Investigation of occurrence, elimination and degradation of pharmaceutical and personal care

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Investigation of occurrence, elimination and degradation of Investigation of occurrence, elimination and degradation of

pharmaceutical and personal care products in drinking water pharmaceutical and personal care products in drinking water

using liquid chromatography-tandem mass spectrometry using liquid chromatography-tandem mass spectrometry

Chuan Wang

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INVESTIGATION OF OCCURRENCE, ELIMINATION AND DEGRADATION

OF PHARMACEUTICAL AND PERSONAL CARE PRODUCTS IN DRINKING

WATER USING LIQUID CHROMATOGRAPHY-TANDEM MASS

SPECTROMETRY

by

CHUANWANG

A DISSERTATION

Presented to the Faculty of the Graduate School of the

MISSOURI UNIVERSITY OF SCIENCE AND TECHNOLOGY

In Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

in

CHEMISTRY

2011

Approved by

Dr. Yinfa Ma, Advisor Dr. Craig D. Adams, Co-advisor

Dr. Philip D. Whitefield Dr. Paul Nam

Dr. Jeffrey G. Winiarz

© 2011

Chuan Wang

All Rights Reserved

PUBLICATION DISSERTATION OPTION

This dissertation consists of the following two m1icles that have been published,

or submitted for publication as follows:

Pages 13-43 were published in WATER RESEARCH

Pages 44-66 were submitted for publication in TOXICOLOGICAL AND

ENVIRONMENTAL CHEMISTRY

iii

iv

ABSTRACT

Pharmaceutical and personal care products have benefited humans and animals

around the world. However, their presence in natural and treated water system as

emerging contaminates may have potentially adverse effects on the aquatic environment

and cause development of bacterial resistance. In this research project, a comprehensive

liquid chromatography tandem mass spectrometry method has been developed and

validated for the analysis of sixteen pharmaceutical compounds using solid phase

extraction. Treated and untreated water samples collected across Missouri from water

treatment facilities were analyzed to access the distribution of sixteen pharmaceutical

compounds in both winter and summer seasons. The results of the occunence study

indicated that these pharmaceutical compounds in different types of water resources were

usually below 80 ng/L, except caffeine. It was also found that the treatment processes in

water facilities were effective to remove pharmaceutical compounds in most cases. The

study of pharmaceutical elimination is still crucial in providing information for the

disinfection strategy in water treatment facilities. The follow-up study was performed to

investigate both the treatability and elimination of eight detected pharmaceuticals in the

occurrence study as a function of treatment approach, types of disinfections (free chlorine,

monochloramine, ozone and permanganate ), and treatment conditions (e.g., pH, contact

time, etc). The results indicated that the degradation levels of pharmaceutical compounds

varied significantly in different oxidation processes. Chlorination at 1 mg/L was found to

be highly effective in the elimination of the selected pharmaceuticals. The pH conditions

also played an important role in pharmaceutical removal, and its effect was conditional

based on the oxidation system and pharmaceutical involved.

v

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. Yinfa Ma for being

an outstanding advisor. The constant support and valuable suggestions from him made

this successful. Besides my advisor, I also want to thank my co-advisor Dr. Craig D.

Adams for his insightful cmru11ents, and my other committee members Dr. Philip D.

Whitefield, Dr. Paul Nam, and Dr. Jeffrey G. Winiarz for their time and effort in

reviewing this work.

My sincere thanks also go to Missouri Department ofNatural Resources for

funding the project I was involved in.

I would also like to thank Dr. Honglan Shi for helping me in the experiments. I

thank my fellow lab mates in Missouri University of Science and Technology for their

kind help and assistance.

At last, I am deeply indebted to my family: my parents and my uncle for their

love, encouragement, and support tlll'oughout my life. I am also grateful to all my friends

in both China and US.

vi

TABLE OF CONTENTS

Page

PUBLICATION DISSERTATION OPTION ................................................................... .iii

ABSTRACT ....................................................................................................................... iv

ACKNOWLEDGEMENTS ................................................................................................. v

LIST OF ILLUSTRATIONS .............................................................................................. ix

LIST OF TABLES ............................................................................................................... X

SECTION

1. INTRODUCTION ...................................................................................................... 1

1.1. PHARMACEUTICALS AND PERSONAL CARE PRODUCTS .................... 1

1.2. ANALYTICAL TRENDS .................................................................................. 2

1.3. PHARMACEUTICAL COMPOUND SELECTION ......................................... 3

1.4. ELIMINATION STRATEGIES ......................................................................... 7

REFERENCES .............................................................................................................. 8

PAPER

1. Investigation ofPharmaceutica1s in Missouri Natural and Drinking Water Using High Performance Liquid Chromatography-Tandem Mass Spectrometry ..................... 13

ABSTRACT ................................................................................................................. 13

1. INTRODUCTION .................................................................................................... 14

2. EXPERIMENTAL.. .................................................................................................. 17

2.1. Pharmaceutical standards and reagents ............................................................ 17

2.2. Sample collection and preservation .................................................................. l7

2.3. Solid phase extraction ....................................................................................... 18

vii

2.4. LC-MS/MS analysis .......................................................................................... 19

2.5. Quality control .................................................................................................. 20

3. RESULTS AND DISCUSSION ............................................................................... 20

3 .1. HPLC separation and mass spectrometty detection .......................................... 20

3.2. Method detection limit and possible interference in water sample matrix ....... 21

3.3. Spike recovery .................................................................................................. 24

3.4. Occurrence of pharmaceuticals in Missouri drinking water systems ............... 25

3.5. The removal efficiency by water treatment facilities ....................................... 35

4. CONCLUSIONS ..................................................................................................... .38

ACKNOWLEDGEMENTS .......................................................................................... 39

REFERENCES ............................................................................................................. 40

2. Investigation of Oxidative and PAC Removal of Selected Pharmaceuticals in Various Oxidation Systems by Using Liquid Chromatography-Tandem Mass Spectrometry .................................................................................................................. 44

ABSTRACT .................................................................................................................. 44

l. INTRODUCTION .................................................................................................... 45

2. EXPERIMENTAL ................................................................................................... .47

2.1. Selection ofphannaceutical standards and reagents ........................................ .47

2.2. Oxidant solution preparations .......................................................................... .47

2.3. Analysis ............................................................................................................. 49

2.4. Treatments ......................................................................................................... 50

3. RESULTS AND DISCUSSION ............................................................................... 5!

3.1. Free chlorine oxidation ..................................................................................... 5!

3.2. Permanganate oxidation .................................................................................... 54

viii

3.3. Ozone oxidation ................................................................................................ 56

3 .4. Monochloramine oxidation .............................................................................. 59

3.5. Removal of pharmaceutical compounds with powdered active carbon ........... 59

4. CONCLUSIONS ..................................................................................................... 63

ACKNOWLEDGEMENTS .......................................................................................... 63

REFERENCES ............................................................................................................. 64

APPENDICES

A. ADDITIONAL INFORMATION ABOUT PPCP OCCURRENCE ...................... 67

B. PESTICIDE STUDY ............................................................................................... 78

VITA .................................................................................................................................. 96

ix

LIST OF ILLUSTRATIONS

Page

INTRODUCTION

Figure 1.1 Compound structure of sixteen pharmaceuticals ............................................... 5

PAPER

2. Investigation of Oxidative and PAC removal of Pharmaceuticals in Various Oxidation Systems by Using Liquid Chromatography-Tandem Mass Spectrometry

Figure 1. The oxidation of selected pharmaceutical compounds by free chlorine in

(a) pH 8.6 buffer and (b) pH 6.6 buffer ............................................................. 52

Figure 2. The concentration of detected pharmaceutical compounds in free chlorine

oxidation experiment in pH 8.6 lake water ......................................................... 53

Figure 3. The oxidation of selected pharmaceutical compounds by permanganate in

(a) pH 8.6 buffer and (b) pH 6.6 buffer .............................................................. 55

Figure 4. The oxidation of selected pharmaceutical compounds by permanganate in

pH 8.6 Missouri river water ................................................................................ 56

Figure 5. The oxidation of selected pharmaceutical compounds by ozone in (a) pH 8.6

buffer and (b) pH 6.6 buffer ............................................................................... 58

Figure 6. The oxidation of selected pharmaceutical compounds by monochloramine in

(a) pH 8.6 buffer and (b) pH 6.6 buffer .............................................................. 60

Figure 7. The concentration of detected pharmaceutical compounds in PAC removal

experiment in (a) pH 8.6 buffer and (b) pH 6.6 buffer ....................................... 61

Figure 8. The concentration of detected pharmaceutical compounds in PAC removal

experiment in pH 8.6 Missouri river water matrix ........................................... 62

X

LIST OF TABLES

Page

INTRODUCTION

Table l.l Selected pharmaceutical compounds and their general information .................. .4

PAPER

l. Investigation of Pharmaceuticals in Missouri Natural and Drinking Water Using

High Performance Liquid Chromatography-Tandem Mass Spectrometry

Table l. Sixteen pharmaceutical compounds and their general information .................... 16

Table 2. LC-MS/MS experimental conditions of the sixteen pharmaceutical

compounds ........................................................................................................... 22

Table 3. Method detection limits (MDL), spike recovery and relative standard

deviation (RSD) of studied pharmaceuticals in reagent water (Deionized (DI)) water and tap water matrix (n=9) ............................................ 23

Table 4. Pharmaceutical concentration of real water samples in winter season ................ 29

Table 5. Pharmaceutical concentration of real water samples in summer season ............. 32

Table 6. Seasonal monitoring ofPPCPs from February to June 2009 .............................. 37

1. INTRODUCTION

Pharmaceuticals, hormones, and endocrine disruption compounds are widely used

around world for the benefit to humans and animals. Recently, the detection of

pharmaceuticals and personal care products (PPCPs) in various natural environmental

water sources (often a result of municipal wastewater discharge), as well as treated

drinking water (incomplete removal during water treatment process), has been

increasingly reported. The presence of this group of compounds as emerging

contaminates in natural and drinking water systems has attracted increasing public

concerns about their potential adverse ecological effects on aquatic organism at trace

levels, possible estrogenic or androgenic effects on human health, as well as the

development of bacterial resistance.

1.1. PHARMACEUTICALS AND PERSONAL CARE PRODUCTS

Pharmaceuticals and personal care products (PPCPs) are a group of compounds

consisting of over-the-counter (OTC) drugs, prescription drugs, drugs used exclusively in

hospitals and active drug ingredients used in research area [1]. It is estimated that around

3000 pharmaceutically active compounds are currently used in drug development [2].

PPCPs are generally made for a wide variety of applications to benefit humans

and livestock. Due to their chemical properties, some compounds can be easily broken

down in a human or animal body, or degraded in the environment. However, some

pharmaceuticals are very resistant to disinfection treatment, bacterial or other natural

conditions. Therefore, the pharmaceuticals that are stable in the environment can be

2

eventually transferred to drinking water systems and promote possible adverse effects on

human health [3-5]. Even though the risk that trace amount of pharmaceuticals pose to

humans is still under investigation, some studies found that the potential adverse effect

from pharmaceuticals on the aquatic environment still exists [6-8]. For example,

estrogenic effects including alteration of sex ratio and decreased egg fertilization in fish

are quite possible from endocrine disruption compounds (ECDs) [9, 10].

1.2. ANALYTICAL TRENDS

Research on pharmaceuticals has grown exponentially in the last several years

(review 2010). The detection of pharmaceuticals in environmental samples usually

employs liquid cluomatography-tandem mass spectrometry (LC-MS/MS) [4-5, 11-20] or

Liquid cln·omatography-mass spectrometry (LC-MS) [21-23]. LC-MS is a more popular

technique than GC-MS in analysis of pharmaceuticals because these compounds are

usually highly polar [10]. Yet, efficient derivatization followed by gas chromatography

mass spectrometly (GC-MS) or gas chromatography-tandem mass spectrometry (GC

MS/MS) was also used [2, 9-1 0]. The detection limit of pharmaceutical measurement

usually can fall at nanogram per liter levels in environmental samples after solid phase

extraction (SPE) [10]. Most widely used LC interfaces coupled with mass spectrometry

are electro-spray ionizaiotn (ESI) and atmospheric pressure chemical ionization (APCI)

[2].

The presence of pharmaceuticals in many different types of natural surface water

systems including rivers, lakes, and reservoirs has been increasingly repmied

[3, 7, 24-34]. Due to the incomplete removal of pharmaceutical in drinking water

treatment process, detection of pharmaceuticals in treated drinking water was reported as

well [3, 35]. Currently, pharmaceutical compounds and hormones are listed on the US

environmental protection agency's (US EPA) final contaminant candidate list (CCL)- 3

[10, www.epa.gov/safewater/ccl].

1.3. PHARMACEUTICAL COMPOUND SELECTION

3

In this study, totaling sixteen pharmaceutical compounds were selected for

occurrence study. The preliminary research indicated that some pharmaceutical

compounds including caffeine, ibuprofen, and acetaminophen were detected in Missouri

river water samples. In addition, these compounds were chosen to represent different

groups of pharmaceutical compounds, including analgesics, antibiotics and antimicrobials,

anticonvulsant/antiepileptics, antidiabetics, antihystamines, anti psychotics,

antidepressants, antianxiety drugs, beta-blockers (~-blockers), cytostatics and

antineoplastics, estrogens and hormonal compounds, lipid-regulators, stimulants, and X

ray contrast media. Table I lists general information about these pharmaceutical

compounds in this study. The structures of these pharmaceuticals are shown in Figure I.

In order to make this study more representative, water sampling location were

also carefully chosen. Water samples were taken from a broad range of resources,

including rivers, reservoirs, lakes, unconsolidated wells, and deep wells. The difference

of distribution among these water sources was also compared.

Table 1.1 Selected phannaceutical compmmds and their general information.

C OJ:q>OUDds Formula CAS# Molecular weight Class acetaminophen C 8H 9N02 103-90-2 15 1.2 analgestts

caffeine CsH1oN402 58- 08- 2 194.2 stinm1ant

carbamazepine C1 5H12N20 298-46- 4 236.3 anticonvulsant

clofibric acid C 10H 11Cl03 8 82 - 09- 07 214.7 lipid-regulator

codeine C1sH21N03 76-57- 3 299.4 analgestts

estradiol C 1sH2402 50- 28-2 272.4 hormone

estriol C1sH2403 50-27- 1 288.4 hormone

estrone C1sH2202 53-16-7 270.4 hormone

ethyny1estradiol C2oH2402 57-63- 6 296.4 hormone

ibuprofen C 13H 180 2 15687-27-1 206.3 analgestts

iopromide C1sH24I3N30s 107793-72-6 791.1 X-ray contrast media

lincomycin C 18H 34N 20 6S 154-21-2 406.5 anttbiotics

sulfamethoxa.znle C 10H 11N 30 3S 723 - 46-6 253.3 annbiotics tri;losan C 12H 7Cl30 2 3380- 34-5 289.5 antibiotics

trimethoprim C14H1sN403 738-70-5 290.3 annbiotics tylosin C46H77N017 1401 - 69-0 916.1 antibiotics

~

Lipid-regulator Stimulant

• Clofibric acid • Caffeine

OH ~ o--l-L Cl~ I "'0

0 II CH3

H3CI(c.__c~ ~c Q I ....:CH o:;:..----. _...,c- # N N

I CH3

0

HO HO

estrone estradiol Figme 1.1 CompOlmd stn1ctlues of sixteen phannaceuticals.

X-ray Contrast Media

• lopromide

I 0 OH

Antiepileptic

• Carbamazepine

~C~OH

H,CO~ ~H I 0

OH

.... o()H

~-<~_CH t_____J__;,~

HO~

estriol ethynylestradiol

l.n

0

_,]'(J•' ·<,

i \ - Co, •t :j . ~ o--c~o\ o~ o•.(.. ··o~1 I

~ lu. ~> I o;>., . ....,_ A/·. c~"" oc-, ( 0 ()<

o-, c•,

Tylosin

~N-H::-:.• O OH

HO~;:"' HO--c_)>

o~ /Jo ~-o\_ (Ys,~)V -

H2NN HO$ ~S--

Lincomycin

Cl OH

b o1i ,~

Cl .o Cl

Triclosan

Figme 1.1 (continued).

Sulfamethoxazole

:.:H,

CO:H, ~

~:.:-:-~, I o:r:.,

Trimethoprim

H y J ~

0

Acetaminophen

.0

Ibuprofen

,....Me

Codeine

0'1

7

1.4. ELIMINATION STRATEGIES

It is known that pharmaceutical compounds are introduced to the environment as

a result from incomplete removal in wastewater treatment. Reliable information for

pharmaceutical elimination from source water is crucial to water treatment facilities. Free

chlorine, ozone, and permanganate are very commonly used oxidants in the drinking

water process. Monochloramine is a disinfectant often used in water treatment plants to

prevent the growth of bacterial. Free chlorine and ozone were found to effectively

remove pharmaceutical compounds [36-40]. Hydrogen peroxide was usually combined

with ozone to work as an advanced oxidation method for pharmaceutical decomposition

in some studies [ 41, 42]. Other chemical oxidations (permanganate, and chloramine)

were reported [43, 44], however, not all pharmaceutical compounds were included.

In this study, a LC-MS/MS method was developed and validated in tap and lab

reagent water for analysis of sixteen pharmaceutical compounds in environmental water

samples collected in the state of Missouri. Validation results indicated that this high

throughput approach was accurate. The distribution of pharmaceutical compounds were

obtained and compared in both cold and hot seasons. The results indicated that reliable

information was needed for elimination of these compounds from drinking water.

Treatability and elimination of pharmaceutical compounds that were found in the

occurrence study by oxidation and PAC was then investigated.

8

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6. Snyder, S.A., Keith, T.L., Verbrugge, D.A., Snyder, E.M., Gross, T.S., Kannan, K., Giesy, J.P., 1999. Analytical methods for detection of selected estrogenic compounds in aqueous mixtures. Environ. Sci. Techno!. 33,2814-2820.

7. Jobling, S., Nolan, M., Tyler, C.R., Brighty, G., Sumpter, J.P., 1998. Widespread Sexual Disruption in Wild Fish. Environ. Sci. Techno!. 32, 2498-2506.

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9

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13. Bossi, R., Vejrup, K.V., Mogensen, B.B., Asman, W.A.H., 2002. Analysis of polar pesticides in rainwater in Denmark by liquid chromatography-tandem mass spectrometry. J. Chromatogr., A 957,27-36.

14. Croley, T.R., Hughes, R.J., Koenig, B.G., Metcalfe, C.D., March, R.E., 2000. Mass spectrometry applied to the analysis of estrogens in the environment. Rapid Commun. Mass Spectrom. 14, 1087-1093.

15. Hirsch, R., Ternes, T.A., Haberer, K., Mehlich, A., Ballwanz, F., Kratz, K.-L., 1998. Determination of antibiotics in different water compartments via liquid chromatography-electrospray tandem mass spectrometty. J. Chromatogr., A 815, 213-223.

16. Jeannot, R., Sabik, H., Sauvard, E., Genin, E., 2000. Application of liquid chromatography with mass spectrometry combined with photodiode array detection and tandem mass spectrometry for monitoring pesticides in surface waters. J. Chromatogr., A 879, 51-71.

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19. Ternes, T., Bonerz, M., Schmidt, T., 2001. Determination of neutral pharmaceuticals in wastewater and rivers by liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr., A 938, 175-185.

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10

22. Lindsey, M.E., Meyer, M., Thurman, E.M., 2001. Analysis of trace levels of sulfonamide and tetracycline antimicrobials in groundwater and surface water using solid-phase extraction and liquid chromatography/mass spectrometry. Anal. Chern. 73, 4640-4646.

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33. Snyder, S.A., Kelly, K.L., Grange, A.H., Sovocool, G.W., Snyder, E.M., Giesy, J.P., 2001a. Pharmaceuticals and personal care products in the waters of Lake Mead, Nevada. ACS Symp. Ser. 791, 116-139.

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35. Wang, C., H. Shi, C.D. Adams, S. Gamagedara, I. Stayton, T. Timmons, and Y. Ma. 2011. Investigation of Pharmaceuticals in Missouri Natural and Drinking water Using High Performance Liquid Chromatography-Tandem Mass Spectrometry. Water Res. 45, 1818-1828.

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41. Huber, M.M., S. Canonica, G.-Y. Park, U. von Gunten. 2003. Oxidation of Pharmaceuticals during Ozonation and Advanced Oxidation Processes. Environ. Sci. Techno!. 37, 1016-1024.

42. Zwiener, C., and F.H. Frimmel, 2000. Oxidative treatment of pharmaceuticals in water. Water Res. 34, 1881-1885.

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13

PAPER

1. Investigation of Phannaceuticals in Missouri Natural and Drinking Water Using High Performance Liquid Chromatography-Tandem Mass

Spectrometry

ABSTRACT

A comprehensive method has been developed and validated in two different water

matrices for the analysis of 16 pharmaceutical compounds using solid phase extraction

(SPE) of water samples, followed by liquid chromatography coupled with tandem mass

spectrometry. These 16 compounds include antibiotics, hormones, analgesics, stimulants,

antiepileptics, and X-ray contrast media. Method detection limits (MDLs) that were

determined in both reagent water and municipal tap water ranged from 0.1 to 9.9 ng/L.

Recoveries for most of the compounds were comparable to those obtained using U.S.

EPA methods. Treated and untreated water samples were collected from 3 1 different

water treatment facilities across Missouri, in both winter and summer seasons, and

analyzed to assess the 16 pharmaceutical compounds. The results showed that the highest

pharmaceutical concentrations in untreated water were caffeine, ibuprofen, and

acetaminophen, at concentrations of 224, 77.2, and 70 ng/L, respect ively. Concentrations

of pharmaceuticals were generally higher during the winter months, as compared to those

in the summer due, presumably, to smaller water quantities in the winter, even though

pharmaceutical loadings into the receiving waters were similar for both seasons.

14

Keywords:

Pharmaceuticals, natural and drinking water, LC-MS/MS

1. INTRODUCTION

Many types of pharmaceuticals are used in this country for a wide variety of

applications to benefit humans and animals. Impmtant classes of human pharmaceuticals

include: analgesics, antibiotics and antimicrobials, anticonvulsant/antiepileptics,

anti diabetics, antihystamines, anti psychotics, antidepressants, antianxiety drugs, beta

blockers CP-blockers), cytostatics and antineoplastics, estrogens and hormonal

compounds, lipid-regulators, stimulants, and X-ray contrast media. These compounds

may be excreted unmetabolized or partially metabolized, resulting in their eventual

passage into the environment.

Pharmaceuticals and personal care products (PPCPs) have been detected globally

in many natural water systems including rivers, lakes, and reservoirs (Benotti et al., 2009;

Daughton and Ternes, 1999; Halling-Sorensen et al., 1997; Jobling et al., 1998; Jones et

al., 2001; Kim et al., 2009; Kolpin et al., 2002; Loraine and Pettigrove, 2006; Moldovan,

2006; Nakada et al., 2007; Snyder et al., 2001a; Snyder et al., 2001b; Yu and Chu, 2009).

The trace amounts of pharmaceuticals that have been detected in natural waters have

attracted more public attention and serious concern because of their potentially adverse

effects on the aquatic environment (Jobling et al., 1998; Keith et al., 2000; Snyder et al.,

1999). Moreover, the pharmaceuticals that enter natural waters can ultimately transfer to

our drinking water and promote unknown, but disastrous impacts on human health

(Benotti et al., 2009; Vanderford and Snyder, 2006; Ye et al., 2007). While the risk that

15

low concentrations of pharmaceuticals pose to humans is still not being adequately

investigated (due to their biologically-active nature), it is important to identify the

concentrations of these compounds in natural and treated drinking waters.

Liquid chromatography-mass spectrometry (LC-MS) (Ahrer et al., 2001; FatTe et

al., 2001; Lindsey et al., 2001) and liquid chromatography-tandem mass spectrometry

(LC-MS/MS) (Asperger et al., 2001; Baronti et al., 2000; Bossi et al., 2002; Croley et al.,

2000; Hirsch et al., 1998; Jeannot et al., 2000; Lagana et al., 2000; Sacher et al., 2001;

Ternes et al., 2001; Ternes et al., 1998; Vanderford and Snyder, 2006; Ye et al., 2007) are

popular techniques currently being used in pharmaceutical analyses. Several published

reviews have discussed various methods for analyzing pharmaceutical compounds that

are found in water resources (Lopez de Aida and Barcelo, 2001; Richardson, 2008;

Richardson, 2010; Ternes, 2001). Since most of pharmaceuticals are present in low

concentrations in surface waters, an extraction process (e.g., solid phase extraction (SPE))

is often needed to concentrate target pharmaceutical compounds for analysis.

Comprehensive studies that focus on PPCPs in Missouri's water resources and finished

drinking water have not been conducted and reported.

In this study, a LC-MS/MS method was developed and applied for simultaneous

analysis of 16 pharmaceutical compounds obtained from 31 treated and untreated

drinking water resources in the state of Missouri during both the winter and summer

seasons. General information about the 16 pharmaceutical compounds is listed in Table 1.

Water samples were taken from a broad range of sources, including the Mississippi River,

Missouri River, reservoirs, lakes, unconsolidated wells, and deep wells.

Table 1. Sixteen phannaceutical compounds and their general infonnation.

Compounds Formula CAS# Molecular weight acetaminophen CgH9N02 103-90-2 1512

caffeine CgH10N402 58-08-2 194_2

carbamazepine C1sH12N20 298-46-4 236.3

clo:fibric acid C10HuCl03 882-09-07 214.7

codeine C18H21N03 76-57-3 299.4

estradiol c18H2402 50-28-2 272.4

estriol c18H2403 50-27-1 288.4

estrone C1sH2202 53-16-7 270.4

ethynylestradio 1 C 2oH240 2 57-63-6 296.4

ibuprofen C13H1s02 15687-27-1 206.3

iopromide C18H24l3N 308 107793-72-6 791.1

lincomycin C18H34N206S 154-21-2 406.5

sulfumethoxazo le CIOH11N303S 723-46-6 253.3

triclosan C12H1Cl302 3380-34-5 289.5

trimethoprim C14H18N403 738-70-5 290.3

tylosin C46H77N011 1401-69-0 916. 1

...... 0"1

17

2. EXPERIMENTAL

2.1. Pharmaceutical standards and reagents

All pharmaceutical standards were purchased from Sigma-Aldrich (St. Louis,

MO), except lincomycin (purchased from MP Biomedicals, Aurora, OH)) and iopromide

(purchased from United States Pharmacopeia, Rockville, MD). Labeled pharmaceutical

standards (i.e., 13C3-caffeine, 13C3-Trimethoprim, 13C2-Estrone and 13C3-Ibuprofen) were

purchased from Cambridge Isotope Laboratories (Andover, MA). Oasis HLB extraction

cartridges were obtained from Waters Corp. (Milford, MA), and extraction was

performed using a vacuum manifold (Supelco, C01p., Bellefonte, P A). All solvents

(methanol, acetonitrile, etc.) were LC-MS grade from Fisher Scientific (Fairlawn, NJ,

USA). Formic acid (MS grade) and ammonium acetate (99.99+%) were purchased from

Sigma-Aldrich (St. Louis, MO). Lab reagent water was purified by the Millipore Elix 3

water purification system (Millipore, Bierica, MA).

2.2. Sample collection and preservation

Sample collection, preservation, and storage were accomplished by following the

US EPA Method Guideline (US EPA method 1694, 2007). Sample bottles used to collect

water samples were new 500-mL amber glass bottles with Teflon® liner screw caps.

Prior to use, bottles were pre-cleaned with Mill-Q water, methanol, acetone, and Mill-Q

water, and then baked at 105°C for at least 2 hr. Forty mg of sodium thiosulfate were

added to each bottle to reduce any residual chlorine that had been added as a disinfectant.

To collect treated tap water samples, the water was allowed to flow from the tap without

an aerator for about 5 min, prior to completely filling the sample bottle, with no

18

headspace. To collect raw (untreated) water, a large pre-cleaned wide-mouth bottle or

beaker was used, and then the water was carefully transfened from the original container

to a sample bottle that was completely filled with no headspace remaining. Sodium

thiosulfate should not be flushed out during the collection of either treated or untreated

water. The bottles were then sealed and agitated by hand for about one minute. Two

bottles of untreated source water sample and two bottles of treated water samples were

collected from each water treatment facility. The samples were kept on ice and shipped

overnight to the laboratory for analysis. Upon arrival, the samples were immediately

preserved by adjusting the pH to 5 with sulfuric acid, and then stored at 4°C until

extraction (normally, within 2 days after an·ival).

2.3. Solid phase extraction

Solid phase extraction was performed by following US EPA method 1694 (US

EPA, 2007), with some modification and validation. Water samples were first filtered

using 0.45-!.tm nylon membrane filters (Whatman, England), and then acidified to pH

2.0±0.2 using HCI. One hundred twenty-five mg ofN84EDTA·2H20 were then added for

each 250 mL of water sample. 50 !.!L internal standard mixture (concentration 1 !.lg/mL )

was spiked in the water. Solid phase extraction was conducted using Oasis HLB 6cc (200

mg) cartridges conditioned with 6 ml of methanol, 2 ml of Milli-Q water, 2 ml of pH 2.0

Milli-Q water, and 6 ml of Milli-Q water. Next, 250 ml of each water sample was

extracted, at a flow rate of 1-2 drops per second. After extraction of the sample, each

cartridge was washed with Sml Milli-Q water to remove any EDT A residue. The

cartridges were dried under vacuum for 5 minutes. Analytes were eluted from the

19

cartridge by using 5ml of methanol and then 3 ml of an acetone-methanol (1: 1) mixture

were placed into a clean test tube. The combined eluent was evaporated to 100 J.lL by

using a Turbovap LV evaporator at 50±5°C. Nine hundred J.lL of 20% acetonitrile in MQ

water with 0.1% formic acid was then added to each sample tube and the contents were

vo1tex mixed. Finally, the samples were transfe1Ted to 2-mL amber glass sample vials,

and stored in a refrigerator until LC/MS/MS analysis.

2.4. LC-MS/MS analysis

A 4000Q TRAP mass spectrometer (AB SCIEX, Concord, ON, CA), equipped

with an Agilent 1100 series HPLC system (Agilent Technologies, Inc.,Santa Clara, CA,

USA), was used for all analyses. A sample volume of 10 J.ll was injected. The HPLC

column was a Supelco C-18 column (150x2.1 mm, 5-J.lm pmticle size, Sigma-Aldrich, St.

Louis, MO). Different mobile phase additives were used for positive electrospray

ionization (ESI+) and negative electrospray ionization (ESI-) mode analyses. The mobile

phase for ESI+ compounds consisted of 0.1% formic acid (v/v) in water (A) and 0.1%

formic acid (v/v) in acetonitrile (B). Elution flow rate was 0.25 mL!min. The mobile

phase for ESI- mode compounds consisted of 5 mM of ammonium acetate in water (C)

and 100% acetonitrile (D). Elution flow rate was 0.25 mL!min. For both ESI+ and EST

mode compounds, the gradient began with 5% solvent B or D for 1.5 min, ramping to

75% B or D at 2.5 min, 15% B or D at 13.5 min, and 100% B or D at 15 min, where it

was held for 1.5 min. Mass spectrometry utilized both ESI+ and ESI- modes in multiple

reaction monitoring (MRM). Mass spectrometers, including mass calibration, polarity of

each compound, compound-dependent parameters, and source-dependent parameters

20

were all optimized for each compound. After optimization, the ion transition with the

most intense signal was selected as the quantification ion pair of the conesponding

compound, while the ion transition with the second highest signal was selected as the

confirmation ion pair of the corresponding compound. In positive mode, the ion source

temperature was set at 650°C with an ion spray voltage of 5000 V. The nebulizer gas, auxiliary

gas, and cmtain gas were 50, 65, and 25 psi, respectively. In negative mode, the ion source

temperature was set at 550°C with an ion spray voltage of -4500 V. The nebulizer gas, auxiliaty

gas, and cmtain gas were 50, 60, and 30 psi, respectively.

2.5. Quality control

During the method development, the liner range of calibration for each compound,

method detection limit, reagent blank, reproducibility, spike recoveries of each

compound in reagent water and in drinking water matrices were all performed. During

the analysis of water samples, at least one blank, one duplicate, and one spike were

proceeded with sample preparation and LC-MS detection for each batch (there were

generally 8 water treatment facilities in each batch). The choices of these sample matrices

represented river water, lake water, well water, and reservoir water.

3. RESULTS AND DISCUSSION

3.1. HPLC separation and mass spectrometry detection

Each sample was injected for both ESI+ and ESI- analyses. Ten of the 16

compounds were detected with ESI+ and six of the compounds were detected with the

ESI- mode. The retention times and compound dependent mass spectrometry parameters

21

of the target pharmaceuticals are listed in Table 2. Most compounds were well separated

chromatographically. Several analytes, that were not well separated chromatographically,

were monitored at different MRM ion pairs. All pharmaceuticals were optimized

individually for maximum sensitivity. The dwell time for each compound was also

optimized, depending on their peak width thereby allowing the peaks to be integrated

accurately and with high sensitivity.

3.2. Method detection limit and possible interference in water sample matrix

The method detection limit (MDL) for each compound was determined by

following U.S. EPA method (EPA-821-R-03-005), spiking 9 replicates of 2-5 times

concentration of the estimated instrumental detection limits (IDL) (before 250x

concentrated by SPE) (The IDL of each PPCP compound was determined based on the

PPCP concentration at signal-to-noise ratio of 3-5). The spiked PPCP standards were

proceeded by SPE and then analyzed by LC-MS/MS. The MDL of each compound was

calculated by multiplying the standard deviation(s) of the replicate analyses by the

Student's T value for eight degrees of freedom at the 99 percent confident level. The

MDL in both reagent water and tap drinking water were determined separately. A sample

matrix was analyzed first to assess the concentrations of the target pharmaceutical

compounds. For the tap water matrix, since some target analytes were found to be

present in the water, each pharmaceutical standard was spiked individually to make the

concentrations of pharmaceuticals fall to 2-5 times the estimated IDL and proceeded to

determine MDL. The method detection limits of studied pharmaceuticals are listed in

Table 3.

Table 2. LC-MS/MS e::q>erimental conditions of the sixteenphannaceutical compotmds.

Compounds Retention ESI

mode Precursor Product Dedustering Collision

time (min) mode ion ion potential (V) energy

~ acetaminophen 5.7 positive 151.8 110 66 25

caffeine 8.2 positive 194.9 138 51 31

carbamazepine 11.7 positive 236.9 194 71 31

clofibric acid 8.1 negative 212.9 126.8 -40 -24

codeine 7.9 positive 300 215 81 37

estradiol 13.1 positive 255 159 76 29

estriol 8.8 negative 286.7 171.1 - 100 -50

estrone 12 negative 268.9 144.8 - 110 -54

ethynylestradiol 13.8 positive 279 133.2 61 27

ibuprofen 9.5 negative 204 .9 159 -55 -10

iopromide 3.5 negative 789.9 126.7 -85 -48

lincomycin 8 positive 407.1 126.2 76 39

sulfumet:OOxazole 10.2 positive 253.9 156 61 25

trick> san 15.8 negative 286.7 35 -50 -110

trimet:OOprim 8.3 positive 291 230.2 81 35

tylosin 10.6 positive 916.5 174.1 136 55

CoUision cell

exit potential (V)

6

8

12

-7

14

10

-9

-7

8

-10

-5

6

10

-3

14

10

Internal standard

--1-3 c affi . 3-c eme

13C3-ca1feine

13c3-~t:OOprim 13C3-ibuprofen

13c . t:OO . 3-trnre pnm

13C 3-caffeine 13C6-estrone 13C6-estrone t3c affe . 3-c me

13 C3-ibuprofen 13C3-ibuprofen

t3cr~t:OOprim t3c . t:OO .

3-trnre pnm 13 C6-estrone

13c3-~t:OOprim 13Crca1feine

N N

Table 3. Method detection limits (MDL), spike recovery and relative standard deviation (RSD) of studied phannaceuticals in reagent water (Deionized (DI)) water and tap water mat:ri~ (n=9).

:\IDL (ng/L) Higb-le,·el spike Low-Je,·el spike

DI water Tap Water Dl \\'liter I~ Water Componnd

DI Tap Spike Recore~· RSD Spike Recorery· RSD Spike Recorery RSD Spike Recovel)· RSD

Water Water (ng/L) (%) (%) (ng/L) (%) (%) (ngiL) (%) (%) (ng/L) (%) (%)

acetamincphen 2.7 1.4 200 95.8 10.4 200 IOO 6.9 IO 106 8.8 10 73.9 6.4 caffeine 0.8 l.l 400 109 9.1 400 ll5 8.5 2 46.4 29 2 50.7 37.4

carOOma.zepae 0.5 01 200 104 4.9 200 I2I 'i ~ .. ) I I25 13 I 125 5.9 clofibric acid 1.3 0.6 400 122 12.9 400 157 7.4 I 72.6 28.1 I 56.7 37.5

codeine 1.0 1.5 400 104 2.6 400 122 4.5 5 I45 4.7 5 I 50 6.7 estradiol 0.8 I1 400 95.6 5 400 106 4.4 5 I02 5.3 5 97.9 8.7 estriol 4.3 5.2 200 I28 4.9 200 115 2.4 20 I20 6.2 20 121 7.4 estrooe 1.4 1.0 200 116 2.6 200 109 1.9 2 891 26 2 123 13.9

ethynylestradid 0.1 0.5 200 92.3 6.2 200 102 2.8 1 108 4.4 1 75.8 22 ibuprofen 1.0 1.6 400 119 1.9 400 110 2.2 20 106 3.5 20 85.9 ' ' :u

ioprooride 3.5 9.9 200 98.1 8.8 200 90.6 7.8 20 136 4.4 20 153 111 lincomycin 0.1 0.1 200 103 9.9 200 97.5 5.6 1 12.6 32.8 1 7.1 55.2

sulfamethoxazole 0.4 0.3 400 90 11.5 400 102 4.7 1 90.5 14.8 1 92.7 9.3 tricbsan 1.0 1.2 400 32 14.1 400 33.6 15.7 10 39.3 8.7 10 29.8 131

trimethoprim 0.3 0.4 400 123 11.3 400 125 4.9 1 91.2 121 1 91.2 14 tylosin 0.3 0.2 200 44.6 14.8 200 48.6 30.8 I 181 511 I 15.1 37.8

N w

24

3.3. Spike recovery

Spike recoveries in both reagent water and tap water were determined separately.

For each type of water, two concentrations (low and high) and nine replicates of each

concentration were performed discretely. High and low concentrations were selected to

cover the most likely range of pharmaceutical concentrations that would be expected to

be observed in actual treated and untreated water samples. Recoveries of all compounds

were determined by adding an appropriate amount of standard solutions of the target

pharmaceutical compounds to 250 ml of reagent and real water samples. The recovery

study consisted of four experiments. For each experiment involved, known

concentrations of target pharmaceutical compounds were prepared in nine replicate water

samples (250 ml water each). The samples were proceeded by SPE and then analyzed by

LC-MS/MS. The relative percent recoveries and relative standard deviations were

calculated, and are reported in Table 3.

Recoveries of most of the high concentration pharmaceutical compounds (200-

400 ng/L) in reagent and tap water ranged between 90-130%. The recoveries of tylosin

and triclosan, however, were relatively low in both water matrices, while the recovery of

clofibric acid was high in the tap water matrix. The results of recovery studies, in most

cases, showed excellent reproducibility with the percent relative standard deviation being

less than 10%. Most of the compounds at the spiked concentrations behaved similarly

between the two different water matrices.

Recoveries of the pharmaceutical compounds at low concentration spikes

(ranging 1.0 -20 ng/L, which was near the MDL for most of the compounds) ranged from

50-150% for most of the compounds. Majority of the compounds also showed good

25

reproducibility, with the percent relative standard deviation being less than 15%. For

most of the studied compounds, the low concentration recoveries did not show significant

differences between the two water matrices. However, some compounds, such as

Lincomycin, triclosan, and tylosin, had low recovery in both reagent water and tap water

matrices. Acetaminophen and ethynylestradiol had relatively lower recoveries in tap

water. Lincomycin also had a lower recovery and much higher percent relative standard

deviation in the tap water matrix than reagent water matrix. This phenomenon was also

reported in US EPA method 1694. Ideally, an isotope-labeled internal standard should be

used for each of the studied compounds. However, only four labeled internal standards

were used in this study due to the limited budget. Different isotope-labeled internal

standards used in this study may also have contributed to the poor recoveries of

pharmaceuticals.

During pharmaceutical occurrence study in Missouri water, spike recoveries were

also tested with each batch of samples and each major water type (river, lake, and well).

The recoveries were similar with or better than the initial recovery studies as shown

above.

3.4. Occurrence of pharmaceuticals in Missoul"i drinking water systems

Treated and untreated waters were sampled from 31 different water treatment

facilities across Missouri in both the cold (winter) and warm (summer) seasons. These

water treatment facilities used varied source waters, including rivers, reservoirs, lakes,

unconsolidated wells, groundwater, and deep wells. The water samples from these water

treatment plants represent the most common tap drinking water in Missouri. The water

26

treatment plants on the Missouri and Mississippi Rivers utilize conventional treatment

(i.e., pre-sedimentation, rapid mix, flocculation, sedimentation, filtration, and disinfection)

including two-stage lime softening. Specifically, the treatment plants utilize ferric

chloride or sulfate coagulants, chlorine as the primary disinfection, and chloramine as the

residual disinfectant. Periodic powdered activated carbon addition is also used. All of the

water facilities that participated in this study used chlorine and chloramines as water

disinfectants. Both untreated and treated water samples were collected from each water

treatment facility at same time to evaluate the effects of the water treatment process on

the studied pharmaceuticals.

In the winter season, 11 pharmaceutical compounds (Table 4) were detected in at

least one of the untreated source waters: Caffeine was found in all of the untreated and

treated water samples, with concentration ranges of 2.5-225 ng/L, and were present at

higher levels than most of the other pharmaceuticals monitored in this study. Caffeine

concentrations were higher in river water than in most of the other types of water. In most

cases, lower concentrations were detected in treated waters (free chlorine or chloramines)

than in untreated waters, indicating some removal of this compound by the water

treatment process. Carbamazepine was found in 11 of the 31 water treatment facilities

studied, in both treated and untreated waters at concentrations up to 8.7 ng/L. The

concentrations were also lower, however, in treated water than in untreated water. This

compound was found in all river water samples. Sulfamethoxazole was present at a

detectable level in 11 out of the 31 water treatment facilities, primarily in untreated river

waters, with a maximum concentration of 38.1 ng/L. This indicated that the water

treatment effectively removed most of this compound. Ibuprofen was also found in both

27

untreated and treated waters in 7 of the 31 water treatment facilities; 6 sources were from

rivers with a highest concentration of 77.2 ng/L. The concentrations of this compound

were found to be about the same before and after water treatment, indicating that the

water treatment procedure used was not effective in removing this pharmaceutical. The

concentrations of the rest of the target compounds were all below method detection limits.

These results indicate that the concentrations of detected pharmaceutical compounds

were water source dependent and were usually higher in river water and other types of

surface water samples than concentrations found in the underground water samples. A

probable reason could be that the rivers receive all of the effluent, and other types of

surface water (such as lakes and reservoirs) receive water flows from surrounding areas.

In the summer season, fewer compounds were detected in water facilities (Table 5).

These compounds were tylosin, lincomycin, thrimethoprim, sulfamethoxazole,

acetaminophen, caffeine, carbamazepine, and triclosan. The relative concentrations of

the detected compounds in different types of water sources in water samples during the

summer shared a trend similar to that of water samples collected during the winter season.

The difference between the two seasonal water samples was the level of concentration of

the detected compounds. Concentrations of pharmaceutical compounds were lower in the

summer than in the winter. One of the possible reasons for this difference might be due to

more degradation of these pharmaceuticals in a warmer environment. The other reason

may be attributed to the normally heavier rainfall during the summer season in Missouri

vs. that in the winter season, since increased rain reduces the concentrations of the target

compounds. In addition, variation of the concentration over time might have some

contributions because grab-sampling method was used. Caffeine is a commonly used

28

tracer pharmaceutical due to its relatively higher concentrations, and due to its relatively

constant daily use. Based on stream flow and concentration data, the caffeine mass flow

rate in the Missouri River was 88.8, 141.6, 97.6, and 131.1 mg/s, respectively, for

January, April, May, and June, 2009. The mean and relative standard deviation were 115

mg/s and 22%. Similarly, the caffeine mass flow rate in the Mississippi River was 281,

238, 284, and 183 mg/s, respectively, for January, April, May and June, 2009. The mean

and relative standard deviation were 247 mg/s and 19%. These relatively small relative

standard deviations in mass flow rate for caffeine are an indication of the great

impot1ance of dilution effects. Similarly, the effect of dilution by increased flow in the

summer vs that in the winter, creates a relatively constant input of a chemical, as seen by

the relatively strong inverse correlations between river flow rates and concentrations. For

example, for sulfamethoxazole and caffeine in the Missouri River, the inverse conelation

coefficients were -0.89 and -0.98. For sulfamethoxazole and caffeine in the Mississippi

River, the inverse correlation coefficients were -0.77 and -0.89. Thus, the effects of

seasonal dilution are clearly, at least pattially, responsible for the trends observed.

By following guidance from Missouri Department of Natural Resources, three

types of water matrixes (Mississippi River water, reservoir water and Missouri River

water were sampled monthly from winter to summer for more detailed assessments of

changes in the trend of applicable pharmaceutical compounds. Assessment results

showed that eight pharmaceutical compounds were detected in Mississippi River and

Missouri River water samples (lincomycin, trimethoprim, sulfamethoxazole,

acetaminophen, caffeine, carbamazepine, triclosan, and ibuprofen), with the caffeine

level being the highest. Pharmaceutical compound levels found in both Mississippi River

Table 4. Phannaceutical concentration of real water samples in winter season.

ID # Water source

MSRiver

2 MSRiver

3 MORivcr

4 ADuvialGW

5 ADuvialGW

Treatment Sample

type

T~-~~

sin

Line~

m~·cin

Free chlorine Untreated <MDL 4.2

Treated <MDL <MDL

Cbloramines Untreated <MDL <MDL

Treated <MDL <MDL

Cbloramines Untreated <MDL 1.8

Treated <MDL <MDL

Cbloramines Untreated <MDL 1.1

Treated <MDL <MDL

Free chlorine Untreated <MDL <MDL

Treated <MDL <MDL

6 Deep rock wells Cbloramines Untreated <MDL <MDL

Treated <MDL <MDL

7 Deeprockwells Freechlorine Untreated <MDL <MDL

Treated <MDL <MDL

8 Reservoirs Freecblorine Untreated <M DL <MDL

Treated <MDL <MDL

9 Reservoir Cbloramines Untreated <MDL 2.5

Treated <MDL <MDL

10 MO River Cbloramines Untreated <MDL 2.9

Treated <MDL <MDL

Concentration (n:JL)

Trimeth~ Sul!.ameth- Acrtamin- Cal-prim oxazole opben feine

4.6

<MDL

7.7

<MDL

2.8

1.7

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

9.1

<MDL

13.7

<MDL

28.8

1.5

18.8

8.2

5.5

<MDL

<MDL

<MDL

2.8

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

38.1

4.8

5.3

<MDL

21.8

<MDL

16.0

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

14.2

<MDL

56.0

<MDL

47.2

13.0

106.0

54.4

49.6

36.0

14.8

2.5

50.0

43.6

11.4

3.4

18.2

4.5

10.3

8.8

157.2

18.4

224.8

180.8

ND =No Data MDL= Mc:t1toclDctcction Limit

Carbama- Code- Trid~ Ibup

rofen Iopr

omide zepine

8.4

21

8.7

39

5.5

4.7

31

ine san

<MDL <MDL 16.6 <MDL

<MDL <MDL 8.8

<MDL <~DL 37.5

<MDL

22.4

<MDL <~DL 26.6 <MDL

<MDL 2.1 27.1 <MDL

<MDL <~DL 23.4 <MDL

<MDL <MDL <MDL <MDL

<MDL <MDL <MDL <MDL <MDL

<MDL <MDL <~DL <MDL <MDL


1.9 <MDL 9.8

<MDL <MDL 2.8

<MDL <MDL

<MDL <MDL



<MDL <MDL 3.4 <MDL <MDL


1.0 <MDL <MDL 2.4 <MDL

<MDL <MDL <~DL 2.0 <MDL

8.1 3.7 <MDL 772 <MDL

6.8 <MDL <MDL 7.2.8 <MDL

N 1.0

Table 4. (continued)

ID # Water source

11 MS River

12 Lake

13 Lake

14 Lake

IS Deep Well

16 Deep Well

17 Deep Well

18 Deep Well

19 Lake

20 Unconsolidated

Wells

Coucentratiou (n:JL)

Sample T~·lo- linc:o- Trimetho- Sulfameth- Acetamin- Caf- Carbama- Code- Triclo- lbup- Iopr-

Treatment type sin m~·c:in prim oxazole ophen feine zepine ine san rofen omide

Freec:hlorine Untreated <MDL 2.8

Treated <MDL <MDL


Chloramines

Chloramines

Treated <MDL <MDL

Untreated <MDL <MDL

Treated <MDL <MDL

Untreated <MDL <MDL

Treated <MDL <MDL


Treated <MDL <MDL

Freechlorine Untreated <MDL ~\l:DL

Treated <MDL <MDL


Treated <MDL <.t\fDL

Free chlorine Untreated <MDL ~\fDL

Treated <MDL <MDL


Treated ND N"D

Free chlorine Untreated ND KD

Treated <MDL ~\fDL

4.0

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

}\1)

ND

<MDL

l\Tl) = Ko Data MDL= MctllodDctcction Limit

18.2

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

1.7

ND

ND

<MDL

11.6

28.0

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

ND

ND

<MDL

39.0

32.9

20.1

8.9

21.3

4.0

29.4

43

61

6.5

3.0

4.4

2.9

2.6

2.7

3.0

11.1

ND

ND

2.8

8.1 3.0 <MDL 13.6 <MDL

3.2 <MDL <MDL 10.4 <MDL








<MDL <~DL <MDL <MDL <MDL

<~DL <MDL <MDL <MDL <~DL






<MDL

~Tl)

:-ill

<MDL <MDL <MDL <MDL

}\1)

ND

}\1)

KD

ND

ND

N"D

}\1)

<MDL <~DL <MDL <MDL <MDL

w 0


ID # Water source

21 Unconsolidated

22

23

24

25

26

27

28

29

30

31

WeDs

Unconsolidated

WeDs

Unconsolidated

WeDs

Lake

Lake

Lake

Reservoir

Lake

River

Lake

Lake

Sample T~·lo- linco-Treatment (Hit sin mycin


Treated <MDL <MDL


Treated <MDL <MDL


Treated <MDL <MDL

Free chlorine Untreated 4.3 <MDL

Treated <MDL <MDL

Chloramines Untreated <MDL 7.7

Treated <MDL <MDL

Freechlorine Untreated <MDL <MDL

Treated <MDL <MDL


Treated <MDL <MDL


Treated <MDL <MDL

Chloramines Untreated <MDL <MDL

Treated <MDL <MDL


Treated <MDL <MDL

Chloramines Untreated <MDL <MDL

Treated <MDL <MDL

Coacentratioo (n:IL)

Trimetho- Sulfameth- Acetamin- Car-prim onzole opbeo feioe

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

2.1

<MDL

<MDL

<MDL

<MDL

<MDL

7.2

<MDL

<MDL

<MDL

<M DL

<MDL

<MDL

<MDL

3.1

<MDL

<M DL

<MDL

6.1

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

70.0

<MDL

<MDL

<MDL

19.4

<MDL

<MDL

<MDL

<MDL

<MDL

5.6

72

34.4

93

39.1

32

6.7

4.8

27.7

3.7

79

7.4

10.0

6.0

11.3

6.5

31.5

20.4

16.9

3.8

10.8

5.2

·No =No Data MDL =Method Detection Limit

Carbama- Code- Triclo- lbup- lopr-zepine ine san rofen omide



<MDL

<MDL

33

<MDL <MDL <MDL <MDL

<MDL <MDL <MDL <MDL

<MDL <MDL <MDL <MDL








4.S <MDL <MDL <MDL <MDL

3.5 <MDL <MDL <MDL <MDL


<MDL <MDL <MDL <M DL <MDL

' ., ~-

1.4

<MDL <MDL 4.4

<MDL <MDL 3.2

<MDL

<MDL





w 1-"

Table 5. Phannaceutical concentJ:ation of real water samples in swmner season.

C.cmtatila(~

Slllllflle TJI-- lac. ~~ SlllfultA- .la:4wrio- c.J.. ea...-1)1 W:der:S~~~Ut:e T~t .. sia .,.... ..- -· .til ra- zepiae

1 MSRm Fm:dabDc: UDiralal -<MDL 2.6 <MDL (.2 -<MDL 15.6 5..7

Tmtal -<MDL <MDL <MDL <MDL <MDL 9.5 LD

2 MSRm UDiralal <MDL 4.6 <MDL lLO -<MDL 134..8 8.6

Tmtal <MDL 2.0 <MllL <MDL -<MDL 22..8 s..o 3 MORivcr Chb•lil•s UDiralal -<MDL 1.9 <MDL 4.6 -<MDL 1:1.6 u

Tmtal -<MDL <MDL <MllL 4J) <MDL 124 1.5

4 AhiiiGW ~ I UDiralal -<MDL 13 <MDL 4.8 <MDL 324 83

Tn:lltal -<MDL <MDL <MDL 13 <MDL &.8 1.4

s Ahiii.GW Fm:d!bime UDiralal -<MDL <MDL <MllL 3.9 <MDL 72 -<MDL

Tmtal -<MDL <MDL <MDL <MDL <MDL 72 <MDL

6 Dap lOCi. wells CJ:obwWn UDiralal -<MDL <MDL <MDL <MDL <MDL 18.4 1.3

T1CIIIal <MDL <MDL <MDL <MDL <MDL 20.9 <MDL

7 Dap lOCi. well Fa: d!biDe UDiralal <MDL <MDL <MDL <MDL <MDL 14.1 <MDL

T1CIIIal -<MDL <MDL <MDL <MDL <MDL 6.1 <MDL

8 Rcavoim F111:dlbiac UDiralal <MDL 3.D <MDL <MDL <MDL S6.0 -<MDL

T1CIIIal <MDL <MDL <MllL <MDL <MDL 17.8 <MDL

9 ~-- UDiralal -<MDL 13 <MDL <MDL <MDL 6.6 <MDL

T1CIIIal -<MDL <MDL <MDL <MDL <MDL 1.2 -<MDL

10 MORivcr UDiralal -<MDL <MDL <MDL au 9.4 46.0 7.3

T1CIIIal <MDL <MDL <MllL 1.6 6.2 35..6 s..o ND=Nolhb. MDL= MdhodDclccti:al.ial

c.4. Tlic:» eiae -<MDL 3_1

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <MllL

<MDL <MDL

<MDL <MllL

<MDL <MllL

<MDL <MDL

<MDL <MDL

<MDL <MllL

<MDL <MllL

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL <XDL

.... faa

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

...._ -<MDL

<MllL

<MllL

<MDL

<MDL

<MDL

<MDL

<MllL

<MDL

<MDL

<VllL

<MDL

<MDL

<MDL

<VllL

<VllL

<MDL

<MDL

<MDL

<MDL

w N


s-.~e T,S.. lac. r.-...s~ 1)1 W.krs.mE Tn:at.mt ~ .. .,a. ..- -· 11 MS:Rift:t FtllC dlbimc l.Tutn::llal 4tmL 3.9 ~ 4.0

Ttatal 4tmL <MDL ~ <MDL

u :r..Kt: FtllCdllarimc l.Tutn::llal 4tmL -<MDL <MDL <MDL

TJatal -MDL -<MDL <MDL <MDL

13 :r..Kt: U!m:llal -MDL <MDL <MDL 1.9

TJatal -MDL <MDL <MDL <MDL

14 :r..Kt: l.Tutn::llal -MDL <MDL <MDL <MDL

TJatal -MDL -<MDL ~ <MDL

IS DapWc:B Fm:dllarimc l.Tutn::llal -MDL -<MDL ~ <MDL

TJatal -MDL <MDL ~ <MDL

16 DapWcD Fm:dllarimc 'Untratal <MDL <MDL -<MDL <MDL


17 DapWcl Fm:dlbimc 'Untratal -MDL <MDL <MDL <MDL

TJatal <MDL <MDL <MDL <MDL

18 Dap'Wdl Fm: dlbimc 'Untratal <MDL <MDL -<MDL <MDL


19 :r..Kt: FKICdllarimc U!m:llal -MDL <MDL <MDL L1

TJatal <MDL <MDL <MDL <MDL

7D lJuc:uauiddal F~~r:dllarimc U!m:llal 3.9 7.0 5.1 3.2

Wdls TJatal <MDL u (.7 LS

ND = Noi>G MDL= Mcthodl>dcdDlliDt

O.CI!IIb-(~ .a. .. _.__ ~ c...-... m Ia- xqja.e

46..0 25.3 u 9.5 11..6 u

328 S9.6 <MDL

9.4 16.4 4tO)L

-MDL 6.D <MDL

-MDL 33 -MDL

-MDL 75.6 -MDL

-MDL 14.6 -MDL

4tmL L7 <MDL

-MDL u -MDL

4tmL 1.6 <MDL

-MDL u <MDL

-MDL 14.9 4tO)L

4tmL 6.S 4tO)L

-MDL 14.5 -<MDL

4tmL 6.3 <MDL

4tmL 16.4 1..6

4tmL 14.9 <MDL

4tmL lll 9.6

4tmL 8..1 6.9

c. Tlid. eiae s-.

-<MDL 7.0

-<MDL <MDL

<MDL 2.9

<MDL 22

<MDL .u -<MDL 2.6

-<MDL -<MDL

-<MDL <MDL

-<MDL <MDL

<MDL <MDL

-<MDL <MDL

<MDL <MDL

<MDL <MDL

<MDL -<MDL

-<MDL <MDL

<MDL -<MDL

<MDL 3..8

<MDL 3.5

<MDL 3.1

<MDL 2.6

..... lal

4tO)L

4tO)L

4tO)L

4tO)L

<MDL

<MDL

-MDL

-MDL

-MDL

-MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

-<MDL

<MDL

<MDL

-<MDL

.._._ ide

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

-<MDL

-<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

-<MDL

w w


ID I Watn"s-ftle

21 lJnoonaoichlal

W4s

22 lJDcoiiMiidllal

WeBs

D lJnoonaoichlal

W&

24 :take

2S L*

16 I.lllke

IT Rmelvoir

28 L*

19 River

30 L*

31 L*

Tftabaalt

F~ee dllotiDe

F~ee dllotiDe

F~ee dllotiDe

Flee dllorme

Ch6••••a

FJeedllotiDe

Flee dllotiDe

Flee c:bbiDe

Flee dlbDe

ND=Nol>aa

S-,le Ty»- lae»-type sill ..,..

UD~ratal <;MDL <MDL

T!Sal <;MDL <MDL

UDirellal <;MDL <MDL

Tmted <;MDL <MDL

UD~ratal <;MDL <MDL

Tmtal <MDL <MDL

UD~rate:l <MDL <MDL

Tmtal <MDL <MDL

UDirelle:l <MDL <MDL

Tmted

UD~rated

<;MDL <MDL

ND ND

Tmted <MDL <MDL

UDirelled <MDL <MDL

T!Sal <MDL <MDL


Tmted <MDL <MDL


Tmted <MDL <MDL


Tmted <MDL <MDL


C..cab-.<-oQ Tm.ea.- Slllflaea.. ~ Cal- C.....

pm -• l(llla ra-e •q*e

<MDL <MDL <MDL S.Z <;MDL

<MDL <MDL <MDL LS <;MDL

<MDL <MDL ~L 152..0 40>L

<MDL <MDL ~L 1..2 <;MDL

<MDL <MDL ~L llU 40>L

<MDL <MDL <MDL 9..6 13

<MDL <MDL <;MDL 8.9 40>L

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<;MDL

~L

ND

<MDL

<MDL

40>L

<MDL

<;MDL

40>L

<MDL

40>L

ND

<MDL

1..6

4.7

<MDL

<;MDL

17.9

3..6

<MDL

40>L

<MDL

Cell.

da.e

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

ND

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

Tddo-<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

S.O

4.9

4.4

4..2

ND

1.3

s.z 3.4

9.1

5.4

<MDL

<MDL

<MDL

<MDL

<MDL

...... fal

~L

~L

~L

~L

<MDL

<;MDL

<MDL

<MDL

<;MDL

<;MDL

ND

~L

<MDL

<MDL

<MDL

<;MDL

<;MDL

<;MDL

<MDL

<MDL

<MDL

Tmted 40>L <MDL

ND

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

ND

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

4..6

<MDL

<;MDL

~L

<;MDL

40>L

2.7

24..0

4.4

ND

S.B

12.4

lLl

4U

6.8

13.1

1Q.6

8.9

2.S

6.4

3.6 <MDL <MDL <MDL <MDL

MDL= MdhodDdecti!D Lilllil

...... illt

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

ND

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

w ~

35

water and Missouri River water were similar. In the reservoir water samples, however,

only four compounds were detected (lincomycin, acetaminophen, caffeine, and triclosan)

and their concentrations were much lower than those detected in Mississippi River and

Missouri River water samples. The monthly monitoring also showed a decrease in the

concentrations of detectable pharmaceutical compounds from February to June. Specific

concentrations of all target pharmaceutical compounds in treated and untreated waters

samples in this seasonal study are reported in Table 6.

One of the largest studies of pharmaceutical occurrence in untreated drinking

water sources from the U.S. Geological Survey (Focazio et. a!., 2008) concluded that the

concentrations of most detected compounds were typically in the sub-iJg/L range. This

matched our results. Our study also indicated that the highest concentrations of the

detected pharmaceuticals in Missouri were lower than the maximum concentrations in

Focazio's repm1. In our study, caffeine was much more frequently detected than in their

national survey. However, the MDL in national survey (14 ng/L) was several times

higher than the MDL in this study (1.1 ng/L). The maximum concentrations of caffeine in

our study were 0.22 11g/L whereas the national survey reported 0.27 iJg/L.

3.5. The removal efficiency by water treatment facilities

Since untreated and treated water samples were collected at the same time from

each water treatment facility, the pharmaceutical concentration difference should

represent the removal efficiency of water treatment. Free chlorine and chloramines were

used as disinfectants in these selected water treatment p !ants. A general decrease in

concentrations of target pharmaceuticals in finished drinking water was observed in

36

occurrence study. Both treatments showed comparable removal efficiencies of

acetaminophen. The main site of transformation of acetamination during chlorine

oxidation was reported as the phenol group (Pinkston and Sedlak, 2004). On the other

hand, results indicated that the removal of carbamazepine was not effective by

chlorination or chloramination treatment. Most water treatment plants failed to remove

this compound to below detection limit. This result matched previous studies by Benotti

et al and Stackelberg et al.. (Benotti et al., 2009; Stackelberg et al. 2007). However, it has

reported that carbamazepine can be efficiently removed by chlorination at low pH

(Westerhoff et al. 2005). The removal efficiency of Caffeine was also found low by

chlorination or chloramination treatment in the occunence study. This was generally in

agreement with observations by Syder et al. It was found that the removal efficiencies of

caffeine by chlorination or chloramination were below 20% after 24 hrs at pH 7.9-8.5

(Syder et al. 2007).

There was also some difference on removal efficiency between chlorination and

chloramination treatments. Occurrence results indicated that 8 of 9 water treatment plants

removed sulfamethoxazole to below detection limit with chlorination treatment. Only 4

of 10 water treatment plants removed sulfamethoxazole to below detection limit with

chloramination treatment. Similar results were also founded by Stackelberg et al.

(Stackelberg et al. 2007), sulfamethoxazole in source water can be effectively removed

after chlorination treatment. The less effective oxidation efficiency of sulfamethoxazole

and tylosin was also reported by Chamberlain et al. (Chamberlain and Adams, 2006).

Other studies repotted that the sulfamethoxazole aniline-nitrogen was directly attacked by

chlorine (Dodd and Huang, 2004).

Table 6. Seasonal monitoring ofPPCPs fi:om Febmru.y to J1.me 2009.

as.. a Water -.a> Tntat.eat

MO ltiver F n:oe chJariDe

Feb R.c::savoir

MSJtiver CbJoiaoiiMs

MO River CbJoiaoiiMs

April R.c::savoir Fn:oe ddaDDe


MOJtiver F n:oe chJariDe

!by R.c::savoir Fn:oe chJariDe


MOJtiver CbJoiaoiiMs

Juuc R.c::savoir Fmc dllaDDe

MSJtiver F n:oe chJariDe

Sa..ple

type

U1llmllll:d

Tn:311al

Untft:atal

Tn:311cd

Uum::2led

Tn:311al

Uum::2led

Tn:311cd

Untft:atal

Tn:311cd

Uum::2led

Tn:311cd

Uum::2led

Tn:311al

Uum::2led

Tn:311cd

Uum::2led

Trc:lllal

Uum::2led

Trc:lllcd

Untft:atal

Trc:lllcd

Untn:dcd

Tn:311cd

Lba~

~ L.8

<MDL

2.5

<MDL

4..2

<MDL

L.1

<MDL

5..9

<MDL

1_8

<MDL

2...3

<MDL

6..9

<MDL

2...0

<MDL

L9

<MDL

L.3

<MDL

2...6

Ceae>e~~taU... (IICIL)

Tdmea- s ..... ea- Ac:e..__-pria oxa&ole .,.._

2..8 18_8 16_0

<MDL

<MDL

<MDL

4_6

<MDL

<MDL

<MDL

<MDL

<MDL

2...6

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

8..2

<MDL

<MDL

13_7

<MDL

14_0

6_8

22

<MDL

9_4

<MDL

8 _4

5_6

2...2

<MDL

6 _7

<MDL

4_6

4 _0

<MDL

<MDL

4..2

<MDL

14..2

<MDL

5.3

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL <MDL <MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

:MDL=M~Dd~L~

Cal

r.ke

49_6

36_0

157.2

18_4

47.2

13_0

38.3

22...0

110_8

2...7

19.9

3-4

17..6

10_6

4_6

3_7

18_7

2...8

27_6

12...4

6_6

4_0

15_6

9S

Ca ... __

s~e

5S

4_7

LO

<MDL

8_4

22

4.2

2...1

L.3

<MDL

4_6

<MDL

3.3

<MDL

L1

<MDL

4 _0

<MDL

4 _4

L.5

<MDL

<:MDL

5_7

LO

Tide:»- .._..,......

- fell 2...1 27_1

<MDL

<MDL

23_4

2...4

<MDL <MDL

<MDL

<MDL

<MDL

16_6

8_8

2...2

<MDL <MDL

<MDL <MDL

<MDL <MDL

4.2

3_4

5_4

<MDL

<MDL

<MDL

<MDL <MDL

&.1 <MDL

<MDL <MDL

8.9

6.9

<MDL

<MDL

<MDL <MDL

<MDL <:MDL

<MDL <MDL

<MDL <MDL

3_1 <MDL

<MDL <MDL

w -...!

38

The distribution of these compounds was water source dependent. River water

and surface water samples had higher concentrations of studied pharmaceutical

compounds, although concentrations of pharmaceutical compounds in well water were

much lower. This is understandable since well water is purified by underground materials,

such as sand and dirt filtration. Results also showed that most of the investigated

phatmaceutical compounds were not detectable after water treatment. However, the

removal efficiency by water treatment plants was compound dependent. Some

pharmaceutical compounds, such as lincomycin, trimethoprim, and sulfamethoxazole,

were relatively easier to remove than other compounds, such as ibuprofen,

carbamazepine, and caffeine. Even though some pharmaceuticals were still detectable

after treatment, the concentrations were much lower than those in the untreated water,

which implied that most phatmaceutical compounds can be effectively removed during

water treatment.

4. CONCLUSIONS

A comprehensive method has been developed and validated for quantitative

analysis of 16 pharmaceutical compounds in two different water matrices, using liquid

chromatography coupled with tandem mass spectrometry. Most selected compounds

investigated by this method showed good recovery and reproducibility in reagent water

and tap water. The results of the occurrence study indicated that the levels of selected

compounds in different types of water resources across Missouri were below 80 ng/L for

all of the pharmaceuticals, except caffeine, which had the highest concentration, while

levels of other compounds were very low. Most of them were below method detection

limits. The levels of the studied pharmaceutical compounds were also water source

39

dependent. Studied pharmaceutical compound concentrations were higher in surface

water than those in well water. Occurrence data also showed that the levels of the most

pharmaceutical compounds in summer water samples were lower than those found in the

winter water samples. The treatment processes in water facilities across Missouri were

found to be effective for removing most pharmaceutical compounds.

ACKNOWLEDGEMENTS

This study was funded by Missouri Department of Natural Resources. We also

thank Applied Biosystems, Inc, Millipore Inc., and the Environmental Research Center at

Missouri University of Science and Technology for the valuable support.

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2. Investigation of Oxidative and PAC Removal of Selected Pharmaceuticals in Various Oxidation Systems by Using

Liquid Chromatography Tandem Mass Spectrometry

ABSTRACT

44

The presence of pharmaceuticals and personal care products (PPCPs) in natural

water resources and drinking water supplies has raised public concern because of their

potential adverse ecological effects on aquatic organism or even on human health. The

study of pharmaceutical elimination is crucial in providing information for the

disinfection strategy in water treatment facilities. This study investigated both the

treatability and removal of eight pharmaceuticals (caffeine, acetaminophen,

carbamazepine, sulfamethoxazole, trimethoprim, erythromycin, lincomycin, and codeine)

as a function of treatment approaches, types of disinfections (free chlorine, ozone,

monochloramine, and petmanganate ), and pHs, by using liquid chromatography- tandem

mass spectrometry (LC-MS/MS). Both deionized (DI) water and natural water were

spiked with 5 to 20 ).I giL of pharmaceuticals and dosed by commonly used oxidants at a

typical concentration followed by analysis using LC-MS/MS. It was found that the

degradation levels of the pharmaceutical compounds varied significantly in different

oxidation processes. Chlorination at 1 mg/L was highly effective in removal of some

phatmaceuticals. Monochloramine at a dose of 3 mg/L was mostly ineffective to all the

pharmaceuticals tested. The pH conditions also played an important role in PPCP

removal, and its effect was conditional based on the oxidation systems and

pharmaceuticals involved. Finally, powered active carbon (PAC) was also evaluated for

its effectiveness in the removal of the pharmaceuticals in drinking water treatment.

45

Key words:

Pharmaceuticals; removal; LC-MS/MS

1. INTRODUCTION

Pharmaceuticals and personal care products (PPCPs) have been detected in many

natural water systems as well as drinking water supplies (Ahrer, Scherwenk, and

Buchberger 2001; Benotti et al. 2009; Hirsch, Ternes, and Haberer 1998; Jones,

Voulvoulis, and Lester 2001; Kolpin et al. 2002; Lindsey, Meyer, and Thurman 2001;

Sacher et al. 2001; Ternes, Bonerz, and Schmidt 2001). The potential occurrence of

pharmaceuticals in drinking water supplies raises public concerns because available

treatment data and the information about combined and synergistic effects of different

types of pharmaceuticals are limited. It is already known that some pharmaceuticals can

cause adverse biological impacts on aquatic and terrestrial organisms at ve1y low

concentrations (Fent, Weston, and Caminada 2006; Jjemba 2006). Due to the possible

existence of pharmaceuticals and their metabolites in water supplies and the food chain,

some risk assessments have been performed to indicate the possible impacts of

pharmaceuticals on ecosystems (Bound and Voulvoulis 2006; Stuer-Lauridsen et a!.

2000). Even though current toxicity data show no acute effects of pharmaceuticals in

surface water on aquatic organisms (Flippin, Huggett, and Foran 2007; Heruy and Black

2008; Henry et al. 2004; Hong et al. 2007; Schreurs et al. 2004), chronic effects and

bacterial resistance to antibiotic pharmaceuticals may exist. As a result, attention should

be given to the long term concerns regarding the accumulation and spread of non

degraded pharmaceuticals in the environment.

46

Reliable information for pharmaceutical elimination from surface water can be

critical to water treatment plant operations because surface water is widely used as a

source for municipal water systems. To minimize the risk of potential chronic harmful

effects, drinking water should be free of pharmaceuticals. Some recent studies have

already reported the oxidation of pharmaceuticals by chlorine and ozone (Stackelberg et

a!. 2007; Westerhoff et a!. 2005). Ozone is an effective oxidant for the effective

degradation of pharmaceuticals and has been well studied (Broseus et a!. 2009; Hua,

Bennett, and Letcher 2006; Vieno et a!. 2007). In some studies, hydrogen peroxide was

combined with ozone to create an advanced oxidation method for pharmaceutical

decomposition (Huber et a!. 2003; Zwiener and Frimmel 2000). There are also some

studies reported the oxidation of pharmaceuticals by permanganate and chloramines

(Chamberlain and Adams 2006; Waldemer and Tratnyek 2006), however, they did not

cover all the compounds we found in Missouri natural and drinking water in our

occurrence study.

In our recent occurrence study on pharmaceuticals in Missouri water resources ,

some pharmaceuticals were detected in both untreated source water and treated drinking

water, including caffeine, acetaminophen, carbamazepine, sulfamethoxazole,

trimethoprim, and ibuprofen (Wang eta!. 2010). The aim of this presented research was

to investigate the treatability of these compounds by using liquid cluomatography -

tandem mass spectrometry (LC-MS/MS). Eight pharmaceuticals were chosen to evaluate

oxidation effectiveness of the following commonly used oxidants in water treatment

plants: free chlorine (HOCl/OCl), monochloramine, ozone (03), and permanganate

(Mn04"). Powered active carbon (PAC) was also evaluated for its effectiveness in the

47

removal of the pharmaceuticals. The PAC removal efficiency in DI water was compound

dependent. The major task of this study was to investigate the concentration x time (CT)

values for the oxidation reactions of selected pharmaceuticals with the four chosen

oxidants in deionized (DI) water. The pH effect on oxidation efficiency was also studied

at pH 6.6 and 8.6. Finally, pharmaceutical oxidation in real surface water was studied to

investigate the effects of sample matrix on reaction efficiency.

2. EXPERIMENTAL

2.1 Selection of pharmaceutical standards and reagents

Total of eight pharmaceuticals were chosen for the oxidation experiments,

including caffeine, acetaminophen, carbamazepine, sulfamethoxazole, trimethoprim,

erythromycin, lincomycin, and codeine. This is because our previous occmTence study

showed these compounds were detected in Missouri natural or drinking water. All

pharmaceutical standards were obtained from Sigma-Aldrich (St. Louis, MO) except

lincomycin, which was purchased from MP Biomedicals (Aurora, OH). All other

treatment chemicals (sodium hypochlorite, sodium dihydrogen phosphate, disodium

hydrogen phosphate and sodium permanganate) were certified ACS grade and were

purchased from Sigma-Aldrich (St. Louis, MO). LC-MS grade mobile phase solvents

(methanol, acetonitrile) were purchased from Fisher Scientific (Fairlawn, NJ, USA).

Formic acid (MS grade) was obtained from Sigma-Aldrich (St. Louis, MO).

2.2 Oxidant solution preparations

Sodium hypochlorite (NaOCl, 5%) served as a source of free chlorine stock

solution. Laboratory DI water was used to make all dilutions. The NaOCl working

48

solution was stored in an amber glass bottle and wrapped with aluminum foil for

protection from light. The concentration of free chlorine solution was determined using

Accuvacs Ampuls from the Hach Company (Loveland, CO, USA) and using the Hach

DPD Method 8021. The free chlorine concentration was measured before every

experiment and DI water served as blank.

Monochloramine stock solution was created from the reaction of sodium

hypochlorite solution and ammonium chloride solution at the molar ratio of 1:1.05 in pH

adjusted 5.0 mM phosphate buffer. Excess ammonia at pH >9 (-5%, determined by

Orion Model9512 Ammonia probe (Thermo-Electron Corporation, Waltham, MA)) was

maintained during monochloramine solution preparation to prevent the presence of free

chlorine. Monochloramine concentration was measured by Accuvacs Ampuls with the

Hach DPD Method 10200 for nitrogen, free ammonia and chloramine (mono). The

concentration of total chlorine in the monochloramine working solution was also

measured by Accuvas Ampuls with the Hach DPD Method 8167 to ensure free chlorine

was indeed absent in the working solution.

Permanganate stock solution was prepared by dissolving sodium permanganate

crystals in lab DI water to the desired concentration. The working solution concentration

was measured by a Cary 50 Cone UV-Visible Spectrophotometer (Varian CO, Australia)

at 525 nm and lab DI water served as blank.

Ozone gas was produced from an ozone generator (Model GLS-1 PCI-WEDECO,

Environmental Technologies, West Caldwell, NJ, USA) with a source of compressed

oxygen. Nonstop gaseous ozone flow bubbled out from a diffuser into deionized Milli-Q

water (Millipore, Bierica, MA) to keep the ozone stock solution saturated for immediate

49

use. The concentration of ozone solution was determined using Accuvas Ampuls with the

Hach Indigo Method 8311 immediately after sampling and lab DI water again served as

blank.

2.3 Analysis

All sample analysis was performed using a triple-quadrupole mass spectrometer

(Applied Biosystems API 4000Q TRAP) equipped with an Agilent (Palo Alto, CA) 1100

series HPLC system. The analytical method was the same as in our previous study [28].

In summary, 10 111 sample solution was injected onto a Supelco C-18 150x2.1 mm

column from Sigma-Aldrich (St. Louis, MO) for the separation of the analytes at room

temperature. The binary gradient for separation of the selected compounds consisted of

0.1% formic acid (v/v) in both water and acetonitrile at a flow rate of 0.25 mL!min.

Multiple reaction monitor (MRM) in positive electrospray ionization (ESI) mode was

used to analyze the selected pharmaceuticals. Mass calibration was performed before

each study. Compound-dependent parameters and source-dependent parameters were

optimized in the mass spectrometer individually. Two pairs of ion transitions were

chosen in the optimization process. The ion transition with the most intense signal was

selected to be the quantification ion pair of the corresponding compound, while the ion

transition with the second highest signal was selected to be the confirmer ion pair of the

corresponding compound.

Instrument detection limits (IDLs) were determined by performing injections of

each individual pharmaceutical at a low concentration (with a signal to noise ratio of

approximately 3-5:1) for 10 consecutive times with the column. The method detection

limits (MDLs) of pharmaceutical compounds in both lab DI water and natural surface

50

water were determined separately, and was calculated by multiplying the standard

deviation(s) of the replicate analyses by the appropriate students' t value at 99 percent

confidence level with nine degrees of freedom. The method reporting levels (MRLs) of

all selected pharmaceuticals in this study were chosen as 0.25 ng/ml except for

acetaminophen, which was I ng/ml.

2.4 Treatments

Phosphate buffer systems were chosen to carry out the oxidation experiments.

Phosphate buffer (5 mM NaH2P04 and Na2HP04) were prepared in lab DI water, and

adjusted to pH 6.6 and 8.6, respectively using different ratios. Natural water samples

were collected from Missouri River and Shuman Lake (Rolla, MO), and were for

immediate use. Both natural water samples were filtered using 0.45-~m nylon filters

(Whatman, England), followed by the addition of solid phosphate salts and adjustment of

pH to 8.6.

The concentrations of working pharmaceutical solutions were 5 ~giL (except for

acetaminophen, which was 20 ~g/L), which were prepared in both buffer systems. Each

working pharmaceutical solution was transferred to an amber glass reactor for initial

concentration determination. Oxidations were initiated by spiking an appropriate amount

of each oxidant solution to the working pharmaceutical solutions at ambient temperature.

The concentrations of free chlorine, permanganate, ozone and monochloramine were I, 1,

1.5, and 3 mg/L, respectively. The reactor was rapidly mixed and put on an orbital shaker

for reaction. Samples were taken at 0, 3, 30, 60, 100, 150, and 300 min (samples were

taken at 0, 2, 5, 10, 15, 30, and 40 min. in ozone oxidation experiment) for analysis to

51

determine the concentrations of target pharmaceutical compounds and the oxidants. All

the experiments were conducted at room temperature of25°C. All reactors were wrapped

in aluminum foil to prevent any light disturbance during the experiments. Each

experiment was conducted in duplicate for accuracy control and calculation of standard

deviation.

For the PAC removal experiment, the pharmaceutical working solutions were

prepared in amber glass reactors at the same concentrations used in the oxidation

experiments. Then 2 mg/L Norit HOB PAC was spiked to initiate the reaction. The

solution was mixed up to 24 hrs for the reaction to take place. Samples were analyzed at

1, 2, 4, and 24 hours.

3. RESULTS AND DISCUSSION

3.1 Free chlorine oxidation

The reactivity of selected compounds with free chlorine in two pH buffer systems

was shown in Figure 1. Error bars on the data points indicated the reproducibility of the

measurements of pharmaceuticals in the study. Under specified oxidation conditions,

both caffeine and carbamazepine were resistant to free chlorine in both pHs. At pH 8.6,

most pharmaceuticals were reactive with free chlorine (OCl-) and >85% removed at aCT

value of 24 mg·min!L. Showing high reactivity, all of lincomycin, 70% of

sulfamamethoxazole and 50% of trimetethroprim were removed at a CT value of 3

mg·min!L at pH 8.6. At pH 6.6, four pharmaceutical compounds were >90% removed at

a CT value of 24 mg·min!L. Erythromycin and acetaminophen were 90% removed at the

CT value of 198 mg·min!L. By comparing the results in two different pH systems, it was

52

found that the oxidation of erythromycin and acetaminophen by free chlorine were a little

faster at pH 8.6 than pH 6.6. Most pharmaceuticals can be removed at the CT value of

200 mg·min/L at both pHs.

a 16

14 - lincom cin - Trimetho rim

- Acetaminophen Carbamazepine

12 .D a.

Codeine FC/ppm Caffeine a. 10 -c 0

~ 8 -c Q}

6 .... c +-----------~---------------------------------------------~--~~r----

::0:: 0 u

4 1

2

0

{;}~

CT (mg/l min)

b 18

- Erythr omycin - Lincomycin - Trimeth oprim 16

- suifamamethoxazoie - Acetaminophen • Carbamazepine

14

.D 12 a. a. -c 10 .2 ~ -c 8 ~ c 0 6 u

4

2

0 -{;}~

CT (mg/l min)

Figure 1. The oxidation of selected pharmaceutical compounds by free chlorine in (a) pH 8.6 buffer and (b) pH 6.6 buffer.

53

The removal experiments were also carried out in the lake water for comparison

(Figure 2). The concentration of free chlorine decreased faster in real water oxidation

than lab DI water. This indicated that the real water matrix had a strong competition with

the selected pharmaceutical compounds for free chlorine oxidant. Most pharmaceuticals

can still be removed during the oxidation study, but the rate was much slower than that in

the lab DI water. The results showed that the oxidation trends for all of the studied

pharmaceutical compounds in lake water were the same as those in the lab DI water.

25

- Erythromycin - Lincomycin - Trimethoprim

Acetaminophen Carbamazepine 20

Caffeine Codei ne Cl2/ mgl -1

..Q D. D.

........ 15 II:

,Q .. I! .. II:

~ 10 II:

8

~ ~ ~ :::: ~

5 ... ... .....,..__

'II ,_ IE

0 - ----,

0.0 2.6 16.7 26 .5 36.3 45.5 62 .0

CT (mg/l min)

Figure 2. The concentration of detected pharmaceutical compounds in free chlorine oxidation experiment in pH 8.6 lake water.

54

3.2 Permanganate oxidation

Permanganate is a highly reactive oxidant and widely used in the water treatment

facilities. When it functions as an oxidizing agent, sodium pennanganate (Mn04-, Mn

VII) is reduced to manganese dioxide (Mn02, Mn IV). Figure 3. showed the oxidation

results of the target pharmaceutical compounds using permanganate as an oxidant in two

different pH buffer systems. It was found that the reactivity was not pH dependent.

Acetaminophen was degraded rapidly at both pHs in the presence of petmanganate.

Codeine and carbamazepine were also reacted readily with pennanganate. Both

compounds were reacted at a CT value of 27 mg·min/L. The reactivity of lincomycin

with permanganate was slow. It was 90% oxidized by permanganate at a CT value of

250 mg·min/L in pH 8.6, but only 30% was oxidized at pH 6.6 at a similar CT value. All

other compounds were not reactive to permanganate. Trimethoprim, erythromycin and

sulfamamethoxazole were removed 20-50% at aCT value of250 mg·min/L at both pHs.

Same oxidation experiments were also carried out in Missouri River water for

comparison (Figure 4). The concentration pattern of permanganate was the same as that

of the oxidation study in DI water. This indicated that the sample matrix (organic matters)

in the surface water was not competitive with selected pharmaceutical compounds in the

permanganate oxidation system, but did interfere with the oxidation of pharmaceutical

compounds in the free chlorine oxidation system (as demonstrated in Figure 2).

55

25 a

- Erythromycin - Lincomycin - Trimethoprim

- sulfamametho>G!Izole - Acetaminophen Carbamazepine 20

Caffeine Codeine Mn04-/ ppm

15 .a D. D.

...... c:

.S2 .. 10 I! T .. T T -- ..L c: ..L ..L - ... ... I ~ ..L ..L ... -~ c:

8 5

-0 ----,

0 .0 3 .0 27.0 51.7 84.5 125.8 248.8 CT (mg/L min)

b 25

- Erythromycin - Lincomycin - Trimethopri m

- su lfamametho>G!Izo le 20

- Acetaminophen Carbamazepine

Caffeine Codeine M n04-/ppm

15 .a D. D.

...... II: .Iii

10 .. I! .. II:

~ - .... I ~ ~ II: .,.

-~ ~ ....

8 - ± 5 !I:

-.o.!: !:: i i :;- :m ::: ~ =

0 --,

0 .0 2.9 26.8 52.6 87.0 129.7 256.5 CT (mg/L min)

Figure 3. The oxidation of selected pharmaceutical compounds by permanganate in (a) pH 8.6 buffer and (b) pH 6.6 buffer.

25 .-------------------------------------------------------

20

.a 8:

...... 15 a ,Q .. I! .. a

I - Erythromycin - lincomycin - Trimethoprim

- Su lfamamethoxazole - Acetaminophen Carbamazep ine

Mn04-/ mgl -l

~ 10 +-----~r------------------------------------------a 8

5

0

0.0 2.1 17.8 34.1 55.5 81.9 160.6

CT (mgjl min)

Figure 4. The oxidation of selected pharmaceutical compounds by permanganate in pH 8.6 Missouri river water.

3.3 Ozone oxidation

56

The reactivity of selected compounds with ozone (1 .5 mg/L) in two different pH

buffer systems was shown in Figure 5. The oxidation of the selected pharmaceuticals by

ozone was quite rapid and completed in about 2 min. In addition, ozone was unstable in

aqueous solution. Due to these two reasons, the concentration of ozone was barely

detected after 2 min. As a result, a CT curve was not used in the ozone oxidation studies.

The concentration of each pharmaceutical compound did not decrease significantly after

two minutes, which indicated that the soluble ozone in the oxidation system was entirely

57

reacted until ozone was totally decomposed (about 2 min). A higher concentration of

ozone was required to completely remove the pharmaceuticals at the experimental

concentrations. The data also showed that ozone oxidation was pH dependent. Since

ozone was more stable in lower pH, some phmmaceuticals were easier to be oxidized at

pH 6.6 than 8.6. In particular, acetaminophen, licomycin, erythromycin, codeine and

carbamazepine showed were very reactive with ozone. The oxidation of some

pharmaceuticals was pH dependent. Carbamazepine, licomycin, codeine, trimetethroprim

and sulfamamethoxazole showed higher oxidation percentages in pH 6.6 buffer than

those in pH 8.6 buffer. A greater percentage (-75%) of carbamazepine removal was

observed in pH 6.6, but only -10% of carbamazepine was removed in pH8.6.

Acetaminophen and erythromycin did not show strong pH dependence on oxidation

between the two pHs. The data in Figure 5 also indicated that there was a strong

competition among the pharmaceuticals for ozone oxidation system. Carbamazepine,

erythromycin and licomycin appeared more reactive with ozone compared to other

pharmaceuticals. Caffeine showed very little change over time. This data may still be in

agreement with the literature when the following factors are considered: in our study,

higher PPCP concentrations were used, half or less of the literature ozone concentration

was used, reactions were performed at approximately 16 oc higher than the literature

work (thus decreasing ozone solubility), and higher pH buffer conditions were used. As

our pH study showed that greater reaction efficiency was observed at lower pH, it is

reasonable that less pharmaceuticals were oxidized in our 6.6 and 8.6 pH systems than

the literature's pH 5 system. Finally, the ozone in our experiment was below the detection

58

limit after 2 min. , while the experiment in the literature repotied 0.2-0.3 mg/L of ozone

after 3 min (Snyder et al. 2006; Westerhoff et al. 2005).

a 20

18 - E..!:llt_hromvcin -Lincom_y~cin - Trimet h oorim

16 ~ - s u ifam am ethoxazol e - Acetam inophen - Carb amaze pina

14 ..a a. a.

12 -a ,Q .. 10 I!

"\. - Caffe ine - Code i ne

" "'il: T ..L .J.. T

T T _ ~-.!: ~ .. ..

fj 8 .. 8

6 T ..,.. ..,.. T ~ I--"" :c .... "' L

4

2

~-~· :.- .,., - ::; - :;: I ""' .....

~ ... ~ !!: . ... ~ - - -- ::: - - -"

0

0 2 5 10 15 30 4 0

Tlme/min

b 20 ~

18

16

..a 14 a. a. -1 2 ..

.Q .. 10 I! ..

- T - Erythromyci n - Li ncomycin - T r imet ho p rim

~ - s u ifamamethoxazoie - Acetamino ph e n - earb am azepi ne

' - Caffe i ne Code i n e

- ' " T~ _T T _T

,.. .... .... .J.. ..... ..

fj 8 -.. 8

6

4

2

~~ ..,.. --~ ..... = -; ];.---

; : : ~ J;;

-==- - .r.

""'' 0

0 2 5 10 1 5 30 40

Tlme/mln

Figure 5. The oxidation of selected pharmaceutical compounds by ozone in (a) pH 8.6 buffer and (b) pH 6.6 buffer.

59

3.4 Monoclzloramine oxidation

Monochloramine is a relatively stable compound and is becoming an increasingly

popular alternative disinfectant to chlorine in municipal drinking water systems because

of its stability in distribution system. The reactivity of selected pharmaceutical

compounds with monochloramine at two different pH buffer systems was shown in

Figure 6. Data were collected after exposures of 0, 2, 30, 60, 120, 240 and 360 minutes.

All compounds were very resistant to monochloramine except lincomycin. No significant

oxidation was observed following exposure of monochloramine up to 1000 mg·min/L.

The results demonstrated that monochloramine at a typical drinking water treatment

dosage was not so effective to remove these selected pharmaceuticals as free chlorine due

to the lower oxidation potential of monochloramine. This also indicated that fewer

pharmaceutical degradates might be generated when monochloramine was used as a

disinfectant in municipal drinking water systems.

3.5 Removal of pharmaceutical compounds with powered active carbon

The removal of selected pharmaceuticals by powdered activate carbon was also

investigated in DI water and in surface water. Figure 7 showed the results of 2 mg/L

Norit HDB PAC to remove the selected pharmaceuticals in DI water in two different

pHs. Even though these compounds were partially removed by PAC in a 24 h

experiment, the removal efficiency was compound dependent. The 24 h contact time was

used because it was close to the equilibrium condition for the PAC. Erythromycin and

trimetethroprim showed approximately 70% and 50% removal at the equilibrium time,

60

and all other pharmaceuticals showed about 20-30% removal at the selected conditions.

No obvious difference in the removal efficiency was found at different pHs.

25

a

20 T T T I 1 --- J ---1 --T T.--- 1 -

-a 1 I 1 a. -15 ..

,Q - Erythromycin - lincomycin - Tri m ethoprim .. I! 1: - Sul famamethoxazole - Acetam i nophen Carbam azepine el 10 -a --Caffeine Co de ine MCA(as Cl2)/ mcl-1 8

-=- -~

5 = = --= 1 -==. == :£~- = :L _ ... - -" = ~

'J: ~

0

0 .0 9 .0 89.4 178.4 354.9 705 .9 1056.0 CT (mg/l min)

b 25

20 T

l. .L .L .L 1. _T 1 .a .L

a. a. -15 .. - Erythromyci n - Li ncomycin - Tr i m ethoprim ,Q .. I! - sul famamethoxazo le - At:etam inophen _. Carbamazapine .. II: Caffe i ne Codeine ~ MCA(as Ci 2 )/ m&L-1

~ 10

8

= 5

3: = -~ ::::=.-.;; : =~ ---= ~

~

0

0 .0 8 .9 87.8 175.2 349.1 694.7 1039.7

CT (mg/l min)

Figure 6. The oxidation of selected pharmaceutical compounds by monochloramine in (a) pH 8.6 buffer and (b) pH 6.6 buffer.

62

The PAC removal experiment was also carried out in Missouri River water to

investigate the effect of sample matrices (Figure 8). The data showed that most

pharmaceuticals were not removed from the river water at the selected dosage of PAC

even when the equilibrium was reached. This comparison indicated that the sample

matrix played an important role in the pharmaceutical removal in natural surface water.

25

l T _l T _T 20 .l. 1 ..... 1 l.

- Erythromycin - lincomycin

J:l 15 - Trimethoorim - su lfamamethoxazole a. a. - Acetaminophen Carbamazepine ..... c:

Caffeine Codeine .Q .. ~ 10 .. c: 8 c: T 8 - ... 'T' ,: a;-,-

"£. - ~ -- a; -5 :;- "'- ~ - ... "" ~

0

Oh 1h 2h 4h 24 h

Time

Figure 8. The concentration of detected pharmaceutical compounds in PAC removal experiment in pH 8.6 Missouri river water matrix.

63

4. CONCLUSIONS

Thorough investigation of pharmaceutical removal using different oxidants was

performed. The oxidation removal of selected pharmaceutical compounds varied

significantly in different oxidation processes. In comparison, chlorination was highly

effective in the elimination of the selected pharmaceuticals. Sample matrix and pH

caused notable differences in oxidation efficiency. Permanganate was also effective to

decompose some pharmaceuticals. Even though different removal efficiency was

observed between two pHs, sample matrix in sutface water did not change the oxidation

efficiency. Ozone was a fast oxidizer but very unstable. The ozone oxidation was

completed in about 2 min under the selected conditions. Chloramination was mostly

ineffective in oxidation removal of all the studied pharmaceuticals at the typical

disinfection dose (3 mg/L). Parallel studies in different water matrices indicated that the

oxidations proceeded similarly but with a relatively slower rate in the surface water due

to the effect of matrices. The results of this work have shown that some oxidation

systems may be useful in eliminating some of the PPCPs in water treatment facilities.

ACKNOWLEDGEMENTS

This study was funded by Missouri Department of Natural Resources. We also

thank Applied Biosystems, Inc, Millipore, Inc., and the Environmental Research Center

at Missouri University of Science and Technology for the valuable suppmi.

64

REFERENCES

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65

Hua, W., E.R. Bennett, and R.J. Letcher. 2006. Ozone treatment and the depletion of detectable pharmaceuticals and atrazine herbicide in drinking water sourced from the upper Detroit River, Ontario, Canada. Water Research 40: 2259-66.

Huber, M.M., S. Canonica, G.-Y. Park, U. von Gunten. 2003. Oxidation of Pharmaceuticals during Ozonation and Advanced Oxidation Processes. Environmental Science and Technology 37: 1016-24.

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Snyder, S. A., E. C. Wert, D. J. Rexing, R. E. Zegers, and D. D. Drury. 2006 Ozone oxidation of endocrine disruptors and pharmaceuticals in surface water and wastewater. Ozone: Science & Engineering 28: 445-60.

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66

Ternes, T., M. Bonerz, and T. Schmidt, 2001. Determination of neutral pharmaceuticals in wastewater and rivers by liquid chromatography-electrospray tandem mass spectrometry. Journal of Chromatography, A 93 8: 17 5-85.

Vieno, N.M., H. Haerkki, T. Tuhkanen, and L. Kronberg. 2007. Occurrence of Pharmaceuticals in River Water and Their Elimination in a Pilot-Scale Drinking Water Treatment Plant. Environmental Science & Technology 41: 5077-84.

Waldemer, R.H. and P.G. Tratnyek. 2006 Kinetics of Contaminant Degradation by Permanganate. Environmental Science and Technology 40: 1055-61.

Wang, C., H. Shi, C.D. Adams, S. Gamagedara, I. Stayton, T. Timmons, andY. Ma. 2011. Investigation of Pharmaceuticals in Missouri Natural and Drinking water Using High Performance Liquid Chromatography-Tandem Mass Spectrometry. Water Research 45:1818-28

Westerhoff, P., Y. Yoon, S. Snyder, and E. Wert, 2005. Fate ofEndocrineDisruptor, Pharmaceutical, and Personal Care Product Chemicals during Simulated Drinking Water Treatment Processes. Environmental Science and Technology 39: 6649-63.

Zwiener, C., and F.H. Frimmel, 2000. Oxidative treatment of pharmaceuticals in water. Water Research 34: 1881-5.

APPENDIX A.

ADDITIONAL INFORMATION ABOUT PPCP OCCURRENCE

68

Total ion chromatogram of pharmaceutical compounds in positive ionization mode:

u

U ti

U ri 9

I.Crl

:.ft .

t .C'tl I llt'l'l ,)lllll\01'"~' 1\

Ur i ') \\,\l~' ll)('

l .(t l 1 ~ lllh'l)ll)\\' II)

Uri -l \\t(i~·m~· ..

!.Col 5 trnn~· tlll'l 'l'lm &

~ 4

l.lrl 5 6 b S\tl(,um•th~'~·IZ\''~'

f 7 Utt ,. trh:m I

l.lrl I 8 ~·.u·h,un.tz~'l'l l\~' ICr l

0 <) ~'SII\hll(ll

: .hi

3 10 ~·thr II\' l l'SII\Idt~'l : ( tl 8 l.lr l

I.( I I 2

10000 l, 00

\_ -I : ) I ' ' l • t 10 II ll I ) II II II tl II I' :o ll .. :I l l

Tt-U,h lt

69

Total ion chromatogram of pharmaceutical compounds in negative ionization mode.

·~· I 1 W)'l'll JIIHll' ..0•

Mit. 2 4 1 dlllthnl· ,\fad

w~•

,. ... u.

I J l'Sinol

.J a hu l'l'll l l'll

~· 5 s l'SII'\l lh' Ult•

;:,:t • (l lndll an

:::. :. ... ... ~· :!-

:.: ~ . ..

Q

3

I I 6

I 1.8t •

·~~ · ..... ~~· 1!)6 i

&«00

(n:IU/

1

I 6(00.0

~ I I~ OJ)

.. _ I : ) • $ " r 8 I) II 1: I) .. 1$ I ) r. 6 U• ::0 :I :: ::I

The composition of water sources in the occurrence study.

The composition of water sources in the occurrence study

• River

• Reservoir

• LJke

• UnconsolidJted well

• Ground wJter

Deep well

70

71

The pharmaceuticals levels in untreated water samples (ng/L). (Winter)

100.00

90.00

80.00 River ::; "M 70.00

• Surf~< e w~ter c - 60.00 c 0 ·;;

50.00 ~£_well 111 .... ... c 40.00 Ql Oii<omolid~ted well _ u c ~~~~~ ground w~ter 0 30.00 u

---.:;.__ -------20.00

10.00

0.00

72

The pharmaceuticals levels in treated water samples (ng/L). (Winter)

70.00

60.00 -::;-tio 50.00 c -------------~

c 40.00 0

·p , --------.J!!..i\!lf~CE'W~ter

~----------· --------... ... c 30.00 C1l -u c 20.00 0 u

10.00

The pharmaceuticals levels in untreated water samples (ng/L). (Summer)

60.00

:I 50.00 ....... Qll c: .... c: 0 ... 1\1 .. ... c Ill u c 0 v

40.00

30.00

20.00

10.00

0.00

...------------~~------ River ~~-----------T--l------_:surtM.._e-1-'la-t~-r ~~~~=-

f JITIIr ------.J..!JOeeJl weu - -----

73

The pharmaceuticals levels in treated water samples (ng/L). (Summer)

20.00

::; lio 15.00 r:: -r:: 0 . ., ~ 10.00 r:: ~ r:: 0 u

---• River

• Sur fa ct> water

--- - ..-o~epwcll

74

--

The pharmaceuticals levels in Mississippi river from February to June 2009 (ng/L).

50

45

40 35

:::1 30 'tio c 25 c 0 20 ... Ill

15 .. ... c QJ 10 u s:: 0 5 u

0

,.,..-....... /

~ ,..../---------~ ~ / .·

// / /' / _,

~---/ _.-· .,., ,.. ,..... . / / /' / /

./ / ,

-1 t.llsslsslppl River Feh-Raw

1 t.tlssisslpj>l River Atlr·Rdl't

I t.tlssilllpj>l River May-Raw

I Mllliii iJllll River Jun-Raw

1 t.tllllllit>lll River Feh-Fin

75

76

The pharmaceuticals levels in Reservoir from February to June 2009 (ng/L).

• R~eo voir F eh -Raw • Reservoir Apr-Raw

160 • Reservoir May-Raw • Reservoir Jun ·Raw

140 • Reservoir Feh-Fin Reservoir Apr-Fin

::;- 120 - - Reservoir May-Fin Reservoir Jun-Fin ...... 00 c c 100 0 ...

80 <11 ..... ... c Gl

60 v c 0 v

40

20

0

- ----- -/\ - -- -- ------

~

The pharmaceuticals levels in Missouri river from February to June 2009 (ng/L).

so

4 5

40 :::i' 'ba 3 5 .:. g 30 ·~ ~ 25 ~

"' ~ 20 s 15

10

5

0

----• Mlnourl Rlvf.>r Fei>·Rd\'1

• Mlnourl Rjyer Apr·Rd\'1

• MI«Otu'l Rlvf.>r Mdy·Rd\'1

• Mh.~ourl Rlv~r Jun-Ra\'J

• Ml«ourl Rlvf.> r Feh -fh>

• Ml«ourl River Apr-Fin

- l\11«otnl River Mdy-fln

Ml«ourl RlverJun·fJn

77

APPENDIX B.

PESTICIDE STUDY

79

Investigation of Oxidative Degradation of Molinate

in Various Oxidation Treatment Systems by Using liquid chromatography- tandem

mass spectrometry

ABSTRACT

Thiocarbamate pesticides, which are widely used in agriculture, have attracted

considerable attention because of their impact on people's health and adverse effect on

environmental safety. In this study, the removal efficiency of molinate by various

oxidants and identification of the de gradate of molinate in water at different oxidation

treatment conditions were investigated using LC-MS/MS. The oxidants tested were, free

chlorine (FC), monochloroamine (MCA), UV radiation (UV), chlorine dioxide (Cl02),

permanganate (Mn04-), ozone (03), and hydrogen peroxide (H202). Hexahydro-1-H

azepane-1-carboxylic acid was detected as being a major degradate of molinate after its

treatment with free chlorine and ozone, while no degradates were present after treatment

by other oxidants. Kinetic results for a typical free chlorine degradation indicated that the

reaction of molinate with free chlorine was extremely fast. Hexahydro-1-H-azepane-1-

carboxylic acid, the molinate degradate, showed more resistant to free chlorine in the

same treatment. A possible degradation pathway of molinate is proposed.

Keywords:

Molinate; Oxidation degradates; Hexahydro-1-H-azepane-1-carboxylic acid;

HPLC/MS/MS.

80

INTRODUCTION

Molinate (S-ethyl perhydroazepane-1-carbothioate) is a herbicide, in the

thiocarbamate chemical class, that controls the water weeds in rice paddies around the

world. Molinate is one ofthe most welcome herbicides in the rice plant area because it is

much easier to use than other herbicides. In California, over 95% of acreage was treated

with molinate in 1989, and approximately 60% of that acreage was still being treated

with molinate in 1997 [1]. In 1997, the amount of molinate applied during the 6-week

spring period in California totalled 1.3 million pounds [2]. This large amount of molinate

usage has generated a lot of concern about the potential adverse impact on human health

and environmental safety. Since the 1970s, several toxicity studies have been conducted

to prove that molinate is toxic to the reproductive systems ofrats and even has an effect

on the ovaries of these rodents [3]. These studies have led to more extensive

investigations of other organs and systems that found that molinate is toxic to the nervous

system and is possibly carcinogenic as well [4].

Due to the harmful effects of molinate on animals and its potential adverse effect

on human, some studies have been conducted to determine the occmTence of molinate in

the environment and its degradation pathway under ce11ain systems. Several gas

chromatographic methods have been developed to study the occmTence of molinate in a

variety of environmental samples [5-8]. The photodegradation ofmolinate in water has

also been investigated, and 4-keto derivative was found to be the predominant degradate

[9, 1 0]. The degradation pathway of molinate in plants has also been repmted, and

several degradates have been identified [11], including lH-Azepane, 1-

[ ( ethylsulfinyl)carbonyl]hexahydro-, 1 H-Azepane-1-carbothioic acid, hexahydro-4-

81

hydroxy-, S-ethyl ester, 1H-Azepane-1-carbothioic acid, hexahydro-2-oxo-, S-ethyl ester,

1 H-Azepane-1-carbothioic acid, hexahydro-4-oxo-, S-ethyl ester. The major metabolic

pathway of molinate degradation in laboratory rats has involved sulfoxidation and

conjugation with glutathione [12]. The oxidative metabolism ofmolinate in humans was

mainly mediated by cytochrome P-450 [13]. The pathway of mineralization of molinate

by a mixed culture of five bacteria was also recently reported [14]. However, information

about the oxidation pathway of molinate in the water treatment process is very limited.

Several occurrence studies have shown that high concentrations of molinate were

detected in different water samples, including four rivers in Japan [6], rain samples from

Greece [15], and ground and surface waters from several countries [15-19]. All of the

occurrence studies indicated that molinate contamination exists in natural water systems.

Therefore, assessments of the removal of molinate by various oxidation systems become

very impm1ant in providing information of value for drinking water and waste water

treatment processes. However, no extensive study has been conducted to date to provide

such information.

In this paper, the efficiency of molinate removal under different treatment

conditions, using various oxidant systems, was investigated and quantified. A new

oxidation de gradate was observed and confirmed by liquid chromatography- UV /mass

spectrometry (LC-UV/MS) and liquid chromatography- tandem mass spectrometry (LC

MS/MS). This finding can be impot1ant because the disinfection process could make the

situation even worse due to the formation of more toxic degradation products. Based on

our study, a predicted degradation mechanism ofmolinate in the oxidation processes is

proposed.

82

MATERIALS AND METHODS

Reagents and chemicals. Molinate (99.0% CAS: 2212-67-1), formic acid (96%,

ACS grade), sodium hypochlorite solution (available chlorine 3 4%), and hydrogen

peroxide solution (30%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Methanol (optimal grade), acetonitrile (HPLC grade), ammonium chloride (certified ACS,

99.5%), sodium phosphate (dibasic, 99%), and potassium permanganate (ACS reagent)

were purchased from Fisher Scientific (Pittsburgh, P A, USA).

A molinate stock solution was prepared in methanol at a concentration of2643.3

11g mL-1, and then diluted to 1000 11g mL-1

, as stock solution 2. Both stock solutions were

stored in a freezer during the experiment. Further spike solutions were prepared from

stock solution 2, depending on the pmticular experiment. All experiments were carried

out at room temperature (25 ± 1 °C). The pH values were measured by an Accumet

AccuCap combination pH electrode from Fisher Scientific (Pittsburgh, P A).

Instruments. The HPLC-UV /MS analysis of molinate and its oxidation de gradate

were carried out by the Hitachi M-8000 LC/3DQMS system, coupled with a UV detector

and an electrospray ion source. A Perkin Elmer series 200 HPLC coupled with an

Applied Biosystem API 4000 Q triple quadrupole mass spectrometer was used to confirm

the structure of an oxidation de gradate of molinate. A Phenomenex Synergy 4u Fusion

RP HPLC column (150 x 3.0 mm i.d., 4 11m) was used to separate molinate and its

degradate with a typical flow rate of0.5 mL min-1 during the whole experiment. Mobile

phase A was composed of water with 0.1% formic acid, while mobile phase B was

acetonitrile. The HPLC separation procedure included two parts: ( 1) cln·omatographic

condition one was for the molinate parent with an isocratic composition of mobile phase

A to B at 55 to 45%, and (2) chromatographic condition two was for the degradate with

an isocratic composition of mobile phase A to B at 80 to 20%.

83

The Hitachi electrospray ion source parameters were optimized as follows: ESI

probe voltage, focus voltage, drift voltage, and detector voltage were set at 4000, 25, 30

and 450 V, respectively. The assistant gas heater temperature, desolvator temperature,

aperture 1 temperature, and aperture 2 temperature were set at 180,200, 160, and 120°C

respectively. Nitrogen sheath gas was set at 300 kPa.

For the MS/MS study of molinate and its de gradate by an API 4000 Q trap triple

quadrupole mass spectrometer, a positive ion electrospray mode was chosen for the

ionisation source and a Multiple Reaction Monitoring (MRM) scan was applied. The first

quadrupole was used to monitor the precursor ion (M + Ht at m/z 188 amu, and the third

quadrupole was used to monitor the quantification ion (M +H)+ at m/z 126 amu and the

identification ion at m/z 83. 'Unit' resolution was set for both Q1 and Q3 during the

whole experiment and the dwell time for each MRM scan was 150 ms. The ion source

temperature was set at 700°C with an ion spray voltage of 4000 V. The nebulizer gas,

auxiliary gas, and curtain gas were 413, 551, and 206 kPa, respectively. The declustering

potential and entrance potential were maintained at 46 and 10 V. The collision energy

and collision cell exit potential were set at 21 and 8 V for the quantification ion m/z 126

and 25 and 6 V for the identification ion m/z 83.1 respectively.

Free chlorine (FC) oxidation system. Free chlorine solutions of desired

concentrations were diluted from a sodium hypochlorite solution and stored in the dark

for later use. Accuvacs, obtained from the Hach Co. (Loveland, CO, USA), with the Hach

DPD Method 8021 were used to measure the concentration of free chlorine every time

84

before use. A 500-J.tL aliquot ofmolinate stock solution (10 mg mL-1 in methanol) was

spiked into a 100-mL Pyrex glass bottle (served as a reactor). Methanol was evaporated

in the hood at room temperature, followed by the addition of 49 mL of Mill-Q water.

After thorough mixing using a vmtex, 1-mL aliquot offree chlorine (5 mg mL-1 in Mill

Q water) was spiked into the reactor to allow both the molinate and the free chlorine to

reach a I 0 mg L-1 concentration. The solution was mixed well with the vo11ex again. The

bottle was wrapped with aluminium foil and kept in the dark. The mixed sample was

shaken for 2 hr on an orbital shaker for a reaction, followed by an injection to the HPLC

system coupled with UV and mass spectrometer detectors.

The concentration-time experiment was conducted by spiking a desired free

chlorine stock solution into the molinate in a reactor to reach an initial concentration of

1.61 mg L-1 for the free chlorine and 3.69 mg L-1 for the molinate. This reactor was

wrapped with aluminium foil and shaken on an orbital shaker. Samples were collected

and quenched using ascorbic acid before being injected into the HPLC-UV /MS system

for analysis at intervals of 0, 10, 20, 30 and 40 min.

Ozone oxidation system. Gaseous ozone was generated from an ozone generator

(Model GLS-1 PCI-WEDECO, Environmental Technologies, West Caldwell, NJ, USA)

using compressed oxygen. Ozone gas was bubbled out from a diffuser into Mill-Q water

to form a saturated ozone solution for immediate use. Concentration of the ozone solution

was measured by a Cary 50 Cone UV-Visible Spectrophotometer (Varian CO, Australia)

at 260 nm immediately after sampling, with the Mill-Q water served as a blank. Ozone

oxidation of the molinate was performed in both a pH 6.6 and a pH 8.6 NaH2P04 buffer

with three different ozone dosages. After the molinate was spiked into the reactors,

different volumes of the saturated ozone solution were added into these three reactors.

The final concentrations were 21.1 mg L-1, 15.8 mg L-1

, and 10.5 mg L-1 for the ozone

and 10 mg L -I for the molinate. This reaction lasted for 4 lu on the orbital shaker,

followed by injection into the HPLC system coupled with UV and mass spectrometer

detectors.

85

Chlorine dioxide (Cl02), monochloroamine (MCA), permanganate (Mn04-) and

hydrogen peroxide (H202) oxidation systems. Gaseous chlorine dioxide was generated

from a CDG Bench Scale chlorine dioxide generator by solid sodium chlorite (CDG,

Bethlehem, PA). Chlorine dioxide gas, that was bubbled out from a diffuser into Mill-Q

water to form a saturated chlorine dioxide solution, was stored in the dark for later use.

The concentration of the chlorine dioxide solution was measured at 359 nm by a Cary 50

Cone UV-Visible Spectrophotometer (Varian CO, Australia) every time before use.

Monoch1oramine was created from the reaction of the sodium hypochlorite

solution and the ammonium chloride solution, with a molar ratio of I : 1.05 [20]. During

the reaction, the pH value was controlled above 9 using pre-adjusted phosphate-buffered

Mill-Q water. The final stock solution had 5% excess ammonia, so there was no free

chlorine in the solution. The concentration of the monochloramine stock solution was

measured by Accuvas with the Hach DPD Method 10200 for nitrogen, free ammonia, and

chloramine (mono).

Proper amounts of potassium permanganate solid crystal were directly dissolved

in Mill-Q water and then diluted to desired concentrations. The concentration of the

potassium permanganate was measured by Accuvacs with the Hach DPD Method 8167.

86

An oxidation reaction was started by spiking a proper amount of the oxidant stock

solution to reactors of the molinate working solution to reach a final concentration of 10

mg L-1 for both the oxidant (3% for H202) and the molinate. The reactions were stopped

after 4 hr on an orbital shaker, followed by injection into the HPLC-UV/MS system.

Ultraviolet radiation (UV) oxidation system. UV radiation was generated by a

254-nm Pen Ray 1 W low-pressure mercury-vapor lamp (Model 90-0004-01, UVP Inc.,

Upland, CA). The diameter of this lamp was 0.9 em and the length was 9 em. All non

submerged parts of the lamp were covered by Teflon tape to prevent radiation from the

sample liquid. Four identical amble glass vials, with a diameter of I. 9 em, served as

reactors. Experiments were carried out by placing a lamp in the middle of the vials which

contained 5 mL of2 mg L-1 molinate in NaH2P04 buffer for 2, 25, 45, and 90s. A small

stirring bar was put in each vial to thoroughly mix the sample buffer during the whole

exposure process. The experimental dosage was calculated to make certain that the

fmthest point from the lamp receiveed the desired dosage. In this study, the UV dosages

were from 134-6030 mW•s cm-2•

RESULTS AND DISCUSSION

HPLC-UV!MS ofmolinate and identification of its degradate. A chromatogram of

molinate is shown in Fig. I a, under chromatographic condition one, and the mass

spectrum of the molinate standard shows that the mass to charge ratio (m/z) of molinate

was 188 (Fig. I b). After the molinate standard was oxidised by various oxidants, the

mixture that contained molinate and its degradate was injected into the HPLC-UV/MS

system for analysis. An unknown degradate was detected after oxidation by free chlorine

87

and ozone. A new chromatographic condition (condition two) was then developed to

separate the molinate and its degradate (as shown in Fig. 2a, b under chromatographic

condition two). Hitachi ion trap HPLC-MS was used to identify the degradate of molinate

with a mass to charge ratio of 144. The mass spectra of the degradate fi"Om the free

chlorine oxidation system and the ozone oxidation system were identical (Fig. 2c). Based

on the total ion chromatogram and mass spectrum of free chlorine and ozone oxidation

degradate ofmolinate, the structure of the oxidation degradate ofmolinate was proposed

to be 1 H-Azepane-1-carboxylic acid, hexahydro-. This oxidation degradate was much

more stable to free chlorine than its parent. Unfortunately, the standard of this oxidation

degradate was not commercially available for fi.uther identification.

An AP14000 Q-trap LC triple-quadrupole mass spectrometer, with a Turbo Spray

ionisation source, was used to confirm the oxidation de gradate of molinate. Two ion pairs

were chosen for the molinate parent: 188/126 was for the quantification and 188/83 was

for identification. The detection limit was 0.10 mg L-1. After injection of a sample

oxidation mixture into the LC-MS/MS, a strong signal at m/z 143.8 was observed in the

mass spectrum, which indicated the molinate oxidation degradate. This result matched

the data from the Hitachi ion trap HPLC-MS (as shown in Fig. 3).

88

L (a) 30

Blank

20

> 10 Molinate -----+ " .. ~ ~~ Control u~

" a 0 ...

-10 .-. Degradate Molinate ~ Sample

- 20

0 3 4 5 6 7 8 9 10 11 12 13 14 15 Retention Tirre (min)

RT: 11.158_12.148 min Bl: m/z 1881nt19720 AT: 100 ms ESI+ DV: lOV

"' "' 20000 (M+1)+ ...

(b)

15000 .. ~ ll

10000 " " c H

"' 5000 ~

0

120 130 140 150 160 170 180 190 200 2 10 220 230 240 2 50 M0.33 (ro/ z)

Figure 1. (a) LC- UV chromatograms of blank, control (standard molinate), and oxidation degradate of molinate under chromatographic condition 1. (b) Mass spectrum ofthe molinate standard (MW: 187).

:,;

" ~ ' :,

.,, I

., 10

"' -,,, -

(a)

\ Oogradato

" 10

Mo lin to st <tndnrd

.o 10

~ Samplo

1

Blank ]

r•~I~[' '' ' I':. ,J •. .,. , , ,f""' 'l'" 'lm ' l ''' '('~~"l'~T ............ "T....,.. '' "r'' 'l' ' 'T '" I" ' 'I" ' ' I '''' ( '''' I' '-('"'I""(' '''I~J • n I U t l l l 1 l 14 1•

. -t- .... ,. , ... ,. 1 \ • "" 19'\\ t\t

l OOC'tJ') -l !~)) I • Oegrodate (b) , ,~,.,

l , ~ .. _,:;)

l i t<>>)

l ~t<->l -OC(>))

' t<no) -

' >)

:co»> '~-~

Control

1 ·"'-~t~~~"'.w.\~~\-\Ni/"".~"·' l-r.jt...,..,.\v.~rJ .,,.,_., ~"'4,•,,t¥N'·A~ \~, . 0

· It())) Blank

~;, "1,-JJI )• •{•'' I , 11 , • ' ' ' I I l • ' ' 1

,. , ·~I I :J ~~..,·~~· ~,.~·-r• ~· r1 ~'~""' "T' ,~•Tt--:--1 ~· ""'' ,,.,, lr-·r-r-r-r-..,...1~·-r-~·~·..,.,....·r

' l Q l l I : ~ ) li I'· 1 f

AT: 5.150_6.230 min Dl: m/Z 1441nl5510 AT: 500 ms ESI• DV: JOV

t OOO ~

sooo

1000

- -- (M+1)+ (c)

120 UO 110 150 160 110 180 1$0 200 210 220 2 l 0 H O 250 1'14~.3 ( n/ t.)

89

Figure 2. (a) LC- UV chromatograms of blank, control (standard molinate), and oxidation de gradate of molinate under chromatographic condition 2. (b) Total ion chromatograms of blank, control (standard molinate) and oxidation degradate of molinate under clu·omatographic condition 2. (c) Mass spectrum for oxidation degradate of molinate by free chlorine and ozone.

90

3.6e4 143.9

3.4e4

3.2e4

3.0e4 oy''-/' 0~/··t

FC, Ozone Oxidation 2.9e4 0 0 2.6e4

2.4e4 Molinate 1H·Azeplne-1-earboxy1 1e acid, hexahydro-

2.2e4 MW: 187 MW: 143

I ~ 2.0e4 u

i- 1.8e4 ~ ~ 1.6e4 £

1.4e4

1.2e4

1.0e4

9000.0 117.2

6000.0 96.2 126.2 102.8 116.

4000.0 94 99.2 129.0

2000.0 76.2

~0 76 eo 95 90 95 105 110 140 146 mlz. •mu

Figure 3. (a) Mass spectrum of the degradation product of molinate by using API 4000 triple quadrupole mass spectrometer. (b) Product ion scans of the degradation product using API 4000 triple quadrupole mass spectrometer.

Quantitative results of the oxidation of molinate using several oxidation systems.

The quantitative studies depicted in Table 1 show that the percentage of molinate removed

by various oxidants had changed significantly. The data demonstrated that molinate can

only be oxidized by free chlorine and ozone, while other oxidants do not show significant

removal (£ 7%). The reaction of molinate with free chlorine was quite fast; 10 mg L - t of

molinate were totally removed by 10 mg L - I of free chlorine within two hours. UV

91

radiation did not show any significant removal under a pH= 8.6 condition, with a dosage

range of 134-6030 mW•s cm-2.

Based on the data in Table l, ozone oxidation of molinate was pH dependent;

molinate was easier to be oxidised and removed in acidic pH rather than an alkaline

condition. The data also demonstrated that removal of molinate by ozone was not

significantly dosage dependent. It showed that the percentages of removals of molinate

at pH 8.6 for 4 hr were about the same as by using a low dose, a medium dose, and a high

dose of ozone.

Experimental results of a concentration-time study of the free chlorine oxidation

system. Due to the strong oxidation ability of free chlorine to molinate, a concentration

time experiment was necessary to determine the detailed degradation process of molinate

by free chlorine. Since the standard of the oxidation de gradate was not available, and

only one major degradate was observed in the LC-UV chromatogram and total ion

chromatogram in LC-MS, it was assumed that molinate was stoichiometrically oxidized

to only one oxidation degradate in 21u·s. The reason for this assumption was that

molinate was undetectable after 2 lu·s and the peak area of molinate de gradate remained

constant at about the same peak area as that of molinate. It was also found that the free

chlorine oxidation of molinate was fast and its oxidation degrade was more stable than

that of its parent compound. Therefore, after appropriate amounts of free chlorine were

spiked to the 2, 3, 4, and 5 mg L-1 of the molinate standard solution, and followed by a 2

hr reaction, a pseudo-calibration curve of molinate de gradate was made by measuring the

peak area of the molinate degradate. In this case, the concentration of the molinate

de gradate we measured served as the real concentration of the oxidation de gradate

92

because of its molecular stability. The sum of molarities of molinate and the oxidation

degradate in the experiment (as shown in Fig. 4) was >87% of the spiked concentration of

molinate in the 40 min oxidation experiment. It was found that at each time interval that

was studied, the sum of the measured molinate concentration and the calculated

concentration of the molinate degradate was almost the same. This result, combined with

the linear curve of the molinate degradate, indicated that our assumption was correct. The

trend line also demonstrated that molinate was quickly oxidized in the first 10 min, and

then reached an equilibrium state with the degradate, as significant drop of free chlorine

concentration.

Table 1. Quantitative results of molinate oxidation using different oxidants

Reaction Conditions

High ozone dosage (21.1 mg/L), pH6.6, 4 h

Medium ozone dosage (15.8 mg/L), pH6.6, 4 h

Low ozone dosage (10.5 mg/L), pH6.6, 4 h

High ozone dosage (21.1 mg/L), pH8.6, 4 h

Medium ozone dosage (15.8 mg/L), pH8.6, 4 h

Low ozone dosage (10.5 mg/L), pH8.6, 4 h

Free chlorine (10 mg/L), 2 h

MCA (10 mg/L), 4 h

Cl02 (10 mg/L), 4 h

Percentage molinate removal

41.9

32.6

33.8

17.3

18.8

19.7

100

7.0

6.7

93

~.b ~. b

3 3

E' /.n 7.. f'i __-<> I' / Q. ,..---- //

.9: // 2 c 2 0

0 // R' = 0.9029 u ·~ I // u .... 1.5 / 1.5 c 1: 'T Q)

// (,) slop•=O 06J5 min·• IS u

---- -- 0. [j

0 I I I I I I I I 0 0 5 10 15 20 25 30 35 40 45

Time/min ---.-concentration of ~t·t,; ~Concentration of ltolinat ~

-lr-Concentr•t ion of llolin.,.te des•·.,.d:ote _._Total of llolinate :>nd ito desr<>d:>te

:r Jn(C/CO)

Figure 4. Concentration-Time curve for FC oxidation of molinate.

ACKNOWLEDGMENTS

This work was funded by the American Water Works Association Research

Foundation (Project #3170: Pesticide Degradates in Water Treatment: Oxidative

Formation and Pa11itioning Parameter Estimation). The authors thank Alice Fulmer

(AwwaRF Project Officer), and the project advisory committee consisting of Dr. Wayne

Koffskey (Jefferson Parish Water Quality Laboratory, Louisiana), Dr. Michelle Hladik

(USGS, California), and Dr. Alistair Boxall (Central Science Laboratory, York, UK) for

their comments and suggestions. The opinions expressed in this work do not necessarily

reflect those of the sponsor.

94

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96

VITA

Chuan Wang was born on April30, 1984 in Pingdingshan, Henan Province,

People's Republic of China. In 2000, he joined Department of Biological Engineering,

Jimei University, Xiamen China. He received scholarships throughout four years of study

in Xi amen. He graduated with a Bachelor of Engineering in Food Science and

Technology from Jimei University in 2006.

In 2006, Chuanjoined Dr. Yinfa Ma's group at the Missouri University of

Science and Technology, Rolla, Missouri for his Ph. D. in Analytical Chemistry. During

his graduate study, he held a Graduate Research Assistantship and Graduate Teaching

Assistantship in the Chemistry Department. His research mainly focuses on GC, LC and

MS application on Emerging Contaminants including PPCPs, pesticides and nitrosamines.

His Ph. D. degree was awarded in May, 2011.

Investigation of occurrence, elimination and degradation of pharmaceutical and personal care

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