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A comp ehensi e s udy o a new e sa ile mic ochip de ice based liquid phase mic oex ac ion
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o s opped- low and double- low condi ions.
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Ma ía Ramos Payána*, Elia San igosa Mu illob, Jo di Coellob, Miguel Ángel Bello Lópeza
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aDepa men o Analy ical Chemis y, Facul y o Chemis y, c/P o . Ga cía González s/n, 41012,
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Se ille, Spain
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bDepa men o Analy ical Chemis y, Uni e si a Au ónoma de Ba celona, 08193 Bella e a,
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Ba celona, Spain
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*co esponding au ho : [email p o ec ed] elephone numbe : +34954557172
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Abs ac
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A new geome y o a e sa ile mic o luidic-chip de ice based liquid phase mic oex ac ion
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was de eloped in o de o enhance he p econcen a ion in mic o luidic chips and also o enable
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double- low and s opped- low wo king modes. The mic ochip de ice was combined wi h a HPLC
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p ocedu e o he simul aneous de e mina ion o wo di e en amilies as model analy es, which we e
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pa abens and non-s e oidal an i-in lamma o ies (NSAIDs): E hyl 4-hyd oxybenzoa e (E -P), P opyl 4-
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hyd oxybenzoa e (P -P), Bu yl 4-hyd oxybenzoa e (Bu-P), IsoBu yl 4-hyd oxybenzoa e (iBu-P),
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salycilic acid (SAC), ke op o en (KET), nap oxen (NAX), diclo enac (DIC) and ibup o en (IBU) in
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u ine samples. The new minia u ized mic ochip p oposed in his wo k allows no only he possibili y
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o wo king in double- low condi ions, bu also unde s agnan condi ions (s opped- low) (SF-µLPME).
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The sample (pH 1.5) was deli e ed o he SF-µLPME a 20 µL min-1 while keeping he accep o phase
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(pH 11.75) unde s agnan condi ions du ing 20 minu es. The highes en ichmen ac o s (be ween 16
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and 47) we e ob ained unde s opped- low condi ions a 20 µL min-1 (sample low a e) a e 20 min
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ex ac ion; whe eas he ex ac ion e iciencies we e wi hin he ange o 27-81% o all compounds.
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The p ocedu e p o ided e y low de ec ion limi s be ween 0.7 and 8.5 µg L-1 wi h a sample olume
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consump ion o 400 µL. Pa abens and NSAIDs ha e success ully been ex ac ed om u ine samples
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wi h excellen clean up and eco e ies o e 90 % o all compounds. In pa allel, he new de ice was
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also es ed unde double low condi ions, ob aining good bu lowe en ichmen ac o s (be ween 9 and
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2
20) and highe ex ac ion e iciencies (be ween 45 and 95) a e 7 min ex ac ion, consuming a olume
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sample o 140 µL .
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The e sa ile de ice o e ed e y high ex ac ion e iciencies and good en ichmen ac o o double
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low and s opped- low condi ions, espec i ely. In addi ion, his new minia u ized SF-µLPME de ice
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signi ican ly educed cos s compa ed o he exis ing analy ical echniques o sample p epa a ion since
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his mic ochip equi e ew mic oli e s o sample and eagen s and i is eusable.
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Keywo ds: mic oex ac ion, minia u iza ion, sample p epa a ion, mic o luidic, d ugs, u ine sample.
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1. In oduc ion
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Liquid Phase mic oex ac ion (LPME) is a e y well-known and popula echnique used o he
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ex ac ion o acid and basic d ugs based on he passi e di usion o he analy es om he sample (dono
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solu ion) in o an accep o solu ion, h ough a memb ane (which suppo an o ganic sol en in o i s
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po ous). LPME has been applied o many di e en ields, conside ing biological, pha maceu ical,
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en i onmen al, ood, oxicology analysis, among o he s [1–7]. The anspo phenomena based on
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passi e di usion depends no only on he na u e o he analy es, and he op imal pa ame e s (as phase’s
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composi ion, o ganic sol en , s i ing speed, low a e, e c), bu also on he geome y o he sys em
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used o LPME. Ano he popula echnique based liquid phase mic oex ac ion, named
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elec omemb ane ex ac ion (EME), has also been equen ly used since i imp o es he ex ac ion o
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compounds in many cases due o an ex e nal elec ical ield c ea ed o bo h sides o he suppo liquid
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memb ane [8–16]. Howe e , EME also o e s some limi a ions since i s equi emen is he use o a
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sui able and conduc o o ganic sol en o ca ying ou he ex ac ions. Bo h echniques ha e been
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widely used o he de e mina ion o pha maceu ical d ugs ei he in biological samples (u ine) o wa e
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samples [17-23] due o he g ea conce n ha exis s ega ding hei con ibu ion as eme gen pollu an s
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in he en i onmen . Also, pa abens ha e been s udied due o he conce n abou hei endoc ine
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dis up ing po en ial [24-29]. This has equi ed he use o powe ul, as and sensi i e echniques ha
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o e be e limi s o quan i ica ion.
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Up o da e, pa abens and non-s e oidal an iin lama o ies ha en been de e mined by adi ional LPME
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and EME p ocedu es esul ing in e y good en ichmen ac o s [1-4,17,19, 20, 30-33]. Those
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p ocedu es allowed good en ichmen ac o s bu low ex ac ion e iciencies. In he las yea s, liquid-
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liquid ex ac ion has been minia u ized in o mic o luidic de ices in o de o add ess he limi a ions
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om adi ional p ocedu es and hese chip de ices a e becoming an a ac i e al e na i e due o he
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many ad an ages ha i p esen s [34-42]. The mic ochip de ices o sample ea men ha e wo
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channels ha allow wo king in wo di e en ways based on he low a e o each phase: double- low
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o s opped- low condi ions. In double- low condi ions, bo h phases (sample and accep o ) a e mo ing
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a some low a e. Howe e , in s opped- low condi ions, he accep o phase keep s agnan while he
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sample solu ion is used a some low a e. Recen mic ochip de ices based LPME, ha e been
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demons a ed o wo k only unde double- low condi ions bu no unde s opped- low condi ions in a
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single s ep since he la e equi ed o collec se e al ex ac s o i s di ec injec ion in o HPLC. This
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was due o he low sample olume a ailable in he accep o channel ( 2 µL) [41,42] and consequen ly,
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he analysis ime inc eased and he ep oducibili y dec eased when an en ichmen ac o was necessa y
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p io o he sample analysis. On he o he hand, he de ices did no allowed high p econcen a ion
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ac o s al hough he sample low a e was signi ican ly inc eased unde double- low condi ions.
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Based on he cu en limi a ions o mic o luidic de ices o mic oex ac ion p ocedu es, he aim o his
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wo k was o de elop a new e sa ile and e ec i e mic o luidic de ice in o de o o e come he
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limi a ions om p e ious mic o luidic de ices, inc easing he p econcen a ion and allowing wo king
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unde s opped- low condi ions compa ible wi h di ec analysis.
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Based on geome y aspec s, an inc ease o he dep h channel would inc ease he olume capaci y
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con ained in he channel bu i could dec ease he anspo phenomena by passi e di usion since he
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analy es a e a he away om he memb ane. Mic o luidic sys ems ha ollow a lamina egimen do
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no ca y agi a ion, so di usion can be slow i he dis ance be ween he analy es and ex ac ion sol en
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is inc eased. Mo eo e , an inc ease o he channel´s wid h would inc ease he con ac su ace be ween
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he sample and he analy es, howe e , e y wide channels could des abilize he lamina low and a ec
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he memb ane s abili y.
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In his wo k, we p esen o he i s ime a new e sa ile and e ec i e mic o luidic chip based LPME
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which allow he possibili y o wo king unde wo di e en wo king modes (double- low o s opped-
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low condi ions). The mic ochip was applied o he simul aneous de e mina ion o wo di e en
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amilies in u ine samples. This way, a comp ehensi e s udy be ween bo h di e en wo king condi ions
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was ca ied ou . The mic ochip dec eased he sample olume and ime o analysis since no collec ing
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samples we e needed o di ec injec ion. The p oposed s opped- low de ice (SF- µLPME) is he
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easies mic o luidic chip o he simul aneous ex ac ions o di e en d ugs esul ing in highe
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en ichmen ac o s wi h lowe cos ins umen a ion, simple handling, eusabili y and is s ill conside ed
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a “g een me hod” by keeping low o ganic sol en (< 5µL) consump ion. The p oposed de ice has been
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success ully applied o u ine samples.
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2. Expe imen al
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2.1. Chemicals and solu ions
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E hyl 4-hyd oxybenzoa e (E -P), P opyl 4-hyd oxybenzoa e (P -P), Bu yl 4-hyd oxybenzoa e (Bu-P),
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IsoBu yl 4-hyd oxybenzoa e (iBu-P), salicylic acid (SAC), ke op o en (KTP), nap oxen (NPX),
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diclo enac (DIC), ibup o en (IBU),1-oc anol, dihexyl e he , 2-ni ophenyl oc hyl e he (NPOE),
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o mic acid, sodium hyd oxide, chlo ide acid,sodium chlo ide and me hanol we e pu chased om
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Fluka–Sigma–Ald ich (Mad id, Spain). 100 mg L-1 s ocks solu ions we e p epa ed in me hanol excep
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SAC, DIC and IBU ha we e p epa ed in Milli-Q Plus wa e (Elga, pu elab op ion S-R 7-15 (Mad id,
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Spain). All wo king dilu ions we e p epa ed using ul apu e wa e om a Milli-Q Plus by adequa e
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dilu ions om s o ed a 4ºC. A memb ane (Celga d 2500) o 25 µm hickness, 55% po osi y, and 0.21
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µm x 0.05 µm po es was ob ained om Celga d (Cha lo e, NC, USA).
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2.2 Fab ica ion o he mic o luidic-chip de ice
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Figu e 1 shows a scheme o he mic o luidic de ice based liquid phase mic oex ac ion. This
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mic o luidic de ice has been e-designed and modi ied in o de o o e come he limi a ions and
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disad an ages om p e ious mic o luidic de ices. The op imal poly(me hyl me hac yla e(PMMA)
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de ice consis ed o wo symme ical pa e ned pla es wi h one channel o 23 mm leng h, 120 µm dep h
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and 3 mm wid h each. Fou holes o 3 mm and 1.35 mm diame e we e d illed o assembling and
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ixing in/ou le s Te lon ubes, espec i ely. A la polyp opylene memb ane piece o 27 mm leng h x
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5 mm wid h sepa a ed he accep o phase (channel 1) and he dono phase (channel 2). Fi s ly, he
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memb ane was placed o e one channel and imp egna ed wi h 4 µL o dihexyl e he . Once he
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ex ac ing sol en was immobilized along he memb ane by capilla y o ces, he channels we e aligned
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and he de ice was closed using ou small c ews. The inal size o a mic o luidic de ice o one single
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ex ac ion was 47×29×6 mm, howe e by inc easing he size o bo h PMMA pla es, an a bi a ily la ge
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numbe o ex ac ion channels can be implemen ed and independen ly add essed. Also, he mic ochip-
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de ice can be opened any ime when exchange memb ane is needed.
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A lase cu e (Epilog Mini 24-30 W) was used o ab ica e his chip. The bes quali y was ob ained
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using a w i ing speed o 40%, powe o 33%, a esolu ion o 1500 and a equency o 5000.
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Inle s Te lon ubes (accep o and dono inle s) we e connec ed o wo sepa a e mic o-sy inge pumps
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(Ce oni GmbH, Ko bussen, Ge many). The sample (pH 1.5) was pumped in o he mic o luidic de ice
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a 20 µLmin-1 while keeping he accep o phase (pH 11.75) cons an . The mic o luidic de ice was also
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es ed unde double- low condi ions as desc ibed below, in o de o compa e di e en wo king modes.
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The accep o phase was collec ed using a mic opipe e and was di ec ly injec ed in o a HPLC o
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analysis.
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2.3. Ch oma og aphic condi ions
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An Agilen 1100 se ies (Ba celona, Spain) liquid ch oma og aphy equipped wi h a G1312A Bipump
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and an au osample G1313A o 5 µL o sample injec ion was used as HPLC sys em. The column used
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o he sepa a ion o he nine compounds was a LiCh oCART® 75-4 Pu osphe ® STAR RP-18e 3 µm
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(75 mm x 4.0 mm i.d.) (VWR, Ba celona, Spain) p oceeded by a gua d column K omasil1 100 Å, C18,
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5 µm (20 mm x 4.6 mm i.d.) (Scha lab S.L., Ba celona, Spain).
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The mobile phase consis ed o 0.1% o mic acid (pH 2.6) (componen A) and me hanol (componen
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B) a a low a e o 0.5 mL min-1. Sepa a ion was pe o med a 25ºC. An ini ial 60% componen B
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was used in isoc a ic mode o 2 min, and hen a linea elu ion g adien was p og ammed om 60% o
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80% (B) o 3.4 min and om 80 % o 86 % B o ano he 2.4 minu es. Th ee minu es we e wai ed
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be ween injec ions which allowed e-equilib a ion o he column o he ini ial condi ions.
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The wa eleng hs used o DAD we e 235, 255, 230, 280 and 225 nm o SAC, KTP, NAX, DIC and
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IBU, espec i ely and 255 nm o all pa abens. The ch oma og am was comple ed in less han 10
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minu es and he e en ion ime was 3.1, 3.3, 4.7, 5.3, 6.3, 6.6, 6.8, 8.9 and 9.1, o SAC, E -P, P -P,
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KTP, NPX, iBu-P, Bu-P, DIC and IBU, espec i ely.
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2.4. P epa a ion o biological samples analysis using µLPME ex ac ion
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Spiked u ine samples we e adjus ed o pH 1.5 wi h HCl and il e ed h ough Pall Nyla loTM nylon
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memb ane il e 0.45 µm (Pall Co po a ion, Ann A bo , Michigan, USA) p io o mic oex ac ion
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p ocedu e.
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2.5. Calcula ions o ex ac ion e iciency and en ichmen ac o
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The en ichmen ac o (EFi) o he analy e i was calcula ed acco ding o he ollowing equa ion (1):
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𝐸𝐹𝑖=𝐶𝑓,𝑎,𝑜𝑢𝑡𝑙𝑒𝑡
𝐶𝑖,𝑠,𝑖𝑛𝑙𝑒𝑡 (1)
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whe e𝐶𝑓,𝑎,𝑜𝑢𝑡𝑙𝑒𝑡 is he concen a ion o he analy e i a he ou le o he accep o channel and 𝐶𝑖,𝑠,𝑖𝑛𝑙𝑒𝑡
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is he ini ial concen a ion o he analy e in he sample. 𝐶𝑓,𝑎,𝑜𝑢𝑡𝑙𝑒𝑡was de e mined by HPLC UV-
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de ec ion using ex e nal calib a ion. The en ichmen ac o is calcula ed using he same equa ion ei he
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using double- low o s opped- low condi ions. The ex ac ion e iciency (EE) was de ined as he
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ac ion o analy e ans e ed o he accep o phase om he sample. Using a double- low wo king
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mode, he ex ac ion e iciency (EE %) was calcula ed acco ding o he ollowing equa ion (2):
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𝐸𝐸 (%)= 𝐶𝑓,𝑎,𝑜𝑢𝑡𝑙𝑒𝑡
𝐶𝑖,𝑠,𝑖𝑛𝑙𝑒𝑡 𝑥 𝑣𝑎
𝑣𝑠 𝑥 100= 𝐸𝐹𝑖 𝑥 𝑣𝑎
𝑣𝑠 𝑥 100 (2)
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Whe e 𝑣𝑎 𝑎𝑛𝑑 𝑣𝑠, a e he accep o and sample low a e, espec i ely.
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Howe e , unde s opped- low condi ions, he ex ac ion e iciency (EE %) was calcula ed by
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subs i u ing he pa ame e “accep o and sample low a e” by he “accep o and sample olume”
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co esponding o each phase sample.
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In o de o ob ain a global EE alue o he 9 analy es, he a e age ex ac ion e iciency index (a EEi)
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was de ined (3):
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𝐴𝑣𝐸𝐸𝑖=1 − √∑(𝐸𝐸𝑖−100)2
𝑛
𝑖=1 𝑛 (3)
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3. Resul s and discussion
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3.1. P inciple o he ex ac ion
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The model analy es co esponded o wo di e en amilies: non-s e oidal an iin lama o ies and
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pa abens. The ex ac ion o he analy es is based on a passi e di usion p ocess due o a s ong pH
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g adien di e ence be ween he accep o and he sample solu ion. Non-s e oidal an iin lama o ies
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con ain acid g oups wi hin a pKa ange alue o 2.5-5, while he pa aben a e es e s o
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pa ahyd oxybenzoic acid and con ain alcohol g oup which pKa alue a e wi hin he ange o 5-8.8. A
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h ee phases liquid phase mic oex ac ion con igu a ion p esen s wo aqueous solu ions (accep o and
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sample) sepa a ed by he suppo liquid memb ane (o ganic sol en ). The analy es o in e es we e in
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neu al o m in he sample solu ion and nega i ely cha ged in he accep o solu ion. This way, a pH
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alue o unde 7 (HCl solu ion) and o e 9 (NaOH solu ion) we e used as sample and accep o solu ion,
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espec i ely.The mic o luidic de ice was es ed using wo di e en wo king modes: double- low mode
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and s opped- low condi ions. The memb ane was eused o consecu i e ex ac ions wi hou obse ing
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memo y e ec s and he accep o phase collec ed was analyzed by HPLC once he ex ac ion was
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comple ed. Unde s opped- low condi ions, he accep o phase was pumped con inuously o a leas
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2-3 minu es be ween ex ac ions o clean he SLM a oiding memo y e ec s.
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3.2. Op imiza ion o he mic ochip´s geome y
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In LPME, he ex ac ion o he analy es depends on a anspo phenomenon based on passi e
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di usion. The design o a new geome y was ocused on he inc easemen o he channel olume
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capaci y (compa ible wi h di ec injec ion in o HPLC) wi hou decele a ing he anspo phenomena.
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Di e en leng h, wide and dep h we e es ed in o de o ob ain an adequa e and s able lamina low
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du ing he ex ac ion, conside ing a inal channel olume capaci y be ween 7 and 10 µL o i s di ec
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analysis by HPLC a e s opped- low condi ions. The leng h was ixed a 23 mm and he wide and
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dep h we e es ed be ween 1-3 mm and 50-300 µm, espec i ely. The dep h was he mos c i ical
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pa ame e and i was limi ed o 120 µm since an inc eased dep h signi ican ly decele a ed he anspo
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phenomena. In one hand, a less deep channel kep high ex ac ion e iciencies unde double- low
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condi ions (o e 90%) bu he channel olume capaci y was no enough o wo king unde s opped-
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low condi ions. On he o he hand, a dep h o e 150 µm dec eased he ex ac ion e iciency unde
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double- low condi ions (less han 70%) and he en ichmen ac o dec eased 20 % o all compounds
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unde s opped- low condi ions. Addi ionally, a wide o 2 mm equi ed a deepe channel in o de o
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inc ease he olume capaci y and i dec eased he ex ac ion e iciencies and a wide o 4 mm did no
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o e good ep oducibili y and s able low a e. Fo his easons, a comp omise be ween dep h, leng h
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and wid h was ca ied ou o inc ease anspo phenomena and channel´s olume bu s ill main aining
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minia u iza ion size and simple handling condi ions. The bes esul s and he mos ep oducible and
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s able low we e ob ained wi h a channel geome y o 23 mm leng h, 120 µm dep h and 3 mm wid h.
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Based on he undamen al basis o LPME, his new geome y p esen s longe and wide channels o
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inc easing he con ac a ea be ween he analy es and he suppo liquid memb ane compa ed o
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p e ious one made on PMMA. Addi ionally, he dep h was inc eased o allow a g ea e olume
198
capaci y compa ed o he only 2 µL olume capaci y om he p e ious µLPME de ice [41].
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3.3. Op imiza ion and e alua ion o expe imen al condi ions
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Acco ding o da a ounded in he bibliog aphy o indi idual mic oex ac ion o pa abens o non-
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s e oidal an iin lama o ies, p elimina y expe imen s we e pe o med o de e mina e he mos sui able
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o ganic sol en o be used as SLM o he simul aneous ex ac ion o bo h amilies. Fo he
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op imiza ion o he o ganic sol en , a pH 3 (HCl) sample solu ion, pH 11.75 (NaOH) as accep o
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solu ion, and 1 µL min-1 as sample and accep o low a e we e used. 2-ni ophenil oc yl e he (NPOE),
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1-oc anol, 1-hep anol and dihexyle he we e es ed by opening he mic o luidic de ice o memb ane
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exchange a e each o ganic sol en es . As seen in Table 1, he ex ac ion e iciency (based on 3
207
eplica e expe imen s) was e y di e en depending on he analy e. Bes a EEi was ob ained when
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dihexyle he was used as suppo liquid memb ane, which was consequen ly used as SLM o he es
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o he s udy.
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Fo op imiza ion o sample and accep o composi ion, he accep o and dono phase we e es ed wi hin
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he anges o pH 10-12 (aqueous NaOH solu ions) and 1-4 (aqueous HCl solu ions), espec i ely. The
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dono phase, con aining 1 mg mL-1 o each analy e, was es ed keeping he accep o phase ixed a pH
213
11.75. As seen in Figu e 2, he highes peak a eas we e ob ained a pH 1.5 a e 7 minu es ex ac ion,
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no obse ing a signi ican dec ease o he es o he pH ange es ed. Then, accep o phase
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composi ion was op imized by keeping he sample solu ion ixed a pH 1.5 o all expe imen s. Figu e
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3 shows ha he highes peak a eas we e ob ained a pH 12 and pH 11.75 o i e non-s e oidal
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an iin lama o ies and ou pa abens, espec i ely. On he o he hand, pa abens we e no s able o e pH
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12 due o a sligh deg ada ion du ing hei ex ac ion, so a comp omised pH o 11.75 was selec ed o
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he ex ac ion o bo h amilies. A ela i e s anda d de ia ion (RSDs %) below 4 % o all analy es
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esul ed based on 3 eplica e expe imen s o each expe imen al poin o Figu e 2 and 3. Consequen ly,
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liquid memb anes. Tuning he memb ane chemis y o di e en ypes o d ug subs ances, J.
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Ch oma og . A. 1124 (2006) 29–34.
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[9] S. Pede sen-Bje gaa d, K.E. Rasmussen, Elec okine ic mig a ion ac oss a i icial liquid
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memb anes: New concep o apid sample p epa a ion o biological luids, J. Ch oma og . A.
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1109 (2006) 183–190.
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[10] S. Pede sen-Bje gaa d, K.E. Rasmussen, Ex ac ion ac oss suppo ed liquid memb anes by use
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o elec ical ields, Anal. Bioanal. Chem. 388 (2007) 521–523.
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o sample p epa a ion in ch oma og aphy, T AC - T ends Anal. Chem. 27 (2008) 934–941.
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460
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461
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462
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463
464
465
466
467
468
469
20
470
471
472
473
474
475
476
477
Leyend o he ables and igu es cap ions
478
Figu e 1. Schema ic o he mic ochip de ice based liquid phase mic oex ac ion
479
Figu e 2. Op imiza ion o he dono phase composi ion. SLM: dihexyle he , low a e (dono and
480
accep o phase): 1 µL min-1, accep o phase composi ion: pH 11.75
481
Figu e 3. Op imiza ion o he accep o phase composi ion. SLM: dihexyle he , low a e (dono phase):
482
1 µL min-1, low a e (accep o phase): 1 µL min-1, dono phase composi ion: pH 1.5
483
Figu e 4. Ex ac ion e iciency e sus sample low a e. SLM: dihexyle he , low a e (accep o phase):
484
1 µL min-1, dono phase composi ion: pH 11.5 and accep o phase composi ion: pH 1.5
485
Figu e 5. Ex ac ion en ichmen e sus sample phase low a e. SLM: dihexyle he , low a e (accep o
486
phase): 1 µL min-1, dono phase composi ion: pH 11.75 and accep o phase composi ion: pH 1.5
487
Figu e 6. Op imiza ion o sample solu ion low a e and ex ac ion ime o SAC, E -P, P -P, NPX,
488
KTP, iBu-P, Bu-P, DIC and IBU.
489
Figu e 7.Ch oma og am o a spiked u ine sample con aining 16 µg L-1 o all compounds excep o
490
IBU ha was 30 µg L-1. Ex ac ion ime: 20 minu es. SLM: diexyle he ; dono phase composi ion: pH
491
11.75 and accep o phase composi ion: pH 1.5. Sample low a e: 20 µL min-1. No sample dilu ion
492
Table 1. Ex ac ion e iciencies (RSD %) o he model subs ances using di e en o ganic sol en s as
493
he SLM o µLPME o acid d ugs.
494
21
Table 2. µLPME calib a ion pa ame e s, me hod de ec ion limi (MLOD), me hod quan i a ion limi
495
(MLOQ), ex ac ion e iciencies and en ichmen ac o o all analy es in a) s opped- low condi ions
496
mode a e 20 min ex ac ion and b) in double- low condi ions mode wi h an ex ac ion ime o 7 min.
497
Table 3. SF-µLPME/HPLC eco e ies (a e age o h ee de e mina ions ± s anda d de ia ion) om
498
non-dilu ed spiked u ine samples.
499
Table 4. Compa ison o igu es o me i o µLPME wi h o he analy ical echniques o de e mina ion
500
o non-s e oidal an i-in lamma o ies and pa abens.
501
502
503
504
505
506
507
508
509
510
Tables
511
Table 1
512
Table 1. Ex ac ion e iciencies (RSD %) o he model subs ances using di e en o ganic sol en s as
513
he SLM o µLPME o acid d ugs.
514
% Ex ac ion e iciency (%RSD, n=3)
NPOE
Dihexyle he
1-hep anol
1-oc anol
Salicylic acid
15 (2)
89 (1)
3 (1)
9 (1)
E hyl 4-hyd oxybenzoa e
82 (1)
100 (2)
77 (2)
85 (1)
P opyl 4-hyd oxybenzoa e
99 (1)
99 (1)
79 (1)
88 (1)
Ke op o en
94 (4)
98 (2)
12 (3)
86 (3)
Nap oxen
81 (2)
93 (2)
17 (1)
58 (2)
IsoBu yl 4-hyd oxybenzoa e
100 (2)
100 (1)
82 (2)
94 (2)
Bu yl 4-hyd oxybenzoa e
70 (2)
98 (2)
81 (3)
97 (1)
Diclo enac
54 (1)
88 (2)
10 (1)
33 (1)

22
Ibup o en
70 (2)
100 (3)
5 (2)
22 (3)
A EEi
64
94
31
51
a Sample: 1 µL min-1 o HCl a pH 3 con aining he nine d ugs each a 1 µg mL-1; accep o : 1 µL min-1 o NaOH a pH
515
11.75; ex ac ion ime: 7 min. NPOE: 2-Ni ophenyl oc yl e he
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
Table 2. µLPME calib a ion pa ame e s, me hod de ec ion limi (MLOD), me hod quan i a ion limi
533
(MLOQ), ex ac ion e iciencies and en ichmen ac o o all analy es in a) s opped- low condi ions
534
mode a e 20 min ex ac ion and b) in double- low condi ions mode wi h an ex ac ion ime o 7 min.
535
536
S ooped- low condi ions a
Double- low
condi ionsb,c
Double- low
condi ionsb,d
MLOD
(µg L-1)
MLOQ
(µg L-1)
R2
EF
EE*
EF
EE*
EF
EE*
Salicylic acid
2.0
6.7
0.9997
47
81
9
44
-
-
-
-
-
-
-
-
-
98 (1)
E hyl 4-hyd oxybenzoa e
1.1
3.7
0.9995
42
73
15
74
100 (1)
P opyl 4-hyd oxybenzoa e
0.7
2.3
0.9992
35
61
17
87
98 (1)
Ke op o en
2.9
9.7
0.9991
34
60
18
89
99 (1)
Nap oxen
1.8
6.0
0.9994
41
71
13
64
100 (1)
IsoBu yl 4-hyd oxybenzoa e
0.9
3.0
0.9990
21
44
14
70
100 (1)
Bu yl 4-hyd oxybenzoa e
1.5
5.0
0.9992
16
27
11
55
99 (1)
Diclo enac
4.2
14.0
0.9989
19
34
15
76
92 (1)
Ibup o en
8.5
28.3
0.9991
35
61
19
94
99 (1)
23
*% Ex ac ion e iciency (%RSD, n=4)
537
a Ex ac ion ime: 20 min and sample low a e 20 µL min-1
538
b Ex ac ion ime: 7 min
539
c Accep o low a e o 1 µL min-1 and sample low a e o 20 µL min-1
540
d Accep o and sample low a e o 1 µL min-1
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
Table 3. SF-µLPME/HPLC eco e ies (a e age o h ee de e mina ions ± s anda d de ia ion) om
568
non-dilu ed spiked u ine samples.
569
Spiked le el
(µg L-1)
SAC
KTP
NAX
DIC
IBU
U ine 1
7
92.5 ± 0.6
N.Q
94.4 ± 0.9
N.Q
N.D
18
94.2 ± 1.2
93.3 ± 0.8
92.4 ± 1.1
86.6 ± 0.7
89.1 ± 0.3*
50
95.5 ± 0.6
95.5 ± 0.8
98.2 ± 0.5
85.0 ± 0.5
90.2 ± 0.4
U ine 2
7
90.1 ± 0.5
N.Q
95.2 ± 0.4
N.Q
N.D
18
92.4 ± 0.8
93.4 ± 0.9
93.3 ± 0.8
86.1 ± 0.7
87.2 ± 1.5*
50
95.9 ± 1.2
94.0 ± 1.0
100.0 ± 0.8
88.8 ± 0.4
91.7 ± 0.6
*Spiked concen a ion: 30 µg L-1
570
571
572
573
574
24
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
Table 4. Compa ison o igu es o me i o µLPME wi h o he analy ical echniques o de e mina ion
590
o non-s e oidal an i-in lamma o ies and pa abens.
591
Analy ical me hod
Analy e
Ma ix
Sample
Volume
(mL)
LOQ
(µg L-1)
EF
EE %
Ex ac ion
ime (min)
Mul i-
ex ac ion
Re e ence
HF(3)-LPME-HPLC/UV
SAC, DIC,
IBU
U ine
50
41-180
70-900
-
15
No
4
HF(3)-LPME-MS/MS
SAC, DIC,
IBU
Was e
wa e
50
0.5-5
-
50-100
15
No
1
SPE-LC-MS/MS
SAC, DIC,
IBU
Was e
Wa e
500
0.1-3
-
70
> 30
No
21
DLLME-SFO-HPLC/UV
KTP, DIC
U ine
5
4-5
-
95-100
5
No
22
µLPME-HPLC/UV
double- low
SAC, KTP,
NAX, DIC,
IBU
U ine
0.007
100-500
-
75-100
5
Yes
41
HF-LPME-GCa
MeP, E P,
P P
Wa e and
u ine
8
100-300
21-154
-
40
No
32
HF-LPME-b
MeP, E P,
P P, BuP,
iP P iBuP.
BzP
Wa e
3.5
0.5
3-16
24-60
30
No
3
25
EME-HPLC/UV
E P, P P,
BuP,
iBuP. BzP
Wa e
10
2.4-5
32-49
 8
40
No
33
DF-µLPME-
HPLC/UV
double- low
E P, P P,
BuP, iBuP
Wa e
0.05
5-12
9-10
84-100
5
yes
42
DF-µLPME- HPLC/UV
(1 µL min-1)
SAC,
KTP,
NAX,
DIC, IBU
E P, P P,
BuP, iBuP
U ine
0.007
55-980
-
92-100
7
Yes
This wo k
DF-µLPME- HPLC/UV
(20 µL min-1)
SAC, KTP,
NAX, DIC,
IBU
E P, P P,
BuP, iBuP
U ine
0.14
4.5-49
9-19
44-94
7
Yes
This wo k
SF- µLPME- HPLC/UV
SAC, KTP,
NAX, DIC,
IBU
E P, P P,
BuP, iBuP
U ine
0.4
2.3-28
21-47
27-81
20
Yes
This wo k
592
a Hollow ibe liquid phase mic oex ac ion- 2 phases
593
b Hollow ibe liquid phase mic oex ac ion- 3 phases
594
595
596
597