Elizabeth I. Hamelin

Evaluation of Ricinine, a Ricin Biomarker, from a Non-Lethal Castor Bean Ingestion

A case is presented of the attempted suicide of a 58-year-old man using castor beans. The patient came to the emergency room complaining of nausea, vomiting and diarrhea for nine hours following the ingestion of six castor beans. Urine samples were taken throughout the hospital stay and submitted to the Centers for Disease Control and Prevention for analysis of ricinine, a castor bean component. The samples were found to be positive for ricinine, with a maximum concentration of 674 µg/g-creatinine excreted approximately 23 h post-exposure. Subsequent samples demonstrated lower ricinine concentrations, with the final sample taken at 62 h post-exposure at a concentration of 135 µg/g-creatinine of ricinine. The estimated urinary excretion half-life was approximately 15 h and the recovery of ricinine in the urine over the three days was estimated to be less than 10%. The patient fully recovered with supportive care and was discharged from the hospital six days after admission.

Comparison of High Resolution and Tandem Mass Spectrometry for the Analysis of Nerve Agent Metabolites in Urine

RATIONALE Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.

Comparison of two automated solid phase extractions for the detection of ten fentanyl analogs and metabolites in human urine using liquid chromatography tandem mass spectrometry

Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC-MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002ng/mL to 0.041ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine.

Analysis of a Ricin Biomarker, Ricinine, in 989 Individual Human Urine Samples

Ricinine (3-cyano-4-methoxy-N-methyl-2-pyridone) is a urinary biomarker that can be measured to confirm human exposure to castor bean products such as ricin. Because many consumer products contain castor oil, another castor bean product, ricinine may be detectable in the general population. The following study characterized urinary ricinine concentrations from 989 individuals who were presumed to be unexposed to ricin. An automated diagnostic method was utilized to simplify the analysis of this large sample set. Sample preparation included a 96-well polystyrene divinylbenzene high throughput extraction and preconcentration step. Purified samples were analyzed by an efficient dual column, reversed-phase liquid chromatography separation and (13)C-isotope dilution tandem mass spectrometry. In this convenience sample set, only 1.2% of the urine specimens had detectable amounts of ricinine, randomly distributed between 0.186 and 4.15 ng/mL.

Quantification of saxitoxin in human blood by ELISA

Abstract Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half‐life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme‐linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost‐effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost‐efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross‐reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross‐reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross‐reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements. HighlightsWe have validated an ELISA for detection of saxitoxin in human whole blood.The ELISA can accurately detect down to 0.02 ng/mL in blood with no additional clean up steps and 0.06 ng/mL in dried blood.Quantitation of saxitoxin in human blood using synthetic blood calibrators is inaccurate.The ELISA cross‐reacts significantly with gonyautoxins 2 and 3.

Development and validation of a high-throughput online solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the detection of gonyautoxins 1&4 and gonyautoxins 2&3 in human urine

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities.

Evaluation of Multiple Blood Matrices for Assessment of Human Exposure to Nerve Agents

Biomedical samples may be used to determine human exposure to nerve agents through the analysis of specific biomarkers. Samples received may include serum, plasma, whole blood, lysed blood and, due to the toxicity of these compounds, postmortem blood. To quantitate metabolites resulting from exposure to sarin (GB), soman (GD), cyclosarin (GF), VX and VR, these blood matrices were evaluated individually for precision, accuracy, sensitivity and specificity. Accuracies for these metabolites ranged from 100 to 113% with coefficients of variation ranging from 2.31 to 13.5% across a reportable range of 1-100 ng/mL meeting FDA recommended guidelines for bioanalytical methods in all five matrices. Limits of detection were calculated to be 0.09-0.043 ng/mL, and no interferences were detected in unexposed matrix samples. The use of serum calibrators was also determined to be a suitable alternative to matrix-matched calibrators. Finally, to provide a comparative value between whole blood and plasma, the ratio of the five nerve agent metabolites measured in whole blood versus plasma was determined. Analysis of individual whole blood samples (n = 40), fortified with nerve agent metabolites across the reportable range, resulted in average nerve agent metabolite blood to plasma ratios ranging from 0.53 to 0.56. This study demonstrates the accurate and precise quantitation of nerve agent metabolites in serum, plasma, whole blood, lysed blood and postmortem blood. It also provides a comparative value between whole blood and plasma samples, which can assist epidemiologists and physicians with interpretation of test results from blood specimens obtained under variable conditions.

Bridging the gap between sample collection and laboratory analysis: using dried blood spots to identify human exposure to chemical agents

Public health response to large scale chemical emergencies presents logistical challenges for sample collection, transport, and analysis. Diagnostic methods used to identify and determine exposure to chemical warfare agents, toxins, and poisons traditionally involve blood collection by phlebotomists, cold transport of biomedical samples, and costly sample preparation techniques. Use of dried blood spots, which consist of dried blood on an FDA-approved substrate, can increase analyte stability, decrease infection hazard for those handling samples, greatly reduce the cost of shipping/storing samples by removing the need for refrigeration and cold chain transportation, and be self-prepared by potentially exposed individuals using a simple finger prick and blood spot compatible paper. Our laboratory has developed clinical assays to detect human exposures to nerve agents through the analysis of specific protein adducts and metabolites, for which a simple extraction from a dried blood spot is sufficient for removing matrix interferents and attaining sensitivities on par with traditional sampling methods. The use of dried blood spots can bridge the gap between the laboratory and the field allowing for large scale sample collection with minimal impact on hospital resources while maintaining sensitivity, specificity, traceability, and quality requirements for both clinical and forensic applications.

Quantitative analysis and stability of the rodenticide TETS (tetramine) in finished tap water

The determination of the rodenticide tetramethylenedisulfotetramine (TETS) in drinking water is reportable through the use of automated sample preparation via solid phase extraction and detection using isotope dilution gas chromatography-mass spectrometry. The method was characterized over twenty-two analytical batches with quality control samples. Accuracies for low and high concentration quality control pools were 100 and 101%, respectively. The minimum reporting level (MRL) for TETS in this method is 0.50 μg L−1. Five drinking waters representing a range of water quality parameters and disinfection practices were fortified with TETS at ten times the MRL and analyzed over a 28 day period to determine the stability of TETS in these waters. The amount of TETS measured in these samples averaged 100 ± 6% of the amount fortified suggesting that tap water samples may be held for up to 28 days prior to analysis.

Detection of α-, β-, and γ-amanitin in urine by LC-MS/MS using 15N10-α-amanitin as the internal standard

ABSTRACT The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect &agr;‐, &bgr;‐, and &ggr;‐amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N10‐&agr;‐amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N10‐&agr;‐amanitin internal standard, precision and accuracy of &agr;‐amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1–200 ng/mL. &bgr;‐ and &ggr;‐Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5‐200 ng/mL and 1.0–200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled &agr;‐amanitin with the ability to detect and confirm human exposures to &agr;‐, &bgr;‐, and &ggr;‐amanitin. HighlightsAn LC‐MS/MS method for the detection of &agr;‐, &bgr;‐, and &ggr;‐amanitin was developed.The development included an evaluation of an 15N10 a‐amanitin internal standard to compensate for matrix effects.The method can accurately detect down &agr;‐, &bgr;‐, and &ggr;‐amanitin to 1.0, 2.5, and 1.0 ng/mL in urine, respectively.The 5N10 &agr;‐amanitin internal standard significantly improved precision for &agr;‐amanitin.