Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability

Abstract

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor

“…To determine the expression level of LvAPN1 gene and EF-1α internal control gene, the qRT-PCR analysis was performed using an appropriate amount of cDNA for each gene, specific oligonucleotide primers (Table 1) and qPCR Master Mix (KAPA Biosystem) in CFX96 Touch RealTime PCR System (Bio-Rad) under the following conditions: 95˚C for 3 min, 40 cycles at 95˚C for 30 s, 60˚C for 30 s, and 72˚C for 30 s. The experiments were done in three biological replicates. Relative expression level was calculated using the mathematical model of Pfaffl (2001) [40]. Data were analyzed using one-way ANOVA followed by Duncan's new multiple ranges test and presented as means ± standard deviations (SD).…”

Solid-phase micro extraction (SPME) and headspace derivatization of clenbuterol followed by GC–FID and GC–SIMMS quantification

“…In athletes, the drug is rapidly metabolized and is often present at only trace levels making it difficult to assay [3,9,10,11,12]. For these reasons there is much interest in methods of assay for clenbuterol that are not only highly sensitive and selective [13,14], but amenable to automation and high throughput as well.…”

Overexpression of glutamate‐gated chloride channel in the integument is mainly responsible for emamectin benzoate resistance in the western flower thrips Frankliniella occidentalis

“…qPCR for each gene was conducted with three biological replicates for each strain. Relative transcription levels of target Cyp s were determined by the 2 −ΔΔCt method 27 …”

“…The relative gene copy number of FoGluClc was determined by the 2 −ΔΔCt method. 27 To investigate any possibility that the FoGluClc CNV observed is driven by its endosymbionts, the relative amount of Pantoea agglomerans, a common endosymbiont in thrips species, was determined by quantifying the 16S ribosomal RNA gene (GenBank: AY616181.1) via qPCR using six pooled specimens, with each containing 15 randomly collected EB-R females. The resulting quantity of P. agglomerans was then compared with the corresponding FoGluClc CNV of each pooled specimen.…”

“…Relative transcription levels of target Cyps were determined by the 2 −ΔΔCt method. 27 2.4 Molecular characterization, cloning and transcription profiling of three GluCl genes Three GluCl genes were identified in F. occidentalis from NCBI: LOC113210645 (FoGluCla), LOC113205570 (FoGluClb) and LOC113210214 (FoGluClc). The full-length fragments of these three GluCl genes from both the RDA and EB-R strains were amplified using sequence-specific primer sets (Table S2).…”