Quantitative effect-directed analysis based on TLC/HPTLC-direct bioautography Irena Choma, UMCS Lublin, Poland HPTLC Berlin, 2017

Effect –directed analysis Extracts extraction fractionation BIOLOGICAL DETECTION Complex mixture Fractions refractionation/ purification Pure constituents Chemical identification LC-MS, GS-MS, IR, NMR Toxicological studies I.M. Choma, W. Jesionek, Instrumental TLC, ed. C.F. Pool, chapter 11

Frequently used TLC-bioassays Estrogenic activity (Yes test). Enzymatic tests (acetylocholinesterase inhibition test). Antioxidant and radical scavenging activity tests (DPPH, ABTS). Antimicrobial tests. Spira D. et al., J. Planar Chromatogr. 26 (2013) 395 Zheng X. et al., J. Planar Chromatogr, 24 (2011) 470 Jesionek, W. et al., J. Liq. Chromatogr. & Rel. Tech., 38 (2015) 1147-1153. Jesionek, W. et al., JPC., 26 (2013) 109-113

contact bioautography immersion/overlay bioautography direct bioautography immersion in agar overlay with agar dipping in broth spraying with broth TLC plate Petri dish (mirror image)

Visualization in TLC-DB Bacillus subtilis Aliivibrio fischeri

TLC-DPPH Lihua Gu et al., J. Planar Chromatogr. 27 (2014) 5-10 TLC chromatogram, scanning profile of Magnoliae officinalis Cortex samples from different locations in China Linear calibration curves of magnolol and honokiol, based on their antioxidant effect Lihua Gu et al., J. Planar Chromatogr. 27 (2014) 5-10

TLC-DB: Estrogenic compounds S. Buchinger, D. Spira, Anal. Chem., 85 (2013) 7248−7256

TLC-DB: Estrogenic compounds M. B. Muller, C. Weins, Chromatography, 60 (2004) 207-211

TLC-DB: Estrogenic compounds HPTLC-pYES dose-response curves (n = 4) for six EDCs I. Klingelhöfer, G. E. Morlock, J. Chromatogr. A., 1360 (2014) 288–295

TLC-DB: Estrogenic compounds D. Schick, W. Schwack, J. Chromatogr.A 1509 (2017) 147-152

CALIBRATION IN TLC-DB Calibration curves: rhombus – enrofloxacin, square – ciprofloxacin I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 27 (2004) 2071  

CALIBRATION IN TLC-DB Exponential calibration curves: rhombus – enrofloxacin, square – ciprofloxacin. The data from four bioautograms. I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 27 (2004) 2071  

NP TLC RP HPLC I.M. Choma, J. Planar Chromatogr., 19 (2006) 104-108

Cefacetril in milk Excretion of cefacetril after intramammary application at 2, 3, 6, 8, 10, 24, 36 and 48 hours. Lower trial - 5 μl, upper trial 10 μl. I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

Cefacetril in milk HPLC TLC - DB I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

Cefacetril in milk Concentration of cefacetril (ppm) in milk as a function of time after administration of the drug to a cow. Circle – HPLC data, rhombus – TLC - DB data. I.M. Choma et al., J. Liquid Chromatogr. & Rel. Technol., 31 (2008) 1903

A. Ramirez et al. J. Chromatogr. B 784 (2003) 315–322

Chlorogenic acid in coffee TLC-DB Bacillus subtilis DPPH NP-PEG

Chlorogenic acid in coffee HPLC 254nm TLC-densitometer 254 TLC-VideoScan 254 nm Coffee C CQA [mg/mL] SD n = 5 RSD % SD n = 4 RSD % SD n = 9 Brazil 2,34 0,10 4,42 0,21 8,87 1,66 0,65 39,21 Salvador 2,22 0,11 4,96 2,14 9,93 1,82 0,71 39,05 Colom-bia 2,79 0,23 8,30 2,39 0,14 5,90 2,06 0,73 35,27 Papua 3,10 0,17 5,53 3,05 0,45 14,85 2,41 0,95 39,38 Papua Black 0,19 0,02 7,90 7,03 0,32 0,03 8,59 Thank you for your kind attention

Chlorogenic acid in coffee [mg/ml] HPLC 325 TLC-densit. 325 HPLC 254 TLC-densit. 254 TLC-Video Scan 254 TLC-Video Scan 366 TLC-DB NP-PEG 366 B.subtilis VIS DPPH VIS Brazil green 2,44 2,93 2,18 2,34 1,66 3,56 4,87 2,05 1,70 Salvador green 2,59 2,71 2,22 2,14 1,82 2,75 5,44 1,94 1,61 Colombia green 3,09 3,24 2,79 2,39 2,06 3,31 6,17 2,50 1,73 Papua green 3,49 4,20 3,10 3,05 2,41 4,27 8,58 3,77 2,62 Papua black 0,18 0,17 0,19 0,21 0,32 0,27 0,58 0,37 0,26

Total amount of polyphenols in coffee TLC ,,dot-blot’’-NP/PEG Spectrophotometry UV-VIS Folin-Ciocalteu Brazil green 4,31 4,53 Salvador green 4,15 4,25 Colombia green 4,34 2,90 Papua green 4,55 5,76 Papua black 1,11 1,99 CQA 20 mg/mL, MeOH, 5 μL 5 μL CQA 0,1 mg/mL, 0,5 mg/mL, 1 mg/mL, 2 mg/mL, 5 mg/mL 10 mg/mL, Brazili G. Salvador G . Colombia G. Papua G. Papua B.

Thank you for your kind attention