Analytical Method Validation for Aceclofenac 100mg and Paracetamol 325mg Tablets

nirvesh400

 

Aceclofenac 100mg and Paracetamol 325mg Tablets

(As per ICH guidelines)

 (BY HPLC)

This document is an exercise on Analytical Method Validation of the various analytical

Methods used in determination of active ingredients in Quality Control Laboratory of

“Company Name ”.

CONTENTS

 

Sr. No Contents Page No.
A Post-Approval 3
B Objective 4
C Method Summary 4
D Scope 4
E Responsibility 4
F Method of Analysis 5
G Summary table 7
1 Specificity 9
2 System Accuracy 9
3 Linearity 10
4 Range 10
5 Accuracy 10
6 (i) Precision-Method Precision 14
6 (ii) Intermediate Precision 15
7 Robustness 18
8 Conclusion 19

 

CONTRIBUTIONS:

This protocol is a team effort of Quality control Laboratory chemists to achieve the objective of validating the analytical methods carried out to estimate the contents of pharmaceutical products manufactured by “Company Name ”.

 

  1. POST APPROVAL:

 

·  Analytical

Method Validation

Protocol Number

 AMV/SCT/T/003
·            Validation Frequency Analytical Methods should be re-validated  after any significant change in analytical method.
  Designation Name of the Person Sign /Date
·  Prepared By  Officer -QC  

 
·  Checked By Executive -QC  

 
·  Checked By  Manager-QC  

 
·  Approved By Head-QA  

 

 

  1. OBJECTIVE: The efficacy & safety of a medicinal product can only be assured by analytical monitoring of its quality. This report is designed to validate the HPLC Assay Method for Aceclofenac and Paracetamol  in Tablets as per ICH Guidelines for routine analysis.
  2. METHOD SUMMARY:

The analytical method for assay of Aceclofenac + Paracetamol Tablets by HPLC shall be validated as per ICH Guidelines for routine analysis.

  1. SCOPE : The Quantity determination of Aceclofenac + Paracetamol Tablets by HPLC shall be validated for following analytical and performance parameters as described in ICH guidelines.
  2. Specificity
  3. System Accuracy
  4. Linearity
  5. Range
  6. Accuracy
  7. Precision
  8. Robustness

 

  1. RESPONSIBILITY:

Quality Control

. To prepare the analytical method validation Report for the assay of Aceclofenac + Paracetamol . To validate the analytical method for the assay of Aceclofenac + Paracetamol tablets

. To prepare analysis record based on the results and observations.

Quality Assurance Department

. To approve the analytical method validation report.

PRODUCT DETAILS:

Product Name

 

 Aceclofenac and Paracetamol  Tablets
Label Claim

 

 

Batch No.

Mfg. Date

Exp. Date

Each Film Coated Tablets Contains :

Aceclofenac IP         100mg

Paracetamol IP         325mg

 

 

 

  1. METHOD OF ANALYSIS (BY HPLC):-

Equipment required:

  • HPLC system UV/Visible detector model- Agilent 1260 auto sampler , Instrument ID   SC/QC/038.
  • Analytical balance, Make RADWAG, Model-AUX220, Instrument ID No. SC/QC/002
  • Ultrasonic cleaner, Instrument ID No. SC/QC/010.

 

Glass wares required:

  • Volumetric Flask 100 & 50 ml
  • Beaker
  • Measuring Cylinder

Reagent required:

  • Water (HPLC grade)
  • Methanol (HPLC grade)

Working standard: 

  • Aceclofenac, WS ID No. WS/002, (Valid upto- 15/06/2019), Purity- 98.84% (on as such basis)
  • Paracetamol WS ID No. WS/0166(Valid upto- 15/03/2019), Purity- 98.84% (on as such basis)
  • Aceclofenac (100mg) and Paracetamol(325mg)(BY HPLC):–

Mobile phase: Mobile phase: – Methanol + Water + G.A.A. = 80: 20 +0.01; degas,

Column: – C-18, (150 x 4.6mm, 5µm); Wavelength: – 280nm; Flow rate: – 1.8ml per minute;

Injection volume: – 20µl;

Standard preparation: –

Weigh accurately about 162.5 mg of Paracetamol ws, and 50 mg of Aceclofenac ws in 100ml volumetric flask, dissolve in and dilute to the mark with mobile phase. Shake well and further dilute 2 ml to 50 ml with mobile phase. Shake well and filter through 0.45µm nylon filter.

 

  • Chromatographic condition:
  • Column: – C-18,( 250 x 4.6mm), 5µm,
  • Wave length: – 271 nm,
  • Flow rate: –   1.0 ml per minute,
  • Injection volume: – 20 µl,
  • Test preparation: –
  • Weigh accurately about equivalent to 250 mg of Paracetamol from crushed powder of 20 tablets in 100 ml volumetric flask, add 50 ml of mobile phase, sonicate for 5 minutes, cool to room temperature.
  • Temperature and dilute to the mark with mobile phase, filter. Shake well and further dilute 2 ml to 50 ml with mobile phase, shake well and filter through 0.45µm nylon filter.
  • Procedure: –
  • Inject separately standard and test preparation. Inject five replicate injection of test and two replicate injection of test preparation.RSD of replicate injection should not more than 2%. Calculate the assay of Paracetamol and Aceclofenac by comparison to peak area of standard preparation.
  • Calculation:
  • Test area      ͌ 162.5mg            2          100          50
  • Paracetamol = ———– x ———— x ——- x ——– x ——- x Av wt of tablet x potency of ws
  • (mg/tablet)       area            100               50        test wt.         2
  • Test area    ͌ 50mg              2            100              50
  • Aceclofenac.=——— x ———– x ——– x ———– x ——- x Av wt of tablet x potency of ws
  • (mg/table)      area       100              50            test wt.          2
  1. SUMMARY TABLE:

                                                              Analytical Performance Parameters

Sr. No Validation Parameters Acceptance Criteria Observations
1. Specificity There should not be any peaks of Blank, Placebo at main peak retention time There is no any peak of Blank, Placebo at main peak retention time.
2. System Accuracy
Tailing Factor       Not More Than 2.0

 

 1.05
% Relative standard deviation Not More Than 2.0 0.1 %
3 Linearity

Coefficient of Correlation (r) Linearity concentration verses detector response (area)

 Not Less Than 0.99 0.99202

 
4 Range

(80 % to 120 % of assay concentration)

 Should be Linear Linear
5 Accuracy

% Relative standard deviation

  

Not More Than 2.0

  

0.10%
6 Precision
System Precision
% Relative standard deviation Not More Than 2.0 0.30%
(i) Method Precision
% Relative standard deviation Not More Than 2.0 1.32%
(ii) Intermediate Precision
% Relative standard deviation Not More Than 2.0 1.34%
Overall % Relative standard deviation Not More Than 2.0 1.45%
7 Robustness
For flow rate 0.8 ml/min
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 16236.7
For flow rate 1.0 ml/min
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 14770.0
For flow rate 1.2 ml/min
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 12950.6
For mobile phase water (20%) : Methanol (80%)
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 17448.7
For mobile phase water (15%) : Methanol (85%)
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 14542.9
For mobile phase water (25%) : Methanol (75%)
Tailing Factor Not More Than 2.0

 

 1.0
Theoretical Plates Not Less Than 2000 12733.6
  1. SPECIFICITY:

Check the interference of diluents and placebo (as preservatives) by individual injections

Blank: as methanol

Placebo Preparation:

A placebo solution was prepared same as the formulation except for the addition of the active ingredients. Here, the product contains no inactive ingredients. So, here the diluents are used as the placebo solution. Area at 271 nm, Observation Result: Nil

Conclusion for Specificity:

We observed that at wavelength 271 nm there is no significant area for Blank and placebo at main peak of Aceclofenac & Paracetamol. And inject the different dilution of Aceclofenac & Paracetamol ws for better clarity to identify the peaks of both. Therefore specificity of the method considered acceptable.

2. SYSTEM ACCURACY:

The system precision of the above method was carried out by taking area for six injection of the standard preparation of exact weight. Text data collection sheet:

Serial No.   Area  of Aceclofenac Assay of Paracetamol
1.
2.
3.
4.
5.
Mean
% RSD

 

Acceptance Criteria: RSD is not more than 2.0%

3, 4, 5. LINEARITY, RANGE AND ACCURACY:

(i)Linearity:

Linearity can be defined as the response of an analytical method conducted as a function for the analyse concentration. In other words, it is the response of an analytical method in proportion to the concentration of analyse in samples.

Linearity can be evaluated by visual inspection of a plot of signals / response as a function of analyte concentration of content. Correlation Coefficient not less than 0.99.

(ii)Range:

The Range of an analytical method is the interval between the upper & lower level of analyte that have been demonstrated with precision, accuracy & linearity using the method as written. The Range is normally expressed in same units as test results e.g. Percent or Parts per million, obtained by the analytical method. All five concentrations (80, 100, and 120 in per cent) will be considered for the linearity and range of the method will be 40 to 60ppm.

(iii)Accuracy:

It describes the deviation from the standard or expected result.

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

The accuracy of method is determined by recovery experiment. The recovery is performed by adding Aceclofenac+Paracetamol tablets standard at five different concentration (40, 45, 50, 55, 60ppm) levels to placebo (excipients mixture) in the range of test concentration.

Text data collection sheet:

Sr. No. Standards   Area  of Aceclofenac Assay of Paracetamol
1. Standard-1
2. Standard-2
3. Standard-3
4. Standard-4
5. Standard-5
6. Mean
7. %RSD

Acceptance Criteria: RSD is not more than 2.0%

Samples Area  of Aceclofenac Mean Area of Paracetamol Mean
A-01   80%  

 

  

 

 

 

 

 
A-02   80%
A-03   80%
C-01 100%
C-02 100 %
C-03 100 %
E-01 120%
E-02 120 %
E-03 120 %

 

Concentration (mg/ml) Conc.

in %

 Mean area % Recovery Corr. Coefficient

(area/recovery)
Aceclofenac Aceclofenac Aceclofenac Aceclofenac
16 80
20 100
24 120

Concentration (%)                                                                                          Recovery%

80                                                                                                                                         81.47

100                                                                                                                                      98.46

120                                                                                                                                      114.44

 

 

Concentration (mg/ml) Conc.

in %

 Mean area % Recovery Corr. Coefficient

(area/recovery)
Paracetamol Paracetamol Paracetamol Paracetamol
16 80  

 

 

Area- 0.99302

Recovery- 0.9980

 
20 100
24 120

 Linearity graph plot of Paracetamol :

Concentration (%)                                                                                          Recovery%

80                                                                                                                                         81.53

100                                                                                                                                      98.49

120                                                                                                                                      119.74

Linearity graph plot of Aceclofenac:

 

Linearity graph plot of Paracetamol :

                                                                              

Linearity graph plot of Aceclofenac:

Concentration (%)                                                                                          Recovery%

80                                                                                                                                         81.47

100                                                                                                                                      98.46

120                                                                                                                                      114.44

 

Linearity graph plot of Paracetamol:

Concentration (%)                                                                      Area

80                                                                                                                                         16521460.3

100                                                                                                                                      20263820

120                                                                                                                                      24263801.6

R-squared value (R2): The R-squared value, also known as the coefficient of determination, is an indicator that ranges in value from 0 to 1 and reveals how closely the estimated values for the trend line correspond to your actual data. A trend line is most reliable when its R-squared value is at or near 1.

Linearity Equation

Equations for calculating trend line

Calculates the least squares fit for a line represented by the following equation:

y = m x + b

Where m is the slope and b is the intercept.

x = concentration

y = Area Value

Sample

Therefore, from Linearity Equation, y = mx + b, m              0.999x

b              0.163

Conclusion for Linearity:

The graphical representation & data collected during this exercise proves Aceclofenac and Paracetamol for demonstrate linearity in the range of 80% to 120% when determined by HPLC.

6.    PRECISION-

6. (i) METHOD PRECISION:

(i) Analyst (I): Dinesh Kumar–

Sample Dilutions:

      (A) Take about 404.3mg of the sample and proceed as per above.

      (B) Take about 405.2mg of the sample and proceed as per above.

      (C) Take about 405.5mg of the sample and proceed as per above.

      (D) Take about 405.3mg of the sample and proceed as per above.

      (E) Take about 405.4mg of the sample and proceed as per above.

      (F) Take about 405.4mg of the sample and proceed as per above.

Test Data Collection:-

Sr. No. Standards   Area  of Aceclofenac Area  of Paracetamol
1. Standard-1
2. Standard-2
3. Standard-3
4. Standard-4
5. Standard-5
6. Mean 4650378.6 12535673.2
7. %RSD 0.23 0.30
Acceptance Criteria: RSD is not more than 2.0%
Samples Sample Area Sample Area Mean Mean
Aceclofenac Paracetamol Aceclofenac Paracetamol
A T1 3805553 10251267 3800378.5 10237086
T2 3795204 10222905
B T1 3762973 10176399 3781116.5 10196259.5
T2 3799260 10216120
C T1 3766156 10178710 3783837 10205700
T2 3801518 10232690
D T1 3778423 10215079 3790844.5 10206685
T2 3803266 10198291
E T1 3801525 10208390 3794786 10216069
T2 3788047 10223748
F T1 3806593 10214874 3788029 10212701.5
T2 3769465 10210529

 

Table for Six Replicate Assays: Aceclofenac 100mg

Sample Number Estimated % Amount Mean RSD
Aceclofenac Aceclofenac Aceclofenac
Sample A  

100.384%

  

 

100.122 %

  

 

1.12%
Sample B 99.949 %
Sample C 99.947 %
Sample D 100.19 %
Sample E 100.22%
Sample F 100.045%

 

Table for Six Replicate Assays: Paracetamol 350mg

Sample Number Estimated % Amount Mean RSD
Paracetamol Paracetamol Paracetamol
Sample A  

100.493%

  

 

100.228 %

  

 

1.10%
Sample B 99.968%
Sample C 99.944%
Sample D 100.85%
Sample E 100.075%
Sample F 100.042%

 

Acceptance Criteria: NMT 2% (% of Relative Standard Deviation)

Conclusion for precision:

The overall % Relative standard deviation for 1.12% respectively of Aceclofenac and Paracetamol in tablets, there is no significant difference. Therefore Repeatability of the method considered acceptable as it well within 2 % Relative Standard Deviation.

 

  1. (ii) INTERMEDIATE PRECISION:

(Within laboratory variations such as different days, analyst & equipments):

 

(ii) Analyst (II): –

Sample Dilutions:

      (A) Take 5.0368gm of the sample and proceed as per above.

      (B) Take 5.0654gm of the sample and proceed as per above.

      (C) Take 5.0373gm of the sample and proceed as per above.

      (D) Take 5.0551gm of the sample and proceed as per above.

      (E) Take 5.0373gm of the sample and proceed as per above.

      (F) Take 5.0320gm of the sample and proceed as per above.

 

Test Data Collection:-

Sr. No. Standards   Area  of Aceclofenac Area of paracetamol
1. Standard-1 4627069 12527057
2. Standard-2 4676479 12602272
3. Standard-3 4658172 12541237
4. Standard-4 4654081 12507089
5. Standard-5 4636092 12500711
6. Mean 4650378.6 12535673.2
7. %RSD 0.23 0.30
Acceptance Criteria: RSD is not more than 2.0%

 
Samples Sample Area Sample Area Mean Mean
Aceclofenac Paracetamol Aceclofenac Paracetamol
A T1 3816972 10222430 3794098 10212870.5
T2 3771224 10203311
B T1 3804314 10193938 3791808 10184115
T2 3779302 10174292
C T1 3804414 10143064 3784826.5 10180360
T2 3765239 10217656
D T1 3771454 10190342 3782254 10195864
T2 3793054 10201386
E T1 3767640 10198919 3786835.5 10208185.5
T2 3806031 10217452
F T1 3760595 10177601 3768631.5 10199051.5
T2 3776668 10220502

 

Table for Six Replicate Assays:

Sample Number Estimated % Amount Estimated % Amount Mean RSD RSD
Aceclofenac Paracetamol Aceclofenac Paracetamol Aceclofenac Paracetamol
Sample A 99.541 % 99.762%  

 

99.849 %

  

100.208%

 

  

 

1.34%

  

1.25%

 

 
Sample B 100.147% 100.048%
Sample C 100.16% 100.21%
Sample D 99.722% 99.99%
Sample E 99.892% 100.52%
Sample F 99.633% 100.29%

Acceptance Criteria: NMT 2% (% of Relative Standard Deviation)

Conclusion for precision:

The overall % Relative standard deviation for 1.34% respectively of Aceclofenac and Paracetamol in Tablets, there is no significant difference. Therefore Repeatability of the method considered acceptable as it well within 2 % Relative Standard Deviation.

 

Table for Six Replicate Assays analyst by two different Analysts & days:

Test Data analyst by :-

Table for Six Replicate Assays:

Sample Number Estimated % Amount % Amount Mean Mean RSD RSD
Aceclofenac Paracetamol Aceclofenac Paracetamol Aceclofenac Paracetamol
Sample A 101.89 %  

 

101.635 %

  

 

 

  

 

1.32%
Sample B 101.96 %
Sample C 103.20 %
Sample D 102.39 %
Sample E 99.28 %
Sample F 101.09 %

 

Test Data analyst by :-

Table for Six Replicate Assays:

Sample Number Estimated % Amount % Amount Mean Mean RSD RSD
Aceclofenac Paracetamol Aceclofenac Paracetamol Aceclofenac Paarcetamol
Sample A 100.10 %  

 

 

  

 

101.12 %

  

 

 

  

 

1.34%
Sample B 98.93 %
Sample C 101.73 %
Sample D 102.51 %
Sample E 101.33 %
Sample F 102.09 %

Acceptance Criteria: NMT 2% (% of Relative Standard Deviation).

Conclusion for Intermediate Precision:

The overall % Relative standard deviation of two different analysts is 1.32% & 1.34% respectively of Aceclofenac & Paracetamol Tablets there is no significant difference between two analysts within laboratory variations such as different days, analyst & equipments. Therefore reproducibility of the method considered to be acceptable.

  1. ROBUSTNESS:

The robustness of an analytical method should show the reliability of an analysis with respect to deliberate variations in method parameters. The variation used for the study are:

  • Change in ratio of the mobile phase. Record the observation in below observation table.
  • Change in Flow rate of mobile phase. Record the observation in below observation table.

Observation Table:-

Change ratio in the mobile phase at 296nm

Mobile Phase Flow rate (ml/min) System suitability
Water Acetonitrile   Retention Time Theoretical Plates Tailing Factor
500ml 500ml 1.0 ml/min 12.001 17448.7 1.0
450ml 550ml 1.0 ml/min 8.577 14542.9 1.0
400ml 600ml 1.0 ml/min 6.612 12733.6 1.0
Change in flow rate at 296nm
Mobile Phase Flow rate (ml/min) System suitability
Water Acetonitrile   Retention Time Theoretical Plates Tailing Factor
450ml 550ml 0.8 ml/min 10.599 16236.7 1.0
450ml 550ml 1.0 ml/min  8.561 14770.0 1.0
450ml 550ml 1.2 ml/min 7.141 12950.6 1.0

 Acceptance criteria:

Analytical method validation shall be robust (i.e. Theoretical Plates is not less than 2000 & tailing factor is not more than 2.0).

Conclusion for Robustness:

There is no significant difference for Fexofinadine & Montelukast in Fexolock-M Tablets for different conditions, such as composition & Flow rate of mobile phase. Therefore Robustness of the method considered acceptable.

  1. CONCLUSION:

 

All the analytical parameter are checked as per the approved validation process and found well within specified acceptance criteria. Hence, it is concluded that, this method is suitable for accurate & precise results for routine analysis.

***

 

Analytical Method Validation