Transferred 20µL of related compound-C



e) Linearity solution-5 (0.247%): Transferred 24.7µL of related compound-C



f) Linearity solution-6(0.297%): Accurately transferred 29.7µL of related

compound-C stock solution into a 10mL volumetric flask, containing 5mL of

diluent dissolved and diluted to volume with diluent. Injected all above

Chapter-7 Page 235

solutions each preparation once and calculated the Linearity parameters i.e.

correlation coefficient, slope and intercept for impurity.

Conclusion:

Linearity established for related compound-C at 0.049%, 0.099%, 0.148%, 0.198%,

0.247% and 0.297%. The correlation coefficient (r) are more than 0.99. The above

result reveal that method is linear, results are summarized in purity wise and

presented in table-7.7.

Table- 7.7: Ziprasidone related compound-C linearity data:

S. No Level (%) Concentration (%) Area of related compound-C

1 LOQ 0.049 5236

2 50 0.099 10845

3 75 0.148 15787

4 100 0.198 21248

5 125 0.247 25794

6 150 0.297 31249

Correlation coefficient(r) 0.9997

Slope 104026.7

Y-Intercept 363.2

% 100 Y-Intercept 1.70

Chapter-7 Page 236

Figure- 7.15: Ziprasidone related compound-C linearity graph.

7.5.1.7. Accuracy:

a) Accuracy solution-1 preparation (LOQ): Accurately weighed 45mg of

sample into 100 mL volumetric flask, dissolved in 30mL of diluent and added

50µL impurity stock solution, dissolved and diluted to volume with diluent.

Three solutions prepared as mentioned above.

b) Accuracy solution-2 preparation- (50%): Weighed 45 mg of sample into

100 mL volumetric flask, dissolved in 30 mL of diluent and added 100µL of

impurity stock solution, dissolved and diluted to volume with diluent. Three

solutions prepared as mentioned above.

c) Accuracy solution-3 preparation (100%): Transferred 45 mg of sample into

10 mL volumetric flask, dissolved in 5mL of diluent and added 200µL of

impurity stock solution, dissolved and diluted to volume with diluent. Three

solutions prepared as mentioned above.

d) Accuracy solution-4 preparation (150%): Accurately weighed 45mg of

sample into 10 mL volumetric flask, dissolved in 5mL of diluent and added

300µL of impurity stock solution, dissolved and diluted to volume with diluent.

Three solutions prepared as mentioned above.

Chapter-7 Page 237

Injected each above preparation once and calculated the % recovery for

Ziprasidone related compound-C.

Conclusion:

The percentage recovery of related compound-C in Ziprasidone hydrochloride

monohydrate samples is shown in table - 7.8.

Table- 7.8: % Recovery in accuracy :

7.5.1.8. Precision:

a) Sample preparation: Weighed 45mg of sample into 100mL volumetric flask

,dissolved and diluted to volume with diluent.

b) Sample + LOQ Solution spiked preparation: Accurately weighed 45mg of

sample into a 100mL volumetric flask, dissolved in 30mL of diluent added

200µL of impurity stock solution dissolved and diluted to volume with diluent.

Prepared the solution six times as mentioned above.

Injected all above sample preparations and calculated the % RSD for impurity.

Conclusion:

The precision of the related substance method was checked by injecting six

individual preparations of Ziprasidone hydrochloride monohydrate spiked with

0.20% related compound-C. The % R.S.D of the area for of related compound-C was

calculated. The results was summarized in the table-7.9,

Concentration Related compound-C (%)

LOQ 105.7

50% 95.8

100% 95.5

150% 95.7

Chapter-7 Page 238

Table- 7.9: Precision data:

7.5.1.9. Robustness:

Flow variation:

a) Sample solution preparation: Weighed 5 mg of sample into 100mL


b) Sample + 0.20% spiked preparation: Transferred 45 mg of sample into

100mL volumetric flask, dissolved in 5 mL of diluent added 200µL of impurity

stock solution dissolved and diluted to volume with diluent.

Injected the above sample solution at flow rates 0.8mL/min and at 1.2mL/min

and observed the system suitability parameters and impurities relative

retention times and compared with 1.0mL/min results.

Temperature variation:

a) Sample solution preparation: Transferred 45mg of sample into 100mL


b) Sample + 0.20% spiked preparation: Accurately weighed 45mg of sample

into 100mL volumetric flask, dissolved in 30mL of diluent added 200µL of

impurity stock solution dissolved and diluted to volume with diluent

Injected the above sample solution at temperature 30°C and at 40°C and

observed the system suitability parameters and impurities relative retention

times and compared with 35°C results.

S. No Preparation Related compound-C area %

1 1 0.1858

2 2 0.1862

3 3 0.1958

4 4 0.1927

5 5 0.1954

6 6 0.1879

Average 3.83099

Standard deviation 0.0909

%RSD 2.37

Chapter-7 Page 239

pH variation:

a) Sample solution preparation: Weighed 45mg of sample into 100mL


b) Sample + 0.20% spiked preparation: Accurately weighed 45mg of sample

into 100mL volumetric flask, dissolved in 30 mL of diluent added 200µL of

impurity stock solution dissolved and diluted to volume with diluent.

Injected the above sample solution at pH 5.8 and at 6.2 and observed the

system suitability parameters and impurities relative retention times and

compared with 6.0 results.

Conclusion:

The results are summarized in the table-7.10.

Table -7.10: Robustness data:

Parametr 30°C 40°C 0.8

mL/min

1.2

mL/min

pH at

5.8

pH at

6.2

As

such

Tailing factor for

Ziprasidone

reference solution

1.14 0.84 1.03 1.02 1.25 1.08 1.10

% RSD for

Ziprasidone

reference solution

0.3 1.4 1.7 2.2 1.8 2.4 0.5

% RSD for

Ziprasidone

related

compound-D

1.9 3.3 1.4 1.0 2.6 2.22 2.4

7.5.1.10. Solution stability:

Sample solution preparation: Accurately weighed 45mg of sample into a 100mL


Injected the solution for 0 hrs(Initial), 12hrs, 24 hrs and 48 hrs and performed the

impurity content.

Conclusion:

Related compound-C is not increased and other impurities are also not observed

during the solution stability and mobile phase stability experiments when

performed using the related substance method. The solution stability and mobile

Chapter-7 Page 240

phase stability experiment data confirms that the sample solutions and mobile

phases used during the related substance determination were stable for at least

48 hours. The results are summarized in the table-7.11.

Table- 7.11: Solution stability data:

Duration Related compound-C (%)

Sample solution initial 0.1858

After 12 hrs 0.1784

After 24 hrs 0.1811

After 48 hrs 0.1802

Table -7.12: Mobile phase stability data:

Duration Related compound-C (%)

Sample solution initial 0.1858

After 12 hrs 0.1794

After 24 hrs 0.1848

After 48 hrs 0.1891

7.5.1.11. Batch analysis:

Using the above validated method, Ziprasidone hydrochloride monohydrate sample

was analyzed and the data is furnished in table- 7.13.

Table- 7.13: Batch analysis data

Lot Number Related compound-C

001 0.1858

7.6. SUMMARY AND CONCLUSION

The simple isocratic HPLC method for quantification of related compound-C

in Ziprasidone hydrochloride monohydrate. Related compound-C is precise,

accurate, rapid and specific. The method was fully validated showing satisfactory

data for all the method validation parameters tested. The developed method can be

used for regular samples and stability samples analysis also.

Chapter-7 Page 241

7.7. REFERENCES

1. Daniel oakowiecki, and Krzysztof cal .Development of rapid and robust

stability-indicating method for analysis of Ziprasidone hydrochloride and

freebase as drug substance and in medicines by UPLC. Pelpioska 19, 83-200

starogard gdaoski, poland. Hallera107, 80-416 Gdaosk, Poland.

2. T.Kaleemullah1, Mansur Ahmed, Hemant Kumar sharma, KVSN Raju ,

M.Narendra Kumar. Development and validation of gas chromatography

method for low level detection of residual methyl chloride, ethyl chloride and

isopropyl chloride in Ziprasidone hydrochloride scholars Research Library Der

Pharma Chemica, 2011, 3 (6):390-399 ISSN 0975-413XCODEN(USA): PCHHAX.

3. B. Sudha Rani and P. Venkata Reddy . Volume 3 (2006), Issue 3, Pages 169-

72,DOI:10.1155/2006/252570.

Volume 3 (2006), Issue 3, Pages 169-172, doi:10.1155/2006/252570.

4. Medikondu Kishorea, Y.Hanumantharao. A validated spectrophotometric

methods for the determination of Ziprasidone as hydrochloride in bulk and

pharmaceutical formulations. International Journal of Chemical and analytical

Science 2010, 1(7),151-153, ISSN: 0976-1209.

5. A. Singh, B. M . Rao, G. R. Deshpande, S. Sangaraju, M.K. Srinivasu, M. Lalitha

Devi, P. V. V. Satyanarayana and K.B.Chandrasekhar. A rapid stability-

Indicating LC method for Ziprasidone hydrochloride. Volume 65,numbers 3-4

(2007),191-196,DOI:10.1365/s10337-006-0125-2.

6. S.Uma Devi, E. Pushpa Latha, C.V.Nagendra Kumar Guptha, P. Ramalingam,

Development and validation of HPTLC method for estimation of Ziprasidone

hydrochloride in bulk and pharmaceutical dosage forms. ISSN 2229 – 6867.

7. Ganji ramanaiah, Dr.D.Ramachandran,G.srinivas, Jayapal gowardhane,

Purnachanda rao. Development and validation of a rapid uv-spectroscopic

method for the estimation of Ziprasidone hydrochloride monohydrate in drug

substance and its dosage forms. Issn- 0975-1491 vol 4, issue 2, 2012.

8. M.Gnana ruba priya, M. Karikalan, S. Asadulla, S. Rajesh, J. Siva rama krishna

and S. Vijay sridhar. Development and method validation using HPLC for assay

Chapter-7 Page 242

of Ziprasidone capsule,IJPSR (2011), vol. 2, issue 9(research), ISSN: 0975-

8232.

9. K. Srinivasa rao, K.Srinivas, S. Satyanarayana raju, new RP-HPLC method For

the estimation of Ziprasidone hydrochloride In pharmaceutical dosage forms.

Vol. 7, Issue 8, 2008.

10. J.D.Chudasama, K.P.Channabasavaraj, J. S. Modiya, T. T. Mani.Development and

validation of three new assay methods for Ziprasidone hydrochloride.

monohydrate using UV-Spectrophotometry, IJPRD, 2011; Vol 3(3): 8; May

2011 (57 - 63) International Standard Serial Number 0974 – 9446.

11. Shilpi agarwal1,Vinod K. Gupta2, Barkha Singhal3, potentiometric assay of

antipsychotic drug (Ziprasidone hydrochloride) in pharmaceuticals, serum and

urine..1, J. Electrochem. Sci., 6 (2011) 3036 – 3056.

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