SLIDE 1 AQUAbase Workshop on Analytical Methods, Aachen, 2006
O O H O H C H
3
O H C H
3
C H
3
O H O H O H C
9 H 1 9
Detection of Pharmaceuticals in Aqueous Environment Detection of Pharmaceuticals in Aqueous Environment An Introduction An Introduction
- M. Möder, Department of Analytical Chemistry
UFZ Centre for Environmental Research Leipzig-Halle in the Helmholtz Association
SLIDE 2
Problems in Detection of Pharmaceutical Residues trace concentration ( ppt=ng/L) pre concentration HPLC-UV/fluorescence, LC-MS2 derivatization GC-MS2, LC-MS2 structure elucidation, stability study,... complex matrix (DOC > 50 mg/L) great variety of structurally different substances different enrichment conditions and analysis techniques remove matrix or separate analytes from polar, high water solubility thermal unstable no chromophores several metabolites, conjugates
SLIDE 3
Solid Phase Extraction SPE (RP-C18/EN) Solid Phase Microextraction SPME (PDMS,PA) Membrane Assisted Extraction e.g. dialysis All methods GC-MS HPLC-MS2, CE GC-MS HPLC-MS2, CE Solvent-free or solvent-reduced extraction methods
Sample Preparation Analysis Methods
SLIDE 4 Sample Preparation
magnetic stir bar sample vial with screw cap injection guide water sample
microporous Polypropylen fibre filled with solvent
Membrane-Assisted Liquid-Liquid Extraction
- simple, fast, low-cost, fexible
- selectivity depends on membrane
- low solvent consume
- adsorption, >200ng/L, ... ??
Polystyrene/ divinylbenzene
C18
SPE
- suited for many drugs
- flexible
currently used
- Clean-up
- time and solvent
consuming
SPME
- simple,
- fast
- solvent free
- automated
- semi-polar compounds
- concentration > 1µg/L
- hard matrix can
influence extraction
SLIDE 5 Derivatization - Gas Chromatography - Mass Spectrometry
Mass spectral features: - often small or no molecular ion,
- RCOOMe
- R´COOMe ]+
- RCOOTMS and ROTMS
- Me]+ ;
- ROCOCF3 - CF3 or –C OCF3 ]+
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) clofibric acid
Cl O O OH
17-∝-ethinylestradiol R= COCF3 Si(Me)3 Me Si(Me)3
O H CH3 OH
R R
SLIDE 6 Derivatization-Gas Chromatography-Negative Chemical Ionization MS
Derivatization (for “H-acidic” compounds) + pentafluorobenzylbromide (PFBBr) CH4
⎤•+
CH4 + e-
+ R
R+H⎤+
Reagent gas
bisphenol A 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%
N Cl Cl CH2 H HOOC
diclofenac
M- + e-
slow electrons
+ 2 e-
PFB
CH3 CH3 O H OH
PFB PFB
Principle of NCI
SLIDE 7
HPLC - Tandem Mass Spectrometry
API Atmospheric Pressure Ionization - MS
Atmospheric Pressure Chemical Ionization APCI or Heated Nebulizer ElectroSpray Ionization ESI or Ion spray IS Atmospheric Pressure Photoionization APPI negative ESI: R-COO- positive ESI: R-NHx+H]+.... Gas phase reactions with e.g. NH4
+
R-OH+H]+ or R-OH+NH4]+ Ionization by UV-radiation e.g. aromatic, conjugated compounds Polarity, molecular weight
SLIDE 8 LC-Tandem MS Electrospray Ionization Atmospheric Pressure Photoionization
+ + + + + +
5 kV
Quadrupol Mass Filter 1
+
UV 10 eV
Collision cell Quadrupol Mass Filter 2 Multiple reaction monitoring MRM
N Cl Cl CH2 H HOOC
M-HCl-COOH M+H]+ m/z 296 m/z 214
NH Cl
SLIDE 9 Comprehensive Protocol for Analysis of Pharmaceutical Residues from Water
von 100-200 mL sample ß-blockers (metoprolol, propranolol) antibiotics (erythromycin, macrocyclides,) lipid regulators (fenofibrate) antiphlogistics (diclofenac, ibuprofen,…) analgetics (phenazon,…) caffeine, carbamazepine Diagnostic reagents (x-ray contrast media: iopamidol, iopromide…) Carboxylic acids: clofibric acid, ibuprofen, gemfibrozil, naproxen, fenofibric acid, diclofenac, indometacin, bezafibrate; Endocrine disrupting compounds (BPA, EE2, techn. nonylphenol)
GC-EI-MS (full scan) GC-NCI-MS
Clean-up/Derivatization with PFBBr
neutral analytes (caffeine, phenazon, carbamazepine, tonalide, galaxolide)
SPE (1 litre sample), filtrated Flow Injection Analysis-ESI-MS (SIM)
Microcystins (also ELISA)
SPME of 4 mL
Higher concentrated compounds e.g. polycyclic musk compounds, phytoestrogens (ß-sitosterol)
LC-ESI+/--MS/MS
SLIDE 10 Method Comparison
GC-MS(EI) Derivatization +/- Enrichment SPE, SPME Membrane Clean-up + LOQ 1-250 ng/La Sensitivity Effort Drugs with RCOOH, ROH (hormones)
aTernes, TRAC,20(2001)419
LC-MS-MS ESI+/- (APPI)
Membrane + 5-50 ng/L RCOOH, RJ, R-NH-CO-R´ β-blocker APCI+
Membrane + 0.6-15 ng/Lb Hormones Antibiotics
bSchlüsener,RCM,
19(2005)3269
GC-MS(NCI) + SPE, Membrane + 0.3-1.5 ng/L RCOOH, ROH,
SLIDE 11 Example
0,0 500,0 1000,0 1500,0 2000,0 2500,0 3000,0 3500,0
ng/l
U H H 1 U H H 2 U H H 3 U H H 5 U H H 6 a U H H 9 U H H 1 U H H 1 1 U H H 1 2 U H H 1 3
Clofibrinsäure Ibuprofen t-Nonylphenol Gemfibrozil Naproxen Diclofenac BPA Ethinylestradiol Bezafibrat Carbamazepin Coffein Galaxolid Tonalid Phenazon
WWTP of Halle Confluence of river Weiße Elster into Saale
SLIDE 12
Summary Summary
Currently used target analysis methods : GC-MS (SIM) with derivatization LC-MS-MS Currently used sample preparation/enrichment methods: SPE (clean-up) off-line or on-line SPME for pollutants at higher concentrations 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
SLIDE 13 Acknowledgement
Steffi Schrader Franziska Lange (DFG project) Toralf Einsle (project sponsored by Ministry of Environment and Agriculture of Saxony-Anhalt)
Pamela Braun UFZ-intern research project “Micropollutants in water and soil in the urban environment” :
- Dr. K. Schirmer
- Dr. G. Strauch
H.-R. Gläser
