Background: The increasing prevalence of xylazine in the illicit drug supply is a growing concern for major health consequences in individuals who use fentanyl mixed with xylazine, but limited data are available regarding the pharmacokinetics of xylazine in humans. Methods: Xylazine was quantified in serial remnant plasmas collected from 28 patients starting at the initial patient encounter and continuing for up to 52 h from presentation, using LC-MS/MS to calculate the terminal half-life for xylazine. Xylazine metabolites were identified by product ion scanning, and multiple reaction monitoring was used to estimate the relative abundance of xylazine metabolites in 74 collected plasma samples. Results: The median terminal half-life for xylazine was calculated to be 12.0 h (range: 5.9–20.8). Oxo-xylazine and sulfone-xylazine metabolites were detected in all plasma specimens that contained xylazine. Conclusions: The half-life of xylazine in humans is longer than previously observed in animal studies, which furthers the current understanding of the expected duration of effects in individuals who use fentanyl mixed with xylazine and the window of detection. Both oxo-xylazine and sulfone-xylazine appear to circulate in plasma for as long as xylazine.

Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.

Mass spectrometry (MS)-based clinical proteomic Laboratory Developed Tests (LDTs) for the measurement of protein biomarkers related to endocrinology, cardiovascular disease, cancer, and Alzheimer's disease are gaining traction in clinical laboratories due to their value in supporting diagnostic and treatment decisions for patients. Under the current regulatory landscape, MS-based clinical proteomic LDTs are regulated by Clinical Laboratory Improvement Amendments (CLIA) under the auspices of the Centers for Medicaid and Medicare Services (CMS). However, should the Verifying Accurate Leading-Edge In Vitro Clinical Test Development (VALID) Act pass, it will grant the FDA greater authority to oversee diagnostic tests, including LDTs. This could impede clinical laboratories' ability to develop new MS-based proteomic LDTs to support existing and emerging patient care needs. Therefore, this review discusses the currently available MS-based proteomic LDTs and their current regulatory landscape in the context of the potential impacts imposed by the passage of the VALID Act.

Xylazine in an anesthetic drug used for the sedation of animals and increasingly appearing as an adulterant in uncontrolled drug supplies, primarily illicit fentanyl. The ability to detect xylazine exposure by urine drug testing may improve monitoring of this drug trend and our understanding of the effects and risks associated with xylazine exposure. Currently, limited information is available regarding the elimination of xylazine or its metabolites in humans. In this study, we report quantification of xylazine and 4-hydroxy-xylazine (4-OH-x) in hydrolyzed urine specimens collected from 109 patients testing positive for fentanyl and xylazine using liquid chromatography tandem mass spectrometry (LC-MS/MS). 4-hydroxy-xylazine was a minor urinary metabolite in most patients with a median metabolite-to-xylazine (MR) concentration ratio 0.09. Additional urinary metabolites were identified including oxo-xylazine (oxo-x), OH-oxo-xylazine (OH-oxo-x), OH-sulfone-xylazine (OH-sulfone-x), and sulfone-xylazine (sulfone-x), with median MR peak area ratios of < 0.01, 0.60, 0.30, and 1.60, respectively. Sulfone-x signal exceeded that of xylazine in more than 70% of urine specimens. Sulfone-x is not glucuronidated and does not appear to form positional isomers. Additional studies are needed to examine whether detection of xylazine metabolites may improve the sensitivity and/or extend the detection time window for xylazine exposure.

A 66-year-old female with a history of smoking, lung adenocarcinoma, and chronic obstructive pulmonary disease presented with dyspnea and light-headedness for 4 months. She was cyanotic on examination and self-reported increased carboxyhemoglobin in the past. Co-oximetry using 2 separate blood gas instruments at 2 separate time points revealed methemoglobin results that were suppressed due to an interfering substance (in one case) or flagged (in the second case) due to increased sulfhemoglobin ( Table 1). Carboxyhemoglobin was elevated at the second time point ( Table 1). She was treated with methylene blue after admission.