Rapid metabolite identification with sub parts‐per‐million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean‐up on a ZipTip and LTQ/Orbitrap mass spectrometry

Abstract

Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100,000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments.

Exploring metallodrug–protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound

“…High-resolution ESI MS has previously been used to study covalent and non-covalent ligand-biomolecule interactions [37,38] and to screen complex mixtures of metabolites [39][40][41], often without the need for chromatographic separation of the adducts prior to analysis. Hence, this method allows the simultaneous screening of complicated mixtures of compounds with the direct (Ub), cytochrome c (cyt c) and superoxide dismutase (SOD) (1:1:1) in tetramethylammonium acetate (TMeAmAc) buffer pH 7.4 and the same protein mixture after 24-h incubation at 37°C with cisplatin, transplatin and RAPTA-C, respectively (5:1 metal-to-protein ratio).…”

Fourier Transform Mass Spectrometry

“…FTMS can reliably deliver mass accuracy below 1 ppm (54,(57)(58)(59)(60). This accomplishment can be aided by exploiting the use of certain ubiquitously present background ions.…”

Spectral accuracy of molecular ions in an LTQ/Orbitrap mass spectrometer and implications for elemental composition determination

“…The commercially available Triversa NanoMate with its electrospray ionization (ESI)-chip employed in the direct infusion mode removes any time constraints to high-resolution measurements because the ESI-chip, which contains 5 m diameter nozzles, leads to flow rates measured in nL per minute so that L volumes can be analyzed for an hour or more. In addition to high-resolution capabilities, the mass accuracies of the Orbitrap are often within 2 ppm when employing internal calibration [22] and sub-ppm mass accuracies have also been reported [23].…”