Isomer Platform

[Lane Stefano]

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One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon-carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS, and high resolution mass spectrometry. Modification of an IMS-capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressure trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities, and in all cases, the positions of carbon-carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions, geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of cis/trans double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes including human plasma.

Every agency needs an Internet front to provide information in the digital age. When there is a need for a web-front to provide for such user centric informational needs in a timely manner, CMS (Content Management System) is at play. Regardless of who you are, in the public or private sector, you are bound to need some form of modern digital content management. Even the Singapore Government Technology Department recognizes this and created the Isomer Platform.

As a locally born and bred solution provider, we have dealt with many content management solutions initiatives by the SG Gov, and are well aware of the stringent needs of the digital system to safeguard national interests. We believe this platform has what it takes to delivery such a solution.

Fluorofentanyl is the prominent fentanyl analog that has stuck around in post-mortem casework since its resurgence in 2020. It was one of the compounds first synthesized by Janssen Pharmaceutica and has popped in and out of the illicit drug market but has never been as persistent as it is now. Our lab first started looking for fluorofentanyl in 2021.

In terms of potency, they are very similar to each other. The para- and meta-fluorofentanyl isomers are about 2.5x and 5x, respectively, less potent than fentanyl. The ortho-fluorofentanyl isomer is slightly more potent than fentanyl at about 2x the potentcy.

There is some speculation as to why this particular analog has persisted. One theory is that a fluorinated precursor is being used in the synthesis process and fluorofentanyl could be a byproduct of illicitly manufactured fentanyl. Another is that the presence of fluorine could inhibit metabolism of the drug and lead to longer lasting effects. Ultimately, the answer is not clear without further information gathered.

Analysis of fluorofentanyl can be tricky. The isomers are hard to separate chromatographically and the fragmentation patterns in a mass spectrometer are nearly indistinguishable. Getting them to separate chromatographically can be beneficial to distinguish them by their retention time. There have been several methods published that have shown separation of ortho-fluorofentanyl from the para- and meta- -isomers is possible.

In our casework, fentanyl was the most common drug that was detected with fluorofentanyl in 96.4% of cases. Methamphetamine (33%) and cocaine (27%) were also commonly found with fluorofentanyl. The most common NPS compound found with fluorofentanyl was metonitazene. Given its continued detection in post-mortem casework, it is beneficial to be looking for it.

In summary, the most common source of chirality in organic chemistry is the "asymmetric carbon", or, better yet, the carbon at a center of asymmetry. We showed that the carbon only serves the capacity of keeping the four different atoms, or colored balls if you wish, where they belong. But an asymmetric carbon is not the only source of chirality. Conformations of molecules, and molecules themselves, can be chiral without having an asymmetric carbon. Consider the gauche conformations 16 and 17 of n-butane as an example of a chiral molecular conformation. [If you need a refresher on this topic, click here.] n-Butane has no asymmetric carbons but yet the two gauche conformations are mirror images of one another, equal in energy and present in equal amounts. These two conformations constitute a racemate whose enantiomers are rapidly interconverting by rotation about the C2-C3 sp3 hybridized bond. The formation of enantiomers by bond rotation in achiral molecules is called stochastic (random) chirality [Mislow]. At ambient temperaturethey cannot be separated. The net racemic gauche conformation and the achiral anti conformation are the major conformations of n-butane, which of course is optically inactive. Gauche butane defines a screw axis, one conformation is left-handed and the other is right-handed.

A similar situation exists in the class of compounds known as allenes, RHC=C=CRH. 1,3-Disubstituted allenes can exist as racemates that can be separated (resolved) into their enantiomers. Structures 18 and 19 are the two enantiomers of 1,3-dimethylallene. Although they define a screw axis, the sp hybridized central carbon prohibits rotation about the central, linear carbon axis (C2-C4). There is no asymmetric carbon to be seen in these structures.

Diastereomers are stereoisomers that are not enantiomers. How is this possible? Consider two gray tetrahedral "asymmetric carbons" that are bonded to each other and each one has three atoms attached: red, yellow and green. Structures 20a and 21a represent one staggered conformation of both possible arrangements. Each one is clearly a stereoisomer of the other because they both have the same atom connectivity. Rotation about the C-C bond of staggered conformation 20a, which is a chiral representation, by 60o gives eclipsed conformation 20b, which clearly is achiral. A mirror plane can be passed through the C-C bond.

Biotransformation activities require the comparison of metabolites across species and studies. In general, chromatographic retention time, accurate mass measurement, and mass spectral data are used to align metabolites. Isomeric metabolite comparison may be more challenging particularly when retention times may differ depending on the analytical conditions used. Additionally, the elemental formulae as well as tandem mass spectrometry (MS/MS) spectra can be identical which significantly increases the complexity of the data interpretation and localization of the biotransformation. The use of collision cross section (CCS) values to compare metabolites analyzed using the SELECT SERIES Cyclic IMS and the SYNAPT G2-Si QTof instruments located in different facilities has been shown here and demonstrates the benefit of this analyte-specific physiochemical property to align metabolites across studies.

Moreover, computational prediction of CCS values may provide an additional data asset, allowing the comparison of predicted with measured CCS values. This can further provide additional insights to differentiate between isomers. The prediction can also be used to suggest when additional cyclic ion mobility separation (cIMS) would be beneficial in the separation of isomers and increase confidence in any assignment with the use of higher ion mobility resolution. Examples are given here where cIMS has been used to separate oxygenated metabolites of ranitidine and imipramine; this alternative separation mechanism adds to the separating power of UltraPerformance Liquid Chromatography (UPLC) and is of benefit when isomers co-elute.

In vitro and in vivo metabolism studies are key elements during drug development. Typically, the level of complexity and study detail increases alongside drug development to address safety aspects with regards to human-specific or disproportionate metabolites and to identify metabolites contributing to pharmacological activity.

At the discovery stage, generic high-throughput liquid chromatography-mass spectrometry (LC-MS) methods with short gradients are used, focusing mainly on major metabolites to identify molecular liabilities within the potential drug candidate. In contrast, dedicated LC-MS methods with long gradients are used at development stage to provide chromatographic separation and structural elucidation of all observed metabolites to ensure safety coverage of any human metabolite in animal species as requested by health authorities.1,2

Here, we compare CCS values for a series of approved drugs and their metabolites obtained using two different IMS enabled mass spectrometers (the SELECT SERIES Cyclic IMS and the SYNAPT G2-Si QTof), which were located in different facilities and operated by different scientists. Moreover, CCSonDemand,3 a machine-learning algorithm, was used to predict CCS values and compare theoretical with measured CCS values from each instrument. The cIMS technology was further used, providing increased IMS resolution, to distinguish isomeric metabolites, which was not possible on a commercially available linear IMS device.

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