Detection of Pharmaceuticals in Aqueous Environment Detection of Pharmaceuticals in Aqueous Environment An Introduction An Introduction Leipzig-Halle in the Helmholtz Association UFZ Centre for Environmental Research O H C H 3 O O H H O H O C H 3 C 9 H 1 9 H O O H C H 3 M. Möder, Department of Analytical Chemistry AQUAbase Workshop on Analytical Methods, Aachen, 2006
Problems in Detection of Pharmaceutical Residues trace concentration ( ppt=ng/L) pre concentration remove matrix or complex matrix (DOC > 50 mg/L) separate analytes from great variety of structurally different enrichment different substances conditions and analysis techniques polar, high water solubility HPLC-UV/fluorescence, LC-MS 2 thermal unstable no chromophores derivatization GC-MS 2 , LC-MS 2 several metabolites, conjugates structure elucidation, stability study,...
Sample Preparation Analysis Methods Solid Phase Extraction All methods SPE (RP-C18/EN) GC-MS Solid Phase Microextraction HPLC-MS 2 , CE SPME (PDMS,PA) GC-MS Membrane Assisted Extraction HPLC-MS 2 , CE e.g. dialysis Solvent-free or solvent-reduced extraction methods
Sample Preparation Membrane-Assisted SPE SPME Liquid-Liquid Extraction - simple, sample vial with C18 screw cap - fast injection Polystyrene/ - solvent free guide divinylbenzene - automated water sample - suited for many drugs microporous - flexible Polypropylen fibre filled with currently used solvent magnetic stir bar - Clean-up - semi-polar compounds - simple, fast, low-cost, fexible - time and solvent - concentration > 1µg/L - selectivity depends on membrane consuming - hard matrix can - low solvent consume - RSD dependent influence extraction - adsorption, >200ng/L, ... ?? on batch
Derivatization - Gas Chromatography - Mass Spectrometry 17- ∝ -ethinylestradiol clofibric acid R OH O CH 3 Cl O Me OH Si(Me) 3 R= COCF 3 Si(Me) 3 R H O Mass spectral features : - often small or no molecular ion, - RCOOMe - R´COOMe ]+ - RCOOTMS and ROTMS - Me] + ; - ROCOCF 3 - CF 3 or –C OCF 3 ] + LOD: 0.4-85 ng/L (depends on substance and matrix) Problems: quantitative reaction in case of multi-functionalized compounds limited stability of derivatives (rearrangements of TMS products)
Derivatization-Gas Chromatography-Negative Chemical Ionization MS Derivatization (for “H-acidic” compounds) + pentafluorobenzylbromide (PFBBr) diclofenac bisphenol A PFB CH 2 Cl HOOC CH 3 H PFB PFB N H O OH CH 3 Cl Principle of NCI M - + e - CH 4 + e - CH 4 ⎤ •+ + 2 e - Reagent gas + R slow electrons -CH 4 R+H ⎤ + Features : - quantitative reaction, stable derivatives - mass spectra marked by [RCO OPFB - PFB or -F ] - - extremely selective detection > high signal/noise > very low chromatogram base line - LOD ranges from 0.01 to 0.5 ng/L , mean RSD = 14%
HPLC - Tandem Mass Spectrometry API Atmospheric Pressure Ionization - MS ElectroSpray Ionization Atmospheric Pressure Chemical Ionization ESI or Ion spray IS APCI or Heated Nebulizer negative ESI: R-COO - Gas phase reactions with e.g. NH 4 + positive ESI: R-NH x +H] + .... R-OH+H] + or R-OH+NH 4 ] + Atmospheric Pressure Photoionization APPI Ionization by UV-radiation e.g. aromatic, conjugated compounds Polarity, molecular weight
LC-Tandem MS Electrospray Ionization 5 kV + + + + + + + Quadrupol UV Quadrupol Collision Mass Filter 2 10 eV Mass Filter 1 cell HOOC CH 2 Cl NH H M-HCl-COOH N Cl Atmospheric Pressure Photoionization Cl m/z 214 M+H] + m/z 296 Multiple reaction monitoring MRM
Comprehensive Protocol for Analysis of Pharmaceutical Residues from Water SPE (1 litre sample), filtrated online-SPE-LC-MS/MS GC-EI-MS (full scan) LC-ESI +/- -MS/MS von 100-200 mL sample neutral analytes ß-blockers (metoprolol, propranolol) (caffeine, phenazon, antibiotics (erythromycin, macrocyclides,) carbamazepine, lipid regulators (fenofibrate) tonalide, galaxolide) antiphlogistics (diclofenac, ibuprofen,…) analgetics (phenazon,…) caffeine, carbamazepine Clean-up/Derivatization Diagnostic reagents (x-ray contrast media: with PFBBr iopamidol, iopromide…) GC-NCI-MS Carboxylic acids: clofibric acid, SPME of 4 mL ibuprofen, gemfibrozil, naproxen, Higher concentrated fenofibric acid, diclofenac, compounds Flow Injection indometacin, bezafibrate; e.g. polycyclic musk Analysis-ESI-MS (SIM) Endocrine disrupting compounds compounds, Microcystins (BPA, EE2, techn. nonylphenol) phytoestrogens (also ELISA) (ß-sitosterol)
Method Comparison LC-MS-MS ESI +/- (APPI) GC-MS(EI) APCI + GC-MS(NCI) Derivatization +/- - - + SPE(online) SPE(online) SPE, Enrichment SPE, SPME Membrane Membrane Membrane Membrane + + + Clean-up + 0.6-15 ng/L b 0.3-1.5 ng/L LOQ 1-250 ng/L a 5-50 ng/L Sensitivity Effort Hormones RCOOH, RCOOH, Drugs with RCOOH, ROH ROH, Antibiotics RJ, (hormones) R-NH-CO-R´ b Schlüsener,RCM, β -blocker a Ternes, TRAC,20(2001)419 19(2005)3269
Example 3500,0 Clofibrinsäure Ibuprofen 3000,0 t-Nonylphenol Gemfibrozil 2500,0 Naproxen 2000,0 Diclofenac ng/l BPA 1500,0 Ethinylestradiol Bezafibrat 1000,0 Carbamazepin Coffein 500,0 Galaxolid Tonalid 0,0 1 2 3 5 a 9 0 1 2 3 H H H H 6 H 1 1 1 1 H H H H H Phenazon H H H H H H H H H H U U U U U U U U U U Confluence of river Weiße WWTP of Halle Elster into Saale
Currently used sample preparation/enrichment methods: � SPE (clean-up) off-line or on-line � SPME for pollutants at higher concentrations Summary Summary Currently used target analysis methods : � GC-MS (SIM) with derivatization � LC-MS-MS Challenges: � increasing variety of pharmaceutical substances prescribed and applied � new kinds of highly bioactive drugs designed � lower concentration � Metabolites, Conjugates � Complete the data base to support toxicity tests and risk assessment
Acknowledgement Steffi Schrader Franziska Lange (DFG project) Toralf Einsle (project sponsored by Ministry of Environment and Agriculture of Saxony-Anhalt) Dr. Sabine Müller Pamela Braun UFZ-intern research project “Micropollutants in water and soil in the urban environment” : Dr. K. Schirmer Dr. G. Strauch H.-R. Gläser
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