Methods for the Separation and Detection of Triclosan in Water

Brandon M. YoungSoper Research Group

Analytical SeminarApril 26, 2010

2

• Triclosan determination in water related to wastewater treatment– Jian-Lin Wu, Ng Pak Lama, Dieter Martens,

Antonius Kettrup, Zongwei Cai*

• Determination of triclosan, triclocarban and methyl-triclosan in aqueous samples by dispersive liquid–liquid microextraction combined with rapid liquid chromatography– Jie-Hong Guoa, Xing-Hong Li*, Xue-Li Caob, Yan Li,

Xi-ZhiWanga, Xiao-Bai Xua

3

Outline

Triclosan (TCS)

5-chloro-2-(2,4-dichlorophenoxy)phenol

I. Introduction

II. Objectives

III. Separation and Detection Method1. Goals

2. Experimental

3. Results

4. Conclusion

IV. Comparison of Methods

V. Summary

VI. Critiques

VII. Acknowledgements

4

Beginning of Triclosan

• White aromatic organic compound

• Its functional groups include phenols and ethers

• First registered as a pesticide in 1969

• Then used as a surgical scrub in 1972

• Triclosan (TCS) has since been marketed as the “Aspirin of anti-bacterials”

• “Lifesaver” in countries with unsanitary conditions.

http://www.epa.gov

5

Reaction Scheme for the Synthesis of Triclosan

PTC = Phase Transfer Catalysis (i.e. Tetra-n-butylammonium bromide)

Potassium 2,4-dichlorophenolate

2,5-dichlorophenol Triclosan (TCS)5-chloro-2-(2,4-dichlorophenoxy)phenol

Novelties of Solid-Liquid Phase Transfer Catalyzed Synthesis of Triclosan from Potassium 2,4-Dichlorophenolate and 2,5-Dichlorophenol

PTCΔ

6

Triclosan Anti-microbial Activity

• Triclosan (TCS) works by blocking the active site of the ENR (enoyl-acyl carrier protein reductase) enzyme

• Enzyme involved with the fatty acid biosynthesis in bacteria, fungi, and mildew with is is necessary for building cell membranes, growth and reproduction

• Since humans do not have this ENR enzyme, triclosan has been thought to be harmless to them

• Triclosan is a very potent inhibitor, and only a small amount is needed for powerful antibiotic action

Figure: Structure of the ternary EnvM complex. Ribbonrepresentation of the EnvM monomer; triclosan andNADH are shown in ball-and-stick representation withthe Cl atoms in green.

Structural Basis and Mechanism of Enoyl Reductase Inhibition by Triclosan

7

Current Uses • Bar, liquid and foaming soaps• Underarm deodorants• Toothpastes• Mouthwashes• Shampoos• Cosmetics• Laundry detergents• Fabric softeners• Facial tissues• Conveyor belts

• Antiseptics for wound care• Medical devices• Plastics• Kitchen utensils• Toys• Bedding• Socks• Trash bags• Fire hoses• Carpeting

http://www.epa.gov

January 11, 2010

Triclosan Raises Alarm!

• Question if TCS products are more effective than regular soap in reducing illness

• Disruption to the endocrine system

• Increasing antibiotic resistance

• Regulation on over-the-counter TCS products

9

Health and Triclosan• Acute health effects - skin irritation

• Severe health problems such as:

– weakening of the immune system– decreased fertility– birth defects – Cancer

• Researchers express concern about link between triclosan and carcinogenic dioxins

• Sunlight could degrade/convert triclosan to dioxins naturally

• Chlorine molecules in tap water mixed with triclosan containing soaps

• Dioxon found as impurities in commercial products

PCDDs (Polychlorinated dibenzodioxins ), or simply dioxins, are a group of

polyhalogenated compounds

http://www.dioxinfacts.org/dioxin_health/dioxin_rumors/triclosan.html

10

Triclosan to form Dioxins

The Chlorine Chemistry Division of the American Chemistry Council

1. 2.

1. 3. 4.

1) 5-chloro-2-(2,4-dichlorophenoxy)phenol (Triclosan)2) 2,8-dichlorodioxion (Dioxin)3) 4,5,6-trichloro-2-(2,4-dichlorophenoxy) phenol4) 1,2,3,7,8-tetrachlorodioxin (Dioxin)

11

Health and Triclosan

http://www.grinningplanet.com/2005/10-04/triclosan-article.htm

• Overuse of triclosan (and other antibacterials) is also linked to allergies.

• The "hygiene hypothesis"

• The concept is that children who are reared in an overly clean environment have immune systems that are not challenged and thus do not develop and mature properly.

• Based on studies that have found an increase in the frequency of allergies and asthma in persons who have been raised in more sterile and hygienic environments.

12

Triclosan Persists in the Environment

Breast Milk

Americans > 6 (urine)

Cord Blood

Rivers and Streams

Tap water and Food

Liquid hand soap

Toothpaste

Over 400 products

75%

97%

58%

47%

43%

?

?

7%

0 100

People

Environment, Food and Water

Consumer Products

Triclosan in consumer products leads to widespread pollution in people and the environment

http://www.ewg.org/reports/triclosan

13

Objective

• Investigate current methods for the separation and detection of triclosan

• Describe the importance of water sample detection

• Compare and contrast different methods

14

To prove that GC–MS/MS is a suitable approach for triclosan analysis in water, allowing the detection limits at low ng/L levels.

Goal

15

Method Overview

Extraction

Separation

Detection

Solid-phase Extraction (SPE)

16

Washing Eluting

Target Compound

Sample

Analyte

Interference

Extraction

SampleConditioning

Solid-phase Extraction (SPE)

http://www.waters.com/webassets/cms/category/media.jpg

17

Method Overview

Extraction

Separation

Detection

Gas Chromatography (GC)

Mass Spectrometry (MS/MS)

Interfaced

18

Separation/Detection

Sample Injection

Carrier Gas

Flow Control

Column

Gas Chromatography (GC)- Mass Spectrometrey

http://hiq.linde-gas.com/International/Web/LG/SPG/like35lgspg.nsf/docbyalias/anal_gaschromhttp://aemc.jpl.nasa.gov/instruments/vcam/

19

Experimental

Reagents and chemicals

GC–MS/MS analysis

Water sample pre-

treatment

20

Reagents and chemicals• Triclosan and 13C12-labeled triclosan

( internal standard ) were used

• HPLC grade solvents

• Stock solution prepared by dissolving 1.0 mg of triclosan in 1.0 mL of ethyl acetate

• Calibration standard solutions containing the analytes and internal standard were prepared by diluting the stock solution

• Milli-Q water was used as clean water

• Kept under 4 ◦C

• Sea Water– Tai Po Harbour – Victoria Harbour

• River water– Fo Tan industrial area– Sha Tin residentail area– Pearl River

• Wastewater – Sha Tin– Kwun Tong – Stonecutters Island

o Raw water o Primary treatmento Secondary treatment

21

Experimental

Reagents and chemicals

GC–MS/MS analysis

Water sample pre-

treatment

22

Water sample pre-treatment

• Amber glass bottles were used

• 2L water samples were collected from each sampling site

• Acidified with concentrated phosphoric acid to pH 2

• Capped lined with Teflon then placed on ice

• Stored at 4 ◦C until sample analysis.

23

SPE for water sample

Absorbent: Florisil

Base: Magnesium Silicate

Porosity: 60Å

Particle Size:75-150μm

Retention Mechanism: Normal-phase

http://www.discoverysciences.com/product.aspx?id=2958

The cartridge was pre-washed and conditioned with 5mL of ethyl acetate, methanol and Milli-Q water

Conditioning

1 liter water sample was filtered, spiked with 40 ng of internal standard (13C12-labeled triclosan) and passed through

the cartridgeSample

The column was rinsed with 5mL of 5% methanol in Milli-Q water to wash the interferences, then aspirated for 15 min to

remove airWashing

The cartridge was eluted with 3mL x 2mL ethyl acetate at a slow rate of around 1 mL/min, and the combined extracts

were then driedEluting

24

GC–MS/MS analysis

Experimental

Reagents and chemicals

Water sample pre-

treatment

25

GC–MS/MS analysis

80 ◦C1 min

280 ◦C6 min

300 ◦C

20 ◦C/min

GC–MS transfer line / ion source

•Thermo Finnigan Trace GC system interfaced with a Polaris Q ion trap mass spectrometer•DB-5MS GC column (30m x 0.25mm x 0.25μm film thickness (5%-phenyl)-methylpolysiloxane)•Carrier gas helium at flow rate of 1 mL/min. •The sample injected volume was 1μL in splitless mode.

•GC–MS/MS analysis showed the molecular ion peaks of triclosan m/z 288 and 13C12-triclosan m/z 300 at retention of time 10.2 min.

•Detection and retention time was confirmed through the NIST EI-mass spectrum library

Injection temp 260◦C

26

Results

27

GC–MS/MS analysis• Predominant ion peak: m/z

218 triclosan and m/z 230 13C12triclosan

• Ions at m/z 218 and 230 were chosen as the precursor ions for the second stage MS/MS analysis

• Most abundant fragment ion peaks at m/z 155 and 166

• Optimization of GC–MS/MS parameters was achieved by monitoring the corresponding peak intensities of the selected quantitation ions.

166 m/z

230 m/z

155 m/z

218 m/z

Triclosan

13C12Triclosan

28

•Optimization based on the determination under the best conditions to detect maximum peak intensity of the selected quantitation ion of triclosan.

MS/MS chromatograms of triclosan (A) and 13C12-triclosan (B) with the quantitation ions at m/z 218 > 155 and 230 > 166, respectively,

GC–MS/MS analysis

29

Results and Discussion•Recovery and Detection limit• Extraction recoveries investigated by using 1L sample water spiked with 70, 120

and 200 ng of triclosan.

•Method accuracy and precision• The obtained results indicate that the 13C12 triclosan internal standard method

provided satisfactory accuracy and precision.

30

Triclosan in environmental waters

• Compare levels in river water from the residential and industrial areas.

• River water collected in two seasons to investigate the seasonal triclosan levels.

• Different wastewater treatment plants and treatment steps were analyzed

31

Conclusion

• A method for the determination of triclosan in water samples was developed.

• The whole procedure combining SPE and GC–MS/MS analysis provided good recoveries and sensitivity

• The triclosan was detected at low concentrations in water samples.

• Triclosan concentration can be lowered during wastewater treatment

32

To show the novel method of DLLME–UHPLC–TUV for the determination of trace amounts of Triclosan in aqueous samples,

which showed a large dynamic range, good repeatability and accuracy.

Goal

33

Method Overview

Extraction

Separation

Detection

Dispersive liquid-liquid microextraction (DLLME)

34

Inject

1 mL Syringe

Solvent droplet

Sample solutionCloudy state Centrifuge Sedimented

phase

Withdraw

ExtractionDispersive liquid-liquid microextraction (DLLME)

ZANG Xiao-Huan, WU Qiu-Hua, ZHANG Mei-Yue, XI Guo-Hong, WANG Zhi*Key Laboratory of Bioinorganic Chemistry, College of Science, Agricultural University of Hebei, Baoding 071001, China

35

Method Overview

Extraction

Separation

Detection

Ultra High Performance Liquid Chromatography.

(UHPLC)

36

Separation

Mobile phase Injector

Solvent Delivery Pump

Column

Waste

Detector

http://hiq.linde-gas.com/international/web/lg/spg/like35lgspg.nsf/docbyalias/anal_hplc

Ultra High Performance Liquid Chromatography (UHPLC)

37

Method Overview

Extraction

Separation

DetectionTunable Ultraviolet (TUV)

38

Detection

• Uses ultraviolet-visible spectroscopy

• Measures the intensity of light passing through a sample

• Compares it to the intensity of light before it passes through the sample

• The ratio is called transmittance which the absorbance is calculated:

A = − log(%T / 100%)

• Tunable, dual wavelength ultraviolet/visible (UV/Vis) detector

• High sensitivity for low-level detectionhttp://www.waters.com/waters/nav.htm?locale=en_US&cid=514228

Tunable Ultraviolet (TUV)

39

Dispersive liquid–liquid

microextraction (DLLME)

Experimental

Materials and reagents

Standard solution UHPLC conditions

Instrumentation

40

Materials and reagents

• Solvents were HPLC grade: Acetonitrile, THF (tetrahydrofuran) and Methanol

• Extraction solvents: Tetrachloroethylene (C2Cl4), 1,4-dichlorobutane (C4H8Cl2), trichloroethylene (C2HCl3), 1,3- dichlorobenzene (C6H4Cl2), 1,3-dichloropropane (C3H6Cl2)

• Clean Water Milli-Q water purification system

• Buffer solution (pH 9) for mobile phase: 0.618 g boracic acid, 0.746 g potassium chloride and 0.168 g sodium hydroxide dissolved in 140mL purified water then diluted 20x before used

• Individual stock solution (1.00 g /L) made by dissolving the solid standard substance in methanol

Triclosan (TCS) Triclocarban (TCC) Methyl-triclosan (M-TCS)

Target analytes purchased at between 99-99.5% purity

41

The 5.00mL water was placed into a 10-mL glass test tube. Mixture of THF (1.00mL) as dispersive

solvent and C6H4Cl2 (15.0μL) as extraction solvent was rapidly injected

Dispersive liquid-liquid microextraction (DLLME) for water sample

The milky cloudy mixture was centrifuged for 5.0 min at 3000 rpm, the dispersed fine particles were sedimented in the bottom of the

test tube, about 20.0nL.

The sediment phase was then transferred to sample bottle by amicrosyringe and blown until dryness.

35.0 μL of methanol was used to dissolve the mixture, and 5.0 μL was injected automatically for UHPLC analysis.

Disperser & ExtractionSolvent Injection

Cloudy state formed &

Centrifugation

Sediment phaseExtraction

Sample to Chromatography

Method

42

Instrumentation & UHPLC conditions• UHPLC analyses were performed on Reversed Phase C18 analytical column (50 mm x

2.1 mm, 1.7 μm).

• Time for analysis was 5 min

• Detection wavelength = 283 nm

• 25 and 30 ◦C adopted as sample temperature and column temperature, respectively.

• The injection volume was 5.0 μL.

• Gradient elution

mobile phase Buffer solution 50% 40% 50% 10%

Acetonitrile 50% 60% 50% 90%

0.4mL/min 0.5mL/min 0.3mL/min 0.3mL/min

43

Results

Volume of extraction solvent

Selection of dispersive solvent

Selection of extraction solvent

Volume of dispersive solvent

Main method parameters

Real water sample analysis

Evaluation of Method performanceOptimization of DLLME

44

Four Extraction Methods to investigate the need and delivery of dispersive:

•Only extraction solvent C6H4Cl2

•Extraction solvent C6H4Cl2 followed by dispersive solvent THF•Dispersive solvent THF followed by extraction solvent C6H4Cl2

•Mixture of extraction solvent and dispersive solvent

Best recoveries was achieved when mixed composition of C6H4Cl2 and THF was employed. While results were relatively worse without THF and separately added of C6H4Cl2 / THF

Optimization of DLLME - Selection of dispersive solvent

45

• Experimental condition: inject (acetonitrile, methanol, acetone and THF (Tetrahydrofuran)) solution containing 1.00mL different dispersive solvents and 25.0μL C6H4Cl2 into water sample

• The different solubility in the two phases gave a sediment volume:

Dispersive solvent in DLLME, the miscibility in organic phase (extraction solvent) and aqueous phase (sample solution) is a key factor, which can disperse extraction solvent into very fine droplets in aqueous phase.

Optimization of DLLME - Selection of dispersive solvent

Dispersive solvent Acetonitrile Methanol Acetone THF

Sedimented Phase 23.0μL 22.0μL 22.5μL 35.0μL

46

Optimization of DLLME – Extraction Solvent type and volume

• Experimental condition: comparing the extraction recoveries of these compounds in the extraction of a 5.00mL water sample with mixture of 25.0μL different extraction solvents and 1.00mL THF as dispersive solvent at ambient temperature.

• Sedimented phase of these extraction solvents differed dramatically:

Obtaining good extraction recoveries in DLLME method some primary requirements must be met: •higher density than water•low water solubility•high extraction capability of target compound

Extraction solvent C2Cl4 C4H8Cl2 C2HCl2 C6H4Cl2 C3H6Cl2

Sedimented Phase 27.0μL 30.0μL 22.0μL 35.0μL 20.0μL

47

•Due to low volatility, C6H4Cl2 maintains a relatively stable sediment phase.

•10.0, 15.0, 20.0, 25.0, 30.0 and 35.0 μL C6H4Cl2were investigated while other methods remained the same

•Recoveries were satisfactory when the volume of C6H4Cl2 was higher than 15.0 μL

•On this basis, 15.0 μL C6H4Cl2 was used to study the performance of DLLME

Optimization of DLLME – Extraction Solvent type and volume

48

Optimization of DLLME - Volume of dispersive solvent

• THF volume was changed from 0.5, 1.0, 1.5 to 2.0 mL, sedimented phase was kept constant adjusting C6H4Cl2

• Recovery was satisfactory with the exception of 0.5 mL THF

• 1.0 mL was selected in the experiment due to minimum use of THF

Dispersive solvent volume is important to make extraction solvent form fine droplets, which has direct effect on the extraction efficiencies:

49

Optimization of DLLME - Effect of extraction time

• Investigation of time influence from 1.00 to 60.0 min optimized parameters described

• Extraction time has little influence on the extraction recovery

• Centrifugation procedure could be done immediately after forming milky solution

• Important element of DLLME for rapid determination of pollutants.

Defined as the interval time after injecting the extraction solution (mixture of dispersive solvent and extraction solvent) and before centrifugation:

50

Evaluation of Method performance - Main method parameters

• Yielded good linearity • Correlation coefficient (r2) • The relative standard deviations varied between 4.60% and 6.01% with

the levels of 100gL−1 for TCS and M-TCS, and 50.0gL−1 for TCC (n = 7).

Optimized conditions: Analytes were extracted by mixed solution of 1.00 mL THF and 15.0 μL C6H4Cl2 in a 5.00 mL water solution and then, the solution was centrifuged at 3000 rpm for 5 min, and finally 20.0 μL sedimented phase was dried for the UHPLC analysis.

Analytes with DLLME–UHPLC method were tested using spiked water samples underthe optimum conditions:

51

Evaluation of Method performance - Real water samples analysis

• Filtered through 0.22 µm filter membrane and stored at 4 ◦C

• TCS was detected in domestic water at a concentration of 2.08 μg/L

• No target compounds were found in the other water samples

Real environmental sample determination of river water, irrigating water, reclaimed water and domestic water were tested for the target compounds:

The blank domestic water (A) and domestic water spiked (B) at concentration levels of 2.00 gL−1 for TCS and M-TCS and 1.00 gL−1 for TCC obtained using DLLME combined with UHPLC–TUV

TCS

TCCM-TCS

52

Conclusion• Novel mode DLLME–UHPLC–TUV has been developed for TCS, TCC

and M-TCS• Showed wide linearity, good repeatability and satisfactory

accuracy. • Small sample volume (5.00 μL)• Minimized consumption of toxic organic solvents (15.0 μL)• Sample preparation time (<1 min)• High sensitivity and repeatability• Convenient extraction procedure• DLLME is expected to extend to the pretreatment of other

pharmaceuticals and personal care products in aqueous environment.

53

Comparison of Methods of Triclosan

Method Sample size Time Cost ($) SamplePre-treatment

1st Article

SPE-GC-MS/MS Large 370.2 min High Simple

2nd Article

DLLME-UHPLC-TUV Small 10 min Low Simple

Accuracy Sensitivity LOD Linearity Repeatability Robustness RSD

1st Article

satisfactory High 0.25 ng/L N/A High satisfactory 11.0%

2nd Article

satisfactory satisfactory 134 ng/L 0.0500-100 μg/L

High High 5.43%

54

Dispersive liquid-liquid

Microextraction

•Initial Volume 5.00μL•<1 min Sample prep.•1.00mL organic solvent (THF)•Glass test tube & syringe

Solid-phase Extraction

•Initial Volume 1.00L•6 hr Sample prep.•5.00mL organic solvent (Methanol)•Florisil SPE column

Extraction

55

Gas Chromatography

(GC)

•10.2 min retention time •Injected volume 1.0μL•Mobile Phase Gas (Helium)

Ultra High Performance

Liquid Chromatography

(UHPLC)•5 min total run time•Injection volume 5.0μL•Mobile Phase Liquid (Buffer solution pH 9)

Separation

56

Tunable Ultraviolet (TUV)•Low cost•Tunable frequency

Mass Spectrometry

(MS/MS)•Cost•Lower LOD

Detection

57

Summary

• Both methods demonstrate the ability to effectively detect triclosan

• Both have reasonably low detection limits• Both methods have high reproducibility• Both provided good recoveries and sensitivity

for the analysis of triclosan• Neither method has a extreme time

requirement

58

Critiques

• Both introduced dioxin compounds but never investigated the triclosan degradation intermediates

• Neither papers spoke about acceptable triclosan levels

• The 2nd Article when looking at DLLME they focused more on the Sediment phase volume more than the actual recovery

• The writing style was sub-pare

59

Acknowledgements

• Dr. Soper• Dr. McCarley• Soper Research Group