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Yüksek Çözünürlüklü Kütle Spektrometrenin Eşzamanlı Çoklu Mantar Toksin Tayinleri Üzerindeki Etkisi

Amatoksinler, insanlarda ishalden organ işlev bozukluğuna kadar ciddi zehirlenmelere neden olan ölümcül yabani mantar toksinleridir. Spesifik bir tedavi uygulanmazsa mortalite %80 kadar yüksek olabilir. Bu çalışmada Amanita phalloides' e ait 7 tane toksinin eş zamanda belirlenmesi için ayırma ve tayin yöntemleri geliştirildi. Amanita phalloides (ölüm kapağı) yabani mantarının ekstraksiyonu ve saflaştırılmasının ardından toksinler, HPLC-ESI MS ve LC-doğru kütle uçuş zamanlı MS (TOF-MS) pozitif iyonizasyon modu kullanılarak tespit edildi. Ayrıca α- ve β-amanitin toksinlerinin LC'de birbirlerine çok yakın alıkonmaları (Rt) durumunda HR-MS özellikteki dedeksiyon sayesinde birbirlerinden ayırt edilmesinin mümkün olduğu görüldü. Kromatogramda aynı alıkonma noktasında iki toksin varlığı HR-MS sayesinde α-amanitinin C13 izotop kütlesi (920.3696 DA) ile β-amanitinin moleküler iyon kütlesinin (920.3514 DA) farklandırılması yapılarak tespit edildi. Optimize edilmiş her iki yöntem birbirleriyle karşılaştırılarak mevcut yöntemlere göre daha hızlı ve geniş spektrumlu toksin tanımlaması geçekleştirildi. Bu çalışmada aynı yöntem ile eş zamanda yedi adet toksinin hızlı ve doğru tanımlanması büyük önem taşımaktadır. Özellikle zehirlenmenin yabani mantardan kaynaklı olduğunun belirlenmesi ile tedavi sürecinde önemli katkılar sağlayacaktır.

Anahtar Kelimeler:

Amanitin, Amatoksin, HPLC ESI/MS, LC-TOF MS, Mantar Zehirlenmesi, Falloidin

The Effect of A High-Resolution Accurate Mass Spectrometer On Simultaneous Multiple Mushroom Toxin Detection

Amatoxins are deadly wild mushroom toxins that cause severe poisoning in humans, from diarrhea to organ dysfunction. Mortality can be as high as 80% if no specific treatment is applied. In this study, separation and determination methods were developed for the simultaneous determination of 7 toxins belonging to Amanita phalloides. After extraction and purification of Amanita phalloides (death cap) wild mushrooms, toxins were detected using HPLC-ESI MS and exact mass with time of flight MS (TOF-MS) in positive ionization mode. In addition, it was observed that the toxins of α- and β-amanitine could be differentiated from each other thanks to HR-MS detection in case of their close retention (Rt) in LC. The presence of two toxins at the same retention point in the chromatogram was detected by differentiating the molecular ion mass (920.3514 DA) of the α-amanitine with the HR-MS (920.3696 DA). By comparing both optimized methods with each other, faster and broad-spectrum toxin identification was achieved compared to existing methods. In this study, the rapid and accurate identification of seven toxins simultaneously with the same method is of great importance. It will make important contributions to the treatment process, especially by determining that poisoning is caused by wild mushrooms.

Keywords:

Amanitin, Amatoxin, HPLC ESI/MS, LC-TOF MS, Mushroom Intoxication, Phalloidin,

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