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Analytical Method Validation protocol

Analytical Method Validation protocol

                                                                                                                                

Product/Material Name                :________________________Tablet 

Strength(s)                                      :NA 

Test  Name                                      :Related substance (By HPLC) 

Protocol No:……………….

Protocol Content

Sr. No. Section Title Page No.
1.0    Protocol approval
2.0    Objective
3.0    Scope
4.0    Introduction and overview
5.0    Validation team
6.0    Methodology
7.0 Instrument/Equipment and materials
8.0 Validation parameters to be perform
9.0 System suitability
10.0 Filter Compatibility
11.0 Filter Saturation
12.0 Specificity
13.0 Limit of Quantification and Limit of Detection
14.0 Linearity and Range
15.0 Precision (System Precision, Method precision and Intermediate precision)
16.0 Accuracy
17.0 Solution Stability
18.0 Robustness
19.0 Validation summary table
20.0 Overall conclusion
21.0 Abbreviation
22.0 Attachment: Test Data Sheet

1.0              Protocol approval

Prepared By

Name Designation Department Signature Date
   

  Reviewed By

Name Designation Department Signature Date
   

     Approved By

Name Designation Department Signature Date
   

1.0  Objective

To validate analytical method for Related Substances of _________________Tablets (Etamsylate Tablets)by GC. This Validation study is intended to show that this method is specific, linear, precise and accurate for its intended purpose. Method reference is STP No. ……….

2.0 Scope

This protocol is applicable for Validation of analytical method for Related Substances of Etamsylate Tablets  at ___________ Ltd.

3.0 Introduction and overview

3.1   This protocol provides detailed information on the aspects of Validation of the Related Substances method.

3.2   All qualified and calibrated instruments/equipments shall be used.

3.3   Acceptance criteria and rationale for assessing Validationare asperSOP No.: SOP/QC/184-current version.

4.0 Validation Team

Sr. No. Responsibility Department
1 Preparation of Validation protocol QA
2 Checking of Validation protocol QC
3 Execution of Validation protocol QC
4 Review of final Validationprotocol QA
5 Approval ofValidation protocol Head Quality / Designee

 5.0 Methodology (Assay):

Reagents:

Potassium dihydrogen orthophosphate (AR Grade)

Sodium hydroxide (AR Grade)

Phosphoric Acid (AR Grade)

Ammonium Acetate (AR Grade)

Glacial Acetic acid (AR Grade)

Acetonitrile (HPLC grade)

Tetrahydrofurn (AR Grade)

Methanol (HPLC Grade)

Water

Preparation of 1.54 gm of Ammonium acetate in 1000ml of water: Accurately weigh and transfer 1.54 g of ammonium acetate in 1000 ml of volumetric flask. Add about 700 ml of water, shake to dissolve and make up the volume with water adjusting the pH 4.0 with glacial acetic acid.

Preparation of mixture of 92.5 volume of acetonitrile and 7.5 volumes of tetrahydrofuran:

Accurately transfer 925 volumes of acetonitrile and 75 volumes of tewtrahydrofuran in 1000 ml of volumetric flask and mix it properly.

Solvent mixture: A solution prepared by dissolving 6.8 gm of potassium dihydrogen orthophosphate and 0.9 gm of sodium hydroxide in 1000ml of water and adjusting the pH 6.8 with phosphoric acid or sodium hydroxide.

Mobile phase: A mixture of 50 volume of a buffer solution prepared by dissolving 1.54 gm of Ammonium acetate in 1000ml of water and adjusting the pH 4.0 with glacial acetic acid, and 50 volume of a mixture of 92.5 volume of acetonitrile and 7.5 volumes of tetrahydrofuran.

Test Solution: Crush 20 tablets and transfer accurately weigh a portion of powder equivalent to about 100 mg OF Atorvastatin in dry 100ml volumetric flask, add about 25 ml methanol and sonicate to dissolve and make up the volume with methanol. Dilute 10 ml of this solution to 100ml of solvent mixture. Centrifuge the solution for 5 minutes at 1500 rpm. Use the supernatant liquid as test.

Reference solution-A: Transfer an accurately weighed 50 mg of Etamsylate working standard into a 100 ml dry volumetric flask. Add 50 ml of purified water, sonicate to dissolve and make up the volume with purified water and mix. Dilute 5 ml of this solution to 250 ml with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate.

Reference solution-B: Weigh accurately about 10 mg of Etamsylate working standard and 10 mg of Hydroquinone impurity-A into a 10 ml dry volumetric flask. Add 5 ml of purified water, dissolve and make up the volume with purified water and mix. Further dilute 1 ml of this solution to 100 ml with purified water. Filter through 0.45μ filter and discard first few ml of the filtrate.

Chromatographic condition.:

Column                                              :Hypersil BDS, C18 (250 x 4.6) mm, 5µm or equivalent

Injection volume                             :10µl

Wavelength                                        : 220 nm

Flow rate                                            : 0.8 ml/minute

Run time                                            :40 minutes

Sample cooler temperature       : 2ºC to 8°C

A gradient program is given below:

Time (minutes) Mobile Phase-A Mobile Phase-B
0 90 10
15 90 10
20 50 50
25 30 70
30 55 45
35 90 10
40 90 10

Procedure:

  1. Inject one injection of water as blank into liquid chromatograph.
  2. Inject one injection of placebo preparation into liquid chromatograph.
  • Inject one injection of reference solution-B into liquid chromatograph.

Calculate the system suitability parameters:

Resolution between Etamsylate and Hydroquinone impurity-A should not be less than 5.0.

  1. Inject five replicate injections of reference solution-A into liquid chromatograph.

The test is not valid unless the tailing factor is not more than 2.0, column efficiency is not less than 1500 theoretical plates and the relative standard deviation for area of Etamsylate for replicate injections is not more than 2.0%.

  1. Inject one injection of each test solution into the liquid chromatograph.
  2. The approximate retention time of Etamsylate peak is about 4.5 minutes and relative retention time of Impurity-A is about 1.5.

Note: Disregard any peak in sample chromatogram if peak observed at the retention time of blank and placebo.

Calculation: Calculate the Impurity-A, Single maximum unknown impurity and total impurities.

  AT1 WS 5 10 P Avg.Wt.
% of Impurity A = ——- x ——x ——- x ——- x ——- x ——- x 100 x 0.5
  AS 100 250 WT 100 Claim

 

  AT2 WS 5 10 P Avg.Wt.
% of Single Maximum = ——- x ——x ——- x ——- x ——- x ——- x 100
unknown impurity   AS 100 250 WT 100 Claim

 

  AT3 WS 5 10 P Avg.Wt.
% of Total = ——- x ——x ——- x ——- x ——- x ——- x 100
unknown impurities   AS 100 250 WT 100 Claim

Total Impurities: Impurity-A + Total unknown impurities

Where,

AT1     = Peak area of Impurity-Aobtained with test solution.

AT2     = Peak area of any single maximum unknown impurity obtained with test solution.

AT3     = Peak area of Total unknown impurities obtained with test solution.

AS       = Peak area of Etamsylate obtained with Reference solution-A.

WS      = Weight of standard in mg.

WT      = Weight of sample in mg.

P          = Potency of working standard on as such basis in %.

0.5       = Correction factor of Impurity-A 

Reference Specification No.:QC/

Reference STP No.:QC/STP/

Acceptance Criteria:

S. No. Impurity Acceptance criteria
1. Impurity-A Not more than 0.3%
2. Single maximum unknown impurity Not more than 0.3%
3. Total Impurities Not more than 1.0%

Assay Methodology for Forced Degradation Study:

Reagents and chemical:

Disodium hydrogen Orthophosphate AR Grade

Phosphoric acid AR Grade

Water

Chromatographic Condition:

Column                       : C18, 250 x 4.6 mm 5 µ (YMC ODS, 250 x 4.6 mm is suitable)

Flow rate                     : 1.0 ml / minute

Wavelength                 : 305 nm

Temperature                : 25o C ± 2o C

Injection volume         : 10 µl

Run time                     : About 5 minutes

Mobile Phase: Dissolve 1.4 g of disodium hydrogen orthophosphate in 1000 ml water; adjustpH to 4.0 with dilute phosphoric acid. Prepare a filtered and degassed mixture of this buffer and acetonitrile in the ratio 73 : 27.

Standard Preparation: Weigh accurately 63.0 mg Etamsylate working standard into a 25 ml dry volumetric flask, sonicate until it gets dissolved and dilute to volume with water. Transfer 5 ml of this solution into a 25 ml volumetric flask and dilute to volume with mobile phase and mix. Filter through 0.45µ filter and discard first few ml of the filtrate.

Test Preparation:As per respective degradation condition mentioned under Specificity study.

System suitability:

Chromatograph the standard preparation and record the peak responses as directed under procedure. The column efficiency is not less than 2000 theoretical plates, the tailing factor not more than 2.0, and the relative deviation for replicate injections is not more than 2.0%.

Procedure:

(i) Inject mobile phase as blank into liquid chromatograph.

(ii) Inject six replicates of standard preparation into liquid chromatograph.

(iii) Inject once test preparation into liquid chromatograph.

(iv) Record the chromatograms. Measure the responses for major peaks. Calculate the assay of Etamsylate in each tablet from the major peak areas of standard and test preparation and percentage potency of working standard used.

(v) Retention time for Etamsylate peak is about 3.0 minutes..

Calculation:

AT         WS          5       10         20        P

= ———x———–x——–x———x——-x——-x Average weight of tablets

AS          25          25        WT         1       100

Where,

AT       =          Peak area due to Etamsylate in the  test preparation

AS       =          Peak area due to Etamsylate in the standard preparation

WS      =          Weight of Etamsylate working standard taken in mg

WT      =          Weigh of sample taken in mg

P          =          Potency of Etamsylate working standard on as such basis.

Assay (In %) =   Average result in mg/tablet x 100

———————————————-

Label Claim

1.0  Instrument/Equipment and materials:

Instrument/Equipments details:

S. No. Instrument/ Equipment Name ID No. Make Calibration due on

Material Details:

S. No. Material Name B.No./Lot No. Potency/Purity Validity

Reagent and Solvent Details:

S. No. Reagent/Solvent B.No./Lot No. Make Grade Validity

2.1 Column Details:

S. No. Column Description Dimension Make Serial No. Column ID

2.0 Validation parameters to be perform:

Analytical method shall be validated as per predefined protocol for the analytical parameters as given below.

  1. System suitability
  2. Filter Compatibility
  3. Filter Saturation
  4. Specificity (Interference Study and Forced Degradation)
  5. Limit of Quantification and Limit of Detection
  6. Linearity and Range
  7. Precision
    1. System Precision
    2. Method Precision
    3. Intermediate Precision
  8. Accuracy
  9. Solution Stability
  10. Robustness

Note: Experimental plan and Data Evaluation

  1. System suitability to be done as per methodology and to be monitored throughout the experiment.
  2. The experiments may be done as sequential or parallel operations.
  3. The dilutions can be altered depending on the quantity of Standards, impurities and Stock solutions but the final concentration should remain same as mentioned in the protocol.
  4. During preparation of solutions and evaluating results, Potency/Purity of Working Standard(s)/ Reference Standard(s)/ Impurity Standard(s) shall be considered and compensated for calculation of concentration of solution as applicable.

3.0  System suitability

To demonstrate and verify that the system suitability parameters of the chromatographic system are adequate to the subjected analysis.

Prepare Mobile phase-A and Mobile phase-Bas mentioned in methodology part.

Reference solution-A: Transfer an accurately weighed 50 mg of Etamsylate working standard into a 100 ml dry volumetric flask. Add 50 ml of purified water, sonicate to dissolve and makeup the volume with purified water and mix. Dilute 5 ml of this solution to 250 ml with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate.

Reference solution-B: Weigh accurately about 10 mg of Etamsylate working standard and 10 mg of Hydroquinone impurity-A into a 10 ml dry volumetric flask. Add 5 ml of purified water, dissolve and makeup the volume with purified water and mix. Further dilute 1 ml of this solution to 100 ml with purified water. Filter through 0.45μ filter and discard first few ml of the filtrate.

Procedure:

Separately inject water as blank (one injection), reference solution (b) (one injection) and reference solution (a)(five replicate injections) into the chromatograph, record the chromatograms and measure the responses for the major peaks.

Observations shall be recorded in table-1.

Table-1 (a): System suitability

Parameter Acceptance Criteria Observations
Resolution (Reference Solution-B) Resolution between Etamsylate and Hydroquinone impurity-A should not be less than 5.0.
Tailing factor (Reference Solution-A) Should not be more than 2.0
Column efficiency (Reference Solution-A) Should not be less than 1500 theoretical plates

Table-1(b): System suitability (Reference solution (A)

S.No. Area of Etamsylate
1.
2.
3.
4.
5.
AVG
STDEV
%RSD

 Acceptance Criteria:

Resolution between Etamsylate and Hydroquinone impurity-A should not be less than 5.0 from Reference solution-B.

The test is not valid unless the tailing factor is not more than 2.0, column efficiency is not less than 1500 theoretical plates and the relative standard deviation for area of Etamsylate for replicate injections is not more than 2.0% from Reference solution-A.

Conclusion: ______________________________________________________________

 4.0  Filter Compatibility:

Procedure: Prepare one spike sample with impurities at 100% of specification level as described in method precision parameter.

At the filtration stage, Filter through 0.45µm nylon, 0.45µm PVDF, 0.45µm PTFE Syringe filter. Discard 3 ml of the sample solution and collect the sample solution for further analysis Centrifuge the sample at about 3000 rpm for 10 minutes. Analyze both the sample solution (filtered and centrifuged) as described in the test procedure and calculate the % impurity results. Determine the absolute difference between the impurity results obtained from the filtered sample and centrifuge sample.

Observations shall be recorded in table-2.

Table-2(a)Filter Compatibility 

____________________________Tablets
Details % Impurities
Impurity-A Absolute Difference Single Maximum unknown impurity Absolute Difference
Filtered through 0.45 µm Nylon filter
Filtered through  0.45 µm PVDF filter
Filtered through 0.45 µm PTFE filter
Centrifuged Sample NA NA

 Table-2(b)Filter Compatibility 

___________________Tablets
Details % Impurities
Total Impurities Absolute Difference
Filtered through 0.45 µm Nylon filter
Filtered through  0.45 µm PVDF filter
Filtered through 0.45 µm PTFE filter
Centrifuged Sample NA

 Acceptance Criteria:

The Absolute difference between the unfiltered (Centrifuged) and filtered samples should not differ by more than 0.05 for each individual impurity and 0.1 for total impurities.

Conclusion: __________________________________________________________________

5.0  Filter Saturation:

Procedure: Prepare single spiked sample solution as described in the method precision parameter.

At the filtration stage, Filter the sample through filter selected in Filter compatibility study, discarding 1 ml, 3 ml and 5 ml of the sample solution and collect the sample solution for further analysis. Determine the absolute difference between the consequent impurity results obtained from the filtered sample.

Observations shall be recorded in table-3. 

Table-3(a)Filter Saturation 

__________________Tablets
Details % Impurities
Impurity-A Absolute Difference Single Maximum unknown impurity Absolute Difference
1 ml Discarded NA NA
3 ml Discarded
5 ml Discarded

 Table-3(b)Filter Saturation 

______________Tablets
Details % Impurities
Total impurities Absolute Difference
1 ml Discarded NA
3 ml Discarded
5 ml Discarded

 Acceptance Criteria:

Absolute difference in the impurity result obtained for two consequent or successive filtered solution should not differ by more than 0.05 for each individual impurity and 0.1 for total impurities.

Conclusion: __________________________________________________________________

6.0  Specificity:

Interference Study:

Objective: To demonstrate the ability of the analytical method to separate the analyte and there is no interference in the Etamsylate peak and other known impurity peak due to other component that may be present in the sample matrix.

Reference solution-A: Transfer an accurately weighed 50 mg of Etamsylate working standard into a 100 ml dry volumetric flask. Add 50 ml of purified water, sonicate to dissolve and makeup the volume with purified water and mix. Dilute 5 ml of this solution to 250 ml with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate.

Reference solution-B: Weigh accurately about 10 mg of Etamsylate working standard and 10 mg of Hydroquinone impurity-A into a 10 ml dry volumetric flask. Add 5 ml of purified water, dissolve and makeup the volume with purified water and mix. Further dilute 1 ml of this solution to 100 ml with purified water. Filter through 0.45μ filter and discard first few ml of the filtrate.

Placebo solution: Weigh accurately 100 mg placebo into a 10 ml dry volumetric flask. Add 5 ml of purified water; sonicate to dissolve and make up the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate.

Test Solution: Weigh and crush 20 tablets and transfer an accurately weighed portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. (Use freshly prepared solution).

Impurity-A Stock solution: Transfer an accurately weighed quantity of about 1.0 mg of Hydroquinone Impurity-A standard into a 10.0 ml volumetric flask, add about 5.0 ml of purified water and sonicate until the substance is completely dissolved. Dilute to mark with purified water and mix.

Impurity-A ID solution:Pipette 3.0 ml of Impurity-A stock solution into 10 ml volumetric flask. Make volume upto the mark with purified water and mix well.

Spiked Sample Preparation:Weigh and crush 20 tablets and transfer an accurately weighed portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes, add 3.0 ml of Impurity-A stock solution and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate.

Procedure:Inject water as blank, placebo solution, reference solution (B) and reference solution (A) into the chromatograph as per methodology followed by impurity ID solution, sample solution and spiked sample into the chromatograph.

Record the observation in table-4. 

Table-4Specificity (Interference Study)

Sample name Retention Time (In minutes) Peak purity (Pass/Fail)
Etamsylate Impurity-A
Blank
Placebo      
Reference solution (B)
Reference solution (A)
Impurity-A ID solution
Sample solution
Spiked sample solution

 Acceptance Criteria:No peak should be observed due to blank, placebo and known impurities at the retention time of the principal (Etamsylate) peak as observed in the reference solutions and sample solution. Peak of Etamsylate and known impurities should be spectrally pure (No impurity should be detected in respective PDA spectrum).

Conclusion: ______________________________________________________________ 

Forced Degradation Study:

Objective: Forced degradation studies are performed to demonstrate that the analytical method is stability indicating. This study will be performed on ______________tablets stored under relevant stress conditions (e.g. Acid, Base, Peroxide, Humidity, Thermal and UV).

Preparation of solution:

Prepare the Mobile phase-A, Mobile phase-B, Reference Solution-B,Reference solution-A and test solution (control sample) as per the proposed method of Analysis given in methodology (6.0).

UV Degradation:

Sample Preparation:

For Related Substances test:Keep the tablet powder about 1.0g in suitable transparent Petri plate and expose under UV light for an overall illumination of not less than 1.2 million lux hours. Weigh and transfer accurately a portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above filtered sample to 20ml volumetric flask with mobile phase. (To be analyzed by Assay methodology).

Placebo preparation:Keep the placebo powder about 1.0g in suitable transparent petri plate and expose under UV light for an overall illumination of not less than 1.2 million lux hours. Weigh and transfer accurately a portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:Prepare as per Methodology. (For Assay and related substances method)

Thermal degradation:

Sample Preparation:

For Related Substances test:Keep the tablet powder about 1.0g in suitable transparent petri plate for 24 hours in oven at 60°C.Weigh and transfer accurately a portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above filtered sample to 20 ml volumetric flask with mobile phase. (To be analyzed by Assay methodology).

Placebo preparation:

Keep the placebo powder about 1.0g in suitable transparent petri plate for 24 hours in oven at 60°C.Weigh and transfer accurately a portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:

Prepare as per Methodology.(For Assay and related substances method) 

Humidity Degradation:

Preparation of saturated sodium chloride solution: Dissolve 35g of sodium chloride in 100ml water in suitable container/beaker. (Prepare this solution as per desiccator capacity, weight of sodium chloride and volume can be adjusted accordingly).

Sample Preparation:

For Related Substances test:Transfer an accurately weighed portion of tablet powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Keep the volumetric flask in open condition (without cap) for 24 hours at humidity 75% in humidity chamber. After 24 hours add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above filtered sample to 20 ml volumetric flask with mobile phase. (To be analyzed by Assay methodology).

Placebo preparation:

Transfer an accurately weighed portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Keep the volumetric flask in open condition (without cap) for 24 hours at humidity 75% in humidity chamber. After 24 hours add 5 ml of purified water, sonicate for 5 minutes and makeup the volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:

Prepare as per Methodology.(For Assay and related substances method) 

Acid Degradation:

Preparation of 1.0N HCl: Slowly add 8.5 ml of concentrated Hydrochloric acid in a 100 ml volumetric flask containing sufficient water & mix further dilute to volume with water.

Preparation of 1.0N NaOH solution: Weigh accurately about 4.0g of sodium hydroxide in a 100ml volumetric flask. Add sufficient water and sonicate to dissolve and further dilute to volume with water.

Sample Preparation:

For Related Substances test: Weigh and transfer accurately a portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 1N Hydrochloric acid and keep for 3 hours at room temperature. After 3 hours add 1.0ml of 1N sodium hydroxide for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above-filtered sample to 20 ml volumetric flask with mobile phase. (To be analyzed by Assay methodology).

Placebo preparation:

Weigh and transfer accurately a portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 1N Hydrochloric acid and keep for 3 hours at room temperature. After 3 hours add 1.0ml of 1N sodium hydroxide for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:

For Related Substances test:In 10 mL of volumetric flask. Add about 5 ml of purified water and add 1.0 ml of 1N Hydrochloric acid and keep for 3 hours at room temperature. After 3 hours add 1.0ml of 1N sodium hydroxide for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Base Degradation:

Preparation of 0.05N HCL: Slowly add 4.3 mL of concentrated Hydrochloric acid in a 1000 ml volumetric flask containing sufficient water & mix further dilute to volume with water.

Preparation of 0.05N NaOH solution: Weigh accurately about 2.0g of sodium hydroxide in a 1000ml volumetric flask. Add sufficient water and sonicate to dissolve and further dilute to volume with water.

Sample Preparation:

For Related Substance test: Weigh and transfer accurately a portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 0.05N sodium hydroxide and keep for 5 minutes at room temperature. After 5 minutes add 1.0ml of0.05N Hydrochloric acid for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above filtered sample to 20 ml volumetric flask with mobile phase. (To be analyzed by Assay methodology). 

Placebo preparation:

Weigh and transfer accurately a portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 0.05 N sodium hydroxide and keep for 5 minutes at room temperature. After 5 minutes add 1.0ml of0.05 N Hydrochloric acid for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:

For Related Substances test:In 10 mL of volumetric flask. Add about 5 ml of purified water and add 1.0 ml of 0.05N sodium hydroxide and keep for 5 minutes at room temperature. After 5 minutes add 1.0ml of0.05N Hydrochloric acid for neutralization. Dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology. 

Peroxide Degradation:

Sample Preparation:

For Related Substances test: Weigh and transfer accurately a portion of powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 30% v/v solution of hydrogen peroxide and keep for 3 hours at room temperature. After 3 hours dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

For Assay test:Further dilute 1.0 ml of above filtered sample to 20 ml volumetric flask with mobile phase. (To be analyzed by Assay methodology).

Placebo preparation:

Weight and transfer accurately a portion of placebo powder equivalent to 100 mg of Etamsylate into a 10 ml dry volumetric flask. Add about 5 ml of purified water and sonicate for 5 minutes. Add 1.0 ml of 30% v/v solution of hydrogen peroxide and keep for 3 hours at room temperature. After 3 hours dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Blank Preparation:

For Related Substances test:In 10 mL volumetric flask. Add about 5 ml of purified water and add 1.0 ml of 30% v/v solution of hydrogen peroxide and keep for 3 hours at room temperature. After 3 hours dilute to volume with purified water and mix. Filter through 0.45μ filter and discard first few ml of the filtrate. Treated sample shall be analyzed as per related substances methodology.

Record the detected impurities (Spectral purity) in PDA spectrum for ______________tablets in Table- 5.

Table: 5a Forced degradation for XXXXXXXXXXXXXXXXXXXTablets

Sample ID % Impurity-A

(by Related Substances method)

% Highest individual unknown Impurity

(by Related Substances method)

% Total Impurities (by Related Substances method) % Assay

(by Assay method)

Mass Balance
Control Sample  
Acid degradation
Alkali degradation  
Peroxide degradation
Thermal degradation
UV light degradation
Humidity degradation

 Table: 5b Forced degradation for __________________Tablets

Sample ID Impurity-A Etamsylate
Retention Time Impurity Detected (Yes/No) Retention Time Impurity Detected (Yes/No)
Control Sample  
Acid degradation
Alkali degradation  
Peroxide degradation
Thermal degradation
UV light degradation
Humidity degradation

 

(Treated sample total impurities (from RS) + Treated sample Assay) X 100
Mass Balance = —————————————————————————————–
                               Untreated Sample Assay + Untreated sample Impurities (from RS)

 Note: Respective degradation conditions may be adjusted to obtain degradation between 5% to 30% on Initial value in any of the one degradation condition. If product/material did not show degradation with worst condition, then it shall be reported as Product/Material did not degrade under specified conditions.

Acceptance criteria:All the known impurity peaks and main peak should be well resolved from each other and should be spectrally pure.Mass balance should not be less than 95%. If Mass balance does not achieved, same shall be justified.

Conclusion: ________________________________________________________________

 7.0   Limit of Quantification and Limit of Detection:

LOD & LOQ shall be determined by residual standard deviation method:

Prepare a series of solution by quantitative dilutions of stock solution of Impurity-A standard and Etamsylate standard to obtain solution of suitable lower concentrations

Standard stock solution:Transfer an accurately weighed quantity of about 3.0 mg each of Hydroquinone Impurity-A standard and Etamsylate standard into a 100.0 ml volumetric flask, add about 50.0 ml of purified water and sonicate until the substance is completely dissolved. Dilute to mark with purified water and mix.

10% solution:Dilute 1.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water.

20% solution:Dilute 2.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water.

30% solution:Dilute 3.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water.

40% solution:Dilute 4.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water.

50% solution:Dilute 5.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water.

60% solution:Dilute 6.0 ml of Standard stock solution into 10 ml volumetric flask and dilute with water. 

Determine the slope and residual standard deviation for Impurity-A and Etamsylateby using the peak area and corrected concentration (ppm) and calculate the value of limit of detection and limit of quantitation for impurity-A by using the following formula. 

Calculation:

3.3 × σ                                                      10 ×  σ

LOD = ——————–                               LOQ = ——————–

S                                                                   S

 Where,

σ                = Residual Standard Deviation of regression line

S               = Slope of regression line

LOD          = Limit of detection

LOQ        = Limit of Quantitation

Table-6 (a) Limit of Detection & Limit of Quantitation (Impurity-A)

Sr.No. Concentration (%) Corrected Concentration (ppm) Area Response
1.0 10 %
2.0 20 %
3.0 30 %
4.0 40 %
5.0 50 %
6.0 60 %
Slope
STEYX (σ)
Predicted LOD (ppm)
Predicted LOQ (ppm)
Predicted LOD (%)
Predicted LOQ (%)

Table-6 (b) Limit of Detection & Limit of Quantitation (Etamsylate for unknown impurity)

Sr.No. Concentration (%) Corrected Concentration (ppm) Area Response
1.0 10 %
2.0 20 %
3.0 30 %
4.0 40 %
5.0 50 %
6.0 60 %
Slope
STEYX (σ)
Predicted LOD (ppm)
Predicted LOQ (ppm)
Predicted LOD (%)
Predicted LOQ (%)

Prepare LOD solution containing Impurity-A and Etamsylate as per predicted concentration and inject in duplicate to confirm the detection limit.

Prepare a solution at LOQ level (as per predicted concentration) and inject in six replicates. Calculate the relative standard deviation of the peak area.

The results shall be tabulated in table-7

Table-7(a): Response at LOD level

Sample No. Area
Impurity-A Etamsylate
1.
2.
Concentration in % (w.r.t. sample)

Table-7(b): Response at LOQ level

Sample No. Area
Impurity-A Etamsylate
1.
2.
3.
4.
5.
6.
Mean
SD
% RSD
Concentration in % (w.r.t. sample)

Table-7(c): Signal to noise ratio at LOQ level

Sample No. Signal to noise ratio
Impurity-A Etamsylate
1.
2.
3.
4.
5.
6.

 Acceptance Criteria:

The detector response should be positive for LOD solution.

%RSD for six replicate injections of LOQ concentration should not be more than 10.0% and signal to noise ratio at LOQ concentration shall not be less than 10. 

Conclusion: ______________________________________________________________

 8.0  (A) Linearity:

Linearity: To demonstrate that the analytical method is capable to obtain test results, which are directly proportional to the concentration of analyte.

Procedure: Prepare linearity solutions to obtain solution at LOQ & minimum five levels between 50 % and 150 % level of the specification limit and Inject each solution into the chromatograph.

Preparation of solution:

Prepare the Mobile phase-A, Mobile phase-B and reference solutions as per the proposed method of Analysis given in methodology (6.0).

Linearity Solution at LOQ level:Prepare as per LOQ Precision solution.

Preparation of Linearity Stock solution: Transfer an accurately weighed quantity of about 3.0 mg each of Hydroquinone Impurity-A standard and Etamsylate standard into a 50.0 ml volumetric flask, add about 25.0 ml of purified water and sonicate until the substance is completely dissolved. Dilute to mark with purified water and mix.

LOQ level linearity solution: Prepare dilution as per LOQ Validation solution.

50 % Linearity solution: Transfer 5.0 ml of Linearity stock solution into 20 ml volumetric flask and dilute with water and mix well.

80% Linearity solution:Transfer4.0 ml of Linearity stock solution into 10 ml volumetric flask and dilute with water and mix well.

100% Linearity solution: Transfer 5.0 ml of Linearity stock solution into 10 ml volumetric flask and dilute with water and mix well.

120% Linearity solution:Transfer 6.0 ml of Linearity stock solution into 10 ml volumetric flask and dilute with water and mix well.

150% Linearity solution: Transfer 15.0 ml of Linearity stock solution into 20 ml volumetric flask and dilute with water and mix well.

Procedure: Perform the system suitability as directed in 9.0. inject the above prepared linearity solutions and Plot a graph of slope, Y-intercept and correlation coefficient of the regression line.

The results shall be tabulated in table-8.

Table-8(a)Linearity 

Etamsylate
Sr. No. % Concentration Corrected concentration Area
1. LOQ
2. 50
3. 80
4. 100
5. 120
6. 150
Correlation Coefficient
% Y-intercept
Slope

 Table-8(b) Linearity 

Impurity-A
Sr. No. % Concentration Corrected concentration Area
1. LOQ
2. 50
3. 80
4. 100
5. 120
6. 150
Correlation Coefficient
% Y intercept
Slope

Acceptance criteria: Correlation coefficient should not be less than 0.980, slope and %Y intercept are for information only.

Conclusion: ________________________________________________________________

 (B)  Range:

Procedure

For range, inject six replicates each of lower (LOQ) and higher (150%) concentration levels and calculate the mean and relative standard deviation and also record the concentration levels over which the result are linear. Record the observation in table-9. 

Table:9 (a)Range

Area of Etamsylate
Sr. No. Lower level (LOQ) Higher level (150%)
1.
2.
3.
4.
5.
6.
Mean
SD
%RSD

 

Table:9 (b) Range

Area of Impurity-A
Sr. No. Lower level (LOQ) Higher level (150%)
1.
2.
3.
4.
5.
6.
Mean
SD
%RSD

 Acceptance criteria:For Range the relative standard deviation should be not more than 10.0% for six replicate injections.

Conclusion:________________________________________________________________

9.0 Precision:

To demonstrate that the analytical method is capable to yield closeness of data values between a series of measurements obtained from analysis of the same sample.

  • System Precision:

Procedure: Separately inject water as blank (one injection), reference solution (b) (one injection) and reference solution (a) (five replicate injections) into the chromatograph, record the chromatograms and measure the responses for the major peaks.

Observations shall be recorded in table-10.

Table-10 System Precision

Reference solution (A)
S.No. Area of Etamsylate
1
2
3
4
5
AVG
STDEV
%RSD

Acceptance Criteria: The relative standard deviation of six replicate injections of reference solution (A)should not be more than 2.0%.

Conclusion: ________________________________________________________________

  • Method Precision:

Separately inject blank, placebo solution,reference solutions and six independent sample preparation of Etamsylatesample prepared as per methodology.

Note: Prepare and inject three as such samples and six independent spiked sample preparation of Etamsylate sample spiked at 100 % specification level (as per spike sample preparation mentioned in Specificity parameter) and calculate the actual content of spiked sample using mean content from three as such samples.

Calculate the %Related substances and calculate the mean and relative standard deviation.

The results shall be tabulated in table-11.

Table- 11Method precision 

Sample No. Impurity-A Single maximum unknown impurity Total Impurities
1.
2.
3.
4.
5.
6.
Mean
SD
RSD (%)

 Acceptance criteria:

Impurity Level %RSD
0.05% to 0.10% Not more than 25.0%
0.11% to 0.50% Not more than 15.0%
0.51% to 1.0% Not more than 10.0%
More than 1.0% Not more than 5.0%

 Conclusion:________________________________________________________________

  • Intermediate Precision:
    1. For Batch Analysis:

Prepare and inject the six spiked sample solution prepared same as described in Method precision parameter (Using same sample Batch/Lot as used in Method Precision) by different analyst, using different instrument and column on different day.

The results shall be tabulated in table-12.

Table- 12 (a)Intermediate precision 

Sample No. Impurity-A Single maximum unknown impurity Total Impurities
1.
2.
3.
4.
5.
6.
Mean
SD
RSD (%)

Table- 12 (b)Intermediate precision 

Details Analyst-1 Analyst-2
Analyst Name
Instrument ID
Column ID
Date of Analysis

 Acceptance criteria: The absolute difference in Individual impurity between normal condition and changed condition should be not more than 0.1% and that of total impurities should be not more than 0.2%.

Conclusion: ______________________________________________________________ 

  1. For LOQ level:

Prepare a solution at LOQ level (as per predicted concentration) and inject in six replicates. Calculate the relative standard deviation of the peak area., to be established on two different instruments, by two different analysts using two different columns on different days.

The results shall be tabulated in table-13.

Table-13(a): Intermediate Precision (Signal to noise ratio at LOQ level)

Sample No. Signal to noise ratio
Impurity-A Etamsylate
A1 A2 A1 A2
1.
2.
3.
4.
5.
6.

 Table-13(b): Intermediate Precision (Response at LOQ level)

Sample No. Area
Impurity-A Etamsylate
A1 A2 A1 A2
1.
2.
3.
4.
5.
6.
Mean
SD
% RSD

A1 = Analyst-1, A2 = Analyst-2

Table- 13 (c)Intermediate precision 

Details Analyst-1 Analyst-2
Analyst Name
Instrument ID
Column ID
Date of Analysis

Acceptance criteria: The RSD of six test results obtained under normal and changed condition should be not more than 10.0%. The S/N ratio for the analyte peak shall not be less than 10 at LOQ concentration.

Conclusion: ______________________________________________________________

10.0  Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Inject as such sample preparation in triplicate and calculate the mean of Impurity-A. Prepare accuracy samples in the range of LOQ to 150% (LOQ, 50 %, 100 % and 150 %) of specification limit in triplicate by spiking the Impurity-A with sample.

The results shall be tabulated in table-14.

Table-14 Accuracy (Impurity-A)

Level no/Spike level in % Added  ppm Observed ppm %Recovery Mean Recovery
Level – 1

(LOQ)

Level – 2

(50%)

Level – 3

(100%)[1]

Level – 4

(150%)

 Acceptance criteria:

Impurity Level % Recovery
Up to 0.10% Between 50.0 to 150.0%
0.11% to 0.50% Between 70.0 to 130.0%
0.51% to 1.0% Between 80.0 to 120.0%
More than 1.0% Between 90.0 to 110.0%

 Conclusion:________________________________________________________________

11.0  Solution Stability

Determination:

Stability in analytical solution to be validate by freshly preparing sample solution and reference solution (A) as per test method and analyze.

Note: Sample solution spiked with all impurities at specification level to be injected initially and at different time intervals.

Procedure:Prepare reference solution (A) as defined in Methodology part (Section-6) of this protocol and sample solution as per spiked solution described in specificity parameter.Keep the prepared solutions at Bench Top (Room Temperature) and 2°C to 8°Ctill completion of study. Use these solutions to fill the HPLC vial at particular time intervals set in batch sequence.

Analyse the reference solution (A) and sample solutions at zero hour (Initial) and minimum up to 48 hours. Time intervals may vary as per sequence run time.Report the results of initial and actual time interval condition. Determine %difference of response at particular time interval with respect to the initial for reference solution (A) and absolute difference in impurities content at a particular time point with respect to the initial for sample solution.

Results shall be recorded in Table-15. 

Table:  15(a)

Stability of Reference solution (A) (At Room temperature)

Time of injection (Hours) Peak Area % difference of response from initial
Initial

 Table:  15(b)

Stability of Reference solution (A) (At 2°C to 8°C)

Time of injection (Hours) Peak Area % difference of response from initial
Initial

 Acceptance criteria.

The response of standard solution should not differ by more than ± 10.0% from the initial response.

Conclusion:  ________________________________________________________ 

Table:  15(c)

Stability of Sample Solution (At Room temperature)

Time of injection (Hours) % Impurity Content Absolute Difference from initial value
Initial

 Table:  15(d)

Stability of Sample Solution (At 2°C to 8°C)

Time of injection (Hours) % Impurity Content Absolute Difference from initial value
Initial

 Acceptance criteria: The individual impurity should not differ by more than ± 0.05 and total impurities by more than ± 0.1 from initial value.

Conclusion:  ________________________________________________________

12.0  Robustness:

To demonstrate that the analytical method is capable to yield reproducible results, under small but deliberate variations in method parameters such as change of column temperature.

Procedure:Perform analysis at each variable condition by following the methodology, for the following parameters:

  • Change in column oven temperature 25 ± 5°C.
  • Change in pH of Mobile phase-A 6.5 ± 0.1.

Note: Sample solution spiked with all the known related substances (Impurity-A) to be injected under each of the variable conditions to monitor the RRT’s.

Table-16 (a) Robustness (Column oven temperature)

S. No. Area
20°C 25°C 30°C
1
2
3
4
5
6
AVG
STDEV
%RSD

 Table-16 (b) Robustness (Column in pH of Mobile phase-A)

S. No. Area
6.4 6.5 6.6
1
2
3
4
5
6
AVG
STDEV
%RSD

 Table-16 (c) Robustness (Column oven temperature)

Component RRT
20°C 25°C 30°C
Etamsylate
Impurity-A

 Table-16 (d) Robustness (Column in pH of Mobile phase-A)

Component RRT
6.4 6.5 6.6
Etamsylate
Impurity-A

 Acceptance Criteria: System suitability parameters should pass.

Conclusion:  ________________________________________________________

13.0  Validation summary table:

Table 17 Validation summary

Validation parameters Results Acceptance criteria
System suitability
Reference solution (B) Resolution: Not less than 5.0.
Reference solution (A) Tailing Factor:

Column Efficiency:

%RSD:

Not more than 2.0.

Not less than 1500.

Not more than 2.0%

Filter Compatibility
Filter Compatibility Absolute Difference

Impurity-A:

Nylon Filter:

PVDF Filter:

PTFE Filter:

Single Maximum unknown impurity:

Nylon Filter:

PVDF Filter:

PTFE Filter:

Total impurities:

Nylon Filter:

PVDF Filter:

PTFE Filter:

The Absolute difference between the unfiltered (Centrifuged) and filtered samples should not differ by more than 0.05 for each individual impurity and 0.1 for total impurities.

 

Filter Saturation
Filter Saturation Absolute Difference

Impurity-A:

3ml discarded:

5ml discarded:

Single Maximum unknown impurity:

3ml discarded:

5ml discarded:

Total impurities:

3ml discarded:

5ml discarded:

Absolute difference in the impurity result obtained for two consequent or successive filtered solution should not differ by more than 0.05 for each individual impurity and 0.1 for total impurities.

 

Specificity (Interference Study):
Component RT Peak purity (Pass/Fail)
Blank No peak should be observed due to blank, placebo and known impurities at the retention time of the principal (Etamsylate) peak as observed in the reference solutions and sample solution. Peak of Etamsylate and known impurities should be spectrally pure (No impurity should be detected in respective PDA spectrum).
Placebo
Reference solution (B)
Reference solution (A)
Impurity-A ID solution
Sample solution
Spiked sample solution
Specificity (Forced Degradation Study):
Condition Mass Balance Peak purity (Pass/Fail)
Control Sample All the known impurity peaks and main peak should be well resolved from each other and should be spectrally pure.Mass balance should not be less than 95%.
Acid degradation
Alkali degradation
Peroxide degradation
Thermal degradation
UV light degradation
Humidity degradation
Limit of Quantification Validation
Etamsylate %RSD: %RSD for six replicate injections of LOQ concentration should not be more than 10.0%.
Impurity-A %RSD:
Linearity
Etamsylate Correlation coefficient-

% Y intercept-

Slope-

Not less than 0.980

For information only

For information only

Impurity-A Correlation coefficient-

% Y intercept-

Slope-

Not less than 0.980

For information only

For information only

Range
Etamsylate %RSD at lower level-

%RSD at higher level-

Not more than 10.0%

Not more than 10.0%

Impurity-A %RSD at lower level-

%RSD at higher level-

Not more than 10.0%

Not more than 10.0%

Precision
System  Precision
Etamsylate %RSD :  Not more than 2.0
Method Precision
Etamsylate  (%Related Substances) % RSD (Impurity-A):

% RSD (Single maximum unknown impurity):

%RSD (Total impurities):

Impurity Level %RSD
0.05% to 0.10% Not more than 25.0%
0.11% to 0.50% Not more than 15.0%
0.51% to 1.0% Not more than 10.0%
More than 1.0% Not more than 5.0%
Intermediate Precision
For Batch Analysis Absolute Difference

Impurity-A:

Single maximum unknown impurity:

Total impurities:

The absolute difference in Individual impurity between normal condition and changed condition should be not more than 0.1% and that of total impurities should be not more than 0.2%.
For LOQ level %RSD

Etamsylate:

Impurity-A:

The RSD of six test results obtained under normal and changed condition should be not more than 10.0%.
Accuracy
Recovery % Recovery

Impurity-A

1.     For Impurity level up to 0.10% : 50.0-150.0%

2.     For Impurity level 0.11% to 0.50% : 70.0-130.0%

3.     For Impurity level 0.51% to 1.0% : 80.0-120.0%

4.     For Impurity level more than 1.0%: 90.0-110.0%

5.

Solution Stability
Reference solution (A)

At Room temperature

% response difference :

(Time-        )

The response of standard solution should not differ by more than ± 10.0% from the initial response.
Reference solution (A)

At 2°C to 8°C

% response difference :

(Time-        )

Sample solution

At Room temperature

Absolute Difference:

(Time-        )

The individual impurity should not differ by more than ± 0.05 and total impurities by more than ± 0.1 from initial value.
Sample solution

At 2°C to 8°C

Absolute Difference:

(Time-        )

Robustness
Column oven temperature 20°C
Reference solution (B) Resolution: Not less than 5.0.
Reference solution (A) Tailing Factor:

Column Efficiency:

%RSD:

Not more than 2.0.

Not less than 1500.

Not more than 2.0%

Column oven temperature 30°C
Reference solution (B) Resolution: Not less than 5.0.
Reference solution (A) Tailing Factor:

Column Efficiency:

%RSD:

Not more than 2.0.

Not less than 1500.

Not more than 2.0%

Mobile Phase pH 6.4
Reference solution (B) Resolution: Not less than 5.0.
Reference solution (A) Tailing Factor:

Column Efficiency:

%RSD:

Not more than 2.0.

Not less than 1500.

Not more than 2.0%

Mobile Phase pH 6.6
Reference solution (B) Resolution: Not less than 5.0.
Reference solution (A) Tailing Factor:

Column Efficiency:

%RSD:

Not more than 2.0.

Not less than 1500.

Not more than 2.0%

14.0  Overall Conclusion: _____________

 15.0          Abbreviation:

RSD Relative standard deviation
Sr. No. Serial number
QC Quality control
QA Quality assurance
mg Milligram
ID Identification
No. Number
HPLC High Performance Liquid Chromatograph
µm Micro meter
µl Micro liter
ml/min Milliliter per minute
RT Retention time
% Percent
mL Milliliter
PDA Photo diode array
nm nanometer
API Active Pharmaceutical Ingredient
LOD Limit of Detection
LOQ Limit of Quantitation

16.0   Attachment:Test Data Sheet

Attachment-I: Test data sheet for Analytical Method Validation

[1] 100% Accuracy results can be calculated from first three spiked samples of Method Precision.

All Post URL of Drugs formulations

About Pharmaceutical Guidanace

Mr. Shiv Kumar is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 14 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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