DETERMINATION OF ETORICOXIB IN HUMAN PLASMA USING AUTOMATED ON-LINE SOLID-PHASE EXTRACTION COUPLED WITH LC-APCI/MS/MS

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1 Quim. Nov, Vol. 31, No. 3, , 2008 DETERMINATION OF ETORICOXIB IN HUMAN PLASMA USING AUTOMATED ON-LINE SOLID-PHASE EXTRACTION COUPLED WITH LC-APCI/MS/MS Artigo Sérgio Luiz Dlmor*, Liberto Brum Junior, Ricrdo Mchdo Ferretto, Pulo Rento de Oliveir, Thigo Brth nd Mximilino d Silv Sngoi Deprtmento de Frmáci Industril, Centro de Ciêncis d Súde, Universidde Federl de Snt Mri, Snt Mri-RS, Brzil Recebido em 24/4/07; ceito em 20/9/07; publicdo n web em 26/2/08 A liquid chromtogrphy-tndem mss spectrometry method with tmospheric pressure chemicl ioniztion (LC-APCI/MS/MS) ws vlidted for the determintion of etoricoxib in humn plsm using ntipyrin s internl stndrd, followed by on-line solidphse extrction. The method ws performed on Lun C 18 column nd the mobile phse consisted of cetonitrile:wter (95:5, v/ v)/mmonium cette (ph 4.0; 10 mm), run t flow rte of 0.6 ml/min. The method ws liner in the rnge of ng/ml (r 2 >0.99). The lower limit of quntittion ws 1 ng/ml. The recoveries were within %. Moreover, method vlidtion demonstrted cceptble results for the precision, ccurcy nd stbility studies. Keywords: etoricoxib; LC-APCI/MS/MS; on-line solid-phse extrction. INTRODUCTION Etoricoxib (5-chloro-6 -methyl-3-[4-(methylsulfonyl)phenyl]- 2,3 -bipyridine) represents second-genertion of cycloxygense-2 (COX-2) inhibitors tht hs been developed for the tretment of mny inflmmtory diseses such s rheumtoid rthritis, osteorthritis, pin relief nd cute gout, cusing fewer gstrointestinl complictions thn conventionl non-steroidl ntiinflmmtory drugs. 1-3 Etoricoxib is well orlly bsorbed, nd following 120 mg single dily dosge to fsted dults, the pek plsm concentrtion (geometric men C mx = 3600 ng/ml) ws observed t pproximtely 1 h (t mx ). 4-6 An nlyticl high performnce liquid chromtogrphy (HPLC) method with photochemicl cyclistion nd fluorescence detection for the quntittion of etoricoxib in humn plsm nd urine ws published using structurl nlogue s internl stndrd nd offline solid-phse extrction (SPE). 7 A liquid chromtogrphy-tndem mss spectrometry (LC-MS/MS) method with tmospheric pressure chemicl ioniztion (APCI) ws vlidted over the concentrtion rnge of ng/ml for the determintion of etoricoxib in humn plsm with stble isotope s internl stndrd, nd the run time of 8 min. 8 The LC-MS/MS method with electrospry ioniztion (ESI) for the determintion of etoricoxib in humn plsm, fter extrction by off-line SPE ws lso developed over the concentrtion rnge of ng/ml. 9 A liquid chromtogrphy (LC) coupled with to ion trp mss spectrometry nd APCI for quntittion of both etoricoxib nd vldecoxib in humn plsm ws lso performed in the liner rnge of ng/ml. 10 The LC method for the quntittion of etoricoxib in humn plsm ws vlidted with the run time of 10 min. 11 The reversed phse LC method ws vlidted for the determintion of etoricoxib in phrmceuticl dosge forms nd compred to the LC-MS/MS method vlidted with detection by ESI, pplied lso for the determintion of etoricoxib in spiked humn plsm. 12,13 The mtrix ion suppression requires tht the mjority of the *e-mil: sdlmor@terr.com.br biologicl mtrix constituents being removed prior to LC-MS/MS nlysis of the nlyte of interest mking smple pre-tretment time consuming step in the development of LC-MS/MS procedure. This cn be performed using either liquid-liquid extrction (LLE) or trditionl off-line SPE, which needs either reltively lrge volumes of plsm (typiclly 1-2 ml) or orgnic solvents (2-4 ml) for the elution of the nlyte from the solid phse. Aprt from full utomtion, the stte-of-the-rt for on-line SPE lso provides high precision nd sensitivity nd higher smple throughput s compred to LLE or off-line SPE. 14,15 The im of the present work ws to develop nd vlidte sensitive nd utomted high throughput on-line SPE-LC-MS/MS method for the determintion of etoricoxib in humn plsm, improving the current published procedures, in order to support humn clinicl studies. EXPERIMENTAL Chemicl nd regents Etoricoxib reference substnce ws generously supplied by Merck Reserch Lbortories (Rhwy, USA) nd ntipyrin, s internl stndrd (IS), ws purchsed from Sigm (St Louis, MO, USA). HPLC-grde cetonitrile, methnol nd formic nd cetic cid were purchsed from Tedi (Firfield, USA). Ammonium cette ws purchsed from Merck (Drmstdt, Germny). All chemicls used were of phrmceuticl or specil nlyticl grde. For ll the nlyses, ultrpure wter ws purified using n Elix 3 coupled to Milli-Q Grdient A10 system (Millipore, Bedford, MA, USA). Preprtion of stock solutions The stock solution of etoricoxib ws prepred by weighing 10 mg of reference mteril into 10 ml volumetric flsk nd diluting to volume with cetonitrile, obtining concentrtion of 1 mg/ml. Antipyrin stock solution ws lso mde t finl concentrtion of 1 mg/ml using cetonitrile. The prepred stock solutions were stored t 2-8 C protected from light.

2 Vol. 31, No. 3 Determintion of etoricoxib in humn plsm 575 Preprtion of clibrtion reference nd qulity control smples The stock solution of etoricoxib ws diluted with cetonitrile to obtin clibrtion reference solutions with the concentrtions of 10000, 1000, 100 nd 10 ng/ml. The corresponding volume tken of the reference solutions were evported under nitrogen strem while immersed in 40 C wter bth, nd the residues reconstituted in 0.5 ml of blnk plsm to prepre the clibrtion reference contining from 1 to 5000 ng/ml (1, 10, 100, 500, 1000, 2000 nd 5000 ng/ml). The qulity control (QC) smples were prepred in pooled plsm, with the concentrtions of 3 (low), 2500 (medium) nd 4000 ng/ml (high), nd then divided in liquots tht were stored t 80 ºC until nlysis. Smple preprtion A totl of 500 μl of the plsm ws trnsferred to glss tube, followed by ddition of 50 μl of internl stndrd solution (250 ng/ ml of ntypirin in cetonitrile). All smples were mixed by vortex gittion for 30 seconds nd trnsferred to utosmpler vils. Smples were then injected onto the system. Crtridges nd on-line SPE system The following crtridges (10 x 2 mm i.d.) with the different prticles size were used for the development of the on-line solidphse extrction: HySphere CN, 8 μm; HySphere C 2, 8 μm; HySphere C 8, 8 μm; HySphere C 8 (EC), 8 μm; HySphere C 18, 8 μm; HySphere C 18 HD, 7 μm; HySphere Resin GP, μm; HySphere Resin SH, μm; BondElut C 18, μm nd BondElut C 8, μm. On-line SPE system Prospekt-2 (Sprk Hollnd, Emmem, The Netherlnds), consisted of n utomted crtridge exchnge (ACE) module for disposble crtridge exchnge; high-pressure dispenser (HPD) module for hndling of solvents, which trnsports the liquids by the 2 ml high pressure syringe (up to 300 br) nd the trithlon utosmpler. The entire system ws operted by SprkLink TM softwre progrm (V Sprk, Hollnd). The injection nd the extrction were completely utomted using Prospekt-2 system nd the method developed consisted of the following steps: the crtridge ws solvted with 1 ml of methnol t 5 ml/min nd equilibrted with 1 ml of wter t sme flow rte. Following the injection of 20 μl of the plsm smple into the crtridge, 750 μl of wter t 2 ml/min were eluted, to retin etoricoxib nd ntipyrin, nd wste plsm proteins. The crtridge ws wshed with 1 ml of wter:methnol (95:5, v/v) t 2 ml/min, nd then, eluted with mobile phse t 0.6 ml/min for 1 min to the nlyticl column nd hence to MS/MS. After the elution nd during LC-MS/MS run, the crtridge ws wshed with 2 ml of cetonitrile:wter (50:50, v/v) t flow rte of 2 ml/min. The utosmpler ws progrmmed to perform wsh of the needle with 500 μl of cetonitrile: 2% formic cid (50:50, v/v), to reduce crryover, including one extr-wsh step. LC-MS/MS conditions The LC-MS/MS method ws performed on Shimdzu LC system (Shimdzu, Kyoto, Jpn) equipped with SCL-10A VP system controller, LC-10 AD VP pump, DGU-14A degsser, nd CTO-10 AD VP column oven. A trithlon utosmpler (Sprk, Emmen, Hollnd) ws used. The experiments were crried out on reversed phse Phenomenex (Torrnce, USA) Lun C 18 column (50 x 3.0 mm i.d.), with prticle size of 3 μm nd pore size of 100 Å). A security gurd holder (4.0 x 3.0 mm i.d.) ws used to protect the nlyticl column. The LC system ws operted isocrticlly t controlled temperture (25 ºC) using mobile phse of cetonitrile:wter (95:5, v/v)/ mmonium cette (ph 4.0; 10 mm). This ws filtered through 0.45 μm membrne filter (Millipore, Bedford, MA, USA) nd run t flow rte of 0.6 ml/min. The injection volume ws 20 µl for both reference nd smples. The triple qudrupole mss spectrometer (Micromss, Mnchester, UK), model Quttro LC, equipped with n APCI source using crossflow counter electrode run in positive mode (APCI+), ws set up in multiple rection monitoring (MRM) mode, monitoring the trnsitions 359.6>280.1 nd 189.6>172, for etoricoxib nd IS, respectively. For the optimiztion of mss spectrometer conditions, mixed reference solution (1000 ng/ml) contining etoricoxib nd IS ws directly introduced nd the following prmeters were selected: Coron voltge, extrctor voltge, RF lens voltge, source temperture nd APCI probe temperture were 2.5 kv, 5 V, 0.5 V, 120 nd 450 ºC, respectively. The cone voltge ws 65 nd 30 V nd the collision energy 35 nd 10 ev, respectively for etoricoxib nd IS. Dt cquisition nd nlysis were performed using the softwre Msslynx (v 3.5). Vlidtion of the bionlyticl method The method ws vlidted by the determintion of the following prmeters: mtrix effects, specificity, linerity, recovery, ccurcy, precision, lower limit of quntittion (LLOQ) nd stbility studies, ccording to the FDA guidelines. 16 To evlute the mtrix effects, three replictes of low, medium nd high qulity control smples were spiked, ech one, with six smples of blnk humn plsm from different sources. The men pek res of ech qulity control were compred to the men pek res of the net reference (etoricoxib nd IS dried nd reconstituted in cetonitrile:wter, 50:50, v/v) t the sme concentrtions. In order to determine the linerity of the method, the clibrtion curves were constructed from blnk plsm, blnk plsm spiked with IS nd seven concentrtions of etoricoxib including the LLOQ, rnging from 1 to 5000 ng/ml. The pek re rtio of the drug to the IS ginst the respective reference concentrtions ws used for plotting the grph nd the linerity evluted by weighted (1/x) lest squres regression nlysis. The cceptnce criteri for ech clculted reference concentrtion ws not more thn 15% devition from the nominl vlue, except for the LLOQ, which ws set t 20%. The lowest reference concentrtion on the clibrtion curve should be selected s t lest five times the response compred to blnk response nd the nlyte pek (response), identifible, discrete nd reproducible with precision of 20% nd ccurcy of %. To evlute the inter-dy precision nd ccurcy, the qulity control smples were nlysed together with one independent clibrtion reference curve for 3 dys, while intr-dy precision nd ccurcy were evluted the nlysis of vlidtion control smples t three different concentrtions in six replictes in the sme dy. Inter- nd intr-dy precision ws expressed s reltive stndrd devition (RSD). The ccurcy ws expressed s the percent rtio between the experimentl concentrtion nd the nominl concentrtion for ech smple. The evlution of precision ws bsed on the criteri 16 tht the devition of ech concentrtion level should be within ± 15%, except for the LLOQ, for which it should be within ± 20%. Similrly for ccurcy, the men vlue should not devite by ± 15% of the nominl concentrtion except the LLOQ where it should not devite by ± 20% of the nominl concentrtion. The nlyticl recovery ws clculted by compring chromtogrphic pek res from un-extrcted reference smples nd from extrcted reference smples t three different

3 576 Dlmor et l. Quim. Nov concentrtions (3, 2500 nd 4000 ng/ml) for the etoricoxib nd 100 ng/ml for the IS. The specificity ws ssessed using six blnk humn plsm smples, rndomly selected, from different sources (including hemolysed nd lipemic plsm), tht were subjected to the extrction procedure nd chromtogrphed to determine the extent to which endogenous plsm components could interfere in the nlysis of etoricoxib or the IS. The results were compred to solution contining 1 ng/ml of etoricoxib. For the stbility studies, the concentrtion of etoricoxib fter ech storge period ws determined t low, medium nd high QC smples (n = 3), nd relted to the initil concentrtion s zero cycle (smples tht were freshly prepred nd processed immeditely). The smples were considered stble if the devition (expressed s percentge bis) from the zero cycle ws within ± 15%. The freeze-thw stbility of etoricoxib ws determined over three freeze-thw cycles within 3 dys. In ech cycle, the frozen plsm smples were thwed t room temperture for 2 h nd refrozen for 24 h, nd then nlysed. The short-term stbility ws evluted using three liquots of the unprocessed QC smples kept t room temperture (25 ± 5 C) for 12 h nd then nlysed. For the longterm stbility the three liquots of the QC smples were frozen t 80 C for 60 dys, nd then nlysed. The processed smple stbility ws tested using six liquots ech one of the QC smples, processed nd plced into the utosmpler t room temperture. Three sets were nlysed fter 24 h nd three sets fter 48 h. RESULTS AND DISCUSSION During the SPE method development different types of crtridges, nd different extrction nd elution conditions were tested. Solvents nd mixtures were lso evluted, including cetonitrile, methnol nd formic cid. The best chromtogrphic seprtion efficiency, MS/MS sensitivity nd recovery for etoricoxib nd IS, were obtined using the HySphere C 18 HD crtridge. In the optimized condition chieved, the crtridge ws solvted, equilibrted nd wshed using methnol, wter nd wter:methnol (95:5, v/v), respectively. The totl cycle time of the Prospekt-2 method ws 4 min. Compring to the trditionl off-line solid-phse nd liquid-liquid extrctions coupled with LC-MS/MS, the proposed method requires smller mounts of plsm, does not need concentrtion of extrcts before injection, nd requires shorter smple preprtion time. When ll the steps of the nlyticl procedure re tken into ccount (SPE nd LC-MS/MS), the totl nlysis time ws 6 minutes, n importnt dvntge when lrge mount of smples need to be quntified, s it is, for exmple in clinicl nd phrmcokinetic studies. The chromtogrphic conditions were studied nd the best signl chieved with the mobile phse selected for the nlysis, performed using short (50 mm) C 18 nlyticl column, which is convenient for high throughput of smples. The low flow rte nd the short run time (2 min) resulted, comprtively, in lower consume of solvents with better cost-effective reltion. The APCI ws used s the LC-MS/MS interfce, which is less susceptible to nlyte-ion suppression compred to the ESI technique. The mss spectrometric response of the nlyte nd the IS were mesured by using selected MRM mode, in which, set of precursor ion/product pirs ws monitored. The specificity is much better thn tht obtined with selected ion monitoring (SIM). The MS/MS trnsition 359.6>280.1 nd 189.6>172 were selected for the etoricoxib nd IS, respectively. The product ion scn, of etoricoxib nd IS, nd the frgments of etoricoxib re illustrted in Figure 1. Mtrix effects were exmined nd the reltive stndrd devition Figure 1. () Mss spectr nd chemicl structures of etoricoxib nd ntipyrin (internl stndrd) obtined by positive tmospheric pressure chemicl ioniztion (APCI+); (b) Product ion scn of etoricoxib (m/z 359.6) nd suggested structure of this frgment (RSD) of the men pek res of etoricoxib nd IS were <6.26%, indicting low difference in ioniztion efficiency using different plsm smples. Besides, the results were higher thn 93.24%, suggesting tht the ion suppression by endogenous components ws not interfering in the repetbility of the method. The linerity determined by six determintions of the concentrtions in the rnge of ng/ml gve the determintion coefficient (r 2 > 0.99, y = 0.28 ± x ± 0.03, where, x is concentrtion nd y is the pek re rtio of the drug to the IS) indicting significnt linerity of the clibrtion curve for the method. For ech stndrd concentrtion clculted, the RSD vlues obtined were lower thn 5.25% devition from the nominl vlue (Tble 1). Tble 1. Results of the stndrd curve for the determintion of etoricoxib in humn plsm Spiking plsm Men concentrtion RSD Accurcy concentrtion found (%) (%) Men of six replictes The LLOQ evluted in n experimentl ssy, with the precision

4 Vol. 31, No. 3 Determintion of etoricoxib in humn plsm 577 of 5.25% nd ccurcy of 106%, ws found to be 1 ng/ml. The coupling of LC with MS/MS detection showed high specificity becuse only the ions derived from the nlytes of interest were monitored nd the comprison of the chromtogrms of the blnk nd spiked humn plsm (1 ng/ml) indicted tht no interferences were detected from endogenous substnces. A typicl chromtogrm obtined by the proposed LC-MS/MS method, with the resolution of the symmetricl pek corresponding to etoricoxib nd ntipyrin, is shown in Figure 2. The low retention times of 0.75 nd 0.70 min llow rpid determintion of the drug, which is n importnt dvntge for the routine nlysis. ccurcy ws between nd % with RSD of % (Tble 4). The dt show tht the method possesses suitble repetbility nd reproducibility. Tble 3. Intr-dy precision nd ccurcy for the determintion of etoricoxib in humn plsm Spiking plsm Men concentrtion RSD Accurcy concentrtion found (%) (%) Men of six replictes Figure 2. Representtive LC-APCI/MS/MS chromtogrm of plsm spiked with 3 ng/ml of etoricoxib nd 25 ng/ml of ntipyrin (internl stndrd) Tble 4. Inter-dy precision nd ccurcy for the determintion of etoricoxib in humn plsm Spiking Dy Men Men b RSD Accurcy plsm concentrtion (%) (%) concentrtion found Men of five replictes. b Men of three dys Solid-phse extrction procedures were reported to extrct the etoricoxib from plsm with recoveries <90%. 7,9 The liquid liquid extrction lso described in the literture showed recoveries of etoricoxib in humn plsm of 96.09, 82.9 nd 76.1%, respectively. 10,11 The proposed SPE extrction method developed nd optimized, enbled high men recoveries of etoricoxib (94.92%) nd IS (92.20%), confirming the suitbility of the method (Tble 2). In the method development significnt crry-over problems were found, nd the introduction of n extr-wsh step, reduced to negligible effect (bout 0.06%) fter the injection of the high QC, followed by the blnk plsm extrct. Tble 2. Recovery of etoricoxib nd ntipyrin fter the extrction procedure Etoricoxib concentrtion % Recovery (men ± RSD%) Etoricoxib Antipyrin ± ± ± ± ± ± 5.2 Men of six replictes The intr-dy ccurcy of the method ws between nd % with precision of % (Tble 3). The inter-dy As shown in Tble 5, the plsm smples were stble for t lest 60 dys t 80 C (long-term) nd lso fter three freezethw cycles without compromising their integrity. Etoricoxib ws stble in net plsm for up to 12 h t room temperture (shortterm), nd lso, fter mintined in the utosmpler for t lest 48 h with n cceptble precision nd ccurcy. The method ws pplied for the quntittive nlysis of etoricoxib in humn plsm, fter single orl dministrtion of 120 mg in pilot study in five helthy humn mle volunteers. The mximum plsm concentrtion (C mx ) of etoricoxib ws found to be 2200 ng/ml nd the time to chieve mximum plsm concentrtion (t mx ) ws 1 h. CONCLUSION An nlyticl method using n on-line SPE system followed by LC-APCI/MS/MS detection ws developed nd vlidted for the determintion of etoricoxib in humn plsm. This method includes n utomted extrction procedure, using ntipyrin, commercilly vilble substnce, s internl stndrd. The short smple preprtion time llows higher smple throughput compred to LLE nd off-line SPE methods. The results of the vlidtion studies show tht the optimized SPE-LC-APCI/MS/MS method possesses specificity, sensitivity, linerity, precision nd ccurcy over the entire rnge of significnt therpeutic plsm concentrtions.

5 578 Dlmor et l. Quim. Nov Tble 5. Summry of stbility of etoricoxib in humn plsm Stbility Zero Concentrtion RSD Bis b cycle found fter (%) (%) concentrtion storge Long term Short term Autosmpler 24 h Autosmpler 48 h Three freeze-thw cycles Men of three replictes. b Bis = (mesured concentrtion - nominl concentrtion/nominl concentrtion) x 100 Therefore, the proposed method cn be pplied to support clinicl nd phrmcokinetic studies in helthy humn volunteers. ACKNOWLEDGMENTS The uthors wish to thnk CNPq (Conselho Ncionl de Desenvolvimento Científico e Tecnológico) nd Merck Reserch Lbortories for the support. REFERENCES 1. Riendeu, D.; Percivl, M. D.; Brideu, C.; Chrleson, S.; Dubé, D.; Ethier, D.; Flgueyret, J. P.; Friesen, R. W.; Gordon, R.; Greig, G.; Guy, J.; Mncini, J.; Ouellet, M.; Wong, E.; Xu, L.; Boyce, S.; Visco, D.; Girrd, Y.; Prsit, P.; Zmboni, R.; Rodger, I. W.; Gresser, M.; Ford-Hutchinson, A. W.; Young, R. N.; Chn, C. C.; J. Phrmcol. Exp. Ther. 2001, 296, Rodrigues, A. D.; Hlpin, R. A.; Geer, L. A.; Cui, D.; Woolf, E. J.; Mtthews, C. Z.; Gottesdiener, K. M.; Lrson, P. J.; Lsseter, K. C.; Agrwl, N. G. B.; Drug Metb. Dispos. 2003, 31, Agrwl, N. G. B.; Mtthews, C. Z.; Mzenko, R. S.; Woolf, E. J.; Porrs, A. G.; Chen, X.; Miller, N.; Michiels, M.; Wehling, A.; Schultz, A.; Gottlie, A. B.; Krft, H. E.; Greenberg, S. A.; Wldmn, S. P.; Curtis, S. P.; Gottesdiener, K. M.; J. Clin. Phrmcol. 2004, 44, Agrwl, N. G. B.; Porrs, A. G.; Mtthews, C. Z.; Woolf, E. J.; Miller, J. L.; Mukhopdhyy, S.; Neu, D. C.; Gottesdiener, K. M.; J. Clin. Phrmcol. 2001, 41, Agrwl, N. G. B.; Porrs, A. G.; Mtthews, C. Z.; Rose, M. J.; Woolf, E. J.; Musser, B. J.; Dynder, A. L.; Mzin, K. E.; Lsseter, K. C.; Hunt, T. L.; Schwrtz, J. I.; McCre, J. B.; Gottesdiener, K. M.; J. Clin. Phrmcol. 2003, 43, Dllob, A.; Hwkey, C.; Greenberg, H.; Wight, N.; De Schepper, P.; Wldmn, S.; Wong, P.; Detor, L.; Gertz, B.; Agrwl, N.; Wgner, J.; Gottesdiener, K.; J. Clin. Phrmcol. 2003, 43, Mtthews, C. Z.; Woolf, E. J.; Lin, L.; Fng, W.; Hsieh, J.; H, S.; Simpson, R.; Mtuszewski, B. K.; J. Chromtogr., B: Anl. Technol. Biomed. Life Sci. 2001, 751, Rose, M. J.; Agrwl, N.; Woolf, E. J.; Mtuszewski, B. K.; J. Phrm. Sci. 2002, 91, Bräutigm, L.; Nefflen, J. U.; Geisslinger, G.; J. Chromtogr., B: Anl. Technol. Biomed. Life Sci. 2003, 788, Werner, U.; Werner, D.; Hinz, B.; Lmbrecht, C.; Brune, K.; Biomed. Chromtogr. 2005, 19, Rmkrishn, N. V. S.; Vishwottm, K. N.; Wishu, S.; Koteshwr, M.; J. Chromtogr., B: Anl. Technol. Biomed. Life Sci. 2005, 816, Brum Junior, L.; Fronz, M.; Ceni, D. C.; Brth, T.; Dlmor, S. L.; J. AOAC Int. 2006, 89, Brum Junior, L.; Ceni, D. C.; Fronz, M.; Oliveir, P. R.; Dlmor, S. L.; J. Liq. Chromtogr. Relt. Technol. 2006, 29, Schellen, A.; Ooms, B.; vn de Lgemt, D.; Vreeken, R.; vn Dongen, W. D.; J. Chromtogr., B: Anl. Technol. Biomed. Life Sci. 2003, 788, Gonçlves, J. C. S.; Monteiro, T. M.; Neves, C. S. M.; Grm, K. R. S.; Volpto, N. M.; Silv, V. A.; Cminh, R.; Gonçlves, M. R. B.; Sntos, F. B.; Silveir, G. E.; Noel, F.; Ther. Drug Monit. 2005, 27, US Deprtment of Helth nd Humn Services, Food nd Drug Administrtion, Center for Drug Evlution nd Reserch (CDER); Guidnce for Industry, Bionlyticl Method Vlidtion, 2001.