Application of LC-MS technique

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1 Application of LC-MS technique in environmental studies Agata KOT-WASIK, Jacek NAMIEŚNIK Department of Analytical Chemistry, Chemical Faculty, Gdańsk University of Technology, G. Narutowicza 11/12, Gdańsk 1

2 AIMS AND GOALS OF ENVIRONMENTAL ANALYTICS AND MONITORING Identification of sources of emission and evaluation of scale and impact assessment of emission Determination D 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 2

3 AIMS AND GOALS OF ENVIRONMENTAL ANALYTICS AND MONITORING 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 xenobiotics concentration 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 3

4 ENVIRONMENTAL POLLUTANTS Regulated pollutants Nonregulated pollutants t Dioxins (PCDD+PCDF) Metal complexes Polyaromatic hydrocarbons (PAH S) Polichlorinated Biphenyls (PCB s) Pesticides Estrogens fitoestrogens Bisfenol A Brominated Flame Retardants (BFR s) Nonionic surfactants Alkilofenole Pharmaceutical residues ENDOCRINE DISRUPTING COMPOUNDS EDC s Derivatives of phtalanes Synthetic musk compounds Personal Care Products (PCP s) 4

5 APPLICATION OF CHROMATOGRAPHIC TECHNIQUES Signal in ntensity t ri t ri+2 y i+2 In typical case chromatographic i+2 analysis constitutes a sources of two- dimensional information (2D) t ri+1 i+1 Generally it is a relationship between detector signal (first dimension) and retention time (secondd dimension). Time Identification is based on comparison of retention times. 5

6 PROBLEMS TWO DIMENSIONAL (2D) analysis Problem no 1 overlapping of peaks Solutions Repeat analysis with use of second chromatographic column (character of interactions between analyte and stationary phase has to be different than in the case of first column) Advantages: - Easy to realize -Low cost Disadvantages: - Time consuming task!! 6

7 TWO DIMENSIONAL (2D) analysis Problem no 2 identification of unknown compounds Solution Apply detector which permits to obtain information on the structure of analyte (HYPHENATED TECHNIQUES) Advantages: - Possibility of identification of unknown species -Availability of information on molecular mass and/or structure of analyte -Possibility y of confirmation of overlapping peaks effect Disadvantages: - High cost of investment -Specific S requirements on the level l of professional knowledge of personnel 7

8 intensywność sygnału HYPHENATED TECHNIQUES Thanks to application of HYPHENATED TECHNIQUES analytical information gains an additional dimension (STRUCTURE) m/z czas

9 APPLICATION RANGE OF DIFFERENT TYPES OF HYPHENATED TECHNIQUES (GC-MS and LC-MS) mass of the analyt tes Molecular APCI GC/MS ESI (electospray) THERMOSPRAY FAB PBI nonpolar Very polar Polarity of the analyte 9

10 Antibiotics Erytromicyn Ofloxacin Chlortetracycline Oxytetracycline Streptomycin Flumequine Ciprofloxacin Trimetoprim Sulfamethoxazole Lincomycin Penicillin Lincomycin Amoxicillin Spiramycin Steroids and related hormones 17-β-estradiolβ Estrone 17 α-ethinyl estradiol Diethylstilbestrol Diethylstilbestrol acetate Antiinflammatory drugs/ analgesics Acetylsalicylic acid (Aspirin) Diclofenac Ibuprofen Acetaminophen Metamizol Codeine Indometacine Naproxen Phenazone Most common pharmaceuticals in the environment Cancer therapeutics Cyclophosphamide Ifosphamide Diuretics Furosemide Antiepleptics Carbamazepine Antidepressants Mianserin Tranquillisers Diazepam THE MOST COMMON PHARMACEUTICAL COMPOUNDS PRESENT IN ENVIRONMENTAL SAMPLES Lipid regulators Bezafibrate Gemfibrozil Clofibric acid Fenofibrate Beta-blockers Metoprolol Propranolol Nadolol Atenolol Sotalol Betaxolol A. Nikolaou, S. Meric, D. Fatta, Anal. Bioanal. Chem., 387, 1225 (2007) 10

11 VETERINARY MEDICAL PRODUCTS In Poland all biologically i ll active compounds used as VETERNARY MEDICAL PRODUCTS can be divided into two groups: GROUP A: substances having ANABOLIC EFFECT and UNAUTORIZED SUBSTANCES GROUP B: VETERNARY DRUGS and CONTAMINANT 11

12 PHARMACEUTICAL RESIDUES IN THE ENVIRONMENT - MILESTONES 1981 confirmation of the presence and quantitative determination of CLOFIBRIC ACID IN ENVIRONMENTAL SAMPLES (USA) 1997 Determination of pharmaceutical residue in hospital sewages (Germany) 1997 Studies related to the antibiotic toxicity in water environment (Denmark) 1998 Monitoring of river waters and sewage in Germany for the presence of pharmaceutical residue (Germany) 1998 Determination of antibiotic compounds in different water samples (Germany) 1999 Elaboration of an analytical method allowing for the confirmation of the presence of estrogens in surface waters (USA) 2002 First method of simultaneous determination of residues of many pharmaceuticals in samples of the environment (Denmark) Determination of pharmaceutical residue in drinking waters (Germany) 2003 Elaboration of appropriate mathematical models for predicting concentration and loss of individual pharmaceuticals in the environment (Belgium) 2005 Determination of pharmaceutical residue in steel samples (Spain Spain) 12

13 HOW PHARMACEUTICALS ENTER DRINKING WATER? PHARMACEUTICALS USED BY MAN PHARMACEUTICAL DISPOSED TO SEWAGE WITH URINE DIRECT DISPOSAL OF PHARMACEUTICAL TO SEWAGE SEWAGE TREATMENT PLANT SLUDGE DISPERSED ON FIELDS WATER USED FOR FIELD IRRIDATION EFFECT ON SOIL MICROORGANISMS Many drugs are distributed, metabolised and finally excreted by the human and/or animal body to the environment. Very often they are only slightly transformed or even unchanged and can contaminate the surface water, groundwater and even drinking water. UNDERGROUND WATER SURFACE WATER DRINKING WATER EFFECT ON LIVING ORGANISMS 13

14 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. 14

15 ANALYTICAL PROCEDURES application of solid phase extraction technique (SPE) off-line SPE passive SPE SPE on-line SPE 15

16 V A L I D A T I O N SAMPLING CONSERVATION ISOLATION/PRECONCENTRATION DERIVATISATION CLEAN-UP STORAGE P R E T R E A T M E N T STEPS IN ANALYTICAL PROCEDURE FOR DETERMINATION OF PHARMACEUTICAL RESIDUES ANALYSIS IDENTIFICATION EVALUATION OF DATA There is no doubt that sample pre-treatment operations are crucial for reliability of analytical results 16

17 SPE in off-line mode MeOH H 2 O Sample H 2 O MeOH conditioning sample deposit (sorption) washing the sorbent deposit solvent evaporation to dryness in the nitrogen stream analyte elution (desorption) dissolution of the dry residue in the mobile phase final indication with the help of HPLC/DAD/MS 17

18 SPE w układzie on-line Analytical column Detectors SPE Column Pump 2 DAD MS Pump 1 SAMPLE Leak Stage 1- analyte and isolation enrichment in water samples 18

19 SPE-HPLC-DAD-MS ADVANTAGES AND DRAWBACKS Advantages Drawbacks off-line on-line 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 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 19

20 DETERMINATION OF NSAID RESIDUE FROM WATER SAMPLES Sample Volume 1000 ml Sample Volume 100 ml Initial sample preparation t= 2 min Initial sample preparation t= 2 min OFF-LINE SPE Column Preparation t= 18 min Column Preparation t= 8 min ON-LINE SPE SPE Extraction t= 70 min SPE Extraction t= 35 min Drying the deposit in the SPE column t= 5 min Valve switch Analyte elution t= 20 min Solvent evaporation t= 200 min HPLC-DAD-MS ANALYSIS t= 25 min Total time: 70 minutes Total time: 70 minutes Dissolution of the residue t= 30 min Total time: 370 minutes Total time: 370 minutes HPLC-DAD-MS ANALYSIS t= 25 min 20

21 METROLOGICAL ASPECTS Proposed analytical approach permits to detect analytes from NSAID group at the level of ppt in water samples Off-line RSD% < 11 < 7 On-line Reproducibility % Limit of detection ,

22 5 O Sign nal intensity (DA AD) F O CH 3 F COOH OH CH 3 COOH OH 2 HO H N Cl Cl STUDIES OF SURFACE WATERS SAMPLES C H 3 MeO O N CH 3 CH 2 COOH H 3 C H 3 C CH 3 O OH min Time Signal intensity (ESI-MS) = tolmetin 2 = naproksen 3 = fenoprofen 4 = diflunisal i l 5 = diklofenak 6 = ibuprofen min Time 22

23 DETERMINATION OF NSAID RESIDUE IN WATER SAMPLE Analyte Drinking water Sample no 1 Sample no 1 Type of water sample Sea water River water Lake water Sample no 2 Sample no 1 Sample no 1 Sample no 1 Ground water 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 n.d. Diflunisal n.d n.d. Diklofenak n.d. n.d. n.d 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. n.d. non detected (below LOQ) n.s. non studied 23

24 DETERMINATION OF VETERNARY DRUGS IN DIFFERENT TYPES OF SAMPLES 24

25 The most commonly used coccidiostatics Ionophore antibiotics (salinomycin, narasin, monensin, lasalocid) Vitamin antagonists (amprolium, trimethroprim, pirymethamine) Sulphonamides (sulfadimethoxine, sulfamethoxine sulfanitron) Nitroimidazoles (nitrofural, nicarbasin,dimetridazol) i id Other coccidiostatics (robenidine, clopidol) 25

26 NAJWAŻNIEJSZE CECHY ZASTOSOWANYCH W TRAKCIE BADAŃ TECHNIK EKSTRAKCJI ANALITÓW Shaking UAE ASE MAE Soxhlet Soxtec Full time of extraction per one sample (include heating and cooling and filtration) 40 min 30 min 20 min 45 min 6.5 h 45 min Volume of solvent used 60 ml 25 ml 30 ml 10 ml 45 ml 25 ml Parameters assisted of extraction shaking ultrasonic temperature pressure microwave temperature temperature Level of difficulty low low medium medium low medium Number of sample, which can be extracted simultaneously One sampl e or series One sample or series One sample or series One sample or series one 6 Level of automatisation low low high high low medium Cost of instruments low low high high low medium 26

27 TECHNIKI EKSTRAKCJI ROBENIDYNY Z PRÓBEK PASZY SHAKING UAE SOXHLET SOXTEC MAE ASE 8 g spiked poultry feed + 8 g pure quartz 5 g spiked poultry feed + 5 g pure quartz 4 g spiked poultry feed 2.5 g spiked poultry feed 0.5 g spiked poultry feed 8 g spiked poultry feed + 5 g pure quartz sample preparation before extraction 60 ml acidified methanol 25 ml acidified methanol 45 ml acidified methanol 25 ml acidified methanol 9 ml acidified methanol 25 ml acidified methanol solvent used for extraction ambient temperature ambient temperature extraction at elevated temperature extraction in boiling solvent extraction at 60, 80, 100, 120, 140 o C extraction at 60, 80, 100, 120, 140 o C extraction temperature microwave power 300W extraction pressure 1000, 1500, 2000, 2500psi additional parameters used for extraction extract + molecular sieve, shaking vigorously 5 min drying step 2 ml aliquot column filled with 1g of aluminium oxide (activity grade I) elution with 10 ml of methanol, careful mixing clean-up step 20 μl aliquot of the purified extract HPLC DAD MS analysis chromatographic analysis 27

28 RECOVERY OF ROBENIDYNE FROM SAMPLES OF ANIMAL FOODSTUF (with addition of standard) 120 Recovery y[%] Samples próbki skażone without standard próbki Samples rzeczyw iste with standard 20 0 ASE MAE Soxtec Soxhlet Shaking UAE 28

29 CHROMATOGRAPHIC SEPARATION OF ROBENIDYNE (LC DAD MS) Chromatographic column: 125 x 4.0, 5 µm LiChrosphere ODS (Merck) Mobile phase flow-rate: 1 ml/min Temperature of the chromatographic column: 30 o C Mobile phase: isocratic conditions: (70% methanol +30% water with addition (1% v/v) oftfaa 29

30 DETECTION Detector DAD Monitored wavelength: 317 nm Mass spectrometer Sygnał Signal of detek MS ktora MS Spectrum widmo SCAN SCAN Ionisation type: electrospray (ESI), mode: positive ions Drying gas flow rate: 12 l/min Pressure: 45psi Drying gas temperature: 350 o C Mode of ions monitoring: SIM, Monitored ions: 334 m/z and 336 m/z m/z min 30

31 PASSIVE TECHNIQUES OF ANALYTES SAMPLING SPMD LDPE strips MESCO POCIS 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. 31

32 CHEMCATCHER PASSIVE SAMPLER DESIGN -Sampler body -Diffusion limiting membrane -Receiving phase SAMPLER has been designed d and evaluated at the University i of Portsmouth within the STAMPS project. STAMPS = Standardised Aquatic Monitoring of Priority Pollutants by Passive Sampling 32

33 CONFIGURATION OF CHEMCATCHER PASSIVE SAMPLER Version of the sampler Chemcatcher 1 Chemcatcher 2 Chemcatcher 3 Chemcatcher 4 Receiving phase (trapping medium) Extraction disc (C18 ) Extraction disc (C18) Extraction disc (SDB-RPS ) Extraction disc (SDB-XC) Membrane polyethersulfone Final determination technique LC-DAD-MS LC-DAD-MS LC-DAD-MS LC-DAD -MS 33

34 CALIBRATION OF CHEMCATCHER IN A FLOW- THROUGH SYSTEM Chemicals in MeOH waste water ov verhead sti rrer Samplers Water reservoir Analytes In MeOH Water Stirrer Peristaltic pump 100 μl/min Exposure tank Peristaltic pump 30 ml/min 34

35 CONDITIONS OF SEPARATION AND FINAL DETRERMINATION (HPLC DAD MS) Elements of measuring system Parameters Liquid id chromatograph h HPLC 1100, Agilent Column Mobile phase Mobile phase flow-rate Altima HPLC - 18 EPS 250mm x 4.6 mm x 5µm A: H2O + 1% HCOOH B: MeOH/ACN (1:1, v:v) + 1% HCOOH 0.9 ml/min Gradient czas %A %B Analysis time 20 min 35

36 MONITORED IONS MS SIM (SCAN ) tolmetin 212 Ionisation mode: ELECTROSPRAY naproxen 229 fenoprofen 241 ibuprofen 205 diklofenak 294 diflunisal 249 DAD 275nm 36

37 CONCLUSIONS Complex and varied composition of the sample matrix SPE Low level concentration of analytes 37

38 CONCLUSIONS DRINKING WATER NATURAL WATERS WASTE WATERS on-line SPE off-line SPE passive sampling (SPE) 38

39 DEPARTMENT OF ANALYTICAL CHEMISTRY Analityczna/analit.htm List of publications Lectures presented during conferences Courses and expertises 39

40 COURSES AND EXPERTISES Individual id courses (on demand) d) HPLC (basic and advanced) GC (basic and advanced) Sample preparation for analysis ABC of the SPE technique Application of HPLC in food analysis Hyphenated techniques Biotests in environmental studies Quality Control and Quality Assurance (QC/ QA) of analytical results 40

41 QUALITY CONTROL AND QUALITY ASSURANCE OF ANALYTICAL L RESULTS EDITED BY: P. Konieczka and J. Namieśnik ISBN:

42 ISEAC 08 42