Challenges in Antibiotics Analysis in Environmental Samples

Diana S. Aga

What Antibiotics Should We Analyze For?

US Food and Drug Administration, 2015. Summary Report: Antimicrobials Sold or Distributed for Use in Food- Producing Animals.

Class Annual Total (Kg)

Tetracyclines 6,514,779

Ionophores (e.g. monensin) 4,434,657

Penicillins 828,721

Macrolides 563,251

Sulfonamides 384,371

Aminoglycosides 270,342

Lincosamides 236,450

Cephalosporins 28,337

Others (e.g. Polypeptides, Streptogramins) 1,527,646

Importance of Transformation Products: Tetracyclines

Tetracycline Oxytetracycline Chlortetracycline

Transformations

Importance of Metabolites: Sulfonamides

Analytical TechniquesLiquid chromatography/mass spectrometry (ion-trap, triple quad, Q-ToF, Orbitrap) vs.

Biological Assays (ELISA, microbial activity assay)

sulfonamides Tetracyclines Macrolides

ELISA Method (striped bar) gave higher concentrations than LC/MS method (solid bar) for tetracyclines in soil

Aga, D. S.; O'Connor, S.; Ensley, S.; Payero, J. O.; Snow, D.; Tarkalson, D., Determination of the Persistence of Tetracycline Antibiotics and Their Degradates in Manure-Amended Soil Using Enzyme-Linked ImmunosorbentAssay and Liquid Chromatography−Mass Spectrometry. Journal of Agricultural

and Food Chemistry 2005, 53, (18), 7165-7171.

Sample Preparation and Clean up

Sample ACN/water

SOLIDS: Lyophilize, pulverize, sonicate with organic solvent

Interference from Co-extracted Matrix

Avoiding “false positives” using tandem mass spectrometry

Batt, A. L.; Aga, D. S., Simultaneous analysis of multiple classes of antibiotics by ion trap LC/MS/MS for

assessing surface water and groundwater contamination. Analytical Chemistry 2005, 77 (9), 2940-2947.

C. Erythromycin-H2O

Spiked Distilled Water

D. Fredonia

Waste Water Effluent

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (min)

05

10152025303540455055606570758085

9095

100

Rela

tive A

bundance

440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800m/z

05

10152025303540455055606570758085

9095

100R

ela

tive A

bundance

653.6

678.6

716.6

568.0

669.5 706.1473.8448.3 492.6 582.4

650.1642.4

528.7 618.7 698.5680.3

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time (min)

05

10152025303540455055

606570758085

9095

100

Rela

tive A

bundance

440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800

m/z

05

10152025

3035404550556065707580859095

100

Rela

tive A

bundance

522.0

558.4

557.5624.3540.6

715.4

m/z 716 522, 558[M-H2O+H]+ = m/z 716 24.6

24.6

9

Sample Collection: Which samples are important?