High throughput and sensitive quantitation of tobacco-specific alkaloids and nitrosamines in wastewater.

Tobacco-specific alkaloids and nitrosamines are important biomarkers for the estimation of tobacco use and human exposure to tobacco-specific nitrosamines that can be monitored by wastewater analysis. Thus far their analysis has used solid phase extraction, which is costly and time-consuming. In this study, we developed a direct injection liquid chromatography-tandem mass spectrometry method for the quantification of two tobacco-specific alkaloids and five nitrosamines in wastewater. The method achieved excellent linearity (R2 > 0.99) for all analytes, with calibration ranging from 0.10 to 800 ng/L. Method limits of detection and quantification were 0.17 ng/L (N-nitrosonornicotine, NNN) and 1.0 ng/L (N-nitrosoanatabine (NAT) and NNN), with acceptable accuracy (100 % ± 20 %) and precision (± 15 %). Analyte loss during filtration was < 15 %, and the relative matrix effect was < 10 %. The method was applied to 43 pooled wastewater samples collected from three wastewater treatment plants in Australia between 2017 and 2021. Anabasine and anatabine were detected in all samples at concentrations of 5.0 - 33 ng/L and 12 - 41 ng/L, respectively. Three of the five tobacco-specific nitrosamines (NAT, NNN, and (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) (NNAL)) were detected, in < 50 % of the wastewater samples, with concentrations nearly ten times lower than the tobacco alkaloids (< 1.0 - 6.2 ng/L). In-sewer stability of the nitrosamines was also assessed in this study, with four (NAT, NNAL, NNN, and N-nitrosoanabasine (NAB)) being stable (i.e. < 20 % transformation over 12 h in both control reactor (CR) and rising main reactor (RM) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) being moderately stable (< 40 % loss over 12 h in RM). This direct injection method provides a high-throughput approach in simultaneous investigation of tobacco use and assessment of public exposure to tobacco-specific nitrosamines.

Direct injection liquid chromatography-tandem mass spectrometry as a sensitive and high-throughput method for the quantitative surveillance of antimicrobials in wastewater.

Environmental antimicrobial pollution and antimicrobial resistance pose a threat to environmental and human health. Wastewater analysis has been identified as a promising tool for antimicrobial monitoring and the back-estimation of antimicrobial consumption, but current pretreatment methods are tedious and complicated, limiting their scope for high-throughput analysis. A sensitive direct injection method for the quantification of 109 antimicrobials and their metabolites in wastewater samples was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated for both wastewater influent and effluent in terms of specificity, calibration range, matrix effect, filtration loss, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Most analytes achieved calibration of R2 > 0.99, and the calibration range was from 0.0002 to 150 µg L-1. Recoveries ranged consistently between ~50 % and ~100 % and losses were attributed to sample filtration. Method LOQs were determined as low as 0.0003 µg L-1, and acceptable accuracy (75 %-125 %) and precision (within 25 %) were achieved for >90 % of the analytes. The method was subsequently further assessed using wastewater of raw influent and treated effluent collected from 6 Australian wastewater treatment plants in 2021. In total, 37 analytes were detected in influent and 22 in effluent. Most of them could be quantified at concentrations ranging from 0.0053 to 160 µg L-1, with benzalkonium chloride-C12, amoxicilloic acid, and cephalexin detected at the highest concentrations. The current study provides a straightforward analytical method for antimicrobial monitoring in wastewater with a fast and simple pretreatment procedure.

Online interactive medical neuroimaging exercise to identify human brain structures.

A persisting need remains for developing methods for inspiring and teaching undergraduate medical students to quickly learn to identify the hundreds of human brain structures, tracts and spaces that are clinically relevant (viewed as three-dimensional volumes or two-dimensional neuroimages), and to accomplish this with the option of virtual on-line methods. This notably includes teaching the essentials of recommended diagnostic radiology to allow students to be familiar with patient neuroimages routinely acquired using magnetic resonance imaging (MRI) and computed tomography (CT). The present article includes a brief example video plus details a clinically oriented interactive neuroimaging exercise for first year medical students (MS1s) in small groups, conducted with instructors either in-person or as an entirely online virtual event. This "find-the-brain-structure" (FBS) event included teaching students to identify brain structures and other regions of interest in the central nervous system (and potentially in head and neck gross anatomy), which are traditionally taught using brain anatomy atlases and anatomical specimens. The interactive, small group exercise can be conducted in person or virtually on-line in as little as 30 min depending on the scope of objectives being covered. The learning exercise involves coordinated interaction between MS1s with one or several non-clinical faculty and may include one or several physicians (clinical faculty and/or qualified residents). It further allows for varying degrees of instructor interaction online and is easy to convey to instructors who do not have expertise in neuroimaging. Anonymous pre-event survey (n = 113, 100% response rate) versus post-event surveys (n = 92, 81% response rate) were attained from a cohort of MS1s in a neurobiology course. Results showed multiple statistically significant group-level shifts in response to several of the questions, showing an increase in MS1 confidence with reading MRI images (12% increase shift in mean, p < 0.001), confidence in their approaching physicians for medical training (9%, p < 0.01), and comfort levels in working online with virtual team-based peers and with team-based faculty (6%, p < 0.05). Qualitative student feedback revealed highly positive comments regarding the experience overall, encouraging this virtual medium as a desirable educational approach.

chi-Conopeptide pharmacophore development: toward a novel class of norepinephrine transporter inhibitor (Xen2174) for pain.

Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.