Prof. Felix G.R. Reyes

DCA / FEA / UNICAMP

E mail: reyesfgr@unicamp.br

Grupo de Toxicologia de Alimentos e Fármacos

São Paulo, SP, Brazil30 August, 2017

Veterinary drugs: development and validation of bioanalytical methods using

LC-MS/MS and MALDI-TOF-MS and its applications in sanitary surveillance

and the development of fish farming in Brazil

Biopharma and Metabolomics - A Fapesp and Agilent Workshop

Summary

Introduction

Activities already performedPapers publishedPapers in the final stage of preparationBook chaptersSymposium organized

Activities been conducted

Other activitiesPapers published

Comments

Felix G. Reyes

Felix G. Reyes

Advancement of analytical techniques (LC-MS/MS and MALDI-

TOF-MS) in the determination of veterinary drugs residues in

foods, fish in particular.

Improvement of fish farming in Brazil, taking into

consideration the protection of the human and environmental

health in relation to the use of veterinary drugs.

GENERAL OBJECTIVE

SPECIFIC OBJECTIVES

1. Production of medicated feed for fish.

2. Development and validation of analytical methods for

the quantification of veterinary drug residues in feed,

plasma and fish fillet (LC-MS/MS and MALDI-TOF-MS).

3. Pharmacokinetic studies of veterinary drugs under the

Brazilian environmental conditions of fish farming.

4. Residue depletion studies of veterinary drug residues

and establishment of the withdrawal period under

Brazilian environmental conditions for fish farming.

Why to use of veterinary drugs in fish farming ?

• Artificial feed

• handling

• High density

• Virus

• Bacteria

• Fungi

• Parasites

Estresse

Felix G. Reyes

Aproved veterinary drugs in fish farming in Brazil

02 Antimicrobials (2008)

Use of veterinary

medicine permitted

for others species

OxitetraciclineFlorfenicol

Masoten / Triclorfon

Felix G. Reyes

01 Parasiticide (2009)

Illegal use

Felix G. Reyes

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Felix G. Reyes

Acknowledgments Funding for this study was provided by São Paulo Research Foundation (FAPESP; Grant # 2013/50452-5) and bythe Brazilian National Council for Scientific and Technological Development (CNPq; Process No. 305390/2013-9). The authors sincerely thank the reviewers whose comments and suggestions helped to improve the manuscript.

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Felix G. Reyes

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Multiresidue method for quantification of sulfonamides and

trimethoprim in tilapia fillet by liquid chromatography

coupled to quadrupole time-of-flight mass spectrometry

using QuEChERS for sample preparation.

Felix G. Reyes

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

De: "Chemosphere" Para: "claudio jonsson" Enviadas: Segunda-feira, 3 de julho de 2017 15:48:50

Bioaccumulation and depletion study of sulfamethazine in tilapia (Oreochromis niloticus) treated with a medicated feed.

Kátia S. D. Nunes, José Henrique Vallim, Márcia R. Assalin, Sonia C. N. Queiroz, Lourival C. Paraíba, Claudio M. Jonsson, Felix G. R. Reyes

Felix G. Reyes

Aspects related to the use of hormones in fish

farming and to the determination of their

residues in fish muscle: a review

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Development and Validation of Analytical Method for the Determination of 17β-Estradiol Residue in Tambaqui by LC-MS/MS

and its Application in a Fish Sexual Reversion Study

17--estradiol

Felix G. Reyes

Paper in the final stage of preparation to be submitted for publication

Celia A. Hoga, Karine V. G. Reche, Fernanda L. Almeida, Vanessa R. Reis, Rafaelle P. Cordeiro, Felix G. R. Reyes

Hormone treatment flowchart.

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Pharmacokinetics of florfenicol and florfenicol amine in tambaqui

(Colossoma macropomum)

Luciana da Conceição Castello Branco, Fabiana Pilarski, Edsandra Campos

Chagas, Vera Lúcia Ferracini, Arturo Anadón, Felix Guillermo Reyes Reyes

Papers in the final stage of preparation to be submitted for publication

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

0,010

0,100

1,000

10,000

0 20 40 60 80 100 120 140 160 180 200

log c

on

cen

trati

on

(µ

g/m

L)

Time (h)

FF FFA

florfenicol Florfenicol amine

Florfenicol and florfenicol amine residue depletion in tambaqui

muscle (Colossoma macropomum)

Castello Branco LC; Pilarski F; Chaga EC; Ferracini VL; Arturo Anadón A,

Reyes FGR

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

Papers in the final stage of preparation to be submitted for publication

florfenicol Florfenicol amine

0,000

0,100

0,200

0,300

0,400

0 2 4 6

x 1

0-3

µg/k

g

Days

FF + FFA

Enrofloxacin and ciprofloxacin residue levels reduction during

fishburgers processing and frying

Branco LCC., Rodrigues, MVN., Reyes FGR.

Papers in the final stage of preparation to be submitted for publication

Act

ivit

y al

read

y p

erf

orm

ed. F

ish

far

min

g

florfenicol Florfenicol amine

uncooked fried

Tilapia feed medicated with florfenicol: study of the use of different

polymers for coating the feed and the leaching of the drug to water

Barreto FM., Silva MR da, Braga PA de C., Reyes FGR.

Act

ivit

y u

nd

er

pro

gres

s. F

ish

far

min

g

12 fish200 g/peixe

Tank 1000 L72 g Feed/day

TOTAL FEED/DAY3% BW

½ morning36 g

½ evening

MEDICATED FEED: 20 gCOMMERCIAL FEED: 16 g

DOSE FLORFENICOL:10 mg/kg BW

24 mg FL/tanK

Capítulo 10: Antimicrobials in fishfarming: use and regulatoryaspects in Brazil. 2014. ISBN: 978-85-8017-098-6.

Chapter 4:Ensuring Food Safety in Developing and Developed Countries: Aspects Associated with the Use of Veterinary Drugs in Fish Farming in Brazil. 2017. ISBN 978-1-4939-6494-9; ISBN 978-1-4939-6496-3 (eBook).

Capítulo 33: Chloranfenicol: quantitation in food of animal origen by LC-ESI-MS/MS. 2016. ISBN: 978-85-277-2833-1.

Felix G. Reyes

Attendance from different units of UNICAMP (FEA, IQ, FCM, FEAGRI, CPQBA)

From different institutions (LANAGRO, University of the State of Mato Grosso, Biological Institute/SP, EMBRAPA, LabFor Laboratory Analyses Ltda, UNESP, ITAL).

Act

ivit

y al

read

y p

erf

orm

ed. C

ou

rse

Development and validation of a UPLC-MS/MS multiresidue

method for quantitation of veterinary drug residues in fish

Target molecules: antimicrobials (n=28)

Fish samples: tilapia file available from the retail market and fish & pay

Act

ivit

y b

een

co

nd

uct

ed

Development and validation of a multiresidue method for

quantitation of anthelmintic veterinary drugs in tambaqui fillets by

LC-MS/MS

TambaquiAct

ivit

y b

een

co

nd

uct

ed

MALDI-TOF/MS application: development of analytical methods for

determination of antimicrobial residues in fish by MALDI-TOF/MS

MALDI-TOF-MS has been timidly employed in the analysis of low

molecular weight compounds, such as veterinary drugs.

MALDI-TOF-MS is a very powerful tool for analysis of biomolecules

such as proteins, polysaccharides, polymers and even in the

identification of intact bacteria .

Target molecules: Quinolones

Collaboration with ThoMSon Mass Spectrometry Laboratory

IQ/UNICAMP Brazil

Act

ivit

y b

een

co

nd

uct

ed

Aditional studies conducted on the AgilentUHPLC-MS/MS

PITE - Research Program in Partnership for

Technological Innovation

Our Partner is Agilent Technologies

UHPLC-MS/MS

Felix G. Reyes

2.5 Method comparisonThe results obtained with the proposed method were compared to those obtained by two different methods. The first was based on an enzymatic detector (AS-310 Biotech Analyser – Sakura, Tokyo, Japan). The second was an UHPLC–MS/MS (Agilent 6460 triple quadrupole tandem mass spectrometercoupled with a UHPLC 1290 system with ESI source in the positive ion mode). Details of the UHPLC method can be found in the Supporting Information.

Oth

er a

ctiv

ity

Felix G. Reyes

AcknowledgmentsThe authors would like to thank the São Paulo Research Foundation-Agilent Technologies for supplying the Agilent UHPLC 1290-MS/MS 6460 system employed in this study (FAPESP-Agilent Grant number 2013/50452-5), and the funding support from the Coordination for the Improvement of Higher Level Education Personnel (Process PROEX/CAPES number 3300301702P1) and the National Council for Scientific and Technological Development (Process CNPq number 483839/2010-8 and 305390/2013-9).

Oth

er a

ctiv

ity

Felix G. ReyesSuffolk Lamb

AcknowledgementsThe authors would like to thank the São Paulo ResearchFoundation-Agilent Technologies for supplying the AgilentUHPLC 1290-MS/MS 6460 system employed in this study(FAPESP-Agilent Grant number 2013/50452-5). We also thankDr. Lenore Kelly, from Agilent Technologies, for her assistancewith the language correction of the manuscript.

Cruz M Del BA, Fernandes MAM, Braga PA de C, Monteiro ALG, Daniela Daniel and Reyes FGR.

Oth

er a

ctiv

ity

Felix G. Reyes

Activities already performed

Papers published 5

Papers submitted for publication 3

Papers in the final stage of preparation 4

Book chapters 3

Ms and PhD Theses 4

Intensive Course 1

Activities been conducted

Multiresidue methods by LC-MS/MS 2

Analytical method by MALDI-TOF 1

Difficulties faced during the progress of the project

Successive repairs of the Agilent LC-MS/MS system.

Unforeseen events.

Suggestion

Allow the partner company to grant additives without FAPESP counterpart.

Comments

http://www.e-science.unicamp.br/gtaf/