ANALYTICAL METHOD VALIDATION PROTOCOL OF OFLOXACIN 200 mg Tablets

nirvesh400

(OFLOXACIN 200 Tablets IP)

 

Product Name Batch No Mfg Date Exp. Date
 

 

 

Pre Approval:

 

PREPARED BY CHECKED BY APPROVED BY
NAME
DESIGNATION
SIGNATURE
DATE

CONTENTS

 

SECTION DESCRIPTION

PAGE
    Report Preparation 1
   

Pre Approval

 2
   

Contents

 3
1   Purpose 4
2   Scope 4
3   References 4
4   Responsibility 4
5   Method used for validation 4
  5.1 Chromatography  conditions 4
  5.2 Standard & Sample Preparation, and Procedure 5
6   Batches used for validation 5
7   Validation 6
7.1 Specificity              6
7.2 Linearity             7
7.3 Precision 8-10
7.4 Accuracy 10-11
7.5 Stability in analytical solution 12-13
8   Acceptance criteria 13-14
9   Compilation of Report 14
10   Summary & Conclusion  15
11   Certification 15
12   Post Approval 16

 1. PURPOSE:

To perform validation of the HPLC method used for the determination of the assay of the active ingredient (Ofloxacin) in Ofloxacin 200 mg tablet.

  1. Scope :

This applies to the testing of all Ofloxacin tablet manufactured at the Solan plant.

  1. References :

Text on validation of analytical procedures – ICH Q2A

Validation of analytical procedures: Methodology – ICH Q2B

Validation of compendial methods USP40

  1. RESPONSIBILITY:
    • Performing validation : Officer/Executive – QA & QC
    • Checking validation data : Head – QC
    • Approval of validation protocol / report : Head – QA
  • Method used for validation:

        ASSAY: (OFLOXACIN)

5.1   CHROMATOGRAPHIC CONDITIONS

       Determined by liquid chromatography.

Test Solution: Weigh and powder 20 tablets. Disperse a quantity of powder containing 25 mg of Ofloxacin in 60 ml of methanol and dilute to 100 ml with methanol and filter.

Reference solution: A 0.025 percent w/v solution of Ofloxacin in reference standard in methanol.

A stainless steel column 15 cm X 4.6 mm packed with octadecylsilane bonded to porous silica (5µm)

Mobile phase: a mixture of 8 volumes of acetonitrile and 92 volumes of phosphate buffer prepare by dissolving 2.72 gm of potassium dihydrogen phosphate in 1000 ml of water, adjust to pH 2.4 with O.P.A.

Flow rate: 2 ml per minute

Spectrometer set at 294 nm

Injection volume: 10 µl

Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 percent.

Inject the reference solution and the test solution.

Calculate the content of Ofloxacin.

Formula for calculation:

Area of test          Weight of STD              100

  —————- X————————- X——————–X Average weight X Potency of STD

Area of STD                 100                    Weight of test

Limits:       180 mg to 220 mg Ofloxacin each tablet

  Batches used for validation :

 Take sample of the Ofloxacin 200 Tablets during the method validation.

 

Batch no. Mfg. date Exp. date              Claimed content each tablet
                          Ofloxacin 200 mg
 

 

 

  1. VALIDATION

Perform validation as per procedure given below. Record all weights and dilutions. Perform calculations             using calculator or Microsoft Excel. Record the results and observations in the given formats. Compare the             results with acceptance criteria.

 7.1      SPECIFICITY

7.1.1   Non-interference from the Excipients

Prepare the placebo granules of the Ofloxacin Tablets except the active ingredients. Inject the                  blank (diluent), solution of the placebo granules in diluent, standard solution of active ingredients (Ofloxacin) and solution of placebo granules with active ingredients in diluent. Record all      the peaks and their corresponding wavelengths.

Placebo solution:

Standard solution:

Ofloxacin

Solutions  details Wavelength maxima (in nm )
Solvent used …………………………………
Placebo……………………………………….
Standard 1 ………………………………………..
Placebo with Active Ingredients………………

 7.2      LINEARITY

Prepare at least 7 different concentrations of standard solution of the active ingredients with the mean final concentration of 300 ppm for  Ofloxacin.(It should be cover 80% to 120 % of  final concentration)  Record the area of each solution at 294 nm, Record the area against the concentration and plot the graph of area against concentration for each solution. Calculate the correlation coefficient.

   Standard 1 Ofloxacin responses:

Level Ofloxacin (%) Final Conc. (ppm) Area (in AU)
1
2
3
4
5
6
7
Correlation coeff. ( r )

7.3       PRECISION

7.3.1    System precision

Make 5 replicate area of the standard solution at working concentration (i.e. 250 ppm for Ofloxacin). Record the area of the standard solution for Ofloxacin and at 294 nm, and Calculate the mean and RSD.

Standard preparation (______ ppm)

 

 

 

Replicate

                        Ofloxacin
Area Wavelength ( λ max)
1.  

 

 294 nm
2.  

 

 294 nm
3.  

 

 294 nm
4.  

 

 294 nm
5.  

 

 294 nm
Mean  

 
SD  

 
%RSD  

 

7.3.2    Precision (Repeatability)

Make 6 independent sample solutions of a pooled sample and analyze by the method. Calculate the assay for each analysis. Calculate the mean and RSD.

Standard preparation –

Mean Area        _____________

Sample preparation –

Calculation –

              Component 1: Ofloxacin

Assay no.  Wight (mg) Area Assay

(mcg)

 Assay

(%)
Std. Sample Std. Sample
1
2
3
4
5
6
Mean
SD
%RSD

  7.3.3    Intermediate precision (Ruggedness)

Analysis of a pooled sample to be performed in triplicate by 2 different chemists. Calculate the assay for each analysis. Calculate the mean and RSD.

Standard preparation (Chemist 1) –

             Mean Area        _____________

Standard preparation (Chemist 2) –

Mean Area       _____________

Sample preparation (Chemist 1)–

         

            Sample preparation (Chemist 2)–

Calculation –

 Component 1: Ofloxacin

 

Chemist 1

 

 

 Assay no. weight (mg) Area Assay

(mcg)

 Assay

(%)
Std. Sample Std. Sample
1

 
2

 
3

 
Chemist 2

 

 4

 
5

 
6

 
Mean
SD
RSD

7.4       ACCURACY

Spike the placebo at label claim with known amount of the active ingredient at 3 levels each in triplicate,     i.e. 3 x 70%, 3 x 100% and 3 x 130% of labeled amount. analyse each sample. Calculate the assay for each     analysis. From the amount found and the amount added, calculate % recovery. Calculate mean recovery and             its RSD.

Standard preparation –

Mean Area – _____________

            Sample preparation –

Calculation –

          Ofloxacin:

 

Recovery Level Standard Area Recovery
mcg % Mean SD %RSD
70%
100%
130%

 7.5       STABILITY IN ANALYTICAL SOLUTION

Prepare a solution of sample and analyse it immediately against standard solution. Keep the sample solution for 24 hours at room temperature and analyse against standard solution freshly prepared. Calculate assay of both the analysis. Calculate the recovery in terms of percentage of the 24 hours result against the initial result.

 Day 1                                                  Standard preparation                      Sample  preparation

Mean Area (Azithromycin)      _____________                                              _____________

 

 Day 2                                                  Standard preparation                   Sample  preparation

             Mean Area (Azithromycin)      _____________                                                     _____________

 

 

 

TIME

 

 

 Wt. (mg) Area              Assay
 

Std.

  

Spl.

 Ofloxacin   mg Recovery %
STD TEST Ofloxacin Ofloxacin
 

 

00Hrs.

 

 

 

 

 Ofloxacin          
 

24 Hrs

 Ofloxacin  

 

 

 

 

 

 

 

 

 

        
 

Mean

 

            
 

Change  %

24hrs. vs. 0 hrs.
  1. Acceptance criteria

     8.1     Specificity

Non-interference from the excipients and solvents in the spectrum of the active

Ingredients   with   placebo   &   solvent,    placebo  (excipients) and   solvent

(I.e. diluent) do not interfere   with   the active drugs (Ofloxacin) peak.

8.2       Wavelength maxima of each active ingredient do not interfere with each other.

8.3       Linearity

Response is linear and correlation coefficient is not less than 0.999

8.4       Precision

8.4.1    System precision

RSD for area of each active ingredient is not more than 2%.

8.4.2    Precision (Repeatability)

RSD for assay values is not more than 2%.

8.4.3    Intermediate precision (Ruggedness)

RSD for assay values is not more than 2%.

8.5       Accuracy

Mean recovery of assay found against added amount is between 98 to 102%. RSD of individual recoveries is not more than 2%.

8.6       Stability in analytical solution

Recovery after 24 hours is between 98 to 102% of the initial value.

Compilation of REPORT

Prepare a report based on all above data. Attach linearity graphs and raw data. Based on the results,             comment on the following –

  • Specificity of the method
  • Suitability of method
  • Linearity, precision and accuracy over the analytical range

Duration for which active ingredient (Ofloxacin) is stable in solution

  • Pertaining to the proposed HPLC method for routine testing of the Ofloxacin Tablet (200 mg)

SUMMARY

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CONCLUSION

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CERTIFICATION

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Post Approval:

 

PREPARED BY CHECKED BY  APPROVED BY
NAME
DESIGNATION
SIGNATURE
DATE

 

Analytical Method Validation