Evaluation of aqueous dimethyl trisulfide as an antidote to a highly lethal cyanide poisoning in a large swine model.

BACKGROUND: Cyanide is a rapid acting, lethal, metabolic poison and remains a significant threat. Current FDA-approved antidotes are not amenable or efficient enough for a mass casualty incident. OBJECTIVE: The objective of this study is to evaluate short and long-term efficacy of intramuscular aqueous dimethyl trisulfide (DMTS) on survival and clinical outcomes in a swine model of cyanide exposure. METHODS: Anesthetized swine were instrumented and acclimated until breathing spontaneously. Potassium cyanide infusion was initiated and continued until 5 min after the onset of apnea. Subsequently, animals were treated with intramuscular DMTS (n = 11) or saline control (n = 10). Laboratory values and DMTS blood concentrations were assessed at various time points and physiological parameters were monitored continuously until the end of the experiment unless death occurred. A subset of animals treated with DMTS (n = 5) were survived for 7 days to evaluate muscle integrity by repeat biopsy and neurobehavioral outcomes. RESULTS: Physiological parameters and time to apnea were similar in both groups at baseline and at time of treatment. Survival in the DMTS-treated group was 90% and 30% in saline controls (p = 0.0034). DMTS-treated animals returned to breathing at 12.0 ± 10.4 min (mean ± SD) compared to 22.9 ± 7.0 min (mean ± SD) in the 3 surviving controls. Blood collected prior to euthanasia showed improved blood lactate concentrations in the DMTS treatment group; 5.47 ± 2.65 mmol/L vs. 9.39 ± 4.51 mmol/L (mean ± SD) in controls (p = 0.0310). Low concentrations of DMTS were detected in the blood, gradually increasing over time with no elimination phase observed. There was no mortality, histological evidence of muscle trauma, or observed adverse neurobehavioral outcomes, in DMTS-treated animals survived to 7 days. CONCLUSION: Intramuscular administration of aqueous DMTS improves survival following cyanide poisoning with no observed long-term effects on muscle integrity at the injection site or adverse neurobehavioral outcomes.

Analysis of the Soil Fumigant, Dimethyl Disulfide, in Swine Blood by Dynamic Headspace Gas Chromatography-Mass Spectroscopy.

Plant parasites and soilborne pathogens directly reduce the overall yield of crops, vegetables, and fruits, negatively impacting the market demand for these products and their net profitability. While preplant soil fumigation helps maintain the consistent production quality of high-value cash crops, most soil fumigants are toxic to off-target species, including humans. Dimethyl disulfide (DMDS) has recently been introduced as a relatively low toxicity soil fumigant. Although DMDS exhibits low toxicity compared to other soil fumigants, it is volatile and exposure can cause eye, nasal, and upper respiratory tract irritation, skin irritation, nausea, dizziness, headache, and fatigue. While there is one analysis method available for DMDS from biological matrices, it has significant disadvantages. Hence, in this study, a dynamic headspace gas chromatography-mass spectroscopy (DHS-GC-MS) method was developed for the analysis of DMDS in swine whole blood. This method is highly sensitive and requires only three steps: 1) acid denaturation, 2) addition of internal standard, and 3) DHS-GC-MS analysis. The method produced a wide linear range from 0.1 - 200 µM with an excellent limit of detection of 30 nM. Intra- and interassay accuracy (100±14% and 100±11%, respectively) and precision (<5% and <6% relative standard deviation, respectively) were also excellent. The method worked well to quantify the DMDS levels in the blood of dimethyl trisulfide (DMTS)-treated swine (i.e., DMDS is a byproduct of DMTS treatment) with no interfering substances at or around the retention time of DMDS (i.e., 2.7 min). This simple, rapid, and extremely sensitive method can be used for the quantification of DMDS levels in blood to verify exposure to DMDS or to monitor levels of DMDS following DMTS treatment (e.g., for cyanide poisoning).

Stability and Hydrolysis of Desomorphine-Glucuronide.

Desomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five ß-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient ß-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using ß-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested.

Quantitative analysis of desomorphine in blood and urine using solid phase extraction and gas chromatography-mass spectrometry.

Desomorphine, a semi-synthetic opioid, is a component of the street drug Krokodil. Despite continued reports of Krokodil use, confirmation via toxicological testing remains scarce. The lack of confirmed desomorphine reports may be in part due to the limited published analytical methodology capable of detecting desomorphine at forensically relevant concentrations. In an effort to assist with identification efforts, a robust analytical method was developed and validated. Solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) were used to determine desomorphine in blood and urine using a deuterated analog as the internal standard. Data was acquired using selected ion monitoring (SIM) mode. Extraction efficiencies in blood and urine were 69% and 90%, respectively. The limits of quantitation in blood and urine were 5 ng/mL and 8 ng/mL, ten-fold lower than previously published methods. Intra- and inter-assay CVs were 2-4% (n = 3) and 3-7% (n = 15), respectively. The method was fully validated in accordance with published guidelines for forensic use. Furthermore, it provides a means by which desomorphine can be identified in toxicology specimens at forensically relevant concentrations, without the need for derivatization.

Analysis of Desomorphine in Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Desomorphine is a primary component of the drug Krokodil. While reports of Krokodil use continue to appear in the literature, analytically confirmed cases remain quite scarce. This might be attributed to trends in geographical use, and limited published analytical methodology to detect its use. A sensitive analytical method to detect desomorphine was developed and validated to assist with identification efforts. Solid phase extraction and liquid chromatography-tandem mass spectrometry were used to quantitatively identify desomorphine in urine. An isotopically labeled analog was used as the internal standard. Assay performance was evaluated in accordance with published guidelines. The extraction efficiency for desomorphine in urine was 90%, and limits of detection and quantitation were 0.5 ng/mL. The calibration range of the assay was 0.5-500 ng/mL. Bias ranged from -1% to 2% (n = 15), and the intra- and inter-assay CVs were 2-3% (n = 3) and 32-6% (n = 15), respectively. Ion suppression was -20% and -10% at low and high concentrations, respectively. Interferences were assessed using common drugs, including 24 opioids and structurally related compounds. Using this approach, the quantitative analysis of desomorphine in urine is described at forensically relevant concentrations.

In vitro metabolism of desomorphine.

Desomorphine is reported to be the principal pharmacologically active opioid in Krokodil, a homemade injectable drug that is perceived to be a cheaper alternative to heroin. There have been limited studies regarding its pharmacology or detection in biological matrices. The goal of this study was to contribute further knowledge regarding its metabolism. Recombinant human cytochrome P450 enzymes (rCYPs) and recombinant uridine 5'-diphospho-glucuronosyltransferases (rUGTs) were used to investigate the biotransformational pathways involved. Samples were analyzed by liquid chromatography/quadrupole-time of flight-mass spectrometry (LC-Q/TOF-MS). Seven rCYP (rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6 and rCYP3A4) enzymes were found to contribute to desomorphine metabolism and eight phase I metabolites were identified, including nordesomorphine, desomorphine-N-oxide, norhydroxydesomorphine, and five hydroxylated species. Inhibition assays were used to confirm individual rCYP isoenzyme activity. Nine rUGTs (rUGT1A1, rUGT1A3, rUGT1A8, rUGT1A9, rUGT1A10, rUGT2B4, rUGT2B7, rUGT2B15, and rUGT2B17) were found to contribute to the formation of desomorphine-glucuronide.

Desomorphine Screening Using Commercial Enzyme-Linked Immunosorbent Assays.

Desomorphine ("Krokodil") is a semi-synthetic opioid that has drawn attention as a recreational drug, particularly in Russia, neighboring former Soviet Republics, Eastern and Central Europe. It has no accepted medicinal uses and is currently a schedule I drug in the United States. In clandestine environments, desomorphine is synthesized from codeine using red phosphorous, hydroiodic acid and gasoline. Residual starting materials in illicit preparations have been associated with severe dermatological effects and extensive tissue necrosis. Desomorphine is not well studied, and there are limited reports concerning its pharmacology or detection in biological matrices. Immunoassays are widely relied upon for both antemortem and postmortem toxicology screening. Although desomorphine is an opioid of the phenanthrene-type, its ability to bind to conventional opioid antibodies has not been described. In this report we describe the cross-reactivity of desomorphine using six commercially available enzyme-linked immunosorbent assays (Immunalysis Opiates Direct ELISA, Immunalysis Oxycodone/Oxymorphone Direct ELISA, Randox Opiate ELISA, OraSure Technologies OTI Opiate Micro-plate EIA, Neogen Opiate Group ELISA and Neogen Oxycodone/Oxymorphone ELISA). Cross-reactivites were highly variable between assays, ranging from 77 to <2.5%. In general, assays directed towards morphine produced greater cross-reactivity with desomorphine than those directed towards oxycodone. The Immunalysis Opiates Direct ELISA produced the greatest cross-reactivity, although several of the assays evaluated produced cross-reactivity of a sufficient magnitude to be effective for desomorphine screening.