Quantitative imaging of cocaine and its metabolites in brain tissue by matrix-assisted laser desorption/ionization linear ion trap tandem mass spectrometry

Detection of drugs in tissue typically requires extensive sample preparation in which the tissue is first homogenized, followed by drug extraction, before the extracts are finally analyzed by liquid chromatography/mass spectrometry (LC/MS). Directly analyzing drugs in intact tissue would eliminate any complications introduced by sample preparation. A matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MSn) method has been developed for the quantification of cocaine and its metabolites present in postmortem brain tissue of a chronic human cocaine user. It is shown that tandem mass spectrometry (MSn) increases selectivity, which is critical for differentiating analyte ions from background ions such as matrix clusters and endogenous compounds found in brain tissue. It is also shown that the use of internal standards corrects for signal variability during quantitative MALDI, which can be caused by inhomogeneous crystal formation, inconsistent sample preparation, and laser shot-to-shot variability. The MALDI-MS n method developed allows for a single MS2 experiment that uses a wide isolation window to isolate both analyte and internal standard target ions. This method is shown to provide improved precision (∼10-20 times reduction in percent relative standard deviation) for quantitative analysis compared to using two alternating MS2 experiments that separately isolate the target analyte and internal standard ions.;A wide isolation window reduces signal variability when the analyte and internal standard signals are ratioed. However, the wide isolation window not only isolates the analyte and internal standard ions, but also other ions that are not of interest. These ions fill up the finite storage capacity of the ion trap and may lead to space-charge effects, which result in reduced resolution and peak shifts that interfere with detection of the target ions. Since the current instrument software only allows for one isolation window during MSn, a multi-notch isolation waveform that selectively isolates the analyte and internal standard ions was created to remove the effects of background interferences and boost the sensitivity for analyte and internal standard ions. A multi-notch stored waveform inverse Fourier transform (SWIFT) pulse was calculated with frequency notches corresponding to the secular frequencies of the [M+H]+ ions of cocaine (COC), benzoylecgonine (BE), cocaethylene (CE), and their trideuterated analogs, COC-d3, BE-d3, and CE-d3. Multi-notch SWIFT isolation was found to have lower precision than wide isolation, which may be caused by frequency shifts of the analyte and internal standard ions from space-charge effects caused by high m/z background ions from the tissue (e.g., lipids).;Finally, a two-stage SWIFT isolation method was developed that uses a high-mass filter to eject high m/z background ions before the multi-notch SWIFT isolation is applied. The twostage SWIFT isolation showed similar precision to wide isolation for the MALDI-MS2 analysis of cocaine and its metabolites in brain tissue. The two-stage SWIFT isolation and wide isolation were used to quantitatively image cocaine and its metabolites in postmortem human brain tissue, and were compared to the quantitative analysis of human brain tissue homogenate using MALDI-MS2.