High-Performance Liquid Chromatography (HPLC) in Pharmacology

High-Performance Liquid Chromatography (HPLC) in Pharmacology

1. Definition, scope & why bioanalysis matters

• High-performance / high-pressure liquid chromatography (HPLC) is a separation technique in which a liquid stream (the mobile phase) is forced under pressure through or over a solid adsorbent (the stationary phase) packed in a column; differential interaction of analytes with the adsorbent resolves them, and the column eluent is passed to a detector whose output is recorded as a chromatogram of peaks (Atkinson 4e Ch.11, p.170).
  • It is the workhorse analytical technique for small-molecule drugs and metabolites in biological matrices — the platform on which therapeutic drug monitoring (TDM), pharmacokinetic (PK) sampling, bioequivalence assay, and metabolite quantitation rest (Atkinson 4e Ch.11, pp.169–70).
• HPLC is both a qualitative (identification by retention time) and quantitative (peak height/area) process — it can separate, identify and quantitate any compound that can be dissolved in a liquid (Medhi Ch.4, pp.84–5).
• Clinical-pharmacology rationale (why the assay underpins everything): every clinical-pharmacology decision — from adjusting a patient's dose by TDM to selecting a safe/effective dose in drug development — depends on accurate, reliable measurement of drug, metabolite or biomarker concentration in the relevant biological matrix; without reliable assays, the application of clinical pharmacology to patient treatment or drug development is "quite limited, if not altogether impossible" (Atkinson 4e Ch.11, p.169).
• Two regulatory contexts for a drug assay (Atkinson 4e Ch.11, p.169):
  • Developmental (pre-FDA approval) assays — generate PK/metabolism/biomarker data to support marketing approval; governed by the FDA Guidance for Industry: Bioanalytical Method Validation (BMV, 2018); these assays are essentially "retired" once the drug is approved.
  • Patient-practice assays — companion / complementary diagnostics and stand-alone diagnostics used to guide treatment; governed by the CDRH (510(k) and/or PMA) and by CLIA (administered by CMS).
• Etymology — chromatography derives from Greek chroma ("colour") + graphein ("to write"); liquid chromatography was first described by the Russian botanist Mikhail S. Tswett (1872–1920) (Medhi Ch.4, p.84).
• Platform-by-analyte logic — small-molecule drugs → chromatographic (HPLC/LC-MS) methods; large therapeutics (e.g. proteins, monoclonal antibodies) → immunoassays / ligand-binding assays; genetic therapeutics (RNA/DNA) → PCR-based assays (Atkinson 4e Ch.11, pp.169–70). HPLC is the dominant platform for the first group.