Introduction
The monitoring of drugs of abuse in biological matrices is a large and growing business for the laboratory testing industry. Testing urine for the presence of drugs of abuse requires methodology sensitive enough to monitor well below therapeutic levels and selective enough for clear identification of specific drugs without false positives. Current methodologies for urine analysis typically involve immunoassay, which is often not specific to an individual drug. This means samples must be subject to confirmation using LC/MS/MS or GC/MS. The importance of sample prep for improving sensitivity and robustness of MS methods is well-known. In this brief report, we will discuss the application of SPME for the extraction of metabolites of illicit drugs from urine samples with subsequent analysis by LC/MS/MS. Applying SPME to this type of analysis has been made possible with the introduction of new fibers that are compatible with HPLC solvents and biological samples.
SPME LC Tips
SPME LC Tips pictured in Figure 1 consist of fiber cores coated with HPLC-type silica particles held in place on the fiber with a special polymeric binder. The coatings used in the SPME LC Tips were designed to overcome the limitations of traditional SPME fiber coatings, making them suitable for extraction in biological matrix and for direct solvent desorption. In the fibers described here, the silica particles are bonded with C18 molecules, allowing for extraction of analytes from highly aqueous samples. The particle binder prevents sample matrix components from co-extracting onto the fiber. In this study, the SPME LC Tips were utilized as a direct sampling method for analysis of illicit drugs in human urine. Cocaine and its metabolites benzoylecgonine and cocaethylene, norfentanyl, and methadone and its metabolite EDDP, were selected as representative drugs and metabolites. Structures appear in Figure 2. Matrix-matched standards with isotopically labeled internal standards were prepared for development of the calibration curve. Patient urine samples were collected at a collaborative testing facility and submitted for SPME extraction. The urine samples had previously tested positive by ELISA techniques for the presence of either cocaine metabolites or methadone metabolites, but actual concentration levels were not determined. These samples were then analyzed using the SPME LC Tips extraction technique for comparison. Sample preparation for the urine samples was minimal, with only the addition of buffer. The SPME LC Tips were placed into the urine samples, then directly desorbed and analyzed by LC/MS/MS. Extraction technique and chromatographic conditions are described.
Figure 2. Structures of Analytes used in the Study>> Download the full Application Note as PDF
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