Hygeia Pharmaceuticals

Quality Control Department

Document No.HP/MVP/001
Method Validation Protocol

Genial (Fluconazole) Capsules 150mg

Method Validation Protocol for:
Batch No.030

Prepared by Reviewed by Approved by Authorized by

QC Analyst Quality Control Manager Quality Assurance Manager Plant Manager

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

1.0 Objective:

Analytical monitoring of a pharmaceutical product is necessary to ensure its efficacy through out all
phases of its shelf life; such monitoring is in accordance with the specifications elaborated during product
development. Method validation ensures that the selective method will give reproducible, reliable and
consistent results adequate for intended purpose, it is therefore necessary to define precisely both the
conditions in which the procedure is to be used and the purpose for which it is intended.
The objective of this validation study is to
 Ensure and justify, through extensive testing, that the precision and consistency are in accordance with
already established Acceptance Criteria.
 Assess the effect of variables (within already set operating limits) on the testing method
 Identify and solve the problem(s), if any, encountered during testing.
 Establish confidence on existing testing process.
 Find ways and means to increase productivity and improve quality.
 Assure that complete process is under control.

2.0 Scope:

The Scope of this document is

i. To describe the work required for the Validation of the Test Method for the determination of
Fluconazole.(Genial 150mg capsules) (SOP No. HP/QC/SAP-0013) This method is routinely
used for the quantitative determination (assay) of Fluconazole in Genial 150mg capsules at the
time of manufacturing and during stability studies.
ii. This Validation Protocol also describes the analytical parameters to be used for the validation
of the test method.
iii. The method (Procedure) (SOP No. HP/QC/SAP-0013) to be used for the determination of
Fluconazole in Genial Capsules 150mg is also explained in this protocol along with the
acceptance criteria.
iv. This Protocol also defines the facilities, responsibilities and equipment, apparatus, glassware,
material and documents that are used for the validation studies.
iv. In this validation protocol the analytical results are evaluated by the application of statistical
techniques and presented by means of graphical techniques.

3.0 Unit Operation:

Validation of testing method, being used for the quantitative determination (Assay) of Fluconazole in
Genial Capsules 150mg.

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

4.0 Periodic Revalidation:

In case there is no change or modification in the Validated Method, the Revalidation will be performed
after every 5 years.

5.0 Change Control:

Any modification or changes in the Validated Test Method HP/QC/SAP-0013being used for the
determination of Fluconazole in Genial Capsules 150mg should be controlled and will be entered into the
change control form in accordance with the change control procedure.
For changes in existing method and proposing new method or proposing procedure for new product,
following data should be established.

i. Justification for proposing a test procedure in comparison with other possible

alternatives.
ii. The proposed procedure should also be validated according to Method Validation Protocol.

6.0 Analytical Parameters: Following analytical parameters are to be considered

6.1 Specificity:

Ability to assess unequivocally the analyte in the presence of components which may be expected to be
present (impurities, degradants, matrix etc.)

6.2 Linearity and Range:

The linearity of an analytical procedure is its ability to produce results that are directly or indirectly
proportional to the concentration of the analyte in the sample within a given range, response to be linear
on at least 5-6 points.
Range is the lowest or highest level of analyte that the method can determine with reasonable accuracy
and precision in the range of 80%, 90%, 100%, 110% and 120 % of claim.
Linearity and range for an analytical procedure can be determine either by
i. Use of reference standard/working standard.
ii. Analysis in duplicate
iii. Determination by least square method/ regression line, slope of curve
iv. Response-concentration curve (Beer’s Law)

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

6.3 Accuracy:

It relates to the closeness of the test results to true value i.e. measure of exactness of analytical method. It
is expressed as % recovery by the assay of known/added amount of analyte in the linearity range. The
accuracy of the test procedure can usually be determined by applying the procedure to the quantitatively
prepared samples of the material to be analyzed. Such samples also include to which analyte have been
added in quantities both above and below the expected range of validation. The usual range being 10 %
above or below the expected range of claim.

6.4 Precision:

The precision of the analytical method is the degree of agreement among individual test results and how
individual test results are scattered from the mean value usually expressed as Standard Deviation or as the
Relative Standard Deviation (Coefficient of Variation). These results are based when the complete
procedure is applied repeatedly to multiple sampling from the same homogenous bulk.
The precision is usually expressed as Standard Deviation or Relative Standard Deviation and is determine
by assaying a sufficient number of portions of homogenous samples and to be subjected to statistical
evaluation. The assays for such cases are independent analysis of samples that have been carried out by
applying the complete analytical procedure from sample preparation to final test results. Precision may
also be explained by the following terms,
i. Repeatability
ii. Intermediate Precision
iii. Reproducibility.

6.4.1. Intermediate Precision:

Intermediate precision expresses within laboratory but

i. Different days
ii. Different analysts,
iii. Different equipment and reagent etc.

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

6.4.2. Repeatability (under same conditions)

Precision of the method when repeated by the same analyst, same test method and under same set of
laboratory conditions (reagents, equipment etc.) within a short interval of time, the only difference being
the sample. The repeatability of any test procedure is required to be assured by carrying out complete
separate determination on separate sample of the same homogenous batch under normal laboratory
conditions.

6.4.3 Reproducibility: (Under Different Conditions)

When the procedure is carried out by different analysts, in different laboratories using different
equipment, reagents by using the sample from the same homogenous batch in each case. Evaluate the
results obtained from the variability of analyst–to–analyst, day-to-day, laboratory-to-laboratory,
equipment-to-equipment etc., this analytical data will provide information about the reproducibility of the
test procedure under validation.

6.5 Robustness:

Robustness is the measure of the capacity of an analytical method to remain unaffected by small but
deliberate variation in procedure. It provides an indication about variability of the method during normal
laboratory conditions.

6.6 Ruggedness:

Ruggedness is the degree of reproducibility of test result obtained by analyzing the same sample under
variety of normal test conditions such as different
i. Analysts
ii. Instruments/ Equipment
iii. Days
iv. Reagents etc.

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

7.0 Acceptance Criteria:

i. Testing Result: 90 – 110 %

ii. Relative Standard Deviation ≤ 2%
iii. Coefficient Correlation 01

8.0 Statistical Techniques:

i. Mean
Mean is the sum of individual values divided by the total number of individual values. Mean is
denoted by X and is calculated by the formula

X = ∑x
n

n= total number of individuals.

∑= summision

ii. Standard Deviation

The standard deviation is a measure of how widely values are dispersed from the average value
(the mean).statistic used as a measure of the dispersion or variation in a distribution, equal to the
square root of the arithmetic mean of the squares of the deviations from the arithmetic mean.

δ = n ∑ x² - (∑ x) ²

n(n – 1)

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

9.0 Sampling Plan:

Analytical Parameters Procedure Criteria

Specificity/ Selectivity For UV Spectrophotometric method RSD ≤ 2%

1. Scan the standard solution and note the λ max.
2. Scan the sample solution and check the
interference at the λ max.
3. Scan the placebo and check the interference at
the specified λ max.

Linearity and Range Solutions of 5 different concentrations (80%, 90%, Response should
100%, 110% and 120%) should be used for linearity, be linear.
one or two concentrations below and above linearity
will cover the range.

Accuracy and recovery Minimum nine determinations over a minimum of three RSD ≤ 2%
concentration levels covering the specified range

Precision (Repeatability) A minimum of six determinations at 100% of the test RSD ≤ 2%

concentration.

Intermediate Precision
Standard and sample solutions of concentration (100%)
(Ruggedness)
should be scanned with different analysts.

Ten samples individually are to be tested by the same

Analysts with small but deliberate changes in the
Robustness procedure e.g. change in temperature, change in
apparatus etc. It provides an indication about variability
of the method during normal laboratory conditions.

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

10.0 Testing Procedure

Product Genial Capsules 150mg SOP Reference No. HP/QC/SAP-0013

Description:
Size “2” hard gelatin shell Yellow Body and maroon color cap containing white powder.
Composition:
Each capsule contains:
Fluconazole ------------------150mg
Limit (Corresponding to 90.0 – 110.0% of the label amount).

Filled weight of conents---------------------190mg/capsule

Shell weight----------------------65mg
Total weight with Shell 255mg
Assay:
Standard preparation:
Accurately weigh working reference standard equivalent to 100mg of Fluconazole in a 100 ml volumetric
flask and sonicate it to dissolve in 0.1N HCl. Make volume up to mark. Dilute 10 ml of stock solution to 50
ml with the same solvent.
Sample preparation:
Take powder sample equivalent to 100 mg of Fluconazole to 100 ml volumetric flask. Add 0.1N HCl to
dissolve. Sonicate it for about 30 minutes to dissolve. Make volume up to mark with same solvent and stir
for about 20-30 minutes. Heat if required.
Pass it through filter paper. Dilute 10 ml of the filtrate to 50 ml with 0.1N HCl.

Procedure: Take reading of both sample and ref solutions in a 1 cm cell on a U.V/ Visible
spectrophotometer at 261 nm

Calculation :
% Age = abs.test × dilution of std × 100 x 190
abs.std ×dilution of sample × 150

Limit: 90 – 110% of label’s claim.

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 Hygeia Pharmaceuticals
Quality Control Department
Document No.HP/MVP/001
Method Validation Protocol

11.0 Facilities/Responsible Personnel/Equipment/Material Documentation

11.1 Facilities:

The validation of the method of Determination of Fluconazole in Genial Capsules 150mg will be carried
out in the Chemical Lab. Hygeia Pharmaceuticals ISLAMABAD, PAKISTAN.

11.2 Identification of Responsible Personnel:

Name Job Title Signature

11.3 Machine/ Equipment

Machine/ Equipment Machine No. SOP Number
UV Spectrophotometer PG T 80
Analytical Balance ATY 224

11.4 Identification of Apparatus/Glassware

Apparatus/Glassware Type Make
Volumetric Flask Glass “A” Pyrex Japan
Pipette Glass Pyrex Germany

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