# Intramolecularly Labeled Reference Standards of Sulfamethoxazole for Fragment-Specific Isotope Analysis by Electrospray Ionization Orbitrap Mass Spectrometry

## TL;DR

This paper introduces a new method to study the environmental breakdown of the antibiotic sulfamethoxazole using isotope analysis with high precision.

## Contribution

The synthesis of intramolecularly labeled reference standards enables fragment-specific isotope analysis of sulfamethoxazole.

## Key findings

- The method achieved 95% confidence intervals of 0.7‰ and 3.4‰ for Δδ13C and Δδ15N in fragment F92.
- Fragment F99 showed 1.3‰ and 2.9‰ confidence intervals for Δδ13C and Δδ15N.
- The approach successfully detected changes in isotopic signatures during SMX degradation.

## Abstract

The
widespread presence of pharmaceuticals, including antibiotics,
in our aquatic environment raises important societal concerns. When
studying their environmental fate, stable isotope analysis of nitrogen
and carbon at natural abundance offers unique insight into source
fingerprinting and degradation-associated kinetic isotope effects.
Here, we synthesized compound-specific reference standards to enable
electrospray ionization (ESI) Orbitrap mass spectrometry (MS) for
fragment-specific carbon and nitrogen isotope analysis (Δδ13C and Δδ15N) of sulfamethoxazole (SMX),
a most frequently detected antibiotic. Fragment-specific isotope analysis
relied on fragmentation of SMX ions in the collision cell, resulting
in two fragment ions representing the aniline part (m/z = 92, F92) and the 3-amino-5-methylisoxazole
ring (m/z = 99, F99) of SMX. Reference
materials were prepared (i) through total synthesis of SMX from labeled
precursors that resulted in specific positions labeled with 13C and 15N, (ii) followed by the mixing of labeled SMX
with SMX at natural abundance. The bulk isotope values of these in-house
standards were determined by elemental analysis isotope ratio mass
spectrometry and used for calibration of the ESI-Orbitrap-MS method.
Injecting standards directly into the ESI-Orbitrap-MS resulted in
95% confidence intervals (CIs) of 0.7‰ and 3.4‰ for
Δδ13C and Δδ15N in F92,
respectively, and 1.3‰ and 2.9‰ for Δδ13C and Δδ15N in F99, for quintuplicate
measurements of standards. A proof-of-principle demonstration shows
that this approach could indeed successfully quantify changes in fragment-specific
isotopic signatures, Δδ13C and Δδ15N, during degradation of SMX.