Bioanalytical determination of unstable endogenous small peptides: RFRP3 and its metabolites in rat blood.

BACKGROUND: Targeting the gonadotropin-releasing hormone pathway for the treatment of endometriosis leads to an interest in monitoring for endogenous modulators of this pathway (RFRP3 and kisspeptin) as baseline controls for treatment development. RESULTS: Stabilization of RFRP3 was shown to be extremely difficult in a highly enzymatically active matrix, such as rat blood. Sample denaturing with solvent at collection was necessary due to enzyme inhibition being unsuccessful at stabilization leading to difficulties in sample processing. Monitoring multiple fragments formed in blood can aid in profiling these peptides once in-source conversion is controlled. CONCLUSION: generic high-sensitivity LC-MS/MS assay was developed for RFRP3 and the fragments formed from it in whole blood. Use of 2D chromatography circumvents concentration and retention issues related to small fragments with a normal flow setup, making a more open-access approach feasible.

Evolution of an open-access quantitative bioanalytical mass spectrometry service in a drug discovery environment.

Increased demand for assays for compounds at the early stages of drug discovery within the pharmaceutical industry has led to the need for open-access mass spectrometry systems for performing quantitative analysis in a variety of biological matrices. The open-access mass spectrometers described here are LC/MS/MS systems operated in 'multiple reaction monitoring' (MRM) mode to obtain the sensitivity and specificity required to quantitate low levels of pharmaceutical compounds in an excess of biological matrix. Instigation of these open-access systems has resulted in mass spectrometers becoming the detectors of choice for non-expert users, drastically reducing analytical method development time and allowing drug discovery scientists to concentrate on their core expertise of pharmacokinetics and drug metabolism. Setting up an open-access facility that effectively allows a user with minimal mass spectral knowledge to exploit the MS/MS capability of triple quadrupole mass spectrometers presents a significantly different challenge from setting up qualitative single stage mass spectrometry systems. Evolution of quantitative open access mass spectrometry within a pharmaceutical drug metabolism and pharmacokinetics group, from its beginnings as a single generic system to a series of specialist fully integrated walk-up facilities, is described.

Characterisation of sulphoxides by atmospheric pressure ionisation mass spectrometry.

An observation that a series of proprietary compounds containing a methyl thiophenyl group all underwent metabolic S-oxidation, and that the product ion spectra of the resulting S-oxides showed methyl radical loss under low-energy atmospheric pressure ionisation tandem mass spectrometry (API-MS/MS) conditions, has led to an investigation of the fragmentation of commercially available sulphoxides. The phenyl methyl sulphoxides studied do lose methyl radicals under MS/MS conditions on triple quadrupole mass spectrometers. In addition, the phenyl sulphoxides, with simple substituents other than a methyl group, also showed a tendency to lose the substituent as a radical. It was concluded that radical loss from these simple sulphoxides was characteristic of S-oxidation of these molecules. Radical losses, such as those reported here, are used in-house to distinguish S-oxidation from N- and C-oxidation in metabolism studies.