PBA-2008, Gdańsk 8-12.06.20081

ADVANCES IN NSAID RESIDUE ANALYTICS IN AQUEOUS

ENVIRONMENT

Agata KOT-WASIK, Jacek NAMIEŚNIKDeopartment of Analytical Chemistry, Chemical Faculty,

Gdańsk University of TechnologyG. Narutowicza 11/12, 80-952 Gdańsk

E-mail: chemanal@pg.gda.pl

PBA-2008, Gdańsk 8-12.06.20082

CELE I ZADANIA ANALITYKI I MONITORINGU ŚRODOWISKA

Identification of sources of emission and evaluation of scale and impact assessment of emission

Determination of mixing ration of pollutants

Studies of environmental fate of xenobiotics

Evaluation of toxicity and ecotoxicity of specific pollutants

Studies of bioaccumulation processes of pollutants by living organisms

PBA-2008, Gdańsk 8-12.06.20083

ANALITYKA I MONITORING ŚRODOWISKA PROBLEMY I WYZWANIA

Low and even very low concentration level of analytes in samples characterized by complex composition of the matrix

Possibility of time and space fluctuations of xenobioticsconcentration

Danger of interferences connected with presence of constituents characterized by similar physico-chemical properties

Lack of information on degradation path pollutants

Necessity of determination not only primary pollutants but also products of degradation and metabolism processes

Lack of suitable standards and reference materials

PBA-2008, Gdańsk 8-12.06.20084

ENVIRONMENTAL POLLUTANTS

Regulated pollutants Nonregulated pollutants

...........

............

.......

.......

Dioxins(PCDD+PCDF)

Polyaromatichydrocarbons

(PAH’S)

PolichlorinatedBiphenyls (PCB’s)

Metal complexes

Brominated FlameRetardants (BFR’s)

Alkilofenole

Estrogens

Pesticides

fitoestrogens

Bisfenol A

Derivatives of phtalanes

ENDOCRINE DISRUPTING COMPOUNDS – EDC’s

Nonionic surfactants

Pharmaceutical residues

Personal Care Products(PCP’s)

Synthetic muskcompounds

PBA-2008, Gdańsk 8-12.06.20085

MOTTO

„Pollution from pharmaceuticals in surface and groundwaters is becoming recognised as an

environmental concern in many countries leading to the area of study labelled PIE”

PHARMACEUTICALS IN THE ENVIRONMENT

S. K. Khetan, T. J. Collins, Chem. Rev., 107, 2319-2364 (2007)

PBA-2008, Gdańsk 8-12.06.20086A. Nikolaou, S. Meric, D. Fatta, Anal. Bioanal. Chem., 387, 1225 (2007)

Antiinflammatorydrugs/ analgesics

Acetylsalicylic acid (Aspirin)DiclofenacIbuprofen

AcetaminophenMetamizolCodeine

IndometacineNaproxenPhenazone

AntibioticsErytromicynOfloxacin

ChlortetracyclineOxytetracycline

StreptomycinFlumequine

CiprofloxacinTrimetoprim

SulfamethoxazoleLincomycinPenicillin

LincomycinAmoxicillinSpiramycin

Lipid regulatorsBezafibrateGemfibrozil

Clofibric acidFenofibrate

Beta-blockersMetoprololPropranolol

NadololAtenololSotalol

Betaxolol

Most common pharmaceuticals in the environment

Steroids and related hormones17-β-estradiol

Estrone17 α-ethinyl estradiol

DiethylstilbestrolDiethylstilbestrol acetate

Cancer therapeuticsCyclophosphamide

Ifosphamide

DiureticsFurosemide

AntieplepticsCarbamazepine

AntidepressantsMianserin

TranquillisersDiazepam

THE MOST COMMON PHARMACEUTICAL

COMPOUNDS PRESENT IN ENVIRONMENTAL SAMPLES

PBA-2008, Gdańsk 8-12.06.20087

MAIN SOURCES OF DISCHARGES OF BIOLOGICALLY ACTIVE COMPOUNDS TO

THE ENVIRONMENT

• Production of pharmaceuticals (human drugs and veterinary drugs)

• Discharge to the environment a large amount of overdue drugs both from households and hospitals

• Excretion of non changed forms of biologically active compounds and their metabolites by humans and animals

PBA-2008, Gdańsk 8-12.06.20088

1. France, 2. Poland, 3. Germany, 4. USA, 5. Japan

AMOUNT OF DRUGS, WHICH ARE BOUGHT IN DIFFERENT COUNTRIES

BY PATIENT IN ONE YEAR

32

22 20

7

29

1 2 3 4 5

PBA-2008, Gdańsk 8-12.06.20089

PHARMACEUTICAL RESIDUES IN THE ENVIRONMENT - MILESTONES

19811981 confirmation of the presence and quantitative determination of CLOFIBRIC ACID IN ENVIRONMENTAL SAMPLES (USAUSA)

19971997 Determination of pharmaceutical residue in hospital sewages (GermanyGermany)

19971997 Studies related to the antibiotic toxicity in water environment (DenmarkDenmark)

1998 1998 Monitoring of river waters and sewage in Germany for the presence of pharmaceutical residue (GermanyGermany)

19981998 Determination of antibiotic compounds in different water samples (GermanyGermany)

1999 1999 Elaboration of an analytical method allowing for the confirmation of the presence of estrogens in surface waters (USAUSA)

2002 2002 First method of simultaneous determination of residues of many pharmaceuticals in samples of the environment (DenmarkDenmark)

2001 2001 –– 20022002 Determination of pharmaceutical residue in drinking waters (GermanyGermany)

2003 2003 Elaboration of appropriate mathematical models for predicting concentration and loss of individual pharmaceuticals in the environment (BelgiumBelgium)

20052005 Determination of pharmaceutical residue in steel samples (SpainSpain)

PBA-2008, Gdańsk 8-12.06.200810

BIOLOGICALLY ACTIVE COMPOUNDS ON THE LIST OF PRIORITY POLLUTANTS

(the Netherlands)

ConcentrationCompounds

Presence in WWTP

effluent min [µg· l-1] max [µg· l-1]

Hormone disruptors

17 α-ethinyloestradiolBisphenol AOestrone

+++++

n.d.0,040,00

<0,014,090,01

Medical substances

IbuprofenAnhydro-erythromecine

SulfomethoxazoleCarbamazepine

SotalolAmidotrizoic acid

++++++++++++++++++

0,120,150,060,330,970,23

0,760,520,131,001,61,2

n.d. non detected+ found in 5% to50% of studied samples of effluent++ found in 50% to 95 % of studied samples of effluent+++ found in more than 95% of studied samples of effluent

P. De Jong, J. F. Kramer, W. F. Slotema, K. A. Third, STOWA REPORT 2005-34, ISBN 90.5773.316.1

PBA-2008, Gdańsk 8-12.06.200811

APPLICATION OF LC-MS TECHNIQUE

Determination of residue of nonsteroid anti-inflammatory drugs (NSAID’s) in environmental samples.

Biologically active compounds from NSAID group are present even in drinking water at ppt level. They are responsible for drug resistance.

Very often this compounds are quantitatively determined in surface waters.

PBA-2008, Gdańsk 8-12.06.200812

STEPS IN ANALYTICAL PROCEDURE FOR

DETERMINATION OF PHARMACEUTICAL

RESIDUESVALIDATION

PRETREATMENT

There is no doubt that sample pre-treatment

operations are crucial for reliability of analytical

results

SAMPLING

CONSERVATION

ISOLATION/PRECONCENTRATION

DERIVATISATION

CLEAN-UP

STORAGE

ANALYSIS

IDENTIFICATION

EVALUATION OF DATA

PBA-2008, Gdańsk 8-12.06.200813

DIFFERENT APPROACHES IN THE FIELD OF APPLICATION OF LC-MS TECHNIQUE

1. SCREENING (direct injection of aqueous sample)

2. SHORT-TERM MONITORING (SPE technique in OFF-LINE or ON-LINE mode)

3. LONG TERM (passive sampling of analytes)

PBA-2008, Gdańsk 8-12.06.200814

SCREENING OF NSAID RESIDUE IN AQUEOUS SAMPLES

• Usual samples volume inRPLC ~20μl.

• LARGE VOLUMES OF WATER SAMPLES (500μl, 1000 μl i 2000 μl) WERE INTRODUCED TO INJECTION PORT

PBA-2008, Gdańsk 8-12.06.200815

COMPARISON OF CHROMATOGRAPHIC PARAMETERS during analysis of large

samples of water with standard addition of analytes from NSAID group

Analit 1 Analit 2 Analit 320 μl 500 μl 20 μl 500 μl 20 μl 500 μl

H 21 11 28 13 47 22w 4 5 5 5 6 5N1 10620 10590 10720 12380 44250 48720N2 9010 9360 11070 11980 36770 45660

Analyte I Analyte II Analyte III

PBA-2008, Gdańsk 8-12.06.200816

Analit 1 Analit 2 Analit 3

Wodawodociągowa

Wodarzeczna

Wodawodociągowa

Wodarzeczna

Wodawodociągowa

Wodarzeczna

LOD[μg/ml]

0,003 0,011 0,003 0,009 0,0015 0,006

LOQ

[μg/ml]0,006 0,021 0,006 0,018 0,003 0,012

SCREENING OF CONCENTRATION LEVEL OF NSAID’s IN REAL SAMPLES

Analyte I Analyte II Analyte III

Tap water river water Tap water river water Tap water river water

PBA-2008, Gdańsk 8-12.06.200817

ANALYTICAL PROCEDURESapplication of solid phase extraction technique(SPE)

SPESPE

off-line SPE

on-line SPEpassive SPE

PBA-2008, Gdańsk 8-12.06.200818

SPE in off-line mode

conditioning

MeOH H2O Sample

sample deposit(sorption)

washing thesorbent deposit

H2O MeOH

analyte elution(desorption)

solvent evaporation to dryness in thenitrogen stream

dissolution of the dry residue in themobile phase

final indication with the help ofHPLC/DAD/MS

PBA-2008, Gdańsk 8-12.06.200819

EXTRACTION OF NSAID’s FROM WATER WITH SPE CARTRIDGE

Influence of volume of sample of recovery of analytes

objętość próbek wody [ml]100 200 300 500 700 1000 2000

AnalitR[%] R[%] R[%] R[%] R[%] R[%] R[%]

tolmetin 50 60 55 70 60 60 45

naproksen 77 88 87 80 87 73 81

fenoprofen 87 90 88 90 90 78 90

diflunisal 40 39 52 75 53 56 31

diklofenak 74 85 82 86 86 69 78

ibuprofen 102 100 115 103 117 106 99

Analyte

Sample volume [ml]

PBA-2008, Gdańsk 8-12.06.200820

DETERMINATION OF NSAID RESIDUE IN DETERMINATION OF NSAID RESIDUE IN REAL WATER SAMPLES (part I)REAL WATER SAMPLES (part I)

Type of water sample

Tap water Sea water River water Lake water

Ground water

Sample no 1

Sample no 1

Sample no 2

Sample no 1

Sample no 1

Sample no 1

Sample no 1

Tolmetin n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Naproksen n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Fenoprofen n.d. n.d. n.d. 55 84 24 n.d.

Diflunisal n.d. 38 62 95 123 28 n.d.

Diklofenak n.d. n.d. n.d. 300 390 528 n.d.

Ibuprofen n.d. n.d. 17 n.d. n.d. 10 n.d.

2,3-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

2,5-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

SA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

Analyte

n.d. – non detected (below LOQ)

n.s. –non studied

PBA-2008, Gdańsk 8-12.06.200821

DETERMINATION OF NSAID RESIDUE IN REAL WATER SAMPLES (part II)

Type of water sample

Tap water Sea water River water Lake water

Ground water

Sample no 1

Sample no 1

Sample no 2

Sample no 1

Sample no 1

Sample no 1

Sample no 1

Tolmetin n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Naproksen n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Fenoprofen n.d. n.d. n.d. 55 84 24 n.d.

Diflunisal n.d. 38 62 95 123 28 n.d.

Diklofenak n.d. n.d. n.d. 300 390 528 n.d.

Ibuprofen n.d. n.d. 17 n.d. n.d. 10 n.d.

2,3-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

2,5-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

SA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

Analyte

n.d. – non detected (below LOQ)

n.s. –non studied

PBA-2008, Gdańsk 8-12.06.200822

SPE w ukSPE w ukłładzie adzie onon--lineline

Pump 2

Leak

Analytical column

SPE Column

DADMS

Detectors

SAMPLE

Stage 1- analyte and isolation enrichment in water samples

Pump 1

PBA-2008, Gdańsk 8-12.06.200823

DETERMINATION OF NSAID’s RESIDUE IN DIFFERENT TYPES OF WATER

Application of SPE-HPLC-DAD-MS

AnalyteRaw waste water

(ng/l)Purified waste water

(ng/l)

Level of reduction of raw waste water in

WWTP [%]

Metformin 151967 8552 94

Cymetydydna 894 90 90

Atenolol 8087 768 90

Ranitydyna <5 33 --

Metoprolol 50 15,1 70

Propranolol 335 97 71

Norfluksetyna <20 <5 --

Paroksetyna 30 0,8 97

Fluksetyna 57 11,4 80

Diklofenak 471 490 0

Felodypina 38,4 1,2 97

Naproksen 2613 150 94

Triklosan ~7500 ~90 ~99

PBA-2008, Gdańsk 8-12.06.200824

SPE-HPLC-DAD-MSADVANTAGES AND DRAWBACKS

Advantages

• The possibility of preparing several samples simultaneously

• The possibility of the optimization of individual stages

• The elasticity of protocol steps• Simple equipment

• The possibility for process automation

• Analyzing the complete sample• The possibility of working with a

smaller sample volume• Shorter analysis time• Lowering the risk of analyte loss and

sample contamination• Improved analysis precision

Drawbacks

• Difficulties with automating the operation

• Work and time consuming• The necessity of working with a

large sample volume• Risk of sample and extract

contamination and analyte loss

• Complicated equipment• Little procedure elasticity• Difficult or impossible optimization

of individual steps

offoff--lineline

onon--lineline

PBA-2008, Gdańsk 8-12.06.200825

DETERMINATION OF NSAID RESIDUE FROM WATER SAMPLES

Sample Volume1000 mL

Initial sample preparationt= 2 min

Column Preparationt= 18 min

SPE Extractiont= 70 min

Drying the deposit in the SPEcolumn

t= 5 min

Analyte elutiont= 20 min

Solvent evaporationt= 200 min

Dissolution of the residuet= 30 min

HPLC-DAD-MSANALYSIS

t= 25 min

Sample Volume100 mL

Valve switch

SPE Extractiont= 35 min

Column Preparationt= 8 min

Initial sample preparationt= 2 min

HPLC-DAD-MSANALYSIS

t= 25 min

OFF-LINE SPE ON-LINE SPE

Total time: 70 minutes

Total time: 370 minutesTotal time:

370 minutes

Total time:

70 minutes

PBA-2008, Gdańsk 8-12.06.200826

METROLOGICAL ASPECTS

Proposed analytical approach permits to detect analytes from NSAID group at the level of ppt in water samples

Off-line On-lineRSD% < 11 < 7

Reproducibility % 30-100 89-105

Limit of detection 20-950 0,7-50

PBA-2008, Gdańsk 8-12.06.200827

min2 4 6 8 10 12 14 16 18Time

Sig

nal i

nten

sity

(ES

I-MS)

1

2

3

4

5 6

Sig

nal i

nten

sity

(DA

D)

min2 4 6 8 10 12 14 16 18Time

1

2

3

4

5

6

N

OHH

Cl

Cl

O

F

F

OH

COOH

OCH3

COOH

CH3

OH

O

CH3

CH3

OHCH3

MeO

NCH3 CH2COOH

O

CH3

1 = tolmetin 2 = naproksen 3 = fenoprofen 4 = diflunisal 5 = diklofenak 6 = ibuprofen

STUDIES OF SURFACE WATERSSAMPLES

PBA-2008, Gdańsk 8-12.06.200828

DETERMINATION OF NSAID RESIDUE IN WATER SAMPLE

Type of water sample

Drinking water

Sea water River water Lake water

Ground water

Sample no 1

Sample no 1

Sample no 2

Sample no 1

Sample no 1

Sample no 1

Sample no 1

Tolmetin n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Naproksen n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Fenoprofen n.d. n.d. n.d. 55 84 24 n.d.

Diflunisal n.d. 38 62 95 123 28 n.d.

Diklofenak n.d. n.d. n.d. 300 390 528 n.d.

Ibuprofen n.d. n.d. 17 n.d. n.d. 10 n.d.

2,3-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

2,5-DHBA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

SA n.s. n.d. n.d. n.s. n.s. n.d. n.s.

Analyte

n.d. – non detected (below LOQ)

n.s. –non studied

PBA-2008, Gdańsk 8-12.06.200829

PASSIVE TECHNIQUES OF ANALYTES SAMPLING

UPTAKE OF ANALYTES occurs as a free transport of mass across the membrane to the receiving medium and results from the difference of CHEMICAL POTENTIALS of analytes in this medium and in the aqueous environment in which the sampler is placed.

SPMD LDPE strips MESCO POCIS

PBA-2008, Gdańsk 8-12.06.200830

CHEMCATCHER PASSIVE SAMPLER DESIGNCHEMCATCHER PASSIVE SAMPLER DESIGN

-Sampler body

-Diffusion limiting membrane

-Receiving phase

SAMPLER has been designed and evaluated at the University of Portsmouth within the STAMPS project.STAMPS = Standardised Aquatic Monitoring of Priority Pollutants by Passive Sampling

PBA-2008, Gdańsk 8-12.06.200831

CONFIGURATION OF CHEMCATCHER PASSIVE SAMPLER

LCLC--DAD DAD --MSMSLCLC--DADDAD--MSMSLCLC--DADDAD--MSMSLCLC--DADDAD--MSMSFinal determination

technique

------polyethersulfonepolyethersulfoneMembrane

Extraction Extraction discdisc

(SDB(SDB--XC) XC)

Extraction discExtraction disc(SDB(SDB--RPS )RPS )

Extraction discExtraction disc(C18)(C18)

Extraction discExtraction disc(C18 )(C18 )

Receiving phase

(trapping medium)

Chemcatcher4

Chemcatcher3

Chemcatcher2

Chemcatcher1

Version of the sampler

PBA-2008, Gdańsk 8-12.06.200832

CALIBRATION OF CHEMCATCHER IN A FLOW-THROUGH SYSTEM

Peristaltic pump30 ml/min

Peristaltic pump100 μl/min Exposure tank

Water

wastewater

Stirrer

Chemicalsin MeOH

Waterreservoir

overhead stirrer

Samplers

AnalytesIn MeOH

PBA-2008, Gdańsk 8-12.06.200833

CONDITIONS OF SEPARATION AND FINAL DETRERMINATION (HPLC – DAD –MS)

Elements of measuring system Parameters

Liquid chromatograph HPLC 1100, Agilent

Column Altima HPLC - 18 EPS 250mm x 4.6 mm x 5µm

Mobile phase A: H2O + 1% HCOOHB: MeOH/ACN (1:1, v:v) + 1% HCOOH

Mobile phase flow-rate 0.9 ml/min

Gradient czas %A %B

0 60 40

7.5 45 55

10 45 55

20 0 100

Analysis time 20 min

PBA-2008, Gdańsk 8-12.06.200834

MONITORED IONS

MS(SCAN 20 - 280) ‏Ionisation mode: ELECTROSPRAY

SIMtolmetin 212

naproxen 229

fenoprofen 241

ibuprofen 205

diklofenak 294

diflunisal 249

DAD 275nm

PBA-2008, Gdańsk 8-12.06.200835

CONCLUSIONS

SPESPE Low levelconcentrationof analytes

Complex and variedcomposition of thesample matrix

PBA-2008, Gdańsk 8-12.06.200836

CONCLUSIONS

DRINKING WATER NATURAL WATERS WASTE WATERS

on-line SPE

passive sampling (SPE) ‏

off-line SPE

PBA-2008, Gdańsk 8-12.06.200837

INTEGRATED APPROACH IN ENVIRONMENTAL MANAGEMENT AND RISK ASSESSMENT

Life cycle of pharmaceutical products

ManufacturersDistributorsConsumers

Sludge from wastewater

treatment plant(WWTP)

Environmental receptors

Organisms’exposure

Type of available data

PharmaceuticalPhysico-chemicalConsumptionAppropriate PPs and their metabolites

Therapy effectivenessEmission of PPsconcentration and products of their transformation

MECs (natural and drinking water, sediments and soil)Environmental fate (bioavailability, durability)

ToxicityBioaccumationOther effects

Integrated approach

AcctionsAiming at decreasing occurrence and impact of PPs (prevention, new ways

of sludge treatment …) C. Coetsier, L. Lin, E. Touraud, Anal. Bioanal. Chem., 387, 1163-1166 (2007)

PBA-2008, Gdańsk 8-12.06.200838

DEPARTMENT OF ANALYTICAL CHEMISTRY

http://www.pg.gda.pl/chem/Katedry/Analityczna/analit.htm

• List of publications• Lectures presented during conferences

• Courses and expertises

PBA-2008, Gdańsk 8-12.06.200839

COURSES AND EXPERTISES

Individual courses (on demand)HPLC (basic and advanced)GC (basic and advanced)Sample preparation for analysisABC of the SPE techniqueApplication of HPLC in food analysisHyphenated techniquesBiotests in environmental studiesQuality Control and Quality Assurance (QC/ QA) of analytical results

PBA-2008, Gdańsk 8-12.06.200840

QUALITY CONTROL AND QUALITY ASSURANCE OF ANALYTICAL RESULTS

EDITED BY: P. Konieczka and J. Namieśnik

ISBN: 978-83-204-3255-8

PBA-2008, Gdańsk 8-12.06.200841

PBA-2008, Gdańsk 8-12.06.200842

PBA-2008, Gdańsk 8-12.06.200843

ISEAC’08

PBA-2008, Gdańsk 8-12.06.200844

DEPARTMENT OF ANALYTICAL CHEMISTRY

Dr inż. Agata KOT-WASIK