What is analytical method validation?
It is the process of demonstrating that analytical procedures are suitable for their intended use. More specifically, analytical method validation is establishing documented evidence that the specified method will consistently provide accurate test results that evaluate a product against its defined specification and quality attributes. The method should be validated, transferable, robust, reliable, accurate, and precise for day-to-day activities in the quality control environment. The method should not enter the validation phase unless it is fully developed. Validation experiments must be properly documented and performed on qualified and calibrated instrumentation and equipment.
There are different types of formulation composition available:
i. Dose-proportional formulation composition.
ii. Pseudo-dose-proportional formulation composition
iii. Look-alike formulation composition
1) Dose-proportional formulation composition:
In these types of formulations, the composition of active and inactive ingredients proportionally increases as the strength increases. In this case, method validation can be performed on any of the strengths.
Pseudo-dose-proportional formulation composition:
In this type of formulation, the composition of the active ingredient proportionally increases as the strength increases, but the average weight of the dosage form remains constant. The weight of constituents of the matrix is modified to keep the average weight constant.
3) Look at formulation composition:
• In these types of formulations, the composition of the active ingredient proportionally increases as the strength increases. Still, the average weight of the dosage form remains constant by a minor change in the weight of one of the excipients.
• The look-alike formulation concept is applicable only for the drug product having less content of active ingredient.
• These current validation characteristics describe the validation parameters stated by the International Conference on Harmonization [ICH] guidelines Q2 (R1).
Different Types of Validation Characteristics:
• System Suitability
• Specificity
• Precision
System Precision
Repeatability
Intermediate Precision
• Accuracy
• Linearity
• Robustness
• Stability Solution
Analytical Method Validation (AMV) of Assay of Ibuprofen 100 mg USP
1.0 PURPOSE
To provide documented evidence that the analytical procedure adopted for the assay of ibuprofen is suitable for its intended purpose and routine testing.
2.0 DESIGNATION & RESPONSIBILITY
QC Executive: To prepare the Analytical Method Validation Report. To organize the Analytical Method Validation Study in coordination with Quality Assurance.
QC Manager: To analyze the sample and prepare the Analytical Method Validation Report and organize the implementation of the recommendations made during the Analytical Method Validation.
QA Manager: To review the entire report for correctness and completeness before approval. To approve and authorize the Analytical Method Validation Protocol as well as the Analytical Method Validation Report.
3.0 MATERIAL, REAGENT
Material: Ibuprofen USP WS, Ibuprofen Tablet USP 400 mg
Reagents: Ammonium hydroxide (AR Grade), acetonitrile (HPLC Grade), water (HPLC Grade)
4.0 Abbreviation
5.0 METHOD OF ANALYSIS.
Mobile phase: Dissolve 4.0 g of chloroacetic acid in 400 mL of water, adjust with ammonium hydroxide to a pH of 3.0 if necessary, add 600 mL of acetonitrile, and mix.
Standard solution: 10.0 mg/mL of USP Ibuprofen RS in mobile phase.
Sample solution: Nominally 10.0 mg/mL of ibuprofen prepared as follows. Transfer NLT 10 tablets to a suitable volumetric flask and add about 50% of the final volume of the mobile phase. Shake on a mechanical shaker for at least 60 min or until the tablets are disintegrated. Dilute with mobile phase to volume. Centrifuge a portion of the solution at about 3000 rpm for about 10 min or until a clear supematant is obtained. Use the supernatant for analysis.
Chromatographic system
[NOTE-It is suggested to use the mixture of methanol and water (90:10) for needle wash.]
Mode: LC
Detectors
Assay: UV 254 nm
Identification test A: Diode array UV 200-400 nm
Column: 4.6 mm × 25 cm; 5-µm packing L1
Flow rate: 2.0 mL/min
Injection volume: 10 µL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.5
Relative standard deviation: NMT 2.0%
Calculate the percentage of the labeled amount of ibIbuprofenC13H18O2) in the portion of tablets taken.
Result (U/tS) × (CS/CU) × 100
U=peak response of ibuprofen from the sample solution
S=peak response of ibuprofen from the standard solution
CS = concentration of USP Ibuprofen RS in the standard solution (mg/ml.)
CU nominal concentration of ibuprofen in the sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%
7.0 VALIDATION PROGRAM DETAILS
7.1 System Suitability
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System suitability parameters, which are generally required to be monitored, include several theoretical plates (column efficiency), internal precision (repeatability, reproducibility, tailing factor (peak asymmetry), resolution, relative retention time, and capacity factors), depending upon the requirement of the analytical method being validated. System suitability is defined by ICH as "the checking of a system, before or during the analysis of unknowns, to ensure system performance. System suitability criteria may include such factors as plate count, tailing, retention, and/or resolution.
System suitability criteria should also include a determination of reproducibility (%RSD) when a system suitability "sample" (a mixture of main components and expected by-products/interferences) is run. System suitability testing is an integral part of analytical procedures.
• If the % RSD specification is below 2.0%, five replicates are used.
• If the % RSD specification is above 2.0%, six replicates are used.
The parameters used in the system suitability tests (SST) report are as follows:
• The number of theoretical plates or efficiency (N).
• Capacity factor (K).
• Separation or Relative Retention (a).
• Resolution (Rs).
• Tailing factor (T).
• Relative Standard Deviation (RSD).
Preparation of System Suitability Solution: Take 100.0 mg of USP Ibuprofen RS in 10 mL of mobile phase.
Run the injections listed below in sequence:
|
S.No. |
Name of solution |
No. of injection |
|
1 |
Blank |
1 |
|
2 |
System Suitability
Solution |
6 |
Result:
7.2 Specificity
Specificity in analytical method validation refers to the ability of a method to accurately measure the target analyte (the substance of interest) without interference from other components that might be present in the sample.
These components could include:
Impurities: Substances present in the sample that are not the target analyte.
Degradants: Substances formed from the degradation of the analyte.
Matrix components: Other substances present in the sample that can potentially interfere with the analysis.
Specificity is the ability to access unequivocally the analyte in the presence of components that may be expected to be present. To prove specificity, the following experiment is carried out. We recorded the placebo and the placebo spiked with ibuprofen outlined in Section 5.0 to check the interference of the placebo with ibuprofen.
Procedure:
Placebo solution: Take 300.0 mg of placebo in 10 mL mobile phase, mix well, and filter.
Preparation of standard Solution: Take 100.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Preparation of sample solution: Nominally 10.0 mg/mL of ibuprofen prepared as follows. Transfer NLT 10 tablets to a suitable volumetric flask and add about 50% of the final volume of mobile phase. Shake on a mechanical shaker for at least 60 min or until the tablets are disintegrated. Dilute with mobile phase to volume. Centrifuge a portion of the solution at about 3000 rpm for about 10 min or until a clear supernatant is obtained. Use the supernatant for analysis.
Run the injections listed below in sequence:
|
S.No. |
Name of solution |
No. of injection |
|
1 |
Blank |
1 |
|
2 |
Placebo |
1 |
|
3 |
Standard Solution |
1 |
|
4 |
Sample Solution |
1 |
Result:
7.2 Precision
Definition: The closeness of agreement between a series of measurements and multiple samplings of the same homogeneous sample under prescribed conditions. The precision of the test method is usually expressed as the standard deviation or relative standard deviation of a series of measurements.
Precision may be considered at three levels:
System Precision, Repeatability, and Intermediate Precision.
The precision of the analytical procedure expresses the closeness of agreement between the value accepted as a conventional true value or an accepted standard value and the value found.
7.2.1 System Precision:
System precision is the closeness of agreement between the detector responses. Usually expressed as the standard deviation (SD) or the relative standard deviation (RSD).
Procedure:
Run separate injections of the mobile phase (blank) and standard solution (six replicates) and record the area responses in the standard solution.
Preparation of standard solution:
Run the injection listed below:
|
S.No. |
Name of solution |
No. of injection |
|
1 |
Blank |
1 |
|
2 |
Standard Solution |
1 |
Result:
Result: Relative Standard Deviation (RSD) for peak area counts was 0.209%, and retention time count was 0.030% (NMT 2.0%).
7.2.2 Repeatability
The preparation of 6 replicates of a test solution was recorded as per the procedure in section 5.0, and the relative standard deviation of the assays was calculated.
Procedure:
Preparation of sample solution: As described in method 5.0
Run the sequence listed below.
|
S.No. |
Name of solution |
No. of injection |
|
1 |
Blank |
1 |
|
2 |
Sample Solution |
1 |
Result:
Result: The % RSD of the % of AsAssayas 0.250% in Ibuprofen, which is less than the acceptance limit, 2.0% RSD.
7.2.3 Intermediate Precision: Ruggedness
Analysis was carried out on different days and by different chemists as per the methodology described in section 5.0.
Preparation of standard solution:
Preparation of sample solution:
Run the sequence listed below:
|
S.No. |
Name of solution |
No. of injections |
|
1 |
Blank |
1 |
|
2 |
Standard Solution |
6 |
|
3 |
Sample Solution |
|
Result:
It expresses laboratory variations: different days, different analysts, different equipment, etc.
Result: Intermediate precision of the analytical method was established by analyzing the difference between RSD results of Day 1 and Day 2. The RSD was 0.261%, which is less than the acceptance limit of 2.0% RSD.
7.3 Accuracy
Definition: It is the closeness of agreement between the actual value and the measured value.
Accuracy is calculated as the percentage of recovery by the assay of the
known added amount of the analyte in the sample or the difference between the mean and the accepted true value together with confidence intervals.
The ICH guidance recommended taking a minimum of 3 concentration levels covering the specified range, and 3 replicates of each concentration are analyzed (total 3 * 3 = 9 determinations).
Accuracy is performed in triplicate at concentrations of 50%, 100%, and 150% of the working concentration. The recovery is calculated against a standard.
Procedure:
Preparation of standard Solution: Take 100.0 mg of USP Ibuprofen RS in 10 mL. Mobile phase.
Solution preparation (50%): Take 50.0 mg of USP Ibuprofen RS and 300.0 mg of placebo in 10 mL mobile phase, mix well, and filter.
Solution preparation (100%): Take 100.0 mg of USP Ibuprofen RS and 300.0 mg of placebo in 10 mL mobile phase, mix well, and filter.
Solution preparation (150%): Take 150.0 mg of USP Ibuprofen RS and 300.0 mg of placebo in 10 mL mobile phase, mix well, and filter.
Run the sequence listed below:
Result:
Result: The accuracy of the analytical method was established by analyzing the concentration range of 50% to 150% of tofacitinib, with recovery found within the limit, between 98% and 102%, and RSD was %, which is within the acceptance
7.4 Linearity and Range:
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 (amount) of analyte in the sample.
Range
The range of the analytical procedure is the interval between the upper and lower concentrations.
At least 6 replicates per concentration are to be studied. Plot a graph of concentration (on the x-axis) vs. mean response (on the y-axis). Calculate the regression equation, Y-intercept, and correlation coefficient. Linearity shall be established across the range. If linearity does not meet the acceptance criteria, establish the range of concentration in which it is linear. 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 standard.
Linearity was studied by preparing a standard of ibuprofen in the concentration range of 50% to 150% of the working concentration, and each concentration was taken and recorded as per the procedure in section 5.0.
Procedure:
Solution preparation 50%: Take 50.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Solution preparation 75%: Take 75.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Solution preparation 100%: Take 100.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Solution preparation 125%: Take 125.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Solution preparation 150%: Take 150.0 mg of USP Ibuprofen RS in 10 mL mobile phase.
Run the below-listed sequence:
S.No. | Name of solution | No. of injections |
1 | Blank | 1 |
2 | Solution 50% | 3 |
3 | Solution: 75% | 3 |
4 | Solution 100% | 3 |
5 | Solution: 125% | 3 |
6 | Solution 150% | 3 |
Result: The HPLC detector area was found linear over the concentration range of 5000 ppm to 15000 ppm ibuprofen with a linear regression coefficient of 1.0, which is within the limit of 0.99.
7.5 Robustness:
Definition: It is a measure of the method's ability to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled, or a precautionary statement should be included in the procedure.
Examples of typical variations are
• pH of buffer in mobile phase: (+/- 0.2 units).
• Mobile phase composition: (+/- 10% of organic phase).
• Column oven temperature: (+/- 5°C).
• Flow rate (+/- 0.2 ml/minutes).
• Wavelength: (+/- 1 nm)
The method shall show the reliability of an analysis concerning deliberate variations in method parameters. If measurements are susceptible to variations in analytical conditions, mention the same in the test method.
7.5.1 At Different Wavelengths: (254 nm)
For 253 nm:
Preparation of Standard Solution: Take 100.0 mg of USP Ibuprofen RS in 10 mL. Mobile phase. Preparation of sample solution: Nominally 10.0 mg/mL of ibuprofen prepared as follows. Transfer NLT 10 tablets to a suitable volumetric flask and add about 50% of the final volume of the mobile phase. Shake on a mechanical shaker for at least 60 min or until the tablets are disintegrated. Dilute with mobile phase to volume. Centrifuge a portion of the solution at about 3000 rpm for about 10 min or until a clear supernatant is obtained. Use the supernatant for analysis.
Run the sequence below. below.
No. |
Name of solution |
No. of injections |
|
1 |
Blank |
1 |
|
2 |
Standard solution |
6 |
|
3 |
Sample Solution |
6 |
Result:
For 255 nm:
Pa Stanae Stana standard sample solution is the same as above at 253 nm.
Run the sequence the same as the above wavelength.
Result
Result: The robustness of the analytical method was established by analyzing the sample at two different wavelengths (1). The % RSD of the assay was 1.047%.
7.6 Solution stability:
It refers to the ability of a sample or standard solution to maintain its concentration and integrity over a specific period. It's a critical factor in ensuring the accuracy and reliability of your analytical results.
Well, if your solution degrades or changes concentration over time, it can lead to significant errors in your analysis. This can impact product quality, patient safety, and regulatory compliance. By determining the stability of your solutions, you can establish appropriate storage conditions and expiration dates."
Several factors can influence solution stability, including:
Temperature: Higher temperatures can accelerate degradation.
Light: Exposure to light can cause photodegradation.
Oxygen: Oxidative reactions can affect stability.
pH: Extreme pH values can destabilize solutions.
Solvents: The type of solvent used can impact stability.
Common acceptance criteria include:
• Percentage change in concentration
• Peak purity
Procedure: Analysis was carried out as per the methodology described in section 5.0 after 24 hrs.
Run the test listed below since:
|
S.No. |
Name of solution |
No. of injections |
|
1 |
Blank |
1 |
|
2 |
Standard solution |
1 |
|
3 |
Sample Solution |
1 |
Initial Result
|
S.No. |
Name of solution |
Area |
% Result |
|
1 |
Standard solution |
3262525 |
100.80% |
|
2 |
Sample Solution |
3284152 |
24-hour result
|
S.No. |
Name of solution |
Area |
% Result |
|
1 |
Standard solution |
3261252 |
100.62% |
|
2 |
Sample Solution |
3277152 |
Result: The solution Stability of the analytical method was established by analyzing the sample over 24 hours. No degradation was observed.
8.0 Summary
9.0 Conclusion:
The assay method for the determination of ibuprofen was validated by HPLC for the analytical performance parameters, viz., interference due to system suitability and specificity and system. Precision, repeatability, intermediate precision, accuracy, linearity, robustness, and solution stability.
Thus, this analytical method is considered validated for its intended use to establish the assay of ibuprofen tablets USP 100 mg with consistent results.
