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High Throughput Analysis of Endocrine Disruptors (Hormones and Parabens) Sébastien Sauvé, Liza Viglino, Paul Fayad and Michèle Prévost Department of Chemistry.

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Presentation on theme: "High Throughput Analysis of Endocrine Disruptors (Hormones and Parabens) Sébastien Sauvé, Liza Viglino, Paul Fayad and Michèle Prévost Department of Chemistry."— Presentation transcript:

1 High Throughput Analysis of Endocrine Disruptors (Hormones and Parabens) Sébastien Sauvé, Liza Viglino, Paul Fayad and Michèle Prévost Department of Chemistry

2 2 Pharmaceuticals & PCPs Parent compounds Soluble Metabolites Incomplete Treatment Introduction Into the environment

3 Estrogens and Progestogens Natural Excretion Anovulants (birth control pills) Hormone remplacement therapy

4 Anovulants (birth control)

5 Hormone remplacement therapy (menopause) The sum of estrogens sold in pharmacy mean that about 17% of women, of all ages are actually consuming them

6 Solid Phase Extraction Sébastien Sauvé, Département de chimie

7 SPE: Enrichment

8 Detection: Tandem Mass Spectrometry (selected reaction monitoring) SPE-LC-MS/MS Analysis ThermoElectron TSQ Quantum Ultra

9 Results Hormones Viglino L, Aboulfadl K, Prévost M, Sauvé S* Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:

10 Principles of the LDTD-APCI source: technique that combines thermal desorption (laser diode) and APCI sample is spotted (1-10 μL) into a 96-well plate and air-dried for 2 min uncharged analytes are thermally desorbed into the gas phase ionization takes place in the corona discharge region by APCI and the charged molecules will be transferred to the MS inlet Source: e - + N 2 N e - Primary ion formation Secondary ion formation Proton transfer N H 2 O N 2 + H 2 O +. H 2 O +. + H 2 O H 3 O + + HO. H 3 O + + M (M+H) + + H 2 O Laser diode thermal desorption (LDTD)

11 LDTD Installation Auto-sampler 960 samples Corona needle position (APCI) IR Laser (980 nm, 20 W) Can ramp up to 3000 o C/sec. Laser power is defined in % Normally ~ o C Laser diode thermal desorption

12

13 LDTD Process (1) Infrared laser (980 nm, 20W) (2) LazWell Plate (96 wells): analyte desorption (1-10 µL spotted) (3) Transfer tube transporting the neutrally desorded analytes to the APCI region (4) Corona needle discharge region (APCI) (5) MS inlet (1) (2) (3) (4) (5)

14 Parameters of the LDTD-APCI source are optimized for signal intensity : solvent choice for analyte deposition in the well cavities laser power (%) carrier gas flow rate (L/min) mass deposition (deposition volume in µL) into plate well laser pattern No need to optimize liquid chromatography - it has been completely eliminated! A minimum of 2 SRM transitions were selected + their ion ratios LDTD Optimization Optimization for MS (precursor) and MS/MS (SRM transitions) conditions in NI and PI mode.

15 Results for Feminizing Hormones Fayad P, Prévost M, Sauvé S. Laser Diode Thermal Desorption-Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry Analysis of Selected Steroid Hormones in Wastewater: Method Optimization and Application. Submitted to Analytical Chemistry

16 Very polar analytes (conjugated drug metabolites) or higher mass analytes (e.g. proteins easily ionized with ESI) can be tough to ionize by APCI. Limits of detection are not as good – may sometime need SPE Solutions to this and other difficulties: 1) No chromatographic separation (no column is used to separate same masses in time) 2) Complexe matrices can reduce signal intensity due to competitive interfering compounds in the APCI region Could de resolved by using two ionization modes (PI and NI) Must utilize a proper washing step in sample preparation or try sample dilution 3) Polar or higher mass analytes usually more suitable for ESI type analyses Different solvants can be used to improve signal response and derivitization Disadvantages of LDTD vs LC

17 Analysis is done in 15 sec opposed to several minutes in LC-MS/MS Small sample volume (1-10 µL) compared to 1+ mL for on-line SPE LC-MS/MS Less contamination sources (tubing, column, injection loop or port, divert valve) Reduction of backround noise resulting from solvant use Reduction of operation costs (solvants, columns, tubing, seringes, pumps) Source: Advantages of LDTD vs LC

18 Compound MW g mol -1 Log K ow Use Estriol Estrogens Estrone Estradiol alpha- ethinylestradiol Synthetic estrogen Progesterone Progestogen Norethindrone Synthetic progestogens Norgestrel Medroxyprogesterone Methylparaben Preservatives Ethylparaben Propylparaben Butylparaben Benzylparaben228.3not found Triclocarban Antibacterial agent For Solids

19 1g of freeze-dried samples (0.2 g for municipal sudge) suspended in in 6 mL of acetonitrile: ethyl acetate (5:1) in a 20-mL Teflon tube. Then, [ 13 C 2 ]-ethynylestradiol (50ng/g) was added as surrogate standard and the sample was vigorously shaken for a few seconds. After a sonication of 30 min at 30ºC using a sequential ultrasonic extraction (USE), samples were stirred for 45 min on a wrist-shaker at room temperature and centrifuged for 10 min at 3500 rpm to recover the organic layer. Before evaporation, under a gentle stream of nitrogen to a volume of 250 L at 20ºC, [ 13 C 2 ]-estradiol and methylparaben-d 4 (100 μ L of 0.2 mg/L) were added as internal standards. Sample Preparation

20 Extract was evaporated to dryness, reconstituted into 5 mL of a heptane-acetone (65/35, v/v) mixture and passed through a silica cartridge Eluted with 6 mL of a heptane-acetone (65/35, v/v) mixture. The final solution was reduced to a volume of 250 L under a nitrogen stream at 20ºC. SPE Clean up for sludge

21 In soils and sediments Feminizing hormones & TCC: 0.9 to 4.0 ng g -1 Parabens: 1.4 to 2.9 ng g -1 In biosolids Feminizing hormones & TCC: 9.0 to 17 ng g -1 Parabens: 2.8 to 6.4 ng g -1 Detection Limits Viglino L, Prévost M, Sauvé S High throughput analysis of solid-bound endocrine disruptors by LDTD-APCI-MS/MS. In revision

22 SEDIMENTSSOILSBIOSOLIDS (ng/g) III IIIIII Estriol Estradiol alpha-ethinylestradiol 30 6 n.d Estrone Norethindrone 90 9 n.d Levonorgestrel 19 5 n.d Medroxyprogesterone 29 3 n.d Progesterone 12 4 LMD TCC 16 4 n.d Methylparaben 56 7 n.d Ethylparaben 12 2 n.d LMD15 3 LMD n.d Propylparaben 15 4 n.d n.d Butylparaben 19 3 n.d LMD n.d Benzylparabenn.d LMD n.d Results Viglino L, Prévost M, Sauvé S High throughput analysis of solid-bound endocrine disruptors by LDTD-APCI-MS/MS. In revision

23 LDTD allows for a very fast and simple analysis for some endocrine disruptors in the environment for water and solids. Conclusions

24 Acknowledgments Natural Sciences and Engineering Research Council of Canada (NSERC) Phytronix Technologies inc.

25 If you want more detailed information, or reprints of published papers, just send me an Questions?

26

27 Tandem Mass Spectrometry Sulfamethoxazole+H + m/z= m/z=156.0 m/z=108.0 m/z=92.0 Argon-induced Fragmentation

28 Tandem Mass Spectrometry Quadrupole 1 Collision Cell Quadrupole 3 Analyte=254.0 m/z Interference=245.2 m/z Filtre=254.0m/z m/z, m/z, etc Filtre=156.0m/z m/z, etc m/z

29 Laser diode thermal desorption (LDTD): Optimization 1) Solvant choice for deposition: example for hormones in NI and PI mode * * * *

30 Laser diode thermal desorption (LDTD): Optimization 2) Laser power (%): example for hormones in PI mode La puissance laser maximale est de 20%.

31 Laser diode thermal desorption (LDTD): Optimisation 3) Carrier gas flow (L/min): example for hormones in NI mode E3 is more polar than other hormones. A lower gas flow allows more time in the corona discharge region for proper ionization. As with LC methods and other techniques, a compromise must be made to combine multiple compounds

32 Laser diode thermal desorption (LDTD): Optimisation 4) Deposition volume (µL): example for PROG in PI mode A clean-up step in sample preparation or a sample dilution will allow for higher S/N ratios, improving the overall sensitivity and reducing the LD of the method.

33 Laser diode thermal desorption (LDTD): Optimisation 5) Laser pattern : example for PROG in PI mode

34 Use an enrichment method to extract and: 1.Isolate the compounds of interest 2.Increase their concentration 3.Remove interfering compounds Solid Phase Extraction (SPE)

35 Filtration Filter = 0.7μm, 0.45 μm

36 Extraction 200 mg Strata-X Cartridges (Phenomenex) PS-DVB modifié Surface Area m 2 /g Pore size nm Pore Volume mL/g Particule size28-34 μm

37 Solid phase extraction Sample ml offline 1 ml online SPE cartridge

38 Solid phase extraction Sample 200 ml SPE Cartridge Wash

39 Solid phase extraction Elution offline SPE Cartridge Into ~250 µl

40 250 μL mobile phase containing internal standard 2 min ultrasonic bath 5 min centrifugation to improve dissolution Injection of 1-10 µl Reconstitution

41 Only a part of the SPE aliquot goes into the MS detector Solid phase extraction offline 10 µl of 250 µl Chromatography MS/MS

42 The entire 1.0 ml goes through the MS detector Solid phase extraction online 1.0 ml Chromatography MS/MS SPE Waste

43 Pharmas/pesticides Chromatograms obtained with the complete method: SPE-LC-MS/MS (ESI/APPI) in milli-Q water 100 ng/l Steroid hormones

44 Compounds with their LODs (using 1ml injections) Pharmaceuticals ng/L Pesticides ng/L Hormones Estrogens ng/L Progestogens ng/L Viglino L, Aboulfadl K, Daneshvar Mahvelat A, Prévost M, Sauvé S* On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters. Journal of Environmental Monitoring 10: On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters Viglino L, Aboulfadl K, Prévost M, Sauvé S* Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76: (doi: /j.talanta )doi: /j.talanta

45 Carba Naproxen Caffeine Trimethoprim Gemfibrozil MOY entrée Lac St Louis MOYFleuve St Laurent Pharmas in the St-Lawrence

46 Hormones Viglino L, Aboulfadl K, Prévost M, Sauvé S* Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:

47 Montréal April 2006 Segura PA, Garcia Ac A, Lajeunesse A, Ghosh D, Gagnon C, Sauvé S* Determination of six anti- infectives in wastewater using tandem solid phase extraction and LC/MS/MS. Journal of Environmental Monitoring 9:

48 Efficiency of wastewater treatment (Montréal - May 2006), amongst the worlds largest ~ m 3 /day N.S. – non significatif 1 ton of antibiotic molecules released every year

49 Pharmaceuticals & PCPs

50 Reproductive issues in fish Multiple or distorted legs in frogs and other amphibians Drug resistance in humans Possible Effects on Humans and Animals in the Environment

51 Develop a simple method, sensitive enough to analyze trace contaminants in waste, raw and drinking waters and eventually solids (soil, sediments and biosolids) Online solid phase extraction preconcentration coupled to liquid chromatography and tandem mass spectrometry (SPE-LC-MS/MS) and Laser Diode Thermal Desorption (LDTD) – APCI (Atmospheric Pressure Chemical Ionization) -MS/MS Pharmaceuticals & PCPs

52 Use an enrichment method to extract and: 1.Isolate the compounds of interest 2.Increase their concentration 3.Remove interfering compounds SPE (solid phase extraction) Solution

53 Filtration Filter = 0.7μm, 0.45 μm

54 Sébastien Sauvé, Département de chimie Solid Phase Extraction

55 SPE: Enrichment

56 LC-MS/MS Analysis Detection: Tandem Mass Spectrometry (selected reaction monitoring) ThermoElectron TSQ Quantum EQuan MAX System

57 Ovarian hormones SPE-LC-MS/MS (ESI/APPI) in milli-Q water 100 ng/l

58 Compounds with their LODs Injection Volume? Pharmaceuticals ng/L Pesticides ng/L Hormones Estrogens ng/L Progestogens ng/L Viglino L, Aboulfadl K, Daneshvar Mahvelat A, Prévost M, Sauvé S* On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters. Journal of Environmental Monitoring 10: On-line solid phase extraction and liquid chromatography/tandem mass spectrometry to quantify pharmaceuticals, pesticides and some metabolites in wastewaters, drinking, and surface waters Viglino L, Aboulfadl K, Prévost M, Sauvé S* Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI- MS/MS. Talanta 76: (doi: /j.talanta )doi: /j.talanta

59 Montréal April 2006 Segura PA, Garcia Ac A, Lajeunesse A, Ghosh D, Gagnon C, Sauvé S* Determination of six anti- infectives in wastewater using tandem solid phase extraction and LC/MS/MS. Journal of Environmental Monitoring 9:

60 Results – Granby April 2006

61 Efficiency of Montréal wastewater treatment - May 2006 (amongst the worlds largest ~ m 3 /day) N.S. – non significant 1 ton of antibiotic molecules released every year

62 What does that mean? A few nanograms per liter represent the equivalent of a sugar cube diluted in the Olympic stadium – really just traces! The concentrations observed at the worst case sites are far from the levels expected to cause impacts on human health (from 0.02 to 3% of the daily therapeutic dose after the consumption of 2 l/d for 70 years)

63 We can certainly detect pharmaceuticals in the environment Concentrations of pharmaceuticals in the environment can be of concern for some issues Especially antibiotic resistance, endocrine disruptors Pharmaceuticals in the Environment

64 What are the effects of chronic exposure to low levels of a mixture of contaminants? What is the contribution of traces of ovarian hormones as endocrine disruptors to human? High seasonal variability – not yet considered for risk assessment What are the risks from chemotherapy agents, and other compounds (surfactants, ionic liquids)? Some other questions:

65 Unreasonable to try and regulate consumption We can improve wastewater treatment to reduce releases – an engineering option We can look at pharmacopeia's redundancy When equivalent drugs are available, we could promote those with the least impact Can we think up « degradable » drugs? More susceptible to oxydation, hydrolysis, UV, microbial degradation, etc. in the natural environment. Tricky considering that they must still remain effective! And Green Chemistry?

66 HRMS (High Resolution Mass Spectrometry) Time of Flight Magnetic Sectors Orbitrap Mass Spec. Accurate mass measurement useful for positive ID when fragmentation is limited or for the screening of unknows Other MS Techniques for PCPPs

67 Most prescribed anti-infectives in Québec in 2004 (IMS Health Canada compilation – 8 million people in Quebec).


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