Carl E. Wolf

Drinking water exposure to cadmium, an environmental contaminant, results in the exacerbation of autoimmune disease in the murine model.

Cadmium is a pervasive environmental contaminant. The primary route of exposure to the general population occurs via contaminated drinking water or food supplies. Our hypothesis was that cadmium could be a trigger for inducing autoimmune disease (AD) in genetically predisposed populations. Therefore, New Zealand Black/White F1 (NZBW) mice were exposed to cadmium via drinking water. Mice were exposed to: 0, 3, 30, 3000 or 10000 parts per billion (ppb) of cadmium in tap water for 2, 4, 28, or 31 weeks. After 4 weeks of exposure, in the group of mice exposed to 10000 ppb cadmium, there was an increased incidence of antinuclear antibodies (ANA). There was also deposition of immune complexes in all groups after 4 weeks of exposure. After 31 weeks, there were increases in IgG2a in mice exposed to low doses of cadmium. In an attempt to establish the progression from an autoimmune reaction to the development of AD, the biological marker for AD, proteinuria, was assessed. Onset of proteinuria was exacerbated by 11 weeks in mice exposed to cadmium. This data suggests that short-term exposure may result in a type of autoimmune reaction since the mice are beginning to produce ANA after only 4 weeks of exposure and there is immune-complex deposition in the kidney. Long-term exposure to cadmium appears to result in the exacerbation of AD as indicated by the development of proteinuria and continued presence of immune complexes in the kidney. The mechanism may involve the increased production of IgG2a, which is capable of forming immune complexes and causing autoimmune glomerulonephritis.

Postmortem tissue distribution of acetyl fentanyl, fentanyl and their respective nor-metabolites analyzed by ultrahigh performance liquid chromatography with tandem mass spectrometry

In the last two years, an epidemic of fatal narcotic overdose cases has occurred in the Tampa area of Florida. Fourteen of these deaths involved fentanyl and/or the new designer drug, acetyl fentanyl. Victim demographics, case histories, toxicology findings and causes and manners of death, as well as, disposition of fentanyl derivatives and their nor-metabolites in postmortem heart blood, peripheral blood, bile, brain, liver, urine and vitreous humor are presented. In the cases involving only acetyl fentanyl (without fentanyl, n=4), the average peripheral blood acetyl fentanyl concentration was 0.467 mg/L (range 0.31 to 0.60 mg/L) and average acetyl norfentanyl concentration was 0.053 mg/L (range 0.002 to 0.086 mg/L). In the cases involving fentanyl (without acetyl fentanyl, n=7), the average peripheral blood fentanyl concentration was 0.012 mg/L (range 0.004 to 0.027 mg/L) and average norfentanyl blood concentration was 0.001 mg/L (range 0.0002 to 0.003 mg/L). In the cases involving both acetyl fentanyl and fentanyl (n=3), the average peripheral blood acetyl fentanyl concentration was 0.008 mg/L (range 0.006 to 0.012 mg/L), the average peripheral blood acetyl norfentanyl concentration was 0.001 mg/L (range 0.001 to 0.002 mg/L), the average peripheral blood fentanyl concentration was 0.018 mg/L (range 0.015 to 0.021mg/L) and the average peripheral blood norfentanyl concentration was 0.002 mg/L (range 0.001 mg/L to 0.003 mg/L). Based on the toxicology results, it is evident that when fentanyl and/or acetyl fentanyl were present, they contributed to the cause of death. A novel ultrahigh performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) method to identify and quantify acetyl fentanyl, acetyl norfentanyl, fentanyl and norfentanyl in postmortem fluids and tissues is also presented.

Concentration of Nicotine and Glycols in 27 Electronic Cigarette Formulations

Personal battery-powered vaporizers or electronic cigarettes were developed to deliver a nicotine vapor such that smokers could simulate smoking tobacco without the inherent pathology of inhaled tobacco smoke. Electronic cigarettes and their e-cigarette liquid formulations are virtually unregulated. These formulations are typically composed of propylene glycol and/or glycerin, flavoring components and an active drug, such as nicotine. Twenty-seven e-cigarette liquid formulations that contain nicotine between 6 and 22 mg/L were acquired within the USA and analyzed by various methods to determine their contents. They were screened by Direct Analysis in Real Time™ Mass Spectrometry (DART-MS). Nicotine was confirmed and quantitated by high-performance liquid chromatography-tandem mass spectrometry, and the glycol composition was confirmed and quantitated by gas chromatography-mass spectrometry. The DART-MS screening method was able to consistently identify the exact mass peaks resulting from the protonated molecular ion of nicotine, glycol and a number of flavor additives within 5 mmu. Nicotine concentrations were determined to range from 45 to 131% of the stated label concentration, with 18 of the 27 have >10% variance. Glycol composition was generally accurate to the product description, with only one exception where the propylene glycol to glycerin percentage ratio was stated as 50:50 and the determined concentration of propylene glycol to glycerin was 81:19 (% v/v). No unlabeled glycols were detected in these formulations.

Analysis of Moonshine for Contaminants

Objectives: In the past, some moonshine products contained potentially toxic contaminants. Although moonshine production continues in the United States, no studies have analyzed the content of moonshine since the early 1960s. We hypothesize that moonshine continues to contain potentially toxic concentrations of contaminants. Methods: Forty‐eight samples of illicitly distilled moonshine were obtained from law enforcement agencies. An independent laboratory, blinded to both the moonshine source and a control sample of ethanol, conducted the analysis. Lead content was determined using atomic absorption spectrophotometry with a graphite tube atomizer. Alcohol content, including ethanol, acetone, isopropanol, methanol, and ethylene glycol, was determined using gas liquid chromatography with flame ionization detection. Results: Ethanol content ranged from 10.5% to 66.0% with a mean value of 41.2%. Lead was found in measurable quantities in 43 of 48 samples with values ranging from 5 to 599 parts per billion (ppb) with a mean value of 80.7 ppb. A total of 29 of 48 (60%) of samples contained lead concentrations above or equal to the EPA water guideline of 15 ppb. Methanol was found in only one sample at a concentration of 0.11%. No samples contained detectable concentrations of acetone, isopropanol, or ethylene glycol. Conclusions: Many moonshine samples contain detectable concentrations of lead. Extrapolations based on the described moonshine lead content suggest that chronic consumers of moonshine may develop elevated lead concentrations. Physicians should consider lead toxicity in the differential diagnosis when evaluating patients consuming moonshine.

Detection and quantification of tricyclic antidepressants and other psychoactive drugs in urine by HPLC/MS/MS for pain management compliance testing.

A sensitive, specific, and rapid high‐pressure liquid chromatography/mass spectrometry/mass spectrometry method was developed for the quantitation of 11 tricyclic antidepressants and/or their metabolites; fluoxetine and norfluoxetine; cyclobenzaprine; and trazodone in urine. Samples were alkalinized with 0.2 N NaOH and extracted into 2 ml of hexane: ethyl acetate (1:1), evaporated to dryness, and reconstituted with 100 μl of 20 mM ammonium formate: methanol (20:80). The chromatographic separation was performed using an Allure Biphenyl 100 × 3.2 mm, 5‐μ column with a mobile phase consisting of 20 mM ammonium formate: methanol (20:80 v/v) at a flow rate of 0.5 ml/min. The detection was accomplished by multiple‐reaction monitoring via electrospray ionization source operating in the positive ionization mode. The calibration curve was linear over the investigated concentration range, 25–2,000 ng/ml, for each analyte using 1.0 ml of urine. The lower limit of quantitation for each analyte was 25 ng/ml. The intra‐ and inter‐day precisions had coefficient of variation less than 15% and the accuracy was within the range from 88% to 109%. The method proved adequate for the tricyclic antidepressants analysis of urine for emergency clinical toxicology and pain management compliance testing. J. Clin. Lab. Anal. 26:286‐294, 2012.

Postmortem Toxicology Findings of Acetyl Fentanyl, Fentanyl, and Morphine in Heroin Fatalities in Tampa, Florida

In the last two years, an epidemic of 40 fatal heroin overdose cases has occurred in the Tampa area of Florida. Of these cases, 14 involved fentanyl and acetyl fentanyl. Victim demographics, case histories, toxicology findings, and causes and manners of death for all 40 deaths are presented. In 26 deaths in which acetyl fentanyl or fentanyl were not involved, free and total peripheral blood morphine concentrations were consistent with fatal heroin intoxications, averaging 0.16 mg/L and 0.35 mg/L, respectively. In the heroin cases with fentanyl present (n=7), the average free morphine concentration was 0.040 mg/L, the average total morphine concentration was 0.080 mg/L, and the average fentanyl concentration was 0.012 mg/L. In the cases with heroin, fentanyl, and acetyl fentanyl (n=3), the average free morphine concentration was 0.010 mg/L, the average total morphine concentration was 0.030 mg/L, the average fentanyl concentration was 0.018 mg/L, and the average acetyl fentanyl concentration was 0.008 mg/L. In the cases involving only acetyl fentanyl (without heroin or fentanyl, n=4), the average acetyl fentanyl concentration was 0.47 mg/L and the average acetyl norfentanyl concentration was 0.053 mg/L. The presented cases, with associated drug concentrations, case histories, demographics, and causes and manners of death may help provide assistance with the interpretation of the postmortem findings. Based on case circumstances, autopsy results, and toxicology results, it is evident that fentanyl and/or acetyl fentanyl, when present, contributed to the cause of death.

Analysis of the First‐ and Second‐Generation Raving Dragon Novelty Bath Salts Containing Methylone and Pentedrone

In recent years, a large number of designer drugs sold as “Bath Salts” have appeared on the market. In July of 2011, Raving Dragon Novelty Bath Salts was obtained over the Internet. This product became unavailable in October of that year coinciding with the DEA issuing a temporarily schedule of mephedrone, methylone, and MDPV. Four months later in February of 2012, a new product was released from the same company under the new name Raving Dragon Voodoo Dust. The contents of both products were identified using spectroscopy methods: nuclear magnetic resonance, infrared, UV–visible, tandem mass spectrometry, and high‐resolution time‐of‐flight mass spectrometry. It was determined that Raving Dragon Novelty Bath Salts contained methylone. The replacement product Raving Dragon Voodoo Dust contained the unscheduled drug pentedrone. The Raving Dragon brand of products illustrates the rapid change of ingredients in these products to circumvent laws restricting availability, distribution, and use.

Evaluation of Two Commercially Available Cannabidiol Formulations for Use in Electronic Cigarettes

Since 24 states and the District of Columbia have legalized marijuana in some form, suppliers of legal marijuana have developed Cannabis sativa products for use in electronic cigarettes (e-cigarettes). Personal battery powered vaporizers, or e-cigarettes, were developed to deliver a nicotine vapor such that smokers could simulate smoking tobacco without the inherent pathology of inhaled tobacco smoke. The liquid formulations used in these devices are comprised of an active ingredient such as nicotine mixed with vegetable glycerin (VG) and/or propylene glycol (PG) and flavorings. A significant active ingredient of C. sativa, cannabidiol (CBD), has been purported to have anti-convulsant, anti-nociceptive, and anti-psychotic properties. These properties have potential medical therapies such as intervention of addictive behaviors, treatments for epilepsy, management of pain for cancer patients, and treatments for schizophrenia. However, CBD extracted from C. sativa remains a DEA Schedule I drug since it has not been approved by the FDA for medical purposes. Two commercially available e-cigarette liquid formulations reported to contain 3.3 mg/mL of CBD as the active ingredient were evaluated. These products are not regulated by the FDA in manufacturing or in labeling of the products and were found to contain 6.5 and 7.6 mg/mL of CBD in VG and PG with a variety of flavoring agents. Presently, while labeled as to content, the quality control of manufacturers and the relative safety of these products is uncertain.

Identification of MDMB-FUBINACA in commercially available e-liquid formulations sold for use in electronic cigarettes

MDMB-FUBINACA (aka MDMB(N)-Bz-F), chemical name Methyl (S)-2-(1-(4-fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate, a designer drug or a new psychoactive substance (NPS), was identified in three commercially available e-liquids formulated for electronic cigarette use. The e-liquids were evaluated using direct analysis in real time ion source attached to a time of flight mass spectrometer (DART-MS) and gas chromatograph mass spectrometer (GC-MS) to identify active ingredients/drugs, flavorants, and other possible constituents. The e-liquids were also evaluated for alcohol content by headspace gas chromatography with flame ionization detector (HS-GC-FID). The aerosol produced from the e-liquids by use of an e-cigarette was analyzed by solid phase micro-extraction gas chromatography mass spectrometry (SPME-GC-MS) to ensure delivery of the active ingredient/drug. Propylene glycol, vegetable glycerin, MDMB-FUBINACA, alcohol content and a flavor profile were determined for each of the e-liquids. MDMB-FUBINACA was determined to be the major active ingredient in all three e-liquids and was successfully detected by SPME-GC-MS in the aerosol generated by a KangerTech Aerotank clearomizer/electronic cigarette.

Evaluation of Two Enzyme Immunoassays for the Detection of the Cocaine Metabolite Benzoylecgonine in 1,398 Urine Specimens

Benzoylecgonine (BE) is the primary urinary metabolite of cocaine. Two enzyme immunoassays were evaluated for the detection of BEin urine with a 300 ng/ml cutoff: the DRI® Cocaine Metabolite Assay and Lin‐Zhi Internationals (LZ) Cocaine Metabolite Enzyme Immunoassay.