RESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Saxitoxina/análogos & derivados , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Saxitoxina/química , Saxitoxina/isolamento & purificação , Saxitoxina/urinaRESUMO
Tetrodotoxin (TTX) is an extremely potent paralytic toxin responsible for yearly illness and death around the world. A clinical measurement is necessary to confirm exposure because symptoms of TTX intoxication cannot be distinguished from other paralytic toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of TTX in human urine with tandem mass spectrometry. The reportable range for the method was 2.80 - 249 ng/mL in urine with precision and accuracy within 15% as determined for all quality control samples. No isotopically-labeled internal standard is available for TTX; thus a surrogate internal standard, voglibose, was investigated to compensate for matrix effects and ionization suppression. However, upon evaluation, voglibose was ineffective for this purpose. This new online method rapidly identifies TTX, facilitating the work of public health authorities and providing support to monitoring programs worldwide.
Assuntos
Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Tetrodotoxina/urina , Calibragem , Humanos , Limite de Detecção , Padrões de ReferênciaRESUMO
Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0 ng/mL to 1.01 ng/mL and from 10.0 ng/mL to 2.62 ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.
Assuntos
Neurotoxinas/urina , Saxitoxina/análogos & derivados , Saxitoxina/urina , Intoxicação por Frutos do Mar/urina , Métodos Analíticos de Preparação de Amostras , Automação Laboratorial , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Neurotoxinas/química , Neurotoxinas/toxicidade , Isótopos de Nitrogênio , Reprodutibilidade dos Testes , Saxitoxina/química , Saxitoxina/toxicidade , Sensibilidade e Especificidade , Intoxicação por Frutos do Mar/diagnóstico , Intoxicação por Frutos do Mar/etiologia , Extração em Fase Sólida , Espectrometria de Massas em Tandem , ToxicocinéticaRESUMO
An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 µg/mL to 10.6 µg/mL, with an average concentration of 6.4 µg/mL.
Assuntos
Análise Química do Sangue/métodos , Substâncias para a Guerra Química/análise , Exposição Ambiental/análise , Compostos Organofosforados/sangue , Biomarcadores/metabolismo , Butirilcolinesterase/metabolismo , Calibragem , Substâncias para a Guerra Química/metabolismo , Cromatografia Líquida , Humanos , Imãs/química , Microesferas , Compostos Organofosforados/metabolismo , Controle de Qualidade , Espectrometria de Massas em TandemRESUMO
A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.
Assuntos
Butirilcolinesterase/sangue , Substâncias para a Guerra Química/análise , Separação Imunomagnética/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Compostos Organotiofosforados/química , Pepsina A/metabolismo , Peptídeos/química , Sarina/química , Espectrometria de Massas em TandemRESUMO
Abrin is a toxic protein found in the jequirity seed. L-Abrine (N-methyl-tryptophan) is also found in the jequirity seed and can be used as a biomarker for abrin exposure. Analysis of L-abrine was added to an existing method for quantifying ricinine as a marker for ricin exposure in human urine and analytically validated. Accuracy and reproducibility were enhanced by including a newly synthesized (13)C(1)(2)H(3)-L-abrine internal standard. One-milliliter urine samples were processed using solid-phase extraction prior to a 6-min high-performance liquid chromatography separation. Protonated molecular ions were formed via electrospray ionization in a triple-quadrupole mass spectrometer and quantified via multiple reaction monitoring. Method validation included the characterization of two enriched urine pools, which were used as quality control materials. Endogenous levels of L-abrine were quantified in a reference range of 113 random urine samples at 0.72 +/- 0.51 ng/mL. Urinary concentrations of L-abrine were monitored in an intentional rat exposure study for up to 48 h. Comparing the results from the human reference range and the animal exposure study indicates that this method is suitable for quantifying L-abrine within 24 h post-exposure. Quantification of L-abrine beyond 24 h is limited by rapid excretion of the biomarker and the level of the L-abrine dose.
Assuntos
Abrina/urina , Alcaloides Indólicos/urina , Abrus/química , Abrus/intoxicação , Animais , Biomarcadores/urina , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Estrutura Molecular , Intoxicação/urina , Ratos , Ratos Wistar , Padrões de Referência , Reprodutibilidade dos Testes , Sementes/química , Sementes/intoxicação , Extração em Fase Sólida , EstereoisomerismoRESUMO
In July 2004, two individuals developed blisters after the destruction of a WWI-era munition. To determine the causative agent, urine samples were collected from both the highly blistered patient (patient 1; 6.5% of total body surface area) and patient 2, who had only one small blister. Their urine was analyzed for metabolites of known vesicants including sulfur mustard (HD), Lewisite (L1), and nitrogen mustards. The urine samples only tested positive for metabolites of HD. Additional metabolites were measured to confirm the exposure of sulfur mustard agent HD, including thiodiglycol (TDG), TDG-sulfoxide, and the bis-mercapturate of mustard sulfone. On day 2 after the exposure, patient 1 had a beta-lyase metabolite level of 41 ng/mL, and patient 2 had a level of 2.6 ng/mL. Detectable levels of the beta-lyase metabolite were observed in patient 1 for 11 days and in patient 2 for 7 days. Levels of TDG and both TDG and its sulfoxide measured together in the urine of patient 1 were found to be 24 ng/mL and 50 ng/mL, respectively, on day 2. The bis-mercapturate of mustard sulfone was detected in patient 1 (3.1 ng/mL) on day 2 but was not detected in samples taken on subsequent days.
Assuntos
Monitoramento Ambiental/métodos , Gás de Mostarda/análise , Biomarcadores/urina , Cromatografia Líquida , Exposição Ambiental/análise , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Liases/metabolismo , Gás de Mostarda/metabolismo , Compostos de Sulfidrila/urina , Sulfóxidos/urina , Espectrometria de Massas em TandemRESUMO
Sesqui- and oxy-mustards pose a significant threat to military forces and civilians because they are potent vesicants. We have developed an isotope-dilution high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry method utilizing negative ion multiple reaction monitoring for the analysis of sesqui-mustard metabolites bis(2-hydroxyethylthio)alkanes (n = 1-5) and oxy-mustard metabolite bis(2-hydroxyethylthioethyl)ether in human urine. Relative standard deviations were < 10% and the reportable limits of detection were 1 ng/mL in 0.5 mL of urine. We applied this method to 100 samples collected from individuals with no known exposure to sesqui- or oxy-mustards, and no urines showed detectable levels of any of the analytes, suggesting that these metabolites may be used for monitoring exposure to sesqui- and oxy-mustards.
Assuntos
Monitoramento Ambiental/métodos , Gás de Mostarda/análise , Espectrometria de Massas em Tandem/métodos , Biomarcadores/urina , Cromatografia Líquida/métodos , Exposição Ambiental/análise , Humanos , Hidróxidos/química , Silicatos de Magnésio/química , Estrutura Molecular , Gás de Mostarda/análogos & derivados , Gás de Mostarda/metabolismo , Compostos de Potássio/química , Reprodutibilidade dos Testes , Extração em Fase Sólida , Titânio/químicaRESUMO
Bis(2-hydroxyethylthio)alkanes and bis(2-hydroxyethylthioalkyl)ethers are important biological and environmental degradation products of sulfur mustard analogs known as sesqui- and oxy-mustards. We used atmospheric pressure chemical ionization mass spectrometry (APCI MS) to acquire characteristic spectra of these compounds in positive and negative ionization modes. Positive APCI mass spectra exhibited [M + H](+); negative APCI MS generated [M + O(2)](-), [M - H](-), and [M - 3H](-); and both positive and negative APCI mass spectra contained fragment ions due to in-source collision-induced dissociation. Product ion scans confirmed the origin of fragment ions observed in single-stage MS. Although the spectra of these compounds were very similar, positive and negative APCI mass spectra of the oxy-mustard hydrolysis product, bis(2-hydroxyethylthiomethyl)ether, differed from the spectra of the other compounds in a manner that suggested a rearrangement to the sesqui-mustard hydrolysis product, bis(2-hydroxyethylthio)methane. We evaluated the [M + O(2)](-) adduct ion for quantification via liquid chromatography-MS/MS in the multiple-reaction monitoring (MRM) mode by constructing calibration curves from three precursor/product ion transitions for all the analytes. Analytical figures of merit generated from the calibration curves indicated the stability and suitability of these transitions for quantification at concentrations in the low ng/mL range. Thus, we are the first to propose a quantitative method predicated on the measurement of product ions generated from the superoxide adduct anion of the sesqui-and oxy-mustard hydrolysis products.
Assuntos
Substâncias para a Guerra Química/química , Gás de Mostarda/análogos & derivados , Gás de Mostarda/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Alcanos/química , Éteres/química , Estrutura MolecularRESUMO
Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.
Assuntos
Alcaloides/urina , Piridonas/urina , Ricina/intoxicação , Alcaloides/administração & dosagem , Alcaloides/isolamento & purificação , Animais , Biomarcadores/urina , Isótopos de Carbono , Ricinus communis/química , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Intoxicação/diagnóstico , Piridonas/administração & dosagem , Piridonas/isolamento & purificação , Ratos , Ratos Wistar , Padrões de Referência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The nitrogen mustards bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2), and tris(2-chloroethyl)amine (HN3) have the potential to be used as chemical terrorism agents because of their extreme vesicant properties. We modified a previously reported method to incorporate automated solid-phase extraction, improve chromatography, and include the urinary metabolite for HN3. The improved method was used to measure levels of the urinary metabolites N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA), and triethanolamine (TEA) in rats dosed with HN1, HN2, and HN3, respectively, and to establish background levels of EDEA, MDEA, and TEA in human urine samples from a population with no known exposure to nitrogen mustards. Rat dosing experiments confirmed that EDEA, MDEA, and TEA could be detected in urine for at least 48 h after exposure to HN1, HN2, and HN3, respectively. Substantial amounts of EDEA (89 ng/mL), MDEA (170 ng/mL), and TEA (1105 ng/mL) were measured in the urine of rats exposed to 10 mg HN1, HN2, and HN3, respectively, 48 h after exposure. The background concentrations for TEA in the human population ranged from below the limit of detection (LOD 3 ng/mL) to approximately 6500 ng/mL. Neither EDEA (LOD 0.4 ng/mL) nor MDEA (LOD 0.8 ng/mL) was detected above the LOD in the human samples.
Assuntos
Substâncias para a Guerra Química/farmacocinética , Exposição Ambiental , Mecloretamina/farmacocinética , Administração Cutânea , Animais , Biomarcadores/urina , Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/intoxicação , Cromatografia Líquida de Alta Pressão , Exposição Ambiental/análise , Humanos , Masculino , Mecloretamina/análise , Mecloretamina/intoxicação , Ratos , Ratos Wistar , Valores de Referência , Espectrometria de Massas por Ionização por Electrospray , UrináliseRESUMO
Nitrogen mustards are a public health concern because of their extreme vesicant properties and the possible exposure of workers during the destruction of chemical stockpiles. A sensitive, rapid, accurate, and precise analysis for the quantitation of ultratrace levels of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) in human urine as a means of assessing recent exposure to the nitrogen mustards bis(2-chloroethyl)ethylamine and bis(2-chloroethyl)methylamine, respectively, was developed. The method was based on solid-phase extraction, followed by analysis of the urine extract using isotope-dilution high-performance liquid chromatography-mass spectrometry with TurbolonSpray ionization and multiple-reaction monitoring. The method limits of detection were 0.41 ng/mL for EDEA and 0.96 ng/mL for MDEA in 1 mL of urine with coefficients of variation < 10% for both compounds.