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

Abstract

The Paterno-Buchi (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 Paterno-Buchi 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.

Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC–Ion Mobility–HRMS

“…Gas-phase IMS analyses are rapid, typically occurring on a time scale of 10–100 ms per spectrum, whereas condensed-phase LC–MS is on the time scale of minutes. Therefore, IMS can be included in existing LC–MS workflows without compromising analytical throughput, providing an additional separation dimension and an associated molecular descriptor (CCS) to support analyte detection and identification. , Previously, IMS has been utilized to characterize intact AASs along with their phase I and phase II metabolites. ,,− Various IMS techniques, such as field asymmetric IMS (FAIMS), traveling wave IMS (TWIMS), and drift tube IMS (DTIMS), have been utilized for AAS analyses. ,− The Yost research group investigated the effect of Group 1 cation adducts on resolving AAS isomers using FAIMS, providing direct separation of metal-coordinated AAS isomers with no increase in analysis time or modifications to the instrumentation, which is useful for high-throughput screening . Hernández-Mesa et al have successfully cross-validated the first TWIMS database for steroids in calves urine and obtained CCS values within 2% of literature databases entries .…”

“…Hernández-Mesa et al have successfully cross-validated the first TWIMS database for steroids in calves urine and obtained CCS values within 2% of literature databases entries . The Chouinard group has demonstrated the application of the Paternò–Büchi reaction for structural modification of steroid isomers, prompting improved identification by DTIMS, which is promising due to the simplicity, low cost, and relatively high efficiency of the Paternò–Büchi reaction for generating structurally informative ions …”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…Yet, the accomplished LODs (1–5 ng/ml) were found to add little value to routine doping controls. Nonetheless, the structure‐specific information obtained from the derivatization can be of particular value when studying the stereochemistry of newly identified metabolites or when enhanced chromatographic separation of isomers is required, which has been shown to be a challenging endeavor particularly in the case of steroidal analytes 53 …”

“…Nonetheless, the structure-specific information obtained from the derivatization can be of particular value when studying the stereochemistry of newly identified metabolites or when enhanced chromatographic separation of isomers is required, which has been shown to be a challenging endeavor particularly in the case of steroidal analytes. 53 Increasing sample throughput and shortening turnaround and reporting times while maintaining and guaranteeing utmost specificity and sensitivity have been long sought-after goals in drug testing laboratories. In that context, the possibility of omitting chromatographic separation and utilizing ultra-fast laser desorption ionization strategies combined with high resolution/high accuracy (tandem) mass spectrometry (HRMS/MS) have been taken into consideration.…”

Targeted glucocorticoid analysis using ion mobility-mass spectrometry (IM-MS)

“…First, simple chemical modifications have been demonstrated to improve resolution of various isomeric steroids; these have included derivatization with p-toluenesulfonyl isocyanate, [23] ozonolysis, [24] and the Paternò-Büchi reaction. [25] Investigation of ion adducts as alternatives to the most common protonated/deprotonated/sodiated has led to interesting separation results, with examples of alkali, alkaline earth, and transition metal complexes as well as negatively charged species, such as chlorides. [5] , [16] , [26] , [27] , [28] , [29] Complexation with other steroids or with larger molecules, such as cyclodextrins and crown eithers (themselves including stereochemical differences), have also promoted improved differentiation.…”