Machine Learning-Assisted Recognition of Environmental Sulfur-Containing Chemicals in Nontargeted Mass Spectrometry Analysis of Inadequate Mass Resolution

Abstract

Sulfur (S)-containing compounds can be unambiguously identified by their distinctive isotope patterns in mass spectrometry (MS) when the instrument has a mass resolution exceeding 500,000. However, many environmental research laboratories that perform nontargeted analysis rely on high-resolution mass spectrometry (HRMS) instruments, such as quadrupole time-of-flight mass spectrometry (QTOF MS). These HRMS instruments typically operate at a mass resolution of less than 50,000. At such limited resolution, confidently recognizing sulfur isotope patterns is challenging. This work develops a machine learning (ML) strategy for recognizing and predicting the number of S present using HRMS at a mass resolution as low as 25,000. We benchmarked our ML strategy on experimental data, where 200 S-containing standard compounds were mixed into complex environmental samples. In positive electrospray ionization (ESI) mode, our ML strategy achieved accuracies ranging from 87.4 to 95.0% for S recognition and accuracies ranging from 86.3 to 96.6% for S number prediction. Notably, the ML method performed similarly well in negative ESI mode. Our ML strategy was further evaluated on an external experimental water dataset where it correctly recognized the presence of S for all 24 previously reported 2-mercaptobenzothiazole disinfection byproducts (DBPs). The developed ML strategy was implemented into SulfurFinder, an R program, to facilitate automated data cleaning, S recognition, and S number prediction in HRMS data. SulfurFinder combined with HPLC-HRMS analysis of a wastewater sample tentatively identified 169 potential S-containing features. Of these, three were confirmed as S-containing pharmaceuticals. An additional S-containing drug was also putatively annotated using molecular networking. The development of SulfurFinder significantly boosts the capability of conventional HRMS to address the challenge of S recognition in the era of exposomics, supporting a wide range of environmental applications.