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

High Throughput Analysis of Endocrine Disruptors (Hormones

and Parabens)

Sébastien Sauvé, Liza Viglino, Paul Fayad and

Michèle PrévostDepartment of Chemistry

[email protected]

2

Pharmaceuticals & PCPs

Parent compoundsSoluble Metabolites

IncompleteTreatment

Introduction Into the

environment

Estrogens and Progestogens

Natural Excretion

Anovulants (birth control pills)

Hormone remplacement therapy

Anovulants (birth control)

19.3

9.8

7.6

3.6

3.4

1.4

0.8

0.1

0.1

0 5 10 15 20 25

ETHINYLESTRADIOL & LEVONORGESTREL

ETHINYLESTRADIOL & NORGESTIMATE

DESOGESTREL & ETHINYLESTRADIOL

DROSPIRENONE & ETHINYLESTRADIOL

ETHINYLESTRADIOL & NORETHINDRONE

NORETHINDRONE

ETHINYLESTRADIOL & ETHYNODIOL

ETHINYLESTRADIOL & NORGESTREL

LEVONORGESTREL

Daily consumption per 1000 people

Hormone remplacement therapy (menopause)

16.1

6.5

6.0

3.5

3.3

1.6

1.0

0.4

0 5 10 15 20

ESTROGENIC SUB,CONJUGATED

MEDROXYPROGESTERONE

ESTRADIOL-TRANSDERMIC

ESTRADIOL

PROGESTERONE

ESTROGENIC SUB,CONJUGATED & MEDROXYPROGESTERONE

ESTROPIPATE

ETHINYLESTRADIOL & NORETHINDRONE

Daily Consumption per 1000 people

The sum of estrogens sold in pharmacy mean that about 17% of women, of all ages are actually consuming them

Solid PhaseExtraction

Sébastien Sauvé, Département de chimie

SPE: Enrichment

Detection: Tandem Mass Spectrometry

(selected reaction monitoring)

SPE-LC-MS/MS Analysis

ThermoElectron TSQ Quantum Ultra

Results Hormones

River Montreal STP

Compound Mille Iles St Lawrence North

collector South

collector Effluent

Estriol n.d. n.d. 243 ± 24 230 ± 13 Trace

Estradiol 9 ± 3 8 ± 4 125 ± 4 120 ± 6 90 ± 4

17-α-ethinylestradiol n.d. n.d. 75 ± 3 90 ± 4 n.d.

Estrone n.d. n.d. Trace Trace n.d.

Norethindrone n.d. n.d. 205 ± 3 70 ± 4 53 ± 3

Levonorgestrel n.d. n.d. 150 ± 7 170 ± 4 30 ± 6

Medroxyprogesterone Trace n.d. 5 ± 3 n.d. n.d.

Progesterone 3 n.d. Trace n.d. n.d.

Viglino L, Aboulfadl K, Prévost M, Sauvé S*. 2008. Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:1088-1096.

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: www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

→ e- + N2 → N2+. +

2e-

Primary ion formation

Secondary ion formation

Proton transfer

→ N2+. + H2O → N2 + H2O+.

→ H2O+. + H2O → H3O+ + HO.

→ H3O+ + M → (M+H)+ + H2O

Laser diode thermal desorption (LDTD)

LDTD

Installation

Auto-sampler

960 samples

Corona needle position (APCI)

IR L

ase

r

(980 n

m,

20 W

)

Can ramp up to 3000oC/sec.Laser power is defined in %Normally ~100-150oC

Laser diode thermal desorption

Laser diode thermal desorption

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)

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.

Results for Feminizing Hormones

Ionisation Linearity range ILD MDLMode (μg/L) (μg/L) (ng/L)

E1 NI 9 - 684 0.0421 0.9999 9 0.0129 0.9984 30E2 NI 18 - 588 0.0084 0.9998 10 0.0030 0.9972 36

PI 18 - 588 0.0384 0.9995 5 0.0035 0.9997 13E3 NI 80 - 749 0.0038 0.9986 24 0.0016 0.9977 30EE2 NI 39 - 915 0.0062 0.9993 17 0.0019 0.9946 42

PI 57 - 915 0.0178 0.9990 8 0.0024 0.9988 14LEVO PI 47 - 780 0.0128 0.9990 11 0.0025 0.9965 20MPROG PI 35 - 677 0.0630 0.9990 6 0.0098 0.9964 30NOR PI 41 - 634 0.0144 0.9989 9 0.0031 0.9950 25PROG PI 21 - 670 0.0320 0.9995 6 0.0080 0.9971 18

Sensitivity R2 Sensitivity R2

In matrixCompound

Matrix-free

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

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

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: www.ldtd-ionsource.com/fra/default.asp

Advantages of LDTD vs LC

Compound MWg mol-1

Log Kow Use

Estriol 288.4 2.81

EstrogensEstrone 270.4 3.43

Estradiol 272.4 3.94

17-alpha-ethinylestradiol 296.4 4.15 Synthetic

estrogen

Progesterone 314.5 3.87 Progestogen

Norethindrone 298.5 2.97

Synthetic progestogensNorgestrel 312.4 3.08

Medroxyprogesterone 344.5 2.69

Methylparaben 152.2 2.00

Preservatives

Ethylparaben 166.2 2.49

Propylparaben 180.2 2.98

Butylparaben 194.2 3.57

Benzylparaben 228.3 not found

Triclocarban 315.6 4.90 Antibacterial agent

For Solids

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, [13C2]-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, [13C2]-estradiol and methylparaben-d4 (100 μL of 0.2 mg/L) were added as internal standards.

Sample Preparation

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

In soils and sedimentsFeminizing hormones & TCC: 0.9 to 4.0 ng g-1

Parabens: 1.4 to 2.9 ng g-1

In biosolidsFeminizing 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

SEDIMENTS SOILS BIOSOLIDS

(ng/g) I II II III I II

Estriol 18 3 6 3 15 3 28 9 69 3 45 8Estradiol 70 7 22 4 93 12 81 11 57 3 41 417-alpha-ethinylestradiol 30 6 n.d 86 12 34 11 55 3 47 12Estrone 16 6 6 3 7 2 10 2 54 3 32 2Norethindrone 90 9 n.d 93 17 91 22 106 3 105 12Levonorgestrel 19 5 n.d 24 12 52 17 53 3 33 7Medroxyprogesterone 29 3 n.d 28 4 13 3 31 3 22 9Progesterone 12 4 LMD 31 9 19 6 89 3 72 9TCC 16 4 n.d 53 9 13 2 59 3 43 7Methylparaben 56 7 n.d 127 12 107 4 72 2 91 9Ethylparaben 12 2 n.d LMD 15 3 LMD n.dPropylparaben 15 4 n.d 5 2 9 1 8 1 n.dButylparaben 19 3 n.d 20 5 23 4 LMD n.dBenzylparaben n.d n.d n.d LMD 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

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

Conclusions

Acknowledgments

Natural Sciences and Engineering Research Council of Canada (NSERC)

Phytronix Technologies inc.

If you want more detailed information, or reprints of

published papers, just send me an e-mail:

[email protected]

Questions?

Tandem Mass Spectrometry

Sulfamethoxazole+H+

m/z= 254.0

NH2 S

O

O

N

NO

HH

S

O

O

NH2

N

ONH2

m/z=156.0

m/z=108.0

m/z=92.0

Argon-induced Fragmentation

Tandem Mass Spectrometry

Quadrupole 1

Collision Cell

Quadrupole 3

Analyte=254.0 m/z

Interference=245.2 m/zFiltre=254.0m/z

156.0 m/z, 108.0 m/z, etc

Filtre=156.0m/z124.2 m/z, etc.

156.0 m/z

Laser diode thermal desorption (LDTD): Optimization1) Solvant choice for deposition: example for hormones in

NI and PI mode

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

3.5E+06

4.0E+06

4.5E+06

5.0E+06

EE2 E2 MPROG LEVO NOR PROG E1 E3

Pea

k A

rea

ACN at 20% laser power

MeOH at 20% laser power

EtOAc at 30% laser power

*

* *

*

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

7.0E+05

8.0E+05

5 10 15 20 25 30 40 50 60 80

Pea

k A

rea

Laser Power (%)

EE2

E2

MPROG

LEVO

NOR

PROG

Laser diode thermal desorption (LDTD): Optimization2) Laser power (%): example for hormones in PI mode

La puissance laser maximale est de 20%.

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Pea

k A

rea

Gas Flow (L/min)

E3

E1

EE2

E2

Laser diode thermal desorption (LDTD): Optimisation3) 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

Laser diode thermal desorption (LDTD): Optimisation4) Deposition volume (µL): example for PROG in PI mode

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

1 2 5 8 10

Ma

trix F

ree

Pe

ak

Are

a

In M

atr

ix P

ea

k A

rea

Deposition Volume (μL)

in matrix

matrix free

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.

Laser diode thermal desorption (LDTD): Optimisation5) Laser pattern : example for PROG in PI mode

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

Pea

k A

rea

Laser Pattern

1 sec – 20%

0.5 sec

1 sec – 20%

0.5 sec

2 sec – 20%

0.5 sec

2 sec – 20%

0.5 sec

1 sec

1 sec

3 sec – 2%

3 sec – 20%

1 sec

1 sec

3 sec – 2%

3 sec – 20%

1 sec

3 sec – 20%

1 sec

3 sec – 20%

1 sec

3 sec – 20%

1 sec

3 sec – 20%

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)

Filtration

Filter = 0.7μm, 0.45 μm

N

O

n

Extraction

200 mg Strata-X Cartridges (Phenomenex)

PS-DVB modifiéSurface Area 760-820 m2/g

Pore size 8.1-9.1 nm

Pore Volume 1.10-1.30 mL/g

Particule size 28-34 μm

Solid phase extraction

Sample100-500 ml offline1 ml online

SPE cartridge

Solid phase extraction

Sample200 ml

SPE Cartridge

Wash

Solid phase extraction

Elutionoffline

SPE Cartridge

Into ~250 µl

250 μL mobile phase containing internal standard

2 min ultrasonic bath

5 min centrifugation to improve dissolution

Injection of 1-10 µl

Reconstitution

Only a part of the SPE aliquot goes into the MS detector

Solid phase extraction offline

10 µl of250 µl

Chromatography MS/MS

The entire 1.0 ml goes through the MS detector

Solid phase extraction online

1.0 ml Chromatography MS/MSSPE

Waste

Pharmas/pesticidesRT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time (min)

0

1000

1000

1000

1000

100

Relati

ve Abu

ndance

0

1000

1000

100 NL: 3.20E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [108.650-109.650] MS 2000ppt2NL: 9.97E3TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [158.560-159.560] MS 2000ppt2NL: 6.70E5TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [122.580-123.580] MS 2000ppt2NL: 1.14E5TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [252.600-253.600] MS 2000ppt2NL: 8.55E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [252.610-253.610] MS 2000ppt2NL: 6.48E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [132.550-133.550] MS 2000ppt2NL: 9.86E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [108.570-109.570] MS 2000ppt2NL: 5.88E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [244.630-245.630] MS 2000ppt2

Progesterone

Estradiol

Medroxyprogesterone

Estriol

Estrone

17-α-ethinylestradiol

19-Northindrone

D(-) Norgestrel

Chromatograms obtained with the complete method:

SPE-LC-MS/MS (ESI/APPI) in milli-Q water doped @ 100 ng/l

Steroid hormones

Compounds with their LODs (using 1ml injections)

Pharmaceuticals2 - 24 ng/L

Pesticides2 - 17 ng/L

HormonesEstrogens

3 - 25 ng/LProgestogens

2 - 7 ng/L

Viglino L, Aboulfadl K, Daneshvar Mahvelat A, Prévost M, Sauvé S*. 2008. 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 10:482-489

Viglino L, Aboulfadl K, Prévost M, Sauvé S*. 2008. Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:1088-1096. (doi:10.1016/j.talanta.2008.05.008)

0

50

100

150

Carb

a

Nap

roxe

n

Caffei

ne

Trim

etho

prim

Gem

fibroz

il

MOY entrée Lac St Louis

MOYFleuve St Laurent

Pharmas in the St-Lawrence

Hormones

River Montreal STP

Compound Mille Iles St Lawrence North

collector South

collector Effluent

Estriol n.d. n.d. 243 ± 24 230 ± 13 Trace

Estradiol 9 ± 3 8 ± 4 125 ± 4 120 ± 6 90 ± 4

17-α-ethinylestradiol n.d. n.d. 75 ± 3 90 ± 4 n.d.

Estrone n.d. n.d. Trace Trace n.d.

Norethindrone n.d. n.d. 205 ± 3 70 ± 4 53 ± 3

Levonorgestrel n.d. n.d. 150 ± 7 170 ± 4 30 ± 6

Medroxyprogesterone Trace n.d. 5 ± 3 n.d. n.d.

Progesterone 3 n.d. Trace n.d. n.d.

Viglino L, Aboulfadl K, Prévost M, Sauvé S*. 2008. Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:1088-1096.

0

50

100

150

200

250

300

SMX TRI CLA AZI

Co

nce

ntr

atio

n / n

gL

-1

Analyzed Anti-infectives

City of Montréal Wastewater Treatment Plant

North Influent

South Influent

Effluent

Montréal April 2006

Segura PA, Garcia Ac A, Lajeunesse A, Ghosh D, Gagnon C, Sauvé S*. 2007. Determination of six anti-infectives in wastewater using tandem solid phase extraction and LC/MS/MS. Journal of Environmental Monitoring 9:307-313.

Efficiency of wastewater treatment (Montréal - May

2006),amongst the world’s largest ~ 2 500 000

m3/dayCompound Removal efficiency (%)

Mass flows in the St-Lawrence River

(g day-1) Sulfamethoxazole 12 ± 1 340 ± 30

Trimethroprim N.S. 310 ± 20

Ciprofloxacin 29 ± 2 320 ± 10

Levofloxacin 31 ± 2 118 ± 2

Clarithromycin N.S. 790 ± 60

Azithromycin N.S. 320 ± 20

N.S. – non significatif

1 ton of antibiotic molecules released every year

Pharmaceuticals & PCPs

•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

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

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

Filtration

Filter = 0.7μm, 0.45 μm

Sébastien Sauvé, Département de chimie

Solid Phase

Extraction

SPE: Enrichment

LC-MS/MS Analysis

Detection: Tandem Mass Spectrometry

(selected reaction monitoring)

ThermoElectron TSQ Quantum EQuan MAX System

RT: 0.00 - 15.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time (min)

0

1000

1000

1000

1000

100

Rel

ativ

e Ab

unda

nce 0

1000

1000

100 NL: 3.20E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [108.650-109.650] MS 2000ppt2NL: 9.97E3TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [158.560-159.560] MS 2000ppt2NL: 6.70E5TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [122.580-123.580] MS 2000ppt2NL: 1.14E5TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [252.600-253.600] MS 2000ppt2NL: 8.55E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [252.610-253.610] MS 2000ppt2NL: 6.48E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [132.550-133.550] MS 2000ppt2NL: 9.86E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [108.570-109.570] MS 2000ppt2NL: 5.88E4TIC F: + c APCI sid=10.00 SRM ms2 [email protected] [244.630-245.630] MS 2000ppt2

Progesterone

Estradiol

Medroxyprogesterone

Estriol

Estrone

17-α-ethinylestradiol

19-Northindrone

D(-) Norgestrel

Ovarian hormones

SPE-LC-MS/MS (ESI/APPI) in milli-Q water doped @ 100 ng/l

Compounds with their LODsInjection Volume?

Pharmaceuticals2 - 24 ng/L

Pesticides2 - 17 ng/L

HormonesEstrogens

3 - 25 ng/LProgestogens

2 - 7 ng/L

Viglino L, Aboulfadl K, Daneshvar Mahvelat A, Prévost M, Sauvé S*. 2008. 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 10:482-489

Viglino L, Aboulfadl K, Prévost M, Sauvé S*. 2008. Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS. Talanta 76:1088-1096. (doi:10.1016/j.talanta.2008.05.008)

Montréal April 2006

0

50

100

150

200

250

300

SMX TRI CLA AZI

Co

nce

ntr

atio

n / n

gL

-1

Analyzed Anti-infectives

City of Montréal Wastewater Treatment Plant

North Influent

South Influent

Effluent

Segura PA, Garcia Ac A, Lajeunesse A, Ghosh D, Gagnon C, Sauvé S*. 2007. Determination of six anti-infectives in wastewater using tandem solid phase extraction and LC/MS/MS. Journal of Environmental Monitoring 9:307-313.

Results – Granby April 2006

City of Granby Wastewater Treatement Plant

0

50100

150

200

250300

350

SMX TRI CLA AZI

Analyzed Anti-infectives

Co

nce

ntr

atio

n /

ng

L-1

Inf luent

Eff luent

Efficiency of Montréal wastewater treatment - May 2006(amongst the world’s largest ~ 2 500 000 m3/day)

Compound Removal efficiency (%)

Mass flows in the St-Lawrence River

(g day-1) Sulfamethoxazole 12 ± 1 340 ± 30

Trimethroprim N.S. 310 ± 20

Ciprofloxacin 29 ± 2 320 ± 10

Levofloxacin 31 ± 2 118 ± 2

Clarithromycin N.S. 790 ± 60

Azithromycin N.S. 320 ± 20

N.S. – non significant

1 ton of antibiotic molecules released every year

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)

•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

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:

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?

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

Most prescribed anti-infectives in Québec in 2004

(IMS Health Canada compilation – 8 million people in Quebec).

0 100 200 300 400 500 600 700 800

Thousands of retail prescriptions dispensed

Amoxicillin

Ciprofloxacin

Azithromycin

Clarithromycin

Penicillin V

Cefprozil

Fluconazole

Sulfamethoxazole + Thrimethoprim

Mesazaline

Minocycline

Moxifloxacin

Clindamycin

Cephalexin

Levofloxacin

Nystatin

Nitrofurantoin

Terbinafine

Cloxacillin

Pivampicillin

Gatifloxacin