Understanding Analytical Method Validation Parameters:Ibuprofen 100 mg USP Assay AMV

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 a matter of 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 comosition

1) Dose proportional formulation composition:

In these types of formulations, 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.

2) Pseudo Dose proportional formulation composition:

In this type of formulations, composition of the active ingredient proportionally increases as the strength increases but the average weight of dosage form remains constant. The weight of constituents of the matrix is modified so as to keep constant average weight.

3) Look like formulation composition:

• In these types of formulations, composition of the active ingredient proportionally increases as the strength increases but the average weight of dosage form remains constant by a minor change in weight of one of the excipient.

• 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 Assay of Ibuprofen is suitable for its intended purpose and routine testing.

2.0 DESIGNATION & RESPONSIBILITY

QC Executive: To prepare Analytical Method Validation Report. To organize the Analytical Method Validation Study in co-ordination 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 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 Abbrevation

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% 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 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-um 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 ibuprofen (C13H1802) 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 a number of theoretical plates (efficiency of the column), internal precision (repeatability), reproducibility, tailing factor (peak asymmetry), resolution, relative retention time, and capacity factor 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 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 Mobile phase.

Run the injection listed below 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, which may be expected to be present. To prove specificity, the following experiment is carried out. The placebo and the placebo spiked with Ibuprofen are recorded as per the procedure laid down 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% 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 injection listed below 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 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:

The system precision is the closeness of agreement between the responses of detector. 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 procedure in section 5.0 and the relative standard deviation of the assay 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 Assay was 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 injection
1	Blank	1
2	Standard Solution	6
3	Sample Solution

Result:

It expresses within 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 Assay was 0.261 % which is less than the acceptance limit, of 2.0% R.SD.

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 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 determination)

Accuracy is performed in triplicate at concentrations of 50%, 100%, and 150% of 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 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 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 Placebo in 10 ml Mobile phase, Mix well, and filter.

Run listed below sequence:

Result:

Result: The accuracy of the analytical method was established by analyzing the concentration range of 50% to 150% Tofacitinib with Recovery found the limit, between 98% and 102% and RSD was %, which is in between 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 which 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 which are directly proportional to the concentration of analyte in the Standard.

Linearity was studied by preparing standard of Ibuprofen at the concentration range of 50% to 150% of 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 injection
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 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 wavelength: (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% 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 sequence listed below

S.No.	Name of solution	No. of injection
1	Blank	1
2	Standard solution	6
3	Sample Solution	6

Result:

For 255 nm:

Prepare Standard and sample solution same as above 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 between two different wavelengths (1). The % RSD of 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 methodology described in section 5.0 after 24 hrs.

Run the Test listed below sequence:

S.No.	Name of solution	No. of injection
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 up to 24 hours. There is no degradation observed.

8.0 Summary

9.0 Conclusion:

The Assay method for the determination of Ibuprofen is validated by HPLC for the analytical performance parameters viz. Interference due to System Suitability, Specificity, and System. Precision, Repeatability, Intermediate Precision, Accuracy, Linearity, Robustness, Solution Stability.

Thus, this analytical method is considered to be validated for its intended use to establish the Assay of the Ibuprofen in Ibuprofen Tablet USP 100 mg with consistent results.