Mushroom poisoning is a common environmental medical emergency. α-Amanitin, a bicyclic peptide, plays a major role in Amanita phalloides poisoning, showing toxic effects on multi-organs, particularly liver and kidneys. Amatoxins, especially α-and β-amanitin, notorious for their high toxicity, interfere with DNA transcription by binding to and inhibiting eukaryotic RNA polymerase II in hepatocytes. Suppressed mRNA synthesis results in cellular necrosis through the inhibition of protein synthesis, and consequently hepatic failure and renal damage can develop, which may lead to death. Amatoxins are not bound to plasma proteins, and are mostly eliminated through urine. After ingestion (Jaeger A. et al., 1993), they are detectable for approximately 30 h in plasma or serum, and up to 72 h in urine. This short time development of symptomatology requires a rapid intervention in order to avoid severe organ damages and to achieve a successful outcome. Unambiguous detection of amanitins in biological fluids, turns to be essential for the early diagnosis of mushroom intoxication due to the invasive and extensive therapy needed for treatment; serum analysis proves to be more accurate and allows a more appropriate treatment, when carried out a short time after ingestion. LC is the method of excellence for amanitin separation. It fills in all the requirements in terms of sensitivity, precision and specificity bounded with operating simplicity and rapid detection of - and - amanitins in biological samples. Methods combining LC with mass spectrometry (MS), such as triple quadrupole tandem MS, for the detection of amanitins, have been reported. Nevertheless, UPLC coupled to tandem mass spectrometry allows a drastic reduction in the time of analysis and the consumption of solvents and consequently lower disposal costs. Single quadrupole mass spectrometry enhances the analytical value and productivity of each analysis, eliminating time-or cost-consuming alternative techniques.. During this thesis work, carried out at the Laboratory of Experimental and Clinical Toxicology of the National Centre for Toxicological Information (CNIT-CAV) of the Istituti Scientifici Maugeri in Pavia, a fast and reliable method for the determination of α-and β-amanitin in serum by UPLC-MS, has been developed and validated. For liquid chromatographic separation, a CORTECS UPLC C18+ column was used. Since α-and β-amanitin have very similar structures and molecular weights, they must be completely separated in LC to avoid interferences from each other. Tilmicosin was used as the internal standard (IS). With regard to the extraction procedures, the lowest background noise and highest recoveries were achieved with the hydrophilic-lipophilic solid-phase extraction (SPE) columns Oasis PRiME HLB. The mixture of serum sample and of 4% H3PO4 in distilled water was slowly loaded into the PRiME HLB cartridge. Then each cartridge was washed with methanol at 5% twice. The retained compounds were eluted into a clean tube with of methanol. The eluent was dried under nitrogen and reconstituted with of methanol before LC-MS analysis. The analytes were identified and quantified on a QDa MS detector (Waters, Milford, MA, USA) coupled to the UPLC ACQUITY system (Waters, Milford, MA, USA). alpha-amanitin, beta-amanitin and tilmicosin were identified and confirmed by their relative retention time (RRT), ion transitions and mass spectrum. The recovery values ranged between 88.2 and 102.1% for alpha-amanitin and between 86.4 and 116.8% for beta-amanitin. The method has been fully validated and successfully applied to real serum samples. These samples were collected from 20 patients with suspected poisoning with wild mushrooms. Six samples only were found positive for alpha-amanitin, with the highest value of 11.4 ng/mL in serum. In summary, this method is suitable for the routine emergency toxicological and clinical determination of amatoxins in human serum.
Le intossicazioni da funghi sono frequenti e strettamente legate alla consuetudine molto diffusa di praticare la raccolta di funghi selvatici e di consumarli senza averli sottoposti allopportuno controllo da parte di esperti. LAmanita phalloides è sicuramente il fungo più pericoloso, per la gravità dellavvelenamento, per la sua diffusione nelle nostre regioni e per il suo elevato polimorfismo che lo rende somigliante a molte altre specie commestibili (ad esempio prataiolo e chiodino). Il riconoscimento dei funghi prima del loro consumo rappresenta oggi lunica misura preventiva nei confronti delle intossicazioni potenzialmente mortali: per tale scopo sono disponibili appositi centri nei quali micologi esperti visionano e controllano, gratuitamente, i funghi raccolti. Le principali sostanze responsabili del grave quadro di intossicazione sono le amatossine, octapeptidi biciclici, presenti a una concentrazione di circa 0,2 - 0,4 mg/g di fungo fresco; la DL50 di questi principi tossici è di circa 0,1 mg/kg di peso corporeo, corrispondenti per un adulto a 20-50 g di fungo fresco. La diagnosi di intossicazione da Amanita phalloides si basa sul riconoscimento del fungo e sullanalisi routinaria dellα-amanitina in urina tramite metodo ELISA. Dopo lingestione, le amanitine sono dosabili sia nelle urine per 72 ore, sia nel siero entro 30 ore. Tuttavia, la positività del test in urina non indica la gravità dellintossicazione e la negatività del test non ha valore di certezza per lesclusione. A fronte di queste problematiche è necessario, quindi, disporre di metodi analitici validati che consentano la determinazione delle amanitine nelle matrici biologiche in tempi brevi e con costi contenuti, garantendo laccuratezza del risultato nellintervallo di concentrazione di interesse clinico. Solo lanalisi di urina o di siero mediante una tecnica altamente specifica come la spettrometria di massa, permette di formulare con certezza la diagnosi di intossicazione falloidea, poiché con il metodo ELISA, comunemente impiegato per lanalisi in urina, sono possibili falsi positivi. Lobiettivo dellinternato di tesi, svolto presso il Laboratorio di Tossicologia Clinica e Sperimentale del Centro Antiveleni di Pavia, è stato sviluppare una metodo di analisi robusto e affidabile per la determinazione di α e β amanitina in siero, utilizzando lUPLC-MS, ovvero la cromatografia ultra-performance abbinata a spettrometria di massa con singolo quadrupolo (Ultra-Performance Liquid Chromatography). Questa tecnica è meno costosa rispetto ai sistemi più comunemente impiegati, come lUPLC-MS/MS a triplo quadrupolo. (Homann J. et al., 1986; Abuknesha R.A. et al., 1993; Bruggemann O. etddo al., 1996; Sgambelluri R.M. et al., 2014; Garcia J. et al., 2015; Helfer A.G. et al., 2015). Il metodo prevede un pretrattamento del campione di siero con acido fosforico, una fase di purificazione ed estrazione in metanolo mediante SPE (estrazione in fase solida) al termine della quale al campione così eluito viene addizionata una quantità nota di standard interno (Tilmicosina). Leluato è successivamente portato a secco sotto flusso di azoto a 40°C mediante concentratore di campioni, risospeso in 100 µL di metanolo, e infine sottoposto ad analisi mediante UPLC/MS. In queste condizioni operative il recupero è risultato compreso tra 88,2 e 102% per α-amanitina e tra 86,4 e 116,8% per β-amanitina. Leffetto matrice, ottenuto dalle curve di calibrazione preparate in 3 giorni non consecutivi, è stato valutato rispettivamente pari all11,8% per α-amanitina e al 15,0% per β-amanitina. La messa a punto e validazione di questo metodo ha permesso lestrazione di α- e β-amanitina e la loro successiva quantificazione nel siero di campioni reali di soggetti con sospetta intossicazione falloidea.
Determinazione di alfa-amanitina e beta-amanitina nel siero mediante UPLC-MS
PUCCI, LUDOVICA
2018/2019
Abstract
Mushroom poisoning is a common environmental medical emergency. α-Amanitin, a bicyclic peptide, plays a major role in Amanita phalloides poisoning, showing toxic effects on multi-organs, particularly liver and kidneys. Amatoxins, especially α-and β-amanitin, notorious for their high toxicity, interfere with DNA transcription by binding to and inhibiting eukaryotic RNA polymerase II in hepatocytes. Suppressed mRNA synthesis results in cellular necrosis through the inhibition of protein synthesis, and consequently hepatic failure and renal damage can develop, which may lead to death. Amatoxins are not bound to plasma proteins, and are mostly eliminated through urine. After ingestion (Jaeger A. et al., 1993), they are detectable for approximately 30 h in plasma or serum, and up to 72 h in urine. This short time development of symptomatology requires a rapid intervention in order to avoid severe organ damages and to achieve a successful outcome. Unambiguous detection of amanitins in biological fluids, turns to be essential for the early diagnosis of mushroom intoxication due to the invasive and extensive therapy needed for treatment; serum analysis proves to be more accurate and allows a more appropriate treatment, when carried out a short time after ingestion. LC is the method of excellence for amanitin separation. It fills in all the requirements in terms of sensitivity, precision and specificity bounded with operating simplicity and rapid detection of - and - amanitins in biological samples. Methods combining LC with mass spectrometry (MS), such as triple quadrupole tandem MS, for the detection of amanitins, have been reported. Nevertheless, UPLC coupled to tandem mass spectrometry allows a drastic reduction in the time of analysis and the consumption of solvents and consequently lower disposal costs. Single quadrupole mass spectrometry enhances the analytical value and productivity of each analysis, eliminating time-or cost-consuming alternative techniques.. During this thesis work, carried out at the Laboratory of Experimental and Clinical Toxicology of the National Centre for Toxicological Information (CNIT-CAV) of the Istituti Scientifici Maugeri in Pavia, a fast and reliable method for the determination of α-and β-amanitin in serum by UPLC-MS, has been developed and validated. For liquid chromatographic separation, a CORTECS UPLC C18+ column was used. Since α-and β-amanitin have very similar structures and molecular weights, they must be completely separated in LC to avoid interferences from each other. Tilmicosin was used as the internal standard (IS). With regard to the extraction procedures, the lowest background noise and highest recoveries were achieved with the hydrophilic-lipophilic solid-phase extraction (SPE) columns Oasis PRiME HLB. The mixture of serum sample and of 4% H3PO4 in distilled water was slowly loaded into the PRiME HLB cartridge. Then each cartridge was washed with methanol at 5% twice. The retained compounds were eluted into a clean tube with of methanol. The eluent was dried under nitrogen and reconstituted with of methanol before LC-MS analysis. The analytes were identified and quantified on a QDa MS detector (Waters, Milford, MA, USA) coupled to the UPLC ACQUITY system (Waters, Milford, MA, USA). alpha-amanitin, beta-amanitin and tilmicosin were identified and confirmed by their relative retention time (RRT), ion transitions and mass spectrum. The recovery values ranged between 88.2 and 102.1% for alpha-amanitin and between 86.4 and 116.8% for beta-amanitin. The method has been fully validated and successfully applied to real serum samples. These samples were collected from 20 patients with suspected poisoning with wild mushrooms. Six samples only were found positive for alpha-amanitin, with the highest value of 11.4 ng/mL in serum. In summary, this method is suitable for the routine emergency toxicological and clinical determination of amatoxins in human serum.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/18954