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.

A Raman-Based Imaging Method for Characterizing the Molecular Adsorption and Spatial Distribution of Silver Nanoparticles on Hydrated Mineral Surfaces.

Although minerals are known to affect the environmental fate and transformation of heavy-metal ions, little is known about their interaction with the heavily exploited silver nanoparticles (AgNPs). Proposed here is a combination of hitherto under-utilized micro-Raman-based mapping and chemometric methods for imaging the distribution of AgNPs on various mineral surfaces and their molecular interaction mechanisms. The feasibility of the Raman-based imaging method was tested on two macro- and microsized mineral models, muscovite [KAl2(AlSi3O10)(OH)2] and corundum (α-Al2O3), under key environmental conditions (ionic strength and pH). Both AgNPs- and AgNPs+ were found to covalently attach to corundum (pHpzc = 9.1) through the formation of Ag-O-Al- bonds and thereby to potentially experience reduced environmental mobility. Because label-free Raman imaging showed no molecular interactions between AgNPs- and muscovite (pHpzc = 7.5), a label-enhanced Raman imaging approach was developed for mapping the scarce spatial distribution of AgNPs- on such mineral surfaces. Raman maps comprising of n = 625-961 spectra for each sample/control were rapidly analyzed in Vespucci, a free open-source software, and the results were confirmed via ICP-OES, AFM, and SEM-EDX. The proposed Raman-based imaging requires minimum to no sample preparation; is sensitive, noninvasive, cost-effective; and might be extended to other environmentally relevant systems.

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.

Analytical-Based Methodologies for Examining the In Vitro Absorption, Distribution, Metabolism, and Elimination (ADME) of Silver Nanoparticles.

The clinical applications of silver nanoparticles (AgNPs) remain limited due to the lack of well-established methodologies for studying their nanokinetics. Hereby, the primary goal is to adapt a suite of analytical-based methodologies for examining the in vitro absorption, distribution, metabolism, and elimination of AgNPs. Vero 76 and HEK 293 cells are exposed to ≈10-nm spherical AgNPs+ and AgNPs- at relevant concentrations (0-300 µg mL-1 ) and times (4-48 h). Absorption: Inductively coupled plasma optical emission spectroscopy (ICP-OES) demonstrates that the two AgNP formulations are not bioequivalent. For example, different bioavailabilities (Cmaximum < 20.7 ± 4% and 6.82 ± 0.4%), absorption times (Tmaximum > 48 and ≈24 h), and absorption rate laws (first- and zeroth-order at 300 µg mL-1 ) are determined in Vero 76 for AgNPs+ and AgNPs- , respectively. Distribution: Raman and CytoViva hyperspectral imaging show different cellular localizations for AgNPs+ and AgNPs- . Metabolism: Cloud point extraction (CPE)-tangential flow filtration (TFF) reveal that ≤ 11% ± 4% of the administered, sublethal AgNPs release Ag+ and contribute to the observed cytotoxicity. Elimination: ICP-OES-CPE suggests that AgNPs are cleared via exocytosis.

Freshwater Crayfish: A Potential Benthic-Zone Indicator of Nanosilver and Ionic Silver Pollution.

Nowadays, silver nanoparticles (AgNPs) are utilized in numerous applications, raising justified concerns about their release into the environment. This study demonstrates the potential to use freshwater crayfish as a benthic-zone indicator of nanosilver and ionic silver pollution. Crayfish were acclimated to 20 L aquaria filled with Hudson River water (HRW) and exposed for 14 days to widely used Creighton AgNPs and Ag(+) at doses of up to 360 µg L(-1) to surpass regulated water concentrations. The uptake and distribution of Ag in over 650 exoskeletons, gills, hepatopancreas and muscles samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) in conjunction with two complementary U.S. EPA-endorsed methods: the external calibration and the standard additions. Reflecting the environmental plasticity of the two investigated species, Orconectes virilis accumulated in a dose-dependent manner more Ag than Procambarus clarkii (on average 31% more Ag). Both species showed DNA damage and severe histological changes in the presence of Ag. However, Ag(+) generally led to higher Ag accumulations (28%) and was more toxic. By the harvest day, about 14 ± 9% of the 360 µg L(-1) of AgNP exposure in the HRW oxidized to Ag(+) and may have contributed to the observed toxicities and bioaccumulations. The hepatopancreas (1.5-17.4 µg of Ag g(-1) of tissue) was identified as the best tissue-indicator of AgNP pollution, while the gills (4.5-22.0 µg g(-1)) and hepatopancreas (2.5-16.7 µg g(-1)) complementarily monitored the presence of Ag(+).

Comparative study of hematological responses to platinum group metals, antimony and silver nanoparticles in animal models.

Research was conducted to examine the hematological effects of heavy metals (platinum (Pt ((IV))), palladium (Pd ((II))), rhodium (Rh ((III))), antimony (Sb ((III)) and Sb ((V))), and silver nanoparticles (AgNPs)) on white blood cells in mammalian (rat) and avian (chick embryo) models. These metals are used in many everyday products and are accumulating in our environment. Six-week old Sprague-Dawley female rats were treated daily by gavage and six-day old, fertile, specific pathogen-free white leghorn strain chick embryos' eggs were injected on days 7 and 14 of incubation with 0.0, 1.0, 5.0 or 10.0 ppm concentrations of Pt ((IV)) and a platinum group metal (PGM) mix of Pt ((IV)), Pd ((II)) and Rh ((III)). Chick embryos were also tested with 1.0 or 5.0 ppm of antimony compounds (Sb ((III)) and Sb ((V))) and 0.0, 15.0, 30.0, 60.0, or 100.0 ppm of silver nanoparticles (AgNPs). After 8 weeks of treatment, blood was obtained from the rats by jugular cut down and from chick embryos on day 20 of incubation by heart puncture. Blood smears were made and stained and a differential white cell count was performed on each. Examination of the smears revealed unconventional dose responses, stimulation of the immune response, and decreases in leukocyte production with various metals and concentrations. Chick embryos responded differently than rats to Pt and the PGM mix; suggesting that species differences and/or stage of development are important components of response to heavy metals. Route of administration of the metals might also influence the response. All of the heavy metals tested affected the immune responses of the tested animals as demonstrated by changes in the types and numbers of leukocytes. Our findings warrant further research to determine the mechanism of these effects and to understand and prevent toxicological effects in humans and other living organisms.

Evaluating the abnormal ossification in tibiotarsi of developing chick embryos exposed to 1.0ppm doses of platinum group metals by spectroscopic techniques.

Platinum group metals (PGMs), i.e., palladium (Pd), platinum (Pt) and rhodium (Rh), are found at pollutant levels in the environment and are known to accumulate in plant and animal tissues. However, little is known about PGM toxicity. Our previous studies showed that chick embryos exposed to PGM concentrations of 1mL of 5.0ppm (LD50) and higher exhibited severe skeletal deformities. This work hypothesized that 1.0ppm doses of PGMs will negatively impact the mineralization process in tibiotarsi. One milliliter of 1.0ppm of Pd(II), Pt(IV), Rh(III) aqueous salt solutions and a PGM-mixture were injected into the air sac on the 7th and 14th day of incubation. Control groups with no-injection and vehicle injections were included. On the 20th day, embryos were sacrificed to analyze the PGM effects on tibiotarsi using four spectroscopic techniques. 1) Micro-Raman imaging: Hyperspectral Raman data were collected on paraffin embedded cross-sections of tibiotarsi, and processed using in-house-written MATLAB codes. Micro-Raman univariate images that were created from the ν1(PO4(3-)) integrated areas revealed anomalous mineral inclusions within the bone marrow for the PGM-mixture treatment. The age of the mineral crystals (ν(CO3(2-))/ν1(PO4(3-))) was statistically lower for all treatments when compared to controls (p≤0.05). 2) FAAS: The percent calcium content of the chemically digested tibiotarsi in the Pd and Pt groups changed by ~45% with respect to the no-injection control (16.1±0.2%). 3) Micro-XRF imaging: Abnormal calcium and phosphorus inclusions were found within the inner longitudinal sections of tibiotarsi for the PGM-mixture treatment. A clear increase in the mineral content was observed for the outer sections of the Pd treatment. 4) ICP-OES: PGM concentrations in tibiotarsi were undetectable (<5ppb). The spectroscopic techniques gave corroborating results, confirmed the hypothesis, and explained the observed pathological (skeletal developmental abnormalities) and histological changes (tibiotarsus ischemia and nuclear fragmentation in chondrocytes).

Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

BACKGROUND/AIMS: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). METHODS: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 µM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. RESULTS: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. CONCLUSION: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

Tangential flow ultrafiltration: a "green" method for the size selection and concentration of colloidal silver nanoparticles.

Nowadays, AgNPs are extensively used in the manufacture of consumer products,(1) water disinfectants,(2) therapeutics,(1, 3) and biomedical devices(4) due to their powerful antimicrobial properties.(3-6) These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication(7) and size-based isolation(4,8) of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.(7-13) Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).(10,14-18) To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 µg ml(-1) down to 10 ml of 198.7 µg ml(-1)) than conventional methods of isolation such as ultracentrifugation.(19) TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.(20,21) Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available. The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 µg ml(-1)) were synthesized using the well-established Creighton method(22,23) by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm(2)) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm(2) and 20 cm(2)) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 µg ml(-1)) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.