Investigation of an individual with background levels of exhaled isoprene: a case study.

Isoprene is one of the most abundant and most frequently evaluated volatile organic compounds in exhaled breath. Recently, several individuals with background levels of exhaled isoprene have been identified. Here, case study data are provided for an individual, identified from a previous study, with this low prevalence phenotype. It is hypothesized that the individual will illustrate low levels of exhaled isoprene at rest and during exercise. At rest, the subject (7.1 ppb) shows background (µ= 14.2 ± 7.0 ppb) levels of exhaled isoprene while the control group illustrates significantly higher quantities (µ= 266.2 ± 72.3 ppb) via proton transfer reaction mass spectrometry (PTR-MS). The result, background levels of isoprene at rest, is verified by thermal desorption gas chromatography mass spectrometry (TD-GC-MS) collections with the individual showing -3.6 ppb exhaled isoprene while the room background containedµ= -4.1 ± 0.1 ppb isoprene. As isoprene has been shown previously to increase at the initiation of exercise, exercise bike experiments were performed with the individual identified with low isoprene, yielding low and invariant levels of exhaled isoprene (µ= 6.6 ± 0.1 ppb) during the exercise while control subjects illustrated an approximate 2.5-fold increase (preµ= 286.3 ± 43.8 ppb, exerciseµ= 573.0 ± 147.8 ppb) in exhaled isoprene upon exercise start. Additionally, exhaled breath bag data showed a significant decrease in isoprene (delta post/pre, p = 0.0078) of the control group following the exercise regimen. Finally, TD-GC-MS results for exhaled isoprene from the individual's family (mother, father, sister and maternal grandmother) illustrated that the mother and father exhibited isoprene values (28.5 ppb, 77.2 ppb) below control samples 95% confidence interval (µ= 166.8 ± 43.3 ppb) while the individual's sister (182.0 ppb) was within the control range. These data provide evidence for a large dynamic range in exhaled isoprene in this family. Collectively, these results provide additional data surrounding the existence of a small population of individuals with background levels of exhaled isoprene.

Detection of Asthma Inhaler Use via Terahertz Spectroscopy.

Inhaled medications are commonplace for administering bronchodilators, anticholinergics, and corticosteroids. While they have a defined legitimate use, they are also used in sporting events as performance-enhancing drugs. These performance enhancers can be acquired via both legal (i.e., at a pharmacy through over-the-counter medications or through a prescription) and illicit (i.e., black market and foreign pharmacies) means, thus making monitoring procurement impossible. While urine tests can detect these pharmacological agents hours after they have been inhaled, there is a significant lag time before they are observed in urine. Direct detection of these inhaled agents is complicated and requires a multiplexed approach due to the sheer number of inhaled pharmacological agents. Therefore, detection of propellants, which carry the drug into the lungs, provides a simpler path forward toward detection of broad pharmacological agents. In this paper, we demonstrate the first use of terahertz spectroscopy (THz) to detect inhaled medications in human subjects. Notably, we were able to detect and quantitate the propellant, HFA-134a, in breath up to 30 min after using an asthma inhaler, enabling the use of a point-of-care device to monitor exhaled breath for the presence of propellants. We also demonstrate via simulations that the same approach can be leveraged to detect and identify next-generation propellants, specifically HFA-152a. As a result, we provide evidence that a single point-of-care THz sensor can detect when individuals have used pressure-mediated dose inhalers (pMDIs) without further modification of the hardware.

The Impact of Nutritional Supplementation on Sweat Metabolomic Content: A Proof-of-Concept Study.

Sweat is emerging as a prominent biosource for real-time human performance monitoring applications. Although promising, sources of variability must be identified to truly utilize sweat for biomarker applications. In this proof-of-concept study, a targeted metabolomics method was applied to sweat collected from the forearms of participants in a 12-week exercise program who ingested either low or high nutritional supplementation twice daily. The data establish the use of dried powder mass as a method for metabolomic data normalization from sweat samples. Additionally, the results support the hypothesis that ingestion of regular nutritional supplementation semi-quantitatively impact the sweat metabolome. For example, a receiver operating characteristic (ROC) curve of relative normalized metabolite quantities show an area under the curve of 0.82 suggesting the sweat metabolome can moderately predict if an individual is taking nutritional supplementation. Finally, a significant correlation between physical performance and the sweat metabolome are established. For instance, the data illustrate that by utilizing multiple linear regression modeling approaches, sweat metabolite quantities can predict VO2 max (p = 0.0346), peak lower body Windage (p = 0.0112), and abdominal circumference (p = 0.0425). The results illustrate the need to account for dietary nutrition in biomarker discovery applications involving sweat as a biosource.

Rate normalization for sweat metabolomics biomarker discovery.

As the demand for real-time exercise performance feedback increases, excreted sweat has become a biosource of interest for continuous human performance assessment. For sweat to truly fulfill this requirement, analyte concentrations must be normalized to adequately assess day-to-day differences within and among individuals. In this manuscript, data are presented highlighting the use of accurate localized sweat rate as a means for ion and global metabolomic data normalization. The results illustrate large sweat rate variability among individuals over the course of two distinct exercises protocols. Furthermore, the data show sweat rate is not symmetrical at similar locations among right and left forearms of individuals (p = 0.0007). Sweat ion conductivity analysis suggest overall sweat rate normalization reduces variability collectively among ion values and participants with principal component analysis showing 77.8% of variation in the data set attributable to sweat rate normalization. Global metabolomic analysis of sweat illustrated overall rate normalization increases the variability among test subjects with 72.7% of the variation explained by sweat rate normalization. Finally, overall rate normalized metabolomic features of sweat significantly correlated (ρ ≥ 0.7, ρ ≤ -0.7) with measured performance metrics of the individual, establishing the potential for sweat to be used as a biosource for performance monitoring. Collectively, these data illustrate the importance of accurate localized sweat rate determination, for analyte data normalization, in support for the use of sweat in biomarker discovery efforts to predict human performance.

Evaluation of a standardized collection device for exhaled breath sampling onto thermal desorption tubes.

The Respiration Collector for In Vitro Analysis (ReCIVA) sampler, marketed by Owlstone Medical, provides a step forward in exhaled breath sampling through active sampling directly onto thermal desorption (TD) tubes. Although an improvement to the issues surrounding breath bag sampling, the ReCIVA device, first released in 2015, is a relatively new research and clinical tool that requires further exploration. Here, data are presented comparing two distinct ReCIVA devices. The results, comparing ReCIVA serial numbers #33 and #65, demonstrate that overall statistically insignificant results are obtained via targeted isoprene quantitation (p > 0.05). However, when the data are parsed by the TD tube type used to capture breath volatiles, either Tenax TA or the dual bed Tenax/Carbograph 5TD (5TD), a statistical difference (p < 0.05) among the two different TD tubes was present. These data, comparing the two ReCIVA devices with both Tenax TA and 5TD tubes, are further supported by a global metabolomics analysis yielding 85% of z-scores, comparing ReCIVA devices, below the limit for significance. Experiments to determine the effect of breathing rate on ReCIVA function, using guided breathing for low (7.5 breaths min-1) and high (15 breaths min-1) breathing rates, demonstrate the ReCIVA device shows no statistical difference among breathing rates for quantitated isoprene (p > 0.05). Global metabolomics analysis of the guided breathing rate data shows more than 87% of the z-scores, comparing high and low breathing rates using both the Tenax and the 5TD tubes, are below the level for significance. Finally, data are provided from a single participant who displayed background levels of isoprene while illustrating levels of acetone consistent with the remaining participants. Collectively, these data support the use of multiple ReCIVA devices for exhaled breath collection and provide evidence for an instance where exhaled isoprene is consistent with background levels.

Characterization of standardized breath sampling for off-line field use.

Due to several sources of potential variability associated with exhaled breath bag sampling procedures for off-line analysis, the Respiration Collector for in vitro Analysis (ReCIVA) sampler was developed. Although designed to improve upon several pitfalls of sampling with exhaled breath bags, the ReCIVA remains a minimally studied research tool. In this manuscript, several attributes of the ReCIVA sampler are investigated among three individual tests, such as background contamination, control software version, performance of different adsorbent tubes, duplicate sample production, and comparison to exhaled breath bags. The data shows greater than a 58% reduction in background siloxanes can be achieved with submersion of ReCIVA masks in ethyl alcohol or baking the masks at a high temperature (200 °C). The results illustrate the ReCIVA control software version plays a key role in the flow rates applied to thermal desorption (TD) tubes. Using exhaled isoprene as a representative analyte, the data suggest duplicate samples among ReCIVA pump banks can be achieved using two different thermal desorption tubes, Tenax TA and Tenax/Carbograph 5TD, when using an updated control software and manually calibrating the ReCIVA pumps to uniform flow rates (Tenax p = 0.3869, 5TD p = 0.3131). Additionally, using the updated control software and manual ReCIVA flow calibration, the data suggest the ReCIVA can produce statistically similar results among TD tube types (p = 0.3824) and compared to standard exhaled breath bags (p = 0.1534). Collectively, these results establish a method for manually calibrating the flow of the ReCIVA device to allow for the most consistent results. These data support further experimentation into the use of the ReCIVA sampler for exhaled breath research.

Metabolomic stability of exercise-induced sweat.

Due to increased interest in the use of excreted sweat for biomarker discovery, data must be generated supporting sample collection and handling methods to allow for controlled, large-scale biomarker discovery studies to be performed. In this manuscript, twelve amino acids were quantitated from exercise-induced excreted sweat held at room temperature or a simulated body temperature of 37 °C for up to 90 min. The data illustrate a large dynamic range exists among amino acids in sweat. Additionally, the amino acid quantities vary across individuals and among the same individual under different storage conditions, with alanine, arginine, and threonine showing a significant statistical difference between sampling events (p < 0.05). Furthermore, the results establish amino acids are relatively invariant, at both storage temperatures tested, for up to 90 min illustrated by <10% (15/156) of the amino acids measurements demonstrating change greater than 10% from the time zero value. An untargeted metabolomics approach was also applied to the data set to evaluate global changes to the metabolome. The results show more than 88% of all data points fall within the established limits, regardless of temperature condition and ionization mode. Collectively, this study demonstrates that sweat is largely invariant at two distinct temperatures for up to 90 min. These results establish sweat collection and sample handling is possible for up to 90 min with minimal changes in metabolite abundances.

Street fentanyl use: Experiences, preferences, and concordance between self-reports and urine toxicology.

BACKGROUND: Conducted in Dayton, Ohio, the study aims to characterize user knowledge and experiences with non-pharmaceutical fentanyl-type drugs (NPFs) and compare self-reports with urine toxicology for NPFs and heroin. METHODS: Between May 2017-January 2018, 60 individuals who self-reported heroin/NPF use were interviewed using structured questionnaire on socio-demographics, NPF and other drug use practices. Unobserved urine samples were collected and analyzed using: 1) liquid-chromatography-tandem mass spectrometry (LC-MS/MS)-based method (Toxicology lab) to identify 34 fentanyl analogues, metabolites, and other synthetic opioids; 2) immunoassay-based method to screen for opiates (heroin). Sensitivity, specificity and Cohen's kappa were calculated to assess agreement between self-reports and urine toxicology. RESULTS: The sample was 52% female, and over 90% white. Almost 60% reported preference for heroin, and 40% for NPF. Participants endorsed a number of ways of distinguishing heroin from NPF, including appearance (88.3%), effects (76.7%), taste (55%), and information provided by dealers (53.3%). Almost 80% felt confident they could distinguish heroin from NPF, but knowledge about fentanyl analogues was limited. LC-MS/MS testing identified 8 types of NPFs. Over 88% tested positive for NPFs, including 86% fentanyl, 48% carfentanil, 42% acetyl fentanyl. About 47% screened positive for opiates/heroin, and all of them were also positive for NPFs. When comparing self-reported use of NPF to urine toxicology, sensitivity and specificity were relatively high (84% and 83.3%, accordingly), while Cohen's Kappa was 0.445, indicating fair agreement. Sensitivity and specificity were lower for heroin (77.8% and 50.0%, accordingly), and Cohen's Kappa was 0.296, indicating low agreement between self-reports of heroin use and urine toxicology. DISCUSSION: Nearly 90% of the study participants tested positive for NPF-type drugs. Participants were more likely to over-report heroin use and underreport NPF use. The majority had little knowledge about fentanyl analogues. Study findings will inform development of novel harm reduction approaches to reduce overdose mortality.

Trends in fentanyl and fentanyl analogue-related overdose deaths - Montgomery County, Ohio, 2015-2017.

INTRODUCTION: There is a lack of information on illicitly manufactured fentanyl and fentanyl analogue-related (IMF) unintentional overdose death trends over time. The study analyzes IMF-related unintentional overdose fatalities that occurred between July 2015 and June 2017 in Montgomery County, Ohio, an area with the highest rates of unintentional overdose mortality in Ohio. METHODS: LC-MS/MS-based method was used to identify fentanyl analogs and metabolites in 724 unintentional overdose death cases. The Chi-square statistic was used to assess differences over time in demographic and drug-related characteristics. RESULTS: The number of unintentional overdose death cases testing positive for IMFs increased by 377% between second half of 2015 and first half of 2017. The majority of decedents were white (82.5%) and male (67.8%). The proportion of fentanyl-only (no other analogs) cases declined from 89.2%-24.6% (p < 0.001), while proportion of fentanyl analogue-containing cases increased from 9.8%-70.3% (p < 0.001) between the second half of 2015 and first half of 2017. The most commonly identified fentanyl analogs were carfentanil (29.7%), furanyl fentanyl (14.1%) and acryl fentanyl (10.2%). Proportion of IMF cases also testing positive for heroin declined from 21.6% to 5.4% (p < 0.001), while methamphetamine positive cases increased from 1.4%-17.8% (p < 0.001) over the same time period. DISCUSSION: Emergence of fentanyl analogs contributed to substantial increases in unintentional overdose deaths. The data indicate a growing overlap between the IMF and methamphetamine outbreaks. Continuous monitoring of local IMF trends and rapid information dissemination to active users are needed to reduce the risks associated with IMF use.

LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogues and Metabolites in Whole Blood at Sub ng mL-1 Concentrations.

The United States and numerous other countries worldwide are currently experiencing a public health crisis due to the abuse of illicitly manufactured fentanyl (IMF) and its analogues. This manuscript describes the development of a liquid chromatography-tandem mass spectrometry-based method for the multiplex detection of N = 24 IMF analogues and metabolites in whole blood at concentrations as low as 0.1-0.5 ng mL-1. These available IMFs were fentanyl, norfentanyl, furanyl norfentanyl, remifentanil acid, butyryl norfentanyl, remifentanil, acetyl fentanyl, alfentanil, AH-7921, U-47700, acetyl fentanyl 4-methylphenethyl, acrylfentanyl, para-methoxyfentanyl, despropionyl fentanyl (4-ANPP), furanyl fentanyl, despropionyl para-fluorofentanyl, carfentanil, (±)-cis-3-methyl fentanyl, butyryl fentanyl, isobutyryl fentanyl, sufentanil, valeryl fentanyl, para-fluorobutyryl fentanyl, and para-fluoroisobutyryl fentanyl. Most IMF analogues (N = 22) could be easily distinguished from one another; the isomeric forms butyryl/isobutyryl fentanyl and para-fluorobutyryl/para-fluoroisobutyryl fentanyl could not be differentiated. N = 13 of these IMF analogues were quantified for illustrative purposes, and their forensic quality control standards were also validated for limit of detection (0.017-0.056 ng mL-1), limit of quantitation (0.100-0.500 ng mL-1), selectivity/sensitivity, ionization suppression/enhancement (87-118%), process efficiency (60-95%), recovery (64-97%), bias (<20%), and precision (>80%). This flexible, time- and cost-efficient method was successfully implemented at the Montgomery County Coroner's Office/Miami Valley Regional Crime Laboratory in Dayton, Ohio, where it aided in the analysis of N = 725 postmortem blood samples collected from February 2015 to November 2016.

Overdose Deaths Related to Fentanyl and Its Analogs - Ohio, January-February 2017.

Ohio is experiencing unprecedented loss of life caused by unintentional drug overdoses (1), with illicitly manufactured fentanyl (IMF) emerging as a significant threat to public health (2,3). IMF is structurally similar to pharmaceutical fentanyl, but is produced in clandestine laboratories and includes fentanyl analogs that display wide variability in potency (2); variations in chemical composition of these drugs make detection more difficult. During 2010-2015, unintentional drug overdose deaths in Ohio increased 98%, from 1,544 to 3,050.* In Montgomery County (county seat: Dayton), one of the epicenters of the opioid epidemic in the state, unintentional drug overdose deaths increased 40% in 1 year, from 249 in 2015 to 349 in 2016 (estimated unadjusted mortality rate = 57.7 per 100,000) (4). IMFs have not been part of routine toxicology testing at the coroner's offices and other types of medical and criminal justice settings across the country (2,3). Thus, data on IMF test results in the current outbreak have been limited. The Wright State University and the Montgomery County Coroner's Office/Miami Valley Regional Crime Laboratory (MCCO/MVRCL) collaborated on a National Institutes of Health study of fentanyl analogs and metabolites and other drugs identified in 281 unintentional overdose fatalities in 24 Ohio counties during January-February 2017. Approximately 90% of all decedents tested positive for fentanyl, 48% for acryl fentanyl, 31% for furanyl fentanyl, and 8% for carfentanil. Pharmaceutical opioids were identified in 23% of cases, and heroin in 6%, with higher proportions of heroin-related deaths in Appalachian counties. The majority of decedents tested positive for more than one type of fentanyl. Evidence suggests the growing role of IMFs, and the declining presence of heroin and pharmaceutical opioids in unintentional overdose fatalities, compared with 2014-2016 data from Ohio and other states (3-5). There is a need to include testing for IMFs as part of standard toxicology panels for biological specimens used in the medical, substance abuse treatment, and criminal justice settings.