enhanced stability and detection window of … · methodologies in analytical chemistry research...

ENHANCED STABILITY AND DETECTION WINDOW OF LARGE AND SMALL MOLECULES PROHIBITED IN SPORTS BY URINE MICROSAMPLING

Pharmaco-Toxicological Analysis Laboratory (PTA Lab) Department of Pharmacy and Biotechnology (FaBiT) Alma Mater Studiorum – University of Bologna

Michele Protti

4th EBF Young Scientists SymposiumThe BioA Brain

Embracing New Ideas15-16 March 2018 - Ghent, Belgium

My progress in bioanalytical research @ PTA Lab

4th EBF Young Scientists Symposium - The BioA Brain: Embracing New Ideas 15-16 March 2018 - Ghent, BelgiumMichele Protti

miniaturised pretreatment of matrix microvolumes dried micromatrix approaches

J Pharm Biomed Anal. 88 (2014) 467-471.

Bioanalysis. 6 (2014) 1487-1495.

J Sep Sci. (2017) in press.

Ph.D.2nd YSS 3rd YSS 4th YSS

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Michele Protti

classic drugs of abuse in alternative specimens NPS in innovative biological microsamples

Ph.D.2nd YSS 3rd YSS 4th YSS

J Pharm Biomed Anal. 76 (2013) 119-125. J Chromatogr B 1044-1045 (2017) 77-86.

J Pharm Biomed Anal. 123 (2016) 186-194. J Pharm Biomed Anal. 152 (2018) 204–214.


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4th EBF Young Scientists Symposium - The BioA Brain: Embracing New Ideas 15-16 March 2018 - Ghent, Belgium

L. Mercolini, M. Protti, Biosampling strategies for emerging drugs of abuse: towards the future of toxicological and forensic analysis, J Pharm Biomed Anal130 (2016) 202-219.L. Mercolini, M.A. Saracino MA, M. Protti, Current advances in biosampling for therapeutic drug monitoring of psychiatric CNS drugs. Bioanalysis. 7 (2015) 1925-1942.

in order to tackle many bioanalytical issues

Ø Minimally invasive sampling

Ø In situ, POC sample collection

Ø Enhanced subject compliance

Ø Minimal sample handling

Ø High-throughput, automated processing

Ø Enhanced sample stability

Ø Simplified sample storage and shipping

Michele Protti


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Recent bioanalytical research @ PTA Lab

Dried microsampling

TDM of CNS drugs

Drugs of abuse

Anti-doping analysis

Alcohol consumption

Application areas

Wet microsampling

DMS

VAMS

MicroSPE

LC-SPME

MEPS

Michele Protti

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Recent bioanalytical research @ PTA Lab

Dried microsampling

TDM of CNS drugs

Drugs of abuse

Anti-doping analysis

Alcohol consumption

Application areas

Wet microsampling

DMS

MicroSPE

LC-SPME

MEPS

VAMS

Michele Protti

4


Critical issues in urine analysis for doping controls

Ø Compound stability

Ø Bacterial degradation

Ø Shipping and storage at controlled

and certified temperatures

Ø Space-demanding samples

Ø Limited detection window

Crucial for:

• ”B sample” delayed reanalysis

• Rapidly degraded compounds

•Microdosing

Ojanperä S., Leinonen A., Apajalahti J., Lauraeus M., Alaja S., Moisander T., Kettunen A. Characterization of microbial contaminants in urine. Drug Testing and Analysis. 2, 576-581, 2010. Kuenen G., Konings W.N. The importance of cooling of urine samples for doping analysis. Accreditation and Quality Assurance 15, 133-136, 2010. Lempiäinen A., Hotakainen K., Alfthan H., Stenman U.H. Loss of human chorionic gonadotropin in urine during storage at -20°C. Clinica Chimica Acta 413, 232-236, 2012.

Michele Protti

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Microsampling approach

Michele Protti

VAMS sampler consists of a polymeric tip

which absorbs an accurate sample volume

(10/20 µL) after few seconds by wicking

Courtesy of Neoteryx LLC, CA, USA

Clamshell Cartridge 96-well format Automation

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Cathinone analogues in plasma, urine and oral fluid VAMS

Michele Protti

Chromatographic separationRP SB C18 column (50 x 2.1 mm, 3.5µm) + guard column0.1% FA in H2O/0.1% FA in ACN, composition gradient

Mass spectrometryTriple quadrupole, MRM, ESI+

VAMS extraction100% MeOHUltrasound-assisted extraction, 15 minVortex agitation, 1 min

StimulantsClass S6. (prohybited in competition)

7L. Mercolini, M. Protti, M.C. Catapano, J. Rudge, A.E. Sberna LC-MS/MS and volumetric absorptive microsampling for quantitative bioanalysis of cathinone analogues in dried urine, plasma and oral fluid samples.Journal of Pharmaceutical and Biomedical Analysis 123 (2016) 123:186


Michele Protti

Cathinone analogues in plasma, urine and oral fluid VAMS

Non-haematic matrix VAMS sampling assaysGravimetric assays performed on plasma, urine and oral fluid sampled by VAMS

Comparative stability assaysMid-term stability on blank spiked urine: fluid matrix storage @ -80 °C vs VAMS @ RT

0

5

10

plasma urine oral fluid

volu

me

(µL)

matrix (n=6)

Pipetted vs. absorbed volume

pipetted volume

absorbed volume

0

5

10

2s 4s 8s 12s

volu

me

(µL)

sampling time

Absorbed volume vs. sampling time

plasmaurineoral fluid

707580859095

100

% p

eak

are

a707580859095

100

0 5 10 15 20 25 30

Time (days)

Fluid urine, -80°C

Urine VAMS, RT

4-MEC Mephedrone MDPV 8L. Mercolini, M. Protti, M.C. Catapano, J. Rudge, A.E. Sberna LC-MS/MS and volumetric absorptive microsampling for quantitative bioanalysis of cathinone analogues in dried urine, plasma and oral fluid samples.Journal of Pharmaceutical and Biomedical Analysis 123 (2016) 123:186


WADA research project 2018

Enhanced urinary stability and detection window doping agents by dried urine microsampling

2017 WADA RESEARCH GRANTSSECTION A:

Detection of doping substances/methods:Methodologies in analytical chemistry

Research project reference number: 17A20LMProject duration: 12 months (starting January 10th 2018)

Michele Protti

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Aims of the research project

Ø Design and optimisation of feasible, reliable protocols for urine microvolumes collectionØ Development of high-throughput, automated DUS and VAMS sample processing and analysis Ø Development and validation of analytical methods for doping substancesØ Stability assessment of dried urine microsamples stored at different conditions Ø Parallel degradation studies in fluid urine stored at temperature-controlled conditions

Dried urine spot (DUS)

Volumetric absorptive microsampling (VAMS)

Michele Protti

WADA research project 2018

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Take-home message

Michele Protti

Microsampling technologies and procedures are very promising for quali-quantitative analysis of doping agents in miniaturised dried urinary samples (as well as haematic matrices and oral fluid)

Miniaturised dried samples allow simplified, yet effective workflow within the bioanalytical laboratory

Effective applications for sport drug testing (both in-competition and out-of-competition) where on-field sampling and high-throughput processing are needed

Promising results from stability assays could enhance actual procedure and extend detection window of substances prohibited in sports

Full bioanalytical validation is required across a wide range of analytes with multi-matrix comparative approach

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Acknowledgements

Michele Protti

LTN Renzo Ferrante

Arma dei Carabinieri Anti-adulteration and Health Unit (NAS)Firenze (Italy)

Angelo E. Sberna

Sports Medicine, Local Health UnitEnna (Italy)

Paolo Sberna

Department of MicroelectronicsDelft University of TechnologyDelft (NL)

James Rudge

Neoteryx® Llc

Roberto Mandrioli

QuVi DepartmentUniversity of BolognaRimini (Italy)

Manuela Larocca

PTA Lab, FaBiT DepartmentUniversity of BolognaBologna (Italy)


Laura Mercolini

Head of PTA Lab, FaBiT DepartmentUniversity of BolognaBologna (Italy)

THANKS!Any questions?

You can find me also at poster [email protected]

4th EBF Young Scientists SymposiumThe BioA Brain

Embracing New Ideas15-16 March 2018 - Ghent, Belgium

enhanced stability and detection window of … · methodologies in analytical chemistry research...

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