AQUAbase Workshop on Analytical Methods, Aachen, 2006

O

O H

OH

C H 3

O H

C H 3

C H 3

OH O H

OH

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

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

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

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

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

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

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

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

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

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,

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

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

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