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.