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Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are small and very polar compounds with carbon chain lengths shorter than C4. Their ubiquitous and high levels of occurrence in environmental aquatic systems are emerging as a significant concern, rivaling the well-established issues associated with long-chain PFAS contamination. Therefore, it is important to analyze both USC and long-chain PFAS together in water samples to comprehensively assess and address the full spectrum of PFAS contamination. The high polarity of USC PFAS poses a challenge for standard chromatographic practices in PFAS analysis, primarily due to insufficient chromatographic retention. In this study, a simple and reliable workflow was developed for the simultaneous analysis of C1 to C14 perfluoroalkyl carboxylic and sulfonic acids, along with other groups of PFAS, in both potable and non-potable waters. The chromatographic separation was conducted using a polar-embedded reversed-phase LC column with an inert coating on the hardware. Three different water matrices (tap water, bottled water, sewage treatment wastewater) were chosen for method evaluation to demonstrate the applicability of the developed workflow for measuring 45 PFAS compounds in diverse water samples. A dilute-and-shoot workflow was evaluated by accuracy and precision analysis at five fortification levels, ranging from 2 to 250 ng/L. Eighteen isotopically labeled PFAS, serving as extracted internal standards, were added to the samples at 100 ng/L to validate the accuracy of the entire workflow. Calibration standards were prepared in reverse osmosis water due to its cleanliness for all analytes. The calibration ranges varied among different analytes, spanning from 1 – 1000 ng/L. All analytes and extracted internal standards exhibited recovery values ranging from 70% to 130% of the nominal concentration across all fortification levels. Satisfactory method precision was demonstrated with %RSD values below 20%. Additional potable and non-potable waters collected from various source waters were tested to further demonstrate that the established workflow is suitable for the accurate quantification of targeted PFAS in a wide range of water matrices.

Millions of deaths caused by Mycobacterium tuberculosis (Mtb) are reported worldwide every year. Treatment of tuberculosis (TB) involves the use of multiple antibiotics over a prolonged period. However, the emergence of resistance leading to multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is the most challenging aspect of TB treatment. Therefore, there is a constant need to search for novel therapeutic strategies that could tackle the growing problem of drug resistance. One such strategy could be perturbing the functions of novel targets in Mtb, such as universal stress protein (USP, Rv1636), which binds to cAMP with a higher affinity than ATP. Orthologs of these proteins are conserved in all mycobacteria and act as “sink” for cAMP, facilitating the availability of this second messenger for signaling when required. Here, we have used the cAMP-bound crystal structure of USP from Mycobacterium smegmatis, a closely related homolog of Mtb, to conduct a structure-guided hunt for potential binders of Rv1636, primarily employing molecular docking approach. A library of 1.9 million compounds was subjected to virtual screening to obtain an initial set of ~2,000 hits. An integrative strategy that uses the available experimental data and consensus indications from other computational analyses has been employed to prioritize 22 potential binders of Rv1636 for experimental validations. Binding affinities of a few compounds among the 22 prioritized compounds were tested through microscale thermophoresis assays, and two compounds of natural origin showed promising binding affinities with Rv1636. We believe that this study provides an important initial guidance to medicinal chemists and biochemists to synthesize and test an enriched set of compounds that have the potential to inhibit Mtb USP (Rv1636), thereby aiding the development of novel antitubercular lead candidates.