1.

0 Method Validation:

Analytical method validation is the process to confirm that the analytical procedure employed for a
specific test is suitable for its intended use.

2.0 Objective:

Analytical monitoring of a pharmaceutical product is necessary to ensure its efficacy throughout 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 the 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 the existing testing process.

• Find ways and means to increase productivity and improve quality.

• Assure that complete process is under control.

3.0 Scope: The Scope of this document is

i. To describe the work required for the Validation of the Test Method for the determination of
Ciprofloxacin, This method is routinely used 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 (SOP No._________) be used for the determination of Ciprofloxacin in 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.

v. In this validation protocol, the analytical results are evaluated by the application of statistical
techniques and presented by means of graphical techniques.

vi. This method validation protocol applies to all test methods performed for release or stability
evaluation of all strengths of Ciprofloxacin Tablets.

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 (SOP
No._________) being used for the determination of Ciprofloxacin should be controlled and will be
entered into the Change Control Form (Form No._________) in accordance with the change control
procedure (SOP No._________). Provide Justification for proposing a test method. The proposed
procedure should also be validated according to this Validation Protocol.
6.0 Validation Parameters: Following analytical parameters are to be considered

6.1 Calibration of Spectrophotometer:

i. Calibration of Wavelength

a. Turn the main power “ON” and both lamps.

b. The instrument will confirm the filter automatically.

c. Set the wavelength from 200 – 400 nm.

d. Set the wavelength from wavelength counter.

e. Fill the cell with blank and check the blank reading.

f. Wash them all with distilled water

g. Fill it with the K2Cr2O7 solution.

h. Note the absorbance from screen

Wavelength (nm) Absorbance

235 nm (minima/valley) 0.748 (0.740 – 0.756)

257 nm (maxima) 0.865 (0.856 – 0.894)

313 nm (minima/valley) 0.292 (0.289 – 0.295)

350 nm (maxima) 0.640 (0.634 – 0.646)

If the instrument shows absorbance at the above-mentioned wavelength with limits, then the
calibration has completed, otherwise, it has to recalibrate.

ii. Calibration of Stray light: • Take 1.2 g of Potassium chloride previously dried at 105 °C for two
hours into the 100 ml volumetric flask. • Add water to dissolve and make up to volume with water. •
Check the absorbance of a 1.2 % w/v solution of potassium chloride at a path-length of 1cm. • The
absorbance should be greater than 2.0 at about 200nm when compared with water as reference
liquid.

iii. Calibration of Resonance Power: • To prepare 0.02 % v/v Toluene, take 0.02 ml of Toluene into
the 100 ml volumetric flask. Add Hexane to volume. • Record the spectrum of a 0.02 % v/v solution of
Toluene in Hexane. • The ratio of the absorbance at the maximum at about 269 nm to that at the
minimum at about 266 nm is not less than 1.5

6.2 System Suitability: Set of parameters and criteria there off to ensure that the system is working
properly. System suitability performed during entire validation of this method, by preparing five
dilutions of the same concentration of Ciprofloxacin Working Standard and shows the results of
system suitability by the application of statistical techniques i.e. Mean, Standard Deviation and
Relative Standard Deviation (%).

6.3 Selection of Solvent: Prepare 50μg/ml of stock solution in four portions by dissolving 5 mg of
Ciprofloxacin in 100 ml of phosphate buffer having pH of 2.5 and pH 7.4 respectively, in 0.1N HCl and
in Distilled water. Then prepare Standard solution of various concentrations (1, 2, 3, 4, 5μg/ml) by
dilution from each stock solution. Found λmax of Ciprofloxacin in each media by scanning them over
the UV range of 190nm to 400nm. Plot concentration vs. absorbance and prepare standard curve by
scanning the standard solutions of Ciprofloxacin at the respective wavelength (λmax) of different
solvents.

6.4 Calibration Curve (Linearity): A calibration curve is a general method for determining the
concentration of a substance in an unknown sample by comparing to a set of standard samples of
known concentration. A calibration curve is simply a graph where concentration is plotted along the x-
axis and absorbance is plotted along the y-axis. After making several Calibration Standards at
different concentrations. After running each one on the instrument and getting the absorbance, the
points are then plotted on the graph. The points are then connected with a line. That line represents
the calibration curve.

Procedure:

i. Prepare solution of Ciprofloxacin Working Standard at concentration 0.01 mg./ ml. in different
media i.e. 0.1 N HCl, Distilled Water, Phosphate Buffer pH 2.5 and Phosphate Buffer pH 7.4
and scan over the UV range 190 to 400 nm to determine the λmax.
ii. Prepare 5 dilutions of Ciprofloxacin Working Standard at conc. i.e. 1, 2, 3, 4 & 5 µg./ ml.
iii. Scan all the 5 solutions of the different concentration of all the 4 media at the determined
λmax using the same media as a blank

iv. Subtract the reading of blank from the reading of the solution to get the correct value. v.
Draw the Calibration Curve.

6.5 Limit of Detection (LOD): The lowest amount of analyte in the sample which can be detected but
not necessarily quantitated. Analysis of sample with a known concentration of the analyte and by
establishing minimum concentration at which analyte can be reliably detected.

Procedure: LOD may also be calculated based on the standard deviation (SD) of the response and
the slope of the calibration curve (S) at levels approximating the LOD according to the formula: LOD
=3.3(SD/S). The standard deviation of the response can be determined based on the standard
deviation of y-intercepts of regression lines. In formula;

S = Slop of Calibration Curve

SD = Standard Deviation of response

6.6 Limit of Quantitation (LOQ): The lowest amount of an analyte in a sample which can be
quantitatively determined with a suitable precision and accuracy. Analysis of sample with a known
concentration of the analyte and by establishing minimum concentration at which analyte can be
reliably quantitated.

Procedure: LOQ may also be calculated based on the standard deviation of the response (SD) and
the slope of the calibration curve (S) at levels approximating the LOQ according to the formula: LOQ
=10(SD/S). The standard deviation of the response can be determined based on the standard
deviation of y-intercepts of regression lines. In formula;

S = Slop of Calibration Curve

SD = Standard Deviation of response

6.7 Accuracy ( % Recovery): The difference between theoretical added amount and the practically
achieved amount is called accuracy of the analytical method. Accuracy was determined at 5 different
level 50%, 75%, 100%, 125% and 150% of the target concentration in triplicate.
 Procedure:

i. Prepare Placebo and draw 6 sample of the same weight

ii. Prepare three (3) replicate for following levels.

• Prepare three samples without addition

• with 50% addition

• with 70% addition

• with 90% addition

• with 100% addition

• with 130% addition

• with 150% addition

iii. Analyze all the samples and calculate the % recovery

Acceptance Criteria:

• % age recovery should be 98 to 102 %

• The coefficient of determination (r2) should be greater than 0.9998.

• The % RSD of the assay or recovery values should not be greater than 2.0%.

6.8 Precision: Precision is the degree of repeatability of an analytical method under normal
operational conditions. Precision may also be explained by the terms, Intermediate Precision and
Repeatability.

6.8.1. Intermediate Precision: Intermediate precision refers to variations within a laboratory as with,
different instruments, by different analysts, and so forth.

Procedure:

• Prepare ten replicate samples solutions from the same composite sample according to the
analytical method by three different analysts keeping the other condition same.

• Analyze the samples according to procedure by the three analysts on the same day

• Calculate the assay results for each sample.

Acceptance Criteria:

• Assay Limit 97 % to 103 %

• The coefficient of determination (r2) should be greater than 0.9998.

• The % RSD of the assay or recovery values should not be greater than 2.0%.

6.8.2. Repeatability Repeatability refers to variations within a laboratory as with different days,
different equipment etc. keeping the other conditions same.
Procedure:

• Prepare ten replicate samples solutions from the same composite sample according to the
analytical method by one analyst keeping the other condition same.

• Analyze the samples according to procedure by the one analyst on ten different days • Calculate the
assay results for each sample.

Acceptance Criteria:

• Assay Limit 97 % to 103 %

• The coefficient of determination (r2) should be greater than 0.9998.

•The % RSD of the assay or recovery values should not be greater than 2.0%.

6.9 Reproducibility: When the procedure is carried out 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 the 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.

Procedure:

• Prepare ten replicate samples solutions from the same composite sample according to the
analytical method by one analyst keeping the other condition same.

• Analyze the samples according to procedure by one analyst on the same day

• Arrange for the testing of the sample from the same batch using the same analytical procedure from
any other laboratory.

Acceptance Criteria:

• Assay Limit 97 % to 103 %

• The coefficient of determination (r2) should be greater than 0.9998.

• The % RSD of the assay or recovery values should not be greater than 2.0%.

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

Procedure:

• Prepare ten replicate samples solutions from the same assay composite sample according to the
analytical method by one analyst keeping the other condition same.

• Prepare another ten replicate samples solutions from the same composite sample according to the
analytical method by one analyst with a minor deviation i.e. 15 minutes sonication instead of 30
minutes stirring, keeping the other condition same.

• Analyze the samples according to procedure by the one analysts on the same day

• Calculate the assay results for each sample.

Acceptance Criteria:
• Assay Limit 97 % to 103 %

• The coefficient of determination (r2) should be greater than 0.9998.

• The % RSD of the assay or recovery values should not be greater than 2.0%.

6.11 Stress Degradation:

Degradation information obtained from stress studies (e.g., products of acid and base hydrolysis,
thermal degradation, photolysis and oxidation) for the drug substance and for the active ingredient in
the drug product.

Procedure (Thermal Degradation):

• Prepare triplicate samples solutions from the same assay composite sample according to the
analytical method by one analyst at room temperature

• Prepare triplicate samples solutions, and standard solution according to the analytical method by
one analyst and subjected to heat treatment at 30 °C, 40 °C, 50 °C, 60 °C & 70 °C for one hour

• Analyze the all samples according to procedure by the one analysts on the same day

Acceptance Criteria: (Not for all samples)

• Assay Limit 97 % to 103 %

• The coefficient of determination (r2) should be greater than 0.9998.

• The % RSD of the assay or recovery values should not be greater than 2.0%.

7.0 Statistical Techniques:

i. Mean Mean is the sum of individual values divided by the total number of individual values.

Mean is denoted by x̄ and is calculated by the formula

∑ x
Ẍ =--------------
n

ii. Standard Deviation

The standard deviation is a measure of how widely values are dispersed from the average value (the
mean). The 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.

ᵹ =√ n ∑ −¿
X2
¿ /N(N-1)
¿ ❑

iv. Relative Standard Deviation:

Standard Deviation divided by the and multiplied by 100

v. Coefficient Correlation

A measure of the interdependence of two random variables that range in value from –1 to +1,
indicating perfect negative correlation at –1, the absence of correlation at zero, and perfect positive
correlation at +1. Also called coefficient of correlation.

vi. Limit of Detection:

LOD = 3.3(SD/S)

Where; S = Slop of Calibration Curve SD = Standard Deviation of response

vii. Limit of Quantitation:

LOQ = 10(SD/S)

Where; S = Slop of Calibration Curve SD = Standard Deviation of response

8.0 Testing Procedure Determination of Ciprofloxacin HCl SOP No. ___________________

Standard Preparation:

Weigh and transfer 58.2 mg. Working Standard (Ciprofloxacin HCl) equivalents to 50 mg.
Ciprofloxacin into 100 ml. volumetric flask add 10 ml. of 0.1 N HCl and shake to dissolve make the
volume up to the mark with 0.1 N HCl and mix thoroughly.

Filter the solution through Whatman filter paper; discard the first 10 ml. of the filtrate. Pipette out 1 ml.
of the filtered solution to a 100 ml. of volumetric flask and make the volume up to 100 ml. with 0.1 N
HCl and mix thoroughly.

Sample Preparation:

Weigh 20 tablets and determine the average weight Powder 20 tablets and mix thoroughly, weigh
and transfer a quantity of powder equivalent to 250 mg. of Ciprofloxacin into 500 ml. volumetric flask
adds 100 ml. of 0.1 N HCl allow stirrer using magnetic stirrer for 30 minutes and make the volume up
to the mark with 0.1 N HCl and mix thoroughly.

Pipette out 1 ml. of the resulting solution to a 100 ml. of volumetric flask and make the volume up to
100 ml. with 0.1 N HCl and mix thoroughly.

Blank preparation: It consists of 0.1 N HCl.

Operate the Spectrophotometer according to SOP and measure the absorbance of the sample and
standard preparation at λmax 276 nm of Spectrophotometer using blank preparation for adjusting the
instrument.

Calculation:

Percentage = Absorbance of sample × P x 100

----------------------------------------------------
Absorbance of standard × 100
Where; P = Potency of working standard (As is basis)

Determined amount =
 Percentage × Labeled amount
100
9.0 Reagent Preparation

Preparation of phosphate buffer of pH 2.5

The buffer solution was prepared by adding 16.3308 g. Potassium Dihydrogen Phosphate (KH2PO4)
and 3.4836 g Dipotassium Hydrogen Phosphate (K2HPO4) in 200 ml distilled water. pH of the
solution was adjusted to 2.5 by Phosphoric Acid (H3PO4)

Preparation of phosphate buffer of pH 7.4

The buffer solution was prepared by adding 2.7218 g. Potassium Dihydrogen Phosphate (KH2PO4)
and 8.709 g. Dipotassium Hydrogen Phosphate (K2HPO4) in 200 ml distilled water. pH of the
solution was adjusted to 7.4 by Sodium Hydroxide (NaOH).

Preparation of 0.1N HCl acid:

0.83 ml of 37% pure concentrated HCl was taken in a 100 ml volumetric flask. The volume was made
up of distilled water and the content is thoroughly mixed it by shaking.

10.0 Facilities/Responsible Personnel/Equipment/Material Documentation

10.1 Facilities: Validation of Test Method (SOP No.__________) being used for the Determination of
Ciprofloxacin HCl in 250, 500 and 750 mg. Tablets will be performed in Chemical Lab. at
____________________ (Company Name and Address).

10.2 Identification of Responsible Personnel:

Name Job Title Signature

10.3 Identification of Documentation

Document Title SOP/QF Number

Validation Master Plan

Method Validation Protocol (SOP)

Method Validation Protocol (QF)

Method Validation Report (QF)

Testing Method Change Control Procedure (SOP)

UV- Spectrophotometer

Analytical Balance

10.4 Machine/ Equipment

Machine/ Equipment Qualification Status Qualification No.

UV. Visible Spectrophotometer Qualified IQ: _________

OQ: _________

PQ: _________

Digital Balance Qualified IQ: _________

OQ: _________

PQ: _________

10.5 Identification of Apparatus/Glassware

Apparatus/Glassware

Volumetric Flask