Verification of chlorine exposure via LC-MS/MS analysis of base hydrolyzed chlorophenols from chlorotyrosine-protein adducts.

Inhalation of chlorine gas, with subsequent hydrolysis in the airway and lungs to form hydrochloric acid (HCl) and hypochlorous acid (HOCl), can cause pulmonary edema (i.e., fluid build-up in the lungs), pulmonary inflammation (with or without infection), respiratory failure, and death. The HOCl produced from chlorine is known to react with tyrosine to form adducts via electrophilic aromatic substitution, resulting in 3-chlorotyrosine and 3,5-dichlorotyrosine adducts. While several analysis methods are available for determining these adducts, each method has significant disadvantages. Hence, a simple and sensitive ultra-high performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) method was developed for the determination of chlorotyrosine adducts. The sample preparation involves base hydrolysis of isolated plasma proteins to form 2-chlorophenol (CP) from monochlorotyrosine adducts and 2,6-dichlorophenol (2,6-DCP), from dichlorotyrosine adducts, as markers of chlorine exposure. The chlorophenols are extracted with cyclohexane prior to UHPLC-MS/MS analysis. The method produced excellent sensitivity for 2,6-DCP with a limit of detection of 2.2 µg/kg, calibration curve linearity extending from 0.054-54 mg/kg (R2 ≥ 0.9997 and %RA > 94), and accuracy and precision of 100 ± 14 %, and <15 % relative standard deviation, respectively. The sensitivity of the method for 2-CP was relatively poor, so it was used only as a secondary marker for severe chlorine exposure. The method successfully detected elevated levels of 2,6-DCP from hypochlorite-spiked plasma protein and plasma protein isolated from chlorine-exposed rats.

Mesna Improves Outcomes of Sulfur Mustard Inhalation Toxicity in an Acute Rat Model.

Inhalation of high levels of sulfur mustard (SM), a potent vesicating and alkylating agent used in chemical warfare, results in acutely lethal pulmonary damage. Sodium 2-mercaptoethane sulfonate (mesna) is an organosulfur compound that is currently Food and Drug Administration (FDA)-approved for decreasing the toxicity of mustard-derived chemotherapeutic alkylating agents like ifosfamide and cyclophosphamide. The nucleophilic thiol of mesna is a suitable reactant for the neutralization of the electrophilic group of toxic mustard intermediates. In a rat model of SM inhalation, treatment with mesna (three doses: 300 mg/kg intraperitoneally 20 minutes, 4 hours, and 8 hours postexposure) afforded 74% survival at 48 hours, compared with 0% survival at less than 17 hours in the untreated and vehicle-treated control groups. Protection from cardiopulmonary failure by mesna was demonstrated by improved peripheral oxygen saturation and increased heart rate through 48 hours. Additionally, mesna normalized arterial pH and pACO2 Airway fibrin cast formation was decreased by more than 66% in the mesna-treated group at 9 hour after exposure compared with the vehicle group. Finally, analysis of mixtures of a mustard agent and mesna by a 5,5'-dithiobis(2-nitrobenzoic acid) assay and high performance liquid chromatography tandem mass spectrometry demonstrate a direct reaction between the compounds. This study provides evidence that mesna is an efficacious, inexpensive, FDA-approved candidate antidote for SM exposure. SIGNIFICANCE STATEMENT: Despite the use of sulfur mustard (SM) as a chemical weapon for over 100 years, an ideal drug candidate for treatment after real-world exposure situations has not yet been identified. Utilizing a uniformly lethal animal model, the results of the present study demonstrate that sodium 2-mercaptoethane sulfonate is a promising candidate for repurposing as an antidote, decreasing airway obstruction and improving pulmonary gas exchange, tissue oxygen delivery, and survival following high level SM inhalation exposure, and warrants further consideration.

Methimazole, an Effective Neutralizing Agent of the Sulfur Mustard Derivative 2-Chloroethyl Ethyl Sulfide.

Sulfur mustard (SM), designated by the military as HD, is a highly toxic and dangerous vesicant that has been utilized as a chemical warfare agent since World War I. Despite SM's extensive history, an effective antidote does not exist. The effects of SM are predominantly based on its ability to alkylate important biomolecules. Also, with the potential for a fraction of SM to remain unreacted up to days after initial contact, a window of opportunity exists for direct neutralization of unreacted SM over the days following exposure. In this study, we evaluated the structure-activity relationship of multiple nucleophilic molecules to neutralize the toxic effects of 2-chloroethyl ethyl sulfide (CEES), a monofunctional analogue of SM, on human keratinocyte (HaCaT) cells. Cell viability, relative loss of extracellular matrix adhesions, and apoptosis caused by CEES were measured via MTT, cell-matrix adhesion (CMA), and apoptosis protein marker assays, respectively. A set of five two-carbon compounds with various functional groups served as a preliminary group of first-generation neutralizing agents to survey the correlation between mitigation of CEES's toxic effects and functional group nucleophilicity. Apart from thioacids, which produced additive toxicity, we generally observed the trend of increasing protection from cytotoxicity with increasing nucleophilicity. We extended this treatment strategy to second-generation agents which contained advantageous structural features identified from the first-generation molecules. Our results show that methimazole (MIZ), a currently FDA-approved drug used to treat hyperthyroidism, effectively reduced cytotoxicity, increased CMA, and decreased apoptosis resulting from CEES toxicity. MIZ selectively reacts with CEES to produce 2-(2-(ethylthio)ethylthio)-1-methyl-1H-imidazole (EEMI) in media and cell lysate treatments resulting in the reduction of toxicity. Based on these results, future development of MIZ as an SM therapeutic may provide a viable approach to reduce both the immediate and long-term toxicity of SM and may also help mitigate slower developing SM toxicity due to residual intact SM.

Concurrent determination of cyanide and thiocyanate in human and swine antemortem and postmortem blood by high-performance liquid chromatography-tandem mass spectrometry.

Cyanide (in the form of cyanide anion (CN-) or hydrogen cyanide (HCN), inclusively represented as CN) can be a rapidly acting and deadly poison, but it is also a common chemical component of a variety of natural and anthropogenic substances. The main mechanism of acute CN toxicity is based on blocking terminal electron transfer by inhibiting cytochrome c oxidase, resulting in cellular hypoxia, cytotoxic anoxia, and potential death. Due to the well-established link between blood CN concentrations and the manifestation of symptoms, the determination of blood concentration of CN, along with the major metabolite, thiocyanate (SCN-), is critical. Because currently there is no method of analysis available for the simultaneous detection of CN and SCN- from blood, a sensitive method for the simultaneous analysis of CN and SCN- from human ante- and postmortem blood via liquid chromatography-tandem MS analysis was developed. For this method, sample preparation for CN involved active microdiffusion with subsequent chemical modification using naphthalene-2,3-dicarboxaldehyde (NDA) and taurine (i.e., the capture solution). Preparation for SCN- was accomplished via protein precipitation and monobromobimane (MBB) modification. The method produced good sensitivity for CN with antemortem limit of detection (LODs) of 219 nM and 605 nM for CN and SCN-, respectively, and postmortem LODs of 352 nM and 509 nM. The dynamic ranges of the method were 5-500 µM and 10-500 µM in ante- and postmortem blood, respectively. In addition, the method produced good accuracy (100 ± 15%) and precision (≤ 15.2% relative standard deviation). The method was able to detect elevated levels of CN and SCN- in both antemortem (N = 5) and postmortem (N = 4) blood samples from CN-exposed swine compared to nonexposed swine.

Identification and determination of phenyl methyl carbamate released from adducted hemoglobin for methyl isocyanate exposure verification.

Methyl isocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, is a toxic industrial chemical that causes irritation and damage to the eyes, respiratory tract, and skin. Due to the high reactivity of MIC, it binds to proteins to form protein adducts. While these adducts can be used as biomarkers to verify exposure to MIC, methods to detect MIC adducts are cumbersome, typically involving enzymatic (pronase) or strong acid (Edman degradation) hydrolysis of hemoglobin. Hence, in this study, a simple method was developed which utilizes base hydrolysis of MIC-tyrosine adducts from isolated hemoglobin to form phenyl methyl carbamate (PMC), followed by rapid liquid-liquid extraction, and liquid chromatography tandem mass spectrometry analysis. The hydrolysis chemistry is the first report of base hydrolysis of a tyrosine-ß-C-hydroxo phenol bond in aqueous solution. The method produced excellent sensitivity (detection limit of 0.02 mg/kg), linearity (R2 = 0.998, percent residual accuracies > 96), and dynamic range (0.06‒15 mg/kg). The accuracy and precision (100 ± 9% and < 10% relative standard deviation, respectively) of the method were outstanding compared to existing techniques. The validated method was able to detect significantly elevated levels of PMC from hemoglobin isolated from MIC-exposed rats.

The Prevalence of Opioids in US Drinking Water Sources Detected Using Direct-Injection High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Active pharmaceutical ingredient (API) contamination of water sources, including opioid contamination, has become more common in recent years. Although drinking water-treatment plants help mitigate API infiltration, API contamination remains in some drinking water sources. Therefore, the ability to detect APIs at ultratrace concentrations is vital to ensure safe drinking water. A method for the ultratrace determination of fentanyl, hydrocodone, and codeine in drinking water via direct injection and high-performance liquid-chromatography tandem mass spectrometry (HPLC-MS/MS) was developed and validated. Drinking water samples (10 ml) are simply syringe-filtered and then analyzed by HPLC-MS/MS. A wide linear range (0.25-100 ng/L) and ultratrace limits of detection (80, 150, and 500 pg/L for fentanyl, hydrocodone, and codeine, respectively) were features of the method. The method produced excellent aggregate accuracies of 90%-115% and precisions of ≤11% for the three analytes tested. This method was used to test drinking water samples from 53 US locations, with hydrocodone and codeine detected in approximately 40% of the samples tested at concentrations between 0.3 and 20 ng/L. Codeine was detected at higher concentrations than hydrocodone (up to 7.3 times) for each sample containing these APIs. Fentanyl was not detected in any field drinking water sample. The detection of opioids in a large fraction of the US drinking water samples tested is cause for concern, and these levels should continue to be monitored to ensure that they do not become a threat to human health. Environ Toxicol Chem 2022;41:2658-2666. © 2022 SETAC.

Analysis of bisaminotetrazole cobinamide, a next-generation antidote for cyanide, hydrogen sulfide and methanethiol poisoning, in swine plasma by liquid chromatography-tandem mass spectrometry.

Cyanide, hydrogen sulfide, and methanethiol are common toxic inhalation agents that inhibit mitochondrial cytochrome c oxidase and result in cellular hypoxia, cytotoxic anoxia, apnea, respiratory failure, cardiovascular collapse, seizure and potentially death. While all are occupational gas exposure hazards that have the potential to cause mass casualties from industrial accidents or acts of terrorism, only cyanide has approved antidotes, and each of these has major limitations, including difficult administration in mass-casualty settings. While bisaminotetrazole cobinamide (Cbi(AT)2) has recently gained attention because of its efficacy in treating these metabolic poisons, there is no method available for the analysis of Cbi(AT)2 in any biological matrix. Hence, in this study, a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the analysis of Cbi(AT)2 in swine plasma. The method is extremely simple, consisting of protein precipitation, separation and drying of the supernatant, reconstitution in an aqueous solvent, and LC-MS/MS analysis. The method produced an LOD of 0.3 µM with a wide dynamic range (2 - 500 µM). Inter- and intraassay accuracies (100 ± 12 % and 100 ± 19 %, respectively) were acceptable and the precision (<12 % and < 9 % relative standard deviation, respectively) was good. The developed method was used to analyze Cbi(AT)2 from treated swine and the preliminary pharmacokinetic parameters showed impressive antidotal behavior, most notably a long estimated elimination half-life (t1/2 = 37.5 h). This simple and rapid method can be used to facilitate the development of Cbi(AT)2 as a therapeutic against toxic cyanide, hydrogen sulfide and methanethiol exposure.

Analysis of sodium 2-mercaptoethane sulfonate in rat plasma using high performance liquid chromatography tandem-mass spectrometry.

Sodium 2-mercaptoethane sulfonate (MESNA) is a thiol-containing compound that has proven to be effective in inactivating acrolein, the toxic metabolite of some anti-cancer drugs (e.g., cyclophosphamide and ifosphamide). Also, it scavenges free radicals which cause numerous disorders by attacking biological molecules. Current methods available to analyze MESNA in biological matrices include colorimetry and high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection. These methods have several limitations including low sensitivity, poor selectivity, a high degree of difficulty, and long analysis times. Hence, a rapid, simple, and sensitive HPLC tandem mass spectrometry (MS/MS) method was developed and validated to quantify MESNA in rat plasma following IP administration. The analysis of MESNA was accomplished via plasma protein precipitation, centrifugation, supernatant evaporation, reconstitution, and HPLC-MS/MS analysis. The method showcases an outstanding limit of detection (20 nM), excellent linearity (R2 = 0.999, and percent residual accuracy >90%) and a wide linear range (0.05-200 µM). The method also produced good accuracy and precision (100 ± 10% and <10% relative standard deviation, respectively). The validated method was successfully used to analyze MESNA from treated animals and will allow easier development of MESNA for therapeutic purposes.

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.

Ultratrace analysis of per- and polyfluoroalkyl substances in drinking water using ice concentration linked with extractive stirrer and high performance liquid chromatography - tandem mass spectrometry.

Detection of drinking water contaminants is vital to the protection of human health. One group of contaminants that have recently generated serious concerns over health risks are per- and polyfluoroalkyl substances (PFAS). These compounds are very bio-persistent, leading to their detection in all types of water sources, including drinking water. While analysis of drinking water for PFAS is important, it is currently arduous to detect ultratrace levels of these contaminants. Specifically, current ultratrace PFAS analysis methods are difficult, costly, require large sample volumes, and consume relatively large volumes of organic solvent. In the present work, an analytical method using Ice Concentration Linked with Extractive Stirrer (ICECLES) and high performance liquid chromatography-tandem quadrupole mass spectrometry (HPLC-MS/MS), was developed and validated to provide simple and ultratrace analysis of drinking water for 14 PFAS. The method featured a relatively low sample volume requirement (10 mL), automated extraction, minimal matrix effects, and minimal organic solvent use (i.e., the method requires only 50 µL of methanol per sample). The method produced a wide linear range of 0.5 to 500 ng/L, ultratrace limits of detection (0.05 to 0.3 ng/L), and good accuracy and precision (i.e., 87 to 108% accuracy and ≤19% relative standard deviation as a measure of precision). This method was tested on drinking water samples from across the United States and detected at least one PFAS compound in 52 of the 53 drinking water samples tested. Perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), and perfluoroheptanoic acid (PFHpA) were detected in 89, 96, and 77% of the samples tested with maximum concentrations of 268 ng/L for PFHxA, 213 ng/L for PFOA, and 75.7 ng/L for PFHpA. Additionally, perfluorononanoic acid, perfluorodecanoic acid, and perfluoroheptanoic acid were each detected in at least one drinking water sample at concentrations > 20 ng/L. The availability of the method presented here allows ultratrace detection of PFAS while circumventing many of the disadvantages of current methods.

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).

Ultratrace analysis of atrazine in soil using Ice Concentration Linked with Extractive Stirrer and High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Atrazine is a widely-used pesticide with a relatively long half-life in the environment. This leads to persistent soil contamination with the potential of migration to ground and surface waters. Analysis of atrazine in soil is difficult due to the inherent complexity of soil as a sample matrix. Moreover, the moderate hydrophobicity of atrazine makes it difficult to extract into typical sorbent phases during sample preparation. Therefore, a method for the ultratrace determination of atrazine in soil using Ice Concentration Linked with Extractive Stirrer (ICECLES) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed to address these issues. For the method, soil samples (10 g) were initially extracted with methanol:water (8:2, v:v), followed by solvent exchange to 100% water. The samples then underwent ICECLES with back-extraction into 100% methanol prior to HPLC-MS/MS analysis. The ICECLES-HPLC-MS/MS method produced a wide linear range of 10 to 1000 ng/kg, featured excellent limits of quantification and detection of 10 and 5 ng/kg, respectively, and good accuracy (100 ± 12%) and precision (≤9.6% relative standard deviation). This method was tested on field soil samples and provided ultratrace detection of atrazine. With this method, previously unachievable low parts per trillion (ppt) detection of atrazine in soil is now possible.

Analysis of TRPA1 antagonist, A-967079, in plasma using high-performance liquid chromatography tandem mass-spectrometry.

The noxious effects from exposure to toxic inhalation hazards (TIHs, such as isocyanates, chlorine, etc.) are known to be triggered by the activation of transient receptor potential ankyrin 1 (TRPA1) ion channel. Antagonists of TRPA1 have shown near complete attenuation of the noxious effects from TIH exposure. One of the TRPA1 antagonists, (1E,3E)-1-(4-fluorophenyl)-2-methyl-1-pentene-3-one oxime (A-967079), has shown impressive efficacy, high selectivity, high potency, and oral bioavailability. Although a validated method to quantify A-967079 in biological matrices is vital for the further development of A-967079 as a therapeutic agent, no method for its analysis from any matrix is currently available. Hence, a rapid and simple HPLC-MS/MS method was developed and validated to quantify A-967079 in rabbit plasma. The method presented here features an excellent LOD of 25 nM and a wide linear range (0.05-200 µM), with good accuracy and precision (100 ± 10.5% and <14.2% relative standard deviation, respectively). The stability of A-967079 in plasma was excellent for most of the storage conditions evaluated. The method was successfully applied to determine A-967079 from treated animals and it may facilitate the development of this TRPA1 antagonist as a therapeutic agent against the noxious effects of TIH exposure.

Comparison of the extraction efficiency of ice concentration linked with extractive stirrer, stir bar sorptive extraction, and solid-phase microextraction for pesticides from drinking water.

With the advent of highly selective analysis techniques (e.g., liquid chromatography-tandem mass spectrometry), and lower limits of detection requirements, extraction efficiency is arguably the most important property of modern sample preparation techniques. In this study, the extraction efficiency of Ice Concentration Linked with Extractive Stirrer (ICECLES) was compared to Stir Bar Sorptive Extraction (SBSE) and Solid-Phase Microextraction (SPME). A direct comparison of these sample preparation techniques was carried out with analysis via both Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) and Thermal Desorption Gas Chromatography - Mass Spectrometry (TD-GC-MS). ICECLES produced 2x and 7x greater TD-GC-MS signals than SBSE and SPME, respectively. When comparing extraction techniques for a suite of 60 pesticides in drinking water, 32, 25, and 13 pesticides were detected via LC-MS/MS at 0.1 ng/mL by ICECLES, SBSE, and SPME, respectively. Overall, ICECLES consistently produced better extraction efficiencies than the other extraction techniques evaluated.

Toxicity of canola-derived glucosinolates in pigs fed resistant starch-based diets.

A study was conducted to determine effects of reducing hindgut pH through dietary inclusion of high-amylose cornstarch (HA-starch) on growth performance, organ weights relative to live body weight (BW), blood thyroid hormone levels, and glucosinolate degradation products of nursery pigs fed cold-pressed canola cake (CPCC). A total of 240 pigs (initial BW: 7.1 kg), which had been weaned at 21 d of age, were housed in 40 pens (6 pigs per pen) and fed 4 diets (10 pens per diet) in a randomized complete block design for 28 d. Four diets were a basal diet with CPCC at 0 or 40%, and with HA-starch at 0 or 40% in a 2 × 2 factorial arrangement. The diets were fed in two phases: Phase 1 from day 0 to 14 and Phase 2 from day 14 to 28 and were formulated to have the same net energy, standardized ileal digestible AA, Ca, and standardized total tract digestible P contents. Dietary inclusion of CPCC and HA-starch was achieved by a partial or complete replacement of corn, soybean meal, and soy protein. At the end of the study, one pig from each pen was euthanized to determine organ weights, blood parameters, hindgut pH, and glucosinolate degradation products. Dietary CPCC reduced (P < 0.05) overall average daily gain (ADG) by 15%; increased (P < 0.05) relative weights of liver and thyroid gland by 27% and 64%, respectively; and reduced (P < 0.05) serum tetraiodothyronine (T4) level from 30.3 to 17.8 ng/mL. Heart, kidney, and gastrointestinal tract weights; serum triiodothyronine level; and hindgut pH of pigs were unaffected by dietary CPCC. Dietary HA-starch reduced (P < 0.05) overall ADG, relative weight of thyroid gland, cecal, and colonic pH; but increased (P < 0.05) relative weight of colon; tended to increase (P = 0.062) serum T4 level. Dietary CPCC and HA-starch interacted (P = 0.024) on relative weight of thyroid gland such that dietary CPCC increased (P < 0.05) weight of thyroid gland for HA-starch-free diet (120 vs. 197 mg/kg of BW) but not for HA-starch-containing diet (104 vs. 130 mg/kg of BW). Dietary CPCC and HA-starch interacted (P = 0.001) on cecal isothiocyanate content such that dietary CPCC increased (P < 0.05) level of isothiocyanates for HA-starch-containing diet but not for HA-starch-free diet. In conclusion, dietary CPCC reduced growth performance, increased liver, size and interfered with thyroid gland functions of pigs. However, the negative effects of dietary CPCC on thyroid gland functions of nursery pigs were alleviated by dietary HA-starch.

Diagnosis of cyanide poisoning using an automated, field-portable sensor for rapid analysis of blood cyanide concentrations.

Cyanide (both HCN and CN- are represented by CN) has multiple industrial applications, is commonly found in some foods, and is a component of fire smoke. Upon exposure, CN blocks production of adenosine triphosphate, causing cellular hypoxia and cytotoxic anoxia, which can eventually result in death. Considering CN's quick onset of action and the long analysis times associated with current techniques, the objective of this study was to develop and validate a rapid and field-portable sensor to detect blood CN concentrations focusing on both concentration and diagnostic accuracy. The sensor takes advantage of the chemical properties of CN by converting it exclusively to HCN via acidification of whole blood. High-speed headspace transfer is used to deliver HCN to a capture solution where it is reacted with naphthalene dialdehyde and taurine to produce a fluorescent ß-isoindole product. Simple spectrofluorometric analysis of the product provides quantitative analysis of CN from whole blood in 60 s and requires only 25 µL of blood (obtainable via fingerstick). A limit of detection of 5 µM, a linear range of 10-200 µM (with ≥15 µM considered CN exposed), and excellent accuracy (100 ± 15%) and precision (≤15.2% relative standard deviation) were obtained. To evaluate the diagnostic accuracy of the sensor, rabbit blood samples (N = 190, including 24 blinded samples) were analyzed by both the sensor and a lab-based spectrophotometric method. An excellent positive correlation was obtained between the sensor and the lab-based method (R2 ˃ 0.995) confirming the concentration accuracy of the CN sensor. Moreover, the sensor produced no false positives or negatives when diagnosing CN poisoning.

Ultratrace analysis of nitrosodipropylamine in drinking water by Ice Concentration Linked with Extractive Stirrer gas-chromatography electron-ionization mass-spectrometry.

Nitrosoamines (NAs), including nitrosodipropylamine (NDPA), are highly toxic drinking water contaminants with minimum reporting levels (MRLs) in the parts-per-trillion range (0.2-20 ng/L). The quantification of NAs at these concentrations is extremely difficult, requiring both sophisticated instrumentation and laborious sample preparation procedures. An advanced sample preparation technique, ICE Concentration Linked with Extractive Stirrer (ICECLES), coupled with gas-chromatography mass-spectrometry, was used to analyze NDPA (MRL = 7 ng/L). ICECLES allowed ultratrace analysis of NDPA, producing an LOD of 0.2 ng/L, will below the MRL, and a linear range of 2-50 ng/L (using NDPA-d14 as an internal standard). Both inter- and intraassay precisions were ≤13%RSD, while the method accuracy was 100 ± 17.5%. The ICECLES method was applied to screen for possible NA contamination in selected drinking water sources. The concentration of NDPA in one drinking water source was 2.38 ± 0.34 ng/L and was detected (i.e., concentrations ≥ LOD), but was not quantifiable, in the other samples.

Determination of free cyano-cobinamide in swine and rabbit plasma by liquid chromatography tandem mass spectrometry.

In recent years, Cobinamide (Cbi) has shown promise as a therapeutic for cyanide poisoning. There are several forms of Cbi based on the identity of the ligands bound to the cobalt in Cbi and these different forms of Cbi have divergent behavior (e.g., the aquo and hydroxo forms of Cbi readily bind to proteins, limiting their distribution significantly, whereas [Cbi(CN)2] does not). While current analysis techniques only measure total Cbi, methods to elucidate the behavior of 'available' Cbi versus cyanide-complexed Cbi would be valuable for biomedical and pharmacokinetic studies. Therefore, a method was developed for the analysis of cyanide-complexed Cbi in plasma via liquid chromatography tandem mass spectrometry (LC-MS-MS). Plasma samples were prepared by denaturing proteins with 10% ammonium hydroxide in acetonitrile. The resulting mixture was centrifuged, and the supernatant was removed, dried, and reconstituted. Cyanide-complexed Cbi was then analyzed via LC-MS-MS. The limit of detection was 0.2 µM, and the linear dynamic range was between 1 and 200 µM. The accuracy was 100 ±â€¯17% and the precision, measured by relative standard deviation (%RSD), was ≤18.5%. Carryover, a severe problem when analyzing Cbi via liquid chromatography was eliminated using a polymeric-based stationary phase (PLRP-S) and a controlled washing protocol. The method allowed evaluation of the cyanide-bound and 'available' Cbi from treated animals and, when paired with a method for total Cbi analysis, allows for estimation of Cbi utilization when treating cyanide poisoning.

Analysis of potential cyanide antidote, dimethyl trisulfide, in whole blood by dynamic headspace gas chromatography-mass spectroscopy.

Cyanide is a rapidly acting and highly toxic chemical. It inhibits cytochrome c oxidase in the mitochondrial electron transport chain, resulting in cellular hypoxia, cytotoxic anoxia and potentially death. In order to overcome challenges associated with current cyanide antidotes, dimethyl trisulfide (DMTS), which converts cyanide to less toxic thiocyanate in vivo, has gained much attention recently as a promising next-generation cyanide antidote. While there are three analysis methods available for DMTS, they each have significant disadvantages. Hence, in this study, a dynamic headspace (DHS) gas chromatography-mass spectroscopy method was developed for the analysis of DMTS from rabbit whole blood. The method is extremely simple, involving only acidification of a blood sample, addition of an internal standard (DMTS-d6) and DHS-GC-MS analysis. The method produced a limit of detection of 0.04 µM for DMTS with dynamic range from 0.2 to 50 µM. Inter- and intraassay accuracy (100 ± 15% and 100 ± 9%, respectively), and precision (<10% and <9% relative standard deviation, respectively) were good. The validated method performed well during pharmacokinetic analysis of DMTS from the blood of rats treated with DMTS, producing excellent pharmacokinetic parameters for the treatment of cyanide exposure. The method produced significant advantages over current methods for analysis of DMTS and should be considered as a "gold standard" method for further development of DMTS as a potential next-generation cyanide countermeasure.

Metabolism of Cyanide by Glutathione To Produce the Novel Cyanide Metabolite 2-Aminothiazoline-4-oxoaminoethanoic Acid.

The direct analysis of cyanide (HCN or CN- inclusively symbolized as CN) to confirm exposure has major limitations due to cyanide's volatility, reactivity, and short half-life in biological fluids. These limitations have led to the exploration of cyanide detoxification products for indirect verification of cyanide exposure. Although cyanide interacts strongly with sulfur-containing molecules, to date, biomarkers resulting from the interaction of cyanide with glutathione (GSH; i.e., a biologically abundant sulfur-donating biomolecule) have yet to be discovered. In this study, we studied the interaction of CN and GSH to produce 2-aminothiazoline-4-oxoaminoethanioc acid (ATOEA). An LC-MS/MS method was developed and validated to analyze ATOEA from plasma, producing a linear range of 0.5-50 µM, a limit of detection of 200 nM, and excellent precision and accuracy. ATOEA concentrations were significantly elevated in the plasma of animals following cyanide exposure. Moreover, the production of ATOEA from cyanide exposure was confirmed by detection of both ATOEA and ATOEA-13C15N in rabbit plasma ( N = 11 animals) following administration of NaCN:K13C15N (1:1), with a similar amount of ATOEA and ATOEA-13C15N formed ( R2 = 0.9924, p < 0.05). The concentration of ATOEA increased with cyanide dose and then decreased rapidly when an antidote was administrated. This study definitively showed that ATOEA is produced from interaction of CN and GSH and can serve as a biomarker of cyanide exposure.