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 in the existing testing process.
- Find ways and means to increase productivity and improve quality.
- Assure that the complete process is under control.
3.0 Scope:
The Scope of this document is
- To describe the work required for the Validation of the Test Method for the testing of Lumenil Drench, This method is routinely used at the time of manufacturing and during stability studies.
- This Validation Protocol also describes the analytical parameters to be used for the validation of the test method.
- This Protocol also defines the facilities, responsibilities and equipment, apparatus, glassware, material, and documents that are used for the validation studies.
- In this validation protocol, the analytical results are evaluated by the application of statistical techniques and presented by means of graphical techniques.
- This method validation protocol applies to all test methods performed for release or stability evaluation of Lumenil Drench.
4.0 Periodic Revalidation:
In case there is any change or modification in the Validated Method, the Revalidation will be performed.
5.0 Change Control:
Any modification or changes in the Validated Test Method (SOP No. /IMS/SAP/QC/018)being used for the testing of Lumenil Drench should be controlled and will be entered into the Change Control Form (Form NoIR/QC/0–) in accordance with the change control procedure (SOP No /IMS/SAP/QC/0-)
Provide Justification for proposing a test method. The proposed procedure should also be validated according to this Validation Protocol.
Provide Justification for proposing a test method. The proposed procedure should also be validated according to this Validation Protocol.
6.0 Facilities/Responsible Personnel/Equipment/Material Documentation
6.1 Facilities:
Validation of Test Method (SOP No. /IMS/SAP/FP/00) being used for the Determination of Oxfendazole & Oxyclozanide in Lumenil Drench will be performed in Chemical Lab.
- Machine/ Equipment
| Machine/ Equipment | Qualification Status | Qualification No. | Calibration Status |
| HPLC | Qualified | IQ: _________
OQ: _________ PQ: _________ |
|
| Analytical Balance | Qualified | IQ: _________
OQ: _________ PQ: _________ |
|
- Identification of Apparatus/Glassware
| Apparatus/Glassware |
| Volumetric Flask |
| Pipette |
7.0 Analytical Method Of Lumenil Drench
As Per FP SOP NO /IMS/SAP/FP/0
Physical Parameters:
| PARAMETERS | SPECIFICATION |
| Physical Form | Suspension |
| Colour | Cream Colored |
| .pH | 3.0 to 6.0 at 25° C |
| Viscosity | 1200 – 1500 C.P.S at 25°C
Spindle # LV 2, 50 rpm,Tarq NLT 40% |
| Wt/ml | 1.04gm/ml |
Chemical Analysis
| PARAMETERS | SPECIFICATION |
| Identification Test of Oxfendazole
By retention time of std and sample in the chromatograph |
The retention time of std and sample in chromatograph should be the same. |
| Identification Test of Oxyclozanide
By retention time of std and sample in the chromatograph |
The retention time of std and sample in chromatograph should be the same. |
Chemical Analysis
- Contents of Oxyclozanide:
Stated Quantity: 62.50 mg / ml
- Contents of Oxfendazole:
Stated Quantity: 22.50 mg / ml
1.1 Sample Preparation:
NOTE: Before taking sample shake well and sonicate suspension for 10minutes in a 250ml glass beaker.
Take 5.20gm of a sample of suspension, previously well mixed, sonicated(10minutes), and free from air bubbles equivalent to 112.5 mg of Oxfendazole and 312.5mg oxyclozanide in 100ml glass beaker, add 20ml distilled water in a beaker in which sample is taken and sonicate it for 10 minutes, then transfer this sample completely in 100ml volumetric flask. rinse the same beaker with a small portion of methanol and transfer to a flask in which the sample is taken.
Add methanol in portions while sonicating and shaking the flask,and makeup volume up to 100ml with methanol.
- Shake well and Sonicate 100ml volumetric flask (step a) for 30minutes till clear solution and filter it.
- c) Take 1 ml from (step b) filtrate in 50 ml volumetric flask and make volume upto 50 ml with methanol.mix well and sonnicate for 5minutes.
1.2 Oxfendazole Standard Preparation:
- Weight accurately 113 mg Oxfendazole and put them in a 100 ml Volumetric flask. Then add 20 to 30 ml methanol ,sonnicate flask for complete solubility and make volume upto 100 ml with methanol.
- Shake well and Sonicate 100ml volumetric flask (step a) for 10 minutes.
- Take 1 ml from (step b) in 50 ml volumetric flask and make volume upto 50 ml with methanol and sonnicate for 5minutes
1.3 Oxyclozanide Standard Preparation:
- Weight accurately 312 mg oxyclozanide and put them in a 100 ml Volumetric flask. Then add 20 to 30 ml methanol.sonnicate flask for complete solubility and make volume upto 100 ml .
- Shake well and Sonicate 100ml volumetric flask (step a) for 10 minutes .
- Take 1 ml from (step b) in 50 ml volumetric flask and make volume upto 50 ml with methanol and sonnicate for 5minutes.
1.3 Chromatographic system
The liquid chromatograph is equipped with octadecyl silane column of C18 250mm X 4.6mm .
Flow Rate: 1 ml / min.
Detector: UV 300 nm.
1.4 Mobile Phase:
1% H3PO4 (OrthoPhosphoric Acid) : Methanol (HPLC grade)
20 ml : 80 ml
Note : 1% H3PO4 (Phosphoric Acid): [ 1ml Con phosphoric acid + Sufficient Distilled water to make total volume 100ml]
1.5 Calculations:
- Contents of Oxyclozanide:
Stated Quantity: 62.50 mg / ml
Calculations:
Ave. Peak area of Sample =__________________________
Ave. Peak area of Standard =___________________________
| Ave. Peak area Sample | X 312 X | 1 X | 100 X | 50 X 1.04(wt/ml) | =–X——– | mg/ml |
| Ave. Peak area Standard | 100 | 50 | 5.2 gm | 1 | ||
| X X 100 | =———% | |||||
| 62.5 |
Limit: The contents of Oxyclozanide should be between 90 -110% of stated amounts.
2.0 Contents of Oxfendazole:
Stated Quantity: 22.50 mg / ml
Procedure: Inject standard 1st.
- Make 2 consecutive injections of standard preparation.
- Make 2 consecutive injections of sample preparation.
Calculations:
Ave. Peak area of Sample =__________________________
Ave. Peak area of Standard =___________________________
| Ave. Peak area Sample | X 113 X | 1 X | 100 X | 50 x 1.04(wt/ml | =–X——– | mg/ml |
| Ave. Peak area Standard | 100 | 50 | 5.2gm | 1 | ||
| X X 100 | =———% | |||||
| 22.50 |
Limit: The contents of Oxfendazole should be between 90 -110% of stated amounts
- Validation Parameters: Following analytical parameters are to be considered
- Linearity:(Calibration Curve )
- Precision
- System precision (Repeatability)
- Method precision (Reproducibility)
- Intermediate Precision:
- Accuracy
- Ruggedness
- Robustness:
- Stress Degradation:
- Limit of Detection (LOD):
- Limit of Quantitation (LOQ):
- Specificity
- system Suitability:
Page Contents
8.1 Calibration Curve (Linearity):
Linearity is the ability of the method to produce results that are directly proportional to the concentration of the analyte in samples with a given range.
8.1.1 Calibration Curve (Linearity) of Oxfendazole
The linearity of the method was evaluated by preparing three series of standard of Oxfendazole in the range of 70 – 130 μg/ml in methanol and note peak area from chromatograph .Excellent correlation between peak area and concentration was observed with R2 = 0.9971 (Figure.1). The regression equation was found to be Y=315706x+472085.Statistical data are presented in table 1and the calibration curve was shown in figure 1.
Table 1: Linearity results for Oxfendazole
| Sr. # | Chrom # | Conc. (ug/ml) | Peak area | MEAN | RSD (%) |
| 1 | AMV 01 | 70 | 775424 | 777985.0
|
0.710% |
| 2 | AMV 02 | 774210 | |||
| 3 | AMV 03 | 784321 | |||
| 4 | AMV 01 | 100 | 1121530 | 1123110.3
|
0.122% |
| 5 | AMV 02 | 1123861 | |||
| 6 | AMV 03 | 1123940 | |||
| 7 | AMV 01 | 130 | 1411486 | 1409397.7
|
0.147% |
| 8 | AMV 02 | 1409355 | |||
| 9 | AMV 03 | 1407352 |
Figure 1: Calibration curve for Oxfendazole
Acceptance Criteria:
- Coefficient of determination (r2) should be greater than 0.9998.
8.1.1 Calibration Curve (Linearity) of Oxyclozanide
The linearity of the method was evaluated by preparing three series of standard of Oxyclozanide in the range of 70 – 130 μg/ml in methanol and note peak area from chromatograph .Excellent correlation between peak area and concentration was observed with R2 = 0.9995 (Figure.1). The regression equation was found to be Y=581220x+ 704039 Statistical data are presented in table 1and the calibration curve was shown in figure 1.
Table 2: Linearity results for Oxyclozanide
| Sr. # | Chrom # | Conc. (ug/ml) | Peak area | MEAN | RSD (%) |
| 1 | AMV 01 | 70 | 1293250 | 1277493.0
|
1.152% |
| 2 | AMV 02 | 1264111 | |||
| 3 | AMV 03 | 1275118 | |||
| 4 | AMV 01 | 100 | 1899469 | 1882009.7
|
1.169% |
| 5 | AMV 02 | 1889269 | |||
| 6 | AMV 03 | 1857291 | |||
| 7 | AMV 01 | 130 | 2449197 | 2439932.7
|
2.255% |
| 8 | AMV 02 | 2489739 | |||
| 9 | AMV 03 | 2380862 |
Figure 1: Calibration curve for Oxyclozanide
Acceptance Criteria:
- Coefficient of determination (r2) should be greater than 0.9998.
-
Precision:
Precision is the degree of closeness of agreement among individual test results when the method is applied to multiple sampling of a homogeneous sample. It is a measure of either the degree of reproducibility (under different conditions) or repeatability (under same conditions) of the method.
8.2.1 System precision (Repeatability)
To study precision, five replicate standard of Oxfendazole (20μg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated. Results of system precision studies were shown in table 3.
Results of system precision for Oxfendazole, Absorbance repeatability
| Table 3 | |||||
| Sr. # | # | Peak area | |||
| 001 | AMV Repeat 01 | 1113443 | |||
| 002 | AMV Repeat 02 | 1106940 | |||
| 003 | AMV Repeat 03 | 1107423 | |||
| 004 | AMV Repeat 04 | 1113836 | |||
| 005 | AMV Repeat 05 | 1113300 | |||
| n = | 5 | ||||
| Mean | 1110988.40 | ||||
| SD | 3484.93 | ||||
| RSD % | 0.314% | ||||
8.2.1 System precision (Repeatability)
To study precision, five replicate standard of Oxyclozanide (20μg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated. Results of system precision studies were shown in table 4.
Results of system precision for Oxyclozanide, Absorbance repeatability
| Table 4 | |||||
| Sr. # | # | Peak area | |||
| 001 | AMV Repeat 01 | 2008905 | |||
| 002 | AMV Repeat 02 | 1973729 | |||
| 003 | AMV Repeat 03 | 1972313 | |||
| 004 | AMV Repeat 04 | 1963391 | |||
| 005 | AMV Repeat 05 | 2002173 | |||
| n = | 5 | ||||
| Mean | 1984102.20 | ||||
| SD | 20107.68 | ||||
| RSD % | 1.013% | ||||
8.2.2 Method precision (Reproducibility)
The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 2 times on the same day and on different days for concentration of standard of 20μg/ml. The result was reported in terms of relative standard deviation (% RSD). Results of method precision studies were shown in table 5.
Results of Results of Method precision for Oxfendazole
Table 5
| Standard preparation 1 | Standard preparation 2 | |||
| Sr. # | Chrom. # | Peak Area | Chrom. # | Peak Area |
| 001 | 1115941 | 1124759 | ||
| 002 | 1119606 | 1125140 | ||
| n = | 2 | 2 | ||
| Mean | 1117773.50 | 1124949.50 | ||
| RSD % | 0.232% | 0.024% | ||
| Grand Mean | 1121361.50 | |||
| RSD % | 0.453% | |||
There were no significant differences between RSD (%) values for intra-day and inter-day precision, which indicates that the method is reproducible.
8.2.3 Method precision (Reproducibility)
The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 2 times on the same day and on different days for concentration of standard of 20μg/ml. The result was reported in terms of relative standard deviation (% RSD). Results of method precision studies were shown in table 6.
Results of Results of Method precision for Oxyclozanide
Table 6
| Standard preparation 1 | Standard preparation 2 | |||
| Sr. # | Chrom. # | Peak Area | Chrom. # | Peak Area |
| 001 | 1918711 | 1930483 | ||
| 002 | 1929929 | 1930649 | ||
| n = | 2 | 2 | ||
| Mean | 1924320 | 1930566 | ||
| RSD % | 0.412% | 0.006% | ||
| Grand Mean | 1927443 | |||
| RSD % | 0.229% | |||
There were no significant differences between RSD (%) values for intra-day and inter-day precision, which indicates that the method is reproducible.
8.2.3 Intermediate precision
The intermediate precision of the proposed method was determined by performing the method by two analysts (Analyst 1 and Analyst 2) for concentration of standard 20μg/ml. The percent relative standard deviation (% RSD) for peak responses was calculated. The results for intermediate precision were shown in table 7,8.
Intermediate precision refers to variations within a laboratory as with, different instruments, by different analysts, and so forth.
Results of Intermediate precision for Oxfendazole
| Table 7 | ||
| ANALYST 1 | ||
| Instrument: | HPLC System 01 | |
| Performed By | (QCM) | |
| Chrom # | ||
| 1127518 | ||
| 1130361 | ||
| n | 2 | |
| Mean | 1128939.500 | |
| SD | 2010.3046 | |
| RSD % | 0.178% | |
| Table 8 | |||
| ANALYST 2 | |||
| Instrument: | HPLC System 02 | ||
| Performed By | (QC analyst) | ||
| Abs # | |||
| 1130742.0 | |||
| 1137243.0 | |||
| n = | 2 | ||
| Mean | 1133992.500 | ||
| SD | 4596.9012 | ||
| RSD % | 0.405% | ||
8.2.4 Intermediate precision
The intermediate precision of the proposed method was determined by performing the method by two analysts (Analyst 1 and Analyst 2) for concentration of standard 20μg/ml. The percent relative standard deviation (% RSD) for peak responses was calculated. The results for intermediate precision were shown in table 9,10.
Intermediate precision refers to variations within a laboratory as with, different instruments, by different analysts, and so forth.
Results of Intermediate precision for Oxyclozanide
| Table 9 | ||
| ANALYST 1 | ||
| Instrument: | HPLC System 01 | |
| Performed By | (QCM) | |
| Chrom # | ||
| 1954688 | ||
| 1917003 | ||
| n | 2 | |
| Mean | 1935845.50 | |
| SD | 26647.32 | |
| RSD % | 1.377% | |
| Table 10 | |||
| ANALYST 2 | |||
| Instrument: | HPLC System 02 | ||
| Abs # | |||
| 1898317.0 | |||
| 1939108.0 | |||
| n = | 2 | ||
| Mean | 1918712.50 | ||
| SD | 28843.5927 | ||
| RSD % | 1.503% | ||
-
Accuracy ( % Recovery):
Accuracy is the closeness of results obtained by a method to the true value. It is the measure of exactness of the method. The accuracy of the method was evaluated by the standard addition method. Recovery of the method was determined by spiking an amount of the pure drug (50%,100%,150%) at three different concentration levels in its Drench has been added to the pre-analyzed working standard Drench of the drug. The sample was analyzed in triplicate at each level. The percent individual recovery and %RSD for recovery at each level are calculated.
- Accuracy ( % Recovery) of Oxfendazole
Peak area of Oxfendazole RS
| 1128939.500
|
|
Sample preparations
| Table 11 | ||||||||||
| Level of recovery | Initial amount of SA (mg) | Amount of std added (mg) |
Total amount present (mg) | Chrom # | Peak area | Mean area | Total amount recovered (mg) | Recovery
(%) |
||
| 50% | 20 | 30 | 50 | 459073 | 457846.000
|
50.69 | 101.39% | |||
| 456619 | ||||||||||
| 100% | 60 | 40 | 100 | 621691 | 623372.000
|
99.39 | 99.39% | |||
| 625053 | ||||||||||
| 150% | 100 | 50 | 150 | 790120 | 787539.500
|
149.98 | 99.99% | |||
| 784959 | ||||||||||
| Mean Recovery | 100.26% | |||||||||
| SD | 1.025 | |||||||||
| RSD % | 1.022% | |||||||||
Acceptance Criteria
Recovery 100 + 2%
The recovery of the method is determined by spiking a placebo with active pharmaceutical ingredient of known potency. The values of recovery (%), RSD (%),in Table 11, above indicate that the method is accurate.
- Accuracy ( % Recovery) of Oxyclozanide
Peak area of Oxyclozanide RS
| 1935845.500
|
|
Sample preparations
| Table 12 | ||||||||||
| Level of recovery | Initial amount of SA (mg) | Amount of std added (mg) |
Total amount present (mg) | Chrom # | Peak area | Mean area | Total amount recovered (mg) | Recovery
(%) |
||
| 50% | 20 | 30 | 50 | 783612 | 776757.000
|
50.16 | 100.13% | |||
| 769902 | ||||||||||
| 100% | 60 | 40 | 100 | 1045308 | 1041968.500
|
99.58 | 99.58% | |||
| 1038629 | ||||||||||
| 150% | 100 | 50 | 150 | 1321999 | 1326721.000
|
148.72 | 99.15% | |||
| 1331443 | ||||||||||
| Mean Recovery | 99.68% | |||||||||
| SD | 0.590 | |||||||||
| RSD % | 0.592% | |||||||||
Acceptance Criteria
Recovery 100 + 2%
The recovery of the method is determined by spiking a placebo with active pharmaceutical ingredient of known potency. The values of recovery (%), RSD (%),in Table 12, above indicate that the method is accurate.
-
Ruggedness:
The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days, etc. Ruggedness is normally expressed as the lack of influence on the test results of operational and environmental variables of the analytical method. Ruggedness is a measure of reproducibility of test results under the variation in conditions normally expected from laboratory to laboratory & from analyst to analyst.
Procedure:
- Analyze two samples from the same composite sample according to the analytical method by two analyst using different lots of reagents keeping the other condition same.
- Analyze the samples according to procedure by one analysts on the same day & the other on second day.
For Oxfendazole
Table 13
|
Day 1 |
ANALYST 1 | |||||
| For Oxfendazole
Standard = 1128939.500 |
||||||
| Sr | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 5246057.000 | 22.50mg/ml | 22.37mg/ml | 99.43% | ||
| 2 | 5277187.000 | |||||
| Mean | 5261622.000 | |||||
| SD | 22012.2341 | |||||
| %RSD | 0.418% | |||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||||
| Assay Limit 97 % to 103 % | ||||||
Table 14
|
Day 2 |
ANALYST 2 |
|||||
| For Oxfendazole
Standard = 1133992.500 |
||||||
| Sr | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 5100169.000 | 22.50mg/ml | 22.65mg/ml | 100.68% | ||
| 2 | 5257771.000 | |||||
| Mean | 5178970.000 | |||||
| SD | 111441.4429 | |||||
| %RSD | 2.1518 | |||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||||
| Assay Limit 97 % to 103 % | ||||||
For Oxyclozanide
Table 15
|
Day 1 |
ANALYST 1
|
|||||
| For Oxyclozanide
Standard = 1935845.500 |
||||||
| Sr | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 10125601.000 | 62.50mg/ml | 63.08mg/ml | 100.93% | ||
| 2 | 10227873.000 | |||||
| Mean | 10176737.000 | |||||
| SD | 72317.2247 | |||||
| %RSD | 0.711% | |||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||||
| Assay Limit 97 % to 103 % | ||||||
Table 16
|
Day 2 |
ANALYST 2 |
|||||
| For Oxyclozanide
Standard = 1918712.500 |
||||||
| Sr | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 9934531.000 | 62.50mg/ml | 62.92mg/ml | 100.67% | ||
| 2 | 9856607.000 | |||||
| Mean | 9895569.000 | |||||
| SD | 55100.5888 | |||||
| %RSD | 0.5568% | |||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||||
|
Assay Limit 97 % to 103 % |
||||||
The ruggedness of the proposed method was determined by two different analysts. The mean, standard deviation, and % RSD values were found to be less than 2 % which shows the ruggedness of the method.
-
Robustness:
The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing parameters like flow rate of mobile phase and detection wavelength Change in the detection wavelength by ± 2nm (298nm and 302nm) Change in flow rate by ± 0.1 ml/minute (0.6 ml/min and 0.8 ml/minute) After each change, sample was injected and % assay with system suitability parameters were checked. Robustness values were given in table 17&18.
Results of Robustness values for Oxfendazole
Table17
|
Changing Parameters |
Detection Wavelength |
||||||
| Instrument | HPLC SYSTEM 01 | ||||||
| Model /Made | WATERS | ||||||
| For Peak area
Standard = 1128939.500 |
|||||||
| Sr | Wavelength | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 298 | 5471089 | 22.50mg/ml | 22.89mg/ml | 101.73% | ||
| 2 | 300 | 5396254 | |||||
| 3 | 302 | 5417940 | |||||
| Mean | 5428427.66 | ||||||
| SD | 38504.06 | ||||||
| %RSD | 0.709% | ||||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | ||||||
| Assay Limit 97 % to 103 % | |||||||
Results of Robustness values for Oxyclozanide
Table18
|
Changing Parameters |
Detection Wavelength |
||||||
| Instrument | HPLC SYSTEM 01 | ||||||
| Model /Made | WATERS | ||||||
| For Peak area
Standard = 1918712.500 |
|||||||
| Sr | Wavelength | Peak area | Theoretical Content |
Amount recovered | Recovery (%) | ||
| 1 | 298 | 10016338 | 62.50mg/ml | 63.51mg/ml | 101.61% | ||
| 2 | 300 | 9970189 | |||||
| 3 | 302 | 9977307 | |||||
| Mean | 9987944.667 | ||||||
| SD | 24845.5733 | ||||||
| %RSD | 0.249% | ||||||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | ||||||
| Assay Limit 97 % to 103 % | |||||||
Robustness studies were made by varying the chromatographic parameters like detector wavelength and flow rate are within a realistic range and the quantitative influence of the variables is determined. The robustness of the proposed method was determined by estimating a drug at a slightly different wavelength from the selected wavelength and a slight change in flow rate. No significant difference was found in the RT & parentage of samples. Therefore, the proposed method was considered robust.
-
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 samples Drench from the same assay composite sample according to the analytical method by one analyst at room temperature
- Prepare samples Drench, and standard Drench according to the analytical method by one analyst and subjected to heat treatment at, 60 °C for one hour
- Analyze both the samples.
For Oxfendazole
Table 19
| UNSTRESS SAMPLE | ||||
| For Oxfendazole
Standard = 1128939.500 |
||||
| Sr | Absorbance | Theoretical Content |
Amount recovered | Recovery (%) |
| 1 | 5281553.000 | 22.50mg/ml | 22.502mg/ml | 100.01% |
| 2 | 5303048.000 | |||
| Mean | 5292300.500 | |||
| SD | 15199.2603 | |||
| %RSD | 0.287% | |||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||
| Assay Limit 97 % to 103 % | ||||
Table 20
| FORCED DEGRADATION | ||||
| For Oxfendazole
Standard = 1128939.500 |
||||
| Sr | Absorbance | Theoretical Content |
Amount recovered | Recovery (%) |
| 1 | 5259868 | 22.50mg/ml | 22.36mg/ml | 99.39% |
| 2 | 5259868 | |||
| Mean | 5259868 | |||
| SD | 0.0000 | |||
| %RSD | 0.000 | |||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||
| Assay Limit 97 % to 103 % | ||||
For Oxyclozanide
Table 21
| UNSTRESS SAMPLE | ||||
| For Oxyclozanide
Standard = 1935845.500 |
||||
| Sr | Absorbance | Theoretical Content |
Amount recovered | Recovery (%) |
| 1 | 9961648.000 | 62.50mg/ml | 62.90mg/ml | 100.64% |
| 2 | 9999654.000 | |||
| Mean | 9980651.000 | |||
| SD | 26874.3003 | |||
| %RSD | 0.269% | |||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||
| Assay Limit 97 % to 103 % | ||||
Table 22
| FORCED DEGRADATION | ||||
| For Oxyclozanide
Standard = 1935845.500 |
||||
| Sr | Absorbance | Theoretical Content |
Amount recovered | Recovery (%) |
| 1 | 9815505 | 62.50mg/ml | 62.62mg/ml | 100.19% |
| 2 | 10056584 | |||
| Mean | 9936044.500 | |||
| SD | 170468.5957 | |||
| %RSD | 1.716 | |||
| Acceptance Criteria:
|
The % RSD of the assay or recovery values should not be greater than 2.0%. | |||
| Assay Limit 97 % to 103 % | ||||
There was no degradation of Oxfendazole & Oxyclozanide and no significant change in % of Oxfendazole & Oxyclozanide.
-
Limit of detection (LOD) & Limit of Quantitation (LOQ)
The LOD and LOQ were predicted based on the parameters of the standard error of estimate and slope, calculated from the linearity of the response data.
- Limit of Detection (LOD):
The lowest amount of analyte in the sample can be detected but not necessarily quantitated. Analysis of sample with the known concentration of 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
LOD = 3.3 X SD
Slope
Limit of Quantitation (LOQ):
The lowest amount of an analyte in a sample can be quantitatively determined with suitable precision and accuracy. Analysis of sample with the known concentration of analyte and by establishing minimum concentration at which analyte can be reliably quantitated.
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
LOQ = 10 X SD
Slope
LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ) OF Oxfendazole & Oxyclozanide
Table 23
| SR # | Compound | SLOPE | STEXY
|
LOD | LOQ |
| (known_y”s,known_X”s,) | (known_y”s,known_X”s,) | = 3.3 X SD
Slope |
= 10 X SD
Slope |
||
| 1 | Oxfendazole
|
315706.350 | 24020.472 | 0.228 | 0.761 |
| 2 | Oxyclozanide | 581219.850 | 19021.798 | 0.098 | 0.327
|
Specificity
The specificity of an analytical method is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components.
- Poor specificity can impact accuracy, precision, and linearity
Acceptance Criteria:
- Assay Limit 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%.
Remarks: Accuracy, precision, and linearity are within the limit, it confirms that the analytical procedure employed for a specific test is suitable for its intended use.
System Suitability:
Set of parameters and criteria there off to ensure that the system is working properly. System suitability conforms to repeatability data. (Mean, Standard Deviation, and Relative Standard Deviation (%).
The performance of the instrument is satisfactory because all the parameters of the system suitability test (Rt, ReDrench, Theoretical Plates,) are within the acceptance range.
CONCLUSION
The developed method was found to be sensitive, accurate, precise, reproducible, and linear over the concentration range studied. The proposed method can be used for the routine quality control
analysis of Lumenil Drench.
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Date:
Q.C Analyst
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Quality Control Manager
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