|Wednesday, August 27||Free Topics|
IMMUNOAFFINITY EXTRACTION OF a- AND ß-AMANITIN FOR THEIR SENSITIVE DETECTION IN BODY FLUIDS BY ATMOSPHERIC PRESSURE IONIZATION ELECTROSPRAY (API-ES) LC-MS|
Schmitt C.J., Weber A.A., Maurer H.H.
After ingestion of toxic amanita mushrooms the amatoxins a- and ß-amanitin may cause severe gastrointestinal disorders and irreversible liver damage. Since diagnosis of an intoxication entails a large scale of invasive and expansive therapy, a highly specific detection of amanitins in body fluids is necessary. A LC-MS procedure for the detection of amanitins in urine after solid phase extraction (RP-18) and isocratic separation was described (1). In the following, improvements using immunoaffinity extraction and gradient elution are described.
Methods. Amanitin antibodies were immobilized on CNBr-activated Sepharose 4B and the immobilizate was packed in columns. These immunoaffinity columns were used to extract amanitins from 2 mL of urine or plasma. The amanitins were separated on an HP ODS Hypersil RP-18 narrowbore column (3 µm) with gradient elution (methanol-ammonium acetate buffer) and detected by an HP API electrospray MS using the SIM mode (ions m/z 918, 919, 920 and 921 for a-amanitin and 919, 920, 921 and 922 for ß-amanitin).
Results. The immunoaffinity extraction allowed selective isolation of amanitins from small volumes of urine or plasma. Using the new gradient HPLC method, the amanitins could be separated in less than 20 min. The ES MS detection was as specific as necessary for clinical and forensic toxicological results. The sensitivity was less than 10 ng/mL in urine.
Conclusions. Immunoextraction followed by gradient API-ES LC-MS resulted in fast, specific and sensitive detection of amanitins in urine, that allowed the diagnosis of intoxications with amanita mushrooms. The procedure also allowed the detection of amanitins in other body fluids.
|Sunday, 24||Monday, 25||Tuesday, 26||Wednesday, 27||Thursday, 28|