Letícia Rocha Guidi

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.

Bioactive amines in soy sauce: validation of method, occurrence and potential health effects.

The objective of this study was to investigate the levels of bioactive amines in soy sauce. A method for the extraction of amines was optimized and an ion pair-HPLC method was validated. Overall, tyramine was the prevalent amine, followed by putrescine, histamine, phenylethylamine and cadaverine. The concentrations of amines varied widely among samples. The brands could be divided into two groups. The first one contained three amines; there was prevalence of cadaverine followed by tyramine and putrescine; and total amine levels were low. The second group contained four amines; there was prevalence of tyramine followed by histamine, phenylethylamine and putrescine; and total amine levels were high. A brand with lower NaCl levels contained significantly higher amine levels. Based on the levels of amines detected, a high percentage of samples could cause adverse effects to human health.

Advances on the chromatographic determination of amphenicols in food

Antibiotics are widely used in veterinary medicine to treat and prevent diseases and their residues can remain in food of animal origin causing adverse effects to human health. Amphenicols (chloramphenicol, thiamphenicol, and florfenicol) may be found in foodstuffs, although the use of chloramphenicol has been prohibited in many countries due to its high toxicity. Since these antibiotics are usually present at trace levels in food, sensitive and selective techniques are required to detect them. This paper reviews analytical methods used since 2002 for the quantitative analysis of amphenicols in food. Sample preparation and separation/detection techniques are described and compared. The advantages and disadvantages of these procedures are discussed. Furthermore, the worldwide legislation and occurrence of these antibiotics in food matrices as well as future trends are also presented.

Quality assurance of histamine analysis in fresh and canned fish.

Histamine determination is relevant for fish safety, quality and trade. Recently a study by the European Union (EU) compared the Codex and the EU mandated methods for the analysis of histamine and observed that they underestimated and overestimated the results, respectively. To solve this problem, a simple and efficient procedure for the extraction and quantification of histamine by ion-pair HPLC method with post-column derivatization and fluorimetric detection is proposed. It was optimized and validated for the analysis of histamine in fish. The method attended the performance criteria established by Commission Decision 2002/657/CE. The method was also submitted to proficiency testing; uncertainty was calculated; and the stability of solutions and standards was investigated. There was no matrix effect. The LOD, LOQ, CCα and CCβ were fit for the purpose. The method was successfully used in the analyses of freshwater fish and fresh and canned tuna.

Synephrine – A potential biomarker for orange honey authenticity

A LC-MS/MS method for synephrine as a biomarker for orange honey authenticity was developed and validated. The sample was extracted with 5% TCA and cleaned up with Florisil providing 83.7% recoveries. Ions transitions for quantification and identification were 168→135.0 and 168→107.0, respectively. The limits of detection and quantification were 0.66 and 1.0ng/g, respectively. Synephrine was detected in orange honey at levels from 79.2 to 432.2ng/g, but not in other monofloral honeys. It was also present in some wildflower honeys (9.4-236.5ng/g), showing contribution of citrus to this polyfloral honey. Results were confirmed by qualitative pollen analysis. No citrus pollen was detected in honey containing synephrine levels ≤43.8ng/g, suggesting that synephrine in honey is more sensitive compared to pollen analysis. Synephrine was found in citrus but not in other apiculture flowers. Therefore, synephrine is a botanical marker to differentiate and attest authenticity of orange honey.

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.

Matrix effect on the analysis of amphenicols in fish by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Matrix effect is an important parameter to be investigated during the development and validation of a method for the quantitative determination of contaminants in food. The objective of this study was to evaluate the matrix effect, through statistical tests, in the quantification of amphenicols in fish by HPLC-MS/MS. The study was performed by comparing the standard curves prepared in solvent solutions and in a fish sample previously known to be free of amphenicols. Since matrix effect was observed for the three analytes, calibration curves for quantification of chloramphenicol, thiamphenicol and florfenicol should be constructed using the matrix.

A simple and rapid LC–MS/MS method for the determination of amphenicols in Nile tilapia

A quantitative method for analysis of amphenicols (chloramphenicol - CAP, thiamphenicol - TAP and florfenicol - FF) in Nile tilapia using LC-MS/MS is described. A simple sample preparation procedure was optimized using a Plackett-Burman design. The method was validated in accordance with Decision 2002/657/EC. Repeatability and reproducibility were less than 10.7% and 16%, respectively, for all compounds. Recoveries varied from 79.8% to 92.0%. CCα was 0.019, 54.81 and 54.93 μg.kg-1 for CAP, FF and TAP, respectively. CCβ was 0.068, 64.88 and 58.91 μg.kg-1 for CAP, FF and TAP, respectively. Limits of quantification (LOQ) were 12.5 μg.kg-1 for FF and TAP and 0.15 μg.kg-1 for CAP. Nile tilapia fillets (n = 32) analyzed did not contain chloramphenicol. Thiamphenicol was detected in one sample (3.1%) and florfenicol was detected in every sample, all of them at concentrations below the maximum residue limit.