Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
History Sheet
Issue No. Effective date Changes made
1 17-01-2005 Prepared for the first time
Prepared by Approved by
Research Scientist Manager (ARD) Head – Technical Services
Date: Date: Date:
Format No.: ARD/F/18/1 Page 1 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
CONTENTS
Section Description Page
1 Purpose 3
2 Scope 3
3 References 3
4 Responsibility 3
5 Method of analysis 4-5
6 Validation 6-9
6.1 Specificity 6
6.2 Linearity 6
6.3 Precision 7
6.4 Accuracy 8
6.5 Stability of analytical solution 8
6.6 Robustness 9
7 Compilation of report 9
Format No.: ARD/F/18/1 Page 2 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
1 PURPOSE
To perform validation of HPLC method used for determination of assay of Zolmitriptan
(API).
2 SCOPE
This applies to validation of the in-house developed assay method as described on page 4.
3 REFERENCES
3.1 Method from SMS Pharma (STP1004)
3.2 Text on validation of analytical procedures – ICH Q2A
3.3 Validation of analytical procedures: Methodology – ICH Q2B
4 RESPONSIBILITY
4.1 Performing validation and preparing validation report – Research Scientist
4.2 Checking validation data and approving validation report – Manager
Format No.: ARD/F/18/1 Page 3 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
5 METHOD OF ANALYSIS
Chromatographic Conditions:
Column : Inertsil C8- 3, 250 x 4.6 mm, 5 µm
Mobile phase : Buffer : Acetonitrile, 90:10, filter through 0.45 µ filter
Detection wavelength : 225 nm
Flow rate : 1.5 ml / min
Column temperature : 40°C
Injection volume : 20 µl
Run time : 20 minutes
Diluent : Mobile phase
Buffer preparation:
Dissolve 7.8 g of sodium dihydrogen orthophosphate in 1000 ml of Milli-Q water. Add to it 1
ml of orthophosphoric acid. Adjust the pH to 7.0+0.05 with triethylamine.
Standard Solution:
Weigh accurately about 25 mg of Zolmitriptan working standard in a 25 ml volumetric flask.
Add about 15 ml of diluent, sonicate to dissolve and dilute to 25 ml with diluent. Further
dilute 5 ml of the above solution to 50 ml with diluent. Prepare in duplicate.
Test Solution:
Weigh accurately about 25 mg of sample in a 25 ml volumetric flask. Add about 15 ml of
diluent, sonicate to dissolve and dilute to 25 ml with diluent. Further dilute 5 ml of the
above solution to 50 ml with diluent. Prepare in duplicate.
Format No.: ARD/F/18/1 Page 4 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
System Suitability:
Inject diluent. Make six replicate injections of first standard solution into the chromatograph
and determine the RSD of peak areas. The RSD should be less than 2.0%.
Inject second standard solution in duplicate. Calculate the response factors for the two
standard solutions using the following formula-
Mean area of first standard solution
Response factor for first standard solution = -----------------------------------------------
Weight of first standard
Mean area of second standard solution
Response factor for second standard solution = ---------------------------------------------------
Weight of second standard
Calculate the RSD for the two response factors. The RSD should be less than 1.0%.
Procedure:
Inject the test solution in duplicate. Calculate the % assay of Zolmitriptan by using following
formula-
AT x WS x P x 100
% Assay = ------------------------------------
AS x WT x (100-L)
Where,
AT = Mean area for Zolmitriptan peak in Test solution.
AS = Mean area for Zolmitriptan peak in first Standard solution.
WS = Weight of Zolmitriptan working standard in mg.
WT = Weight of sample in mg.
P = Purity of Zolmitriptan working standard on as such basis in %.
L = Loss on drying of sample in %.
Format No.: ARD/F/18/1 Page 5 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
6 VALIDATION
Perform validation as per procedure given below. Record all weights and dilutions in
worksheet `Validation of HPLC assay method for API’. Perform calculations using
calculator or Microsoft Excel. Compare the results with acceptance criteria.
6.1 SPECIFICITY:
6.1.1 Blank interference
Inject diluent and observe for any peaks at the RT corresponding to the peak of
Zolmitriptan.
Acceptance Criteria:
There should be no peaks in the chromatogram of blank solution at the RT corresponding
to Zolmitriptan.
6.1.2 Peak Purity
Inject 20 µl of standard solution as given in method into the chromatograph. Determine the
peak purity of Zolmitriptan peak using Photodiode Array Detector.
Acceptance criteria:
The peak should be pure as seen on PDA.
6.2 LINEARITY
Stock standard solution:
Weigh accurately about 100 mg of Zolmitriptan working standard in a 100 ml volumetric
flask. Add about 50 ml of diluent, sonicate to dissolve and dilute to 100 ml with diluent.
From the Stock standard solution prepare various levels as given below:
Format No.: ARD/F/18/1 Page 6 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
Linearity Stock standard Diluted to Conc. Nominal conc.
Levels solution volume (ppm) (%)
(ml) (ml)
1 1.0 100 10 10
2 2.0 100 20 20
3 5.0 100 50 50
4 10.0 100 100 100
5 12.0 100 120 120
6 15.0 100 150 150
Inject each solution in duplicate and record the chromatograms. Calculate the mean peak
area for duplicate injections. Plot graph of mean peak area against concentration (in ppm)
and determine the slope, Y- intercept and correlation coefficient.
Acceptance criteria:
The response should be linear and correlation coefficient should be more than 0.997.
6.3 PRECISION
6.3.1 System precision
Make six replicate injections of a 100 ppm standard solution into the chromatograph.
Calculate the RSD for peak area and retention time for the peak of Zolmitriptan.
Acceptance Criteria:
RSD for peak area and retention times should be less than 2.0% and 0.5% respectively.
Format No.: ARD/F/18/1 Page 7 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
6.3.2 Repeatability
Carry out six replicate determinations (inject each determination in duplicate) of a batch of
Zolmitriptan. From the assay calculate mean, SD, RSD and confidence interval.
Acceptance Criteria:
The RSD for assay should be less than 2.0%.
6.4 ACCURACY
Accuracy will be inferred once precision, linearity and specificity have been established.
6.5 STABILITY OF SAMPLE SOLUTION
Prepare a solution of sample and analyze it immediately against standard solution.
Keep the solution for 24 hrs at room temperature. Withdraw samples at 2, 6, 12 & 24 hrs
and analyze against standard solution. Calculate the assay for each sample.
[Note: Store the standard in refrigerator and use it for 24 hrs interval sample after it attains
room temperature.]
Acceptance Criteria:
The solution is said to be stable upto a given time period if the change in assay at that time
interval is less than 0.5% of the initial value.
Format No.: ARD/F/18/1 Page 8 of 9
Analytical R & D
PROTOCOL
Title : Analytical Method Validation for Assay of Zolmitriptan by HPLC
Document No. : ARD/PRO/17 Issue No. :1
Effective date : 17/01/2005 Supersedes : Nil
Issue no.
6.6 ROBUSTNESS
Make the following changes in method. With each of the changes determine assay of
sample.
- Change the pH of buffer solution by +0.2, i.e. 6.8 and 7.2.
- Change the composition of mobile phase by varying minor component by +10%, i.e.
91:9 and 89:11.
- Change the flow rate by +0.2 ml / min, i.e. 1.3 and 1.7 ml/min.
For each of the changes, calculate separately the % RSD for assay obtained in
repeatability and assay obtained with changed condition.
Acceptance Criteria:
The RSD should be less than 2.0%.
7 COMPILATION OF REPORT
Prepare a report based on all above data. Attach linearity graphs and chromatograms for
specificity and typical chromatogram of sample preparation.
Based on the results, comment on the following:
- Specificity of the method.
- Linearity, precision and accuracy over the analytical range.
-
Duration for which active is stable in solution.
- Robustness of the method. Any critical parameters should be mentioned and their
restrictive limits should be specified.
This is the end of the document
Format No.: ARD/F/18/1 Page 9 of 9
