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Current approaches to discovery-stage drug metabolism studies (pharmacokinetics, microsomal stability, etc.) typically use triple-quadrupole-based approaches for quantitative analysis. This necessitates the optimization of parameters such as Q1 and Q3 m/z values, collision energy, and interface voltages. These studies detect only the specified compound and information about other components, such as metabolites, is lost. The ability to perform full-scan acquisition for quantitative analysis would eliminate the need for compound optimization while enabling the detection of metabolites and other non-drug-related endogenous components. Such an instrument would have to provide sensitivity, selectivity, dynamic range, and scan speed suitable for discovery-stage quantitative studies. In this study, a prototype benchtop Orbitrap-based mass analyzer was used to collect both quantitative and qualitative data from human microsomal incubation samples as well as rat plasma from pharmacokinetic studies. Instrumental parameters such as scan speed, resolution, and mass accuracy are discussed in relation to the requirements for a quantitative-qualitative workflow. The ability to perform highly selective quantitative analysis while simultaneously characterizing metabolites from both in vitro and in vivo studies is discussed.

A noncovalently bound complex, the heme-apomyoglobin protein system (M, 17 568), was detected using electrospray ionization on a double-focusing mass spectrometer with an array detector. The kilovolt energy conditions used for ion transmission and focusing did not cause significant amounts of fragmentation of the weaklybound complex. With a high-performance array detector, ferntomolar sensitivity was achieved for myoglobin in water.Noncovalent interactions play many significant roles in biochemistry. Proteins are folded into specific molecular conformations maintained by intramolecular noncovalent interactions. Protein activity is highly dependent on this 'secondary structure.' Many types of superstrate/substrate binding are examples of intermolecular noncovalent interactions, including antibodylantigen, protein/cofactor, receptor/ligand, and enzyme/substrate pairings. Intermolecular noncovalent interactions are also responsible for aggregation of proteins into multimers (quaternary structure). These weak interactions can be disrupted by a variety of conditions, such as pH, temperature, solvent composition, and the presence of denaturing agents such as guanidine hydrochloride or urea.Since the first reports of electrospray (ES) ionization mass spectra of biomolecules by Fenn et ale,] ES has become an almost indispensable method in the determination of peptide and protein primary ~tructure.~" In electrospray, highly charged liquid droplets containing the analyte are formed at atmospheric pressure. Ionization takes place by attachment (or detachment) of charge-carrying species (typically protons) to the molecular entity. By virtue of its ability to. form stable gas-phase ions with a paucity of fragmentation, even from nonvolatile polar molecules such as polypeptides and proteins, ES has been classified as an 'ultrasoft' ionization method. However the typical solution conditions used in electrospray ionization (i.e., water + methanol or water + acetonitrile with 1-5% (v) acetic acid for positive ionization) are rather harsh from the standpoint of maintaining native conformations and noncovalent interactions necessary for biological activity. Thus, there is usually little information about secondary structure or noncovalently bound complexes in the electrospray mass spectrum.The denaturing effect of pH$' organic solvent^'.^ and ternperat~re'.~ on the ES mass spectra of proteins has been reported for a few systems. Recently, several groups have shown that ES mass spectrometry has the capabiliti18 of examining noncovalently bound systems, such as myogl~bin,~~-'~ by adjustment of solution and pH conditions. Author to whom correspondence should be addressed.Because noncovalent bond energies are about an order of magnitude lower than typical covalent bond strengths, the multiply charged molecular entities generated by ES from such weakly bound biological systems are very susceptible to collisionally activated dissociation (CAD).*' Previous ES work on noncovalently bound systems has been performed on quadrupole mass spe...

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