Analysis of steroid estrogens in water using liquid chromatography/tandem mass spectrometry with...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 1973–1983

) DOI: 10.1002/rcm.3050
Published online in Wiley InterScience (www.interscience.wiley.com
Analysis of steroid estrogens in water using liquid

chromatography/tandem mass spectrometry with

chemical derivatizations

Ying-Hsuan Lin1, Chia-Yang Chen1* and Gen-Shuh Wang1,2

1Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei 10055, Taiwan2Department of Public Health, College of Public Health, National Taiwan University, Taipei 10055, Taiwan

Received 28 February 2007; Revised 21 April 2007; Accepted 25 April 2007

*CorrespoHealth, CTaipei 10E-mail: dContract/tract/gra

Even in trace amounts, estrogens such as 17b-estradiol (E2), estrone (E1), estriol (E3), and 17a-ethinyl

estradiol (EE2) may have adverse effects on humans and the aquatic ecosystem. Therefore, it is

essential to be able to measure trace amounts of steroid estrogens in water. To date, most instruments

are not sensitive enough to detect these chemicals in small samples of water. Sensitivity, however,

may be improved by using appropriate derivatization reagents to modify the structures of these

estrogens so that their ionization efficiency is increased, making them more detectable by liquid

chromatography/mass spectrometry (LC/MS). This study uses dansyl chloride, 2-fluoro-1-methyl-

pyridinium p-toluenesulfonate (FMPTS), and pentafluorobenzyl bromide (PFBBr) as derivatization

reagents to react with the phenolic estrogens tomake themmore detectable inwater.We also test how

environmental matrices (wastewater effluent, river water, and drinking water) influence the detect-

ability of these estrogens. Both qualitative and semi-quantitative comparisons of these derivatization

methods were made. We found that dansyl chloride derivatives created signal intensities one or two

orders of magnitude greater than those normally found in underivatized estrogen standards. The

signals derived by FMPTS were analyte-dependent, and the products derived from E1, E2, and EE2

produced 2.19 to 12.1 times the signal intensity of underivatized E1, E2, and EE2. The product derived

from E3 produced weaker signals than that produced by underivatized E3. The PFBBr derivatives

produced signals that were as much as 5.8 times those found in the underivatized estrogens. When

these derivatizationmethodswere applied to river water, drinkingwater and effluents from a sewage

treatment plant (STP), the different matrices were found to significantly suppress the signals if we

used electrospray ionization, though this influence became less significant if we used atmospheric

pressure chemical ionization. This study suggests that PFBBr derivatization can best be used for the

detection of these estrogens in complex environmental matrices such as river water and STP effluents

and that the dansyl chloride derivatization is best used for clean samples such as drinking water.

Copyright # 2007 John Wiley & Sons, Ltd.

Over the last two decades concern has grown over the issue

of environmental pollution by endocrine disruptors. Some

feminizing compounds are thought to have many different

adverse effects, not only in humans where they suspected to

affect the production of sperm, but also in the environment

where, for example, sex ratios in fish can be reversed. Of

these feminizing compounds, steroid estrogens have been

found to have much greater estrogenic potency.1 Natural

steroid estrogens and synthetic steroid estrogens used in

medicine and veterinary medicine most often find their way

into the environment through human and animal urine.

Although both estrogens are mainly excreted in the form of

biologically inactive conjugates, these conjugates are likely to

ndence to: C.-Y. Chen, Institute of Environmentalollege of Public Health, National Taiwan University,055, [email protected] sponsor: National Science Council, Taiwan; con-nt number: NSC94-2314-B002-278.

be deconjugated and reverted to active free steroids by the

enzymatic reaction of microorganisms in sewage treatment

systems and some other natural environments.2

Trace concentrations of these chemicals in water have been

found to trigger adverse biological effects, particularly in an

aquatic environment.3,4 However, due to the limitations

associated with the analytical techniques used to detect these

estrogens, it is difficult to measure them and assess their

impact on the environment, particularly in complex environ-

mental matrices. Many biological assays and methods of

chemical analysis have been used to measure steroid estro-

gens in different types of water samples. However, the esti-

mates of biological assays, such as immunoassays, often are

confoundedby cross-interactions.1,5 Chemical analysis, includ-

ing gas chromatography/mass spectrometry (GC/MS) or


1974 Y.-H. Lin, C.-Y. Chen and G.-S. Wang

gas chromatography/tandem mass spectrometry (GC/MS/

MS),6–10 has its own set of limitations, particularly the need

for labor-intensive sample preparation and pretreatment.

Although liquid chromatography (LC)-based methods

provide another choice, they may not be sensitive enough

to measure estrogens in complex environmental matrices.11

Complex environmental matrices not only come with

increased background noise, but also may contain molecules

that could suppress ionization of analytes in the atmospheric

pressure ionization (API) source, especially when electro-

spray ionization (ESI) is used.12

Many factors can affect the generation of ions and affect the

intensity of signals in LC/API-MS. Recently, some research-

ers have modified structures into more ionizable derivatives

to improve the detection of poorly or non-ionizable com-

pounds.13 Because steroid estrogens all contain a phenolic

group (Fig. 1), this group can be used to connect

API-responsive moieties. Most derivatization techniques

for ESI couple a readily ionized group or a permanently

charged moiety with analytes; on the other hand, the

derivatization techniques for atmospheric pressure chemical

ionization (APCI) usually couple moieties that either have

high proton affinities or electron affinities with analytes.14

In this study, we experiment with three derivatization

reagents: dansyl chloride, 2-fluoro-1-methylpyridinium

p-toluenesulfonate (FMPTS), and pentafluorobenzyl bro-

mide (PFBBr). Dansyl chloride, because it has a secondary

amine moiety, is the derivatization reagent commonly used

to improve the detection of steroids with phenolic groups in

the ESI mode.15 FMPTS connects a permanently charged

pyridinium moiety to alcohols through nucleophilic substi-

tution.16 Both derivatization methods may render steroid

estrogens readily detectable in ESI-MS. Another reagent,

PFBBr, can react with the phenolic group of the steroid

estrogens and enhance their electron-capturing capability in

the APCI interface.17

When LC/API-MS is used in the quantitative analysis of

complex mixtures (e.g. environmental samples or biological

fluids), matrix effects must be evaluated. MS may respond

differently to analytes in a complex matrix than it does to

those in a standard solution. In LC/ESI-MS, co-eluting

Figure 1. Structures of the steroid estrogens.


matrix components can compete with the target analytes to

access the droplet surface for forming gas-phase ions in the

ionization interface and thus reduce the ionization efficiency

of target compounds.18 In addition to their ability to suppress

ionization, matrix components may also present added

background noise and deteriorate detection limits. Several

approaches have been used to evaluate matrix effects.19–22

Matuszewski et al. used post-extraction addition to deter-

mine ion suppression caused by co-eluting matrix.22 In that

study, they compared signal intensities found in neat

standard solutions with those of standards spiked into

biofluids after extraction. The degree of matrix effects under

chromatographic conditions can be assessed by using the

ratio of the responses of analytes in the samples spiked after

extraction to those of pure standards.22

The objectives of this study were to use three chemical

derivatization methods (dansyl chloride, FMPTS, and

PFBBr) with LC/API-MS/MS to qualitatively and semi-

quantitatively analyze steroid estrogens in drinking water,

river water and effluents from a sewage treatment plant

(STP). Signal intensity or limits of detection (LODs) were

found to be improved by derivatization methods used. By

lowering the detection limits in this way we may be able to

usemuch smaller samples of water than is presently required

to monitor these estrogens in clean waters and in more

complex matrices. By reducing the amount of water to be

analyzed, we may also be able to reduce matrix effects.

This study expands the applications of the three

derivatizations. Previously, dansyl chloride has been

used to measure steroid estrogens (17a-ethinyl estradiol

(EE2),15,23,24 17b-estradiol (E2) and estrone (E1)25) in plasma;

FMPTS has been applied to react with alcohols to form

N-methylpyridyl ether ions,16 which might make it suitable

for derivatizing phenolic estrogens; PFBBr derivatization of

E1 and E2 has only been used in neat standard solutions.17

EXPERIMENTAL

Chemicals and reagentsEstrone (E1), 17b-estradiol (E2), estriol (E3), and 17a-ethinyl

estradiol (EE2) were obtained from Sigma (St. Louis, MO,

USA; purity >98%). Dansyl chloride (5-(dimethylamino)

naphthalene-1-sulfonyl chloride, �95% in purity), FMPTS

(2-fluoro-1-methylpyridinium p-toluenesulfonate, technical

grade, �90%), pentafluorobenzyl bromide (PFBBr, purity

>99%), 4-methylmorpholine (purity >99.5%), triethylamine,

formaldehyde, sodium hydrogen carbonate, and potassium

hydroxide were purchased from Sigma-Aldrich (St. Louis,

MO, USA), and formic acid from J. T. Baker (Phillipsburg, NJ,

USA). Solvents, including methanol, acetone, heptane,

acetonitrile and dichloromethane, were all HPLC grade

and purchased from J.T. Baker.

Synthesis of estrogen derivatives

Derivatization with dansyl chlorideThe method of derivatization with dansyl chloride was

modified from Anari et al.15 and Nelson et al.25 A volume of

20mL of the estrogen mixture in acetone (2.5 ng/mL) was

vortexed with 50mL of sodium bicarbonate buffer (100mM,


DOI: 10.1002/rcm

Analysis of steroid estrogens in water by LC/MS/MS 1975

pH adjusted with NaOH (aq) to 10.5) for 1min. Then 50mL of

1mg/mL dansyl chloride solution was added, and the

mixture was vortexed for another 1min. It was then

incubated at 608C for 3min and then allowed to cool down

to room temperature. The solution was evaporated to

dryness using a SpeedVac concentrator (Thermo Savant

SPD 1010, Holbrook, NY, USA), and the residue was

reconstituted with 20mL of methanol. A volume of 4mL of

the reconstituted solution (representing 10 ng of the original

analyte) was injected onto the LC/MS/MS system for

analysis.

Derivatization with FMPTSThe derivatization with FMPTS was adopted from

Mukaiyama et al.26 and Bald.27 The estrogenmixture solution

in acetonitrile (20mL; 2.5 ng/mL) was vortexed with 50mL of

0.1M triethylamine in acetonitrile for 1min. Then, 50mL of

0.1M FMPTS solution in acetonitrile was added and the

mixture was vortexed for another 1min. It was then shaken

in a reciprocal water bath (RB-120; Cherng Huei, Taiwan) at

room temperature for 1 h (110 rpm). The mixed solution was

evaporated to dryness using the SpeedVac concentrator, and

the residue was redissolved with 20mL of 50:50 (v/v)

methanol/water solutions. A volume of 4mL of the

reconstituted solution was injected onto the LC/MS/MS

system for analysis.

Derivatization with PFBBrThe derivatization with PFBBr was performed according to a

method used by Singh et al.17 The estrogen mixture in

acetonitrile (50mL; 1.0 ng/mL) was vortexed with 50mL of

potassium hydroxide in anhydrous ethanol (8:1000, w/v)

for 1min. Then, 50mL of PFBBr in acetonitrile (1:19, v/v)

was added. It was vortexed for another 1min. The solution

was incubated at 608C for 30min, and then evaporated to

dryness using the SpeedVac concentrator. The residue was

reconstituted with 50mL of methanol. A volume of 10mL of

the reconstituted solution (representing 10 ng of the analyte)

was injected onto the LC/MS/MS system for analysis.

ChromatographyA Waters 616 LC system equipped with an ECO-1 column

oven (Analab, Taipei, Taiwan) and a DU2001 on-line

degasser (Sanwa Tsusho, Tokyo, Japan) was used for liquid

chromatography. Five chromatographic conditions were

optimized for different estrogen derivatives and under-

ivatized parent compounds. A total of 10 ng of each analyte,

both derivatized and underivatized, was injected onto the LC

system for both qualitative and quantitative analysis.

Steroid estrogens without derivatization in ESI(�)Underivatized steroid estrogens in negative ESI mode were

separated using a BetaBasic C18 column (150� 2.1mm, 3mm;

Hypersil-Keystone, Bellefonte, PA, USA). Column tempera-

ture was maintained at 408C. We used a binary gradient

consisting of 10mM 4-methylmorpholine (aq) (pH 9.5–9.6)

(A) and 100% acetonitrile (B) at a flow rate of 0.2mL/min.

Gradient was increased from 10% to 50% of solvent B in

2min, from 50% to 100% in another 11.5min, and decreased


back to its initial composition in 2.5min. The system was

re-equilibrated for 6min before the next injection.

Steroid estrogens without derivatization in APCI(�)Underivatized estrogens in negative APCI mode were

separated using a BetaBasic C18 column (150� 4.6mm,

3mm; Hypersil-Keystone). Column temperature was main-

tained at 408C. Water (A) and methanol (B) were used to

perform a gradient elution at a flow rate of 1.0mL/min.

Gradient was increased from 10% to 50% of solvent B in

3min, from 50% to 70% in another 12min, and decreased

back to its initial composition in 3min. The system was

re-equilibrated for 4min before the next injection.

Dansyl-estrogens in ESI(þ)Dansyl chloride derivatives were separated using a BetaBasic

C18 column (150� 2.1mm, 3mm; Hypersil-Keystone). Col-

umn temperature was maintained at 408C. Ten mM formic

acid (pH 2.9–3.0) (A) and 100% acetonitrile (B) were used as

the mobile phase at a flow rate of 0.2mL/min. Gradient was

increased from 50% to 80% of solvent B in 2min, from 80% to

100% in another 11.5min, and decreased back to its initial

composition in 2.5min. The system was re-equilibrated for

6min before the next injection.

FMP-estrogens in ESI(þ)FMPTS derivatives were separated using a polystyrene-

divinylbenzene (SDB) PRP-1 column (150� 2.1mm, 3mm;

Hamilton, Reno, Nevada, USA). Column temperature was

maintained at 408C. The mobile phase contained 10mM

formic acid (pH 2.9–3.0) (A) and 100% acetonitrile (B) at a

flow rate of 0.2mL/min. Gradient was increased from 5% to

40% of solvent B in 3min, from 40% to 95% in another 12min,

held at 95% for 2min, and then decreased back to its initial

composition in 2min. The system was re-equilibrated for

3min before the next injection.

PFB estrogens in APCI(þ)PFBBr derivatives were separated using a BetaBasic C18

column (150� 4.6mm, 3mm; Hypersil-Keystone). Column

temperature was maintained at 408C. Water (A) and 100%

methanol (B) was used as the mobile phase at a flow rate of

1.0mL/min. Gradient wasmaintained at 60% of solvent B for

2min, increased from 60% to 100% in 10min, held at 100% for

another 1min and then decreased back to its initial

composition in 2min. The system was re-equilibrated for

3 minu before the next injection.

Mass spectrometryWe used a Finnigan TSQ 7000 triple-quadrupole mass

spectrometer (Finnigan MAT, San Jose, CA, USA) controlled

by the XcaliburTM Home Page version 1.1, which we

operated in the selected reaction monitoring (SRM) mode.

Collision-induced dissociation (CID) was performed using

argon as the collision gas at a pressure between 2.9 to

3.1mTorr. The operation parameters are summarized in

detail in Table 1.


DOI: 10.1002/rcm

Table 1. Instrumental parameters of the mass spectrometer

Ionsource

ESI sprayvoltage

Corona dischargecurrent

Vaporizertemperature

Heatedcapillary

Sheathgas

Auxiliarygasa

Underivatized estrogens ESI 4.5 kV — — 2508C 70 psi 20Dansyl-estrogens ESI — —FMP-estrogens ESI — —Underivatized estrogens APCI — 5mA 5008C 2008C 80 psi offPFB-estrogens APCI — 10mA 5008C 2008C 40 psi 10

aAuxiliary gas (nitrogen) is presented in arbitrary units.

Figure 2. Sample preparation procedures for water samples.


Water sampling and analysisThree types of water samples (river water, STP effluents and

drinking water) were analyzed to evaluate the feasibility of

these derivatization methods. The river water was sampled

from the Danshui River, one of three major rivers in Taiwan.

That river flows through the Taipei metropolitan area,

where it was sampled at Taipei Bridge (E 121830.433 and

N 25803.829). Effluents were taken from Neihu STP, where

they had gone through a secondary treatment process using

the activated sludge units. The drinking water we used was

Taipei tap water. All samples were collected in March 2006.

Samples were first collected in a 20-L pre-cleaned tank at the

sampling site, mixed well, and then subsampled into amber

glass jars with Teflon-liner caps in the laboratory. Formal-

dehyde (1%, v/v) was added to prevent biological degra-

dation and samples were stored at 48C until analysis.

The sample extraction procedures were adopted from the

work of Chen et al.28 and are illustrated in Fig. 2. Before

extraction, 2-L samples were filtered using 90-mm PVDF

(polyvinylidene fluoride) membranes with a pore size of

0.45mm to remove suspended solids. Soluble matrices were

extracted with 50-mm Bakerbond PolarPlus C18 Speedisks

(J.T. Baker). After extraction, disks were washed with 40%

MeOH/60% H2O (v/v). The disks were moved to a vacuum

manifold (J.T. Baker) and dried for 10min under a vacuum of

around �25 kPa before being eluted with three portions of

5mL 50% methanol/50% dichloromethane. The collected

eluates were filtered through 25mm PTFE syringe filters

(pore size 0.2mm) to remove particulates and then divided

into five aliquots. Eluates were concentrated to dryness at

458C in a 1.8-mL vial by the SpeedVac concentrator and were

reconstituted by appropriate solvents, with or without

spiking of 50 ng steroid estrogens, and reacted with the

derivatization reagents.

Evaluation of method performanceAbsolute peak heights (as signal intensity) and signal-

to-noise (S/N) ratios (as relative sensitivities) were used to

evaluate the performance of the three derivatization methods.

Steroid estrogens in three types of environmental mat-

rices were detected using appropriate LC and ionization

methods. Deionized/distilled water from a Milli-Q unit

(Millipore, Bedford, MA, USA) was used as the matrix-free

blank for comparison. Eluates from extraction disks were

equally divided into five aliquots to ensure each aliquot

contained the same amount of endogenous estrogens and

matrix residues. We evaluated the matrix effect on each

method by analyzing estrogens in the drinking water, river

water and STP effluents. A matrix effect factor (MEF) was

defined as the quotient of averaged S/N value between


environmental matrices and the Milli-Q (MQ) water

(i.e. MEF¼B/A, A: averaged S/N value of analytes in

MQ water, B: averaged S/N value of analytes in drinking

water, river water or STP effluents) expressed by a

percentage, while MQ water was used as a matrix-free

reference. Low MEF values indicated serious matrix effects.


DOI: 10.1002/rcm


Each analysis was conducted with four duplicates and

consisted of two sets. In the first set, 50 ng of each steroid

estrogen was spiked into the concentrated extracts of water

samples to simulate derivatizations of real samples. The

other set, which was not spiked, was taken as environmental

backgrounds containing endogenous analytes and matrices.

The environmental backgrounds were deducted from the

responses of the first set before the comparisons. Reagent

blanks were included in each batch for analysis. No

experimental contamination was observed.

Data acquisition and analysisMass spectrawere obtainedusingXcaliburHomePage version

1.1 software (Finnigan MAT). Further data processing was

completed with Microsoft Excel 2003. Matrix effects among

various waters, signal intensity and sensitivity enhancement

between different analytical methods were compared statisti-

cally with one-way analysis of variance (ANOVA) followed by

Tukey’s test (p< 0.05) using SPSS for Windows 11.5.0.

Figure 3. Scheme of selected derivatization reactions: (a) d

(c) PFBBr derivatization.


RESULTS AND DISCUSSION

Qualitative analysis of derivatized productsDansyl chloride, FMPTS, and PFBBr react with the phenolic

group of steroid estrogens, resulting in the formation of

ethers (Fig. 3). The amount of reagents in excess of those

theoretically needed to react with analyte standard compen-

sated for matrix components and ensured the completion of

reactions. The derivatives of estrogen standards were

injected into the mass spectrometer by direct infusion to

optimize the sensitivity of the instrument. Following The

Commission of the European Communities guidelines

suggesting four identification points to improve data

reliability, we monitored one precursor and two product

ions for each analyte.29 The most intense product ion was

used for quantification, and the second most abundant

product ion was used to confirm the analyte identity. The

liquid chromatograms of the five analytical methods are

shown in Fig. 4.

ansyl chloride derivatization, (b) FMPTS derivatization


DOI: 10.1002/rcm

Figure

4.Liquid

chromatogramsoffive

analyticalsystems:(a)native

estrogensunderESI(�);(b)dansyl-estrogensunderESI(þ);(c)FMP-estrogensunderESI(þ);

(d)native

estrogensunderAPCI(�);and(e)PFB-estrogensunderAPCI(�);10ngofeachanalyte

wasinjected.

Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 1973–1

DOI: 10.1002/


983

rcm


Analysis of dansyl-estrogen derivatives

Derivatization processAlthough the derivatization efficiencies were not determined

in this study because authentic derivative standardswere not

available, previous reports30,31 have shown them to be close

to 90%, and the variations in yield could be corrected if stable

isotope labeled internal standards were used. The overall

reaction was carried out in acetone; dried residues were

reconstituted with methanol. Practically, the excess reagents

could not be removed after derivatization, so the recon-

stituted solution was used to extract the derivatives from the

reaction mixture. Although derivatized products reconsti-

tuted with the initial mobile phase, 50% ACN/

50% H2O, produced higher analyte signals, the precipitation

of the redissolved bicarbonate salts during the gradient

elution blocked LC tubing. However, when CH3OH was

used for reconstitution, most of the excess reagent, especially

the inorganic slats, was left over.

Product ion characterizationThe four dansyl-estrogens had the same product ions and

similar optimized collision energies. [Mþ233.8]þ was found

to be the most suitable precursor ion, and yielded an intense

product ion m/z 171þ. Another product ion, m/z 156þ,

supposedly resulting from loss of one methyl group from the

product ion ofm/z 171þ, was selected as the confirmatory ion.

The best collision energy was determined to be 42 eV for

dansyl-E1, 40 eV for dansyl-E2, 44 eV for dansyl-E3 and 43 eV

for dansyl-EE2 (Table 2).

LC/MS/MS conditionsAcidic additives of 10mM formic acid (pH 2.9–3.0) were used

as the aqueous mobile phase to enhance the ionization of

dansyl derivatives.15 The signal intensity of dansyl deriva-

tives in MQ water was significantly higher (38–101 times)

than that produced by underivatized estrogens in ESI(–)

(Table 3).

Table 2. SRM transition of native and derivatized estrogens

Derivatization reagent Target compound Precursor ion (m/z)

— E1 269.4�

E2 271.4�

E3 287.4�

EE2 295.4�

Dansyl chloride Dansyl-E1 504.0þ

Dansyl-E2 506.0þ

Dansyl-E3 522.0þ

Dansyl-EE2 530.0þ

FMPTS FMP-E1 362.0þ

FMP-E2 364.0þ

FMP-E3 380.0þ

FMP-EE2 388.0þ

PFBBr PFB-E1 269.4�

PFB-E2 271.4�

PFB-E3 287.4�

PFB-EE2 295.4�


Analysis of FMP-estrogen derivatives

Derivatization processWe tried to carry out FMPTS derivatization in dichloro-

methane according to the reported literature26 but the

reaction failed. We observed that FMPTS remained sus-

pended in dichloromethane. Replacing dichloromethane

with acetonitrile completely dissolved both the analytes

and the reagent. For the derivatization, it is critical to ensure

that the reagent, which is hygroscopic,32 stays dry and that a

fresh solution is always used. Besides, the best commercial

grade of this reagent is only 90% pure, and there may be a

source of impurities from itself in derivatization. An intense

signal at m/z 375þ from unknown origins was observed even

in the reagent blank by full-scan mass spectra of derivatives

of FMPTS. Fortunately, this ion did not interfere with our

experiments because it could be filtered out in selected

reaction monitoring (SRM) mode.

Product ion characterizationFMPTS reacted with steroid estrogens and formed ether ions

ofm/z [Mþ92]þ, an ion pattern also reported byQuirke et al.16

The product ion of m/z 128þ was used to quantify FMP-E2,

FMP-E3, and FMP-EE2. Another product ion ofm/z 110þwas

used for confirmation. The quantitative and confirmatory

ions of FMP-E1 were m/z 252þ and m/z 238þ, respectively

(Table 2). When the method was applied to real water

samples, product ions of m/z 110 resulting from the

derivatization reagent seemed susceptible to matrix effects;

higher noise levels and co-eluting impurities were observed

when m/z 110 was monitored. On the other hand, though

another product ion of m/z 128 had a lower intensity,

the lower noise levels led to a higher S/N ratio than that of

m/z 110. FMP-E2, FMP-E3 and FMP-EE2 all showed similar

results.

LC/MS/MS conditionsWe used a SDB polymer column and 10mM formic acid as

the aqueous mobile phase. Although adding 10mM formic

Product ion (m/z)

Collision energy (eV)Quantification Confirmation

143� 145� 65183� 145� 46145� 171� 50145� 159� 40171þ 156þ 42171þ 156þ 40171þ 156þ 44171þ 156þ 43252þ 238þ 44128þ 110þ 52128þ 110þ 50128þ 110þ 50143� 145� 65183� 145� 46145� 171� 50145� 159� 40


DOI: 10.1002/rcm

Table 3. Signal intensities of different estrogen derivatives (mean�SD, n¼ 4)

Native estrogensin ESI(�) Dansyl-estrogens FMP-estrogens

Native estrogensin APCI(�) PFB-estrogens

MQ waterE1 1061� 181a,b 41150� 11458b,c,d,e 12850� 1201a,c 2990� 281a 6165� 802a

E2 1313� 330a 41650� 8458b,c,d,e 2873� 376a 1475� 121a 4833� 884a

E3 1030� 134a 66050� 19725b,c,d,e 244� 55a 1255� 66a 3515� 402a

EE2 467� 124a 47150� 12445b,c,d,e 1800� 110a 479� 8.1a 2623� 361a

Drinking waterE1 946� 125a 36250� 14769b,c,d,e 12250� 1518a 2568� 204a 8793� 498a

E2 988� 392a 42225� 21855b,c,d,e 2730� 340a 1348� 111a 7065� 559a

E3 671� 224a 52750� 17577b,c,d,e 248� 47a 1280� 167a 4923� 484a

EE2 412� 56a 45700� 20703b,c,d,e 1710� 236a 505� 63a 3933� 297a

River waterE1 162� 60a 18167� 4881b,c,d,e 1755� 441a 2590� 1139a 3080� 370a

E2 11.5� 1.2a 18475� 6236b,c,d,e 355� 114a 1397� 671a 2087� 544a

E3 25.3� 1.5a 29775� 4145b,c,d,e 398� 194a 2343� 373a 2863� 322a

EE2 65.5� 8.3a 22650� 8118b,c,d,e 196� 55a 609� 194a 1303� 115a

STP effluentsE1 518� 46a,b 30425� 2069b,c,d,e 11300� 1100a,c,d 3943� 937a,b 4820� 260a

E2 299� 75a,e 22750� 1690b,c,d,e 1487� 55a 1386� 361a 2288� 302a,c

E3 34.6� 9.3a 27300� 2302b,c,d,e 260� 94a 1563� 192a 2125� 127a

EE2 80.3� 12.9a 27650� 2042b,c,d,e 766� 339a 575� 149a 1428� 276a

Note. Signal intensity is defined as absolute peak height from the detector; 10 ng equivalent of each parent chemical was injected theoretically.a Statistically different from dansyl-estrogens.b, Statistically different from FMP-estrogens.c, Statistically different from native estrogens in ESI(�).d, Statistically different from native estrogens in APCI(�).e Statistically different from PFB-estrogens.


acid slightly suppressed analyte signal compared with the

use of pure deionized water, it produced better peak shapes

and reproducible retention times. We found that silica-based

columns were not able to retain cationic FMP-derivatives

well, which is consistent with previous reports.32–34 The

solution used for reconstitution also influenced signal

intensity. Residues of pyridinium salts redissolved more

readily in a 50:50 (v/v) CH3OH/H2O solution and produced

about twice the signal intensity of those redissolved with

pure methanol.

Analysis of PFB-estrogen derivatives

Derivatization processThe reaction of PFBBr derivatization is well described by

Singh et al.,17 and we used it without modification.

Product ion characterizationSingh et al.17 reported that PFB-estrogen derivatives of E1

and E2 could undergo electron-capture dissociation and

generate negative ions through the loss of a PFB radical. We

observed similar results in not only PFB-E1 and PFB-E2 but

also in PFB-E3 and PFB-EE2, and the produced negative

precursor ions were identical to those of underivatized ones.

Therefore, the product ions and collision energies for the

derivatized estrogens were identical to the underivatized

ones (Table 2). The position of the corona discharge needle

and its current were critical in the electron-capture

process;17,35 the intensity increased as the current increased.

However, the maximum discharge current of the mass

spectrometer (TSQ 7000) was 10mA, and the corona probe

position fixed. These two parameters could be further

optimized for other types of mass spectrometers if they


provid higher corona discharge currents and needle

positions can be adjusted.

LC/MS/MS conditionsChromatographic separation of PFB derivatives carried out

with a column of 4.6mm i.d. at a flow rate of 1.0mL/min

provided 10-fold higher signal intensity than that with a

2.1mm i.d. column at a flow rate of 0.2mL/min. In addition,

derivatized PFB-estrogens became more non-polar than the

parent chemicals, and required higher organic portions in

the mobile phase (60–100% methanol) to elute them out,

while native estrogens were eluted at lower organic fractions

(50–70% methanol).

Evaluation of method performance

Comparison of absolute signal intensities observedby different analytical methodsSignal intensity was defined as the absolute peak height of

analytes on the MS detector. We compared these intensities

under five analytical conditions: (1) dansyl-estrogens in

ESI(þ), (2) FMP-estrogens in ESI(þ), (3) PFB-estrogens in

APCI(�), (4) underivatized estrogens detected under ESI(�),

and (5) underivatized estrogens detected under APCI(�). In

previous studies, ESI(�) has been reported to be the most

common method for analyzing steroid estrogens in water.

Thus, the signal intensity of native estrogens in ESI(�) was

designated as a reference to evaluate the performance among

different methods (Table 3).

In simple matrices such as MQ water and drinking water,

dansyl-estrogens produced the highest signal intensity of all

five methods, reaching as high as 101 times the reference


DOI: 10.1002/rcm


values in MQ water and 111 times the reference values

in drinking water. FMP-estrogens produced better signals

for FMP-E1, FMP-E2 and FMP-EE2, while FMP-E3 did not.

E3 possesses three hydroxyl groups, and FMPTS may not

react specifically with the phenolic one. The byproducts

might not produce identical fragment ions and, therefore, we

might not have detected them. Underivatized E2, E3 and EE2

detected under APCI(�) showed similar intensities to that of

ESI(�), while E1 had 3 times the intensity. PFB-estrogen

signals were enhanced by 3 to 9 times that of the under-

ivatized analytes using ESI(�).

In complex matrices such as river water and STP effluents,

dansyl-estrogen signals were increased significantly up to

2 or 3 orders of magnitude greater than those of under-

ivatized estrogens using ESI(�). Signal intensity of native

estrogen using APCI(�) and PFB derivatization increased by

1 to 2 orders of magnitude compared with the reference

value. The signal intensity of FMP-estrogens ranged from

3–31 times the reference value.

Relative sensitivity between different methodsDifferent from absolute responses, the noise levels were

considered when comparing relative sensitivities. Within the

samematrix, S/N values of underivatized estrogens detected

under ESI(�) were taken as the reference to compare the

relative sensitivities of different analytical methods (Table 4).

In simple matrices such as MQ water and drinking water,

dansyl-estrogens and PFB-estrogens produced significantly

higher sensitivities than the references. In addition, dansyl-

estrogens provided much better sensitivities than PFB-

estrogens. In MQ water, sensitivities of dansyl-estrogens

Table 4. Comparison of sensitivity in different estrogen derivative

Native estrogensin ESI(�) Dansyl-estrogens

MQ waterE1 199� 24a,b 2269� 506b,c,d,e

E2 257� 56a,b 1963� 404b,c,d,e

E3 281� 21a,b 1780� 393b,c,d,e

EE2 188� 26a,b 1068� 190b,c,d,e

Drinking waterE1 156� 23a,b 1145� 70b,c,d,e

E2 193� 42a,b 1436� 238b,c,d,e

E3 173� 46a,b 1133� 198c,d,e

EE2 149� 27a,b 1423� 380b,c,d,e

River waterE1 43.6� 14.6b,e 144� 47b,e

E2 ND 166� 32E3 5.42� 0.36a,b,e 272� 33c,d,e

EE2 ND 158� 39b,d,e

STP effluentsE1 80.6� 11.0b 171� 14b

E2 31.0� 7.5a,b,e 87.7� 14.7b,c,d

E3 5.23� 1.58a,b,e 104� 5.3b,c,d,e

EE2 8.47� 1.17a,b,e 59.5� 5.7b,c

Note. Sensitivity is defined as the signal-to-noise (S/N) ratio; 10 ng equivbND: not detectable (S/N ratio <3).a, Statistically different from dansyl-estrogens.b, Statistically different from PFB-estrogens.c, Statistically different from native estrogens in ESI(�).d, Statistically different from FMP-estrogens.e Statistically different from native estrogens in APCI(�).


were 5.7–11.4 times higher than the reference values,

whereas those of PFB-estrogens were 3.3–5.2 times higher.

APCI(�) produced slightly higher intensities (1.1–3.1 times)

than ESI(�), though the difference was not significant.

Except for E3 (only one-tenth of the reference value), the

performance of FMP-estrogens was similar to that of the

references. In drinking water, dansyl, FMP, underivatized in

APCI(�) and PFB increased S/N by 7.3–9.6, 0.1–2.2, 1.8–3.3

and 4.0–5.6 times, respectively, compared to underivatized

estrogens in ESI(�).

Significant enhancement of sensitivity was observed in

dansyl-estrogens, PFB-estrogens and underivatized estro-

gens under APCI(�) compared with the reference levels

measured when the five methods were applied to the

complex matrices. Different from that observed in cleaner

matrices, in most cases the sensitivities of PFB-estrogens in

river water and STP effluents were better than those of

dansyl-estrogens. Because native E2 and EE2 were not

detectable (S/N<3) in river water under ESI(�), we assigned

them a S/N value of 1.5 (half of the detection limit) to

facilitate method comparisons. In river water, compared to

the reference values, the enhanced sensitivities of

PFB-estrogens were 11.5 times for PFB-E1, 202 times for

PFB-E2, 49.2 times for PFB-E3 and 264 times for PFB-EE2. The

performances of dansyl-estrogens were 3.3 times greater for

dansyl-E1, 110 times for dansyl-E2, 50.1 times for dansyl-E3

and 105 times for dansyl-EE2. The relative sensitivities of

FMP-estrogens were 1.4–11.8 times higher than reference

values. Under APCI(�), the sensitivity of underivatized

estrogens increased 13.2 times for E1, 139 times for E2, 32.8

for E3 and 33.7 times for EE2. In STP effluents, the sensitivity

of PFB-estrogens was increased 9.59–31.9 times and that of

s (S/N, mean�SD, n¼ 4)

FMP-estrogensNative estrogens

in APCI(�) PFB-estrogens

354� 61a,b 615� 79a 1030� 206a,c,d

259� 41a,b 489� 23a 890� 171a,c,d

29.5� 6.4a,b 306� 20a,b 918� 95a,c,d,e

222� 24a,b 266� 19a,b 642� 108a,c,d,e

335� 58a 515� 75a 881� 146a,c

161� 12a,b,e 444� 79a,b,d 774� 66a,c,d,e

23.8� 4.0a,b 316� 23a,b 860� 277c,d,e

162� 42a,b 262� 70a 627� 125a,c,d

60.2� 6.7b,e 578� 70a,c,d 501� 179a,c,d

16.9� 3.6b 208� 138 303� 138d

31.3� 10.9a,b,e 178� 24a,b,c,d 267� 78c,d,e

17.6� 5.2a,b 50.6� 16.9a,b 395� 8.9a,d,e

114� 14b 569� 70 976� 523a,c,d

33.0� 4.8a,b,e 116� 6.2b,c,d 298� 44a,c,d,e

16.5� 2.5a,b,e 72.6� 3.6a,b,c,d 129� 10a,c,d,e

33.6� 7.2b 50.6� 16.9b,c 270� 34a,c,d,e

alent of each parent chemical was injected theoretically.


DOI: 10.1002/rcm


dansyl-estrogens 2.13–19.9 times. The increases in sensitivity

of FMP-estrogens and underivatized estrogens under

APCI(�) ranged from 1.06–3.97 times and 3.73–13.9 times

the reference values, respectively.

Matrix effects in analytical methodsEstrogens detected under ESI(�) were all susceptible to

matrix effects. The signals of underivatized analytes were

seriously suppressed in river water and STP effluents. In

river water, the MEFs were below 1% except for E1 (19.4%).

In STP effluents, the MEFs were below 1% for E3, 3.2% for

EE2, 11.6% for E2 and 28.6% for E1. MEFs of analytes in

drinking water ranged from 61.3–79.1% (Table 5).

Although dansyl derivatization gave good signal intensity,

the MEFs were similar to those of underivatized ones. For

dansyl-estrogens, MEFs ranged from 5.85–14.2% in river

water, under 1–5.50% in STP effluents and 50.4–72.9% in

drinking water (except for 30% of signal enhancement in

EE2), respectively. Thematrix effects on FMP-estrogens were

also severe. In river water, MEFs of FMP-estrogens ranged

from 6.46–13.8%, with the exception of FMP-E3, which had

an MEF of 83.6%. In STP effluents, MEF was higher (42.4%)

for FMP-E3, while for the other three analytes the MEFs

ranged between 11.2–17.1%. The reason that the MEFs of

FMP-E3 were higher than those of other three FMP-

derivatives might be ascribed to its much lower S/N ratio

in MQ water, which was the denominator to calculate the

MEFs (Table 4). The MEFs of FMP-E3 in drinking water

ranged from 61.7–94.9% (Table 5).

Table 5. Matrix effect factors (MEFs, %) of the analytical

methods

MQwater

Drinkingwater

Riverwater

STPeffluents

Native ESI(�)E1 100% 78.2% 19.4% 28.6%E2 100% 74.8% 0.59% 11.6%E3 100% 61.3% 0.54% 0.54%EE2 100% 79.1% 0.80% 3.20%

Dansyl chlorideE1 100% 50.4% 5.85% 0.86%E2 100% 72.9% 8.00% 3.96%E3 100% 63.7% 11.5% 5.50%EE2 100% 133% 14.2% 5.27%

FMPTSE1 100% 94.9% 13.8% 17.1%E2 100% 61.7% 5.29% 11.2%E3 100% 84.1% 83.6% 42.4%EE2 100% 72.5% 6.46% 13.7%

Native APCI(�)E1 100% 82.9% 84.7% 61.3%E2 100% 90.9% 41.0% 22.9%E3 100% 103% 50.1% 23.3%EE2 100% 98.6% 16.4% 18.1%

PFBBrE1 100% 86.0% 45.3% 52.9%E2 100% 87.4% 33.5% 32.1%E3 100% 94.1% 25.9% 13.7%EE2 100% 98.2% 61.5% 41.2%

Note. Matrix effect factors (MEFs) are defined as the quotient of theS/N value between environmental matrices and MQ water; n¼ 4;10 ng equivalent of each parent chemical was injected theoretically.


Analytes detected using the APCI interface suffered less

ion suppression than when using the ESI source. Under-

ivatized estrogens produced MEFs ranging from 16.4–84.7%

in river water, 18.1–61.3% in STP effluents and 82.9–103% in

drinking water. E1 was less susceptible to matrix effects than

the other estrogens; its ion suppression was relatively minor

in river water and STP effluents. PFB-estrogens were also less

sensitive tomatrix components. TheMEFs of analytes ranged

from 25.9–61.5% in river water and from 13.7–52.9% in STP

effluents. Higher MEFs were observed in drinking water,

where they ranged between 86.0–98.2% (Table 5).

Feasibility of derivatization methods to realwater samplesTo evaluate the feasibility of derivatization methods to real

water samples, limits of detection (LODs) (based on S/N¼ 3)

were estimated under the assumption that the signal

responses were linear up to the concentrations we used.

Amounts of 50 ng of steroid estrogen standards were spiked

in real water samples (from 2-L samples that were equally

divided into five aliquots for testing five analytical methods).

Each 400-mL sample was equal to a concentration of 125 ng/

L of each analyte. The estimated LODs of PFB-estrogenswere

0.38–2.9 ng/L in STP effluents, 0.75–1.4 ng/L in river water

and 0.43–0.60 ng/L in drinking water. These detection limits

are low enough that they may be used to measure typical

concentrations of estrogens in environmental waters. The

estimated LODs of dansyl-estrogens were 2.2–6.3 ng/L in

STP effluents, 1.4–2.6 ng/L in river water and 0.26–0.33 ng/L

in drinking water. Without complex matrices, dansyl

derivatization may provide low LODs, which is useful for

monitoring human exposure to estrogens in drinking water.

The estimated LODs of FMP-estrogens were 3.3–23 ng/L in

STP effluents, 6.2–22 ng/L in river water and 1.1–16 ng/L in

drinking water. Reactivity of FMPTS to hydroxyl groups

other than those specific to phenolic ones would be the

primary cause of higher LODs of steroid estrogens. Chemical

derivatization can reduce the required sample volume,

which will not only facilitate the extraction of phenolic

chemicals, but also mitigate the matrix effect.

CONCLUSIONS

This study demonstrates that derivatization with dansyl

chloride and PFBBr improves signal intensity in mass

spectrometry and can be used to access exposure to estrogens

in environmental water samples. While dansyl chloride

derivatization produced more intense signals in a simple

matrix such as drinking water, significant ion suppression

occurred when it was used to measure estrogens in river

water and STP effluents. APCI-based PFBBr derivatization

would be more suitable in the detection of estrogens in

complex matrices because they are less affected by their

complexity. Although FMP-derivatives can be used to

enhance ionization in the ESI source, the reagent did not

exclusively react with the phenolic group,whichmight lower

derivatization yields or possibly increase background noises

in complex environmental matrices.

PFB-estrogens possess both qualitative and quantitative

benefits in the detection of estrogens because their product


DOI: 10.1002/rcm


ions originate from the fragmentation of parent steroid

estrogens. They were also found to be more sensitive than

native estrogens. Although dansyl chloride derivatization

significantly enhanced signal intensity, the product ions of

dansyl-estrogens originate from the bonded derivatization

reagents, making it possible that false positive results would

occur when there are phenols with identical molecular

weights to target steroid estrogens, since all dansyl-

derivatized precursors will produce intense product ions of

m/z 171þ. Therefore, in this case, better sample preparation to

remove interferences or optimized LC conditions to avoid

co-eluting components will be needed. Based on our results,

PFBBr derivatization under APCI(�) with methanol and

water as its mobile phase composition can best be used to

detect phenolic chemicals in complex water samples, such as

river water and STP effluents. Dansyl chloride derivatization

under ESI(þ) with 10mM formic acid and acetonitrile as its

mobile phase can best be used to detect them in simple

matrices, such as MQwater and drinking water. However, if

dansyl chloride is used in complex matrices, then the sample

would need more cleanup to mitigate the matrix effect.

This study describes in detail the derivatization steps,

matrix effect evaluations, chromatographic and mass

spectrometric parameters which can be applied to future

research involving the detection of phenolic chemicals.

Further research should focus on validating this method of

quantifying steroid estrogens and other phenolic pollutants

in different waters.

AcknowledgementsThe authors are grateful for kind comments from Drs. Guor-

Rong Her and Pao-Chi Liao. The LC/MS/MS system

was provided by the College of Medicine, National Taiwan

University. This work is supported by the National Science

Council, Taiwan (NSC94-2314-B002-278).

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DOI: 10.1002/rcm

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