The diagnostic fragmentation of N-oxides resulting from loss of the oxygen atom (MH+ --> MH+-O) in electrospray and atmospheric pressure chemical ionization (APCI) mass spectra was investigated. When the temperature of the heated capillary tube was elevated, the ratio of the intensity of the [MH+ -16] fragment to the precursor ion (MH+) increased. This 'deoxygenation' process was associated with thermal activation and did not result from collisional activation in the desolvation region of the API source. Although the extent of 'deoxygenation' is compound-dependent, it can provide evidence for the presence of an N-oxide in a sample and can be used to distinguish N-oxides from hydroxylated metabolites (Ramanathan et al. Anal. Chem. 2000; 72: 1352). To demonstrate the practical application of thermal fragmentation of N-oxides, liquid chromatography (LC)/APCI-MS was used to distinguish an N-oxide drug from its hydroxylated metabolite in an unprocessed rat urine sample, despite the fact that the drug and its metabolite were not fully resolved by HPLC.

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run . LC-MS/MS techniques for quantitation require reference and internal standard(s), so that LC-MS/MS responses from unknown quantities of relevant drug-related peaks can be related back to responses obtained using known quantities of each analyte. Usually a standard curve is generated for each and every analyte to be quantified [6]. Although reference standards may be available for the parent drug, it is very unlikely to have standards for metabolites available in early stage of drug discovery and development thereby making conventional LC-MS/MS based quantitation of metabolites impractical.Nanospray ionization (NSI), introduced by Wilm and Mann in the 1990s [8], has been used to identify proteins [9 -13] and drug metabolites [14], quantify pharmaceutical drugs [15,16], study absorption, distribution, metabolism and excretion (ADME) properties of drugs [17], and employed in various other applications [12, 13, 15, 16, 18 -34]. NSI-MS has the following advantages over conventional flow ESI-MS: (1) higher sensitivity, (2) lower rate of sample consumption, and (3) improved signal-to-noise ratio (S/N). Although both on-and off-line NSI-MS have been developed, off-line NSI-MS methods have been more frequently used because of the technical difficulties associated with interfacing conventional flow LC systems with NSI and

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