Laboratory validation of an LC-MS/MS method for the detection of ractopamine, clenbuterol and salbutamol in bovine and swine muscle at sub-µg kg-1 regulatory limits.

Ractopamine (RAC), is a ß-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic ß-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three ß-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg-1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d5, CLB-d9 and SLB-d6 as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCß, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCß, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg-1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCß values were 0.03, 0.22 and 5.8 µg kg-1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05-7.5 µg kg-1 of the studied ß-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.

Liquid chromatography/tandem mass spectrometry method to determine boldenone in bovine liver tissues.

Boldenone, an androgenic steroid, is forbidden for use in meat production in most countries worldwide. Residues of this drug in food present a potential risk to consumers. A sensitive LC/MS/MS method for analysis of 17ß-boldenone using boldenone-d3 as an internal standard was developed. An enzymatic hydrolysis and extraction using ethyl acetate, methanol, and hexane were performed in the sample preparation. Parameters such as decision limit (CCα), detection capability (CCß), precision, recovery, and ruggedness were evaluated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC) and International Organization for Standardization/International Electrotechnical Commission 17025:2005. CCα and CCß were determined to be 0.17 and 0.29 µg/kg, respectively. Average recoveries from bovine liver samples fortified with 1, 1.5, and 2 µg/kg were around 100%. A complete statistical analysis was performed on the results obtained, including an estimation of the method uncertainty. The method is considered robust after being subjected to day-to-day analytical variations and has been used as a standard method in Brazil to report boldenone levels in bovine liver.

Determination and confirmation of metronidazole, dimetridazole, ronidazole and their metabolites in bovine muscle by LC-MS/MS.

Nitroimidazoles are a class of veterinary drugs used for the treatment and prevention of certain bacterial and protozoal diseases in poultry, swine dysentery and genital trichomoniasis in cattle. Since the safety assessment of nitroimidazoles showed them to be genotoxic, carcinogenic and mutagenic, a number of nitroimidazoles have been banned for therapeutic purposes in different countries. Moreover, nitroimidazoles have also been extensively used as growth promoters in food-producing animals. Due to their efficacious improvement in meat production and feed conversion, deliberate use still exists. Therefore, the illegal use of nitroimidazoles in animal husbandry must be monitored. A sensitive method based on LC-MS/MS for the simultaneous determination and confirmation of five banned nitroimidazole drugs including metronidazole, ronidazole, dimetridazole, metronidazole-OH (metabolite of metronidazole), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (metabolite of ronidazole and dimetridazole) in bovine muscle, using ronidazole-d3 as an internal standard, was developed and validated. After extraction with ethyl acetate and evaporation, the nitroimidazoles were reconstituted in petroleum ether and purified, and LC-MS/MS analysis was performed. The method was validated according to Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Parameters such as decision limit (CCα), detection capability (CCß), precision, accuracy, uncertaincy and ruggedness were determined. Average accuracy of the five nitroimidazoles from bovine muscle fortified at 5 levels (0.5, 1.0, 1.5, 2.0 and 2.5 µg kg(-1)) ranged from 96% to 103%. The calculated CCα ranged from 0.0 to 0.17 µg kg(-1); CCß ranged from 0.08 to 0.41 µg kg(-1). A complete statistical analysis was performed and the results indicate that the method is robust when subjected to day-to-day analytical variations.

Prediction of drug dissolution profiles from tablets using NIR diffuse reflectance spectroscopy: a rapid and nondestructive method.

A comparison between dissolution profiles obtained by using a dissolution apparatus (conventional method) and the NIR diffuse reflectance spectra of a series of clonazepam-containing batches is reported. Ten different formulations with fixed amount of clonazepam and varying proportions of excipients were analyzed at seven dissolution times and three different media. The percentages of dissolution of each sample were correlated with the NIR spectra of three tablets of each batch, through a multivariate analysis using the PLS regression algorithm. The squared correlation coefficients for the plots of percentages of dissolution from the equipment laboratory (dissolution apparatus and HPLC determination) versus the predicted values, in the leave-one-out cross-validation, varied from 0.80 to 0.92, indicating that the NIR diffuse reflectance spectroscopy method is an alternative, nondestructive tool for measurement of drug dissolution from tablets.