Determination of biomarkers of chlorine exposure from biological samples: a review of analysis techniques.

Introduction: Chlorine gas can be toxic when inhaled or absorbed at high concentrations through the skin. It can cause pulmonary edema, pulmonary inflammation, respiratory failure, and potentially death. Monitoring chlorine exposure helps in determining treatment regimens and may inform safeguards, such as personal protective equipment and ventilation systems. Therefore, verification of chlorine exposure is crucial to protecting human health. This has led to identification of multiple biomarkers of Cl2 exposure with associated innovations in methods of analysis to monitor these markers.Materials and methods: In this review of the last 30 years of literature, biomarkers and associated methods of detection for the determination of chlorine exposure from biological samples are detailed and critically evaluated.Results and discussion: From the 36 included studies, the most useful biomarkers for Cl2 exposure include tyrosine adducts, chlorohydrin, chloro-fatty-acids, chloro-fatty-aldehydes, and chloro-fatty-alcohols. The most common sample preparation methods for these markers are hydrolysis and extraction and the most common analysis techniques are chromatographic separation with mass spectrometric detection.Conclusion: The findings of this review emphasize the need for continued research into biomarkers and stronger evaluation of proposed analytical methods, including validation, to allow more appropriate comparison, which will ultimately improve patient outcomes.

Can the cyanide metabolite, 2-aminothiazoline-4-carboxylic acid, be used for forensic verification of cyanide poisoning?

PURPOSE: Forensic verification of cyanide (CN) poisoning by direct CN analysis in postmortem blood is challenging due to instability of CN in biological samples. CN metabolites, thiocyanate (SCN-) and 2-aminothiazoline-4-carboxylic acid (ATCA), have been proposed as more stable biomarkers, yet it is unclear if either is appropriate for this purpose. In this study, we evaluated the behavior of CN biomarkers in postmortem swine and postmortem blood to determine which serves as the best biomarker of CN exposure. METHODS: CN, SCN-, and ATCA were measured in postmortem swine (N = 8) stored at 4 °C and postmortem blood stored at 25 °C (room temperature, RT) and 37 °C (typical human body temperature, HBT). RESULTS: Following CN poisoning, the concentration of each CN biomarker increased well above the baseline. In postmortem swine, CN concentrations declined rapidly (t1/2 = 34.3 h) versus SCN- (t1/2 = 359 h, 15 days) and ATCA (t1/2 = 544 h, 23 days). CN instability in postmortem blood increased at RT (t1/2 = 10.7 h) and HBT (t1/2 = 6.6 h). SCN- and ATCA were more stable than CN at all storage conditions. In postmortem swine, the t1/2s of SCN- and ATCA were 15 and 23 days, respectively. While both the t1/2s of SCN- and ATCA were relatively lengthy, endogenous levels of SCN- were much more variable than ATCA. CONCLUSION: While there are still questions to be answered, ATCA was the most adept forensic marker of CN poisoning (i.e., ATCA produced the longest half-life, the largest increase above baseline levels, and most stable background concentrations).

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.