A SEMINAR ON METHOD DEVELOPMENT AND VALIDATION OF ZIPRASIDONE

1. ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ZIPRASIDONE BY
REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
THE DISSERTATION SUBMITTED TO OSMANIA UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
AWARD OF THE DEGREE OF
MASTER OF PHARMACY
IN
PHARMACEUTICALANALYSIS & QUALITYASSURANCE
BY
J.Shraddha kiran
(ROLL NO:170616885007)
Under the guidance of
Dr.M.Sumakanth
Prof& Principal
RBVRR WOMEN’S COLLEGE OF PHARMACY
(AFFILIATED TO OSMANIA UNIVERSITY)
BARKATPURA, HYDERABAD-500007-TELANGANA-INDIA
APRIL -2019

1

2. CONTENTS
 ABSTRACT
 INTRODUCTION
 DRUG PROFILE
 AIM & OBJECTIVES
 LITERATURE REVIEW
 METHOD DEVELOPMENT
 VALIDATION
 SUMMARYAND CONCLUSION
 REFERENCES
2

3. ABSTRACT
 A rapid and precise reverse phase high performance liquid chromatographic
method has been developed and validated for ziprasidone , in its pure form as
well as in Capsule dosage form. Chromatography was carried out on a sunsil
C18 (150×4.6mm 5µ ) column using a mixture of water:methanol (55:45) as
the mobile phase at a flow rate of 1.0ml/min, the spectrometric detection was
carried out at 261nm. The retention time of the Ziprasidone was 3.08min. The
method produced linear responses in the concentration range of 2-40µg/ml of
Ziprasidone .The method is useful in the quality control of bulk and
pharmaceutical formulations.
3

4. INTRODUCTION
4

5. BLOCK DIAGRAM
5

6. DRUG PROFILE
 Drug : Ziprasidone hydrochloride
 Drug category : Antipsychotic
 Stucture:
 Chemical name :5-[2-[4-(l, 2-Benzisothiazol-3-yl)-l-piperazinyl] ethyl]-6-chloro-l, 3-dihydro2//-indol-
2-one
 Molecular Formula : C21H21CIN4OS
 Molecular Weight : 412.899 gm/mole.
 Melting point : 226.64°C
 Pka : 6.5
 Description(Physical State): pale pink color powder form
 Solubility: Soluble in methanol.
 Storage Conditions: store at room temperature protect from light and moisture .
6

7. MARKETED FOMULATIONS
S.No Drug name Label Claim Brand name Company
1
2
Ziprasidone
hydrochloride
Ziprasidone
hydrochloride
20,40,80 mg
(capsule )
20,40,80mg
(Capsule )
Azona
Zipsydone
Torrent
pharmaceuticals ltd
Sun pharma
laboratories ltd
7

8. AIM AND OBJECTIVE
 To develop new simple, specific, Precise ,accurate and economical
analytical method for ziprasidone and validate the method by RP-HPLC
 To perform all the validation parameters according to the ICH guidelines.
 The proposed method is in accordance with ICH guidelines is intended for
analytical application i.e., to apply the proposed method for analysis of
ziprasidone in marketed formulation..
8

9. TITLE AUTHOR YEAR Parameters
Estimation of
Ziprasidone
hydrochloride in
bulk and in capsules
by RP-HPLC
(AZONA )
B.Sudha rani,
P.Venkata reddy
, et al.,
2006 Column - Rp C18 (150mm×4.6mm
5µ),Mobile phase: Methanol:
phosphate buffer (pH 3.2), (55:45)
Wavelength : 314nm
flow rate : 1mI/min
Rt: 4.5 min
Validation Parameters : intraday &
Interday : % RSD : <2 % Linearity
range: 0.5- 30 µg/mlAssay : 100.5%
LITERATURE REVIEW
9

10. TITLE AUTHOR YEAR Parameters
Development and
Validation of Rp-
hplc method for
ziprasidone HCL
monohydrate
Abhay R.shirode
,Arpita P. nath ,
Vilaj raj kadam et
al.,
2016 C18 column (250 mm×4.6 mm
5µ), Mobile phase-Water :
Methanol
(45: 65 ),Wavelength 317 nm Rt:
4.8 min,
Validation Parameters :
Intra and interday : %RSD -
<2.0 %,r2 – 0.998 , Linearity
range : 2 to 12µg/ml %recovery
: 100.46
LOD - 0.25 µg/ml
LOQ : 0.10 µg/ml
10

11. TITLE AUTHOR YEAR PARAMETERS
Development and
method validation
Using HPLC for
Assay Of
ziprasidone Capsule
M.Gnana ruba
priya, M.kali kolan
, S.asadulla,
S.rajesh et al.,
2011 Column - C18 ( 150 ×4.6mm
5µ),Mobile Phase :Potassium
hydroxide buffer : ACN:
Methanol (45:40:15V/V/V) .
Wavelength :230nm,
flowrate : 1.5 ml /min , Rt :7-9
min , Linearity range : 50-
150µg/mL
11

12. Title Author Year PARAMETERS
RP-HPLC method for
Estimation of
ziprasidone
K.Srinivasa rao,
Nagesh kumar et al.
2013 C18 column (250 mm × 4.0 mm, 5
μm) Mobile phase -: Ammonium
acetate : Meoh
(30:70 v/v), Wavelength : 225 nm
flowrate 0.8– 1 ml,
Rt : 4.76 ml/min ,precision-RSD :
<2% , Linearity range : 1-500µg/ml.
LoQ:1.0 µg/ml, LOD: 0.43µg/ml,
Assay : 99.32%
12

13. LITERATURE SUMMARY
 All the retention times in the reported methods were with in the range 4.8 -9
min in different mobile phase compositions
 In the present method by using economical mobile phase the retention times
were reduced.
13

14. METHOD DEVELOPMENT
14

15. INSTRUMENTS USED
S.NO Instruments Model
1
HPLC , DETECTOR , WEIGHING
MACHINE , DIGITAL ULTRA
SONICATOR, UV VISIBLE
SPECTROMETER
WATERS
SOFTWARE: EMPOWER
3.0, WATERS DUAL
ABSORBANCE 2487
DETECTOR, CONTECH
LTD , LABMAN , ELICO
210 ,SOFTWARE
SPECTRA TREATS
15

16. DETECTION OF LAMBDA MAX
 10 mg of Ziprasidone standard was accurately weighed and transferred into a
10ml of clean dry volumetric flask and about 7ml of Methanol was added and
sonicated to dissolve and volume was made up to the mark with the methanol,
which is 1000µg/ml solution –stock -I
 1ml from stock –I was pipetted out into a 10ml of volumetric flask and made
up to the mark with diluent which is 100µg/ml solution – Stock - II
 1ml from stock -II solution was pipetted in a 10ml of volumetric flask and
was diluted up to the mark with diluent which is 10µg/ml solution
 Lambda max was checked for the above solution and was found at 261nm with
methanol as blank
16

17. UV SPECTRUM OF ZIPRASIDONE
17

18. HPLC METHOD DEVELOPMENT
 HPLC grade water and HPLC grade Methanol were taken and filtered through 0.45 µ
and were degassed in digital ultrasonicater for 10 minutes.
 Diluent Preparation:
 HPLC methanol filtered and sonicated was used as a diluent.
TRAILS
 Preparation of standard solution:
 10 mg of Ziprasidone standard was accurately weighed and transferred into a
10ml of clean dry volumetric flask and about 7ml of Methanol was added and
sonicated to dissolve and volume was made up to the mark with the same
Methanol, which is 1000µg/ml solution –Stock I
 1ml of solution from stock -I was pipetted out in a 10ml of volumetric flask
and was diluted up to the mark with diluent which is 100µg/ml solution –
stock II
 From stock II 0.5ml of solution was pipetted out in a 10ml of volumetric flask
and was diluted up to the mark with diluent which is 5 µg/ml solution
18

19. TRIAL - 1
 Mobile phase : Water: Methanol (60:40 v/v)
 Column : phenomenex LC column (250mm×4.6mm, 5µ)
 Flow rate : 0.8 ml/min
 Wavelength : 261nm
 Column temp : Ambient Temperature
 Injection Volume : 10 µl
S. No Peak name Rt Area Height USP Tailing
USP plate
count
1
Ziprasidone
7.9 36741 4642 2.4 526
INFERENCE : : Peak was eluting later
19

20. TRIAL- 2
 Mobile phase : water: Methanol (50:50 % v/v)
 Column : phenomenex column (250mm×4.6mm 5µ)
 Flow rate : 1ml /min
 Wavelength : 261 nm
 Column temp : Ambient temperature
 Injection Volume : 10 µl
S. No Peak name Rt Area Height USP Tailing
USP plate
count
1 Ziprasidone 3.212 36741 4642 2.4 526
INFERENCE: Improper separation of analyte was observed
20

21. TRIAL -3
 Mobile phase : Water:Methanol (55:45)
 Column : Sunsil C18 (150×4.6mm 5µ)
 Flow rate : 1 ml/min
 Wavelength : 261 nm
 Column temp : Ambient temperature
 Injection Volume : 10 µl
S. No Peak name Rt Area Height
USP
Tailing
USP plate
count
1 Ziprasidone 3.082 204754 23850 1.3 4558
INFERENCE : from the above chromatogram it was observed that the ziprasidone showed proper
retention time ,no additional peaks were observed the plate count was also with in the acceptable limit
21

22. OPTIMIZED CHROMATOGRAM
 Mobile phase : Water:Methanol (55:45)
 Column : Sunsil C18 (150×4.6mm 5µ)
 Flow rate : 1 ml/min
 Wavelength : 261 nm
 Column temp : Ambient temperature
 Injection Volume : 10 µl
S. No Peak name Rt Area Height
USP
Tailing
USP plate
count
1
Ziprasidon
e
3.082 204754 23850 1.3 4558
INFERENCE : from the above chromatogram it was observed that the ziprasidone showed proper
retention time ,no additional peaks were observed the plate count was also with in the acceptable limit
22

23. VALIDATION
 Validation is the process of establishing documentary evidence demonstrating that a procedure,
process, or activity carried out in testing and then production maintains the desired level of
compliance at all stages.
VALIDATION PARAMETERS:
 Specificity
 Precision
 Method precision /repeatability
 Intermidate precision /ruggedness
 Linearity
 Accuracy
 Limit of detection
 Limit of Quantification
 Robustness
23

24. CHROMATOGRAM FOR STANDARD
 SPECIFICITY:
 Mobile phase : Water:Methanol (55:45)
 Column : Sunsil C18 (150×4.6mm 5µ)
 Flow rate : 1 ml/min
 Wavelength : 261 nm
 Column temp : Ambient temperature
 Injection Volume : 10 µl
S. No Peak name Rt Area Height USP Tailing
USP plate
count
1 Ziprasidone 3.082 204754 23850 1.3 4558
INFERENCE : from the above chromatogram it was observed that the they were no
additional peaks eluting at the RT of ziprasidone hence the method was specific and all
the system suitability parameters were found to be within limits
Chromatogram showing blank
24

25.  METHOD PRECISION/REPEATABILITY
 Preparation of Ziprasidone Solution for Precision:
 10 mg of Ziprasidone standard was accurately weighed and transferred into a 10ml
clean dry volumetric flask and about 7ml of Methanol was added and sonicated to
dissolve and volume was made up to the mark with the same Methanol, which is
1000µg/ml solution –Stock I
 1ml from stock-I solution was pipeted in a 10ml of volumetric flask and was diluted
up to the mark with diluent which is 100µg/ml solution –stock II
 From stock II -0.5ml of solution was pipeted out in a 10ml of volumetric flask and
dilute up to the mark with diluent which is 5 µg/ml solution
25

26. precision
Day 1 injection 1
Day 1 injection 2
Day 1 injection 3
Day 1 injection 4
Day 1 injection 5
26

27. S no Name Rt Area Height USP plate count
USP
Tailing
1 Zipraasidone 3.082 204754 23850 4506 1.3
2 Ziprasidone 3.082 204332 24032 4674 1.2
3 Ziprasidone 3.082 204754 23850 4298 1.2
4 Ziprasidone 3.082 204332 24032 4032 1.0
5 Ziprasidone 3.082 204754 23850 4812 1.3
Mean 204584.2
Std. Dev 1412
% RSD 0.68
PRECISION DAY - 1
27

28.  INTERMEDIATE PRECISION:
 To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision
was performed on different days by maintaining same conditions.
 Procedure:
 The standard solution was injected for five times and measured the area for all five injections
in HPLC on day 2 The %RSD for the area of five replicate injections was found to be within
the specified limits.
28

29. Ruggedness –day 2
 Day 2 injection 1
Day 2 injection 3
Day 2 injection 2
Day 2 injection 4
Day 2 injection 5
29

30. S no Name Rt Area Height
USP plate
count
USP Tailing
1 Ziprasidone 3.082 204753 23850 4506 1.3
2 Ziprasidone 3.099 204331 24032 4674 1.2
3 Ziprasidone 3.082 204753 23850 4298 1.2
4 Ziprasidone 3.099 204331 24032 4032 1.0
5 Ziprasidone 3.082 204753 23850 4812 1.3
Mean 204584.2
Std. Dev 206.7369
% RSD 0.1
 Ruggedness day 2
The method precision and intermidate precision were performed and the % RSD
was found with in limits hence the method was precise and rugged
30

31. LINEARITY
 PREPARATION OF DRUG SOLUTIONS FOR LINEARITY:
 Stock I Solution :
 10 mg of Ziprasidone standard was accurately weighed and transferred into a
10ml of clean dry volumetric flask and about 7ml of Methanol was added
and sonicated to dissolve and volume was made up to the mark with the same
Methanol, which is 1000µg/ml solution
 Stock II Solution :
 1ml from stock I solution was pipeted in a 10ml of volumetric flask and was
dilute d up to the mark with diluent which is 100µg/ml solution
31

32. LINEARITY SOLUTIONS
 Preparation of Level – I (2µg/ml of Ziprasidone ):
 0.2ml from stock II solution was pipeted in a 10ml of volumetric flask and was
diluted up to the mark with diluent which gives 2µg/ml solution.
 Preparation of Level – II (10µg/ml of Ziprasidone):
 1ml from stock II solution was pipetted out in a 10ml of volumetric flask dilute up to
the mark with diluent, which gives 10µg/ml solution
 Preparation of Level – III (20µg/ml of Ziprasidone):
 2ml of from stock II solution was pipetted out in a 10ml of volumetric flask and was
diluted up to the mark with diluent, which gives 20µg/ml solution
32

33. LINEARITY SOLUTIONS
• Preparation of Level – IV (30µg/ml of Ziprasidone):
 3ml from stock solution II was pipetted out in a 10ml of volumetric flask and was diluted
up to the mark with diluent which gives 30µg/ml solution
 Preparation of Level – V (40µg/ml of Ziprasidone):
 4ml from stock solution II was pippeted out in a 10ml of volumetric flask was diluted up
to the mark with diluent ,which gives 40µg/ml solution
 Procedure: Each level were injected into the chromatographic system and Peak area was
measured . graph was plotted of peak area versus concentration (on X-axis concentration and on
Y-axis Peak area) and correlation coefficient was calculated.
33

34. Chromatograms for linearity concentrations
Chromatogram for linearity concentration-2µg/ml
Chromatogram for linearity concentration-10µg/ml
Chromatogram for linearity concentration-20µg/ml
Chromatogram for linearity concentration-30µg/ml
Chromatogram for linearity concentration-40µg/ml
34

35. LINEARITY PLOT FOR ZIPRASIDONE
s.no
Concentration
g/ml Peak Area
1
2
16265
2 10 85535
3 20 159277
4 30 246375
5
40
327185
35

36. ACCURACY
 Preparation of sample solution :
 Average weight of 10 capsules was taken and capsule powder was weighed which was 68.75
(10mg equivalent )of Ziprasidone sample and transferred into a 10mL clean dry volumetric flask
and a about 7mL of Diluent was added and sonicated to dissolve completely and volume was
made up to the mark with the same diluent.- stock 1 sample
 1ml from stock-I solution was pipetted out in a 10ml of volumetric flask and was diluted up to
the mark with diluent which is 100µg/ml solution –Stock - II Sample
 2ml from Stock -II was pipetted out in a 10ml of volumetric flask and was diluted up to the mark
with diluent which was 20µg/ml solution
 Preparation of standard Stock solution :
 10 mg of Ziprasidone standard was taken into a 10ml of clean dry volumetric flask and
about 7ml of Methanol was added and sonicated to dissolve Completely and volume was
made up to the mark with the same Methanol, which is 1000µg/ml solution Stock I standard
 1ml from stock –I standard was pipetted out in 10ml of volumetric flask and was diluted
up to the mark with diluent which is 100µg/ml solution - Stock II standard
36

37.  For preparation of 50% Standard solution:
 From the above standard stock II solution 1ml of solution was pipetted out in 10ml volumetric flask
and was diluted up to the mark with diluent which was 10µg/ml solution
 10µg/ml standard stock solution was spiked with 20µg/ml sample solution
 and was make up to the mark with the diluent.
 For preparation of 100% Standard stock solution:
 From the above standard stock II solution 2ml of solution was pipetted out in 10ml of volumetric
flask and was diluted up to the mark with diluent which is 20µg/ml solution
 20µg/ml standard stock solution was spiked with 20 µg/ml of sample solution and was make up to
the mark with the diluent.
 For preparation of 150% Standard stock solution:
 From the above standard stock II solution 3ml of solution was pipetted out in a 10ml of volumetric
flask and was diluted up to the mark with diluent which is 30µg/ml solution
 30µg/ml solution of standard stock solution was spiked with 20 µg/ml of sample and was make up to
the mark diluent.
37

38. ACCURACY INJECTIONS -50 %
Chromatogram showing accuracy 50% injection -1 Chromatogram showing accuracy 50% injection -2
Chromatogram showing accuracy 50% injection -3
38

39. ACCURACY INJECTIONS - 100%
Chromatogram showing accuracy 100% Injection -1 Chromatogram showing accuracy 100% Injection -2
Chromatogram showing accuracy 100% Injection -3
39

40. ACCURACY INJECTIONS -150%
Chromatogram showing accuracy 150 % Injection -1 Chromatogram showing accuracy150 % Injection -2
Chromatogram showing accuracy 150 % Injection -3
40

41. Accuracy results for ZIPRASIDONE
%Concentration
(at specification
Level)
Mean
area
Amount
Added
(ppm)
Amount
Found
(ppm)
% Recovery
Mean
Recovery
50% 79890 10 10.2 98%
99%
100% 159780 20 20 100%
150% 239679 30 30.1 99%
Accuracy at different concentrations (50%, 100%, and 150%) were
prepared and the % recovery was calculated.
41

42.  LIMIT OF DETECTION
 The detection limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be detected but not necessarily
quantitated as an exact value.
 LOD= 3.3 × σ / s
 Where
 σ = Standard deviation of the response
 S = Slope of the calibration curve
 Result:
 Ziprasidone :
 =3.3 × 1412/7989
 0.5=µg/ml
42

43.  LIMIT OF QUANTITATION
 The quantitation limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be quantitatively determined.
 LOQ=10×σ/S
 Where
 σ = Standard deviation of the response
 S = Slope of the calibration curve
 Result:
 Ziprasidone :
 =10×1412/7989
 =1.76µg/ml
43

44. ROBUSTNESS
 Preparation of Ziprasidone Solution for Robustness:
 Accurately weighed and transferred 10 mg of Ziprasidone standard into a 10ml of clean dry
volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air
completely and volume was made up to the mark with the same Methanol, which is 1000µg/ml
solution –stock I
 Pipetted out 1ml from stock-I solution in a 10ml of volumetric flask and was diluted up to the
mark with diluent which is 100µg/ml solution –stock II
 From stock II 0.5ml of solution was pippeted out in a 10ml of volumetric flask and dilute up to the
mark with diluent which is 5 µg/ml solution
CHROMATOGRAM SHOWING
VARIATION IN FLOW 1.1 ml
CHROMATOGRAM SHOWING
VARIATION IN FLOW 0.9 ml
44

45. ROBUSTNESS RESULTS FOR ZIPRASIDONE
Parameter used for
sample analysis
Retention Time
Peak Area
Theoretical plates
Less Flow rate of 1.1
ml/min
3.081 207331
4558
More Flow rate of 0.9
ml/min
3.099
207332
7036
Mean 207331.5
SD 0.70
%RSD 0.3
The %RSD was found to be with in limits after deliberate changes
in the developed method hence the method was found robust.
45

46. ASSAY FOR ZIPRASIDONE
 Average weight of 10 capsules was taken and capsule powder was weighed which
was 68.75 (10mg equivalent) of Ziprasidone sample and transferred into a
10mL clean dry volumetric flask and a about 7mL of Diluent was added and
sonicated to dissolve completely and volume was made up to the mark with the
same diluent.- stock 1 sample
 1ml from stock-I solution was pippetted out in a 10ml of volumetric flask and
was diluted up to the mark with diluent which is 100µg/ml solution –Stock -II
Sample
 0.5 ml from Stock –II was pipeeted out in a 10ml of volumetric flask and was
diluted up to the mark with diluent which was 5µg/ml solution
46

47. ASSAY FOR ZIPRASIDONE
CHROMATOGRAM SHOWING ASSAY SAMPLE
S. No Peak name Rt Area Height USP Tailing
USP plate
count
1
Ziprasidone
Formulation
3.137 206361 24637 1.3 4657
47

48.  Average weight of 10 capsules =0.275g
 0.275 ×1000 = 275mg
 275×10÷40 = 68.75
 %ASSAY =
 Sample area /standard area × Weight of standard / Dilution of standard ×Dilution of
sample /weight of sample ×purity /100 ×weight of the tablet /label claim ×100
 = 206361/ 204754× 10/1000× 1000/68.675 × 100/100 × 275/40× 100
 = 100.2%
 The % purity of Ziprasidone in pharmaceutical dosage form was found to be 100.2 %.
CALCULATIONS
48

49. SUMMARY & CONCLUSION
.A rapid and precise reverse phase high performance liquid chromatographic method has been
developed and validated for ziprasidone , in its pure form as well as in Capsule dosage form.
Chromatography was carried out on a sunsil C18 (150×4.6mm 5µ ) column using a mixture
of water:methanol (55:45) as the mobile phase at a flow rate of 1.0ml/min, the spectrometric
detection was carried out at 261nm. The retention time of the Ziprasidone was 3.08min. The
method produced linear responses in the concentration range of 2-40µg/ml of Ziprasidone
,the % recovery was 99%, and % assay was 100.2 % ,the % RSD were <2 % .The method is
useful in the quality control of bulk and pharmaceutical formulations.
From the above it was concluded that the method developed was simple, specific, precise and
accurate for ziprasidone by RP-HPLC and all the validation parameters were with in limits .
49

50. REFERENCES
1. Sudha rani ,venkata reddy et al, (2006) A reverse phase HPLC method is described for the
determination of Ziprasidone HCl mono hydrate in bulk and pharmaceutical dosage forms.-[journal
of chemistry]volume 2(4)169-172
2. Abhay R.shirode et al., (2016) A new isocratic simple and rapid reverse phase high performance
liquid chromatographic (RP-HPLC) method was developed and successively validated for the
estimation of ziprasidone hydrochloride monohydrate (ZHM)-[International journal of
pharmaceutical sciences and drug research] volume 8 ( 2) (121-127)
3.Gnana ruba priya.M.kali kolan , S.asadulla,S.rajesh et al., (2011) A reverse phase HPLC method
is described for the determination of Ziprasidone HCl capsule by analytical methanol-[International
journal of pharmaceutical sciences and Research]-Volume 2 (9)2325-232
4.K.Srinivasa rao, Nagesh kumar et al. (2013) A simple, sensitive, accurate and precise LC assay
method was developed for the quantitative determination of Ziprasidone Hydrochloride (ZSH) in
pharmaceutical dosage form.-[International journal of pharma medicine and biological sciences ] –
volume 2 (1)
50

51. REFERENCES
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(1996), PP 1-2
7.Andrea Weston and Phyllisr. Brown, HPLC Principle and Practice, 1st edition,
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51

52. THANK YOU
52