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Quantitation of 9 different coccidiostats in poultry using LC-MS/MS on a LCMS 8060 triple quadrupole Jana Rykl1; Ty Kahler2 1Shimadzu Switzerland GmbH, Reinach Switzerland; 2Restek, Bellefonte USA 1. Introduction 3. Results Coccidiosis is parasitic disease of the intestinal tract of animals caused by protozoan parasites. It is a common disease in poultry and can be fatal or leave the birds compromised digestion. In order to prevent the disease birds are fed with mixtures that contain a coccidiostats. The application of these substances is regulated by European law in order to protect human and and the environment. In this poster we present a LC-MS/MS methods for the quantification of authorized coccidiostats in meat. There are two classes of coccidiostats: small synthetic chemical molecules ( e.g. decoquinat, diclurazil) and ionophoric glycosides (e.g. maduramicin, salinomycin). The results of the quantitation were analyzed using the Insight data review software. The upper concentration limits (ppb or ug/kg) for the coccidiostats were set according to the maximum residue level of the Eurpean commision for food safety. Table 2: Maximum residue level MRL [ppb] in food Fig 1 Structure of the analyzed coccidiostats 2. Materials and Methods A mix of nine different coccidiostat standards was analyzed on a Restek Raptor Bipyhenyl column (100x 2.1 mm, 2.7µm). The flow rate was set to 0.6 mL/min. Fig 2 Chromatogram of the coccidiostats standards (20 ppb) Solvent A: 2 mM Ammonium Acetate, 10% Methanol, 1% Acetic Acid, 89% Water Solvent B: 2mM Ammonium Acetate, 97% Methanol, 1% Acetic Acid, 2% Water 1 g of meat (chicken, chicken liver, goose, turkey and duck) was minced and extracted for 10 min in a ultrasonic bath using 20 mL of solvent A. The sample was centrifuged and the supernatant was filtered. 10 uL external standard was added to 490 uL of the extracted meat solution. 10 uL of this dilution was directly injected into the LCMS system. The LCMS system consisted of a Nexera-i UHPLC system, coupled to a LCMS 8060 Triple quadrupole. Since there is no isotopic labled internal standard available for the coccidiostats, quantitation was done in the «Standard Addition» mode, that means different amounts of the coccidiostats mix were directly added to the solution of the extracted meat. Each sample was measured in triplicates. Below are the settings for the UHPLC gradient and the MRM transition. Time [min] 0 1 15 15.5 15.51 18 Pump B conc. [%] 20 20 100 100 20 20 Nebulizing gas flow 3L/min Heat gas flow 5L/min Drying gas flow 10L/min Interface Temp. 300°C Desolvation Line Temp. 250°C Heat Block Tem. 400°C Table 3 LOQ, LOD and Linear Range, * detector saturated at 50 ppb, ** detector saturated at 20 ppb, (1ppb= 1ng/mL= 1ug/kg) We found a significantly high amount of maduramicin in turkey and chicken liver. The concentration in turkey was around 13 ppb. The concentration in chicken liver was 7 ppb. Also we found a very high concentration of Narasin (187 ppb) and Salinomycin (890 ppb) in chicken liver. This is almost 100 times (for Narasin) and 450 times (Salinomycin) higher than the allowed maximum residue level in meat. 4. Discussion and Conclusion Most chicken receive a coccidiostat during a part or all of their life. It is added to the feed at a maximum allowed concentration of 70 mg/kg. Its use is prohibited 5 days before slaughter. Salinomycin is also a drug candiate for the treatment of cancer. During preclinical drug metabolism and pharmacokinetics tests it has been shown that substance is metabolized by the enzyme CYP3A4, which is mainly found in the liver. It seems that the drug therefore accumulates in the liver. Table 1 MRM Transitions, 10 msec dwell time for each transition Using an UHPLC coupled to a triple quadrupole MS system we seperated 9 different coccidiostats in poultry on a Restek Biphenyl column. The quantitation limit for the substances was in in the sub ppb range.