Cobalt(III)- and rhodium(III)-porphyrin complexes for the effective degradation of fentanyl: Biological inactivation and mechanistic insights.

Cobalt(III) and rhodium(III) complexes containing the water-soluble porphyrin ligand meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphine (C1S3TPP), [Rh(C1S3TPP)]Nax•nH2O (1) and [Co(C1S3TPP)]Nax•nH2O (2) were prepared from the direct reaction of free porphyrin and metal chloride salts in refluxing MeOH/DMF or EtOH/H2O. Compounds 1 and 2 were characterized using UV-vis and 1H NMR spectroscopies, and high-resolution mass spectrometry. Cell culture based assays of opioid receptor activation showed that while the rhodium complex reduced fentanyl opioid activity 113-fold to an IC50 value of 1.7 µM, the cobalt complex reduced fentanyl activity by 160-fold to an IC50 value of 2.4 µM. An oxidative mechanism for fentanyl breakdown is proposed.

Toward Routine Analysis of Anabolic Androgenic Steroids in Urine Using Ion Mobility-Mass Spectrometry.

Anabolic androgenic steroids (AAS) make up one of the most prevalent classes of performance-enhancing drugs banned by the World Anti-Doping Agency (WADA) due to the competitive advantage they can afford athletes. Mass spectrometry-based methods coupled with chromatographic separations have become the gold standard for AAS analysis because of the superior sensitivity and selectivity provided. However, emerging analytical techniques including ion mobility spectrometry (IMS) have been demonstrated in recent applications as a means to further characterize and identify potential unknowns while simultaneously delivering improved sensitivity by filtering noise. Herein we outline the next crucial steps in bringing IMS to the routine drug testing workflow by combining it with established chromatographic and mass spectrometry methods (i.e., LC-IM-MS) for the detection of AAS in human urine. In addition to robust measurement of collision cross sections which can be used for identification purposes, functional group microtrends provide a structural basis on which to elucidate the structure of future novel anabolic agents. Lastly, the developed workflow is tested by analysis of testosterone in a realistic matrix (human urine) and demonstrates a limit of detection of 524 pg/mL, which surpasses the WADA Minimum Required Performance Levels for anabolic steroids. This work is expected to pave the way toward routine incorporation of IMS into analytical drug testing workflows to augment both qualitative and quantitative measure of performance enhancing drugs in the future.

Native Ubiquitin Structural Changes Resulting from Complexation with ß-Methylamino-l-alanine (BMAA).

The objective of this research was to investigate potential changes to unfolding energy barriers for ubiquitin in the presence of the noncanonical amino acid ß-methylamino-l-alanine (BMAA). Although BMAA has been implicated in neurodegenerative disease, its specific role remains unclear. We hypothesized that formation of a ubiquitin + BMAA noncovalent complex would alter the protein's unfolding dynamics in comparison with native ubiquitin alone or in noncovalent complexes with other amino acids. Ion mobility-mass spectrometry (IM-MS) revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, and similar complexes were identified for a range of additional amino acids. Collision-induced unfolding (CIU) was used to interrogate the unfolding of native ubiquitin and these Ubq-amino acid complexes, showing a major transition from its compact native state (∼1200 Å2) to an unfolded state (∼1400 Å2) at activation energies in the range from 8.0 to 9.0 V (entrance grid delta). The Ubq-BMAA complex, on the other hand, was observed to have a significantly higher energy barrier to unfolding, requiring more than 10.5 V. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location worthy of further study for its potential role in the onset of neurodegenerative disease.

Improved Identification of Isomeric Steroids Using the Paternò-Büchi Reaction with Ion Mobility-Mass Spectrometry.

The Paternò-Büchi (PB) reaction is a common organic reaction in which a carbonyl radical formed by exposure to UV radiation reacts with an alkene to form an oxetane ring. Recent analytical applications of this reaction have included the determination of C═C bond position in lipid fatty acyl tails using tandem mass spectrometry. Our group has recently investigated methods for structurally modifying steroid isomers to improve their identification and resolution using ion mobility spectrometry. Herein, we report the first application of the Paternò-Büchi reaction to form steroid oxetanes using a simple, low-cost, and high efficiency method with a low pressure mercury lamp. This methodology is performed on several endogenous steroid isomers, resulting in unique ion mobility spectra that provide a unique fingerprint for each. These fingerprint spectra can add confidence in identification of those compounds, especially in complex biological matrixes. Testosterone and epitestosterone, an epimer pair commonly interrogated in a number of applications such as for their use as performance enhancing drugs, displayed one and three unique ion mobility peaks, respectively. These spectra and their measured collision cross sections (CCS) allow for unambiguous differentiation of these and several other steroid isomer groups analyzed in this work. Finally, multiple anabolic androgenic steroids prohibited by the World Anti-Doping Agency were tested with this method and resulted in unique CCS for their PB reaction products. This approach can offer improved confidence in their identification as well as for many other banned substances.

Ozone-Induced Cleavage of Endocyclic C═C Double Bonds within Steroid Epimers Produces Unique Gas-Phase Conformations.

Herein we demonstrate the first application of ozone-induced cleavage of endocyclic C═C double bonds for improved steroid isomer separation using ion mobility-mass spectrometry. Steroids represent a challenging biomolecular class for ion mobility (IM) separations due to their structural rigidity and subtle stereochemical differences. In this work, we compare the effects of ozonolysis on the relative mobilities of a model stereoisomer pair, testosterone and epitestosterone. A solution-phase ozonolysis approach is used due to its simplicity, relatively low cost, and potential for rapid, online analysis. Despite the presence of solvent-based addition products, we observe that these steroids undergo an ozone-based cleavage resulting in unique, stable gas-phase conformations. The resulting resolution between testosterone and epitestosterone, with collision cross section values of 176.6 and 193.3 Å2, respectively, demonstrates a significant improvement in comparison with previous IM-based approaches. The significantly smaller conformation observed for epitestosterone is stabilized by a three-point interaction between the oxygen-containing functional groups and a sodium ion; this same conformation cannot be sterically achieved by testosterone. Identification of this specific structural difference is strengthened by experimental results showing the disappearance of this conformation following in-source water loss, which eliminates the potential for that three-point interaction. Computational modeling of the lowest energy gas-phase structures for these ozone products corroborates the experimental results. In conclusion, this approach provides tremendous potential as a rapid IM separation method for steroid isomers and other endocyclic C═C double bond containing molecules.