1 Liquid chromatography-tandem mass spectrometry as a powerful tool for the determination of pharmaceuticals in environmental samples Meritxell Gros, Mira Petrovic and Damià Barceló IIQAB-CSIC, Environmental Chemistry Department, Barcelona, Spain 1 st Thematic Workshop of the EU project NORMAN CHEMICAL ANALYSIS OF EMERGING POLLUTANTS OUTLINE Introduction Sources of pharmaceuticals Suitability of LC-tandem MS for drug analysis in environmental samples according to EC directives Analytical protocols based on off line SPE -LC-tandem MS for the identification of pharmaceuticals in natural waters: HPLC – QqQ HPLC – Q-TRAP UPLC-Q-TOF Performance comparison between the LC-tandem MS techniques Conclusions
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Liquid chromatography-tandem massspectrometry as a powerful tool for the
determination of pharmaceuticals in environmental samples
IntroductionSources of pharmaceuticalsSuitability of LC-tandem MS for drug analysis in
environmental samples according to EC directives
Analytical protocols based on off line SPE -LC-tandem MS for the identification of pharmaceuticals in natural waters:
HPLC – QqQ HPLC – Q-TRAPUPLC-Q-TOF
Performance comparison between the LC-tandem MS techniques
Conclusions
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INTRODUCTIONSources and fate
Human Drugs
Excretion
Sewage
STP
Fish farmsSurface water
Drug manufacturer
Disposal
Waste
Landfill
Groundwater
(low removal forsome compounds)
Excretion
Veterinary Drugs
Manure
Soil(run-off)
Drinking water
INTRODUCTIONSuitability of LC-tandem MS
Identification and confirmation criteria for the analysis of drugs andother contaminants are defined in Directive 96/23/EC and CommisionDecision 2002/657/EC, requiring a minimum of 3 identification points
4.51 precursor 1 product (MS/MS)2 for precursor ion2.5 for transition
product
2One ion from a full scan2
LC-Q-TOF-MS
7.51 precursor 2 products (MS/MS)
52 precursors, each with 1 product (2 SRM)
41 precursor 2 products (2 SRM)
2.51 precursor 1 product (SRM)
1 for precursor ion1.5 for transition
productLC-MS-MS (QqQ)
1SIM1LC-MS (Q)
IP earnedExample per ionsNumber of IP earned per ionTechnique
Resolving (Scan Offset)FragmentResolving (Scan)Neutral Loss Scan (NL)
Resolving (Fixed)FragmentResolving (Scan)Precursor Ion Scan (PI)
Resolving (Scan)FragmentResolving (Fixed)Product Ion Scan (PIS)
Resolving (Scan)RF-onlyRF-onlyQ3 Scan
RF-onlyRF-onlyResolving ScanQ1 Scan
Q3q2Q1Scan Type
Q3 as aquadrupole analyzer
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TARGET COMPOUNDS
O NH
CH3
CH3
ROH
COMMON STRUCTURE OF THE ß-BLOCKERS STUDIED
Used to treat cardiovascular disorders. Included in the list of prohibited substances of the IOC due to their sympathomimetic properties, similar to other central nervous system stimulants
hypertensionarrhythmia
HN
NH
Pindolol
Propranolol
Carazolol
RCompounds
heart failurerelief intraocular pressure
NH2
O
O
H3C
N N
S
N
O
Betaxolol
Metoprolol
Timolol
Atenolol
RCompounds
HNS
O O
H3C NH
OH
CH3
CH3
Sotalol
ANALYTICAL PROTOCOL
SPE EXTACTION
INSTRUMENTALANALYSIS
SAMPLE PRE-TREATMENT
ELUTION
100mL (WWTP influent), 200mL (WWTP effluent)
500mL river water
MIP (25mg) OASIS® HLB(60mg)
25mL (WWTP influent), 25mL (WWTP effluent)
100mL river water
FILTRATION1µm glass fiber filters
Conditioning5mL MeOH
5 mL HPLC water
Conditioning1mL MeOH
1 mL HPLC water
2x4 mL MeOH2x1 mL MeOH with 10% HAc2x1mL MeOH
1mL AcN1mL CH2Cl2
LC (ESI) tandem MS (Q-TRAP)
1mL H2O 5% MeOH
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HPLC-QTRAPHPLC Conditions
0 min (15%B), 3 min (15%B), 20 min (75%B), 25 min (15%B), 40 min(15%B) TOTAL RUN: 40min
There is an important dilution factor: Levels found in surface watersdownstream WWTP are in the lowng/range, whereas in WWTP effluentconcentrations of target compounds are between low µg/L-high ng/L range.
Environmental risks are reduced in river water due to the important
dilution
0
1
2
3
4
5
6
I E I E I E I E I E I E I E
Tota
l loa
ds (g
/day
/100
0 in
habi
tant
s)
Overall removal efficiency
91% 76% 0% 88% 44% 29% 77%
WWTP1 WWTP2 WWTP3 WWTP4 WWTP5 WWTP6 WWTP7
0
1
2
3
4
5
6
I E I E I E I E I E I E I E
Tota
l loa
ds (g
/day
/100
0 in
habi
tant
s)
Overall removal efficiency
91% 76% 0% 88% 44% 29% 77%
WWTP1 WWTP2 WWTP3 WWTP4 WWTP5 WWTP6 WWTP7
Total loads of pharmaceuticals were normalized for population equivalents andexpressed as g/day/1000 inhabitants
OCCURRENCEEbro river basin (NE Spain)
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CONCLUSIONSLC-MS-MS permits unequivocal identification of studied pharmaceutical
classes. Pharmaceutical residues can be traced from wastewaters (“hot spots” are WWTP) and receiving waters (surface waters) down to coastal and drinkingwaters
UPLC showed very good performances - reduced analysis time, improvedseparation and sensitivity than HPLC
Q-TOF – high quality structural information for unequivocal identification oftarget compounds; rather good sensitivity and selectivity in full scan mode; lower capacity for trace quantitation
QqQ– powerful tools for quantitative target analysis (LOD in low ng/L level. QTRAP is suitable for both quantitation and identification of pharmaceuticals(MRM combined with several scans in the same experiment), providing highersensitivity than QTOF and QqQ.
UPLC-Q-TOF is a complimentary tool for QqQ and QTRAP providing verygood performances for pharmaceutical analysis in environmental matrices
ACKNOWLEDGEMENTS
This work has been supported by:EU project EMCO (INCO CT 2004-509188) Reduction of environmental risks, posed by Emerging Contaminants, through advanced treatment of municipal and industrial wastes
Spanish Ministerio de Ciencia y TecnologiaProject CTM2004-06255-CO3-01
Thanks to MIP Technologies for providing the MIP cartridges, to Applied Biosystems for the 4000 QTRAP, to Waters Corporation for the SPE cartridges and to Merck for the gift of HPLC columns
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INTRODUCTIONConcern of drugs as pollutants
Due to their continuous introduction via WWTP effluents, they are referred to as "pseudo" persistent contaminants (i.e. high transformation/removal rates are compensated by their continuous introduction into environment)
They often have the same type of physico-chemical behavior as other harmful xenobiotics (persistence in order to avoid the substance to be inactive before having a curing effect, and lipophilicity in order to be able to pass membranes).
They are used by man in rather large quantities (i.e. similar to those of many pesticides).
Some pharmaceuticals (antidepressants, anti-inflamamtories, ß-blockers) showed chronic toxicity to several aquatic organisms at the levels found in WWTP effluents.