HPLC Method Validation Overview

Overview

Chapter 9 outlines the purpose, scope, and execution of HPLC method validation for pharmaceutical analysis across development stages, detailing parameters, protocols, acceptance criteria, and supporting studies.

Validation Purpose and Scope

• Goal: Confirm a method is reliable and suitable for intended use.
• Applies to drug substance (DS) and drug product (DP) for identity, assay, impurities, CU, dissolution.
• Pharmacopeial methods: verify (not necessarily validate), as needed.

Development Stages and Strategy

• Early phase (to Phase IIa): preliminary validation; ICH Q2A/B not yet binding.
• Full development (after Phase IIa/NDA/market): extensive validation with protocol and QA approval.
• Methods evolve with project; validation extent and documentation increase over time.

Validation Protocol and Report

• Protocol includes: method, test(s), parameters, solutions/injections count, acceptance criteria, batch lists, excipient grades, reference materials, instruments, responsibilities.
• Full protocols may detail solution prep, design, calculations, and software.
• Reports: reference method IDs, DS/DP batches, standards, instruments; present results vs criteria, pass/fail, deviations, changes; QA approval required.
• Revalidation: assess impact of changes (method/process); document rationale or perform partial/full revalidation per updated protocol.

Assignment of Validation Parameters

• Required parameters vary by test type and development stage.
• All analytical procedures require validation appropriate to use (GLP tox, stability, in-process, release).

Early vs Full Development Requirements

Early Development (Table)

• Notes: b four points adequate; c DP only; d spike if impurities available; e at least triplicate; f LOD not required but recommended; g LOQ needed for 0-mg placebo identity.

Full Development (Table)

• Notes: a lack of specificity may be compensated by support methods; c exceptional cases; d LOQ for placebo identity if needed; e LOD not ICH-required but recommended; f depends on test.

Acceptance Criteria Highlights (Selected, DP/DS)

• Identity (selectivity): all known peaks separated; API peak pure (peak purity angle < threshold).
• Dissolution (DP): recovery 95–105%; Srel recovery ≤2.5%; repeatability Srel ≤2.0% (n=6 at Q time).
• Linearity (DP): n≥6; r ≥0.990; |y-intercept| ≤5%; residual SD ≤2.5%.
• Assay (DP) accuracy: 98.0–102.0%; Srel ≤2.0% (n=9 over ≥3 levels).
• Weight percent (DS) accuracy: % difference vs second method ≤2.0%.
• Precision: DP Srel ≤2.0% (n=6); DS Srel ≤1.0% (n=6); intermediate Srel ≤2.0% (n=4, two analysts).
• Range: Assay DP/DS at least 80–120% (DP CU 70–130%).
• Solution stability: DS samples/standards change ≤1.0%; DP ≤2.0%; impurity-level dependent thresholds.
• Impurity precision and accuracy: tiered by level; LOD S/N≈3:1; LOQ S/N≈10:1 and Srel ≤10% (n=5).

Accuracy

• Definition: closeness to true/accepted value; inferred from precision, linearity, specificity.
• Demonstrate via:
  • Comparison to independent/orthogonal methods (e.g., RP vs SCX; HPLC vs CEC/CE; HPLC vs NMR/DSC/PSA/titration with caveats).
  • Recovery studies: spike API into placebo (DP) or spike impurities into DS/DP matrix.
• Design:
  • Use ≥3 levels (e.g., 70%, 100%, 130%), 3 reps each (n=9).
  • For impurities: from reporting level to ≥120% of largest impurity spec; account for impurity purity and existing levels in DS.
• Filter check:
  • Compare filtered (after defined discard volumes) vs centrifuged aliquots; establish minimum preflush volume; repeat for formulation/vendor changes.
• Completeness of extraction:
  • Kinetic (time t1–t3) and thermodynamic (volume V1–V3) studies vs method t0/V0; adjust method if recovery increases with time/volume.
  • Homogenizer can drastically reduce extraction time vs sonication/shaking; example pulse program provided.

Precision

• Repeatability: same analyst, same day; includes injection precision and analysis precision (multiple preps).
• Intermediate precision: different analysts/systems/days; indicates method transfer readiness.
• Assess via %RSD (Srel); compare manual vs automated if automation used.

Linearity

• Purpose: proportional response across relevant range; perform separately for API and each related substance.
• Ranges:
  • Assay DS/DP: 80–120% (CU 70–130%; dissolution 30% of specified range).
  • Impurities: LOQ/reporting level to ≥120% of spec.
• Practice:
  • ≥5 concentrations; include low-level series for impurities.
  • If using 100% standard to quantify impurities, compare slopes of low-level vs assay-level curves; consider dilute standard if slopes differ.
• Evaluation:
  • r and y-intercept limits; residual SD; inspect residuals and response factor vs concentration.
  • Response factor %RSD targets: ≤2.0% (assay 80–120%); ≤10% (low-level impurities).
  • Additional checks: response factor differences between LOQ and 5×LOQ, and LOQ vs max low-level ≤10%.

Low-Level vs High-Level Quantitation Strategy

• If deviation from linearity at low levels, quantify impurities using a dilute standard (e.g., ~1%) rather than 100% standard or area normalization.
• Compare slopes and response factors across low/high ranges; if criteria unmet, adopt dilute standard to avoid under/overestimation and aid mass balance.

LOD/LOQ

• LOD: lowest detectable; S/N ≈ 3:1.
• LOQ: lowest quantifiable with acceptable accuracy/precision; S/N ≈ 10:1; %RSD ≤10% (n≈5).
• LOQ must be ≤ reporting threshold (depends on MDD per ICH Q3B).
• Determine via serial dilutions; typical HPLC LOD/LOQ ranges: non-peptides 0.01–0.2%; peptides/proteins 0.1–0.5%.
• Include LOQ point in low-level linearity and meet linearity criteria.

Relative Response Factors (RRF) and Variant Ratios

• RRF ~1: similar responses; outside 0.8–1.2 requires correction to avoid bias.
• Mass% impurity calculation includes RRF; for two-actives products, include variant ratio (VR) of actives in formula.

Stability of Solutions

• Demonstrate sample/standard stability over analysis time (e.g., 24–72 h) at room temperature and 5°C.
• Analyze initially and at time t against fresh standards; apply acceptance criteria (assay/CU ±2.0% DP; DS ±1.0%; impurities level-based).
• Consider light sensitivity; define storage conditions (e.g., protect from light).

Ruggedness and Robustness

• Ruggedness: reproducibility across labs/analysts/instruments; typically assessed via intermediate precision and pooled %RSD; may include mean differences limits.
• Robustness: deliberate single-parameter variations; ensure system suitability maintained.
• Typical HPLC parameters to vary: mobile phase composition/pH/ionic strength/additives, gradient slope/hold, flow, column (lot/supplier), temperature, injection volume, autosampler temperature, wavelength.
• Examples:
  • Modifier concentration (phosphoric acid 0.4–0.8% v/v): changes >±0.1% from 0.6% affected selectivity/system suitability for a basic impurity.
  • Temperature (17–25°C): acceptable system suitability only within 19–23°C; co-elution observed at 25°C; define system suitability for critical pairs.

Specificity

• Demonstrate for identity, impurities, assay; confirm no placebo interference and API peak purity (PDA, MS).
• Forced degradation supports specificity by generating potential degradants and confirming resolution and peak purity.

Forced Degradation Studies (Stress Testing)

• Purpose: develop stability-indicating methods; understand pathways/products; target ~5–10% degradation without over/under-stressing.
• Conduct on DS, DP blends, and placebo in parallel; not part of formal stability program.
• Solid-state conditions:
  • Thermal (50–80°C, closed); thermal/oxidative (open); thermal/humidity (40°C/75% RH); light (ambient up to 1.2 M lux·h and 200 Wh/m²), with/without oxygen exposure.
• Solution conditions:
  • pH (water, 0.1 N HCl/NaOH), oxidation (H2O2; metal-catalyzed with Fe3+/Ni2+/Cu2+; bubbled O2; pressurized O2; free-radical initiators like AIBN/AMVN); heat; light.
• Oxidation considerations:
  • Select systems per structure (amines, sulfides, alkenes); peroxide vs peroxy-radical; singlet oxygen for susceptible motifs; assess excipients for singlet oxygen generation.
• Hydrolysis: assess neutral/acid/base; escalate temperature if needed; consider functional group and substituent effects.
• Photolysis: assess multiple reaction types; include controls protected from light; excipients may mediate reactions.
• Reporting (late phase/NDA): isolate/characterize significant degradants (LC-MS, NMR, UV); provide mechanisms/kinetics; distinguish DS-related vs excipient-related products.

Distinguishing Drug-Related vs Non-Drug-Related Degradants

• Stress placebo, DS, and DP under similar conditions.
• Key questions:
  • Origin of new peaks (DS impurity, degradant, or placebo).
  • Levels present; process step origin; stress condition origin.
• For combination products: assess inter-active reactions and physical impacts; stress combined actives in solid/solution.

Structured Ranges and Criteria (Selected)

Minimal Accuracy Ranges (Table)

Recommended Linearity Ranges (Table)

Key Terms & Definitions

• Accuracy: Agreement with true/accepted value (often via recovery/orthogonal methods).
• Precision: Random error indicator; includes repeatability and intermediate precision; reported as %RSD.
• Linearity: Proportional response over concentration range; assessed via regression, residuals, response factors.
• LOD: Lowest detectable analyte (S/N ≈ 3:1).
• LOQ: Lowest quantifiable analyte with acceptable accuracy/precision (S/N ≈ 10:1).
• Specificity: Ability to assess unequivocally the analyte in presence of components.
• Robustness: Reliability under small deliberate variations in parameters.
• Ruggedness: Reproducibility under varied normal conditions (labs, analysts, instruments).
• RRF: Relative response factor of impurity vs API; used to correct quantitation.
• Variant ratio (VR): Ratio adjustment for multi-active products in impurity calculations.
• Reporting threshold (RTH): Minimum reportable impurity level, based on MDD (ICH Q3B).

Action Items / Next Steps

• Draft validation protocol with test-specific parameters and acceptance criteria; obtain QA approval.
• Generate preliminary validation data in early development; expand to full validation post-Phase IIa.
• Establish system suitability criteria during development; use in robustness testing.
• Design and execute accuracy, precision, linearity, LOD/LOQ, specificity, solution stability, and robustness studies per test.
• Implement filter checks and extraction completeness assessments for DP sample prep.
• Perform forced degradation on DS, DP blends, and placebo; ensure stability-indicating capability.
• Decide impurity quantitation approach based on low-level linearity; adopt dilute standards when needed.
• Define revalidation triggers; document rationale and changes; update reports and methods with QA approval.