Production of “in house” reference materials for ELISA screening of bovine urine and liver samples for clenbuterol

Abstract

Clenbuterol screening of bovines is done by analysis of urine, for monitoring living animals, and liver, for monitoring animals after slaughter. ELISA has generally been used as the main method for these purposes. Nevertheless, in Europe, methods must be validated according to Commission Decision (EC) 657/2007 criteria, i.e. by use of reference materials. Production of ''in house'' reference materials is a possibility, but the homogeneity, storage temperature, and period of stability of these materials must be investigated in the laboratory itself. This paper reports GC-MS evaluation of an ''in-house''-produced batch of aliquots of bovine urine and liver, fortified with 10.0 ng/ml and 10.0 ng/g clenbuterol, respectively, and stored at -20°C and at -60°C. For urine stored for 20 weeks at -20°C and at 60°C the stability of clenbuterol was proved at the 95% confidence level. For liver, however, it was demonstrated at the same confidence level that clenbuterol was highly unstable during storage for 20 weeks at either of the temperatures studied.

Broad screening and identification of β-agonists in feed and animal body fluid and tissues using ultra-high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry combined with spectra library search

“…These methods include liquid chromatography (Chang, Chou, & Hwang, 2005;Meng et al, 2012;Turberg, Macy, Lewis, & Coleman, 1994), gas chromatography (Bocca, Fiori, Cartoni, & Brambilla, 2003), liquid chromatography tandem mass spectrometry (LC-MS/MS) (Shishani, Chai, Jamokha, Aznar, & Hoffman, 2003;Wu, Liu, & Peng, 2014), gas chromatography-mass spectrometry (Montrade, Lebizec, Monteau, & Andre, 1995;Wang, Li, Zhang, & Shen, 2006), enzyme-linked immunoassay (Hahnau & Julicher, 1996;Harkins, Woods, Lehner, Fisher, & Tobin, 2001;Pinheiro et al, 2008), colloidal gold test strip (Khamta, Pattarawarapan, Nangola, & Tayapiwatana, 2009;Zhang et al, 2009), surface plasmon resonance immunosensors (Traynor, Crooks, Bowers, & Elliott, 2003), and electrochemical immunosensors (He, Wang, Zhang, & Yang, 2009). Table 1 Chemical formula, accurate mass, retention time and characteristic fragment ions of 32 b-agonists.…”

Clenbuterol Storage Stability in the Bovine Urine and Liver Samples Used for European Official Control in the Azores Islands (Portugal)

“…The same conclusions were found for liver samples, when stored at -30 °C without any prehomogenization (16). Nevertheless, in a previous study it was also found that in prehomogenated liver clenbuterol was unstable at both -20 and -60 °C (14).…”

“…This study was performed with incurred lyophilized samples from which conclusions cannot be extrapolated to laboratory samples. Two other studies, using fortified urine samples instead of incurred urine, were found: in the first it was found that clenbuterol recovery diminished in urine during frozen storage at -15 °C (13), but in the second one no clenbuterol degradations occurred during storage at -20 °C or at -60 °C (14).…”