High-Performance Liquid Chromatography (HPLC) in Pharmacology

Introduction & rationale

• Definition — High-performance / high-pressure liquid chromatography (HPLC) is a separation technique in which a liquid mobile phase, forced under pressure through a column packed with a solid adsorbent (stationary phase), resolves analytes by their differential interaction with the adsorbent; the column eluent passes to a detector whose output is recorded as a chromatogram of peaks.
• Dual function — it is both a qualitative tool (identification by retention time) and a quantitative tool (peak height/area) — it can separate, identify and quantitate any compound that can be dissolved in a liquid.
• Why bioanalysis matters — it is the workhorse analytical technique for small-molecule drugs and metabolites in biological matrices — the platform on which therapeutic drug monitoring (TDM), pharmacokinetic sampling, bioequivalence assay and metabolite quantitation rest. Every clinical-pharmacology decision depends on accurate, reliable measurement of drug/metabolite/biomarker concentration; without reliable assays the application of clinical pharmacology is "quite limited, if not altogether impossible."
• Two regulatory contexts — a drug assay is either developmental (pre-approval PK/metabolism data, governed by the FDA Bioanalytical Method Validation guidance) or a patient-practice assay (companion/complementary or stand-alone diagnostics governed by CDRH 510(k)/PMA and CLIA).
• Platform-by-analyte logic — small-molecule drugs → chromatographic (HPLC / LC-MS) methods; large therapeutics (proteins, mAbs) → immunoassays / ligand-binding assays; nucleic-acid (RNA/DNA) therapeutics → PCR-based assays. HPLC dominates the first group. (Liquid chromatography was first described by the Russian botanist Mikhail Tswett.)