On-Line Immunoaffinity Extraction-Coupled Column Capillary Liquid Chromatography/Tandem Mass Spectrometry: Trace Analysis of LSD Analogs and Metabolites in Human Urine

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Abstract
An on-line immunoaffinity extraction-coupled column capillary liquid chromatography/tandem mass spectrometry (IAE/LC/LC/MS/MS) method is described. The system involves three columns, a 2.1-mm-i.d. protein G immunoaffinity column with noncovalently immobilized antibody specific to the analytes of interest, a packed capillary trapping column, and a packed capillary analytical column. With use of a short packed capillary trapping column, the protein G column could be operated at flow rates of 2.5−4 mL/min while the packed capillary analytical column was maintained at a flow rate of 3.5 μL/min. Human urine diluted 1:1 with phosphate-buffered saline was pumped directly onto the immunoaffinity column without pretreatment and was analyzed by electrospray mass spectrometry following the column switching process. Sample handling and transfer procedures were eliminated. The system was optimized and evaluated for the determination of LSD, its analogs, and metabolites in spiked human urine at low part-per-trillion (ppt) levels using mass spectrometric detection. LSD-positive human urine specimens from LSD users were also analyzed. Concentrations as low as 2.5 ppt of LSD and several of its analogs were detected in spiked human urine using IAE/LC/LC/MS/MS. This is 20-fold below our previous limit of detection using solid phase extraction and LC/MS/MS.