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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations
Guang-Wen Lien, Chia-Yang Chen *, Gen-Shuh Wang
I n st i tu te o f E nv i ro n me nt al H eal th , C o ll eg e of Pu b l i c H ea l th , Na ti o na l T ai wa n U n iv er si t y, Tai p ei 10 05 5 , Ta i wan

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abstract
This study compared the sensitivities and matrix effects of four ionization modes and four reversedphase liquid chromatographic (LC) systems on analyzing estrone (E1), 17 -estradiol (E2), estriol (E3), 17 -ethinylestradiol (EE2), 4-nonylphenol (NP), 4-tert-octylphenol (OP), bisphenol A (BPA) and their derivatives of dansyl chloride or penta uorobenzyl bromide (PFBBr) in water matrixes using a triple-quadrupole mass spectrometer with selected reaction monitoring (SRM). The four probes were electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and APCI/APPI; the four LC systems were ultra-performance liquid chromatography (UPLC) with or without post-column split, a mixed-mode column and two-dimensional LC (2D-LC). Dansylated compounds with ESI at UPLC condition had the most intense signals and less matrix effects of the various combinations of ionization and LC systems. The on-column limits of detection (LODs) of dansylated estrogens by SRM were 0.050.20pg, and the LODs in sewage treatment plant ef uent and in river water were 0.230.52 and 0.560.91ng/L, respectively. The LODs using selected ion monitoring (SIM) reached low ng/L levels in real samples and measured concentrations were comparable with those of SRM. 2008 Elsevier B.V. All rights reserved.

Keyw or ds : D ans y l chl o r i d e Ma tr i x e ffec t P ent a u o ro b e nzyl b r om id e U PL C St er o i d e s tr og en 2 D -L C

1. Introduction Feminizing contaminants of steroid estrogens, detergent degradates and plasticizers have caused a worldwide concern. They may in uence the ecosystem at trace levels and affect human health through their contamination of drinking water. Natural estrogens 17 -estradiol (E2) and its synthetic analogue 17 -ethinylestradiol (EE2), an ingredientin oral contraceptives, are the most estrogenic. Moreover, their major metabolites, estrone (E1) and estriol (E3), are still bioactive. These steroid estrogens enter the water environmentvia the urineof humans and animals in theform of hydrophilic glucuronide and sulfate conjugates [1], which are biologically inactivated [2]. However, they are likely to be deconjugated in sewage treatment systems and converted to estrogenicallyactivefreeforms

xenoestrogens are released into the water environment from daily usage of non-ionic surfactants and plasticizers. Atmospheric pressure photoionization (APPI) is an emerging source, which is capable of ionizing nonpolar compounds and is possibly less susceptible to matrix effects. In addition, dualsource ionization (e.g. atmospheric pressure chemical ionization (APCI)/APPI combo in this study) expands the range of compounds that can be simultaneously analyzed. Although most studies determined feminizing chemicals with electrospray ionization (ESI) coupled with LC/MS(/MS) [79], the suitability of APCI and APPI deserve further exploration. Matrix effect, which co-eluting components from the matrix or the mobile phase may enhance or suppress signals, is animportant issue in using LC/MS/MS. Selective extraction, additional clean-up, ef cientLC separationorchange of mobilephase compositions may reduce matrix effects [10]. Furthermore, while the use of suitable internal standards (e.g. isotope-labeled chemicals)may correct signal irreproducibility, this approach will not be able to overcome the loss in sensitivity caused by matrix effects. Some studies utilized direct online extraction or post-column split to minimize matrix effects and simplify the sample preparation. A novel column developed on September 2006 combines both size exclusion and

[3]. 4-nonylphenol (NP), 4-tert-octylphenol (OP) and bisphenol A (BPA), which are all xenoestrogens, can affect normal endocrine functions. Although they are less potent, they are usually found in much higher concentrations in water (ng/L g/L) [36]. These

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G .- W. Li en et al . / J . C h ro m ato gr . A 1216 (2 0 09 ) 9 5 6 9 66

9 57

reverse-phase chemistry to separate small molecules from complex matrix [11]; to the best of our knowledge, one study has analyzed drugs in bovine serum using the mixed-mode column [11]. A restricted access material (RAM) pre-column, with a similar separation mechanism to the mixed-mode column, has also been applied onanalyzing food, biological and environmental samples [12,13]. However, the RAM pre-column is an alkyl-diol silica (ADS) column and provides little chromatographic separation for small molecules; it requires a column switch to connect it with an additional analytical column for chromatographic separation (a two-dimensional LC, 2D-LC). In addition, a post-column split delivers only a portion of LC ow into the MS, which may substantially decrease matrix effects, especially when a ow rate into ESI interface was decreased to nano ow of 0.1 L/min [14,15]. This nanosplit requires special nanospray probes, whichis notamenable to a conventional ESI interface, whose owrates can be only as low as 2050 L/min. Reports on the mixed-mode column, 2D-LC and post-column split are very limited in environmental analysis and so little is known about their ability to reduce matrix effects.

[24,25]. However, the conclusions based on the results using nonoptimized parameters of various ionization methods could be controversial. The main purpose of the study was to nd out the best combination of a chromatographic system and an ionization method with satisfactory sensitivity using low volumes of water samples or single quadrupole MS. The nal method was validated using river water and ef uents from a sewage treatment plant (STP).

2. Experimental 2.1. Chemicals and reagents Estrone, 17 -estradiol, estriol, 17 -ethinylestradiol, 4-tertoctylphenol, bisphenol A, and bisphenol A-d16 (as a recovery standard) were obtained from Sigma/Aldrich (Saint Louis, MO, USA; purity > 98%). The technical mixture of nonylphenol was supplied by Riedel-de Han (Seelze, Germany; purity > 94%). 2,4,16,16-2 D4 -estrone, 2,4,16,16-2 D4 -17 -estradiol, 2,4,17-2 D3 16 -hydroxy- 17 -estradiol, 2,4,16,16-2 D4 -17 -ethinylestradiol and 4-n-Octyl- d17-phenol were bought from C/D/N Isotopes (Pointe-Claire, Quebec, Canada; purity > 98%). Bisphenol A-13 C1 2

Recently, there has been an increase in the number of studies using ultra-performance liquid chromatography (UPLC) combined with MS/MS. UPLC takes advantage of smaller packing particles (<2.0 m) that enable high ow rates for fastchromatography without sacri cing separationef ciency, and signal-to-noise (S/N) ratios of analytes are increased because of sharp peaks. However, to best of our knowledge none have used UPLC/MS/MS to study estrogenic compounds in water. Steroid estrogens and phenolic xenoestrogens are weak acids and their ionization on ESI and APCI are not very ef cient compared with other more polar chemicals. Chemical derivatization can add on moieties improving ionization and enhance signals. Forexample, dansyl chlorideor penta uorobenzyl bromide (PFBBr) can react with phenolic groups, signi cantly improving sensitivity [1618]. By adding the dansyl moiety with ESI interface, signal intensity may be increased as much as three orders of magnitude [16,19,20]. This technique has been also found to improve the sensitivity in APCI interface when used to measure steroid estrogens [21]. To date no dansyl derivatives have been analyzed with APPI interface. PFBBr derivatives can capture soft electrons in APCI, resulting in unstable metastic ions, and cause subsequent dissociation to generate negative ions through the loss of penta uorobenzyl radical (electron-capture atmospheric pressure negative ionization, EC-APNI). For estrone, the use of PFBBrderivatives inECAPNI can enhance ef ciency of ionization as much as 25 times that of APCI alone [18]. This method has been also used in APPI with a high toluene dopant ow rate (e.g. 200 L/min or higher) and was found to be able to detect as little as 0.17 pg of 2,4-dinitrophenol

was purchased from Cambridge Isotope Laboratories (Andover, MA, USA; purity > 99%). Dansyl chloride (5-(dimethylamino) naphthalene-1-sulfonyl chloride, ~95% purity), penta uorobenzyl bromide (PFBBr, purity > 99%), 4-methylmorpholine (purity > 99.5%), sodium hydrogen carbonate, and potassium hydroxide were purchased from Sigma/Aldrich. Milli-Q water was obtained from a Millipore water puri cation system (Milford, MA, USA). Formic acid (purity > 88%) and formaldehyde (purity> 37%) were provided by J.T. Baker (Phillipsburg, NJ, USA). Solvents, including methanol, acetone, n-heptane, acetonitrile and toluene, were all HPLC grade from J.T. Baker.

2.2. Extraction The procedure used to extract estrogenic compounds from the water sample has beenpreviously described [9]. Brie y, water samples were spiked with internal standards and then ltered through 90-mm PVDF membranes (pore size 0.45 m) to remove suspended solids before extraction. Extraction was performed using 50-mm Bakerbond PolarPlus C18 Speedisks (J.T. Baker), followed by a cleanup using 40% methanol/60% Milli-Q water (v/v). The disks were dried for 10 min under a vacuum of about -25 kPa. Analytes were eluted with three portions of 5-mL 50% methanol/50% dichloromethane (v/v). The eluates were ltered through 25-mm PTFE syringe lters (pore size 0.2 m) and concentrated to dryness at45 C by a SpeedVac concentrator (Thermo Savant SPD 1010, Holbrook, NY, USA). Theresidues were re-dissolved by spiking recovery standard and then reacted with dansyl chloride reagents.

[22], whereas for PFBBr-derivatized estrone, signal enhancement (1.49.8 times) was less than that using EC-APNI [18]. Our group previously reported that dansylated estrogens with ESI interface provided better signal intensities than that PFBBr derivatives with EC-APNI, but obvious signal suppression was encountered with ESI when analyzing complex matrixes suchas river water and ef uents from sewage treatment plants [23]. In this study, we investigated signal intensity and matrix effects on various chromatographic systems (UPLC with or without ow split, mixed-mode column, 2D-LC) and several ionization modes (ESI+, ESI-, APCI+, APCI-, APPI+, APPI-, APCI/APPI+, APCI/APPI-) for bothestrogenic compounds and their derivatives of dansyl chlorine and PFBBr. In addition, the study is unique in that it rst optimized the operation conditions speci c for each ionization methods, including those for LC columns, mobile phase ow rates and compositions. Previous studies usually compared the performance of different ionization sources under only one analytical column kept at a constant solvent ow rate, isocratic chromatography, the same injection volume, or ow injection analysis alone

2.3. Derivatization 2.3.1. Dansyl chloride derivatization The procedure used to derive dansyl chloride was based on EE2
derivatization method used by Penzes and Oertel [26] and Shou etal. [19]. Brie y, 0.9 mL of 100 g/mL analytes in acetone was vortexed for 1.0 minwith0.1 mL of 1 mg/mL dansyl chloride indry acetone followed by mixing with0.01 mL of 0.1N sodium hydroxide for 1.0 min. Themixturewas kept at 50 C for 30 min. 5 mL of n-heptane was added to the mixture which was then shaken for 3 min. It was centrifuged at3000 rpm for 10 minand refrigerated at-20 C. Once ithad separated into two layers, the organic layer was collected and ltered through0.20- m PTFE into another glass tube. The aqueous layer was discarded. The organic layer was evaporated to dryness at 45 C by a SpeedVac concentrator. The residue was re-dissolved

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G. -W. Li en et a l. / J . C h r om at og r. A 121 6 (2 0 0 9) 9 5 6 9 6 6

F ig. 1. S chem e of t he 2D - LC s ys t em. A t the s t ar t o f a nal y s i s ( a), the s am p l e wa s i nje cte d a nd w ent t hro u g h t he RAM co l u mn, a nd t he mo b i le p has e wa s 5 % ac eto ni t ri l e /w at er. T hre e mi nu te s l a te r, v al v e wa s au t o mat i cal l y s w i tc hed to ( b ); t he ana ly t es we re b a ck u s he d to a n a nal y ti ca l c ol u mn w i th a s u i ta b le mob i l e p ha s e gr ad i e nt fr o m p u mp B and the n i ntr od u c ed to the t and em ma s s s p e ct ro met er .

with 0.9 mL of methanol to optimize the parameters of operations on the MS. However, the above protocol cannot be directly applied to the derivatization of water samples and needs another protocol that was modi ed from Anari etal. [16] and Nelsonetal. [21].100 L of 0.9 ng/ L (for ESI analysis) and 250 L of 0.36 ng/ L (for APPI, APCI and APCI/APPI analysis), both in acetone, were vortexed with 250- and 625- L sodium bicarbonate buffer (10 mM, pH adjusted withNaOH(aq ) to 10.5), respectively. To these standards were added 250 and 625 L of 1-mg/mL solution of dansyl chloride, respectively. They were thenincubated at60 C for 3.0 minand evaporated to dryness in a SpeedVac concentrator. The residues were reconstituted with 100 and 250 L of methanol, respectively. Then, 4 L (for ESI mode)and 10 L (for APPI, APCI and APCI/APPI mode) were injected into LC/MS/MS for comparative analysis.

2.4.2. The mixed-mode column A Shodex ODP 2 HP-2D (2.0 mm 150 mm, 5 m) was used for ESI and APPI at a ow rate of 0.2 mL/min, while dansylated analytes in APPI were set at 0.5 mL/min. A Shodex ODP 2 HP-4D column (4.6 mm 150 mm, 5 m)was used for APCI and APCI/APPI at a ow rate of 1.0 mL/min. 2.4.3. 2D-LC system with RAM pre-column This system was composed of a VICI six-port switching valve (Valco Instruments Inc., Houston, TX, USA) and an extra isocratic pump (Jasco PU-980, Tokyo, Japan) (Fig. 1). The elution pro les of the RAM pre-column (LiChrosphere RP-4ADS (25 mm 2mm, 25 m)) for ef uent of sewage treatment plants and river water were monitored with a UV detector set at 280 nm. The injection volume was 50 L of each extract, and the ow rate of mobile phase of Milli-Q water-acetonitrile (95:5, v/v) through RAM pre-column was set at 1 mL/min. The time required to elute major matrix components was less than 3 min (pro le not shown). Based on this result, the valve was switched after 3 min to back ush the analytes into an analytical column. A Thermo Hypersil Gold column (2.1 mm 50 mm, 1.9 m, Bellefone, PA, USA) was used for ESI and APPI, which ow rate was 0.2 mL/min, while dansylated analytes in APPI was set at 0.5 mL/min. A Thermo BetaBasic C18 column (4.6 mm 150 mm, 3 m) was used for APCI and APCI/APPI set at a ow rate of 1.0 mL/min. All chromatographic separations including UPLC, mixed-mode column and 2D-LC were performed at 60 C (Table 1).

2.3.2. PFBBr derivatization PFBBr was derived based ona procedure reported by Singh et al. [18]. We vortexed 250- L mixture standards of native (0.36 ng/ L) analytes in methanol with 250 L of potassium hydroxide in anhydrous ethanol (8:100 0; w/v). We then added 250 L of 5% PFBBr in acetonitrile. The mixture was baked at 60 C for 30 min and then evaporated to dryness ina SpeedVac concentrator. The residue was reconstituted with 250 L of methanol, and 10 Lwas injected into LC/MS/MS for comparative analysis.

2.4. LC systems and analytical columns 2.4.1. The UPLC with or without post-column split
A Waters BEH C18 column (2.1 mm 100 mm, 1.7 m) was used

2.5. Instruments and parameters The separation and detection were performed on a Waters Acquity UPLC system (Waters Corporation, Milford, MA, USA) coupled with a Waters Quattro Premier XE triple quadrupole mass spectrometer. The UPLC/MS/MS system was controlled by

for ESI and APPI at a ow rate of 0.5 mL/min, while native analytes in APPI were set at 0.2 mL/min for better signal intensities. A Sepax GP-C18 column (3.0 mm 100 mm, 1.7 m) was used for APCI and APCI/APPI at a ow rate of 1.0 mL/min. Post-column split (split ratio = 1:5) was tested on ESI.

Ta ble 1 D i ffe re nt LC s ys t ems and i o ni zat i o n m od e s f or bo t h na ti v e co mp o u nds a nd d e ri v at iv es w i th P FBBr o r d a ns yl chl o r i d e. C o l u mn t y p e U PL C w i t h o r w i t hou t s p l i t


BE H C 18 (2 .1 mm 10 0 mm, 1 .7 m) ES I G P -C 1 8 ( 3 .0 mm 10 0 mm, 1 .7 m) A P C I ( /AP P I) AP C I A P C I ( /AP P I)

Na ti v e (- )

PF BBr ( - ) Da ns yl chl o r i de (+ )

AP P I

ES I

AP P I

Mi x ed - mo d e LC O D P 2 H P- 2 D ( 2. 0 mm 1 5 0 m m, 5 m) ES I O D P 2 H P- 4 D ( 4. 6 mm 1 5 0 mm, 5 m) A P C I ( /AP P I) AP C I A P C I ( /AP P I) 2 D -L C w i th RA M H y p er s i l Go l d (2 .1 mm 5 0 mm, 1.9 m ) ES I Be ta Bas i c C 18 (4 . 6 m m 15 0 mm, 3 m ) A P C I ( /AP P I) AP C I A P C I ( /AP P I)

AP P I

ES I

AP P I

AP P I

ES I

AP P I

G .- W. Li en et al . / J . C h ro m ato gr . A 1216 (2 0 09 ) 9 5 6 9 66 Ta ble 2 Majo r MS p ar ame te rs fo r d i ff er ent a nal y te s a nd i o ni za ti o n met ho ds . Pa ra met er N ati v e PF BBr D ans y l chl o r i d e E SI- AP C I- A PP I- A PC I/ AP P I- AP C I- E SI+ A PC I+ AP P I+ AP C I/A PP I+ So u r ce te mp er at u r e ( C ) 1 20 15 0 15 0 15 0 1 50 1 20 12 0 1 2 0 1 20 De s o l vat i o n t emp e ra tu r e ( C ) 40 0 6 0 0 6 0 0 6 0 0 40 0 4 50 5 0 0 7 0 0 50 0 C on e ga s o w ( L/h) 50 0 0 0 0 0 0 5 0 0 De s o l vat i o n g as o w ( L/h) 90 0 7 5 2 0 0 7 5 1 50 10 0 0 6 0 0 2 0 0 60 0 C or o na ( A) 3 0 3 0 30 2 .8 2 . 8 C ap i l l a ry (k V) 3 Rep e l l er ( k V) 22

9 59

2.8 33

MassLynx V4.1 with QuanLynx Application Manager and the data were acquired and processed using MassLynx V4.1. Instrumental parameters in various ionization methods were optimized to achieve maximal analyte signal intensities.
Changes of the desolvation gas (N2 ) ow rate and source tem-

at100% Bfor 1.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min. 2.5.1.3. APCI (+) and APCI/APPI (+). 10 mM formic acid (A) and methanol (B) were used as the mobile phase. There were three LC conditions. (1) A GP-C18 column had an initial gradient of 50% B, followed by a linear gradient to 85% B for 0.5 min, and then to 100% B in 1.7 min, at which point it was held at 100% B for 0.3 minbefore being returned to initial condition. The column was re-equilibrated for 2 min. (2) An ODP 2 HP-4D column had an initial gradient of 50% B, followed by a linear gradient to 85% B for 3.0 min, and then to 100% B in 3.0 min. It was held at 100% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min. (3) A RAM coupled with a BetaBasic C18 column had a gradient of 30% B for 3 min, followed by a linear gradient to 50% B in 1.0 min, then to 85% B in 2.0 min, and then to 100% B in 2.0 min, at which point it was held at 100% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min.

perature did not produce signi cantly different signals in either ESI or APPI interface when ow rates of mobile phase were at either 0.2 or 0.5 mL/min, so these two parameters were kept the same for these two ow rates. Major parameters are summarized in Table 2. Extractor voltage was 3.0 V and RF lens voltage was 0 V. Collision gas was argon at 3 10- 3 mbar. Ion energy 1 and 2 were set at 0.3 and 3, respectively. Both LM 1 and LM 2 resolution were set at 15. The multiplier voltage was set at 650 V. Ions were monitored by selected reactionmonitoring (SRM)as shown in Table 3. Dansylated analytes produced intense precursor ions with m/z [M+233.8]+ , and the collision-induced dissociation produced intense product ions with m/z 171+ and m/z 156+ , corresponding to the 5-(dimethylamino)-naphthalene moiety and the loss of one methyl group from the m/z 171+ , respectively [19,23]. PFBBr derivatives produced the same precursor ions as the underivatized ones with m/z [M-H]- [23]. Several LC mobile phase compositions were tested to obtain good separation and peak shapes. Data points across the peak were no less than 20 to ensure the integration precision.

2.5.1. Dansyl derivatives 2.5.1.1. ESI (+). 10 mM formic acid (pH 2.9) (A) and acetonitrile (B) were used as the mobile phase. There were three LC column conditions. (1) A BEH C18 columnwith and withoutsplit had a gradient of 50% B for 0.2 min, followed by a linear gradient to 85% B in 0.8 min, and then to 100% B in 1.5 min, at which point it was held at 100% B for 0.7 min before being returned to initial condition. The column was re-equilibrated for 1.0 min. (2) An ODP 2 HP-2D column had a gradient of 50% B for 2.0 min, followed by a linear gradient to 85% B in 1.0 min, and then to 90% B in 2.0 min, at which point it was held at 90% B for 1.5 min before being returned to initial condition. The column was re-equilibrated for 4.5 min. (3) A RAM coupled with a Thermo Hypersil Gold column had a gradient of 30% B for 3.0 min, followed by a linear gradient to 50% B in 1.0 min, then to 85% B in 2.0 min, and then to 100% B in 4.0 min, at which point it was held at 100% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min.

2.5.2. PFBBr derivatives 2.5.2.1. APCI (-). Water (A) and methanol (B) were used as the mobile phase. There were three LC conditions. (1) A GP-C18 column had a gradient of 70% B for 0.2 min, followed by a linear gradient to 90% B in 0.8 min, and then to 100% B in 2.2 min. It was held at 100% B for 0.2 minbefore being returned to initial condition. The column was re-equilibrated for 1.0 min. (2) An ODP2 HP-4D column had a gradient of 70% B for 2 min, followed by a linear gradient to 95% B in 3.0 min. It was held at 95% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min. (3) A RAM coupled with a BetaBasic C18 column had a gradient of 30% B for 3.0 min, followed by a linear gradient to 75% B in 1.0 min, and then to 100% B in 4.0 min, at which point it was held at 10 0% B for 4.0 min before being returned to initial condition. The column was re-equilibrated for 3.0 min.

2.5.1.2. APPI (+). 10 mM formic acid (A) and acetonitrile (B) were used as the mobile phase. There were three LC conditions. (1)A BEH C18 column had a gradient was setthesame as ESI mode. (2) An ODP 2 HP-2D column had a gradient of 50% B for 1.0 min, followed by a linear gradient to 70% B in 1.0 min, and then to 85% B in 2.0 min, at which point it was held at 85% B for 1.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min. (3) A RAM coupled with aThermo Hypersil Gold column had agradient of 30% Bfor 3.0 min, followed by alinear gradient to 50% Bin1.0 min, then to 85% B in 4.0 min, and then to 100% B in 4.0 min. It was held

2.5.3. Native analytes 2.5.3.1. ESI (-). 10 mM 4-methylmorphline (pH 9.5) (A) and acetonitrile (B) were used as mobile phase. There were three LC conditions. (1) A BEH C18 column with and without split had a gradient of 10% B for 0.2 min, followed by a linear gradient to 40% B in 0.8 min, then to 70% B in 1.7 min, and then to 95% B in 0.5 min. It was kept at 95% B for 0.6 min before being returned to the initial condition. The column was re-equilibrated for 1.7 min. (2) An ODP 2 HP-2D column had a gradient of 10% B for 2.0 min, followed by a linear gradient to 50% B in 3.0 min, and then to 70% B in 8.0 min, at which point it was held at 70% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 4.0 min. (3) A RAM coupled with a Thermo Hypersil Gold column had a gradient of 30% B for 3.0 min, followed by a linear gradient to 50% B in 1.0 min, then to 75% B in 3 min, and then to 100% B in 2 min. It was held at100%B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min.

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Ta ble 3 Se l ec ted r ea cti o n mo ni to ri ng (S RM) tr ans i t i o ns , i nd i vi d u a l co l l i s i o n e ner g y (C E , V ) and co ne vo l t age (C V, V) o f ana ly te s , an d l i ne ar r ang es w i th cal i b r at i on cu r ve eq u at i ons of d ans y l at ed co mp o u nd s.
Ana l yt e MW [M - H ]- Na ti v e P FBBr [ M+2 3 3 .8 ] + D a ns yl chl o ri d e

C E C V C E C V C E C V Li ne ar r ang e (ng / L ) E 1 2 70 . 4 2 6 9 .1 > 1 45 . 0 >1 43 . 0 36 5 0 4 0 55 >1 5 6 .0 5 4 5 0


E 1 -d 4 2 74 . 4 2 73 . 4 > 1 46 . 8

E q u at i o n 5 0 4 .1 > 1 71 .1 3 4 0 .0 0 0 1 1 Y = 3 .8 9 9 X + 0. 0 10 ,
R 2 = 0 . 99 9

5 0 8 .1 > 1 71. 0 3 4 5 0 6 .1 > 171 .1 4 6 0 .0 0 1 1 Y = 2 .8 4 5 X + 0. 0 2 8,


R 2 = 0 . 99 8

E 2 2 72 . 4 2 71. 2 > 18 3 .0 >1 4 4. 8 40 6 5 3 8 60 >1 5 6 .2 5 8 5 5


E 2 -d 4 2 76 . 1 2 74 .6 > 14 7. 0

5 10 .1 > 171 .0 4 6 5 2 2 .2 > 17 1.1 3 4 0 .0 0 0 1 1 Y = 3 .5 4 2 X + 0. 0 19 ,


R 2 = 0 . 99 7

E 3 28 8 . 4 2 8 7.2 > 17 0 .9 >1 45 . 0 38 5 0 3 6 50 >1 5 6 .1 6 2 6 0


E 3 -d 3 2 91 . 4 2 9 0. 6 > 17 3. 0

5 2 5 .2 > 17 1. 0 3 4 5 3 0 .2 > 17 1.1 3 4 0 .0 0 0 1 1 Y = 3 .0 3 0 X - 0 .0 0 3 ,


R 2 = 0 . 99 8

E E2 29 6 . 4 2 9 5 .2 > 14 4 .8 >1 59 . 0 38 5 5 4 0 55 >1 5 6 .0 5 8 6 0


E E2 - d 4 3 0 0 . 4 2 9 8 .9 > 14 7.0

5 3 4 .2 > 17 1. 0 3 4

N P 22 0 . 4 2 19 .2 > 13 3 . 0 30 3 5 3 0 35 4 5 4 .2 > 17 1. 2 3 2 4 5 0 .0 5 3 Y = 6 .9 0 9 X + 0. 5 73 ,
R 2 = 0 . 99 9

>1 47. 0 O P 20 6 . 3 2 0 5. 0 > 13 3 .2 >1 47. 0 25 3 5 2 4 45 >1 5 6 .1 5 4 5 0 4 -n - O cty l - d 17 -p he no l 22 3 . 4 2 2 2 .1 > 1 07 .6 BPA 22 8 . 3 2 27 .2 > 21 2 .1 >1 32 . 8 18 3 5 1 8 35 >1 5 6 .0 5 0 4 5
B PA -1 3 C 12 24 0 . 2 2 3 8 .9 > 2 23 . 5

>1 5 6 .1 4 4 4 4 0 .2 > 17 1.1 3 4 0 .1 2 Y = 11.17X + 4 . 82 3 ,


R 2 = 0 . 99 5

4 5 7.2 > 17 1. 0 3 4 4 6 2 .1 > 1 71 .2 3 8 0 .0 5 3 Y = 3 .0 5 0 X + 0. 6 8 4 ,


R 2 = 0 . 99 7

4 74 .1 > 171 .0 3 8

BPA -d 1 6 ( RS) 24 4 . 4 2 41. 0 > 22 2 . 6 22 3 5 4 7 8. 0 > 171 .0 3 8 4 5 N ote: 4-n - Oct yl - d 17 - p heno l wa s u s e d a s i nter na l s t and ar d of N P and OP .

2.5.3.2. APPI (-). Water (A) and methanol (B) were used as mobile phase. There were three LC conditions. (1) A BEH C18 column had a gradient of 30% B for 1.0 min, followed by a linear gradient to 50% B in 2.0 min, then 75% B in 4.0 min, and then 100% B in 2.0 min. It was held at 100% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 4.0 min. (2) An ODP 2 HP-2D column had a gradient of 50% B for 1.0 min, followed by a linear gradient to 75% B in 1.5 min, and then to 90% B in 5.0 min, where it was held at 90% B for 1 min before being returned to initial condition. The column was re-equilibrated for 4.0 min. (3) A RAM coupled with a Thermo Hypersil Gold column had a gradient of 30% B for 3.0 min, followed by a linear gradient to 50% B in 1.0 min, then to 75% B in 3.0 min, and then to 100% B in 2.0 min, at which point it was held at 10 0% B for 2.0 min before being returned to initial condition. The column was re-equilibrated for 2.0 min.

B in 4.0 min, where it was held at 100% B for 4.0 min before being returned to initial condition. The column was re-equilibrated for 3.0 min. 2.6. Method comparisons Because estrogenic compounds are frequently observed in sewage or surface water, it is dif cult to obtain a matrix without estrogenic compounds. Raw water from a drinking water treatment plant (WTP) in Taipei City, which only contains analytes at trace levels, was used as the matrix. Equal aliquots from extracts of one-liter samples were used for each method. Eluates were concentrated to dryness at 45 C using a SpeedVac concentrator and were reconstituted by appropriate solvents, with or without the spiking of 90-ng native compounds for the following analyses: (1) native chemicals in ESI (-), APPI (-), APCI (-) and APCI/APPI (-); (2) dansyl derivatization in ESI (+), APPI (+), APCI (+) and APCI/APPI (+); (3) PFBBr derivatization in EC-APNI (-). The best method was chosen for method validation based on signal intensities and matrix effects. The percentage matrix effect (%ME) was used to assess matrix effects: peak area of post-extraction spiking/peak area of standard 100. Before calculation, the areas of samples without spiking were subtracted from the areas of samples.

2.5.3.3. APCI (-) and APCI/APPI (-). Water (A) and methanol (B) were used as mobile phase. There were three LC conditions. (1) A GP-C18 column had an initial gradient of 30% B, followed by a linear gradient to 50% B for 0.5 min, then to 75% B in 2.0 min, and then to 100% B in 0.5 min. It was held at 100% B for 0.6 min before beingreturned to initial conditions. The columnwas re-equilibrated for 2.0 min. (2) An ODP 2 HP-4D column had a gradient of 50% B for 2.0 min, followed by a linear gradient to 75% B in 1.5 min, and then to 90% B in 3.5 min, at which point it was held at 10 0% B for 1.0 min before being returned to initial condition. The column was re-equilibrated for 4.0 min. (3) A RAM coupled with a BetaBasic C18 column had a gradient of 30% B for 3.0 min, followed by a linear gradient to 75% B in 1.0 min, and then to 10 0%

2.7. Method validation Two types of water, ef uents and river water, were used for method validation. The ef uents were sampled from a sewage treatmentplant in Taipei. Thatplant is a secondary treatment facility with an activated sludge units. The samples were collected in

G .- W. Li en et al . / J . C h ro m ato gr . A 1216 (2 0 09 ) 9 5 6 9 66

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F ig . 2 . S i gna l i nt ens i t i es o f na ti v e ana l yt es fo r v ar i ou s i o n so u r ce s u s i ng ( a) U P LC (* co u p l ed w i th p o s t- co l u mn s p l i t ), (b ) the mi xe d -m od e c ol u mn a nd ( c) 2 D -L C.

January 2008 (pH 7.06, temperature = 21.0 C, DO = 4.29 mg/L). The river water was taken from the Kee-Lung River in Taipei in April 2008 (pH7.0, temperature= 22.4 C, DO = 1.2 mg/L). Threesolutions, 0.25 ng/ L of the four estrogens standards (20, 100, or 200 L), 1 ng/ L of the three xenoestogens standards (50, 250, or 500 L) and 100 L of 0.5 ng/ L internal standards, were spiked into 0.5-L water samples before extraction. Eluates from the SPE disk were concentrated to dryness at 45 C using the SpeedVac concentrator and were re-dissolved with 200- L anhydrous acetone containing 0.25 ng/ L recovery standard, and then reacted with dansyl chlo-

ride derivatization reagent. Four-microliter solution was injected into LC/MS/MS. 2.8. QA/QC, quanti cation and data analysis All glassware was rinsed with acetone, n-heptane, dichloromethane and methanol before being used for experiments. A blank sample spiked with the internal standards was run with each batch of samples to check experimental contamination and provide background levels of the native analytes. A

Fig . 3 . S i gna l i nt ens i t i es of d ans y l at ed ana l yt es for va ri o u s i o n s ou r ce s u s i ng ( a) U PL C ( * cou p l e d w i t h p o s t- co l u mn s p l i t ), ( b ) t he mi x ed - mo d e co l u mn a nd ( c) 2D - LC . E C - AP N I (- ) w as fo r P FBBr d er i va ti ve s .

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F ig . 4 . C hr oma to g ra ms o f d ans y l at ed co mp o u nd s ( 0 .2 ng / L o f s tand a r ds , 4- L i nje cti o n) i n E SI mo d e u s i ng ( a) U P LC , ( b) the mi x ed - mo d e co l u mn and (c) 2D - LC .

calibration curve was built at each analysis. The linear range were 0.00011 ng/ L for steroid estrogens (except E2 0.0011 ng/ L) and 0.053 ng/ L for xenoestrogens (except OP 0.12 ng/ L) at weighted (1/x). Isotope-dilution techniques were used to correct variations in sample preparation and instrumental performance; peak areas of dansylated analytes were normalized to their deuterium-labeled internal standard for quanti cation. The squares of correlation coef cients (r2 ) were 0.995 or above for all calibration curves as shown in Table 3. A one-way analysis of variance (ANOVA) with Tukeys post hoc comparison was used to compare the signal intensities and matrix effects associated with the different methods. Students t-test was used to evaluate the

difference of quantitative results between SRM and selected ion monitoring (SIM) method. SAS 9.1 was used to perform statistical analysis. 3. Results and discussion 3.1. Effects of dopant, mobile phase ow rates and compositions on APPI sensitivity We found that excess dopant ow may or may not improve analyte signals, and optimal dopant portions were compounddependent. Differentamountof thetoluene dopantwere tested on a

F ig . 5 . Re l at iv e r es p o ns es (i n l o ga ri t hmi c s cal e ) o f d er i va ti ve s to na ti v e ana l yt es i n d i f fer ent L C s y s te ms : (a ) U PL C , (b ) t he mi x ed - mo d e co l u mn and (c) 2 D - LC .

G .- W. Li en et al . / J . C h ro m ato gr . A 1216 (2 0 09 ) 9 5 6 9 66 Ta ble 4 Mat r i x ef fect fact o r (%) ob t ai ne d fr o m raw wa te r s am p l es u s i ng p o st - ex tr act i o n s p i k i ng ( n = 4 ). E 1 E 2 E 3 E E2 N P O P BPA N ati v e at ES I ( - ) U P LC (f = 0 .5 mL /mi n) 8 8. 1 6 6 .6 8 6 . 0 75 . 1 9 2 .5 8 7 .6 7 8 . 0 U P LC wi t h s p l i t ( f = 0 .1 m L/mi n) 72 . 3 7 3. 7 6 1 .1 69 . 1 8 4 .4 8 2 .7 8 2 . 2 Mi x ed - mo d e co l u mn (f = 0 .2 mL /mi n) 7 5. 9 7 6. 5 7 4. 1 73 . 5 7 7 .3 7 8 .2 76 . 2 2 D -L C ( f = 0. 2 mL/ mi n) 72 . 0 7 1. 3 8 3 . 4 73 . 3 1 13 8 6 .3 6 8 . 4 N ati v e at AP PI ( -)
U P LC (f = 0 .2 mL /mi n) 5 6. 6 a 6 0 .3 a 4 8 . 5a 59 . 1 a 5 9 .4 a 7 0 .8 c 5 3 . 4 Mi x ed - mo d e co l u mn (f = 0 .2 mL /mi n) 51 . 7a 4 6 .7 a 3 5 .9 a 56 . 5 a 5 9 .3 a 4 4 .3 a ,b 5 5 . 5 2 D -L C ( f = 0. 2 mL/ mi n) 8 5. 4 b , c 8 1 .7 b , c 11 2 b , c 95 . 5 b ,c 12 2 b , c 8 8 .3 b 5 0 . 5

9 63

N ati v e at A P C I ( - )
U P LC (f = 1. 0 mL/mi n) 5 2. 0 a ,c 5 7. 4 a ,c 9 5 . 9a 58 . 7 a ,c 5 3 .7 c 5 6 .2 c 71 . 3 Mi x ed - mo d e co l u mn (f = 1. 0 mL/m i n) 8 4. 2 b 8 6 .7 a , b 8 2 . 3 82 . 6 b 9 6 .1 b 8 4 .4 a ,b 8 2 . 9 a 2 D -L C ( f = 1.0 mL /mi n) 76 . 4 b 7 2. 3 b , c 6 9 . 8b 75 . 5 b 8 5 .5 7 2 .0 b 6 9 . 7 b

N ati v e at AP C I/AP P I (- )
U P LC (f = 1. 0 mL/mi n) 51 . 5c 6 2 .5 c 8 2 . 6 58 . 0 c 51 .8 5 3 .9 c 6 8 . 5 c Mi x ed - mo d e co l u mn (f = 1. 0 mL/m i n) 8 8. 8 a ,b 8 9 .1 a , b 9 3 . 0 91 . 6 a ,b 8 8 .3 8 3 .3 s, b 9 5 . 5 s,b 2 D -L C ( f = 1.0 mL /mi n) 5 4. 6 c 5 6 .6 c 9 2 . 2 55 . 5 c 7 0 .9 6 0 .9 c 6 2 . 5 c

Da ns yl a t ES I (+ ) U P LC (f = 0 .5 mL /mi n) 9 4 .6 9 6 .0 9 2 . 2 92 . 0 6 3 .5 5 9 .6 7 8 . 6 U P LC wi t h s p l i t ( f = 0 .1 m L/mi n) 7 5. 4 7 1. 6 6 2 . 9 74 . 9 7 7 .5 6 3 .3 6 9 . 3 Mi x ed - mo d e co l u mn (f = 0 .2 mL /mi n) 8 2. 3 7 9 .8 7 7 .3 81 . 8 6 0 .8 5 8 .9 7 9 . 4 2 D -L C ( f = 0. 2 mL/ mi n) 9 3. 6 9 3 .8 9 1 .0 83 . 7 6 3 .1 6 4 .5 8 9 . 6 Da ns yl a t AP P I ( +)


U P LC (f = 0 .5 mL /mi n) 8 5. 7 8 5 .8 1 0 4a , c 86 . 0 111 c 10 9 a, c 67 . 8 Mi x ed - mo d e co l u mn (f = 0 .5 mL /mi n) 7 5. 2 6 9 .7 6 3 . 6b 75 . 7 6 4 .2 a , b 7 2 .2 b 5 2 . 8 2 D -L C ( f = 0. 5 mL/ mi n) 8 2. 1 7 7 .6 5 4 . 8b 79 . 4 1 16 c 7 8 .5 b 4 2 . 9

Da ns yl a t AP C I ( + ) U P LC (f = 1. 0 mL/mi n) 119 12 1 11 9 1 22 12 1 10 3 10 9 Mi x ed - mo d e co l u mn (f = 1. 0 mL/m i n) 111 11 2 1 2 0 113 1 31 10 1 8 1 . 3 2 D -L C ( f = 1.0 mL /mi n) 12 1 12 5 1 3 2 1 22 1 15 9 9 .9 8 8 . 6 Da ns yl a t AP C I/A PP I (+ ) U P LC (f = 1. 0 mL/mi n) 10 9 10 9 1 07 115 10 3 8 8 .9 61 . 9 Mi x ed - mo d e co l u mn (f = 1. 0 mL/m i n) 10 1 9 7 .7 1 0 9 10 0 10 6 8 6 .3 6 5 . 4 2 D -L C ( f = 1.0 mL /mi n) 10 7 10 8 1 2 0 1 08 9 0 .4 1 2 2 71 . 6 PF BBr a t AP C I (- ) U P LC (f = 1. 0 mL/mi n) 112 114 11 1 110 10 2 1 2 4 1 35 Mi x ed - mo d e co l u mn (f = 1. 0 mL/m i n) 110 11 0 9 9 . 6 113 10 6 9 7 .9 10 8 2 D -L C ( f = 1.0 mL /mi n) 97 . 5 10 3 9 7 .2 1 05 12 1 11 6 10 4 No te: 3 . 6 ng eq u i va l ent o f ea ch ana l yt es wa s i nje cte d .
a Sta ti s t i cal l y di f fer e nt fro m 2 D -L C . b Sta ti s t i cal l y di f fer e nt fro m U P LC . c Sta ti s t i cal l y di f fer e nt fro m t he mi xe d - mod e co lu m n. d S ta ti s t i cal l y d i f fer ent f ro m U PL C w i th s p l i t .

Waters BEHC18 column, ranging from5% to 25% of a constant mobile phase ow rate at 500 L/min. The mobile phase compositions for native and dansylated compounds were Milli-Q water/methol and 10 mM formic acid/acetonitrile, respectively, which were the optimalcombinations for theirseparation on thecolumn. Based onpeak areas, the optimal amount of dopant for native analytes was 5% of the mobile phase ow, 25 L/min. The intensities of dansylated analytes were gradually enhanced as toluene was increased to 20% of the mobile phase ow (corresponding to 100 L/min). All intensities were enhanced except for dansyl-NP and dansyl-OP, whose signals dropped signi cantly once the dopant exceeded 5%. Therefore, a ve-percent dopant ow was used in this study. Robb et al. used acridine and 9-methylanthracene as model compounds and reported that the signals are close to plateaus when the dopant amount ranges from 5% to 10% of the mobile phase ows, which were at 50, 200, or 1000 L/min [27] and signal intensities are slightly raised when a higher dopant percentage is given [27],a nding similar to ours in native and dansylated steroid estrogens and dansylated BPA; however, in our study, signals of dansylated NP and OP dropped when the dopant portion was higher than 5%.

The suitable ow rate of mobile phase for APPI signals was compound-dependent, which the best ow rates were different between native and dansylated analytes. The mobile phase ow rates of 100, 200, 500 and 1000 L/min were evaluated at a constant dopant of 5% mobile phase ows using an isocratic liquid chromatography; the mobile phasecompositions of native and dansylated analytes were 20% Milli-Q water/80% methanol (v/v) and 12% 10-mM formic acid/88% acetonitrile (v/v), respectively. A Sepax GP-C18 column (3.0 mm 100 mm, 3 m)wasusedata owrate of 1.0 mL/min and a Waters BEH C18 column (2.1 mm 100 mm, 1.7 m) was performed at equal or less than 500 L/min. The best sensitivities of nativeand dansylated analytes wereobtained at ow rates of 200 and 500 L/min, respectively. The signal intensities of native analytes at a ow rate of 200 L/min were 1.6- to 2.8-fold, 1.0- to 1.6-fold, and 2.7- to 6.4-fold higher than those at ows of 10 0, 500, and 1000 L/min, respectively. The signal intensities of dansylated analytes at a ow rate of 50 0 L/min were 4.9- to 18fold, 3.1- to 9.4-fold, and 2.2- to 6.0-fold higher than those at ows of 100, 200, and 1000 L/min, respectively.

Some studies have demonstrated that a low ow rate (e.g. =100 L/min) may improve the ionization ef ciency of APPI

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resulting from lower photoabsorption by solvent [2729].However, our data showed that a lower ow rate may not provide a better ef ciency in APPI. To con rm these observations, we further utilized a BEH C18 column with a smaller I.D. (1.0 mm 100 mm, 1.7 m). Its optimal ow rate is 100 L/min. We found that the signal response was even weaker than that used a larger column I.D. (2.1 mm 100 mm, 1.7 m). Solvent molecules (e.g. methanol) may be involved inthe ionizationprocess, especially in the dopantassisted APPI, and the ions formed by proton transfer are less affected by high ow rates than those formed via charge exchange [25,28,30,31]. This would explain the reason that the optimal mobile phase ow rates on APPI ef ciencies were compounddependent. Mobile phase composition is also critical to APPI sensitivity, and is also compound-dependent. For native analytes, a Milli-Q water-methanol combination gave better responses with better peak shapes than those using water-acetonitrile; however, for dansylated analytes, a composition of 10 mM formic acid-acetonitrile provided two to three times higher sensitivity than a composition of 10 mM formic acid-methanol. Cai et al. indicated that methanol has a lower photoabsorption cross-section than acetonitrile, and its dimmers can be ionized by a Kr Lamp (acetonitrile cannot) [25]; consequently, use of methanol would theoretically provide a better response, whichwas correctfornative analytes inthis studybut was not the case for dansylated analytes. However, Cal et al. also proposed that the APPI mechanism is much more complex, and other factors, such as the ionizationpotential of analytes and the relative proton af nity of solvents and analytes, may affect APPI ef ciencies as well [25].

3.2. Comparison of signal intensity between derivatized and underivatized analytes The best combinations of LC systems and ion sources for native and dansylated analytes were 2D-LC and UPLC coupled with ESI mode, respectively. Signals of all native analytes in ESI (-) mode were better than those in APPI (-), APCI (-) and APCI/APPI (-) except for NP and OP; there was no signi cant difference in signal intensities between APCI/APPI dual mode and APCI or APPI alone (Fig. 2). Within the ESI mode, 2D-LC was superior to UPLC and mixed-modecolumnintermsof signal intensities, butUPLC outperformed both 2D-LC and mixed-mode columns when APPI (-), APCI (-) and APCI/APPI (-) were put on. For PFBBr derivatives using ECAPNI (-), itwas better to use 2D-LC than UPLC for the analysis of E1, E2, E3 and EE2, but it was better to use UPLC rather than 2D-LC for the analysis of NP, OP and BPA. Withregard to the signal intensities of the dansylated analytes, ESI (+)were much better than other ion sources (except for NP); the APCI/APPI dual mode produced similar signal intensities with those of APCI alone, but was much inferior to those of APPI alone. In addition, use of UPLC produced much stronger responses of dansylated analytes relative to other two LC systems in all ion sources (Fig. 3). Based on the results, the on-line cleanup of the mixed-mode column and the RAM pre-column did notgave them a decisive advantage over UPLC ondetecting theanalytes in raw water samples, especially for dansylated derivatives. The mixed-mode column and 2D-LC, a polymer-based column and a coupled-column system, respectively, offered good peak shapes at widths <0.3 min (Fig. 4); nevertheless, UPLC provided sharp peaks at a widthof about 0.06 min(except for NP, whichwas 0.14 mindue to a mixture of isomers).

Derivatization with dansyl chloride and PFBBr signi cantly improved the detection sensitivity relative to underivatized analytes, and the dansylated analytes produced much better responses in four ionization methods than PFBBr derivatives in EC-APNI (Fig. 3). The trends of signal enhancement of derivatization among various ionization methods were similar, which were

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KOH/anhydrouson the LC systems (Fig. 5). For example, onpH was independent EtOH) were prepared in plastic containers, UPLC adjusted using plastic droppers, and plastic8598460 times, 3544030 column, the signal enhancements were tips were utilized to spike solutions and reagents. On times andhand, the blankESI, APPI, times, 23472 times, 21344 the other 541 times in levels were insigni cant to those found in environment samples. to the native APCI, APCI/APPI and EC-APNI, respectively, relative analytes. In other words, the order of responses of derivatized The method was used with samples from STP ef uents and river analytes versus underivatized ones in ionization methods was ESI > APPI The mean concentrations of steroid estrogens in water (Table 5).> APCI APCI/APPI > EC-APNI. In addition, post-column split were 4.3 ng/L for E1, 1.8 ng/L for in 1.9 study did not ef uents (a ow rate at 100 L/min after split)E2, this ng/L for E3, and 2.8 increase response in ESI mode (Fig. 3). In contrast, Kloepferconng/L for EE2; NP, OP and BPA were found in much higher et al. reported that a lower ow rate (e.g. down to 20 L/min) can centrations withmeanvalues of 657, 227, and 138 ng/L, respectively. Fordramatically increase signal intensities ofand E3analytes, although steroid estrogens in river water, only E1 some were detected some other ng/L, respectively; the concentrations of NP, OP and at 2.0, and 0.72analytes were unaffected [32]. BPA in river water were higher than those in ef uents with mean values of 4022, 246, and 6558 ng/L, respectively. High levels of NP and BPA in river water in Taiwan have also been observed by Ding et al. [34,35]. 3.3. Matrix effect Threewas inconclusive to determine which ng/Lforsteroid estroIt differentconcentrations (10, 50, 100 LC systems and ion gens and 10were least susceptible to matrix effects (i.e., higher values sources 0, 500 and 1000 ng/L for xenoestrogens) were spiked intoof matrix effect factors) when using the raw water as the matrix the water samples from the same sources to evaluate the method accuracy andis a source (four duplicates each level). be cleaner (Table 4), which precision for drinking water and may The reported usual surface water; however, we did observe that derivatized than concentrations of spiked samples did not deduct the background levels in the water. In our study, RSD% of underivatized ones. analytes were less prone to matrix effects than all spiked samples were allnative analytes, we found nofor NPcant differencesat100 ng/L For smaller than 15.5% except signi and OP spiked in matrix (and 500 ng/L foramongwhich were close systems with ESI mode; howeffect factors NP), the different LC to the endogenous levels of the ever, the (Table 5). Wewas lower on2D-LC for APPI, and itwas lower samples matrix effect found that the measured concentrations were very mixed-modespiked levelsAPCI and APCI/APPI. There were no on the close to the column for if the backgrounds (no spike) were deducted (Table 5). inmatrix effectfactors among LC systems for signi cant differences dansylated analytes in all sources (except for APPI) and for PFBBr derivatives in EC-APNI. Because of the existing backgrounds of NP, OP, and BPA, it is impractical to calculate their limits of detection (LODs); therefore, we only reported the intensities of 3:1) of E1, analytes using ESI Because the signal LODs (S/N= dansylated E2, E3 and EE2, which ranged were much more intense thanfor STP ef uents and and APPI between 0.23 and 0.52 ng/L others, we further investibetween 0.56matrix effects of these two sources ondansylated analytes gated the and 0.91 ng/L for river water (Table 5). Qin et al. also reported good methodwhich is a limits complexof 0.0380.13 ng/L using river water, detection more (MDL) matrix comparing for with the raw water mL using contain chloride derivatization [20]. river water of 500 and may dansyl higher levels of analytes. The In addition, Vulliet et al. in the river water may in of 0.010.20 ng/L endogenous analytes showed excellent LODs uence our determiwithout chemical derivatization forto avoid this potential problem, we nation on matrix effect factors; 1-L groundwater, which may result fromstable isotope-labeled aanalytes to the injection after extracspiked good recoveries and large-volume residues (100 L) tion (the levels were equivalent to 80 ng/L in the water) before [33]. Although tandem-MS methods (SRM) provide better sensitivity and selectivity than those of single-MS methods (SIM), most labs cannot afford tandem-MS instruments. However, this study, for example, was able to detect steroid estrogens inall real samples of STP ef uents with SIM after dansyl derivatization, and the measured levels of either spiked or non-spiked samples were similar to those using SRM (Fig. 6). The LODs of steroid estrogens in ef uents using SIM were 1.031.75 ng/L. The on-column detection limits of dansylated steroid estrogens with SRM and SIM were0.050.20 and 0.441.48 pg, respectively.

time. We exhaustively dansyl chloride. performance of common of derivatization with investigated the The matrix effect factors ionizationprobes and UPLC with post-column split, mixed-mode colESI using UPLC, differentLC systems. ESI is usually reported to more subjectto ion suppressionthanAPCIand APPI, but this was not umn and 2D-LC were 17.7%70.3%, 22.0%57.3%, 40.2%60.4% and the 16.1%62.1%, respectively; a mixed-mode column or the RAM case in this study. Although the factor values of APPI using UPLC, pre-column did not substantially reduce the matrix effects25.9%49.9% mixed-mode column and 2D-LC were 15.7%46.7%, better than UPLC for the water matrixes we tested, their use witheffect of ESI and 22.1%63.2%, respectively. Therefore, the matrix other matrixes, such as food or tissues, are worth conditions, and none of the and APPIwere similarunderthe sameLC further explorations. four LC systems could signi cantly eliminate ion suppression. The cutoff of molecular mass for RAM pre-columnis about 15 kDa; consequently, the RAM column could not eliminate the matrix effect Acknowledgement molecules [32]. In a previous study, Kloepfer et caused by small al. reported that a post-column split (down to 20100 L/min) not only enhances sensitivity but also reduces ion suppression by This work is supported by the National Science Council, Taiwan 4060% inwastewater samples (NSC 96-2314-B-002-101-MY2). [32]; However, the UPLC with postcolumn split did not reduce matrix effects signi cantly comparing with that References without split in our study.
[ 1 ] B. R. C ar r , J.E . G ri f n, i n: J .D . W i l s on, D . W. F os t er, H . M. Kr o nenb e rg , L. P. Ree d ( E d s. ), W i l l i a ms T ex tb o ok o f E nd ocr i no l o gy , W. B. Sa u nd er s C o mp a ny , Phi l a d el p hi a , 19 9 8 , p . 9 0 1. [ 2 3.4..C . Johns o n, A . Be l fro i d , A . D i C o r ci a, Sci . Tot al E nvi r o n. 2 5 6 ( 20 0 0 ) 1 6 3. ] A Method validation [ 3 ] C . Bar o nti , R. C u ri ni , G. D As c enzo , A . D i C o rci a , A . G ent i l i , R. S amp e ri , E nv i r on. S ci . T echno l . 3 4 ( 2 0 0 0 ) 5 05 9 . [ 4 ] B. Because U . E hr ho rn, O .P. H ee mk en, UPLC o m, H . Re i ncke , G . Saw al , N . T heo b al d , St ache l , dansyl derivatizationunder P. L ep coupled withESI proE nv ro best l u t. 1 2 4 ( 20 0 3 ) 4 9 7. vided ithen. P o lperformance based on the sensitivity and matrix [ 5 effect, we validatednmai e r, W. Ko Good E nvi r o n. Po l l precision were ) 2 91. ] U . Bo l z, H . H a ge this method. r ner , accuracy and u t . 115 (2 0 0 1 [ 6 ] H . M. Ku ch, K. Ba l l s chmi te r, E nv i ro n. S ci . Te chno l . 3 5 ( 20 0 1 ) 3 2 01 . obtained rfor calibration ,curves.eThedintra-daycel o ,inter-day accu- A 10 4 5 ( 2 0 0 4 ) [ 7 ] S . Ro d i gu e z- Mo zaz M. J. d A l a, D. Bar and J. C hr om ato g r. racy . were within 2% and 9%, respectively; the inter-day variations 85 [ 8 (RSD%)s ranged fromne n, M . V i 14.6% K ro nb er g , S ciresponsesnv i r on. 3 7 8 (2 0 0 7 ) ] L . S al t e, P. Le s ki 0.88% to r ta, L. and intra-day . To ta l E were almost identical. The linearity of calibration curves of steroid estro343. [ 9 gensY. C he n, T .Y. We n, G . S. Wa ng, Hsimilarhengbetter than those Li e n, S ci . To ta l E nv i r on. ] C . (10 00-fold to 10,000-fold) are . W. C or , Y. H . Li n, G. W. of Qin( et0al. (0.2200 ng/mL, 1000-fold) [20] and Vulliet et al. 3 7 8 2 07 ) 3 5 2 . [1 0 (0.0520 ng/mL, ams , W. La [33]. t, A. D e Le enhe er , J. C hro ma to gr . A 10 2 9 ( 2 0 0 4 ) ] T . Be nijt s , R. D 400-fold) mb er 1 5 3. [ 11 ] H . Ko nd o , Y. Ok ad a , K. S hi mb o, Y. Fu s ho , E as t er n A nal y ti ca l S ymp o s i u m and E xNobsteroid estrogens s et , Ndetectedey , U SAlaboratory blank, hi i t (E AS ), S o mer were ew Jer s in any , 2 0 0 6. [1 2 ] J. C hi co , S. Meca , R . C o mp any o, M. D . P ra t, M. Gr ana d o s , J. C hro ma to gr . A 118 1 but NP, OP and BPA were detected using dansyl derivatization; ( background the2 0 0 8) 1 . levels in the reagent blanks of Milli-Q water were [1 3 60130 ng/L. The possible sources man, B.OP and BPA C . H ibe a l go , J.V . Sa ncho, F. H er na nd ez, ] E . A. H o ge nd oo r n, E. D i jk of NP, Bau ma nn, could d A nal . C he m. 7 1 19 9 ) 1111. the speedisk for solid( phase9extraction (packed in a plastic disk [1 4 vessel), G ang l , M. water itself (plastic materials in Vo u r os , Anal . C hem. 7 3 (2 0 0 1) 5 6 3 5 . ] E . T. the Milli-Q M. A nnan, N . S p o one r, P . the waterpuri .ed system) or the derivatizationprocess.Vo uexample,C hro ma to gr . A 10 5 3 (2 0 0 4 ) 15 1. [1 5 ] C L. A nd re ws , C .P. Yu , E. Ya ng, P. For ro s , J. during [1 6 the derivatizationR. Ba k hti areagentshu , S . H u/NaOH bufferFr a nkl i n, D . C . E va ns , Ana l . C hem. ] M .R. A nar i , procedure, r, B. Z (NaHCO3 s ke y, R. B. and [ 17 [ 18 [1 9 [2 0 [2 1 [2 2 ] ] ] ] ] ] 7 4 ( 2 0 02 ) 4 13 6 . A . S al va d o r, C . Mo r et to n, A . P i ra m, R. Fa u re , J. C hr o mat o gr . A 114 5 (2 0 0 7) 10 2 . G . S i ngh, A. G u ti e r re z, K . Xu , I. A. Bl ai r, Anal . C hem. 7 2 (2 0 0 0 ) 3 0 07 . W .Z . S ho u , X. Y. Ji ang , N .D . W e ng, Bi o me d . C hr om ato g r. 1 8 ( 20 0 4 ) 4 14 . F. Qi n, Y. Y. Zha o , M.B. Saw y er, X. F. Li , A nal . C hi m. Act a 6 27 (2 0 0 8 ) 91 . R. E . N el s o n, S .K . Gr e be , D .J . O Ka ne, R .J. S i ngh , C l i n. C hem . 5 0 ( 20 0 4 ) 3 7 3. L . S o ng, A .D . We l l man, H . Yao , J. A d co ck, Ra p i d C o mmu n. Mas s Sp e ctr o m. 21

4. Conclusions In this study, we present a quantitative method for the analysis of seven estrogenic compounds with dansyl derivatization in both SIM and SRM. Withthe improvement in sensitivity using UPLC and chemical derivatization method, environmental levels of these chemicals can be determined using a single MS instead of the more expensive tandem-MS. The instrumental throughput was signi cantly increased, with a run in 3.2 min plus 1-min re-equilibrium

( 2 0 07 ) 1 3 43 . [2 3 ] Y. H . L i n, C . Y. C he n, G . S. Wa ng, Ra p i d C o mmu n. Mas s S p ect ro m. 2 1 (2 0 0 7 ) 1 97 3 . [2 4 ] R. M oha me d, Y. A. H am mel , M. H . Le Bre to n, J.C . T ab e t, L. Ju l l i e n, P.A . G u y, J. C hro m ato g r. A 11 6 0 ( 20 0 7 ) 1 94 . [2 5 ] S .S . C ai , L. C . Sho r t, J. A. S ya ge , M. Po tv i n, J. M. C u rt i s , J. C hro ma to gr . A 1 173 (2 0 0 7 ) 88. [2 6 ] L .P. Pe nze s , G. W. Oe r te l , J. C hr om ato g r. 5 1 ( 19 7 0) 3 2 5 . [2 7 ] D . B. Ro bb , M.W . Bl ad e s , J. A m. S oc. Mas s Sp e ctr o m. 1 6 ( 20 0 5 ) 1 27 5 . [2 8 ] T .J. Ka u p p i l a, A. P. Br u i ns, R. Ko s ti a i nen, J. Am. S oc . M as s S p ect ro m. 16 (2 0 0 5 ) 1 3 99 . [2 9 ] D . B. Ro bb , M.W . Bl ad e s , J. A m. S oc. Mas s Sp e ctr o m. 17 (2 0 0 6 ) 1 30 . [3 0 ] L .C . Sho r t, K .A . H a nol d , S. S. C ai , J.A . S yag e, Rap i d C o mmu n. Mas s S pe ct ro m. 21 ( 2 0 07 ) 1 5 61. [3 1 ] L .C . S ho rt , S .S . C ai , J .A . Sy ag e, J. Am. S o c. Ma s s S p ect r om. 18 ( 2 0 0 7) 5 8 9 . [3 2 ] A . Kl o e p fer , J.B. Qu i nt ana , T. Re em ts ma , J. C hr om ato g r. A 1 0 67 (2 0 0 5 ) 15 3 . [3 3 ] E . V u l l i et , L. W i e s t, R . Bau d o t, M .F. G r eni e r- Lo u s ta l ot , J. C hr o mat og r. A 12 10 ( 2 0 0 8) 8 4 . [3 4 ] C . Y. C heng , C . Y. W u , C . H . W ang , W .H . D i ng , C hemo s p he re 6 5 (2 0 0 6 ) 2 27 5 . [3 5 ] W .H . Di ng , C . Y. Wu , J. C hi n. C he m. So c- Tai p . 47 (2 0 0 0 ) 11 55 .

Fi g. 6 . C hro ma to gr am s o f d ans y l -E 1 i n e f u e nts fr om a s e wa ge t re at ment p l ant b et we en: ( a) S RM and (b ) S IM mo d e.