SOP for Procedure for Analytical Method Validation and Verification

SOP for Procedure for Analytical Method Validation and Verification

1.0 OBJECTIVE

To establish a documented procedure for Analytical Method Validation and Verification in the Quality Control (QC) Department to ensure analytical methods are suitable for their intended purpose and comply with regulatory requirements.

2.0 SCOPE

This SOP applies to all analytical method validation and verification activities performed in the Quality Control Department for:

• Drug Products (Assay, Related Substances, Content Uniformity, Preservative Content)

• Drug Substances (Assay)

• Compendial and In-house developed analytical methods

3.0 RESPONSIBILITY

3.1 Officer and above

• Preparation of SOP

• Allocation of Analytical Report (AR) number

• Execution of validation/verification studies

3.2 Executive and above

• Review and checking of SOP and validation documents

3.3 Head – QA & QC

• Approval of SOP

4.0 ACCOUNTABILITY

QC Head

• Implementation and compliance of this SOP

5.0 DEFINITIONS

5.1 Validation
Demonstration that an analytical method is suitable for its intended purpose.

5.2 Verification
Confirmation that a validated compendial method performs adequately under actual laboratory conditions.

5.3 Specificity/Selectivity
Ability to measure the analyte unequivocally in the presence of components such as impurities, degradants, and excipients.

5.4 Limit of Detection (LOD)
Lowest amount of analyte that can be detected but not necessarily quantified.

5.5 Limit of Quantitation (LOQ)
Lowest amount of analyte that can be quantified with acceptable accuracy and precision.

5.6 Linearity
Ability of the method to produce results directly proportional to analyte concentration within a given range.

5.7 Range
Interval between upper and lower concentrations where accuracy, precision, and linearity are demonstrated.

5.8 Precision
Closeness of agreement between multiple measurements of the same homogeneous sample.

• System Precision

• Method Precision (Repeatability)

• Intermediate Precision

5.9 Accuracy
Closeness of agreement between the accepted true value and the value found.

6.0 PROCEDURE

6.1 General Requirements

6.1.1 Validation/verification shall be conducted after method development and before routine use.

6.1.2 Validation shall comply with ICH guidelines. Any deviation must be scientifically justified.

6.1.3 Prior to initiation:

• Verify instrument qualification and calibration status.

• Use AR/HPLC/GR grade reagents.

• Use valid reference/working standards.

• Use Class A glassware.

• Identify unstable or hazardous materials and define storage conditions.

6.1.4 If impurity standards are unavailable, use RRT/RRF from pharmacopoeia, DMF, or API technical package.

6.1.5 For compendial methods, verification typically includes:

• Specificity

• Precision

• Accuracy

• Solution stability

• Filter study (if applicable)

6.2 Sample and Standard Selection

6.2.1 Sample Selection

• For scaled strengths → Validate highest strength.

• For look-alike formulations → Validate lowest strength.

• Preferably use a single batch.

• If two batches are used → establish intermediate precision.

6.2.2 Standards and Impurities

• Use characterized reference/working standards.

• Include COA in documentation.

• Spike impurities at specification limits where applicable.

6.3 VALIDATION/VERIFICATION – ASSAY & PRESERVATIVE CONTENT (DRUG PRODUCT)

6.3.1 Parameters for In-House Method Validation

• System Suitability

• Specificity (including Forced Degradation)

• Linearity and Range

• Precision (System, Method, Intermediate)

• Accuracy

• Robustness

• Stability in Analytical Solutions

• Mobile Phase Stability

• Filter Study (if applicable)

6.3.2 Parameters for Compendial Method Verification

• System Suitability

• Specificity

• Accuracy

• Method Precision

• Stability in Analytical Solutions

• Filter Study (if applicable)

6.3.3 System Suitability

Acceptance Criteria:

• As per method.

• If unspecified → 5 replicate injections.

• %RSD ≤ 2% for assay/preservative.

6.3.4 Specificity

Interference Study

• Analyze blank, placebo, standard, sample.

Acceptance Criteria (HPLC):

• No interference at analyte RT.

• Peak purity ≥ 0.995 (Lab solution).

• Empower: purity angle < threshold.

Acceptance Criteria (UV):

• Blank interference ≤ 1%.

• Placebo interference ≤ 2%.

Acceptance Criteria (Titration):

• Blank/placebo volume ≤ 1%.

6.3.5 Forced Degradation (Stability Indicating)

Stress Conditions:

• Acidic

• Basic

• Oxidative

• Thermal (≥80°C, 24 hr)

• Hydrolytic

• Photolytic

• Humidity (40°C/75% RH)

Acceptance Criteria:

• 5–30% degradation.

• Peak purity compliant.

• Mass balance: 95–105%.

6.3.6 Linearity and Range

• 50–150% concentration.

• Minimum 5 levels, triplicate injections.

Acceptance Criteria:

• r ≥ 0.995

• %RSD ≤ 2%

• Report slope, intercept, %Y-intercept.

6.3.7 Precision

Method Precision

• 6 preparations.

• %RSD ≤ 2% (Assay)

• %RSD ≤ 10% (Preservative)

Intermediate Precision

• Different analyst, instrument, day.

• Overall 12 results within criteria.

6.3.8 Accuracy

• 50%, 100%, 150% levels.

• Triplicate at each level.

Acceptance Criteria:

• Assay: 97–103%

• Preservative: 90–110%

• %RSD ≤ 2% (Assay), ≤10% (Preservative)

6.3.9 Filter Study

• Compare filtered vs unfiltered.

• Absolute difference ≤ 2% (Assay) or 5% (Preservative).

6.3.10 Solution Stability

• Evaluate up to 24 hours.

Acceptance Criteria:

• %RSD ≤ 2%

• Assay difference ≤ 2%

6.3.11 Robustness

Evaluate deliberate changes:

• pH ±0.2

• Flow ±0.2 mL/min

• Temperature ±5°C

• Wavelength ±2 nm

• Mobile phase ±2%

Acceptance Criteria:

• System suitability compliant

• Difference ≤ 2%

6.4 CONTENT UNIFORMITY (CU)

Parameters similar to Assay with following key criteria:

• 10 units tested

• AV ≤ 15

• %RSD ≤ 5%

• Individual results: 85–115%

• Accuracy: 95–105%

6.5 RELATED SUBSTANCES (DRUG PRODUCT)

Validation Parameters (In-House)

• System Suitability

• Specificity (including Forced Degradation)

• LOD & LOQ

• Linearity & Range

• Precision

• Accuracy

• RRF (if applicable)

• Filter Study

• Robustness

• Stability

LOD & LOQ

Signal-to-Noise:

• LOD ≥ 3:1

• LOQ ≥ 10:1

Statistical Method:

LOD = 3.3 σ / S
LOQ = 10 σ / S

Acceptance:

• LOQ %RSD ≤ 15%

Precision (RS)

• %RSD ≤ 15% for impurities ≥ LOQ

• Recovery: 80–120%

Accuracy (RS)

• LOQ level: 70–130%

• Other levels: 80–120%

• %RSD ≤ 15%

Filter Study (RS)

• Difference ≤ 10% of specification limit.

Robustness (RS)

• % Difference ≤ 15%

• Proper resolution maintained

6.6 ASSAY – DRUG SUBSTANCE

In-House Validation

• System Suitability

• Specificity

• Linearity & Range

• Precision

• Accuracy

• Robustness

• Solution Stability

• Mobile Phase Stability

• Filter Study (if required)

Verification (Compendial)

• System Suitability

• Specificity

• Accuracy

• Method Precision

• Solution Stability

Acceptance Criteria:

• %RSD ≤ 2% (if unspecified)

6.7.2 Specificity / Selectivity

6.7.2.1 Interference Study

Determination:

• Prepare and analyze the blank, system suitability solution, standard solution, and sample solution as per the validated methodology.

• For isocratic HPLC methods, specificity may be confirmed by extending the chromatographic run time.

• For UV spectrophotometric methods:

  • Scan blank, standard, and sample from 200–400 nm (UV range).

  • For UV-Visible methods, scan from 200–700 nm.

  • Record absorbance at the specified working wavelength.

Acceptance Criteria:

For HPLC:

• System suitability criteria shall comply with the method requirements.

• No peak from blank or known impurity shall co-elute at the retention time of the analyte.

• All peaks of interest shall be well resolved from interfering peaks (impurities or internal standards).

• Peak purity index shall be positive.

• Peak purity index for analyte ≥ 0.995 (Lab Solutions) or purity angle < purity threshold (Empower).

For UV Spectrophotometry:

• No interference from blank.

• If present, interference shall not exceed 1.0% at the specified wavelength.

For Titration:

• Blank consumption shall not exceed 1.0% of the sample titre value.

6.7.3 Linearity and Range

Determination:

• Prepare at least five concentration levels ranging from 50% to 150% of target concentration (or as per ICH guidelines).

• Inject each level in triplicate.

• Plot concentration (ppm) vs. peak area/absorbance.

• Report slope, intercept, %Y-intercept, and correlation coefficient.

• For range, inject six replicates at lowest and highest levels and calculate %RSD.

Acceptance Criteria:

• System suitability shall comply.

• Correlation coefficient ≥ 0.995.

• %RSD for range ≤ 2.0%.

• Slope, intercept, and %Y-intercept at 100% response shall be reported.

6.7.4 Precision

Precision expresses agreement between repeated measurements and is reported as %RSD.

6.6.4.1 System Precision

Determination:

• HPLC: Six replicate injections of standard.

• UV: Three replicate absorbance measurements.

Acceptance Criteria:

• System suitability criteria shall comply.

6.7.4.2 Method Precision (Repeatability)

Determination:

• Prepare six independent sample preparations from the same homogeneous sample.

• Analyze on the same day, same instrument, same analyst, same column (if applicable).

• Calculate assay results, mean, and %RSD.

Acceptance Criteria:

• System suitability shall comply.

• Results within specification.

• %RSD ≤ 2.0%.

6.7.4.3 Intermediate Precision

Determination:

• Six sample preparations analyzed by different analyst, different instrument, different day, and different column (if available).

• Calculate mean and %RSD.

• Compare overall 12 results (6 repeatability + 6 intermediate precision).

Acceptance Criteria:

• System suitability shall comply.

• Results within specification.

• %RSD ≤ 2.0%.

• Overall %RSD (12 samples) ≤ 2.0%.

• Absolute difference between average results ≤ 2.0%.

6.7.5 Accuracy (% Recovery)

Determination:

• Prepare recovery samples at 50%, 100%, and 150% levels.

• If spike amount <10 mg, prepare stock solution.

• Analyze triplicate samples at each level.

• Calculate % recovery and mean recovery.

Acceptance Criteria:

• System suitability shall comply.

• Individual and mean recoveries: 98.0%–102.0%.

• %RSD ≤ 2.0%.

6.7.6 Filter Study

Determination:

• Prepare sample solution.

• Filter through recommended filters (e.g., GF/C, 0.45 µm nylon, 0.45 µm PVDF).

• Discard first 1 mL; collect filtrate after 1 mL, 3 mL, and 5 mL.

• Compare filtered vs. unfiltered (or centrifuged) sample.

Acceptance Criteria:

• System suitability shall comply.

• Absolute difference ≤ 2.0%.

6.7.7 Stability in Analytical Solutions

Determination:

• Store blank, standard, and sample solutions at controlled room temperature or refrigerated conditions (2–8°C or as specified).

• Analyze up to 48 hours (minimum).

• Evaluate %RSD and assay differences vs. initial.

Acceptance Criteria:

• System suitability shall comply.

• Cumulative %RSD ≤ 2.0%.

• Assay difference ≤ 2.0%.

6.7.8 Mobile Phase Stability

Determination:

• Evaluate mobile phase stability up to 24 hours.

• Inject blank, system suitability, and standard solutions.

Acceptance Criteria:

• System suitability shall comply.

• No haziness observed.

6.7.9 Robustness

Determination:

Deliberately vary:

• pH (±0.2)

• Mobile phase composition (±2% absolute or 30% relative)

• Column temperature (±5°C)

• Flow rate (±0.2 mL/min; ±0.1 if <1.0 mL/min)

• Wavelength (±2 nm)

Acceptance Criteria:

• System suitability shall comply.

• Assay difference ≤ 2.0%.

• If out-of-specification, adjust to determine critical parameters.

6.8 ANALYTICAL METHOD VALIDATION FOR RELATED SUBSTANCES – DRUG SUBSTANCE

Parameters for In-House Validation:

• System Suitability

• Specificity

• Forced Degradation

• LOD & LOQ

• Linearity & Range

• Precision

• Accuracy

• Filter Study

• Stability in Analytical Solutions

• Mobile Phase Stability

• Robustness

6.8.1 System Suitability

• Perform prior to validation.

• If %RSD not specified, inject five replicates.

• %RSD ≤ 5.0%.

6.8.2 Specificity

Interference Study

• Prepare blank, sensitivity, resolution, impurity standards, spiked samples.

• No co-elution.

• Peak purity ≥ 0.990.

• Dilute if peak saturation occurs.

Forced Degradation Study

Perform stress under:

• Acid

• Base

• Oxidation

• Thermal (≥80°C)

• Hydrolysis

• Photolysis

• Humidity (40°C / 75% RH)

Acceptance Criteria:

• 5–30% degradation achieved.

• Peak purity ≥ 0.990.

• Mass balance ≥ 95%.

• Degradants well resolved.

6.8.3 LOD and LOQ

Methods:

1. Signal-to-noise (LOD ≥ 3:1; LOQ ≥ 10:1)

2. Slope method:

   • LOD = 3.3σ/S

   • LOQ = 10σ/S

Acceptance Criteria:

• %RSD at LOQ ≤ 15%.

6.8.4 Linearity and Range

• LOQ to 150% of specification.

• Minimum 5 levels in triplicate.

• Correlation coefficient ≥ 0.95.

• %RSD ≤ 10% (≤15% at LOQ).

6.8.5 Precision

Repeatability & Intermediate Precision:

• Six preparations.

• %RSD ≤ 10%.

• Recovery 80–120%.

• Cumulative %RSD ≤ 10%.

6.8.6 Accuracy

• LOQ to 150% levels.

• LOQ recovery: 70–130%.

• Other levels: 80–120%.

• %RSD ≤ 15%.

6.8.7 Filter Study

• Compare filtered vs. unfiltered sample.

• Absolute difference ≤ 10% of specification limit.

6.8.8 Solution Stability

• Evaluate up to 24 hours.

• %RSD ≤ 5%.

• Difference ≤ 10% of specification limit.

6.8.9 Mobile Phase Stability

• Stable for 24 hours.

• No haziness.

• System suitability shall comply.

6.8.10 Robustness

Evaluate deliberate variations:

• pH

• Composition

• Flow

• Temperature

• Wavelength

Acceptance Criteria:

• System suitability meets criteria.

• Peaks well resolved.

• Recovery 80–120%.

• %RSD ≤ 10%.

6.9 Numbering of Validation / Verification Protocol & Report

Each validation protocol/report shall have a unique identification number:

Format:
QCD / AMV / AA / BBB – C

Where:

• QCD = Quality Control Department

• AMV = Analytical Method Validation/Verification

• AA = Product Category

  • FP = Finished Product

  • RM = Raw Material

• BBB = Sequential number (001 onwards)

• C = P (Protocol) or R (Report)

Addendum documents shall retain the same number with addendum reference.

6.10 Revalidation

Revalidation is required for:

• Method changes

• Addition of impurities

• Manufacturing formula changes

Types:

• Minor changes → Partial validation

• Major changes → Full validation

Revision number updated (e.g., 00 → 01).

6.11 Validation Documentation

• Prepare protocol prior to execution.

• Document all weights, reagents, standards.

• Record chromatograms and raw data.

• Method feasibility study required for critical analysis.

• Validation performed by Officer/Executive or above.

• Soft copy retained for 5 years.

• Validation plan to be discussed with Department Head prior to execution.