Carolina M. Santiago da Silva 1, Gustavo Habermann 2, Mary R. R. Marchi 3, Guilherme J. Zocolo 3 * ABSTRACT INTRODUCTION RESEARCH ARTICLE

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1 2012 BRAZILIAN SCIETY F PLANT PHYSILGY RESEARCH ARTICLE The role of mtrix effects on the quntifiction of bscisic cid nd its metbolites in the leves of Buhini vriegt L. using liquid chromtogrphy combined with tndem mss spectrometry Crolin M. Sntigo d Silv 1, Gustvo Hbermnn 2, Mry R. R. Mrchi 3, Guilherme J. Zocolo 3 * 1 Progrm de Pós-Grdução em Ciêncis Biológics (Biologi Vegetl), Deprtmento de Botânic, IB, Universidde Estdul Pulist (UNESP), Rio Clro, SP, Brzil. 2 Deprtmento de Botânic, IB, Universidde Estdul Pulist (UNESP), Rio Clro, SP, Brzil. 3 Deprtmento de Químic Anlític, IQ, Universidde Estdul Pulist (UNESP), Arrqur, SP, Brzil. *Corresponding uthor: gjzocolo@yhoo.com.br Received: 31 July 2012; Accepted: 5 November 2012 ABSTRACT Phytohormone nlysis using liquid chromtogrphy combined with tndem mss spectrometry is very importnt tool for studies involving plnt metbolism; however, this nlysis cn be ffected by mtrix composition. Although it is necessry to understnd this effect to produce relible results, it hs been widely neglected in nlyses of plnt hormones. Lef extrcts from Buhini vriegt were obtined by solvent extrction followed by solid-phse clenup. The extrct ws nlyzed with liquid chromtogrphy-electrospry ioniztion-tndem mss spectrometry (LC-ESI-MS/MS) in negtive ioniztion mode, using the multiple rection monitoring mode with two or three trnsitions to enhnce nlyticl qulity control. Although deuterted stndrds were used s surrogtes, pronounced mtrix effects were detected for phseic cid (PA), bscisic cid (ABA) -glycosyl ester (ABA-GE) nd dihydrophseic cid (DPA), wheres ABA, neopa nd 7`-hydroxy-ABA (7 HABA) showed negligible mtrix effects. The method ws vlidted using spiked smples nd ws pplied to monitor ABA, PA, DPA, neopa, 7 HABA nd ABA-GE on dily bsis. Anlyte recoveries from spiked smples rnged from 67% to 87%. The highest lef concentrtion of phytohormones ws found t 2:00 pm nd 5:00 pm, which represent typicl dy times relted to the decrese of stomtl conductnce. Despite the use of deuterted phytohormones s internl stndrds, we showed tht the use of clibrtion curve constructed with mtrix extrct is mndtory for the relible quntifiction of ABA nd its metbolites, especilly PA, ABA-GE nd DPA. Dily vritions in endogenous ABA lef concentrtion were discussed. Keywords: ABA-β-D-glucosyl ester, ABA dily vritions, dihydrophseic cid, LC-ESI-MS/MS, phseic cid. INTRDUCTIN Plnts exposed to dily increse in ir temperture, typiclly peking t middy, lso experience n elevtion in vpor pressure deficit (VPD) (Hbermnn nd Rodrigues, 2009). Becuse stomtl conductnce (gs) diminishes with incresing VPD, even well-hydrted plnts of some species tend to trnspire more under these conditions (Hbermnn et l., 2003). Plnts cn minimize such excessive trnspirtion rtes (E) by reducing gs, which is regulted by bscisic cid (ABA), plnt hormone found in the poplst of cells in the mesophyll (Assmnn, 2010). The biosynthesis of ABA occurs in chloroplsts nd other plstids (Schwrtz nd Zeevrt, 2010). The metbolism of ABA differs mong species nd lso Brz. J. Plnt Physiol., 24(3): , 2012

2 224 SILVA C. M. S. et l. ccording to plnt development stge nd tissue type (Schwrtz nd Zeevrt, 2010). The ctbolism of ABA is bsed on three rections: oxidtion, conjugtion nd reduction. The min oxidtive route of nturl ABA is 8` hydroxyltion, which results in the production of 8`-hydroxyABA nd isomerizes spontneously to phseic cid (PA). Then, it my be reduced to form dihydrophseic cid (DPA), the min product, nd lesser quntities of epidihydrophseic cid (epidpa) (Sik et l., 2007). A second oxidtive route involves oxidtion of the 9-methyl group of ABA, resulting in cyclic product clled neophseic cid (neopa). The minor oxidtive route results in the formtion of (+)-7`-hydroxy-ABA (7`HABA), wheres the minor reductive pthwy produces the unstble 1`,4`-diol ABA. ABA nd its metbolites cn lso be conjugted with glucose to form glycosylted esters corresponding to C1, ABA-β-D-glucosyl ester (ABA-GE) or glycosides t C-1`or C-4` (Zhri et l., 2005). It is widely recognized tht ny deficiency in ABA biosynthesis cn led to plnt desicction (Schwrtz nd Zeevrt, 2010; Assmnn, 2010). Therefore, nlyticl methods tht re cpble of determining vritions in the lef ABA content with high sensitivity under vrying conditions re of scientific interest. In mny cses, stisfctory nlyticl results for ABA in plnt tissues cn be obtined using gs chromtogrphy (Duffield nd Netting, 2001; Birkemeyer et l., 2003), immunossys or liquid chromtogrphy (LC) s seprtion methods. These re lwys used in combintion with n identifiction tool, typiclly mss spectrometry (MS). Derivtiztion of the compounds is criticl step tht is essentil for gs chromtogrphy (GC) nlyses (Pn nd Wng, 2009). Becuse immunossys generlly disply high error rtes due to cross-rectivity, liquid chromtogrphy is the preferred nlyticl seprtion method for hormonl nlysis in plnt tissues (Chiwoch et l., 2003; Schwrtz nd Zeevrt, 2010; Suswwngsup et l., 2011). The complexity of the mtrix nd the smll mounts of nlytes present in the plnt turn the study of mtrix effects into n importnt step to enble the collection of relible dt (Thompson et l., 2002). When using liquid chromtogrphy-electrospry ioniztion-tndem mss spectrometry (LC-ESI-MS/MS) for plnt hormone nlysis, co-extrcted constituents in the mtrix cn interfere with the nlysis nd cuse ioniztion enhncement or suppression (Fn et l., 2011). The type nd mgnitude of the mtrix effect my vry for different concentrtion rnges (Zrostlíková et l., 2001); thus, the use of surrogtes (typiclly deuterted phytohormones) cnnot effectively correct for interference in ll cses. In generl, specific surrogtes re dded to the smples t single concentrtion; therefore, the nture nd mgnitude of the mtrix effect must be determined for ech compound to be quntified. We developed LC-ESI-MS/MS method for the rpid determintion of the quntities of ABA nd its metbolites, which re present in Buhini vriegt leves smpled t different times over 24-h period. We investigted the effects of the mtrix on the nlyticl results to determine whether ny corrective precutions were necessry to yield n ccurte nlysis of ABA nd its metbolites. Bsed on the hypothesis tht Buhini vriegt leves show higher levels of ABA in the fternoon compred with the night nd predwn periods, we evluted vritions in the concentrtions of ABA nd its metbolites over the course of the dy. MATERIAL AND METHDS Plnt mteril: To evlute the bility of LC-ESI- MS/MS to discriminte between ABA nd its metbolites (ABA-GE, DPA, PA, 7 -HABA nd neopa) nd to detect trce quntities of these compounds, we used lef smples from three Buhini vriegt plnts hrvested t 2:00 m, 5:00 m, 8:00 m, 11:00 m, 2:00 pm, 5:00 pm, 8:00 pm nd 11:00 pm during the dry seson (August) of The plnts were grown t the Universidde Estdul Pulist (UNESP), cmpus Rio Clro (22 25 S, W) in Brzil. The cnopy of ech plnt ws subdivided into qudrnts (North, South, Est nd West), nd leves were rndomly hrvested from ech qudrnt. After hrvesting, the leves were wrpped immeditely in luminum foil, frozen in liquid nitrogen nd stored t -80 C until nlysis. Regents nd stndrds: Methnol nd formic nd cetic cids (ll HPLC grde) were purchsed from J.T.Bker (Xlostoc, Mexico). High-purity wter ws produced by the Millipore Milli-Q System (Billeric, MA, USA). Solid-phse extrction crtridges (Strt X, 200 mg, 6 ml) were purchsed from Phenomenex (Torrnce, CA, USA), nd membrne filters (Nylon TM, 0.22 µm porosity) were purchsed from Srtorius (Goettingen, Germny). Authentic ABA ws purchsed s solid stndrd from Sigm-Aldrich (kville, Cnd, 98.5% purity). The stndrds for DPA, ABA-GE, PA, 7 -HABA nd neopa nd the deuterted surrogtes d 3 -PA, d 4-7 -HABA, d 5 -ABA-GE, d 3 -DPA, d 3 -neopa nd d 6 -ABA were purchsed from the Plnt Biotechnology Institute (Ssktoon, Ssktchewn, Cnd) nd were synthesized s necessry to t lest 98% purity. Brz. J. Plnt Physiol., 24(3): , 2012

3 THE RLE F MATRIX EFFECTS N THE QUANTIFICATIN F ABSCISIC ACID AND ITS METABLITES IN THE LEAVES F BAUHINIA VARIEGATA L. USING LIQUID CHRMATGRAPHY CMBINED WITH TANDEM MASS SPECTRMETRY 225 Extrction nd purifiction: The used method ws first proposed by Chiwoch et l. (2003) nd modified by Tureckov et l. (2009). In our nlysis, we used n dpted version of Tureckov s method. Leves were ground using porcelin mortr nd pestle. The smple (50 mg) ws extrcted with 750 µl of methnol:wter:cetic cid (10:89:1) solution for 1h t 4 C. A solution contining ech lbeled stndrd (30 ng ech of d 6 -ABA, d 5 -ABA-GE, d 3 -DPA, d 3 -PA, d 4-7 -HABA nd d 3 -neopa) ws dded s n internl stndrd nd surrogte. The solvent ws subsequently removed with syringe, nd the plnt tissue ws extrcted gin for 30 min in the sme conditions s the initil extrction. Both extrcts were combined [to void lef prticles in the extrct, we filtered the solvent in 0.45 mm x 47 mm glss microfiber filter (Srtorius AG, Germny) membrne] nd loded onto solid phse extrction (SPE) crtridge tht hd been conditioned with 2 ml methnol nd blnced with 2 ml of methnol:wter:cetic cid (10:89:1) solution. After the smple ws loded, the crtridge ws wshed with 1 ml of the methnol:wter:cetic cid (10:89:1) solution nd eluted using 3 ml methnol:wter:cetic cid (80:19:1). The elute ws dried under gentle nitrogen flow nd reconstituted with 300 µl of methnol:wter (30:70) contining 0.1% formic cid, filtered through 0.22-µm nylon membrne filter nd nlyzed. The extrction ws performed in duplicte, nd the LC-ESI-MS/MS nlyses were performed in triplicte for ech extrct. LC-ESI-MS/MS nlysis: Abscisic cid nd its metbolites were nlyzed with liquid chromtogrph (Agilent 1200 series) coupled to n ESI-MS/MS system (3200 QTrp; Applied Biosystems/MDS Sciex) using reversed-phse column (Agilent, C18, 150 mm µm). The mobile phse consisted of methnol (A) nd wter (B), both contining 0.1% formic cid in the grdient elution mode. The A:B elution strted with 50% B nd incresed to 80% B over 7.5 min, nd then switched to 100% A over 2.5 min nd remined t 100% A for 2.0 min. The re-equilibrtion time ws 5.0 min. The flow rte ws 500 µl min -1, nd the injection volume ws 20 µl. To prevent the contmintion of the MS detector, divert vlve ws used, which directed the column elution flow directly to the wste before 4 min nd fter 12 min in ech smple run. The instrument detection limit (the concentrtion tht results in signl:noise rtio bove 1:3) ws determined. The limit of quntifiction (LQ) ws defined s the lowest concentrtion in the liner response portion of the clibrtion curve (Eurchem, 1998). Mss spectrometry operting prmeters: To select suitble dignostic precursor-to-product ion trnsitions, stndrd solutions with 200 ng ml -1 of ech compound in methnol:wter (1:1) contining 0.1% formic cid were injected directly into spectrometer (API 3200, Applied Biosystems/MDS Sciex) equipped with TurboV TM ioniztion source operting in negtive ioniztion mode. All dt were processed by the Anlyst softwre (version 1.5.1). A Hrvrd infusion pprtus (Holliston, MA, USA) ws used to optimize the MS conditions. A hybrid triple qudrupole/liner ion trp mss spectrometer with n electrospry (ESI) ion source ws used. MS prmeters were optimized by flow injection nlysis (FIA) of the solutions contining DPA, ABA-GE, PA, 7 -H-ABA, neopa, nd ABA nd their respective deuterted stndrds. The optimized ion source prmeters were s follows: curtin gs (CUR), 10 psi; ion spry voltge (IS), -4,000 V; nebulizer gs (GS1), 45 psi; turboheter gs (GS2), 45 psi; nd temperture (TEM), 450 C. CAD (collisionlly ctivted dissocition) gs ws set to medium pressure. In Tble 1, we list the optimized MS prmeters; the declustering potentil (DP), the entrnce potentil (EP, Q 0 lens), the collision entrnce potentil (CEP), the collision energy (CE) nd the cell exit potentil (CXP) were optimized for ech compound. N 2 (99.999% purity) ws used s the collision gs. The trnsitions selected for the nlysis re lso described in Tble 1. Method performnce evlution: Method performnce ssessment ws conducted with spiked smples. Becuse ll plnt mtrices contin these compounds, smple extrct ws nlyzed, nd the re obtined for ech compound ws subtrcted from the corresponding res obtined for the spiked smples. The spiking solution contined ll of the compounds (nturl nd deuterted) t the sme concentrtion. Mtrix effect: To evlute the effect of the lef compound mtrix extrcted with ABA nd its metbolites, dry extrcts were prepred contining the sme concentrtion of stndrd mixture s used in the clibrtion curve prepred from net regent. The extrcts were reconstituted in methnol:wter (30:70) contining 0.1% formic cid. As noted by Thompson (2002), the mtrix effect cn influence the sensitivity of the nlyticl response either positively or negtively. Thus, the effect ws clculted s the percentge difference between the slopes of the nlyticl curves tht were constructed for the mtrix nd solvent. Using this pproch, the effect cn be considered s importnt when greter thn 10%. The clibrtion curve in solvent ws prepred using eight concentrtions diluted in methnol: wter (30:70) solution with 0.1% formic cid. Dt were obtined by injecting 10 µl of ech solution Brz. J. Plnt Physiol., 24(3): , 2012

4 226 SILVA C. M. S. et l. Tble 1. Specific ion rtios or re rtios collected by MS/MS for ccurte confirmtion. Retention Time (min) 4,65 DPA Anlyte Structures MRM trnsitions H H H MRM1 281 > 237 MRM2 281 > 171 Clculted Ion Rtio* MRM1/MRM2 MRM1/MRM ,93 ABAGE H H H H H H MRM1 425 > 261 MRM2 425 > ,60 PA 7,08 7-H-ABA 7,51 neo-pa H H H H H H H MRM1 279 > MRM2 279 > 205 MRM3 279 > MRM1 279 > MRM2 279 > 217 MRM3 279 > MRM1 279 > MRM2 279 > 122 MRM3 279 > ,61 ABA 1 H H 263 > MRM1 MRM2 263 > 219 *n=3 (spiked smples); Coefficient of vrition rnged from 3 to 12% contining 1, 2.5, 5, 10, 25, 50, 100 or 250 ng ml -1 of the stndrd mixture. The response ws clculted s the rtio between the nturl stndrd pek re nd the lbeled stndrd pek re. Recovery study: Lbeled nd unlbeled stndrds were dded t four concentrtions (0.3, 1.5, 15 or 75 ng) to 50 mg of ground plnt tissue t the beginning of the extrction. In prllel, nother portion of the plnt tissue ws extrcted without spiking, nd its finl dry extrct ws reconstituted with stndrd solution contining the compounds t the sme concentrtion s tht used to spike the originl smples. All procedures were performed in triplicte, nd the extrcts were lso nlyzed in triplicte. The recovery nd the coefficient of vrition were determined by compring the pek res of the nlyticl stndrd spiked before nd fter extrction. Precision nd ccurcy: Precision ws evluted with the reltive stndrd devition determined from repeted extrct injections nd triplicte extrctions. Accurcy ws evluted s the difference between the spiked nlyte mss nd the nlyte mss obtined on nlysis, i.e., the recovery. To quntify ABA nd its metbolites from the smple clibrtion curve, the clibrtion curve prepred using the mtrix extrct ws used insted of the clibrtion curve prepred with the net solvent when the clculted mtrix effect ws higher thn 10% (Thompson et l., 2002). The method quntifiction limit ws set s the lowest spiking concentrtion tht yielded recoveries between % nd CV below 20% (Brito et l., 2003). Dt nlysis: A one-wy nlysis of vrince (ANVA) ws performed to test the significnce of vritions in the lef contents of ech ABA metbolite over the course of the dy (t 2, 5, 8, 11, 14, 17, 20 nd 23 h). For ech ABA metbolite, Dunn s method (α=0.05) ws used to perform post-hoc men result comprisons. RESULTS The optimized chromtogrphic conditions voided coelution of the nlytes, especilly PA, neopa nd 7 HABA, which hve similr structures (Figure 1). A comprison between their retention times showed tht the nlytes Brz. J. Plnt Physiol., 24(3): , 2012

5 THE RLE F MATRIX EFFECTS N THE QUANTIFICATIN F ABSCISIC ACID AND ITS METABLITES IN THE LEAVES F BAUHINIA VARIEGATA L. USING LIQUID CHRMATGRAPHY CMBINED WITH TANDEM MASS SPECTRMETRY 227 X ICof-MRM (30 pirs): 425,174/261,400 D ID: ABAGE 1 from Smple 29 (500ppb010) of Dt Curv nlitic_08_08_20011_2.wiff (Turbo... Mx. 3,9e4 cps. 9,5e4 9,0e4 6 8,5e4 8,0e4 7,5e4 5 7,0e4 6,5e4 6,0e4 3 5,5e4 5,0e4 4,5e4 4,0e4 2 3,5e4 3,0e4 4 2,5e4 2,0e ,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5 1 10,5 11,0 Figure 1. LC-ESI-MS/MS chromtogrm for stndrd solution of ech compound (500 ng ml -1 ). Different lines represent the trnsitions monitored for ech substnce. Peks in elution order: 1, DPA; 2, ABA-GE; 3,PA; 4, 7 H ABA; 5, neopa; 6, ABA. were clerly differentited in the chromtogrm. However, due to the similrities of the compounds, nother form of identifiction, such s mss spectrometry, ws necessry (Pn nd Wng, 2009). Depending on the nlyte, the method described here uses two or three multiple rection monitoring (MRM) trnsitions, which includes dditionl confirmtory informtion to our results (Tble 1). In this study, we estblished rtios mong trnsitions s the key for the relible identifiction of these phytohormones. We determined these rtios using surrogte compounds in stndrd solutions (for ll nlyticl curves) nd using spiked smples. For the confirmtion of nlytes in plnt smples, the chromtogrphic retention time of the smple plnt hormones could not vry by more thn 2%, nd the reltive bundnce of the monitored MRM trnsitions ws required to be within ± 25% of both the clibrtion stndrds nd the spiked smples (Europen community, 2002). As recommended by Eurchem (Eurchem, 1998), our quntifiction dt re within the recovery limits for the given concentrtion zone (Tble 2). An dditionl recovery study of cluster spiked with 75 ng of stndrd mixture, which yielded coefficient of vrition (CV) of pproximtely 40% (dt not shown), indicted tht this concentrtion zone ws not vlidted. The most significnt mtrix effects were observed for ABA-GE, DPA nd PA, with 63%, 29% nd 27%, respectively (Tble 3). n inspection of these reltionships, if solvent curve hd insted been used for quntifiction in these cses, the results would hve been grossly inccurte. Due to the presence of nturl ABA nd its metbolites in plnt tissues, the detection limit cnnot be used s prmeter to vlidte this method (Eurchem, 1998). Thus, we obtined limits of quntifiction of 4 ng g -1 for ABA, neopa nd 7 -HABA nd 20 ng g -1 for PA, DPA nd ABA-GE. Brz. J. Plnt Physiol., 24(3): , 2012

6 228 SILVA C. M. S. et l. Tble 2. Recovery percentge nd precision dt obtined with spiked smples in the recovery study. Spiked smples Compound 6 ng g ng g ng g -1 Recovery CV Recovery CV Recovery CV DPA ABA-GE PA H-ABA neopa ABA The method used for the recovery study is described in the text (Method Performnce Evlution Section, n=9). ng ech compound per g plnt tissue (dry mss) Tble 3. Mtrix effect results showing curve equtions in the solvent nd in the mtrix extrcts, differences between ngulr coefficients nd curve intervls Solvent Mtrix extrct Difference in ngulr Concentrtion rnge R 2 Curve eqution R 2 Curve eqution coefficients (%) (ng ml -1 ) DPA y=1.6925x y=1.1959x PA y=0.882x y=1.1231x ABA-GE y=1.2888x y=0.4779x neopa y=2.9893x y=2.7x HABA y=5.5002x y=5.7308x ABA y=0.1484x y=0.1386x As nticipted, the concentrtion of ABA in B. vriegt leves rnged with the time of dy. The highest lef concentrtions were found t 2:00 pm nd 5:00 pm, intermedite concentrtions were observed t 800h, 8:00m, 8:00 pm, 11:00 pm nd 2:00 m, nd the lowest concentrtions were observed t 5:00 m nd 11:00 m (Figure 2A). There were high lef concentrtions of PA t 2:00 pm nd 5:00 pm (Figure 2C), nd the concentrtions of neopa, 7 -HABA, ABA-GE nd DPA showed no significnt vritions ccording to the time of dy (Figure 2B, D-F). DISCUSSIN Chromtogrphy nd mss spectrometry: ne should consider tht coelution of substnces with similr frgmenttion profiles cn result in flse positives. Tndem nlysis with mss spectrometry is widely used to identify ABA nd its metbolites, with one (Turecková et l., 2009; Zhou et l., 2003; Ross et l., 2004; Lopez- Crbonell et l., 2009) or t most two trnsitions (Gómez-Cdens et l., 2002; Vilró et l., 2006) for ech compound. However, even with different retention times, the use of only one MRM trnsition for ech nlyte my lso be problemtic. Phseic cid nd neo-pa, under our optimized chromtogrphic conditions, showed good pek resolution, but it demonstrted to hve MRM trnsitions with identicl frgmenttion ptterns: PA, 1 st trnsition 279 > 139; neo-pa, 3 rd trnsition 279 > 139; PA, 2 nd trnsition 279 > 205; neo-pa, 1 st trnsition 279 > 205 (Figure 3). Thus, in this cse, it is necessry to use t lest two trnsitions long with retention time for relible identifiction of these nlytes. When determining nlytes (pollutnts) in environmentl wter, it is recommended tht one use retention time plus t lest two trnsitions, with suitble ion-intensity rtio between them, for robust compound identifiction; in ddition, the use of s mny specific trnsitions s possible my help void flse positives due to common losses (e.g., H 2 or C 2 ) (Pozo et l., 2006). Therefore, the sme cn be pplied to plnt tissues, becuse the chievement of relible identifiction of compounds t low concentrtions my be much more complex in plnt mtrices. During the recovery study using spiked smples, we observed peks tht coeluted with the phytohormones, especilly with PA t m/z 279.0, neo-pa t m/z nd 7 -H-ABA t m/z These peks my hve been other plnt constituents with similr structures. ne of the most bundnt chemicl clsses in plnt tissues is phenolics. Phenolic compounds show nonspecific losses of C 2, C nd Brz. J. Plnt Physiol., 24(3): , 2012

7 THE RLE F MATRIX EFFECTS N THE QUANTIFICATIN F ABSCISIC ACID AND ITS METABLITES IN THE LEAVES F BAUHINIA VARIEGATA L. USING LIQUID CHRMATGRAPHY CMBINED WITH TANDEM MASS SPECTRMETRY A B 2.5 ABA (ng mg -1 f. w.) b bc b b b b c b b b b DPA (ng mg -1 f.w. lef) 0.00 C D 0.0 PA (ng mg -1 f.w.) cde e b e bc bd de neo-pa (ng mg -1 f.w.) ABA-GE (ng mg -1 f.w.) E c bcde d b e F c b bc 'H ABA (ng mg -1 f.w.) 0 8h 11h 14h 17h 20h 23h 2h 5h 8h 11h 14h 17h 20h 23h 2h 5h 0.0 Figure 2. Buhini vriegt lef contents of ABA (A), DPA (B), PA (C), neopa (D), ABA-GE (E) nd 7 -HABA (F) over the course of dy. Similr lowercse letters or bsence of letters indictes no significnt differences (p < 0.05) mong dy times. Brs=SD H 2 tht re widely used for their identifiction in plnt extrcts (M et l., 1997; Vessecchi et l., 2011). However, using only these losses could led to mistken compound identifictions. Mtrix effects: Given tht coeluting compounds cn interfere with the ioniztion process, which is essentil for quntifiction, the mtrix effect is directly ffected by the composition of the tissue under nlysis. Therefore, it is essentil to perform similr study using dditionl tissues from the sme species or tissues from different species. In contrst to our results, Fn et l. (2011) reported no mtrix effects on the nlysis of severl phytohormones (including ABA) in cnol. Their results re in ccordnce with our findings, becuse ABA showed 6.6% mtrix effect (Tble 3), llowing its quntifiction by the solvent curve method (i.e., mtrix effect <10%). This lck of mtrix effect my underlie the common prctice of quntifying ABA without nlyzing other compounds from the ABA metbolic route, which re of similr importnce for understnding wter stress. Brz. J. Plnt Physiol., 24(3): , 2012

8 230 SILVA C. M. S. et l. 5,60 MRM1 500 H H PA 6,66 7,50 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 7,50 MRM2 500 H PA H 5,60 6, ,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 H H PA 6,66 MRM3 5,59 7,50 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 1,8e4 7,51 MRM ,61 6,67 H neopa H 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 7,51 MRM2 H 500 5,60 H neopa 6,67 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 1,7e4 5,60 MRM ,51 H H 6,67 neopa 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 1 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 Figure 3. Chromtogrm of smple PA (t r =5.60 min) nd neopa (t r =7.51 min) hve identicl frgmenttion ptterns t 279 >139 nd t 279>205, which cn led to identifiction errors. Brz. J. Plnt Physiol., 24(3): , 2012

9 THE RLE F MATRIX EFFECTS N THE QUANTIFICATIN F ABSCISIC ACID AND ITS METABLITES IN THE LEAVES F BAUHINIA VARIEGATA L. USING LIQUID CHRMATGRAPHY CMBINED WITH TANDEM MASS SPECTRMETRY 231 Considering quntifiction limits, our results re consistent with previously published vlues (Zhou et l., 2003; Turecková et l., 2009; Lopez-Crbonell et l., 2009). However, in severl reports, the uthors did not confirm if the reported limit of quntifiction referred to the nlyticl system or to the entire method. The mtrix effect in most cses depends on the mount of smple used, becuse in more concentrted extrcts coextrcted compounds it will be, consequently, more concentrted, nd incresed effects on ioniztion processes will lso be expected, generting unrelible results. n the other hnd, in the present study the reltionship between the mount of smple used nd consequences for mtrix effects ws not the min objective. Hormone quntifiction nd physiologicl implictions: The bscisic cid lef content reported over the course of the dy re rre for ntive plnts, nd for crop species ABA lef contents re described following wter deficient tretments, insted of during the course of the dy. n the other hnd, our findings fit the physiologiclly expected pttern of vrition in the concentrtion of this plnt hormone throughout the dy. As estblished in other studies, the stomtl conductnce (gs) of leves of mny species diminishes when exposed to high vpor pressure deficit, or VPD (Khiri nd Hll, 1976; Levy, 1980; Sinclir nd Allen, 1982; Grntz, 1990; Assmnn, 2010). Citrus plnts respond to elevted VPDs by lowering both trnspirtion rte nd gs (Hbermnn et l., 2003). In this study, we did not mesure gs, but the VPD ws highest t middy nd erly fternoon nd decresed in the evening in correltion with ir temperture (dt not shown). However, in mple plnts ABA lef content hs been suggested s substitute for gs (Buerle et l., 2006). The lef content of PA exhibited dytime pttern similr to tht of ABA (Figure 2C). Although both exert physiologicl effects, PA is considered s the first compound to be degrded in biochemicl pthwy tht ends with the biosynthesis of the inctive DPA (Schwrtz nd Zeevrt, 2010). The inctive compounds (DPA, neopa, 7 -HABA nd ABA-GE) mesured in B. vriegt leves ppered to be mintined in high concentrtions throughout the dy (Figure 2B,D-F). Becuse we did not mesure these compounds in root smples or in xylem exudtes, it ws not possible to determine whether the root system contributed to the chnges in concentrtion of these compounds. However, ABA lef concentrtion mesured in mple plnts ws described s better indictor of gs responses to soil wter deficit, in comprison with xylem collected ABA (Buerle et l., 2006). Although we were unble to determine the specific mechnisms underlying the fluctutions in the level of ech mesured compound, we performed n ccurte quntittive nlysis of the compounds in the bscisic cid biosynthetic pthwy. Moreover, the endogenous concentrtions of ABA nd PA fit the physiologiclly expected dytime vritions ccurtely. In prior literture, discussion of the mtrix effect on phytohormone quntifiction hs focused on ABA or other plnt hormones exclusively (Fn et l., 2011). In our study, we included ll ABA metbolites. Plnt tissue is very complex mtrix, nd studies of mtrix effects re criticl for hormone quntifiction. ABA does not present significnt mtrix effect; however, if these nlyses were not performed, we would hve obtined PA vlues smller thn the ctul ones. For ABA-GE nd DPA, the concentrtions obtined without ccounting for the mtrix effect would hve been greter thn the ctul vlues. To our knowledge, no other study hs nlyzed dytime vrition in ABA nd its metbolites. We selected Buhini vriegt s subject for the profiling of ABA hormones. Although used only s confirmtion of well known ABA dily lef concentrtions, since our investigtion ws minly devoted to the ABA methodologicl concepts, including the mtrix effects, our results corroborte the fct tht the highest ABA nd PA lef concentrtions were found t 14:00 nd 17:00 h, when VPD typiclly increses nd stomtl conductnce normlly decreses, which is in ccordnce with clssicl ecophysiologicl response in plnts. Acknowledgments: We cknowledge Fundção de Ampro à Pesquis do Estdo de São Pulo (FAPESP) for the post-doctorl fellowship to G. J. Zocolo (Proc: 2011/ ). We extend our cknowledgements to Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq) for finncil support (Proc /2008-4) nd for fellowships to G. Hbermnn, M. R. R. Mrchi (reserch productivity) nd to C. M. S. Silv (Msters). We especilly thnk Prof. M. V. B. Znoni for the lon of the LC-ESI-MS/MS. 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