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Contents lists availa le at !"ien"e#ire"t

Journal of Chromatography A
$ournal homepage% &&&'elsevier'"om(lo"ate("hroma

Comparison of ele"trospray ioni)ation, atmospheri" pressure "hemi"al ioni)ation an* atmospheri" pressure photoioni)ation for *etermining estrogeni" "hemi"als in &ater y li+ui* "hromatography tan*em mass spe"trometry &ith "hemi"al *erivati)ations
,uang-.en /ien, Chia-0ang Chen 1, ,en-!huh .ang
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 1 ! ! , Ta i wan

arti"le info
"r ti c l e h i s to ry# =e" ei ve * 26 !e p te m er 20 0 B =e" ei ve * i n r ev i s e* fo r m 5 # e"e m er 2 0 0B A"" ep te * 1 1 # e" em e r 2 0 0 B Ava i l a l e o nl i ne 2 7 # e "em e r 2 0 0 B

a stra"t
2his stu*y "ompare* the sensitivities an* matri3 effe"ts of four ioni)ation mo*es an* four reverse*phase li+ui* "hromatographi" (/C) systems on analy)ing estrone (41), 15 -estra*iol (42), estriol (46), 15 -ethinylestra*iol (442), 7-nonylphenol (89), 7-tert-o"tylphenol (:9), isphenol A (;9A) an* their *erivatives of *ansyl "hlori*e or penta uoro en)yl romi*e (9<;;r) in &ater matri3es using a triple-+ua*rupole mass spe"trometer &ith sele"te* rea"tion monitoring (!=>)' 2he four pro es &ere ele"trospray ioni)ation (4!?), atmospheri" pressure "hemi"al ioni)ation (A9C?), atmospheri" pressure photoioni)ation (A99?) an* A9C?(A99?@ the four /C systems &ere ultra-performan"e li+ui* "hromatography (A9/C) &ith or &ithout post-"olumn split, a mi3e*-mo*e "olumn an* t&o-*imensional /C (2#-/C)' #ansylate* "ompoun*s &ith 4!? at A9/C "on*ition ha* the most intense signals an* less matri3 effe"ts of the various "om inations of ioni)ation an* /C systems' 2he on-"olumn limits of *ete"tion (/:#s) of *ansylate* estrogens y !=> &ere 0'050'20pg, an* the /:#s in se&age treatment plant ef uent an* in river &ater &ere 0'260'52 an* 0'560'91ng(/, respe"tively' 2he /:#s using sele"te* ion monitoring (!?>) rea"he* lo& ng(/ levels in real samples an* measure* "on"entrations &ere "ompara le &ith those of !=>' C 200B 4lsevier ;'D' All rights reserve*'

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1. Introduction <emini)ing "ontaminants of steroi* estrogens, *etergent *egra*ates an* plasti"i)ers have "ause* a &orl*&i*e "on"ern' 2hey may in uen"e the e"osystem at tra"e levels an* affe"t human health through their "ontamination of *rinEing &ater' 8atural estrogens 15 -estra*iol (42) an* its syntheti" analogue 15 -ethinylestra*iol (442), an ingre*ientin oral "ontra"eptives, are the most estrogeni"' >oreover, their ma$or meta olites, estrone (41) an* estriol (46), are still ioa"tive' 2hese steroi* estrogens enter the &ater environmentvia the urineof humans an* animals in theform of hy*rophili" glu"uroni*e an* sulfate "on$ugates F1G, &hi"h are iologi"ally ina"tivate* F2G' Ho&ever, they are liEely to e *e"on$ugate* in se&age treatment systems an* "onverte* to estrogeni"allya"tivefreeforms

3enoestrogens are release* into the &ater environment from *aily usage of non-ioni" surfa"tants an* plasti"i)ers' Atmospheri" pressure photoioni)ation (A99?) is an emerging sour"e, &hi"h is "apa le of ioni)ing nonpolar "ompoun*s an* is possi ly less sus"epti le to matri3 effe"ts' ?n a**ition, *ualsour"e ioni)ation (e'g' atmospheri" pressure "hemi"al ioni)ation (A9C?)(A99? "om o in this stu*y) e3pan*s the range of "ompoun*s that "an e simultaneously analy)e*' Although most stu*ies *etermine* femini)ing "hemi"als &ith ele"trospray ioni)ation (4!?) "ouple* &ith /C(>!((>!) F59G, the suita ility of A9C? an* A99? *eserve further e3ploration' >atri3 effe"t, &hi"h "o-eluting "omponents from the matri3 or the mo ile phase may enhan"e or suppress signals, is animportant issue in using /C(>!(>!' !ele"tive e3tra"tion, a**itional "lean-up, ef "ient/C separationor"hange of mo ilephase "ompositions may re*u"e matri3 effe"ts F10G' <urthermore, &hile the use of suita le internal stan*ar*s (e'g' isotope-la ele* "hemi"als)may "orre"t signal irrepro*u"i ility, this approa"h &ill not e a le to over"ome the loss in sensitivity "ause* y matri3 effe"ts' !ome stu*ies utili)e* *ire"t online e3tra"tion or post-"olumn split to minimi)e matri3 effe"ts an* simplify the sample preparation' A novel "olumn *evelope* on !eptem er 2006 "om ines oth si)e e3"lusion an*

F6G' 7-nonylphenol (89), 7-tert-o"tylphenol (:9) an* isphenol A (;9A), &hi"h are all 3enoestrogens, "an affe"t normal en*o"rine fun"tions' Although they are less potent, they are usually foun* in mu"h higher "on"entrations in &ater (ng(/ g(/) F66G' 2hese

1 C or r es p o n* i ng au t ho r' 2 el ' % IB B 6 2 6 66 6 B 10 1 @ fa3 % IB B 6 2 2 6 51 95 5 5' E &m a i l a %% r ess # % b ms 'ntu ( e % u( t w )C ( &*( C hen+ ( 0 0 21 - 9 6 56 (J s ee fro nt ma tt er C 2 0 0 B 4l s e vi e r ;' D' A l l r i g hts re s er v e* ' * o i %10 '1 01 6 ($'" hro ma '2 0 0 B '12 ' 02 6

, (& -( .i en et al ( / 0 ( C h ro m ato gr ( " 1112 )1

3 + 3 ! 24 3 22

9 55

reverse-phase "hemistry to separate small mole"ules from "omple3 matri3 F11G@ to the est of our Eno&le*ge, one stu*y has analy)e* *rugs in ovine serum using the mi3e*-mo*e "olumn F11G' A restri"te* a""ess material (=A>) pre-"olumn, &ith a similar separation me"hanism to the mi3e*-mo*e "olumn, has also een applie* onanaly)ing foo*, iologi"al an* environmental samples F12,16G' Ho&ever, the =A> pre-"olumn is an alEyl-*iol sili"a (A#!) "olumn an* provi*es little "hromatographi" separation for small mole"ules@ it re+uires a "olumn s&it"h to "onne"t it &ith an a**itional analyti"al "olumn for "hromatographi" separation (a t&o-*imensional /C, 2#-/C)' ?n a**ition, a post-"olumn split *elivers only a portion of /C o& into the >!, &hi"h may su stantially *e"rease matri3 effe"ts, espe"ially &hen a o& rate into 4!? interfa"e &as *e"rease* to nano o& of 0'1 /(min F17,15G' 2his nanosplit re+uires spe"ial nanospray pro es, &hi"his notamena le to a "onventional 4!? interfa"e, &hose o&rates "an e only as lo& as 2050 /(min' =eports on the mi3e*-mo*e "olumn, 2#-/C an* post-"olumn split are very limite* in environmental analysis an* so little is Eno&n a out their a ility to re*u"e matri3 effe"ts'

F27,25G' Ho&ever, the "on"lusions ase* on the results using nonoptimi)e* parameters of various ioni)ation metho*s "oul* e "ontroversial' 2he main purpose of the stu*y &as to n* out the est "om ination of a "hromatographi" system an* an ioni)ation metho* &ith satisfa"tory sensitivity using lo& volumes of &ater samples or single +ua*rupole >!' 2he nal metho* &as vali*ate* using river &ater an* ef uents from a se&age treatment plant (!29)'

2. Experimental 1(1( Chemicals an% reagents 4strone, 15 -estra*iol, estriol, 15 -ethinylestra*iol, 7-terto"tylphenol, isphenol A, an* isphenol A-*16 (as a re"overy stan*ar*) &ere o taine* from !igma(Al*ri"h (!aint /ouis, >:, A!A@ purity K 9BL)' 2he te"hni"al mi3ture of nonylphenol &as supplie* y =ie*el-*e HaMn (!eel)e, ,ermany@ purity K 97L)' 2,7,16,16-2 #7 -estrone, 2,7,16,16-2 #7 -15 -estra*iol, 2,7,15-2 #6 16 -hy*ro3y- 15 -estra*iol, 2,7,16,16-2 #7 -15 -ethinylestra*iol an* 7-n-:"tyl- *15-phenol &ere ought from C(#(8 ?sotopes (9ointe-Claire, Nue e", Cana*a@ purity K 9BL)' ;isphenol A-16 C1 2

=e"ently, there has een an in"rease in the num er of stu*ies using ultra-performan"e li+ui* "hromatography (A9/C) "om ine* &ith >!(>!' A9/C taEes a*vantage of smaller pa"Eing parti"les (O2'0 m) that ena le high o& rates for fast"hromatography &ithout sa"ri "ing separationef "ien"y, an* signal-to-noise (!(8) ratios of analytes are in"rease* e"ause of sharp peaEs' Ho&ever, to est of our Eno&le*ge none have use* A9/C(>!(>! to stu*y estrogeni" "ompoun*s in &ater' !teroi* estrogens an* phenoli" 3enoestrogens are &eaE a"i*s an* their ioni)ation on 4!? an* A9C? are not very ef "ient "ompare* &ith other more polar "hemi"als' Chemi"al *erivati)ation "an a** on moieties improving ioni)ation an* enhan"e signals' <ore3ample, *ansyl "hlori*eor penta uoro en)yl romi*e (9<;;r) "an rea"t &ith phenoli" groups, signi "antly improving sensitivity F161BG' ;y a**ing the *ansyl moiety &ith 4!? interfa"e, signal intensity may e in"rease* as mu"h as three or*ers of magnitu*e F16,19,20G' 2his te"hni+ue has een also foun* to improve the sensitivity in A9C? interfa"e &hen use* to measure steroi* estrogens F21G' 2o *ate no *ansyl *erivatives have een analy)e* &ith A99? interfa"e' 9<;;r *erivatives "an "apture soft ele"trons in A9C?, resulting in unsta le metasti" ions, an* "ause su se+uent *isso"iation to generate negative ions through the loss of penta uoro en)yl ra*i"al (ele"tron-"apture atmospheri" pressure negative ioni)ation, 4C-A98?)' <or estrone, the use of 9<;;r*erivatives in4CA98? "an enhan"e ef "ien"y of ioni)ation as mu"h as 25 times that of A9C? alone F1BG' 2his metho* has een also use* in A99? &ith a high toluene *opant o& rate (e'g' 200 /(min or higher) an* &as foun* to e a le to *ete"t as little as 0'15 pg of 2,7-*initrophenol

&as pur"hase* from Cam ri*ge ?sotope /a oratories (An*over, >A, A!A@ purity K 99L)' #ansyl "hlori*e (5-(*imethylamino) naphthalene-1-sulfonyl "hlori*e, P95L purity), penta uoro en)yl romi*e (9<;;r, purity K 99L), 7-methylmorpholine (purity K 99'5L), so*ium hy*rogen "ar onate, an* potassium hy*ro3i*e &ere pur"hase* from !igma(Al*ri"h' >illi-N &ater &as o taine* from a >illipore &ater puri "ation system (>ilfor*, >A, A!A)' <ormi" a"i* (purity K BBL) an* formal*ehy*e (purityK 65L) &ere provi*e* y J'2' ;aEer (9hillips urg, 8J, A!A)' !olvents, in"lu*ing methanol, a"etone, n-heptane, a"etonitrile an* toluene, &ere all H9/C gra*e from J'2' ;aEer'

1(1( E5traction 2he pro"e*ure use* to e3tra"t estrogeni" "ompoun*s from the &ater sample has eenpreviously *es"ri e* F9G' ;rie y, &ater samples &ere spiEe* &ith internal stan*ar*s an* then ltere* through 90-mm 9D#< mem ranes (pore si)e 0'75 m) to remove suspen*e* soli*s efore e3tra"tion' 43tra"tion &as performe* using 50-mm ;aEer on* 9olar9lus C1B !pee*isEs (J'2' ;aEer), follo&e* y a "leanup using 70L methanol(60L >illi-N &ater (v(v)' 2he *isEs &ere *rie* for 10 min un*er a va"uum of a out -25 E9a' Analytes &ere elute* &ith three portions of 5-m/ 50L methanol(50L *i"hloromethane (v(v)' 2he eluates &ere ltere* through 25-mm 92<4 syringe lters (pore si)e 0'2 m) an* "on"entrate* to *ryness at75 C y a !pee*Da" "on"entrator (2hermo !avant !9# 1010, HolrooE, 80, A!A)' 2heresi*ues &ere re-*issolve* y spiEing re"overy stan*ar* an* then rea"te* &ith *ansyl "hlori*e reagents'

F22G, &hereas for 9<;;r-*erivati)e* estrone, signal enhan"ement (1'79'B times) &as less than that using 4C-A98? F1BG' :ur group previously reporte* that *ansylate* estrogens &ith 4!? interfa"e provi*e* etter signal intensities than that 9<;;r *erivatives &ith 4C-A98?, ut o vious signal suppression &as en"ountere* &ith 4!? &hen analy)ing "omple3 matri3es su"has river &ater an* ef uents from se&age treatment plants F26G' ?n this stu*y, &e investigate* signal intensity an* matri3 effe"ts on various "hromatographi" systems (A9/C &ith or &ithout o& split, mi3e*-mo*e "olumn, 2#-/C) an* several ioni)ation mo*es (4!?I, 4!?-, A9C?I, A9C?-, A99?I, A99?-, A9C?(A99?I, A9C?(A99?-) for othestrogeni" "ompoun*s an* their *erivatives of *ansyl "hlorine an* 9<;;r' ?n a**ition, the stu*y is uni+ue in that it rst optimi)e* the operation "on*itions spe"i " for ea"h ioni)ation metho*s, in"lu*ing those for /C "olumns, mo ile phase o& rates an* "ompositions' 9revious stu*ies usually "ompare* the performan"e of *ifferent ioni)ation sour"es un*er only one analyti"al "olumn Eept at a "onstant solvent o& rate, iso"rati" "hromatography, the same in$e"tion volume, or o& in$e"tion analysis alone

1(6( 7erivati8ation 1(6(1( 7ansyl chlori%e %erivati8ation 2he pro"e*ure use* to *erive *ansyl "hlori*e &as ase* on 442
*erivati)ation metho* use* y 9en)es an* :ertel F26G an* !hou etal' F19G' ;rie y, 0'9 m/ of 100 g(m/ analytes in a"etone &as vorte3e* for 1'0 min&ith0'1 m/ of 1 mg(m/ *ansyl "hlori*e in*ry a"etone follo&e* y mi3ing &ith0'01 m/ of 0'18 so*ium hy*ro3i*e for 1'0 min' 2hemi3ture&as Eept at 50 C for 60 min' 5 m/ of n-heptane &as a**e* to the mi3ture &hi"h &as then shaEen for 6 min' ?t &as "entrifuge* at6000 rpm for 10 minan* refrigerate* at-20 C' :n"e itha* separate* into t&o layers, the organi" layer &as "olle"te* an* ltere* through0'20- m 92<4 into another glass tu e' 2he a+ueous layer &as *is"ar*e*' 2he organi" layer &as evaporate* to *ryness at 75 C y a !pee*Da" "on"entrator' 2he resi*ue &as re-*issolve*

95B

,( &-( .i en et a l( / 0 ( C h r om at og r( " 111 2 )1

3+ 3 ! 24 3 2 2

F ig. 1. S chem e of t he 2D - LC s ys t em. t the s t ar t o f a nal y s i s ! a"# the s am p l e $a s i n%e cte d a nd $ ent t hro u g h t he & ' co l u mn# a nd t he mo ( i le p has e $a s ) * ac eto ni t ri l e +$ at er. , hre e mi nu te s l a te r# - al - e $a s au t o mat i cal l y s $ i tc hed to ! ( ". t he ana ly t es $e re ( a c/ u s he d to a n a nal y ti ca l c ol u mn $ i th a s u i ta ( le mo( i l e p ha s e gr ad i e nt fr o m p u mp 0 and the n i ntr od u c ed to the t and em ma s s s p e ct ro met er .

&ith 0'9 m/ of methanol to optimi)e the parameters of operations on the >!' Ho&ever, the a ove proto"ol "annot e *ire"tly applie* to the *erivati)ation of &ater samples an* nee*s another proto"ol that &as mo*i e* from Anari etal' F16G an* 8elsonetal' F21G'100 / of 0'9 ng( / (for 4!? analysis) an* 250 / of 0'66 ng( / (for A99?, A9C? an* A9C?(A99? analysis), oth in a"etone, &ere vorte3e* &ith 250- an* 625- / so*ium i"ar onate uffer (10 m>, pH a*$uste* &ith8a:H(a+ ) to 10'5), respe"tively' 2o these stan*ar*s &ere a**e* 250 an* 625 / of 1-mg(m/ solution of *ansyl "hlori*e, respe"tively' 2hey &ere thenin"u ate* at60 C for 6'0 minan* evaporate* to *ryness in a !pee*Da" "on"entrator' 2he resi*ues &ere re"onstitute* &ith 100 an* 250 / of methanol, respe"tively' 2hen, 7 / (for 4!? mo*e)an* 10 / (for A99?, A9C? an* A9C?(A99? mo*e) &ere in$e"te* into /C(>!(>! for "omparative analysis'

1(9(1( The mi5e%&mo%e column A !ho*e3 :#9 2 H9-2# (2'0 mm Q150 mm, 5 m) &as use* for 4!? an* A99? at a o& rate of 0'2 m/(min, &hile *ansylate* analytes in A99? &ere set at 0'5 m/(min' A !ho*e3 :#9 2 H9-7# "olumn (7'6 mm Q 150 mm, 5 m)&as use* for A9C? an* A9C?(A99? at a o& rate of 1'0 m/(min' 1(9(6( 17&.C system with :"; pre&column 2his system &as "ompose* of a D?C? si3-port s&it"hing valve (Dal"o ?nstruments ?n"', Houston, 2R, A!A) an* an e3tra iso"rati" pump (Jas"o 9A-9B0, 2oEyo, Japan) (<ig' 1)' 2he elution pro les of the =A> pre-"olumn (/iChrosphere =9-7A#! (25 mm Q 2mm, 25 m)) for ef uent of se&age treatment plants an* river &ater &ere monitore* &ith a AD *ete"tor set at 2B0 nm' 2he in$e"tion volume &as 50 / of ea"h e3tra"t, an* the o& rate of mo ile phase of >illi-N &ater-a"etonitrile (95%5, v(v) through =A> pre-"olumn &as set at 1 m/(min' 2he time re+uire* to elute ma$or matri3 "omponents &as less than 6 min (pro le not sho&n)' ;ase* on this result, the valve &as s&it"he* after 6 min to a"E ush the analytes into an analyti"al "olumn' A 2hermo Hypersil ,ol* "olumn (2'1 mm Q 50 mm, 1'9 m, ;ellefone, 9A, A!A) &as use* for 4!? an* A99?, &hi"h o& rate &as 0'2 m/(min, &hile *ansylate* analytes in A99? &as set at 0'5 m/(min' A 2hermo ;eta;asi" C1B "olumn (7'6 mm Q 150 mm, 6 m) &as use* for A9C? an* A9C?(A99? set at a o& rate of 1'0 m/(min' All "hromatographi" separations in"lu*ing A9/C, mi3e*-mo*e "olumn an* 2#-/C &ere performe* at 60 C (2a le 1)'

1(6(1( P<==r %erivati8ation 9<;;r &as *erive* ase* ona pro"e*ure reporte* y !ingh et al' F1BG' .e vorte3e* 250- / mi3ture stan*ar*s of native (0'66 ng( /) analytes in methanol &ith 250 / of potassium hy*ro3i*e in anhy*rous ethanol (B%100 0@ &(v)' .e then a**e* 250 / of 5L 9<;;r in a"etonitrile' 2he mi3ture &as aEe* at 60 C for 60 min an* then evaporate* to *ryness ina !pee*Da" "on"entrator' 2he resi*ue &as re"onstitute* &ith 250 / of methanol, an* 10 /&as in$e"te* into /C(>!(>! for "omparative analysis'

1(9( .C systems an% analytical columns 1(9(1( The UP.C with or without post&column split
A .aters ;4H C1B "olumn (2'1 mm Q 100 mm, 1'5 m) &as use*

1(!( Instruments an% parameters 2he separation an* *ete"tion &ere performe* on a .aters A"+uity A9/C system (.aters Corporation, >ilfor*, >A, A!A) "ouple* &ith a .aters Nuattro 9remier R4 triple +ua*rupole mass spe"trometer' 2he A9/C(>!(>! system &as "ontrolle* y

for 4!? an* A99? at a o& rate of 0'5 m/(min, &hile native analytes in A99? &ere set at 0'2 m/(min for etter signal intensities' A !epa3 ,9-C1B "olumn (6'0 mm Q100 mm, 1'5 m) &as use* for A9C? an* A9C?(A99? at a o& rate of 1'0 m/(min' 9ost-"olumn split (split ratio S 1%5) &as teste* on 4!?'

,a (le 1 # i ffe re nt /C s ys t ems an* i o ni )at i o n m o* e s f or o t h na ti v e "o mp o u n*s a n* * e ri v at iv es & i th 9 <;;r o r * a ns yl "hl o r i * e' C o l u mn t y p e A 9/ C & i t h o r & i t hou t s p l i t
;4 H C 1B (2 '1 mm Q 10 0 mm, 1 '5 m) 4! ? , 9 -C 1 B ( 6 '0 mm Q 10 0 mm, 1 '5 m) A 9 C ? ( (A9 9 ?) A9 C ? A 9 C ? ( (A9 9 ?)

8a ti v e (- )

9< ;;r ( - ) #a ns yl "hl o r i *e (I )

A9 9 ?

4! ?

A9 9 ?

>i 3 e* - mo * e /C : # 9 2 H 9- 2 # ( 2' 0 mm Q 1 5 0 m m, 5 m) 4! ? : # 9 2 H 9- 7 # ( 7' 6 mm Q 1 5 0 mm, 5 m) A 9 C ? ( (A9 9 ?) A9 C ? A 9 C ? ( (A9 9 ?) 2 # -/ C & i th =A > H y p er s i l ,o l * (2 '1 mm Q 5 0 mm, 1'9 m ) 4! ? ;e ta ;as i " C 1B (7 ' 6 m m Q 15 0 mm, 6 m ) A 9 C ? ( (A9 9 ?) A9 C ? A 9 C ? ( (A9 9 ?)

A9 9 ?

4! ?

A9 9 ?

A9 9 ?

4! ?

A9 9 ?

, (& -( .i en et al ( / 0 ( C h ro m ato gr ( " 1112 )1 ,a (le 2 >a$o r >! p ar ame te rs fo r * i ff er ent a nal y te s a n* i o ni )a ti o n met ho *s ' 9a ra met er 8 ati v e

3 + 3 ! 24 3 22

9 59

9< ;;r # ans y l "hl o r i * e 4 !?- A9 C ?- A 99 ?- A 9C ?( A9 9 ?- A9 C ?- 4 !?I A 9C ?I A9 9 ?I A9 C ?(A 99 ?I

!o u r "e 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 #e s o l vat i o n t emp e ra tu r e ( C ) 70 0 6 0 0 6 0 0 6 0 0 70 0 7 50 5 0 0 5 0 0 50 0 C on e ga s o & ( /(h) 50 0 0 0 0 0 0 5 0 0 #e s o l vat i o n g as o & ( /(h) 90 0 5 5 2 0 0 5 5 1 50 10 0 0 6 0 0 2 0 0 60 0 C or o na ( A) 6 0 6 0 60 2 'B 2 ' B C ap i l l a ry (E D) 6 =ep e l l er ( E D) 22

2'B 66

>ass/yn3 D7'1 &ith Nuan/yn3 Appli"ation >anager an* the *ata &ere a"+uire* an* pro"esse* using >ass/yn3 D7'1' ?nstrumental parameters in various ioni)ation metho*s &ere optimi)e* to a"hieve ma3imal analyte signal intensities'
Changes of the *esolvation gas (82 ) o& rate an* sour"e tem-

at100L ;for 1'0 min efore eing returne* to initial "on*ition' 2he "olumn &as re-e+uili rate* for 2'0 min' 1(!(1(6( "PCI )>+ an% "PCI/"PPI )>+( 1 m; formic aci% )"+ an% methanol )=+ were use% as the mobile phase( There were three .C con%itions( )1+ " ,P&C1? column ha% an initial gra%ient of ! @ =, followe% by a linear gra%ient to ?!@ = for (! min, an% then to 1 @ = in 1(A min, at which point it was hel% at 1 @ = for (6 minbefore being returne% to initial con%ition( The column was re&eBuilibrate% for 1 min( )1+ "n C7P 1 HP&97 column ha% an initial gra%ient of ! @ =, followe% by a linear gra%ient to ?!@ = for 6( min, an% then to 1 @ = in 6( min( It was hel% at 1 @ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min( )6+ " :"; couple% with a =eta=asic C1? column ha% a gra& %ient of 6 @ = for 6 min, followe% by a linear gra%ient to ! @ = in 1( min, then to ?!@ = in 1( min, an% then to 1 @ = in 1( min, at which point it was hel% at 1 @ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min(

perature *i* not pro*u"e signi "antly *ifferent signals in either 4!? or A99? interfa"e &hen o& rates of mo ile phase &ere at either 0'2 or 0'5 m/(min, so these t&o parameters &ere Eept the same for these t&o o& rates' >a$or parameters are summari)e* in 2a le 2' 43tra"tor voltage &as 6'0 D an* =< lens voltage &as 0 D' Collision gas &as argon at 6 Q 10- 6 m ar' ?on energy 1 an* 2 &ere set at 0'6 an* 6, respe"tively' ;oth /> 1 an* /> 2 resolution &ere set at 15' 2he multiplier voltage &as set at 650 D' ?ons &ere monitore* y sele"te* rea"tionmonitoring (!=>)as sho&n in 2a le 6' #ansylate* analytes pro*u"e* intense pre"ursor ions &ith m/8 D;>166(?E> , an% the collision&in%uce% %issociation pro%uce% intense pro%uct ions with m/8 1A1> an% m/8 1!2> , correspon%& ing to the !&)%imethylamino+&naphthalene moiety an% the loss of one methyl group from the m/8 1A1> , respectively D13,16E( P<==r %erivatives pro%uce% the same precursor ions as the un%erivati8e% ones with m/8 D;&HE& D16E( Feveral .C mobile phase compositions were teste% to obtain goo% separation an% peaG shapes( 7ata points across the peaG were no less than 1 to ensure the integration precision(

1(!(1( 7ansyl %erivatives 1(!(1(1( EFI )>+( 1 m; formic aci% )pH 1(3+ )"+ an% acetonitrile )=+ were use% as the mobile phase( There were three .C column con%i& tions( )1+ " =EH C1? columnwith an% withoutsplit ha% a gra%ient of ! @ = for (1 min, followe% by a linear gra%ient to ?!@ = in (? min, an% then to 1 @ = in 1(! min, at which point it was hel% at 1 @ = for (A min before being returne% to initial con%ition( The col& umn was re&eBuilibrate% for 1( min( )1+ "n C7P 1 HP&17 column ha% a gra%ient of ! @ = for 1( min, followe% by a linear gra%i& ent to ?!@ = in 1( min, an% then to 3 @ = in 1( min, at which point it was hel% at 3 @ = for 1(! min before being returne% to ini& tial con%ition( The column was re&eBuilibrate% for 9(! min( )6+ " :"; couple% with a Thermo Hypersil ,ol% column ha% a gra%i& ent of 6 @ = for 6( min, followe% by a linear gra%ient to ! @ = in 1( min, then to ?!@ = in 1( min, an% then to 1 @ = in 9( min, at which point it was hel% at 1 @ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min(

1(!(1( P<==r %erivatives 1(!(1(1( "PCI )&+( -ater )"+ an% methanol )=+ were use% as the mobile phase( There were three .C con%itions( )1+ " ,P&C1? column ha% a gra%ient of A @ = for (1 min, followe% by a linear gra%ient to 3 @ = in (? min, an% then to 1 @ = in 1(1 min( It was hel% at 1 @ = for (1 minbefore being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min( )1+ "n C7P1 HP&97 column ha% a gra%ient of A @ = for 1 min, followe% by a linear gra%ient to 3!@ = in 6( min( It was hel% at 3!@ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min( )6+ " :"; couple% with a =eta=asic C1? column ha% a gra%ient of 6 @ = for 6( min, followe% by a linear gra%ient to A!@ = in 1( min, an% then to 1 @ = in 9( min, at which point it was hel% at 1 @ = for 9( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 6( min(

1(!(1(1( "PPI )>+( 1 m; formic aci% )"+ an% acetonitrile )=+ were use% as the mobile phase( There were three .C con%itions( )1+" =EH C1? column ha% a gra%ient was setthesame as EFI mo%e( )1+ "n C7P 1 HP&17 column ha% a gra%ient of ! @ = for 1( min, followe% by a linear gra%ient to A @ = in 1( min, an% then to ?!@ = in 1( min, at which point it was hel% at ?!@ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min( )6+ " :"; couple% with aThermo Hypersil ,ol% column ha% agra%ient of 6 @ =for 6( min, followe% by alinear gra%ient to ! @ =in1( min, then to ?!@ = in 9( min, an% then to 1 @ = in 9( min( It was hel%

1(!(6( Native analytes 1(!(6(1( EFI )&+( 1 m; 9&methylmorphline )pH 3(!+ )"+ an% ace& tonitrile )=+ were use% as mobile phase( There were three .C con%itions( )1+ " =EH C1? column with an% without split ha% a gra& %ient of 1 @ = for (1 min, followe% by a linear gra%ient to 9 @ = in (? min, then to A @ = in 1(A min, an% then to 3!@ = in (! min( It was Gept at 3!@ = for (2 min before being returne% to the initial con%ition( The column was re&eBuilibrate% for 1(A min( )1+ "n C7P 1 HP&17 column ha% a gra%ient of 1 @ = for 1( min, followe% by a linear gra%ient to ! @ = in 6( min, an% then to A @ = in ?( min, at which point it was hel% at A @ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 9( min( )6+ " :"; couple% with a Thermo Hypersil ,ol% column ha% a gra%i& ent of 6 @ = for 6( min, followe% by a linear gra%ient to ! @ = in 1( min, then to A!@ = in 6 min, an% then to 1 @ = in 1 min( It was hel% at1 @= for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min(

960

,( &-( .i en et a l( / 0 ( C h r om at og r( " 111 2 )1

3+ 3 ! 24 3 2 2

,a (le 1 !e l e" te* r ea "ti o n mo ni to ri ng (! =>) tr ans i t i o ns , i n* i vi * u a l "o l l i s i o n e ner g y (C 4 , D ) an* "o ne vo l t age (C D, D) o f ana ly te s , an * l i ne ar r ang es & i th "al i of * ans y l at e* "o mp o u n* s'
Ana l yt e >. F> - H G- 8a ti v e 9 <;;r F >I2 6 6 'B G I # a ns yl "hl o ri * e

r at i on "u r ve e+ u at i ons

C 4 C D C 4 C D C 4 C D /i ne ar r ang e (ng ( / ) 4 1 2 50 ' 7 2 6 9 '1 K 1 75 ' 0 K1 76 ' 0 66 5 0 7 0 55 K1 5 6 '0 5 7 5 0

4 1 -* 7 2 57 ' 7 2 56 ' 7 K 1 76 ' B

4 + u at i o n 5 0 7 '1 K 1 51 '1 6 7 0 '0 0 0 1 1 0 S 6 'B 9 9 R I 0' 0 10 ,

:1H ( 33 3

5 0 B '1 K 1 51' 0 6 7 5 0 6 '1 K 151 '1 7 6 0 '0 0 1 1 0 S 2 'B 7 5 R I 0' 0 2 B,

:1H ( 33 ?

4 2 2 52 ' 7 2 51' 2 K 1B 6 '0 K1 7 7' B 70 6 5 6 B 60 K1 5 6 '2 5 B 5 5

4 2 -* 7 2 56 ' 1 2 57 '6 K 17 5' 0

5 10 '1 K 151 '0 7 6 5 2 2 '2 K 15 1'1 6 7 0 '0 0 0 1 1 0 S 6 '5 7 2 R I 0' 0 19 ,

:1H ( 33 A

4 6 2B B ' 7 2 B 5'2 K 15 0 '9 K1 75 ' 0 6B 5 0 6 6 50 K1 5 6 '1 6 2 6 0

4 6 -* 6 2 91 ' 7 2 9 0' 6 K 15 6' 0

5 2 5 '2 K 15 1' 0 6 7 5 6 0 '2 K 15 1'1 6 7 0 '0 0 0 1 1 0 S 6 '0 6 0 R - 0 '0 0 6 ,

:1H ( 33 ?

4 42 29 6 ' 7 2 9 5 '2 K 17 7 'B K1 59 ' 0 6B 5 5 7 0 55 K1 5 6 '0 5 B 6 0

4 42 - * 7 6 0 0 ' 7 2 9 B '9 K 17 5'0

5 6 7 '2 K 15 1' 0 6 7

8 9 22 0 ' 7 2 19 '2 K 16 6 ' 0 60 6 5 6 0 65 7 5 7 '2 K 15 1' 2 6 2 7 5 0 '0 5 6 0 S 6 '9 0 9 R I 0' 5 56 ,

:1H ( 33 3

K1 75' 0 : 9 20 6 ' 6 2 0 5' 0 K 16 6 '2 K1 75' 0 25 6 5 2 7 75 K1 5 6 '1 5 7 5 0 7 -n - : "ty l - * 15 -p he no l 22 6 ' 7 2 2 2 '1 K 1 05 '6 ;9A 22 B ' 6 2 25 '2 K 21 2 '1 K1 62 ' B 1B 6 5 1 B 65 K1 5 6 '0 5 0 7 5
; 9A -1 6 C 12 27 0 ' 2 2 6 B '9 K 2 26 ' 5

K1 5 6 '1 7 7 7 7 0 '2 K 15 1'1 6 7 0 '1 2 0 S 11'15R I 7 ' B2 6 ,

:1H ( 33 !

7 5 5'2 K 15 1' 0 6 7 7 6 2 '1 K 1 51 '2 6 B 0 '0 5 6 0 S 6 '0 5 0 R I 0' 6 B 7 ,

:1H ( 33 A

7 57 '1 K 151 '0 6 B

;9A -* 1 6 ( =!) 27 7 ' 7 2 71' 0 K 22 2 ' 6 22 6 5 7 5 B' 0 K 151 '0 6 B 7 5 N ote# 9&n & Cct yl & % 1A & p heno l wa s u s e % a s i nter na l s t an% ar % of N P an% CP (

1(!(6(1( "PPI )&+( -ater )"+ an% methanol )=+ were use% as mobile phase( There were three .C con%itions( )1+ " =EH C1? column ha% a gra%ient of 6 @ = for 1( min, followe% by a linear gra%ient to ! @ = in 1( min, then A!@ = in 9( min, an% then 1 @ = in 1( min( It was hel% at 1 @ = for 1( min before being returne% to initial con& %ition( The column was re&eBuilibrate% for 9( min( )1+ "n C7P 1 HP&17 column ha% a gra%ient of ! @ = for 1( min, followe% by a linear gra%ient to A!@ = in 1(! min, an% then to 3 @ = in !( min, where it was hel% at 3 @ = for 1 min before being returne% to ini& tial con%ition( The column was re&eBuilibrate% for 9( min( )6+ " :"; couple% with a Thermo Hypersil ,ol% column ha% a gra%i& ent of 6 @ = for 6( min, followe% by a linear gra%ient to ! @ = in 1( min, then to A!@ = in 6( min, an% then to 1 @ = in 1( min, at which point it was hel% at 1 @ = for 1( min before being returne% to initial con%ition( The column was re&eBuilibrate% for 1( min(

; in 7'0 min, &here it &as hel* at 100L ; for 7'0 min efore eing returne* to initial "on*ition' 2he "olumn &as re-e+uili rate* for 6'0 min' 1(2( ;etho% comparisons ;e"ause estrogeni" "ompoun*s are fre+uently o serve* in se&age or surfa"e &ater, it is *if "ult to o tain a matri3 &ithout estrogeni" "ompoun*s' =a& &ater from a *rinEing &ater treatment plant (.29) in 2aipei City, &hi"h only "ontains analytes at tra"e levels, &as use* as the matri3' 4+ual ali+uots from e3tra"ts of one-liter samples &ere use* for ea"h metho*' 4luates &ere "on"entrate* to *ryness at 75 C using a !pee*Da" "on"entrator an* &ere re"onstitute* y appropriate solvents, &ith or &ithout the spiEing of 90-ng native "ompoun*s for the follo&ing analyses% (1) native "hemi"als in 4!? (-), A99? (-), A9C? (-) an* A9C?(A99? (-)@ (2) *ansyl *erivati)ation in 4!? (I), A99? (I), A9C? (I) an* A9C?(A99? (I)@ (6) 9<;;r *erivati)ation in 4C-A98? (-)' 2he est metho* &as "hosen for metho* vali*ation ase* on signal intensities an* matri3 effe"ts' 2he per"entage matri3 effe"t (L>4) &as use* to assess matri3 effe"ts% peaE area of post-e3tra"tion spiEing(peaE area of stan*ar* Q 100' ;efore "al"ulation, the areas of samples &ithout spiEing &ere su tra"te* from the areas of samples'

1(!(6(6( "PCI )&+ an% "PCI/"PPI )&+( -ater )"+ an% methanol )=+ were use% as mobile phase( There were three .C con%itions( )1+ " ,P&C1? column ha% an initial gra%ient of 6 @ =, followe% by a lin& ear gra%ient to ! @ = for (! min, then to A!@ = in 1( min, an% then to 1 @ = in (! min( It was hel% at 1 @ = for (2 min before beingreturne% to initial con%itions( The columnwas re&eBuilibrate% for 1( min( )1+ "n C7P 1 HP&97 column ha% a gra%ient of ! @ = for 1( min, followe% by a linear gra%ient to A!@ = in 1(! min, an% then to 3 @ = in 6(! min, at which point it was hel% at 1 @ = for 1( min before being returne% to initial con%ition( The col& umn was re&eBuilibrate% for 9( min( )6+ " :"; couple% with a =eta=asic C1? column ha% a gra%ient of 6 @ = for 6( min, fol& lowe% by a linear gra%ient to A!@ = in 1( min, an% then to 1 @

1(A( ;etho% vali%ation 2&o types of &ater, ef uents an* river &ater, &ere use* for metho* vali*ation' 2he ef uents &ere sample* from a se&age treatmentplant in 2aipei' 2hatplant is a se"on*ary treatment fa"ility &ith an a"tivate* slu*ge units' 2he samples &ere "olle"te* in

, (& -( .i en et al ( / 0 ( C h ro m ato gr ( " 1112 )1

3 + 3 ! 24 3 22

9 61

F ig . 2 . S i gna l i nt ens i t i es o f na ti - e ana l yt es fo r - ar i ou s i o n so u r ce s u s i ng ! a" 2 3 LC !4 co u p l ed $ i th p o s t- co l u mn s p l i t "# !( " the mi xe d -m od e c ol u mn a nd ! c" 2 D -L C.

January 200B (pH 5'06, temperature S 21'0 C, #: S 7'29 mg(/)' 2he river &ater &as taEen from the Tee-/ung =iver in 2aipei in April 200B (pH5'0, temperatureS 22'7 C, #: S 1'2 mg(/)' 2hreesolutions, 0'25 ng( / of the four estrogens stan*ar*s (20, 100, or 200 /), 1 ng( / of the three 3enoestogens stan*ar*s (50, 250, or 500 /) an* 100 / of 0'5 ng( / internal stan*ar*s, &ere spiEe* into 0'5-/ &ater samples efore e3tra"tion' 4luates from the !94 *isE &ere "on"entrate* to *ryness at 75 C using the !pee*Da" "on"entrator an* &ere re-*issolve* &ith 200- / anhy*rous a"etone "ontaining 0'25 ng( / re"overy stan*ar*, an* then rea"te* &ith *ansyl "hlo-

ri*e *erivati)ation reagent' <our-mi"roliter solution &as in$e"te* into /C(>!(>!' 1(?( I"/IC, Buanti cation an% %ata analysis All glass&are &as rinse* &ith a"etone, n-heptane, *i"hloromethane an* methanol efore eing use* for e3periments' A lanE sample spiEe* &ith the internal stan*ar*s &as run &ith ea"h at"h of samples to "he"E e3perimental "ontamination an* provi*e a"Egroun* levels of the native analytes' A

Fig . 1 . S i gna l i nt ens i t i es of d ans y l at ed ana l yt es for -a ri o u s i o n s ou r ce s u s i ng ! a" 2 3L C ! 4 cou p l e d $ i t h p o s t- co l u mn s p l i t "# ! ( " t he mi x ed - mo d e co l u mn a nd ! c" 2D - LC . E C - 3 5 I !- " $ as fo r 3 F00r d er i -a ti -e s .

962

,( &-( .i en et a l( / 0 ( C h r om at og r( " 111 2 )1

3+ 3 ! 24 3 2 2

F ig . 6 . C hr oma to g ra ms o f d ans y l at ed co mp o u nd s ! 7 .2 ng + L o f s tand a r ds # 6- L i n%e cti o n" i n E SI mo d e u s i ng ! a" 2 3 LC # ! (" the mi x ed - mo d e co l u mn and !c" 2D - LC .

"ali ration "urve &as uilt at ea"h analysis' 2he linear range &ere 0'00011 ng( / for steroi* estrogens (e3"ept 42 0'0011 ng( /) an* 0'056 ng( / for 3enoestrogens (e3"ept :9 0'12 ng( /) at &eighte* (1(3)' ?sotope-*ilution te"hni+ues &ere use* to "orre"t variations in sample preparation an* instrumental performan"e@ peaE areas of *ansylate* analytes &ere normali)e* to their *euterium-la ele* internal stan*ar* for +uanti "ation' 2he s+uares of "orrelation "oef "ients (r2 ) &ere 0'995 or a ove for all "ali ration "urves as sho&n in 2a le 6' A one-&ay analysis of varian"e (A8:DA) &ith 2uEeyUs post ho" "omparison &as use* to "ompare the signal intensities an* matri3 effe"ts asso"iate* &ith the *ifferent metho*s' !tu*entUs t-test &as use* to evaluate the

*ifferen"e of +uantitative results et&een !=> an* sele"te* ion monitoring (!?>) metho*' !A! 9'1 &as use* to perform statisti"al analysis' 1. &esults and discussion 6(1( Effects of %opant, mobile phase ow rates an% compositions on "PPI sensitivity .e foun* that e3"ess *opant o& may or may not improve analyte signals, an* optimal *opant portions &ere "ompoun**epen*ent' #ifferentamountof thetoluene *opant&ere teste* on a

F ig . ) . &e l at i- e r es p o ns es !i n l o ga ri t hmi c s cal e " o f d er i -a ti -e s to na ti - e ana l yt es i n d i f fer ent L C s y s te ms 8 !a " 2 3L C # !( " t he mi x ed - mo d e co l u mn and !c" 2 D - LC .

, (& -( .i en et al ( / 0 ( C h ro m ato gr ( " 1112 )1 ,a (le 6 >at r i 3 ef fe"t fa"t o r (L) o t ai ne * fr o m ra& &a te r s am p l es u s i ng p o st - e3 tr a"t i o n s p i E i ng ( n S 7 )' 4 1 4 2 4 6 4 42 8 9 : 9 ;9A 8 ati v e at 4! ? ( - ) A 9 /C (f S 0 '5 m/ (mi n) B B' 1 6 6 '6 B 6 ' 0 55 ' 1 9 2 '5 B 5 '6 5 B ' 0 A 9 /C &i t h s p l i t ( f S 0 '1 m /(mi n) 52 ' 6 5 6' 5 6 1 '1 69 ' 1 B 7 '7 B 2 '5 B 2 ' 2 >i 3 e* - mo * e "o l u mn (f S 0 '2 m/ (mi n) 5 5' 9 5 6' 5 5 7' 1 56 ' 5 5 5 '6 5 B '2 56 ' 2 2 # -/ C ( f S 0' 2 m/( mi n) 52 ' 0 5 1' 6 B 6 ' 7 56 ' 6 1 16 B 6 '6 6 B ' 7 8 ati v e at A9 9? ( -)
A 9 /C (f S 0 '2 m/ (mi n) 5 6' 6 a 6 0 '6 a 7 B ' 5a 59 ' 1 a 5 9 '7 a 5 0 'B " 5 6 ' 7 >i 3 e* - mo * e "o l u mn (f S 0 '2 m/ (mi n) 51 ' 5a 7 6 '5 a 6 5 '9 a 56 ' 5 a 5 9 '6 a 7 7 '6 a , 5 5 ' 5 2 # -/ C ( f S 0' 2 m/( mi n) B 5' 7 , " B 1 '5 , " 11 2 , " 95 ' 5 ," 12 2 , " B B '6 5 0 ' 5

3 + 3 ! 24 3 22

9 66

8 ati v e at A 9 C ? ( - )
A 9 /C (f S 1' 0 m/(mi n) 5 2' 0 a ," 5 5' 7 a ," 9 5 ' 9a 5B ' 5 a ," 5 6 '5 " 5 6 '2 " 51 ' 6 >i 3 e* - mo * e "o l u mn (f S 1' 0 m/(m i n) B 7' 2 B 6 '5 a , B 2 ' 6 B2 ' 6 9 6 '1 B 7 '7 a , B 2 ' 9 a 2 # -/ C ( f S 1'0 m/ (mi n) 56 ' 7 5 2' 6 , " 6 9 ' B 55 ' 5 B 5 '5 5 2 '0 69'5

8 ati v e at A9 C ?(A9 9 ? (- )
A 9 /C (f S 1' 0 m/(mi n) 51 ' 5" 6 2 '5 " B 2 ' 6 5B ' 0 " 51 'B 5 6 '9 " 6 B ' 5 " >i 3 e* - mo * e "o l u mn (f S 1' 0 m/(m i n) B B' B a , B 9 '1 a , 9 6 ' 0 91 ' 6 a , B B '6 B 6 '6 s, 9 5 ' 5 s, 2 # -/ C ( f S 1'0 m/ (mi n) 5 7' 6 " 5 6 '6 " 9 2 ' 2 55 ' 5 " 5 0 '9 6 0 '9 " 6 2 ' 5 "

#a ns yl a t 4! ? (I ) A 9 /C (f S 0 '5 m/ (mi n) 9 7 '6 9 6 '0 9 2 ' 2 92 ' 0 6 6 '5 5 9 '6 5 B ' 6 A 9 /C &i t h s p l i t ( f S 0 '1 m /(mi n) 5 5' 7 5 1' 6 6 2 ' 9 57 ' 9 5 5 '5 6 6 '6 6 9 ' 6 >i 3 e* - mo * e "o l u mn (f S 0 '2 m/ (mi n) B 2' 6 5 9 'B 5 5 '6 B1 ' B 6 0 'B 5 B '9 5 9 ' 7 2 # -/ C ( f S 0' 2 m/( mi n) 9 6' 6 9 6 'B 9 1 '0 B6 ' 5 6 6 '1 6 7 '5 B 9 ' 6 #a ns yl a t A9 9 ? ( I)
A 9 /C (f S 0 '5 m/ (mi n) B 5' 5 B 5 'B 1 0 7a , " B6 ' 0 111 " 10 9 a, " 65 ' B >i 3 e* - mo * e "o l u mn (f S 0 '5 m/ (mi n) 5 5' 2 6 9 '5 6 6 ' 6 55 ' 5 6 7 '2 a , 2 # -/ C ( f S 0' 5 m/( mi n) B 2' 1 5 5 '6 5 7 ' B 59 ' 7 1 16 " 5 B '5 7 2 ' 9 5 2 '2 52'B

#a ns yl a t A9 C ? ( I ) A 9 /C (f S 1' 0 m/(mi n) 119 12 1 11 9 1 22 12 1 10 6 10 9 >i 3 e* - mo * e "o l u mn (f S 1' 0 m/(m i n) 111 11 2 1 2 0 116 1 61 10 1 B 1 ' 6 2 # -/ C ( f S 1'0 m/ (mi n) 12 1 12 5 1 6 2 1 22 1 15 9 9 '9 B B ' 6 #a ns yl a t A9 C ?(A 99 ? (I ) A 9 /C (f S 1' 0 m/(mi n) 10 9 10 9 1 05 115 10 6 B B '9 61 ' 9 >i 3 e* - mo * e "o l u mn (f S 1' 0 m/(m i n) 10 1 9 5 '5 1 0 9 10 0 10 6 B 6 '6 6 5 ' 7 2 # -/ C ( f S 1'0 m/ (mi n) 10 5 10 B 1 2 0 1 0B 9 0 '7 1 2 2 51 ' 6 9< ;;r a t A9 C ? (- ) A 9 /C (f S 1' 0 m/(mi n) 112 117 11 1 110 10 2 1 2 7 1 65 >i 3 e* - mo * e "o l u mn (f S 1' 0 m/(m i n) 110 11 0 9 9 ' 6 116 10 6 9 5 '9 10 B 2 # -/ C ( f S 1'0 m/ (mi n) 95 ' 5 10 6 9 5 '2 1 05 12 1 11 6 10 7 No te# 6 ( 2 ng eB u i va l ent o f ea ch ana l yt es wa s i nJe cte % (
a !ta ti s t i "al l y *i f fer e nt fro m 2 # -/ C ' !ta ti s t i "al l y *i f fer e nt fro m A 9 /C ' " !ta ti s t i "al l y *i f fer e nt fro m t he mi 3e * - mo* e "o lu m n' * ! ta ti s t i "al l y * i f fer ent f ro m A 9/ C & i th s p l i t '

.aters ;4HC1B "olumn, ranging from5L to 25L of a "onstant mo ile phase o& rate at 500 /(min' 2he mo ile phase "ompositions for native an* *ansylate* "ompoun*s &ere >illi-N &ater(methol an* 10 m> formi" a"i*(a"etonitrile, respe"tively, &hi"h &ere the optimal"om inations for theirseparation on the"olumn' ;ase* onpeaE areas, the optimal amount of *opant for native analytes &as 5L of the mo ile phase o&, 25 /(min' 2he intensities of *ansylate* analytes &ere gra*ually enhan"e* as toluene &as in"rease* to 20L of the mo ile phase o& ("orrespon*ing to 100 /(min)' All intensities &ere enhan"e* e3"ept for *ansyl-89 an* *ansyl-:9, &hose signals *roppe* signi "antly on"e the *opant e3"ee*e* 5L' 2herefore, a ve-per"ent *opant o& &as use* in this stu*y' =o et al' use* a"ri*ine an* 9-methylanthra"ene as mo*el "ompoun*s an* reporte* that the signals are "lose to plateaus &hen the *opant amount ranges from 5L to 10L of the mo ile phase o&s, &hi"h &ere at 50, 200, or 1000 /(min F25G an* signal intensities are slightly raise* &hen a higher *opant per"entage is given F25G,a n*ing similar to ours in native an* *ansylate* steroi* estrogens an* *ansylate* ;9A@ ho&ever, in our stu*y, signals of *ansylate* 89 an* :9 *roppe* &hen the *opant portion &as higher than 5L'

2he suita le o& rate of mo ile phase for A99? signals &as "ompoun*-*epen*ent, &hi"h the est o& rates &ere *ifferent et&een native an* *ansylate* analytes' 2he mo ile phase o& rates of 100, 200, 500 an* 1000 /(min &ere evaluate* at a "onstant *opant of 5L mo ile phase o&s using an iso"rati" li+ui* "hromatography@ the mo ile phase"ompositions of native an* *ansylate* analytes &ere 20L >illi-N &ater(B0L methanol (v(v) an* 12L 10-m> formi" a"i*(BBL a"etonitrile (v(v), respe"tively' A !epa3 ,9-C1B "olumn (6'0 mm Q 100 mm, 6 m)&asuse*ata o&rate of 1'0 m/(min an* a .aters ;4H C1B "olumn (2'1 mm Q 100 mm, 1'5 m) &as performe* at e+ual or less than 500 /(min' 2he est sensitivities of nativean* *ansylate* analytes &ereo taine* at o& rates of 200 an* 500 /(min, respe"tively' 2he signal intensities of native analytes at a o& rate of 200 /(min &ere 1'6- to 2'B-fol*, 1'0- to 1'6-fol*, an* 2'5- to 6'7-fol* higher than those at o&s of 10 0, 500, an* 1000 /(min, respe"tively' 2he signal intensities of *ansylate* analytes at a o& rate of 50 0 /(min &ere 7'9- to 1Bfol*, 6'1- to 9'7-fol*, an* 2'2- to 6'0-fol* higher than those at o&s of 100, 200, an* 1000 /(min, respe"tively'

!ome stu*ies have *emonstrate* that a lo& o& rate (e'g' S100 /(min) may improve the ioni)ation ef "ien"y of A99?

967

,( &-( .i en et a l( / 0 ( C h r om at og r( " 111 2 )1

3+ 3 ! 24 3 2 2

resulting from lo&er photoa sorption y solvent F2529G'Ho&ever, our *ata sho&e* that a lo&er o& rate may not provi*e a etter ef "ien"y in A99?' 2o "on rm these o servations, &e further utili)e* a ;4H C1B "olumn &ith a smaller ?'#' (1'0 mm Q 100 mm, 1'5 m)' ?ts optimal o& rate is 100 /(min' .e foun* that the signal response &as even &eaEer than that use* a larger "olumn ?'#' (2'1 mm Q100 mm, 1'5 m)' !olvent mole"ules (e'g' methanol) may e involve* inthe ioni)ationpro"ess, espe"ially in the *opantassiste* A99?, an* the ions forme* y proton transfer are less affe"te* y high o& rates than those forme* via "harge e3"hange F25,2B,60,61G' 2his &oul* e3plain the reason that the optimal mo ile phase o& rates on A99? ef "ien"ies &ere "ompoun**epen*ent' >o ile phase "omposition is also "riti"al to A99? sensitivity, an* is also "ompoun*-*epen*ent' <or native analytes, a >illi-N &ater-methanol "om ination gave etter responses &ith etter peaE shapes than those using &ater-a"etonitrile@ ho&ever, for *ansylate* analytes, a "omposition of 10 m> formi" a"i*-a"etonitrile provi*e* t&o to three times higher sensitivity than a "omposition of 10 m> formi" a"i*-methanol' Cai et al' in*i"ate* that methanol has a lo&er photoa sorption "ross-se"tion than a"etonitrile, an* its *immers "an e ioni)e* y a Tr /amp (a"etonitrile "annot) F25G@ "onse+uently, use of methanol &oul* theoreti"ally provi*e a etter response, &hi"h&as "orre"tfornative analytes inthis stu*y ut &as not the "ase for *ansylate* analytes' Ho&ever, Cal et al' also propose* that the A99? me"hanism is mu"h more "omple3, an* other fa"tors, su"h as the ioni)ationpotential of analytes an* the relative proton af nity of solvents an* analytes, may affe"t A99? ef "ien"ies as &ell F25G'

6(1( Comparison of signal intensity between %erivati8e% an% un%erivati8e% analytes 2he est "om inations of /C systems an* ion sour"es for native an* *ansylate* analytes &ere 2#-/C an* A9/C "ouple* &ith 4!? mo*e, respe"tively' !ignals of all native analytes in 4!? (-) mo*e &ere etter than those in A99? (-), A9C? (-) an* A9C?(A99? (-) e3"ept for 89 an* :9@ there &as no signi "ant *ifferen"e in signal intensities et&een A9C?(A99? *ual mo*e an* A9C? or A99? alone (<ig' 2)' .ithin the 4!? mo*e, 2#-/C &as superior to A9/C an* mi3e*-mo*e"olumnintermsof signal intensities, utA9/C outperforme* oth 2#-/C an* mi3e*-mo*e "olumns &hen A99? (-), A9C? (-) an* A9C?(A99? (-) &ere put on' <or 9<;;r *erivatives using 4CA98? (-), it&as etter to use 2#-/C than A9/C for the analysis of 41, 42, 46 an* 442, ut it &as etter to use A9/C rather than 2#-/C for the analysis of 89, :9 an* ;9A' .ithregar* to the signal intensities of the *ansylate* analytes, 4!? (I)&ere mu"h etter than other ion sour"es (e3"ept for 89)@ the A9C?(A99? *ual mo*e pro*u"e* similar signal intensities &ith those of A9C? alone, ut &as mu"h inferior to those of A99? alone' ?n a**ition, use of A9/C pro*u"e* mu"h stronger responses of *ansylate* analytes relative to other t&o /C systems in all ion sour"es (<ig' 6)' ;ase* on the results, the on-line "leanup of the mi3e*-mo*e "olumn an* the =A> pre-"olumn *i* notgave them a *e"isive a*vantage over A9/C on*ete"ting theanalytes in ra& &ater samples, espe"ially for *ansylate* *erivatives' 2he mi3e*-mo*e "olumn an* 2#-/C, a polymer- ase* "olumn an* a "ouple*-"olumn system, respe"tively, offere* goo* peaE shapes at &i*ths O0'6 min (<ig' 7)@ nevertheless, A9/C provi*e* sharp peaEs at a &i*thof a out 0'06 min(e3"ept for 89, &hi"h&as 0'17 min*ue to a mi3ture of isomers)'

#erivati)ation &ith *ansyl "hlori*e an* 9<;;r signi "antly improve* the *ete"tion sensitivity relative to un*erivati)e* analytes, an* the *ansylate* analytes pro*u"e* mu"h etter responses in four ioni)ation metho*s than 9<;;r *erivatives in 4C-A98? (<ig' 6)' 2he tren*s of signal enhan"ement of *erivati)ation among various ioni)ation metho*s &ere similar, &hi"h &ere

966

,( &-( , (& .i -(en .iet en a et l( / al 0 (( /C 0h (C r om h ro atm og ato r( gr " 111 ( " 1112 2 )1 )1 3+ 3 + !3 24 !3 24 23 2 22

9 65

T:H(anhy*rous in*epen*ent on 4t:H) the /C &ere systems prepare* (<ig' in5)' plasti" <or e3ample, "ontainers, onpH A9/C &as a*$uste* "olumn, using theplasti" signal *roppers, enhan"ements an* plasti" &ere B59B760 tips &ere times, utili)e* 6577060 to spiEe times, solutions 26752 an* times, reagents' 21677 :n times the other an*han*, 571 the times lanE in 4!?, levels A99?, &ere A9C?, insigni A9C?(A99? "ant to those an*foun* 4C-A98?, in environment respe"tively, samples' relative to the native analytes' ?n other &or*s, the or*er of responses of *erivati)e* 2he metho* &as use* &ith samples from !29 efmetho*s uents an* river analytes versus un*erivati)e* ones in ioni)ation &as 4!? K A99? A9C? A9C?(A99? K 4C-A98?' ?n a**ition, &ater (2a le 5)'K 2he mean "on"entrations of steroi* estrogenspost-"olumn in split (a o& rate at 100 split) in this stu*yfor *i*46, notan* ef uents &ere 7'6 ng(/ for /(min 41, 1'Bafter ng(/ for 42, 1'9 ng(/ 4!? mo*e (<ig' 6)' ?n "ontrast, Tloepfer et 2'B in"rease ng(/ for response 442@ 89,in :9 an* ;9A &ere foun* in mu"h higher "onal' reporte* that a lo&er of o& rate 225, (e'g'an* *o&n tong(/, 20 /(min) "an "entrations &ithmeanvalues 655, 16B respe"tively' in"rease signal intensities ofan* some although <or*ramati"ally steroi* estrogens in river &ater, only 41 46analytes, &ere *ete"te* some other &ere unaffe"te* F62G' at 2'0, an* 0'52analytes ng(/, respe"tively@ the "on"entrations of 89, :9 an* ;9A in river &ater &ere higher than those in ef uents &ith mean values of 7022, 276, an* 655B ng(/, respe"tively' High levels of 89 an* ;9A in river &ater in 2ai&an have also een o serve* y #ing et al' F67,65G' 6(6( ;atri5 effect 2hree *ifferent"on"entrations (10, 50, 100 ng(/forsteroi* estro?t &as in"on"lusive to *etermine &hi"h /C systems an* ion gens sour"es an* 10&ere 0, 500 least an* sus"epti 1000 ng(/ le to for matri3 3enoestrogens) effe"ts (i'e', &ere higher spiEe* values intoof the matri3 &atereffe"t samples fa"tors) from &hen the same using sour"es the ra& to &ater evaluate as the thematri3 metho* (2a a""ura"y le 7), &hi"h an*is pre"ision a sour"e (four for *rinEing *upli"ates &ater ea"h an* level)' may 2he e "leaner reporte* than usual "on"entrations surfa"e &ater@ of spiEe* ho&ever, samples &e *i* *i* o not serve *e*u"t that the *erivati)e* a"Egroun* analytes levels &ere in the less &ater' prone ?n toour matri3 stu*y, effe"ts =!#L than of un*erivati)e* all spiEe* samples ones' &ere <or allnative smaller analytes, than 15'5L &e foun* e3"ept nofor signi 89"ant an* *ifferen"es :9 spiEe* at100 in matri3 ng(/ (an* effe"t 500 ng(/ fa"tors foramong 89), &hi"h the *ifferent &ere "lose /C systems to the en*ogenous &ith 4!? mo*e@ levels ho&of the ever, samples the (2a matri3 le 5)' effe"t .e&as foun* lo&er thaton2#-/C the measure* for A99?, "on"entrations an* it&as lo&er &ere onvery the mi3e*-mo*e "lose to the spiEe* "olumn levels for A9C? if thean* a"Egroun*s A9C?(A99?' (no2here spiEe)&ere no &ere signi *e*u"te* "ant *ifferen"es (2a le 5)' inmatri3 effe"tfa"tors among /C systems for *ansylate* analytes in all sour"es (e3"ept for A99?) an* for 9<;;r *erivatives in 4C-A98?' ;e"ause of the e3isting a"Egroun*s of 89, :9, an* ;9A, it is impra"ti"al to "al"ulate their limits of *ete"tion (/:#s)@ therefore, &e only reporte* the intensities /:#s (!(8S of 41, analytes 42, 46 an* 442, ;e"ause the signal of 6%1) *ansylate* using 4!? &hi"h et&een 0'26 an* 0'52 ng(/ !29 ef an* an*range* A99? &ere mu"h more intense thanfor others, &euents further investiet&een 0'56 an* 0'91 ng(/ for river &ater (2a le 5)' Nin et al' also gate* the matri3 effe"ts of these t&o sour"es on*ansylate* analytes reporte* *ete"tion (>#/) of 0'06B0'16 ng(/ using goo* river metho* &ater, &hi"h is a limits more "omple3 matri3 "omparing for &ith river the &ater of 500 m/ *ansyl "hlori*e *erivati)ation F20G' ra& &ater an*using may "ontain higher levels of analytes' 2he ?n a**ition, Dulliet et al' in sho&e* e3"ellent /:#s 0'010'20 ng(/ en*ogenous analytes the river &ater may in of uen"e our *etermi&ithout "hemi"al *erivati)ation for 1-/ groun*&ater, &hi"h may nation on matri3 effe"t fa"tors@ to avoi* this potential pro lem, &e result fromsta goo* re"overies an* large-volume (100 e3tra"/) spiEe* le isotope-la ele* aanalytes to the in$e"tion resi*ues after tion (the levels &ere e+uivalent to B0 ng(/ in the &ater) efore F66G' Although tan*em->! metho*s (!=>) provi*e etter sensitivity an* sele"tivity than those of single->! metho*s (!?>), most la s "annot affor* tan*em->! instruments' Ho&ever, this stu*y, for e3ample, &as a le to *ete"t steroi* estrogens inall real samples of !29 ef uents &ith !?> after *ansyl *erivati)ation, an* the measure* levels of either spiEe* or non-spiEe* samples &ere similar to those using !=> (<ig' 6)' 2he /:#s of steroi* estrogens in ef uents using !?> &ere 1'061'55 ng(/' 2he on-"olumn *ete"tion limits of *ansylate* steroi* estrogens &ith !=> an* !?> &ere0'050'20 an* 0'771'7B pg, respe"tively'

time' *erivati)ation .e e3haustively &ith *ansyl investigate* "hlori*e' the performan"e 2he matri3 effe"t of "ommon fa"tors of ioni)ationpro 4!? using A9/C, es an* A9/C *ifferent/C &ith post-"olumn systems' 4!?split, is usually mi3e*-mo*e reporte* "olto more umn su an* $e"tto 2#-/C ion suppressionthanA9C?an* &ere 15'5L50'6L, 22'0L55'6L, A99?, ut this 70'2L60'7L &as not an* the 16'1L62'1L, "ase in this stu*y' respe"tively@ Although a the mi3e*-mo*e fa"tor values "olumn of A99? or the using =A> A9/C, pre-"olumn mi3e*-mo*e *i* not "olumn su stantially an* 2#-/C re*u"e &ere the 15'5L76'5L, matri3 effe"ts25'9L79'9L etter than an* A9/C 22'1L66'2L, for the &ater respe"tively' matri3es &e 2herefore, teste*, their theuse matri3 &itheffe"t other of 4!? matri3es, an* A99?&ere su"h as foo* similarun*erthe or tissues, are same/C &orth "on*itions, further e3plorations' an* none of the four /C systems "oul* signi "antly eliminate ion suppression' 2he "utoff of mole"ular mass for =A> pre-"olumnis a out 15 E#a@ "onse+uently, the =A> "olumn "oul* not eliminate the matri3 effe"t c/no$ledgement "ause* y small mole"ules F62G' ?n a previous stu*y, Tloepfer et al' reporte* that a post-"olumn split (*o&n to 20100 /(min) not only enhan"es sensitivity ut also re*u"es ion suppression y 2his &orE is supporte* y the 8ational !"ien"e Coun"il, 2ai&an 7060L in&aste&ater samples F62G@ Ho&ever, the A9/C &ith post(8!C 96-2617-;-002-101->02)' "olumn split *i* not re*u"e matri3 effe"ts signi "antly "omparing &ith that &ithout split in our stu*y' &eferences
F 1 G ;' =' C ar r , J'4 ' , ri f n, i n% J '# ' . i l s on, # ' .' < os t er, H ' >' Tr o nen e rg , /' 9' =ee * ( 4 * s' ), . i l l i a ms 2 e3 t o oE o f 4 n* o"r i no l o gy , .' ;' !a u n* er s C o mp a ny , 9hi l a * el p hi a , 19 9 B , p ' 9 0 1' F 2 6(9( G A 'C ' Johns o n, A ' ;e l fro i * , A ' # i C o r "i a, !"i ' 2ot al 4 nvi r o n' 2 5 6 ( 20 0 0 ) 1 6 6' ;etho% vali%ation F 6 G C ' ;ar o nti , =' C u ri ni , ,' #U As " en)o , A ' # i C o r"i a , A ' , ent i l i , =' ! amp e ri , 4 nv i r on' ! "i ' 2 e"hno l ' 6 7 ( 2 0 0 0 ) 5 05 9 ' F 7 G ;' ;e"ause !t a"he l , A *ansyl ' 4 hr ho *erivati)ationun*er rn, : '9' H ee mE en, A9/C 9' / ep o "ouple* m, H ' =e &ith4!? i n"Ee , , pro' !a& al , 8 ' 2 heo al * , 4 nv ithe ro n' 9 o lperforman"e l u t' 1 2 7 ( 20 0ase* 6 ) 7 on 9 5'the sensitivity an* matri3 vi*e* est F 5 effe"t, G A ' ;o &e l ), vali*ate* H ' H a ge nmai r, .' To ,oo* r ner , 4 nvi r o n' 9o l l pre"ision u t ' 115 (2 &ere 0 0 1 ) 2 91' this emetho*' a""ura"y an* F 6 G H ' >' Tu "h, T' ;a l l s "hmi te r, 4 nv i ro n' ! "i ' 2e "hno l ' 6 5 ( 20 0 1 ) 6 2 01 ' o taine* for "ali ration "urves' 2he intra-*ay an* inter-*ay a""uF 5 G ! ' =o * r i gu e )- >o )a) , >' J' * e A l * a, #' ;ar "el o , J' C hr om ato g r' A 10 7 5 ( 2 0 0 7 ) ra"y B 5 ' &ere &ithin 2L an* 9L, respe"tively@ the inter-*ay variations 0'BBL toi 17'6L an* &ere F B (=!#L) G / ' ! al s range* t e, 9' /efrom s Ei ne n, > ' D r ta, /' T ro nintra-*ay er g , ! "iresponses ' 2o ta l 4 nv i r on' 6 5 B (2 0 0 5 ) almost 6 7 6 ' i*enti"al' 2he linearity of "ali ration "urves of steroi* estro(10 00-fol* to 10,000-fol*) are or ,etter F 9 gens G C ' 0' C he n, 2 '0' .e n, , ' !' .a ng, Hsimilar ' .' C heng 0' H than ' /i n,those ,' .' /i e n, ! "i ' 2o ta l 4 nv i r on' of ng(m/, 1000-fol*) F20G an* Dulliet et al' 6 Nin 5 B ( et 2 0al' 05 (0'2200 )652' F1 0 (0'0520 G 2 ' ;e ni$tng(m/, s , =' # ams , .' /a F66G' m er t, A' # e /e enhe er , J' C hro ma to gr ' A 10 2 9 ( 2 0 0 7 ) 700-fol*) 1 5 6' F 11 G H ' To n* o , 0' :E a* a , T' ! hi m o, 0' <u s ho , 4 as t er n A nal y ti "a l ! ymp o s i u m an* 4 38o hi steroi* i t (4 A! ), ! o mer s et , 8*ete"te* e& Jer s ey !Ala , 2 oratory 0 0 6' estrogens &ere in, A any lanE, F1 2 G J' C hi "o , !' >e"a , = ' C o mp any o, >' # ' 9 ra t, >' ,r ana * o s , J' C hro ma to gr ' A 11B 1 ut 89, :9 an* ;9A &ere *ete"te* using *ansyl *erivati)ation@ ( 2 a"Egroun* 0 0 B) 1 ' levels in the reagent lanEs of >illi-N &ater &ere the F1 6 60160 G 4 ' A' ng(/' H o ge n* possi oo r n,le 4' # i $E man, ;':9 ;au ma nn, C 'Hi* 2he sour"es of 89, an* ;9A "oul* e a l go , J'D ' !a n"ho, <' H er na n* e), A spee*isE nal ' C he m'soli* 5 1 ( phase 19 9 9e3tra"tion ) 1111' (pa"Ee* in a plasti" *isE the for the >illi-N the u &aterF1 7 vessel), G 4 ' 2' , ang l , >' &ater >' A itself nnan,(plasti" 8 ' ! p materials o one r, 9in ' Do r os , Anal ' C hem' 5 6 (2 0 0 1) 5 6 6 5 ' <orue3ample, *uring F1 5 puri G C 'e* /' system) A n* re or &sthe , C*erivati)ationpro"ess' '9' 0u , 4' 0a ng, 9' Do ro s , J' C hro ma to gr ' A 10 5 6 (2 0 0 7 ) 15 1' pro"e*ure, (8aHC:6 uffer an* F1 6 the G >*erivati)ation '=' A nar i , =' ;a E hti areagents r, ;' V hu , ! ' H u (8a:H s Ee y, =' ;' <r a nEl i n, # ' C ' 4 va ns , Ana l ' C hem' F 15 F 1B F1 9 F2 0 F2 1 F2 2 G G G G G G 5 7 ( 2 0 02 ) 7 16 6 ' A ' ! al va * o r, C ' >o r et to n, A ' 9 i ra m, =' <a u re , J' C hr o mat o gr ' A 117 5 (2 0 0 5) 10 2 ' , ' ! i ngh, A' , u ti e r re ), T ' Ru , ?' A' ;l ai r, Anal ' C hem' 5 2 (2 0 0 0 ) 6 0 05 ' . 'V ' ! ho u , R' 0' Ji ang , 8 '# ' . e ng, ;i o me * ' C hr om ato g r' 1 B ( 20 0 7 ) 7 17 ' <' Ni n, 0' 0' Vha o , >';' !a& y er, R' <' /i , A nal ' C hi m' A"t a 6 25 (2 0 0 B ) 91 ' =' 4 ' 8 el s o n, ! 'T ' ,r e e , # 'J ' :U Ta ne, = 'J' ! i ngh , C l i n' C hem ' 5 0 ( 20 0 7 ) 6 5 6' / ' ! o ng, A '# ' .e l l man, H ' 0ao , J' A * "o "E, =a p i * C o mmu n' >as s !p e "tr o m' 21

6. Conclusions ?n this stu*y, &e present a +uantitative metho* for the analysis of seven estrogeni" "ompoun*s &ith *ansyl *erivati)ation in oth !?> an* !=>' .iththe improvement in sensitivity using A9/C an* "hemi"al *erivati)ation metho*, environmental levels of these "hemi"als "an e *etermine* using a single >! instea* of the more e3pensive tan*em->!' 2he instrumental throughput &as signi "antly in"rease*, &ith a run in 6'2 min plus 1-min re-e+uili rium

( 2 0 05 ) 1 6 76 ' F2 6 G 0' H ' / i n, C ' 0' C he n, , ' !' .a ng, =a p i * C o mmu n' >as s ! p e"t ro m' 2 1 (2 0 0 5 ) 1 95 6 ' F2 7 G =' > oha me *, 0' A' H am mel , >' H ' /e ;re to n, J'C ' 2 a e t, /' Ju l l i e n, 9'A ' , u y, J' C hro m ato g r' A 11 6 0 ( 20 0 5 ) 1 97 ' F2 5 G ! '! ' C ai , /' C ' !ho r t, J' A' ! ya ge , >' 9o tv i n, J' >' C u rt i s , J' C hro ma to gr ' A 1 156 (2 0 0 5 ) BB' F2 6 G / '9' 9e n)e s , ,' .' :e r te l , J' C hr om ato g r' 5 1 ( 19 5 0) 6 2 5 ' F2 5 G # ' ;' =o , >'. ' ;l a* e s , J' A m' ! o"' >as s !p e "tr o m' 1 6 ( 20 0 5 ) 1 25 5 ' F2 B G 2 'J' Ta u p p i l a, A' 9' ;r u i ns, =' To s ti a i nen, J' Am' ! o" ' > as s ! p e"t ro m' 16 (2 0 0 5 ) 1 6 99 ' F2 9 G # ' ;' =o , >'. ' ;l a* e s , J' A m' ! o"' >as s !p e "tr o m' 15 (2 0 0 6 ) 1 60 ' F6 0 G / 'C ' !ho r t, T 'A ' H a nol * , !' !' C ai , J'A ' ! yag e, =ap i * C o mmu n' >as s ! pe "t ro m' 21 ( 2 0 05 ) 1 5 61' F6 1 G / 'C ' ! ho rt , ! '! ' C ai , J 'A ' !y ag e, J' Am' ! o "' >a s s ! p e"t r om' 1B ( 2 0 0 5) 5 B 9 ' F6 2 G A ' Tl o e p fer , J';' Nu i nt ana , 2' =e em ts ma , J' C hr om ato g r' A 1 0 65 (2 0 0 5 ) 15 6 ' F6 6 G 4 ' D u l l i et , /' . i e s t, = ' ;au * o t, > '<' , r eni e r- /o u s ta l ot , J' C hr o mat og r' A 12 10 ( 2 0 0 B) B 7 ' F6 7 G C ' 0' C heng , C ' 0' . u , C ' H ' . ang , . 'H ' # i ng , C hemo s p he re 6 5 (2 0 0 6 ) 2 25 5 ' F6 5 G . 'H ' #i ng , C ' 0' .u , J' C hi n' C he m' !o "- 2ai p ' 75 (2 0 0 0 ) 11 55 '

Fi g. 9 . 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 $a ge t re at ment p l ant ( et $e en8 ! a" S &' and !( " S I' mo d e.