A QUEST FOR OLEANDRIN IN DECAYED HUMAN TISSUE

This forensic case taught us several lessons. First, there is a need for improved sample cleanup and treatment of severely decayed tissue samples when trace determinations of target analytes are needed. With the exception of a few reports {15, 16), the literature is lacking in information with regard to the most modern sample preparation techniques. Second, the coupling of LC/LC with tandem MS provides an effective means of “on-line” sample cleanup for complex sample matrices. The improvements in selectivity shown in Figure 3 reveal the analytical power available when these techniques are combined. Third, once we decided to use LC/LC/MS/MS, we were able to analyze more than 50 samples in a semi-automated fashion over approximately three days. The reliability and ruggedness of the combined techniques and equipment suggest this approach may have merit for common applications in which large numbers of biological samples (e.g., plasma and urine) must be analyzed. As a postscript, when this project was completed we proposed that the use of antibodies for isolating oleandrin and its relatives might be a more selective means for trace enrichment of the target analytes (17). For example, a high-pressure immunoaffinity column could have been coupled on line as column 1 in Figure 4. After pumping a relatively high volume of aqueous tissue extract through an immunoaffinity column during trapping and trace enrichment conditions, the column could be rinsed with phosphate-buffered saline. Then the pH could be lowered to unfold the antibody protein and allow release of the trapped analyte from this column with subsequent trapping on column 2 in Figure 4. Back-flushing column 2 with an appropriate eluent could then elnte the trapped analytes and any additional matrix components resulting from nonspecific binding onto column 3, which would provide an analytical separation followed by MS/MS detection. We have demonstrated the feasibility of an immunoaffinity chromatography (IAC) and LC/MS approach with several simple drug and environmental applications. For example, a polyclonal antibody specific for propranolol was used to trace-enrich this drug from human urine. When we use IAC/LC/MS on a modified single quadrupole mass spectrometer operated in the SIM mode, we determined propranolol at a concentration of 2.5 ng/mL directly from urine (18). Other applications in our laboratory include the IAC/LC/MS determination of lysergic acid diethylamide (LSD) in urine as well as carbofuran in environmental water and food matrix samples (19). This approach appears to have significant advantages for particularly complex matrices and may be a modern alternative to current liquid-liquid or SPE sample preparation techniques. It also could be an alternative to the analysis of decayed tissue samples in future legal cases such as the one described here. © 1993, American Chemical Society. All rights reserved.