Detection of Pharmaceuticals in Aqueous Environment An ... · - mass spectra marked by [RCO OPFB...
Transcript of Detection of Pharmaceuticals in Aqueous Environment An ... · - mass spectra marked by [RCO OPFB...
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AQUAbase Workshop on Analytical Methods, Aachen, 2006
O
O H
OH
C H 3
O H
C H 3
C H 3
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C 9 H 1 9
Detection of Pharmaceuticals in Aqueous EnvironmentDetection of Pharmaceuticals in Aqueous EnvironmentAn IntroductionAn Introduction
M. Möder, Department of Analytical Chemistry
UFZ C
entre for Environm
ental Research
Leipzig-Halle in the
Helm
holtz Association
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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
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Solid Phase ExtractionSPE (RP-C18/EN)
Solid Phase MicroextractionSPME (PDMS,PA)
Membrane Assisted Extractione.g. dialysis
All methods
GC-MSHPLC-MS2, CE
GC-MSHPLC-MS2, CE
Solvent-free or solvent-reduced extraction methods
Sample Preparation Analysis Methods
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Sample Preparation
magnetic stir bar
sample vial withscrew capinjectionguidewatersamplemicroporousPolypropylenfibre 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- RSD dependent on batch
SPME
- simple, - fast- solvent free- automated
- semi-polar compounds- concentration > 1µg/L- hard matrix can influence extraction
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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 OO
OH
17-∝-ethinylestradiol
R= COCF3Si(Me)3
MeSi(Me)3
OH
CH3
OHR
R
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Derivatization-Gas Chromatography-Negative Chemical Ionization MSDerivatization (for “H-acidic” compounds)
+ pentafluorobenzylbromide (PFBBr)
CH4⎤•+CH4 + e-
+ R-CH4
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
HHOOC
diclofenac
M- + e-
slow electrons
+ 2 e-
PFBCH3
CH3
OH OHPFBPFB
Principle of NCI
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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 PhotoionizationAPPI
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-radiatione.g. aromatic, conjugated compounds
Polarity, molecular weight
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LC-Tandem MS
Electrospray Ionization
Atmospheric Pressure Photoionization
+
++
+
+
+
5 kV
QuadrupolMass Filter 1
+
UV 10 eV
Collision cell
QuadrupolMass Filter 2
Multiple reaction monitoring MRM
N
Cl
Cl
CH2
HHOOC
M-HCl-COOH
M+H]+ m/z 296 m/z 214
NHCl
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Comprehensive Protocol for Analysis of Pharmaceutical Residues from Water
online-SPE-LC-MS/MSvon 100-200 mL sample
ß-blockers (metoprolol, propranolol)antibiotics (erythromycin, macrocyclides,)lipid regulators (fenofibrate)antiphlogistics (diclofenac, ibuprofen,…)analgetics (phenazon,…)caffeine, carbamazepineDiagnostic 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/Derivatizationwith 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 mLHigher concentrated compoundse.g. polycyclic musk compounds, phytoestrogens(ß-sitosterol)
LC-ESI+/--MS/MS
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Method Comparison
GC-MS(EI)
Derivatization +/-
Enrichment SPE, SPMEMembrane
Clean-up +
LOQ 1-250 ng/La
Sensitivity
Effort
Drugs with RCOOH, ROH (hormones)
aTernes, TRAC,20(2001)419
LC-MS-MS
ESI+/- (APPI)
-
SPE(online)Membrane
+
5-50 ng/L
RCOOH,RJ,R-NH-CO-R´β-blocker
APCI+
-
SPE(online)Membrane
+
0.6-15 ng/Lb
HormonesAntibiotics
bSchlüsener,RCM,19(2005)3269
GC-MS(NCI)
+
SPE, Membrane
+
0.3-1.5 ng/L
RCOOH, ROH,
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Example
0,0
500,0
1000,0
1500,0
2000,0
2500,0
3000,0
3500,0
ng/l
UHH1
UHH2
UHH3
UHH5
UHH6a
UHH9
UHH10
UHH11
UHH12
UHH13
ClofibrinsäureIbuprofent-NonylphenolGemfibrozilNaproxenDiclofenacBPAEthinylestradiolBezafibratCarbamazepinCoffeinGalaxolidTonalidPhenazon
WWTP of HalleConfluence of river WeißeElster into Saale
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SummarySummaryCurrently 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
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Acknowledgement
Steffi SchraderFranziska 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