Validation of an UHPLC-MS/MS Method for Screening of Antimicrobial Residues in Eggs and Their Application to Analyses of Eggs from Laying Hens Subjected to Pharmacological Treatment.

A multiresidue method by UHPLC/MS-MS was optimized and validated for the screening and semiquantitative detection of antimicrobials residues from tetracyclines, aminoglycosides, quinolones, lincosamides, ß-lactams, sulfonamides, and macrolides families in eggs. A qualitative approach was used to ensure adequate sensitivity to detect residues at the level of interest, defined as maximum residue limit (MRL), or less. The applicability of the methods was assessed by analyzing egg samples from hens that had been subjected to pharmacological treatment with neomycin, enrofloxacin, lincomycin, oxytetracycline, and doxycycline during five days and after discontinuation of medication (10 days). The method was adequate for screening all studied analytes in eggs, since the performance parameters ensured a false-compliant rate below or equal to 5%, except for flumequine. In the analyses of eggs from laying hens subjected to pharmacological treatment, all antimicrobial residues were detected throughout the experimental period, even after discontinuation of medication, except for neomycin, demonstrating the applicability of the method for analyses of antimicrobial residues in eggs.

Evaluation of the alpha-amylase activity as an indicator of pasteurization efficiency and microbiological quality of liquid whole eggs.

In order to evaluate the efficiency of the pasteurization process in liquid whole eggs, an UV/visible spectrophotometric method was developed and validated for the assessment of alpha-amylase activity. Samples were collected from 30 lots of raw eggs (n = 30) and divided into three groups: one was reserved for analysis of the raw eggs, the second group was pasteurized at 61.1°C for 3.5 minutes (n = 30), and the third group was pasteurized at 64.4°C for 2.5 minutes (n = 30). In addition to assessing alpha-amylase activity, the microbiological quality of the samples was also evaluated by counting total and thermotolerant coliforms, mesophilic aerobic microorganisms, Staphylococcus spp., and Salmonella spp. The validated spectrophotometric method demonstrated linearity, with a coefficient of determination (R2) greater than 0.99, limits of detection (LOD) and quantification (LOQ) of 0.48 mg kg-1 and 1.16 mg kg-1, respectively, and acceptable precision and accuracy with relative standard deviation (RSD) values of less than 10% and recovery rates between 98.81% and 105.40%. The results for alpha-amylase activity in the raw egg samples showed high enzyme activity due to near-complete hydrolysis of the starch, while in the eggs pasteurized at 61.1°C, partial inactivation of the enzyme was observed. In the samples of whole eggs pasteurized at 64.4°C, starch hydrolysis did not occur due to enzyme inactivation. The results of the microbiological analyses showed a decrease (P < 0.0001) in the counts for all the studied microorganisms and in the frequency of Salmonella spp. in the pasteurized egg samples according to the two binomials under investigation, compared to the raw egg samples, which showed high rates of contamination (P < 0.0001). After pasteurization, only one sample (3.33%) was positive for Salmonella spp., indicating failure in the pasteurization process, which was confirmed by the alpha-amylase test. It was concluded that the validated methodology for testing alpha-amylase activity is adequate for assessing the efficiency of the pasteurization process, and that the time-temperature binomial used in this study is suitable to produce pasteurized eggs with high microbiological quality.

Evaluation of the Presence and Levels of Enrofloxacin, Ciprofloxacin, Sulfaquinoxaline and Oxytetracycline in Broiler Chickens after Drug Administration.

The depletion times of enrofloxacin and its metabolite ciprofloxacin as well as sulfaquinoxaline and oxytetracycline were evaluated in broiler chickens that had been subjected to pharmacological treatment. The presence and residue levels of these drugs in muscle tissue were evaluated using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method that was validated in this work. The results showed the presence of all antimicrobial residues; however, the presence of residues at concentrations higher than the drugs' maximum residue limit (MRL) of 100 µg kg-1 was found only during the treatment period for oxytetracycline and until two days after discontinuation of the medication for enrofloxacin, ciprofloxacin and sulfaquinoxaline. It was concluded that the residues of all antimicrobials were rapidly metabolized from the broiler muscles; after four days of withdrawal, the levels were lower than the limit of quantification (LOQ) of the method for the studied analytes.

HPLC-UV method validation for the identification and quantification of bioactive amines in commercial eggs.

A quantitative and confirmatory high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of bioactive amines in the albumen and yolk of commercial eggs was developed, optimized and validated by analyte extraction with trichloroacetic acid and pre-column derivatization with dansyl chloride. Phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine standards were used to evaluate the following performance parameters: limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, precision, recovery and ruggedness. The LoD of the method was defined from 0.2 to 0.3 mg kg(-1) for the yolk matrix and from 0.2 to 0.4 mg kg(-1) for the albumen matrix; the LoQ was from 0.7 to 1.0 mg kg(-1) for the yolk matrix and from 0.7 to 1.1 mg kg(-1) for the albumen matrix. The validated method exhibited excellent selectivity and separation of all amines with coefficients of determination higher than 0.99. The obtained recovery values were from 90.5% to 108.3%, and the relative standard deviation (RSD) was lower than 10% under repeatability conditions for the studied analytes. The performance parameters show the validated method to be adequate for the determination of bioactive amines in egg albumen and yolk.