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Chapter-4 30

CHAPTER – 4

Analytical method for Piperazine in an active

pharmaceutical ingredient using chemical

derivatization and High Performance Liquid

Chromatography with UV detection

Chapter-4 Page 131

4.1. INTRODUCTION

This chapter describes the method development and validation of trace

analysis of Piperazine content in pharmaceuticals by High Performance Liquid

Chromatography with UV detection. Review of literature, materials and methods,

development trials, validation results and summary and conclusion were covered.

Piperazine(diethylenediamine) is widely used in medicine as an effective

antihelminthic agent. However, the method recommended in the State

Pharmacopoeia for determining Piperazine in medicinal forms is very cumbersome

and time-consuming, since the procedure is based on precipitation processes.

Therefore, it is important to develop a simple, rapid, and sufficiently precise

technique for Piperazine determination. Diethylenediamine is commonly applied in

pharmacy, both as a native compound known under the common name Piperazine,

and as the starting substance for the synthesis of methyl and hydroxyl derivatives,

used for the production of drugs such as estropin, clozapine, or cinarazine. Thus, it

should be assayed in many cases, during industrial synthesis as an intermediate

product, and as technological impurity of the final products, as well as in the

pharmacological and environmental analyses. Diethylenediamine does not possess

chromophores. It absorbs UV light only at a wavelength of 205 nm, and its specific

absorption coefficient is very low (0.01). Therefore, the determination of

diethylenediamine at a level below 1mg/kg is possible only after its transformation

into a derivative with sufficiently high optical density, or emitting induced light.

Various primary and secondary aliphatic amines are present in considerable

amounts in biological and environmental samples. Their selective removal is

theoretically possible, but time- and labour-consuming. The chromatographic

properties of coupling products of aliphatic diamines with several carbonatoms are

affected, to a great degree, by the substituent, whose molecular weight is several

times higher and which usually possesses unsaturated bonds and an additional

heteroatom. Hydrophobic interactions of derivatives of primary and secondary

Chapter-4 Page 132

amines do not differ significantly. Additionally, in contrast to secondary amines,

derivatives of primary amines may form hydrogen bonds with relevant groups of

stationary phases or solid support. This method has been developed for

pharmaceuticals, which do not contain potential co-eluents. Further more, the

results of Chromatographic separation may also be reproducible. The objective of

the present study was the selection of HPLC adsorbents, enabling good separation of

NBD Cl (4-chloro-7-nitro benzofurazan) -diethylenediamine and potential co-

eluents. The research was carried out for determination of Piperazine (1-10) content

in active pharmaceutical ingredients and intermediates.

Table-4.1:Piperazine and Ziprasidone key starting material(KSM) details:

S. No Impurity structure Chemical name Molecular weight

Piperazine

1

NH

HN

diethylenediamine C4H10N2

Mol Wt. 86.14

Ziprasidone KSM

2

SN

N

HN

3-piperazine-1-yl-1,

2-benzisothiazole

C11H13N3S

Mol.Wt.: 219.31

4.2. REVIEW OF LITERATURE

A novel method for the determination of Piperazine in pharmaceutical drug

substances was developed using high performance liquid chromatography (HPLC)

with evaporative light scattering detection (ELSD). This method uses the

hydrophilic interaction chromatography (HILIC) mode on a cyanopropyl (CN)

Chapter-4 Page 133

bonded stationary phase. Optimization of organic modifier and acid composition in

the mobile phase resulted in robust Chromatography conditions with excellent

resolution, peak shape, and retention time for the Piperazine peak. The method was

further evaluated with respect to linearity, precision, selectivity, limit of detection

(LOD), and reproducibility. Based on the data provided, this HPLC–ELSD method

demonstrated acceptable levels of linearity, precision, LOD, and selectivity for

determination of Piperazine.

Simultaneous determination of N-benzylpiperazine and 1-(3-

trifluoromethylphenyl)piperazine in rat plasma by HPLC-fluorescence detection and

its application to monitoring of these drugs. An HPLC-fluorescence detection

method for simultaneous determination of N-benzyl piperazine (BZP) and 1-(3-

trifluoromethylphenyl)piperazine (TFMPP) labeled with 4-(4,5-diphenyl-1H

imidazol-2-yl)benzoyl chloride (DIB-Cl) was described. DIB-BZP and -TFMPP were

well separated with in 13min without interference of peaks from plasma

components. The lower detection limits of BZP and TFMPP at a signal-to-noise ratio

of 3 were 0.9 and 4.6ng/mL, respectively. Precisions of the proposed method for

intra- and inter-day assays were less than 4.8 and 9.1% as % RSD (n=5).

Furthermore, the method could be successfully applied to monitor both compounds

in plasma after their sole or co-administration to rats (each dose, 2mg/kg).

Clearance of TFMPP was significantly different under the conditions (P=0.047).

Copyright © 2011 John Wiley & Sons, Ltd.

An HPLC-fluorescence detection method for simultaneous determination of

N-benzyl piperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP)

labeled with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was

described. DIB-BZP and -TFMPP were well separated within 13min, without

interference of peaks from plasma components. The lower detection limits of BZP

and TFMPP at a signal-to-noise ratio of 3 were 0.9 and 4.6ng/mL, respectively.

Precisions of the proposed method for intra- and inter-day assays were less than 4.8

and 9.1% as % RSD (n=5). Furthermore, the method could be successfully applied to

monitor both compounds in plasma after their sole or co-administration to rats

Chapter-4 Page 134

(each dose, 2mg/kg). Clearance of TFMPP was significantly different under the

conditions (P=0.047). Copyright © 2011 John Wiley & Sons, Ltd. Owing to the

remarkable difference in the chromatographic, behaviors of both compounds on a

reversed-phase column, their, satisfactory separation from the interfering peaks

within, acceptable analysis time could not be obtained by an isocratic elution. Thus,

a gradient elution was required in all previous methods (Vorce et al., 2008; Elliott

and Smith, 2008). We also used a gradient elution with the combination of 0.1 M

acetate buffer(pH 3.5) : acetonitrile (Mobilephase-A: Mobilephase-B) and a good

separation of DIB labels without interference of peaks from reagent and plasma

components could be achieved. The retention times of DIB-BZP and -TFMPP labels

were 9.1 and 12.3 min, respectively. Total running time including washing and

equilibration steps after eluting of DIB-TFMPP was ca 25 min.

4.3. OBJECTIVE

The main objective of this research work is to develop simple accurate

analytical method for quantification of Piperazine content in pharmaceutical

products. HPLC method for the quantification of Piperazine in Ziprasidone

KSM(PBI). Review of literature is reveals that the reported methods were chemical

and internal standard methods with high analysis time and qualitative analysis.

Developed and validated a simple, accurate method for the trace Analysis of

Piperazine content in Active Pharmaceutical ingradients by High Performance

Liquid Chromatography with pre derivatization UV detection by using NBD

Chloride as pre derivative reagent.

4.4. MATERIALS AND METHODS

4.4.1.Reagents & Chemicals:

a. NBD Chloride : Sigma Aldrich

b. Acetonitrile : Sigma Aldrich

c. Methanol : Sigma Aldrich

d. Diethylenediamine : Sigma Aldrich

Chapter-4 Page 135

4.4.2. Drug substance:

Ziprasidone key starting material are gift samples received from M/S

Vegesna Laboratories , Vizag , India.

4.4.3. Instrument details:

The High performance Liquid Chromatography using HPLC instrument

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

detector, make waters instrument. All the components are controlled with

Empower2 software.

4.4.4. Method development:

Development trials were performed with all derivatization reagent and

different make HPLC columns but finally the Chromatographic conditions were

optimized with the diethyl amine, NBD Chloride and acetonitrile with simple

isocratic method.

4.4.4.1. Wave length Selection:

The UV spectrums were generated for Piperazine using with Photo diode

array detector (PDA). Diethylenediamine does not possess chromophores. It

absorbs UV light only at a wavelength of 205 nm, and its specific absorption

coefficient is very low (0.01). Therefore, the determination of diethylenediamine at

a level below 1mg/kg is possible only after its transformation into a derivative with

sufficiently high optical density, or emitting induced light. Various primary and

secondary aliphatic amines are present in considerable amounts in biological and

environmental samples. Their selective removal is theoretically possible, but time-

and labour-consuming. The Chromatographic properties of coupling products of

aliphatic diamines with several carbon atoms are affected, to a great degree, by the

substituent, whose molecular weight is several times higher and which usually

possesses unsaturated bonds and an additional heteroatom. Hydrophobic

interactions of derivatives of primary and secondary amines do not differ

significantly. Piperazine is found to have varying absorption maxima over a range of

wavelength after derivatization reagent NBD Cl. But it was found that at about 336

Chapter-4 Page 136

nm, Piperazine is found to have optimum UV absorption. Therefore, 340 nm was

selected for the study and quantification of Piperazine in Ziprasidone key starting

material.

Figure- 4.1: Chemical structure of Piperazine.

Chemical name : Diethylenediamine

CAS Registry Number : 110-85-0

Molecular formula : C4H10N2

Molecular weight : 86.14

Figure- 4.2: UV Spectra of Piperazine.

4.4.4.2. Selection of mobile phase and stationary phase:

Chapter-4 Page 137

Piperazine and Ziprasidone key starting material 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 Piperazine and Ziprasidone key starting

material i.e., Selection of the mobile phase and column.

4.4.4.3. Selection of Mobile phase:

Piperazine was clearly eluted in Ziprasidone key starting material sample

using with different mobile phases. Piperazine is having wide range of polarities

and the separation of Piperazine mainly depends on the column stationary phase.

An isocratic method was mobile phase of buffer is acetonitrile, methanol and

diethylamine was suitable for the separation of Piperazine content in Ziprasidone

key starting material. Mobile phase was degassed and filtered through 0.22µm

millipore filter paper.

4.4.4.4. Selection of stationary phase:

The objective of this work was to evaluate the piperazine content for

accurate quantification .The preliminary trails carried out in reverse phase columns

were not fruitful in the separation of the separation of piperazine from API.

Different chiral stationary phases were employed during the method development

namely Chiralpak IA, Chiralpak IB, Chiralpak IC and Chiral pak AD-H. In Chiralpak IC

column was found to be good separation between Piperazine and API peak. Very

good resolution was achieved on Chiralpak IC column using mobile phase contains

the mixture of acetonitrile: methanol: diethylamine(90:10:0.1 v/v/v).The additions

of methanol and diethylamine to the mobile phase plays an important role on

enhancing the chromatographic efficiency and resolution between the Piperazine

and Ziprasidone key starting material. Separation was achieved with Chiralpak IC,

250 X 4.6mm I.D., 5.0µm column. Different stationary phases were studied for the

separation of Piperazine and Ziprasidone key starting material such as Chiralpak IA,

Chiralpak IB and Chiralpak AD-H 250X4.6mm I.D., 5.0µm using the mobile phase

Chapter-4 Page 138

specified. The experimentation was started using Chiralpak IA, 250X4.6mm I.D.,

5.0µm column.

Trail-1:

The complete experiment details are as follows.

Column : Chiralpak IA,250 X 4.6mm I.D., 5.0µm column

Mobile Phase : A degassed mixture of acetonitrile: methanol:

Diethylamine in the ratio of 80:30:0.1 (v/v/v)

Sample preparation : 0.5mg/mL

Wave length : 340 nm

Flow rate : 1.0 mL/ min

Oven temperature : 40oC

Diluent

Elution

Runtime

:

:

:

NBD Chloride solution in water

Isocratic

20min

Figure-4.5: Typical HPLC Chromatogram of Piperazine using Chiralpak IA,

250X 4.6mm I.D., 5.0µm.

Observation: Piperazine and main compound and other peaks all are merging.

Hence, Chiralpak IA, 250 X 4.6mm I.D.,5.0µm column is not suitable for the

separation of Piperazine and Ziprasidone key starting material.

Pip

era

zin

e -

5.5

07

AU

0.00

0.50

1.00

1.50

2.00

2.50

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Chapter-4 Page 139

Trail-2:


Column : Chiralpak -ADH,250 X 4.6mm I,D., 5,0µm column

Mobile Phase : A degassed mixture of acetonitrile: methanol:


Sample preparation

Injection volume

:

:

0.5 mg/mL

10µL

Wave length : 340 nm



Figure-4.6: Typical HPLC Chromatogram of Piperazine using Chiralpak ADH,

250X4.6mm I.D., 5.0µm.

Observation: Piperazine are eluting and Ziprasidone key starting material and

Piperazine peaks shape not good. Hence, Chiralpak AD-H, 250X4.6mm I.D., 5.0µm

column is not suitable for the separation of Piperazine.

Pip

era

zin

e -

6.4

15

Peak2 -

7.0

76

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Chapter-4 Page 140

Trail-3:


Column

Mobile phase

:

:

Chiralpak IB, 250 X 4.6mm I.D., 5.0µm column

A degassed mixture of acetonitrile:methanol:


Diluent

Sample preparation

Elution

Runtime

Flow rate

Wave length

:

:

:

:

:

:

NBD Chloride solution in water

0.5mg/mL

Isocratic

20min

1.0 mL/min

340nm

Figure-4.3: Typical HPLC Chromatogram of Piperazine using Chiralpak IB,

250X4.6mm I.D., 5.0µm column.

Observation: Piperazine is eluting and Ziprasidone key starting material resolution

not good and base line not good. Hence, Chiralpak IB 250X4.6mm I.D., 5.0µm column

is not suitable for the separation of Piperazine in Ziprasidone key starting material.

Trail-4:


Column : Chiralpak IC, 250X4.6mm I.D., 5.0µm column

Mobile Phase : A degassed mixture of acetonitrile:methanol:

Peak1 -

5.7

97

Pip

era

zin

e -

6.8

68

PB

I -

7.3

46

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Chapter-4 Page 141


Sample preparation 0.5mg/mL

Wave length 340 nm


Oven temperature

Diluent

:

:

35 oC

NBD Chloride solution in acetonitrile

Figure- 4.4: Typical HPLC Chromatogram of Piperazine using Chiralpak IC,

250X4.6mm I.D., 5.0µm column.

Observation: Piperazine and Ziprasidone key starting material eluting. Piperazine

shape also good. Hence Chiralpak IC, 250 X 4.6mm I.D., 5.0µm column is suitable for

the separation of Piperazine, but buffer composition change is required.

Trail-5:


Column : Chiralpak IC,250 X 4.6mm I.D., 5.0µm column

Buffer preparation : A degassed mixture of Acetonitrile: Methanol:


Mobile phase

Sample preparation

:

:

Buffer and acetonitrile in the ratio of 50:50 v/v

0.5mg/mL

Wavelength : 340 nm

Flow rate : 1.0 mL/min

Pea

k1

- 5

.540

Peak2 -

6.4

33

Pip

era

zin

e -

6.5

65

Peak4 -

7.0

53

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Chapter-4 Page 142

Diluent

Elution

Runtime

Oven emperature

:

:

:

:

NBD Chloride solution in acetonitrile

Isocratic

20 min

35 oC

Injection volume

Elution

Runtime

:

:

:

10µL

Isocratic

20min

Figure-4.7:Typical HPLC Chromatogram of Piperazine trail-5 method

conditions using Chiralpak IC, 250 X 4.6mm I.D.,5.0µm column.

Observation: Piperazine and Ziprasidone key starting material peak shape good.

Hence, Chiralpak IC, 250X4.6mm I.D.,5µm column is suitable for the separation of

Piperazine.

4.4.5. OPTIMISED METHOD:

Chromatographic conditions: The Liquid Chromatograph is equipped with UV

detector.

Column : Chiralpak IC, 250 X 4.6mm I.D., 5.0µm column


Wavelength : 340 nm

Pip

era

zin

e -

6.5

38 P

eak2 -

7.0

61

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Chapter-4 3

Flow rate : 1.0 mL/min

Injection volume : 10 µL

Run time : 20 minutes

Diluent : NBD Chloride solution in acetonitrile

Sample concentration : 0.5 mg/mL

Elution : Isocratic

Mobile phase: A degassed mixture of acetonitrile: methanol: diethylamine in the

ratio of 90:10:0.1 (v/v/v).

Diluent Preparation:

Weigh accurately about 500 mg of NBD Chloride into a 500 ml volumetric

flask, dissolve and dilute to volume with acetonitrile.

Preparation of system suitability solution(0.10%):

Weigh accurately about 20 mg of Piperazine standard into 10 mL Volumetric

flask ,dissolved and make up to the mark with diluent.

Transfered 12.5µL of this solution into 50 mL volumetric flask, dissolve and

dilute to volume with diluent.

Test sample preparation: Weigh accurately about 5mg of test sample in to 10 mL

volumetric flask, dissolved and make up to the mark with diluent.

Note: Prepare & Inject fresh sample solution.

Note: Prepare Piperazine stock on the same day of analysis

Procedure: After equilibrating the column, inject separately 10µL of diluent blank

system suitability solution for six times and test sample into the liquid

chromatographic system and run the chromatograph for 20 minutes and report the

Piperazine content by using below formula.

Calculation:

Area of Piperazine in sample

Piperazine content % = -------------------------------------------------------------- X 0.10

Avg. area of Piperazine in system suitability solution

Note: Integrate only piperazine peak.

Chapter-4 Page 144

System suitability criteria: The % RSD for peak area of Piperazine from system

suitability solution should not be more than 10.

Table- 4.2: Specifications.

Figure- 4.8: Typical HPLC Chromatogram of Piperazine using Chiralpak IC, 250

X 4.6mm I.D., 5.0µm column.

Conclusion:

Piperazine and Ziprasidone key starting material peak shape also very

sharp, resolution and separation also good. Hence, Chiralpak IC, 250 X 4.6mm I.D.,

5.0µm column is suitable and optimized methods conditions for the separation of

Piperazine content in Ziprasidone key starting material.

Pip

era

zin

e -

6.3

18

Peak2 -

6.8

12

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Name of the impurity Specification

Piperazine Not more than 0.10%

Chapter-4 Page 145

Figure-4.9: Blank Chromatogram of diluent.

Figure-4.10: LOD Chromatogram of Piperazine.

Chapter-4 Page 146

Figure-4.11: LOQ Chromatogram of Piperazine.

Figure- 4.12: Sample Chromatogram of Ziprasidone key starting material.

Chapter-4 Page 147

Figure- 4.13: Spiked Chromatogram of Piperazine and Ziprasidone key

starting material.

4.5. RESULTS AND DISCUSSION

4.5.1. Method validation:

Analytical method validation was performed as per ICH and USFDA guide lines

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

quantification , ruggedness and robustness.

4.5.1.1 Piperazine content by HPLC:

4.5.1.2 System suitability:

a) Preparation of Piperazine stock solution: Transferred 20 mg of Piperazine

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

b) Preparation of Sample solution: Accurately weighed 5mg of sample into

10mL volumetric flask ,dissolved and diluted to volume with diluent.

Chapter-4 Page 148

c) Preparation of 0.1% Piperazine system suitability solution : Transferred

12.5µL of Piperazine stock solution dissolved and diluted to volume with 50

mL of diluent.

d) Diluent preparation: Transferred 500 mg of NBD Chloride into 500 mL

volumetric flask, dissolved and diluted to volume with acetonitrile.

Conclusion: Under optimized chromatographic conditions, Piperazine content were

separated well, retention times being about 7.86min respectively. The system

suitability results are given in table-4.3.

Table-4.3: System suitability results:

S. No Name Retention time(min) % RSD

1 Piperazine 7.86 1.13

4.5.1.3 Limit of Detection and Limit of Quantification:

a) LOQ solution preparation (0.035%):Transferred 3.5µL of Piperazine stock

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

diluent.

c) LOD solution preparation: Transferred 3.3mL of above LOQ solution stock

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

diluent.

Injected Piperazine solution and calculated the limit of detection and limit of

quantification for Piperazine.

Conclusion:

The LOD for Piperazine was found to be 0.012 % respectively. The LOQ for

Piperazine was found to be 0.035 % respectively. The results are summarized in the

table-4.4

Table-4.4: Limit of detection and Limit of Quantification data:

Conc. Piperazine

LOD 0.015%

LOQ 0.035%

Chapter-4 Page 149

4.5.1.4 Precision and accuracy at Limit of Quantification level:

a) Solution preparation: Transferred 3.5µL of Piperazine stock solution into

10mL volumetric flask, containing 5mL 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

Piperazine area.

Accuracy:

b) Sample + Piperazine Solution preparation: Accurately weighed 50mg of

sample into 100 mL volumetric flask, dissolved in 50 mL of diluent and added

35µL of Piperazine stock solution dissolved and diluted to volume with diluent.

c) Sample solution preparation: Transferred 50mg of sample into 100 mL

volumetric flask, dissolved and diluted to volume with diluent.

Prepared three times the solution as mentioned above and inject each

preparation once and calculated the % recovery for Piperazine at Limit of

Quantification level.

Conclusion:

The repeatability and recovery at the LOQ concentration for Piperazine were 0.30%

100% respectively. The results are summarized in the table- 4.5.

Table-4.5: Precision and accuracy at Limit of Quantification level data:

S. No Impurity % RSD (n=10) % Recovery (n=3)

1 Piperazine 0.30 100

4.5.1.5 Linearity:

a) Linearity solution-1(0.035%): Taken 3.5 µL of Piperazine stock solution into

10 mL volumetric flask, containing 5mL of diluent dissolved and diluted to

volume with diluent.

Chapter-4 Page 150

b) Linearity solution-2(0.05%):Transferred 5µL of Piperazine stock solution

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

to volume with diluent.

c) Linearity solution-3(0.075%): Transferred 7.5µL of Piperazine stock

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

diluted to volume with diluent.

d) Linearity solution-4 (0.10%): Transferred 10µL of Piperazine stock solution



e) Linearity solution-5 (0.125%): Transferred 12.5µL of Piperazine stock

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

diluted to volume with diluent.

f) Linearity solution-6 (0.15%) :Transferred 15 µL of Piperazine stock solution



Injected all above solutions each preparation once and calculated the linearity

parameters i.e. correlation coefficient, slope and intercept for Piparazine.

Conclusion:

Linearity established for Piperazine at 0.035%, 0.05%, 0.075%, 0.10%, 0.125%,

0.15% The correlation coefficient (r) are more than 0.99. The above result reveal

that method is linear results are summarized in purity wise.

Table-4.6: Piperazine linearity data:

S. No Level (%) Concentration (%) Area of Piperazine

1 LOQ 0.035 1919

2 50 0.0503 2998

3 75 0.07545 4322

4 100 0.1006 6156

5 125 0.12575 7405

Chapter-4 Page 151

6 150 0.1509 9194

Correlation coefficient(r) 0.999

Slope 61862.39

Y-Intercept -214.75

%Y-Intercept -3.49

Figure-4.14:Piperazine linearity graph.

Accuracy:

a) Accuracy solution-1 preparation (0.05%): Transferred 5mg of sample into

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

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

solutions prepared as mentioned above.

b) Accuracy solution-2 preparation- (0.10%): Taken 5mg of sample into 10

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

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

prepared as mentioned above.

c) Accuracy solution-3 preparation (0.15%): Transferred 5mg of sample into

10 mL volumetric flask, dissolved in 5mL of diluent and added 15µL of

y = 61862x - 214.7

R² = 0.997

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0 0.05 0.1 0.15 0.2

A

r

e

a

Concentration (%)

Linearity of Piperazine

Chapter-4 Page 152

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

solutions prepared as mentioned above.

Injected each above preparation once and calculated the recovery for

Piperazine at each level.

Conclusion:

The percentage recovery of Piperazine in Zipraidone key starting material

samples is shown in table-4.7.

Table-4.7: % Recovery data:

Concentration Piperazine(%)

50% 108.06

100% 105.19

150% 104.8

4.5.1.6 Precision:

a) Sample preparation: Weighed 5 mg of sample into 10mL volumetric flask,

dissolved and diluted to volume with diluent.

b) Sample + 0.10% spiked preparation: Transferred 5mg of sample into 10 mL

volumetric flask, dissolved in 5mL of diluent added 10µL of Piperazine 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 Piperazine.

Conclusion:

The precision of the Piperazine content method was checked by injecting six

individual preparations of Ziprasidone key starting material spiked with 0.10% of

Piperazine. The % RSD of the area for Piperazine stock solution was calculated. The

results was summarized in the table-4.8.

Table-4.8: Precision data:

Parameter Piperazine area

% RSD 1.13

Chapter-4 Page 153

4.5.1.7 Robustness:

Flow variation:

a) Sample solution preparation: Weighed 5mg of sample into 10mL volumetric

flask, dissolved and diluted to volume with diluent.

b) Sample + 0.10% spiked preparation: Weighed 5mg of sample into 10 mL

volumetric flask, dissolved in 5mL of diluent added 10µL of Piperazine 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 parameter was Piperzine relative

retention time compared with 1.0 mL/min results.

Temperature variation:

a) Sample solution preparation: About 5mg of sample into 10mL volumetric

flask, dissolved and diluted to volume with diluent.

b) Sample + 0.10% spiked preparation: Accurately weighed 5mg of sample

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

Piperazine 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 parameter was Piperazine relative retention

time compared with results 35°Cresults.

Conclusion:

The results are summarized in the table-4.9.

Table-4.9: Robustness data

Parameter 30°C 40°C 0.8 mL/min 1.2mL/min As such

% RSD for Piparazine 3.56 2.52 0.70 1.35 1.13

Note: For methanol change the parameter cannot be performed, because the

Piperazine peak is merging with Ziprasidone key starting material.

4.5.1.8 Solution stability:

Chapter-4 Page 154

Sample solution preparation: Accurately weighed 5 mg of sample into 10mL

volumetric flask , dissolved and diluted to volume with diluent.

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

performed the Piperazine content.

Conclusion:

Piperazine was 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

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-4.10.

Table-4.10: Solution stability data(0.1% spiked sample).

Duration Piperazine (%)

Sample solution initial 0.10

After 12 hrs 0.11

After 24 hrs 0.11

After 48 hrs 0.11

Table-4.11: Mobile phase stability data.

Duration Piperazine (%)

Sample solution initial 0.10

After 12 hrs 0.11

After 24 hrs 0.11

After 48 hrs 0.11

4.5.1.9 Batch analysis :

Using the above validated method, Ziprasidone key starting material sample was

analyzed and the data is furnished in table -4.12.

Chapter-4 Page 155

Table-4.12: Batch analysis data.

Lot Number Piperazine content

001 Not detected

4.6 SUMMARY AND CONCLUSION

Based on the above experimental data on the various method validation

parameters, it is proved that this method which was designed to quantification of

Piperazine content by HPLC by using pre derivative method for Ziprasidone Key

starting material is precise, accurate, linear, rugged and robust, this method linear

from LOQ to 150% of specification level. Hence, the method can be used for routine

analysis of Piperazine content .

Chapter-4 Page 156

4.7 REFERENCES

1. Fatma M Abdel-Gawad. J. Pharm. Biomed. Anal, 1997, 15: 1679–1685

2. Adela Arranz; Camilo Echevarr´ıa; Jose Mar´ıa Moreda; Adolfo Cid; Juan

Francisco Arranz. J. Chromtogr A, 2000, 871: 399–402

3. Hiroe Tsutsumi; Munehiro Katagi; Akihiro Miki; Noriaki Shima; Tooru

Kamata; Mayumi Nishikawa; Kunio Nakajima; Hitoshi Tsuchihashi. J.

Chromatogr B, 2005, 819: 315–322.

4. Mitsuhiro Wada; Kozue Yamahara; Rie Ikeda; Ruri Kikura-Hanajiri; Naotaka

Kuroda; Kenichiro Nakashima. Biomed. Chromatogr. 2012; 26: 21–25.

5. Bogumila BYRSKA; Dariusz ZUBA; Ro man STANASZEK. Problems of Forensic

Sciences 2010; 81: 101–113.

6. M.I. Walash; F. Belal; F. Ibrahim; M. Hefnawy; M. Eid. J. Pharm. Biomed. Anal,

2001; 26: 1003–1008.

7. Henry S.I. Tan; Jianling Xu; Yaohan Zheng.J. Chromatogr A, 1995; 693: 307-

314.

8. Renata Gadzala-Kopciuch. J. Liq. Chromatogr., 2005; 28: 2211–2223.

9. Jyoti Patel; Eric Loeser; Rudy Kircher; Hanumantha Rao Marrepalli; Steven

Fazio; Donald Drinkwater; Patrick Drumm. J. Liq. Chromatogr., 2010;

33:712–719.

10. Carlie McClintic; David M. Remick; Jeffrey A. Peterson; Donald S. Risley. J. Liq.

Chromatogr., 2003; 26: 3093–3104.

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