VALIDATION OF HPLC ANALYTICAL METHOD FOR

“DETERMINATION OF CLORSULON FROM VETERINARY

MEDICINAL PRODUCT IVERMECTIN-CLORSULON INJ
SOLUTION FOR INJECTION”
 Table of Contents
I. INTRODUCTION .................................................................................................... 4
II. PURPOSE............................................................................................................... 4
III. METHOD ASSAY OVERVIEW ......................................................................... 4
1. Label claim ...................................................................................................................................... 4
2. Drug Profile ..................................................................................................................................... 5
3. Method description ......................................................................................................................... 5
3.1. Equipment................................................................................................................................. 5
3.2. Chemicals.................................................................................................................................. 5
3.3. Preparation of test solutions .................................................................................................... 6
3.4. Calculation of the results ......................................................................................................... 7
3.5. Details on methods and procedures ......................................................................................... 8
3.6. Specification and acceptance criteria ...................................................................................... 8
IV. VALIDATION CHARACTERISTICS AND REQUIREMENTS ................... 8
V. PARAMETERS FOR METHOD VALIDATION ............................................. 8
1. System suitability ............................................................................................................................ 8
1.1. Test procedure .......................................................................................................................... 8
1.2. Acceptance criteria ................................................................................................................... 9
1.3. Documentation ......................................................................................................................... 9
2. Intermediate precision (ruggedness) ............................................................................................. 9
2.1. Test procedure .......................................................................................................................... 9
2.2. Acceptance criteria ................................................................................................................... 9
2.3. Documentation ......................................................................................................................... 9
3. Specificity ........................................................................................................................................ 9
3.1. Test procedure .......................................................................................................................... 9
3.2. Acceptance criteria ................................................................................................................. 10
3.3. Documentation ....................................................................................................................... 10
4. Accuracy ........................................................................................................................................ 10
4.1. Test procedure ........................................................................................................................ 10
4.2. Acceptance criteria ................................................................................................................. 10
4.3. Documentation ....................................................................................................................... 10
5. Precision- Repeatability ............................................................................................................... 11
5.1. Test procedure ........................................................................................................................ 11
5.2. Acceptance criteria ................................................................................................................. 11
 5.3. Documentation ....................................................................................................................... 11
6. Linearity ........................................................................................................................................ 11
6.1. Test procedure ........................................................................................................................ 11
6.2. Acceptance criteria ................................................................................................................. 11
6.3. Documentation ....................................................................................................................... 11
7. Range ............................................................................................................................................. 11
7.1. Test procedure ........................................................................................................................ 11
8. Robustness ..................................................................................................................................... 12
8.1. Test procedure ........................................................................................................................ 12
8.2. Acceptance criteria ................................................................................................................. 12
8.3. Documentation ....................................................................................................................... 12
9. Solution stability ........................................................................................................................... 12
9.1. Test procedure ........................................................................................................................ 12
9.2. Acceptance criteria ................................................................................................................. 12
9.3. Documentation ....................................................................................................................... 12
VI. RESULTS ACCORDING TO THE REQUIREMENTS VALIDATION
PLAN .............................................................................................................................. 13
1. System suitability .......................................................................................................................... 13
2. Specificity ...................................................................................................................................... 14
3. Accuracy ........................................................................................................................................ 14
4. Precision -Repeatability ............................................................................................................... 16
5. Intermediate precision ................................................................................................................. 17
6. Linearity ........................................................................................................................................ 19
7. Range ............................................................................................................................................. 20
8. Robustness ..................................................................................................................................... 22
a. Evaluation of results without variation of test parameters ................................................... 22
b. Evaluation of results with variation of test parameters ........................................................ 22
9. Solution stability ........................................................................................................................... 23
VII. CONCLUSION ................................................................................................. 25
VIII. DEFINITION AND ABREVIATIONS .......................................................... 26
 VALIDATION PROTOCOL:

I. INTRODUCTION
Method validation is the process used to confirm that the proposed analytical
procedures is suitable for its intended use. Results from method validation it is used to
judge the quality, reliability and consistency of analytical results; it is an integral part of
a good analytical practice. The further documentation it is the process of defining an
analytical requirement and confirms that the method under consideration has
performance capabilities consistent with what the application requires.
The analytical method validation has been performed as per ICH (International
Commission of Harmonization) guideline Q2A and Q2B

II. PURPOSE
The objective of the present work was to develop validated analytical method
with the help of which we can quantify CLORSULON from the injectable solution
IVERMECTIN-CLORSULON INJ.
Therefore, there is always a need to develop validated analytical method which is
precise, accurate, selective, and sensitive and can be used for routine analysis and
stability studies of the drug product IVERMECTIN-CLORSULON INJ.

III.METHOD ASSAY OVERVIEW

The method is intended to be used in identification and quantification of
CLORSULON from medicinal veterinary product IVERMECTIN-CLORSULON INJ.

1. Label claim
table no.01 IVERMECTIN-CLORSULON INJ composition per one milliliter of
solution:

Constituent Quantity per milliliter Function

Active medicinal substances
Ivermectin 10.00 mg anthelmintic
Clorsulon 100.00 mg anthelmintic
Excipients add to 1 ml
Glycerinformaldehyde *** mg solubilizer
Propylene glycol q.s *** mg solvent
Glycerine *** mg solubiliser
 2. Drug Profile
Clorsulon, one of the two active substances from injectable solution
IVERMECTIN-CLORSULON INJ for veterinary usage is a compound belonging to the
benzensulphonamide family which is recommended for the treatment and control
(Fasciola hepatica and Fasciola gigantica) in cattle as injectable formulation for
subcutaneous administrations.

fig.1-the structure of clorsulon

3. Method description
The ingredient Clorsulon is identified and quantitatively assayed
with a high-performance liquid chromatographic method. This permits the identification
and quantification of the ingredient in one run, also separation and identification of the
impurities. Clorsulon is identified by comparing the retention time with the reference
standard. Quantifications followed by measurement of the peak area of Clorsulon.

3.1. Equipment
a) HPLC system
-flow: 1.0 ml/min
-UV detector 254 ±10 nm; D2 -lamp
-Runtime 70 minutes
-column temperature 30°C± 2°C
- Column Symmeetry C8, 250x4 mm,5µ
-sample loop 20µl
3.2. Chemicals
b) Mobile phase
• Phase A: weight 700 g water HPLC grade in a beaker. Add 236 gram of
acetonitril HPLC grade. Homogenise. Add 1.05 g acetic acid, homogenise.
 • Phase B: Acetonitril HPLC grade

• Ratio Time A: B (v/v)

at 0 min 100.0: 0.0

at 15 min 100.0: 0.0
at 20 min 30.0: 70.0
at 50 min 30.0: 70.0
at 55 min 100.0: 0.0
at 60 min 100.0: 0.0
at 70 min stop
c) Solvent
Add 700 g of demineralised water in a beaker. Add 236 gram of acetonitril
HPLC grade. Homogenise. Add 1.05 g acetic acid, homogenise
d) Glass
glass manufactured of borosilicate, low actinic glassware
Volumetric glass works minimum class B
e) Standards
Clorsulon std
f) Retention time
- Clorsulon ± 12 min

3.3. Preparation of test solutions

i. Standard solution
64,85, and 106 mg of Clorsulon is accurately weighed into three 50 ml volumetric
flasks. Add solvent to volume (50.0 ml). Pipette 5.0 ml of each flask into a clean 50,0
ml volumetric flask. Add solvent to volume (50.0 ml), to obtain concentrations of
respectively 128, 170 and 212 µg of Clorsulon per 1 ml. These solutions are injected
onto the column.
ii. Sample solution
Pipet 1,7 ml into a 50.0 ml volumetric flask. Add solvent (50.0 ml). Pipette 1.0 ml of
each flask into a clean 20.0 ml volumetric flask. Add solvent to volume (20.0 ml).
This solution is injected onto the column over a 0.45 µm membrane filter.
iii. Blank solution
Solvent -injected in the analysis system in the same quantity as sample
solution
iv. Placebo solution
Pipet 1.7 ml of placebo into a 50.0 ml volumetric flask. Add solvent (50.0 ml). Pipette
1.0 ml of each flask into a clean 20.0 ml volumetric flask. Add solvent to volume (20.0
ml).
This solution is injected onto the column over a 0.45 µm membrane filter.

v. Placebo spiked with ivermectin solution.

Pipet 1.7 ml of placebo spiked with ivermectin into a 50.0 ml volumetric flask. Add
solvent (50.0 ml). Pipette 1.0 ml of each flask into a clean 20.0 ml volumetric flask.
Add solvent to volume (20.0 ml).
This solution is injected onto the column over a 0.45 µm membrane filter.
vi. Placebo spiked solution
Pipet 1.7 ml of placebo spiked (with interest active ingredient -clorsulon) into a 50.0 ml
volumetric flask. Add solvent (50.0 ml). Pipette 1.0 ml of each flask into a clean 20.0
ml volumetric flask. Add solvent to volume (20.0 ml).
This solution is injected onto the column over a 0.45 µm membrane filter
vii. Placebo spiked
Placebo in which is added an adequate quantity of interest active ingredient
(clorsulon). The quantity added is adequate to intended use.
viii. Placebo spiked with ivermectin
Placebo in which is added an adequate quantity of active ingredient other than
clorsulon, that will be ivermectin. The quantity added is adequate to intended use.
ix. Placebo preparation:
Mix 8.05 g of glycerinformaldehyde,15.5g glycerin and 75.5 g propylene glycol.

3.4. Calculation of the results

𝑨 𝒔 𝒙 𝑪 𝒔𝒕𝒅 𝑭𝟏
Label claim of the CLORSULON (mg/ml) = 𝒙 , where
𝑨 𝒔𝒕𝒅 𝑭𝟐
As - peak area of clorsulon in sample solution
C std - concentration of the standard solution (µg/ml)
A std – peak area of clorsulon in standard solution

𝐕𝐝𝟐 𝐕𝐝𝟏
Dilution factor, F1 = 𝐱 , where
𝐕𝐩 𝐕𝐬
Vs – the volume of assay sample (ex,1,7 ml IVERMECTIN-CLORSULON INJ)
Vd1 – the volume the first volumetric flask (ex,50ml)
Vp – the volume transferred by pipetting (ex, 1 ml of sample)
V d2 – the volume of second volumetric flask (ex, 20 ml)
F1 = 588,2353 (20/1x50/1,7), if the dilution of the sample is identical as described in
this document
Transformation factor, F2 =1000 (transformation from µg to mg),
𝑭𝟏
so = 𝟎. 𝟓𝟖𝟖𝟐𝟒
𝑭𝟐
 𝒍𝒂𝒃𝒆𝒍 𝒄𝒍𝒂𝒊𝒎 𝒐𝒇 𝒄𝒍𝒐𝒓𝒔𝒖𝒍𝒐𝒏 (𝒎𝒈/𝒎𝒍) 𝒐𝒃𝒕𝒂𝒊𝒏𝒆𝒅
% Assay of Clorsulon = 𝒙𝟏𝟎
𝒍𝒂𝒃𝒆𝒍 𝒄𝒍𝒂𝒊𝒎 𝒐𝒇 𝒄𝒍𝒐𝒓𝒔𝒖𝒍𝒐𝒏 (𝒎𝒈/𝒎𝒍) 𝒕𝒂𝒓𝒈𝒆𝒕𝒆𝒅

3.5. Details on methods and procedures

The ingredient Clorsulon is identified and quantitatively assayed with a high-
performance liquid chromatographic method. This assay permits the identification and
quantification of the ingredient in one run.
Clorsulon is identified by comparing the peak retention time with the reference
standard.
Quantification is followed by measurement of the peak area of Clorsulon.

3.6. Specification and acceptance criteria

Clorsulon
Specification: 100.0 mg/ml
Acceptance criteria: 95-105 mg/ml

IV. VALIDATION CHARACTERISTICS AND REQUIREMENTS

Validation Assay Identification

Characteristic
Accuracy Yes No
Precision-Repeatability Yes No
Precision-Intermediate precision Yes No
Specificity Yes Yes
Detection limit No No
Quantification limit No No
Linearity Yes No
Range Yes No
Robustness Yes No
table no.02 References: In accordance with ICH Q2A and ICH Q2B guidelines

V. PARAMETERS FOR METHOD VALIDATION

1. System suitability
System suitability is used to verify that the system is adequate for the analysis to
be performed.

1.1. Test procedure

The system suitability test was performed automatically by the soft, based on
clorsulon repeatability sample sequence, and consist in automatically calculation of a
suitable parameters as, repeatability, peak asymmetry (tailing), theoretical plates.
 1.2. Acceptance criteria
Every test performed with aid of HPLC system soft must be passed and
parameters from USP Clorsulon monograph 29 for column efficacy and injection
precision must conform (see table no 3)
SYSTEM SUITABILITY Acceptance criteria (Clorsulon-USP)
PARAMETER
Column efficiency NLT 7400- theoretical plates
NMT 1,4 - Tailing factor
Injection precision for R.T. RSD -NMT 1.0%
Injection precision for peak area RSD -NMT 1.0%
table no.03
1.3. Documentation
The report of system suitability generated at the end of the test is attached in this
document.

2. Intermediate precision (ruggedness)

Intermediate precision is established by demonstrating the effects of random
events on the precision of the method. Such random events include days, analysts and
instrumentation

2.1. Test procedure

Six different sample solutions shall be prepared from the drug product and shall
be analyzed using the proposed method by different analyst and on different days. The
results shall be calculated
The absolute difference in the assay results obtained (Mean value of six results)
for Group 1 (analyst 1/day 1) and the results obtained (Mean value of six results) for
Group 2 (analyst 2/day 2) shall be calculated and the confidence interval (95%).
2.2. Acceptance criteria
The absolute difference in the assay results obtained in group 1 (Mean
value of six results) and group 2 (Mean value of six results) should
not be more than 2.0 %.
Absolute difference =|𝒙 − 𝒚|
2.3. Documentation
Chromatograms obtained for INTERMEDIATE PRECISION of sample solution are
annexed to this document.
3. Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of
components which may be expected to present.

3.1. Test procedure

 The specificity of the assay method will be investigated by injecting the extracted
placebo to demonstrate the absence of interference with the elution of analyte.
For this study, 20 μl of blank control (solvent) and placebo solution were injected
first, then placebo spiked with ivermectin solution (the active ingredient present in
formulation other then clorsulon). Also, were injected standard solution of clorsulon
and then the sample solution (from IVERMECTIN-CLORSULON INJ) using the
current HPLC method.
3.2. Acceptance criteria
The excipients and the other active ingredient must not interfere with the analysis
of the targeted analyte.
3.3. Documentation
Chromatograms are annexed to this document
4. Accuracy
The accuracy of an analytical method is defined as the degree to which the
determined value of analyte in a sample corresponds to the true value.

4.1. Test procedure

Accuracy was measured using spiked – placebo (product matrix) recovery
method. In the spiked – placebo recovery method, a known amount of pure active
constituent is added to formulation blank [sample that contains all other ingredients
except the active(s)], the resulting mixture is assayed, and the results obtained are
compared with the expected result.
Accuracy was carried out at 50.0%, 100.0% and 150.0% of the target
concentration of the target concentration of clorsulon (170.0 μg per ml). Solutions were
prepared in triplicate at each level and the calculation was made using a calibration
curve at seven levels of concentrations that include this range. The seven-points
calibration curve was carried out at 25%,50%,75%,100%,125%,150% and 175% of the
target concentration of clorsulon (170.0 μg per ml).
For each sample, is reported the theoretical value, assay value, and percent
recovery. It will be calculated the mean, standard deviation, RSD, and percent recovery
for all samples.
4.2. Acceptance criteria
All the individual recoveries should be within 97.0 % to 103.0 %, the mean
recovery should be within 98.0 % to 102.0 % for assay, and RSD of recovery of all
recovery levels should not be more than 2.0%.
4.3. Documentation
Chromatograms for accuracy calibration curve are annexed to this document,
also the chromatograms for spiked – placebo recovery method. Results was calculated,
recorded and reported in this material.
 5. Precision- Repeatability
The precision of an analytical procedure expresses the closeness of agreement
(degree of scatter) between a series of measurements obtained from multiple sampling
of the same homogeneous sample under the prescribed conditions.

5.1. Test procedure

It will be made 6 determination at level 100% of test solution and the area
retentions time and peaks area will be recorded. It will be calculated the mean, standard
deviation and RSD.
5.2. Acceptance criteria
RSD≤ 2% for area of peaks and retentions time.
5.3. Documentation
Chromatograms obtained for precision-repeatability of sample solution are
annexed to this document.

6. Linearity
The linearity of an analytical procedure is its ability (within a given range) to
obtain test results that are directly proportional to the concentration of analyte in
the sample.

6.1. Test procedure

Linearity was carried out with minimum 5 concentration levels within a range of
50 to 150% of the target concentration of analyte (170 µg/ml) by using test sample.
Linearity solutions shall be prepared from the stock solution of working standard
to obtain the solutions of 86,128,170,212 and 254µg/ml. A graph of peak area vs.
concentration (µg/ml) will be plotted.
The slope, intercept and correlation coefficient of the regression line shall be reported
6.2. Acceptance criteria
Correlation Coefficient should be not less than 0.999.
The y-intercept must ≤ 2% of the target concentration response.
6.3. Documentation
The chromatogram for each level of concentration and the linearity graphic are annexed
to the present document.
7. Range
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including concentrations from
linearity) for which it has been demonstrated that the analytical procedure has a suitable
level of precision, accuracy and linearity.
7.1. Test procedure
For range, was recorded the concentration levels over which the results are linear. The
data obtained during the linearity and accuracy studies will be used to assess the range
of the method.
8. Robustness
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters and provides an
indication of its reliability during normal usage.

8.1. Test procedure

One set of analysis shall be carried out using the same sample by making
individual small deliberate changes in the analytical procedure.
Those changes shall be done as described below.
a. Change in flow rate (Flow rate specified in method + 0.1 ml)
b. Change in column oven temperature (Temperature specified in method + 5°C)
The result of assay shall be calculated for each set of analysis. The absolute
difference in the results obtained in robustness study and method precision
(repeatability) shall be calculated.
8.2. Acceptance criteria
The absolute difference in the results obtained in robustness study and method
precision (repeatability) should not be more than 2.0 %.
8.3. Documentation
Chromatograms obtained for ROBUSTNESS are annexed to this document
9. Solution stability
A typical approach to establish solution stability is to analyze the solution
following the method, and then store it under the required conditions. It is re-analyzed
after a specified period of time. The original results and results obtained for the stored
samples are compared to evaluate the stability

9.1. Test procedure

Standard solution and sample solution shall be prepared as described in proposed
method. The prepared solutions shall be tightly closed and stored in low actinic
glassware (as prescribed in USP -Clorsulon monograph 29),at room temperature, on
bench top at normal illuminated laboratory condition and analyzed at 6 hours,
12,24,36,48 and 72 hours.
The % difference in response at time point with respect to initial response shall be
calculated for standard solution and test solution.
9.2. Acceptance criteria
The % difference in response at time point with respect to initial response should
not be more than 2.0 %. If it is out of the set criteria, appropriate recommendation shall
be made.
9.3. Documentation
Chromatograms for stability are annexed to this document.

VI. RESULTS ACCORDING TO THE REQUIREMENTS VALIDATION

PLAN
1. System suitability
System suitability testing, originally conceived by the pharmaceutical industry to
determine if a chromatographic system is suitable for a particular analysis, is now used
routinely in laboratories where quality of results is important. System suitability testing
is often used in conjunction with method validation to provide a complete picture of
laboratory quality and reproducibility of results.
Suitability repeatability- Injection of the same sample multiple times lets you
determine if your chromatograph is providing reproducible results. 6 repeatability
samples are necessary to provide adequate data for meaningful results. Repeatability
can be determined through examination of the % relative standard deviation (%RSD)
for such parameters as peak area, height, or concentration.
Suitability Peak Asymmetry (Tailing)- The quantitative values for peak
asymmetry are important, especially when dealing with trace components. Also, since
asymmetry can vary through the life of a column, it can be important to track this data
on an ongoing basis.
Suitability Theoretical Plates- Calculation of plate count is an important
indication of column efficiency.
The system suitability test was performed automatically by the soft, based on
clorsulon repeatability sample sequence.

SYSTEM SUITABILITY Acceptance criteria (Clorsulon- RESULTS

PARAMETER USP)
Column efficiency NLT 7400- theoretical plates Passed
NMT 1,4 - Tailing factor Passed
Injection precision for R.T. RSD -NMT 1.0% Passed
Injection precision for peak area RSD -NMT 1.0% Passed
table no.04 System suitability requirements and results

Conclusion:
The acceptance criteria were fulfilled
 2. Specificity

table no.05.Results of specificity test

SAMPLE CLORSULON-peak of interes other peaks
r.t (min) AREA r.t. (min)
blank - - -
placebo - - -
placebo spiked with IVM - - ~ 34
Clorsulon std sol 11.978 353567028 -
IVERMECTIN- 11.963 343844110 ~ 34
CLORSULON INJ

Conclusion:
Clorsulon assay method for IVERMECTIN-CLORSULON INJ, solution for
injection, is said to specific as per assessment of the specificity study.

3. Accuracy
Table No 06: Preparation of accuracy levels as per following table
level no %level prep. First dilution Second dilution
no. Qty of Wt. Of Diluted Pipetted Pipetted
placebo API to
(mL) (mg) (mL)
LEVEL 1 1,7 85 50 1 20
1 50 2 1,7 85 50 1 20
3 1,7 85 50 1 20
LEVEL 1 1,7 170 50 1 20
2 100 2 1,7 170 50 1 20
3 1,7 170 50 1 20
LEVEL 1 1,7 255 50 1 20
3 100 2 1,7 255 50 1 20
3 1,7 255 50 1 20
Tab
le No. 07: % Recovery at difference levels for CLORSULON
Level % Level wrt. Concentration Concentration %
No. working added in μg per recovered in μg Recovery
conc. mL per mL
85 85.20 100.23
1 50 85 85.23 100.27
85 85.17 100.20
170 166.56 97.98
2 100 170 166.79 98.11
170 166.50 97.94
255 247.76 97.16
3 150 255 247.95 97.23
255 248.45 97.43
Mean % Recovery 98,51
Minimum % Recovery 97,23
Maximum % Recovery 100,27
StDev 1,34
RSD 1,3575

Concentrations recovered (μg per mL) were calculated using seven points standard
calibration curve (peaks areas versus concentrations at this levels).
The results and the equation of this calibration curve it is attached to this document.
Using the calibration curve equation, the formula by which were calculated
concentration recovered is:

(𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎 𝐶𝐿𝑂−778238.2806

Concentration recovered (μg/mL) =
2103775.1454

𝐶𝑜𝑛𝑐𝑒𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 ( 𝜇𝑔/𝑚𝐿)

Recovery% = 𝑥100
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑑𝑑𝑒𝑑 ( 𝜇𝑔/𝑚𝐿)
Table No. 08: Mean results of accuracy study for CLORSULON
Sr.no Parameter Results Acceptance criteria
1 Individual % recovery % Recovery obtained Between 97.0%-103.0%
between 97.23 to 100.27
2 Mean% recovery of 98,51 Between 98.0%-102.0%
all recovery levels
3 RSD of recovery of all 1,3575 Not more than 2.0%
five recovery levels

Conclusion:
Assay method for CLORSULON is found accurate as recovery results and
individual recoveries are well within the acceptance criteria.

4. Precision -Repeatability

Table No. 09: Repeatability test results obtained for sample solution of IVERMECTIN-
CLORSULON INJ, solution for injection.

IVERMECTIN- CLORSULON
CLORSULON Area Retention
INJ time
sample no.
1 353506251 11.987
2 354090278 11.983
3 354005999 11.99
4 353417974 11.98
5 354683895 11.978
6 353657977 11.977
AVERAGE 353893729 11.9825
SD 470671.7319 0.0052
RSD 0.1330 0.0431
Conclusion:
Assay method for CLORSULON in IVERMECTIN-CLORSULON INJ , solution
for injection has precision due to RSD < 2% obtained in sample solution
repeatability study.

5. Intermediate precision
The results for this intermediate precision were calculated with standard solution
of clorsulon 170 µg per mL prepared for each analyst/each day of assay.

Table No. 10: Results of Intermediate precision study for clorsulon for analyst 1
ANALYST 1 Area Label Claim of % ASSAY OF
Clorsulon CLORSULON CLORSULON
sample 1 350186351 99.2946 99.29
sample 2 353009621 100.0952 100.10
sample 3 353426966 100.2135 100.21
sample 4 353569546 100.2539 100.25
sample 5 352667158 99.9981 100.00
sample 6 353034335 100.1022 100.10

Table No. 11 Results of Intermediate precision study for clorsulon for analyst 2
ANALYST 2 Area Label Claim of % ASSAY OF
Clorsulon CLORSULON CLORSULON
sample 1 340197770 100.1162 100.12
sample 2 338196597 99.5273 99.53
sample 3 338779171 99.6988 99.70
sample 4 339480415 99.9051 99.91
sample 5 338554322 99.6326 99.63
sample 6 338630230 99.6549 99.65

Table No. 12: Comparison between results of two analysts

% ASSAY OF % ASSAY OF
Sr.no. CLORSULON CLORSULON
(ANALYST 1) (ANALYST 2)
1 99.29 100.12
2 100.10 99.53
3 100.21 99.70
4 100.25 99.91
5 100.00 99.63
 6 100.10 99.65
Confidence Level (95.0%) 0.3717 0.2266
Average 99.993 99.756
SD 0.3232 0.197
RSD 0.3232 0.198
% Absolute difference 0.237

The acceptance criteria were fulfilled

The results for this intermediate precision were calculated with standard solution
of clorsulon 170 µg per mL prepared for each day of assay.

Table No. 13: Results of Intermediate precision study for clorsulon / day 1
DAY 1 Area Label Claim of % ASSAY OF
Clorsulon CLORSULON CLORSULON
sample 1 349978714 99.4334 99.43
sample 2 353003611 100.2928 100.29
sample 3 352917255 100.2683 100.27
sample 4 353829334 100.5274 100.53
sample 5 352525112 100.1569 100.16
sample 6 351923466 99.9859 99.99

Table No. 14: Results of Intermediate precision study for clorsulon /day 2
DAY 1 Area Label Claim of % ASSAY OF
Clorsulon CLORSULON CLORSULON
sample 1 349521806 100.1650 100.16
sample 2 348697530 99.9288 99.93
sample 3 348058796 99.7457 99.75
sample 4 348224125 99.7931 99.79
sample 5 348144315 99.7702 99.77
sample 6 347844110 99.6842 99.68

Table No. 15: Comparison between results of two analysts

% ASSAY OF % ASSAY OF
Sr.no. CLORSULON CLORSULON
(DAY 1) (DAY 2)
 1 99.43 100.16
2 100.29 99.93
3 100.27 99.75
4 100.53 99.79
5 100.16 99.77
6 99.99 99.68
Confidence Level (95.0%) 0.394 0.184
Average 100.111 99.848
SD 0.3435 0.1599
RSD 0.343 0.160
% Absolute difference 0.263

The acceptance criteria were fulfilled

Conclusion:
Clorsulon assay method and for IVERMECTIN-CLORSULON INJ is found rugged
during analysis of precision and intermediate precision as % absolute difference is well
within acceptance criteria

6. Linearity
Linearity was carried out with 5 concentration levels within a range of 50 to
150% of the target concentration of analyte (170 µg/ml) by using test sample.
Linearity was prepared from the stock solution of working standard; concentration of
the solutions are 86,128,170,212 and 254µg/ml.
Evaluation:
Table No. 16: Linearity study data for clorsulon
No of Linearity % Level w.r.t. Concentration Area
levels Working conc. µg/ml Clorsulon
1 50% 86 182898506
2 75% 128 266426383
3 100% 170 350065789
4 125% 212 436620568
5 150% 254 528039316

Linearity graph of concentration in μg per mL against area response, plotted on

basis of above information is as follows,

graph.no 1 Clorsulon Linearity plot of concentration in μg per mL against area

responce in assay
 y = 2,048,751.9167x + 4,522,286.5667
LINEARITY CALIBRATION CURVE CLORSULON R² = 0.9996
560000000

510000000

460000000

410000000
AREA

360000000

310000000

260000000

210000000

160000000
70 90 110 130 150 170 190 210 230 250 270
CONCENTRATION µg/ml

Table No. 17: Results of Linearity study

S.R. function results
no.
1 The linear regression equation 2,048,751.9167x + 4,522,286.5667
2 correlation coefficient 0.9996
3 slope 2,048,751.9167
4 intercept 4,522,286.5667

The correlation coefficient of the regression line is 0.9996, greater than 0.999.
The y-intercept is 1,3 % of target concentration response, less than 2%.
The acceptance criteria were fulfilled.
Conclusion:
Clorsulon method assay for IVERMECTIN-CLORSULON INJ is found linear within
the specified range.

7. Range
The data obtained during the linearity and accuracy studies will be used to assess the
range of the method.

Table No. 18: Results of range

No of levels % Level w.r.t. Concentration Area response of clorsulon
Working conc. µg/ml
1 25 44 93595287
 2 50 86 182898506
3 75 128 266426383
4 100 170 350065789
5 125 212 436620568
6 150 254 528039316
7 175 296 629160893

graph no. 2 Clorsulon linearity plot of concentration in μg per mL against area

response in assay for range

CLORSULON -RANGE y = 2,097,935.9039x - 1,390,997.6650

R² = 0.9991
699000000
646000000
593000000
540000000
487000000
AREA

434000000
381000000
328000000
275000000
222000000
169000000
116000000
63000000
10000000
0 44 88 132 176 220 264 308 352

CONCENTRATION LEVELS

Table No. 19: Range results -linearity study

S.R. function results
no.
1 The linear regression equation 2,097,935.9039x + 1,390,997.6650
2 correlation coefficient 0.9991
Conclusion:
Good linearity was observed in the range of 44 to 296 mg/mL.

8. Robustness
a. Evaluation of results without variation of test parameters

For this test the results from precision- repeatability data are used. The results for
this set of data was calculated with standard solution of CLORSULON -170 µg/ml;
Peak area of clorsulon= 352673937, and they are presented in table no.20.

Table No. 20: Results for Precision -repeatability data for robustness
IVERMECTIN- AREA Label Claim of % ASSAY OF
CLORSULON CLORSULON CLORSULON CLORSULON
INJ sample no.
1 353506251 100.236 100.24
2 354090278 100.402 100.40
3 354005999 100.378 100.38
4 353417974 100.211 100.21
5 354683895 100.570 100.57
6 353657977 100.279 100.28
MEAN 353893729 100.346 100.35

b. Evaluation of results with variation of test parameters

Table No. 21: List of parameters studied with deliberated changes

Sr no Parameters Deliberate changes Parameters Deliberate changes
 1 Flow rate of mobile phase. 1.1 ml/min
2 Change in column temperature. 35 °C

Robustness 1
Table No. 22 Results of Robustness at the flow rate of 1.1 ml /min for sample solution
IVERMECTIN- AREA Label Claim of % ASSAY OF
CLORSULON CLORSULON CLORSULON CLORSULON
INJ sample no.
1 331023704 99.236 99.24
2 334963121 100.417 100.42
3 332982315 99.823 99.82
MEAN % assay value for robustness 99.83
MEAN % Assay value for Precision Study 100.35
% Absolute difference 0.525

Robustness 2
Table No. 23 Results of Robustness at the column oven temperature 35°C sample
solution.
IVERMECTIN- AREA Label Claim of % ASSAY OF
CLORSULON CLORSULON CLORSULON CLORSULON
INJ sample no.
1 335560427 99.316 99.32
2 337238268 99.813 99.81
3 338630230 100.225 100.23
MEAN % assay value for robustness 99.78
MEAN % Assay value for Precision Study (repeatability) 100.35
% Absolute difference 0.565
Conclusion:
Robustness studies signified that the results of the method remained unaffected by
small, deliberate changes in the flow rate and column temperature.

9. Solution stability
Solution stability study for standard solution has been conducted for 72 hours by
keeping the solution on bench top at normal illuminated laboratory condition stored in
tightly closed low actinic glassware (as prescribed in USP -Clorsulon monograph 29),
Sample solution stability at bench top condition (at normal illuminated laboratory
condition). This study has done for 72 hours for standard and sample solutions
Standard and test solution stability summary:
Solution stability study for standard and test solution has been established by
calculating %relative difference of the standard area (clorsulon) and test solution area at
each time interval with respect to initial solutions area.
Evaluation:

Table No. 24: Solution stability study of standard solution at bench top condition for
CLORSULON (i.e. at normal illuminated laboratory condition) as given below:
Solution stability for Standard solution
Status for Area Recovery Relative %
standard Clorsulon difference
solution
initial 353567028 100.00 -
6 hours 354138567 100.16 0.16
12 hours 355890151 100.66 0.66
24 hours 358631808 101.43 1.43
36 hours 357871579 101.22 1.22
48 hours 357351823 101.07 1.07
72 hours 358070283 101.27 1.27

Table No. 25: Solution stability study of sample solution at bench top condition for
CLORSULON (i.e. at normal illuminated laboratory condition) as given below:

Solution stability for Sample solution

Status for Area Recovery Relative %
standard CLORSULON difference
solution
initial 350263165 100.00 -
6 hours 350031594 99.93 0.07
12 hours 348632862 99.53 0.47
24 hours 343844110 98.17 1.83
36 hours 341918099 97.62 2.38
48 hours 339424603 96.91 3.09
72 hours 338394841 96.61 3.39
Results:
-% Relative difference between area of standard solution up to 72 hours on bench top
under normal illuminated laboratory condition stored in tightly closed low actinic
glassware, with respect to area of initial area is within limit i.e. less than 2.0%.
 - % Relative difference between areas of test solution up-to 24 hours on bench top
under normal illuminated laboratory condition with respect to area of initial test
solution area is within limit i.e. less than 2.0%.
After 24 h the % relative difference of clorsulon areas with respect to area of initial area
is more than 2%.

Conclusion:
-Standard solution is stable up-to 72 hours at normal illuminated laboratory condition
on bench top stored in tightly closed low actinic glassware.
-Test solution is stable up-to 24 hours at normal illuminated laboratory condition on
bench top stored in tightly closed low actinic glassware.

VII. CONCLUSION

The approach described in this document was used to develop and validate a
liquid chromatographic analytical method that can be used for determination of
Clorsulon in a pharmaceutical dosage form (injection).
The proposed method was validated by testing its linearity, accuracy, precision,
robustness and stability of solutions in accordance with ICH Q2A and ICH Q2B
guidelines.
The results of the analysis of solution for injection by the proposed method are
highly reproducible, reliable, and are in good agreement with the label claims of the
drug.
The excipients that are present in the pharmaceutical formulation of the assayed
samples did not interfere with Clorsulon.
It may be said that the proposed method is precise, sensitive, and accurate, so that
these can be used as standard method for determination of Clorsulon in IVERMECTIN-
CLORSULON INJ solution for injection, using the HPLC systems with PDA detector.
VIII. DEFINITION AND ABREVIATIONS
1. USP - United States Pharmacopoeia
2. NLS -not less then
3. NMT -not more then
4. Std sol -standard solution
5. CLO -clorsulon
6. R.T. -retention time
7. A -peak area
8. wt -weight
9. API -active pharmaceutical ingredient
10. Qty -quantity
11. w.r.t. -with respect to
12. Placebo -dosage form that contains all other ingredients except the
active ones
13. SD -standard deviation, is given by
1
SD= σ =√ ∑𝑁
i=1(𝑥𝑖 − 𝑥)
2
𝑁
 𝑆𝐷
14. %RSD -Relative Standard Deviation= × 100 ,is the standard
𝑥
deviation as a fraction of the mean multiplied by 100.

15. Calibration curve - relationship between the signal and the concentration of
analyte in multiple point standardization, when a
. calibration curve is a straight-line, we represent it using
. the following mathematical equation: y= a + bx, where y
. is the signal (Astd) and x is the analyte’s concentration
(Cstd) .The constants a and b are, respectively, the
. calibration curve’s expected y-intercept and its expected
. slope.
2
16.Correlation coefficient (R ) - is a statistical measure that calculates the strength of
the relationship between variables (signal-concentration)
The formulas return a value between -1 and 1, where:
• 1 indicates a strong positive relationship.
• -1 indicates a strong negative relationship.
• A result of zero indicates no relationship at all.
𝑐ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑦 𝑦2−𝑦1
17. Slope = =
𝑐𝑎𝑛𝑔𝑒 𝑖𝑛 𝑥 𝑥2−𝑥1

18. Intercept -is the point where the function crosses the y-axis.
19. Spiking - The addition of a small known amounts of a known
compound to a standard, sample, or placebo, typically for
the purpose of confirming the performance of an analytical
method.
20. Excipient - All other intentionally added components of a medicinal
veterinary product except the active(s) constituent(s)
21. Acceptance criteria - Numerical limits, ranges, or other suitable measures for
acceptance of the results of analytical procedures.
22. Validation - the procedures involved in checking data or programs for
correctness, compliance with standards and conformance with
the requirement specifications.