Flávio Alves Santos

Multiresidue determination of fluoroquinolones in poultry muscle and kidney according to the regulation 2002/657/EC. A systematic comparison of two different approaches: Liquid chromatography coupled to high-resolution mass spectrometry or tandem mass spectrometry

This work involved the optimization and validation of two methods according to the Commission Decision 2002/657/EC directives for determining fluoroquinolones residues in samples of poultry muscle and kidney: ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pipemidic acid and sarafloxacin. The extraction procedure was based on a QuEChERS approach, whose optimization employed a Box-Behnken 3(3) factorial design. A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for determining the twelve analytes using the multiple reaction monitoring mode (MRM). Accuracy, evaluated by recovery studies, varied from 88.8 to 112.2% for the selected levels with RSD values lower than 12.3%. The second validated method employed high resolution mass spectrometry (HRMS) performed in the single ion monitoring mode (SIM), determining nine among twelve analytes. The validation parameters were evaluated as satisfactory, with recoveries from 82.5 to 114.4% and RSD lower than 8.7%. Decision limits and detection capabilities for both methods were reported. The two methods were statistically compared using the Students t test, at 95% confidence level, resulting in no significant difference.

A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish

The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg-1), suggesting possible contamination or illegal use of that antibiotic.

Development and validation (according to the 2002/657/EC regulation) of a method to quantify sulfonamides in porcine liver by fast partition at very low temperature and LC-MS/MS

A novel multi-residue method for the quantification of 15 sulfonamides in porcine liver is described. It involves the application of a liquid-liquid extraction with fast partition at very low temperature (LLE-FPVLT) procedure followed by HPLC-MS/MS (high performance liquid chromatography coupled to tandem mass spectrometry) analysis. By this innovative method, acetonitrile is added to a minced porcine liver sample and the resulting suspension centrifuged and immersed in a container with liquid nitrogen for 15 s. The acetonitrile phase, which remains liquid under these conditions, is isolated, evaporated to dryness, recomposed with formic acid 0.1% v/v, and injected into the liquid chromatograph. The whole analytical procedure was validated according to the European Commission Decision 2002/657/EC and acceptable values (except for sulfanilamide) were obtained for the following parameters: linearity (0.97 < R2 < 0.99), decision limit (107.70 µg kg-1 < CCα < 128.65 µg kg-1), detection capability (115.40 µg kg-1 < CCβ < 157.29 µg kg-1), limit of detection (5.58 µg kg-1 < LOD < 16.75 µg kg-1), limit of quantification (18.41 µg kg-1 < LOQ < 55.26 µg kg-1), accuracy (recovery rates), precision (repeatability, intermediate precision, and measurement uncertainty tests), selectivity, and robustness. Recoveries higher than 70%, at three concentration levels (0.5, 1.0 and 1.5 of the maximum residue limit, MRL), were attained for the majority of the sulfonamides. These very promising results point to the inclusion of the present methodology into the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.

Validation of an LC-MS/MS method for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in fish and shrimp

A quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous analyses of malachite green (MG), crystal violet (CV) and its major metabolites, leucomalachite green (LMG) and leucocrystal violet (LCV) residues in fish and shrimp samples has been validated. Fish and shrimp samples were extracted with citrate buffer/acetonitrile, and the extracts were purified on strong cation-exchange (SCX) solid-phase extraction (SPE) cartridge. After conversion of LMG into MG using a post column oxidation reactor containing lead (IV) oxide (PbO2), the effluents were analysed. Residues were analysed using positive-ion electrospray ionisation (ESI). Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). Validation of the method was carried out in accordance with the Decision 2002/657/EC, which establishes criteria and procedures for the validation of methods. The following parameters were determined: decision limit (CCα), detection capability (CCβ), linearity, accuracy, precision, selectivity, specificity and matrix effect. The decision limits (CCα) for MG, LMG, CV and LCV were 0.164, 0.161, 0.248 and 0.860 µg kg–1. The respective detection capabilities (CCβ) were 0.222, 0.218, 0.355 and 1.162 µg kg–1. Typical recoveries (intermediate precision) in shrimp, for MG, CV, LMG and LCV for 2.0 µg kg–1 level fortified samples using the optimised procedure were in the range 69%, 97%, 80.3% and 71.8%, respectively. The findings demonstrate the suitability of the method to detect simultaneously MG, CV and its metabolite (LMG and LCV) in fish and shrimp.

Development and validation of an efficient and innovative method for the quantification of multiclass veterinary drugs in milk by using LC–MS/MS analysis

An efficient analytical method for the simultaneous quantification of 27 veterinary drugs from five different classes (benzimidazoles, betalactams, quinolones, sulfonamides and tetracyclines) in bovine milk using a novel extraction procedure (liquid–liquid extraction with fast partition at very low temperature) and liquid chromatography coupled to tandem mass spectrometry is described. The full analytical method was validated according to Brazilian legislation (normative instruction SDA/MAPA 24/2009) and suitable values were achieved for all the parameters evaluated. Hence, the recoveries were within the established ranges (>70%), except for sulfathiazole (67.4% at the 0.5 × MRL level) and marbofloxacin and thiabendazole (112.5 and 114.6% respectively, at the 1.0 × MRL level). The repeatability and intermediate precision for all the analytes, expressed as relative standard deviations, were lower than 20 and 25%, respectively. The decision limits (CCα), detection capabilities (CCβ) and expanded uncertainties were also calculated and satisfactory results were obtained. Finally, the method was applied to 15 actual samples and traces of oxytetracycline and sulfamethazine were detected in three of them.

Quinolones and tetracyclines in aquaculture fish by a simple and rapid LC-MS/MS method

The determination of antimicrobials in aquaculture fish is important to ensure food safety. Therefore, simple and fast multiresidue methods are needed. A liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of 14 antimicrobials (quinolones and tetracyclines) in fish. Antimicrobials were extracted with trichloroacetic acid and chromatographic separation was achieved with a C18 column and gradient elution (water and acetonitrile). The method was validated (Decision 2002/657/EC) and it was fit for the purpose. Linearities were established in the matrix and the coefficients of determination were ≥0.98. The method was applied to Nile tilapia and rainbow trout (n = 29) and 14% of them contained enrofloxacin at levels above the limit of quantification (12.53-19.01 µg.kg-1) but below the maximum residue limit (100 µg.kg-1). Even though prohibited in Brazil and other countries, this antimicrobial reached fish. Measures are needed to ascertain the source of this compound to warrant human safety.

Multiresidue method for identification and quantification of avermectins, benzimidazoles and nitroimidazoles residues in bovine muscle tissue by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using a QuEChERS approach

A quantitative and confirmatory multiresidue method for determining the presence of avermectins, benzimidazoles and nitroimidazoles in bovine muscle tissue by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed, optimized and validated, using a QuEChERS extraction. The evaluated performance parameters were linearity, selectivity, matrix effect, decision limits (CCα), detection capability (CCβ), limits of detection (LOD), limits of quantification (LOQ), accuracy, precision and robustness. The validated method exhibited linearity with coefficient of determination (R2) higher than 0.90 in the working range from 0.5 to 2.0 times the maximum residue limit (MRL) or the minimum required performance level (MRPL) for the studied analytes, except for closantel, for which the linear study range was defined from 50 to 200µgkg-1. The method was selective in the presence of macrolides and lincosamides for all the studied analytes. The LOD varied from 0.007 to 66.715µgkg-1, whereas LOQ values ranging from 0.011 to 113.674µgkg-1 were found. The results of the evaluation of the accuracy and precision were satisfactory for all the studied analytes, and according to the assessment of the robustness, the method was not robust only for the analytes abamectin, moxidectin, doramectin fenbendazole sulfone, closantel, thiabendazole, hydroxyl-metronidazole and ronidazole. The performance parameters demonstrated total method adequacy for the detection and quantification of avermectins, benzimidazoles and nitroimidazoles residues in bovine muscle tissues.

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, β-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.

Validation of a LC-MS/MS Multiresidue Methodology Based on a QuEChERS Approach for the Determination of Fluoroquinolones, Sulfonamides and Trimethoprim in Poultry and Porcine Kidney According to the Normative Instruction 24/2009-MAPA

This work involved the validation of a multiresidue method according to the Normative Instruction 24/2009-MAPA for determining 25 analytes, among fluoroquinolones, sulfonamides and trimethoprim in samples of poultry and porcine kidney. The extraction procedure was based on a QuEChERS approach. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed using the selected reaction monitoring mode (SRM) and ESI+ ionization. All of the validated figures of merit were evaluated as satisfactory. Accuracy was assessed by recovery studies, varying from 82.7 to 115.5% for porcine kidney and from 91.5 to 110.4% for poultry kidney. Relative standard deviations were lower than 25.5% for porcine kidney, and 29.8% for poultry kidney. Decision limits (CCα) comprised values from 10.37 to 3298.43 µg kg-1 for porcine kidney and 10.08 to 3176.59 µg kg-1 for poultry kidney. Detection capabilities (CCβ) varied from 10.73 to 3396.86 µg kg-1 for porcine kidney and 10.67 to 3253.19 µg kg-1 for poultry kidney. The developed method has been successfully employed in the routine analysis of incurred samples.

Application of a 33 Box-Behnken Design to Optimize the Extraction of Eleven Fluoroquinolones from Poultry Muscle and Kidney Using a QuEChERS Approach via Liquid Chromatography Tandem Mass Spectrometry: the Easy Use of Microsoft Excel® in Multivariate Analysis

This work presents an optimization of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction approach of eleven fluoroquinolones in poultry muscle and kidney using a 33 Box-Behnken factorial design. All the data treatment was performed using Microsoft Excel® 2010. The suitability of the developed method was confirmed by two proficiency tests for ciprofloxacin and enrofloxacin.