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

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.