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HPLC Coupled with Quadrupole Mass Spectrometry and Forensic Analysis of Cocaine Analysis of Cocaine • Important for forensics • Samples • An unknown powder • Could contain other components • A human specimen • Fluids (urine, blood saliva) • Hair C17H21NO4 Molecular weight: 303.353 g/mol HPLC: High performance liquid chromatography • Sample injected using pressurized system • Compounds travel through column at different rate – Interact with stationary phase • Detection – Traditionally by UV absorption – More recently Mass spectrometry • Better identification of unknowns Typical Silica bead with C18 chains projecting off Reverse Phase HPLC • Stationary Phase: small silica particles with long hydrocarbons chains attached (non polar) • Mobile Phase: solvent, typically a water/alcohol solution (polar) Red compound is less polar than Blue compound Red compound is present in a lower quantity Example HPLC Chromatograms Retention time differences for cocaine and some of its metabolites Mass spectrometry • Ion Source – Compound vaporized into gas phase – Energy added to ionize the compound • Excess energy may also fragment compound – Most Common Method for cocaine: electrospray • Mass Analyzer – Fragments sorted based on mass/charge ratios – Ions detected Quadrupole Mass Spectrometry • Apply voltages to 4 parallel rods • Create an electric field • Can change voltages to scan through m/z ratios Resonant ions will make it through to the detector Non-resonant ions will be neutralized by hitting rod Mass Spectrometric identification of unknowns •Quadrupole MS allows distinction of compounds with similar m/z •Compounds can also be distinguished by fragmentation patterns Parent compound Daughter ions after fragmentation Example Mass Spectra Results Cocaine and its metabolites have different mass spectra and fragmentation patterns Oral Samples (Subject’s Saliva) Applications • Blood Gas Analysis • Allows for in vivo testing of gases present in the blood • • • • Analysis of stomach contents of overdose victims Drug Profiling Hair testing Identification of unknown crime scene specimens Recent Studies • Detection in Human Specimens – Optimize separation and codetection of several different drugs in urine • Detection in Unknown Powder – Cocaine was identified in presence of metabolites and other drugs from benzoylecgonine