Pharmaceutical Analytical Method Validation

(Step-by-Step Guideline- Analytical Method Validation)

A. Accuracy

Definition:

Accuracy expresses the closeness of agreement between the test result
and the true (accepted) value.

[Short Note: Accurate result for different concentration.]

Procedure:

Accuracy is evaluated by recovery studies at different concentration levels:

80%, 100%, 120%

Known quantities of standard drug substance are spiked into the placebo
or sample matrix and analyzed.

Calculation:

% Recovery = (Amount found / Amount added) × 100

Acceptance Criteria:

Recovery: 98.5% – 101.5%

Limit: NMT ± 1.5%

B. Precision

Definition:

Precision indicates the degree of agreement among individual test results
when the method is applied repeatedly to multiple samplings of a
homogeneous sample.

[Short Note: Same result for same concentration]

Acceptance Criteria:

%RSD: NMT 2.0%

i) Repeatability

Prepare 100% working standard and Inject/analyze six (6) times

Calculate: Mean, Standard Deviation (SD), Relative Standard
Deviation (RSD)

[Short Note: Take 100% working standard 6 times. And calculate SD and
RSD]

C. Specificity

Definition:

Specificity is the ability of the analytical method to measure the
analyte response in the presence of components such as impurities,
degradants, and placebo.

Procedure:

Analyze: Blank, Placebo, Standard, Sample

Confirm:

No interference at the analyte wavelength (UV)

Same retention time (HPLC) or same wavelength (UV)

Acceptance Criteria:

Analyte peak should be well resolved and free from interference.

[Short Note: Same Concentration taken in same solvent. Give the same
result in same weave length or same retention time.]

D. Linearity

Definition:

Linearity is the ability of the method to elicit test results that are
directly proportional to analyte concentration within a given range.

i) Standard Linearity

Prepare working standards at: 80%, 90%, 100%, 110%, 120%

Plot Concentration vs Response

ii) Sample + Fixed Placebo

Different concentrations of sample with a fixed amount of placebo.

Plot linearity graph

iii) Fixed Sample + Variable Placebo

Fixed sample concentration, Vary placebo levels (80–120%)

Confirm linear response

Acceptance Criteria:

Correlation coefficient (r² ≥ 0.999)

Linear visual distribution

[Short Note: i) Different concentration of working standard given
linear graph.

ii) different concentration of sample with fixed amount of placebo
given linear graph.

iii) Fixed amount of sample with different amount of placebo. The
result given linier graph.]

E. Sensitivity

Definition:

Sensitivity is the ability of the method to detect and quantify small
amounts of analyte.

Sample Concentrations Used: 1, 2, 4, 8, 16 µg/mL

i) Limit of Detection (LOD):

LOD = 3.3 * (SD/Slop)

ii) Limit of Quantification (LOQ)

LOQ = 10 * (SD/Slop)

Where:

SD = Standard deviation of the response

Slope = Slope of calibration curve

Noise Level in Sensitivity Testing

Signal-to-Noise Ratio (S/N):

LOD: ≈ 3:1

LOQ: ≈ 10:1

Noise level determination is based on: ➡ ICH Q2 guideline

Noise is measured from the baseline fluctuation near the analyte peak
region in chromatographic methods.

F. Robustness

Definition:

Robustness measures the capacity of a method to remain unaffected by
small, deliberate variations in method parameters.

Solvent Composition Variation:

Methanol : Water = 50:50

Methanol : Water = 60:40

Methanol : Water = 40:60

Methanol : Water = 30:70

Methanol : Water = 70:30

Each condition is analyzed and %RSD is calculated.

Acceptance Criteria: %RSD: NMT 2.0%

[Short Note: Robustness is bone by changing the ratio of the diluting
solvent]

Conclusion

If all validation parameters meet acceptance criteria, the analytical
method is considered:

- Accurate

- Precise

- Specific

- Linear

- Sensitive

- Robust

and suitable for routine pharmaceutical analysis.

References (Guidelines)

1. ICH Q2(R1) – Validation of Analytical Procedures: Text and
Methodology

2. ICH Q2(R2) – Updated Analytical Validation Guideline

3. USP – Validation of Compendial Procedures

4. *FDA Guidance for Industry – Analytical Method Validation