Method Validation for Simultaneous Determination of Cholesterol and Cholesterol Oxides in Milk by RP-HPLC-DAD

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1 Article A J. Brz. Chem. Soc., Vol. 25, No. 1, , Printed in Brzil Sociedde Brsileir de Químic $ Method Vlidtion for Simultneous Determintion of Cholesterol nd Cholesterol Oxides in Milk by RP-HPLC-DAD Lucin C. Buer, Débor de A. Sntn, b Mrinne dos S. Mcedo, b Alexndre G. Torres, c Nilson E. de Souz d nd Jullin I. Simionto *,,d Grdute Progrm in Food Engineering nd b Bsic nd Instrumentl Studies Deprtment, University of Southwest Bhi (UESB), Itpeting-BA, Brzil c Institute of Chemistry - Grdute Progrm in Food Science - Federl University of Rio de Jneiro (UFRJ), Rio de Jneiro-RJ, Brzil d Federl Technologicl University of Prná (UTFPR), Londrin-PR, Brzil O escopo d presente pesquis foi desenvolver e vlidr um metodologi pr extrção simultâne de colesterol e óxidos de colesterol utilizndo sponificção diret em mostrs de leite fluido pr posterior nálise por cromtogrfi líquid de lt eficiênci em fse revers com detecção por rrnjo de diodos (RP-HPLC-DAD). Form vlidos os prâmetros nlíticos de seletividde, lineridde e fix de plicção, precisão inter e intrdi, recuperção, limites de detecção e quntificção do método. Os resultdos demonstrrm que metodologi pode ser empregd com segurnç e confibilidde pr nálise do colesterol e óxidos do leite. The im of the present work ws to develop nd vlidte reversed-phse highperformnce liquid chromtogrphy with diode rry detection (RP-HPLC-DAD) method for determintion of cholesterol nd cholesterol oxides extrcted from milk smples. The method consisted of direct smples sponifiction nd nlytes extrction, followed by their determintion using RP HPLC DAD. Suitble nlyticl selectivity, linerity nd rnge of ppliction, intr-ssy nd intermediry precision, recovery, detection nd quntifiction limits (LOD nd LOQ) were chieved nd, the results showed tht the developed RP-HPLC-DAD method cn be pplied to routine determintion of cholesterol nd cholesterol oxides in milk smples. Keywords: direct sponifiction, 7-ketocholesterol, 25-hydroxycholesterol, liquid chromtogrphy, diry ft Introduction Cholesterol is nturl steroidl lipid present in niml fts responsible for severl functions in mmmls. Cholesterol is essentil to mmmlin cell membrne structure nd function, it is substrte for the synthesis of bile cids nd steroid hormones. 1 Although it is essentil to humns, high blood levels of cholesterol cn increse the risk of development of severl diseses, including hypertension, therosclerosis myocrdil infrction, nd stroke. 2 Generlly, most prt of the cholesterol in the humn orgnism is produced endogenously, nd pproximtely *e-mil: jusimionto@gmil.com 30% is obtined by dietry intke (exogenous cholesterol) coming from foods of niml origin, such s eggs, mets, milk nd whole-ft diry products. The mount of cholesterol obtined by dietry intke depends on the hbitul diet nd, n elevted intke of niml fts leds to high levels of cholesterol intke nd, consequently, to high blood levels of cholesterol. 3,4 Consequently, dietry intke of cholesterol must be controlled both t the individul nd the popultion levels. Cholesterol is unsturted, nd therefore susceptible to oxidtion. Cholesterol oxidtion is fvored during therml-processing of food, nd lso during long-term storge. Severl fctors ffect the rte of cholesterol oxidtion, such s foods wter ctivity, ph, composition nd structure, oxygen concentrtion, light exposure,

2 162 Method Vlidtion for Simultneous Determintion of Cholesterol nd Cholesterol Oxides in Milk J. Brz. Chem. Soc. temperture, rdition, free rdicls, nd metllic ions, mong others. 5 Cholesterol oxidtion products (COP) cn be deleterious to humn helth s cn be responsible by therogenic, cytotoxic, crcinogenic nd mutgenic properties. 6 In ddition, COP re potent inhibitors of cholesterol synthesis nd they re ssocited to inflmmtory processes, such s those observed in rheumtic rthritis nd degenertive diseses s Prkinson s nd Alzheimer s. Concerning determintion of cholesterol nd cholesterol oxides in food, gs chromtogrphy (GC) hs been the most used technique. However, quntittive GC determintion of cholesterol nd COP requires purifiction steps, nd tricky derivtiztion (silyltion) to void rtifct peks nd to improve the stbility of the nlytes. Thus, GC hve been slowly replced by other methods, 7 since even when crefully executed, cholesterol nd COP cn suffer thermolysis leding to the formtion of rtifcts. 8 The most common rtifcts re oxides tht re originted during the preprtion nd/or determintion steps, resulting in overestimtion of the COP contents in the smples. 9 To our knowledge, most of the recently published ppers on quntittive determintion of cholesterol nd cholesterol oxides exploits high performnce liquid chromtogrphy (HPLC). 10,11 In spite of some drwbcks, such s elevted volumes of solvents nd limits of detection nd of quntifiction, smple preprtion is simple nd required smll number of steps; bsiclly sponifiction nd the choice of extrction solvents re needed for dequte seprtion nd quntifiction of nlytes by HPLC. Additionlly, HPLC usully does not need high tempertures during the determintion, minimizing degrdtions, which results in higher recovery of nlytes nd in limited or no formtion of rtifcts. 12 Becuse of potentil deleterious helth effects of cholesterol nd COP s intke, the im of the present work ws to develop nd vlidte reversed-phse HPLC with diode rry detector (RP-HPLC-DAD) method for the simultneous determintion of cholesterol nd COP s (7-ketocholesterol nd 25-hydroxycholesterol) in whole milk. Experimentl Smpling Twelve milk smples were nlyzed, collected by mnul milking of crossbred Holstein x Gir cows from the Cmpus of Itpeting-BA of the Stte University of Southwest Bhi. Simultneous extrction of cholesterol nd cholesterol oxides from fluid milk After vrious tests to verify the best conditions to extrct cholesterol nd the oxides (7-ketocholesterol nd 25-hydroxycholesterol), the extrction ws performed in duplicte by direct sponifiction nd posterior extrction with hexne, ccording to Sldnh et l. 13 with modifictions on the type nd time of sponifiction which were bsed on Sldnh et l. 14 For the extrction of the unsponifible mtter, 10 ml of milk smple were tken nd 8 ml of queous potssium hydroxide solution (KOH) 50% (m/v) nd 12 ml of ethyl lcohol P.A were dded. Following, the smple ws vortex mixed for 1 min, the mixture ws rested for 22 h in the drk nd t room temperture to complete the sponifiction rection. Afterwrds, 10 ml of distilled wter were dded long with 10 ml of n-hexne nd the mixture ws vortex mixed for 5 min. After the complete phse-seprtion, the upper lyer ws collected nd n-hexne ws evported t room temperture in rotry evportor. The residue ws suspended in 2.5 ml of crylonitrile nd isoproponlin (95:5, v/v), the sme phse used s HPLC mobile phse. Smples were filtered through membrne of polyvinylydene fluoride (PVDF) with pore dimeter of 0.45 µm nd injected in the RP-HPLC-DAD. Chromtogrphic determintion A Shimdzu (Jpn) liquid chromtogrph ws used for ll nlyses, equipped with quternry pump system, n on-line degsser, Rheodyne injection vlve with 20 µl smple loop, column oven, nd spectrophotometer with diode rry detector. The cholesterol nd cholesterol oxides were seprted in reversed-phse nlyticl column C 18 (Restek C 18, 15 cm 6 mm i.d. 5µm), using n isocrtic binry solvent consisting of cetonitrile nd isopropnol 95:5 (v/v) s mobile phse, which ws filtered nd degssed prior to chromtogrphic runs. Solvent flow-rte nd oven temperture were selected s 2 ml min -1 nd 35 ºC, respectively, iming to get complete resolution of chromtogrphic peks with totl run of 15 min. All nlyzes were performed in duplicte nd chromtogrphic dt were processed with the LC Solution softwre (Shimdzu). Chromtogrphic peks of the cholesterol nd cholesterol oxides from the smples were tenttively identified bsed on the retention times of commercil stndrds of cholesterol, 7-ketocholesterol (5-Cholesten- 3b-ol-7-one), nd 25-hydroxycholesterol, ll supplied by

3 Vol. 25, No. 1, 2014 Buer et l. 163 Sigm-Aldrich. Pek identities were confirmed by spiking smples with stndrds nd lso on the chrcteristic UV-spectrum of ech substnce, with or without stndrd co-elution. For quntittive determintion, cholesterol nd 25-hydroxycholesterol were monitored t 202 nm nd 7-ketocholesterol in 227 nm. concentrtions: 75, 150 e 300 µg ml -1 for cholesterol nd, 50, 75 nd 100 µg ml -1 for cholesterol oxides. The injection of control smples ws performed without strengthening the stndrd. After the quntifiction of the nlytes in the fortified smples nd in the control, the recovery percentge (% REC) ws clculted ccording to eqution 1. Quntifiction of cholesterol nd cholesterol oxides in fluid milk The quntifiction of the cholesterol nd oxides ws ccomplished by externl stndrdiztion. Anlyticl curves were built for ll nlytes by injection of stndrd solutions of the compounds, relting the solution s concentrtion with the equipment s response (pek re), 15 nd the nlytes smples concentrtions were clculted by interpoltion of their nlyticl signls in the nlyticl curves. HPLC method vlidtion The vlidtion of the proposed method ws done ccording to the description provided by Ribni et l., 15 using the following nlyticl prmeters: selectivity, linerity nd rnge of ppliction, repetbility, intermediry precision, recovery, limits of detection nd quntifiction. The evlution of the results ws performed s recommended by the Resolution ANVISA RE number 899, 16 of 05/29/2003, which is bsed on the interntionl norms of IUPAC. For the evlution process, commercil psteurized skim milk products (n = 3) were purchsed in the city of Itpeting-BA, Brzil. Low ft content products were chosen in order to chieve mtrix similr to those of the smples, but with the minimum quntity of the focused nlyte, to minimize interferences during the process steps. Selectivity ws evluted in triplicte by using the spectr provided by the spectrophotometer with photodiode rry detector by comprison of the peks present in the chromtogrms of the products with those peks in the chromtogrms of the stndrds, verifying the resolution nd seprtion of the pure compounds. Prior to the injection, the products were enriched with 50 µg of cholesterol, 7-ketocholesterol, nd 25-hydroxycholesterol for ech 1 ml of milk, then homogenized nd extrcted. After obtining the chromtogrms of the products nd the purity spectrum of ech pek, these dt were compred to those ones collected when processing the stndrd solutions. Recoveries were evluted in triplicte by dding to milk smple liquots stndrd solutions of the nlytes for reching the following The dditionl prmeters were evluted by using the dt obtined for nlyticl curves. Their construction ws ccomplished by injecting stndrd cholesterol nd cholesterol oxide solutions dissolved in the mobile phse in the following concentrtions: 1, 10, 20, 100, 200, 1000, nd 2000 µg ml -1 for cholesterol nd 0.1, 1, 10, 100, e 500 µg ml -1 for the 7-ketocholesterol nd 25-hydroxycholesterol. These solutions were nlyzed with two repetitions of four replics ech, in three non consecutive dys, with the solutions stored in freezer t 18 ºC during the intervls between the dys. The linerity ws evluted ccording to the correltion coefficient by Person (R²) for liner regression. This prmeter llowed estimtion of the curve s qulity, nd shll present vlue close to 1.0, which indictes smller dispersion of the experimentl points nd greter relibility of the estimted regression coefficients. The ppliction rnge of the nlyticl curve ws chieved by the reltive response method. In this method, grph is constructed with the reltive responses on the xis of the coordintes nd the correspondent concentrtions on logrithmic scle on the xis of the bscisss. The ppliction rnge is defined s being the rnge between the points where the reltive response intercepts the lines of the relibility intervls, t 95% nd 105% of the liner rnge. The precision ws evluted point-to-point, for the repetbility (intrdy) s well s for the intermediry precision (interdys) using the estimte of the verges nd coefficients of vrition (CV). The limits of detection (LOD) nd quntifiction (LOQ) were clculted considering the signl-to-noise rtio ccepted for ech limit nd the prmeters estimted for the nlyticl curve, ccording to equtions 2 nd 3: (1) LOD s/s (2) LOQ = 10 s/s (3) where s is the estimte of the stndrd devition of the eqution s liner coefficient, nd S is the ngulr coefficient of the nlyticl curve.

4 164 Method Vlidtion for Simultneous Determintion of Cholesterol nd Cholesterol Oxides in Milk J. Brz. Chem. Soc. Results nd Discussion Choice of method for the extrction of cholesterol nd cholesterol oxides When the determintion of the cholesterol nd the COP is crried out by HPLC, the criticl smple preprtion steps re the sponifiction nd the extrction of the non sponifible mteril, in which re included the cholesterol nd oxides. The sponifiction of the lipids hs the primordil objectives of removing cylglycerols from the extrct of the lipids (trnsforming them into sop nd free glycerol) nd hydrolyzing the esters of cholesterol. 12 The rection cn be done fter the extrction of the lipids, 17 or by direct sponifiction of the smple. 18 The direct sponifiction technique ws selected becuse it uses significntly lower quntity of solvents nd lso requires shorter preprtion time. Additionlly, when previous ft extrction from foods is dopted, higher volumes of toxic solvents re required nd therefore must be voided. Another relevnt spect is the conditions tht sponifiction rection is crried out since rtifcts cn be produced. Thus, the option dopted ws to crry out the process t room temperture (cold sponifiction), seeking to minimize the genertion of these compounds nd voiding submitting the nlytes to high tempertures. Dionisi et l. 19 compred four COP extrction methods in powder milk, including direct cold sponifiction of smples, nd the other three methods with previous lipid extrction nd posterior cold sponifiction, concluding tht the best procedure ws direct sponifiction becuse it presented good repetbility, precision, minimum formtion of rtifcts (< 0.05% of totl cholesterol), nd lower preprtion time nd solvent consumption. Busch nd King 9 studied the stbility of cholesterol nd 7-ketocholesterol in different sponifiction conditions nd observed loss of pproximtely 50% of the oxide when it ws exposed to more elevted tempertures (37 nd 45 ºC) in comprison to room temperture (24 ºC). Furthermore, the formtion of the dehydrtion product colest-3,5-dieno- 7-one ws up to 398% higher when the sponifiction ws performed t higher tempertures, demonstrting the oxide s elevted therml susceptibility. Cholesterol presented better therml stbility nd losses were only observed when the sponifiction ws performed t 45 ºC, nd in this condition the recovery ws pproximtely 70%. Concerning the extrcting solvent, different solvents cn be used, but most uthors use n-hexne which is cpble of extrcting the cholesterol s well s oxides becuse of its polrity. 13,14,19 Therefore, this solvent ws lso selected for use in the present study. Confirmtion of pek identity Peks were identified using smples spiked with nlyticl stndrds of cholesterol nd cholesterol oxides. Stndrds co-eluted with nlytes peks in milk smples (Figure 1), nd lso the UV-spectr of the stndrds nd smples, both before nd fter stndrd spiking, were highly similr (Figure 1). Figure 1. Representtive chromtogrm of the milk smples: (1) 25-hydroxycholesterol; (2) 7-ketocholesterol, nd (3) cholesterol. Vlidtion Selectivity The chosen chromtogrphic conditions proved to be dequte for the seprtion of cholesterol nd cholesterol oxides in milk smples. The method s selectivity ws confirmed using the LC Solution softwre, showing good resolution of the peks nd the seprtion of the pure compounds (Figure 1), confirming tht in the retention times of cholesterol, 7-ketocholesterol, nd 25-hydroxycholesterol there were no co-elution of other compounds in the food mtrix. Linerity nd ppliction rnge The dt reltive to linerity show tht there is strong correltion between cholesterol concentrtions nd the oxides within the studied rnge nd the respective res. The coefficients of determintion superior to found for ll curves confirm tht the method genertes results proportionl to the concentrtions of the nlytes, within specific rnge, being possible to correlte res nd nlytes concentrtions. Furthermore, these results re in

5 Vol. 25, No. 1, 2014 Buer et l. 165 greement with the requirements of the Ntionl Agency of Snitry Vigilnce (ANVISA) nd the Ntionl Institute of Metrology, Stndrdiztion, nd Industril Qulity (INMETRO), which recommend correltion coefficient equl to 0.99 or vlue over 0.90, respectively. 16,20 For the evlution of the ppliction rnges of the chieved nlyticl curves, grphs were plotted relting the logrithm of the concentrtion of stndrds with the respective reltive response of the equipment (pek re divided by the respective concentrtion of the nlyte). Relibility intervls inferior nd superior to 5% between the verges vlues of the injections were ccepted s the cceptble rnge of ppliction. Furthermore, with regrds to the reltive response method, the rnge of ppliction cn be obtined by precision dt, in which the concentrtion rnge where the precision is gurnteed is considered s the ppliction rnge of the method proposed for the extrction nd determintion of the nlytes. 15,21 Results of the method s precision, which re presented hereinfter, confirm the obtined ppliction rnge. Repetbility nd intermediry precision The coefficients of vritions chieved in the study of repetbility nd intermediry precision were dequte for ll concentrtions of cholesterol, 7-ketocholesterol, nd 25-hydroxycholesterol, except for the lowest cholesterol concentrtion, following the criterion of cceptnce for the vrition coefficient of Brzil. The legisltion considers CV vlues of up to 15% cceptble, lthough recommends mximum vrition of 5% for micro constituents. For micro constituents such s cholesterol oxides it dmits CV vlues of up to 20%, depending on the complexity of the mtrix. 22 The vlues obtined for the precision of the nlyticl method for the nlytes re found in Tbles 1 nd 2. Similr results for precision were found by other uthors nd contribute to the confirmtion of the precision of the proposed method for the determintion of cholesterol nd cholesterol oxide in milk smples. Tble 1. Precision of the nlyticl method for the determintion of cholesterol in milk expressed by the vrition coefficient Concentrtion / (µg ml -1 ) Repetbility Cholesterol Intermediry Precision b CV / % CV / % n = 8; b n = 24. Dneshfr et l. 11 vlidted n HPLC-UV method for cholesterol determintion in milk nd obtined precision CV% lower thn to 5%, while Morles-Aizpúru nd Tenut-Filho, 23 using HPLC-DAD system, obtined CV% of 7.25 to 9.29% for repetbility in evluting the content of cholesterol, 7-ketocholesterol, nd 25-hydroxycholesterol in myonnise. According to the results presented for intermediry precision, one notices reduction of precision for 7-ketocholesterol over the dys, which my be relted to the stbility of this compound during nlysis or storge, s demonstrted by Busch et l.. 22 These uthors observed tht the stbility of this oxide diminished over the studied period of 7 dys, in ddition to decresing ccording to the number of injections. Becuse in the present study the sme stndrd solutions were used for injection in different dys, totlizing 6 storge dys, it cn be suggested tht some type of ltertion in these solutions my hve occurred, such s, for exmple, trnsformtion of the 7-ketocholesterol into colest-3,5-dieno-7-on, which even so does not dmge the vlidtion process of the method, since even being reduced, the precision ws gurnteed over the entire period. Tble 2. Precision of the nlyticl method for the determintion of cholesterol oxides in milk expressed by the vrition coefficient 7-ketocholesterol 25-hydroxycholesterol Concentrtion / (µg ml -1 ) Repetbility Precision Intermediry b Repetbility Precision Intermediry b CV / % CV / % CV / % CV / % n = 8; b n = 24.

6 166 Method Vlidtion for Simultneous Determintion of Cholesterol nd Cholesterol Oxides in Milk J. Brz. Chem. Soc. Detection limit nd quntifiction limit The limits of detection (LOD) nd quntifiction (LOQ) obtined by nlyticl curves were e µg ml -1 for cholesterol; 1.35 nd 4.10 µg ml -1 for 7-ketocholesterol, nd 2.35 nd 7.10 µg ml -1 for 25-hydroxycholesterol, respectively. Furthermore, ccording to Denobile e Nscimento, 24 the quntifiction limit corresponds to the smllest concentrtion of the nlyte tht cn be estblished with proper precision; tking this spect into considertion one cn lso consider s LOQ for the cholesterol 10 µg ml -1 nd for the oxides 1 µg ml -1, s seen in Tbles 1 nd 2. Different results were obtined by other uthors. Stroher et l., 25 using n HPLC system similr to the one here used for determintion of cholesterol in met products, found LOD equivlent to mg g 1 nd LOQ of mg g 1. Bggio e Brggnolo 8 obtined, respectively, LOD of 4.8 µg g 1 nd LOQ of 16 µg g -1 for cholesterol nlyses, nd n LOD of 0.09 µg g -1 nd LOQ of 0.3 µg g 1 for ketocholesterol nlyses. These differences re probbly coming from the different methods employed for smple preprtion. Different HPLC systems used for smple nlyses s well s distinct forms for the clcultion of limits did not hrm the results of the cholesterol determintion of the smples of this work, since the milk exhibits higher concentrtions of this substnce. Regrding the oxides, these limits cn be reduced by improving the opertionl conditions during the nlyses, minly in terms of the electricl stbility of the chromtogrphic system, becuse this interferes directly with the equipment s signl-to-noise rtio, lso influencing the devitions of the nlyticl response. Recovery test The results of the recovery test for the milk smples re shown in Tble 3. The obtined results prove the efficiency of the proposed method for the cholesterol determintion nd the oxides, 7-ketocholesterol nd 25-hydroxycholesterol. Anlyte recoveries close to 100% re idel, but smller vlues re dmitted if the precision is good, 16 nd in this cse CV / % of up to 5% is cceptble. Results similr to those found for the cholesterol determintion were found by Sldnh et l. 13 while compring two methods for determintion of cholesterol in milk, one being n enzymtic method nd the other one liquid chromtogrphic method. Other uthors lso chieved similr results for cholesterol, studying extrction methods on vrious smples, such s fish, 26 myonise, 23 nd eggs. 27 Tble 3. Results for the recovery / % of cholesterol nd oxides in milk smples Added nlyte / (µg ml -1 ) Recovery / % ± SD CV / % Cholesterol ± ± ± dded hydroxycholesterol ± ± ± dded ketocholesterol ± ± ± n = 9; SD: stndrd devition; CV: vrition coefficient. In reltion to the cholesterol oxides, Sldnh et l., 14 using the sme extrction method s this work, found superior results for smples of fish (slted cod nd fresh hke), between 95 nd 104% of recovery for oxides with moderte polrity, in which re included the 7-ketocholesterol nd 25-hydroxycholesterol; this my be relted to the differences in the mtrix of the studied foods. Determintion of cholesterol nd cholesterol oxides in milk The oxide 7-ketocholesterol ws not detected in ny of the nlyzed smples; only cholesterol nd 25-hydroxycholesterol were detected in the fluid milk smples. The obtined men vlues nd the respective vrition coefficients for the content of cholesterol in the milk smples cn be seen in Tble 4. The quntities of 25-hydroxycholesterol re not shown, since the detected quntities were inferior to the LQ of the method, mking the relible determintion of these vlues impossible. On verge, the cholesterol content of whole milk is 12 mg per 100 ml, higher thn those found in this work. However, this content my suffer from vrition. Piironem et l., 28 evluted Finnish commercil smples nd found vlues between 5.6 nd 6.4 mg of cholesterol in 100 g of milk; Sldnh et l. 13 evluted Brzilin commercil smples nd found verge vlues for cholesterol of 9.75 mg per 100 g of milk; nd Mnzi nd Pizzoferrto, 29 evluting Itlin commercil milk smples, found vlues of 10.4 nd 11.3 mg per 100 g of whole milk. These vritions cn be ttributed to vritions in the processing of the milk s well s to differences in the niml breeds, individul chrcteristics, nd intervls between

7 Vol. 25, No. 1, 2014 Buer et l. 167 Tble 4. Totl cholesterol content in mg per 100 ml of milk, in the nlyzed smples Smple Cholesterol / Averge ± SD CV / % ± ± ± ± ± ± ± ± ± ± ± ± n = 2; SD: stndrd devition; CV: coefficient of vrition. milking, lcttion phse, composition of the niml s diet, nd sesonl nd climtic interferences, thereby explining the divergence of results. In reltion to the results presented for cholesterol oxides, these were expected becuse the fresh foods did not present COP or presented extremely smll quntities, t trce levels, of these compounds. 29 Furthermore, for being considered s n indictor of the cholesterol oxidtion in foods nd secondry product of the oxidizing rection, 9 the non detection of the 7-ketocholesterol cn be ccepted s positive result. Additionlly, the detection of 25-hydroxycholesterol in low nd not quntifible concentrtions indictes tht the conditions of obtention, storge, nd preprtion of the smple did not contribute significntly to the oxidtion of the cholesterol, concurring with Orczewsk Dudek et l.. 30 These uthors stted tht milk my be more resistnt to uto oxidtion of cholesterol, even under unfvorble conditions of storge nd/or processing, becuse it contins metls of low vlence, high content of sturted fts, nd nturl nti-oxidizers such s vitmins nd sulfhydryl groups, thereby chrcterizing the lcteous products by lowe COP content, the lowest within ll products of niml origin. 30 Conclusions The method proposed here for the simultneous extrction of cholesterol nd cholesterol oxides, 7-ketocholesterol, nd 25-hydroxycholesterol proved to be suitb, nd cn be used with relibility for the determintion of these compounds in milk. The method could be vluble for the routine determintion of cholesterol nd cholesterol oxides in milk. Acknowledgements The uthors would like to thnk Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES) nd Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq) for reserch funds. References 1. Ary, S. K.; Dtt, M.; Mlhotr, B. D.; Biosens. Bioelectron. 2008, 23, Shih, W. C.; Yng, M. C.; Lin, M. S.; Biosens. Bioelectron. 2009, 24, Hrper, A. E.; Feedstuffs 1993, 2, Scherr, C.; Ribeiro, J. P.; Arq. Brs. Crdiol. 2009, 92, Sieber, R.; Int. Diry J. 2005, 15, Peng, S. K., Morin, R. J.; Biologicl Effects of Cholesterol Oxides, 1 st ed.; Crc Press: London, Gurdiol, F.; Botell, J.; Codony, R. In Cholesterol nd Phytosterol Oxidtion Products: Anlysis, Occurrence, nd Biologicl Effects; Gurdiol, F.; Dutt, P. C.; Codony, R.; Svge G. P., eds.; Aocs Press: Chmpign, 2002, ch Bggio, S. R.; Brggnolo, N.; Ciênc. Tecnol. Aliment. 2004, 24, Bush, T. P.; King, A. J.; J. Am. Oil Chem. Soc. 2010, 87, Plozz, T.; Crige, V.; Trenerry, A.; Cridi, D.; Food Chem. 2012, 134, Dneshfr, A.; Khezeli, T.; Lofti, H. J.; J. Chromtogr. B 2009, 877, Ulberth, F.; Buchgrber, M.; In Gurdiol, F., Dutt, P. C., Codony, R, Svge, G. P. Cholesterol E Phytosterol oxidtion products: nlysis, occurrence, nd biologicl effects. Chmpign: Aocs Press; Sldnh, T.; Mzlli, M. R.; Brggnolo, N.; Ciênc. Tecnol. Aliment. 2004, 24, Sldnh, T.; Swy, A. C. H. F.; Eberlin, M. N.; Brggnolo, N.; J. Agric. Food Chem. 2006, 54, Ribni, M.; Bottoli, C. B. G.; Collins, C. H.; Jrdim, I. C. S. F.; Melo, L. F. C.; Quim. Nov 2004, 27, Brsil. ANVISA - Agênci Ncionl De Vigilânci Snitári. Resolução Re Nº 899, De 29 De Mio De Gui pr Vlidção de Métodos Anlíticos e Bionlíticos. ANVISA: Brsíli, Rith, K.; Brenner, C.; Frwnh, H.; Muller, G.; Eder, K.; Neubert, R. H. H.; J. Chromtogr. A 2005, 1067, Tlpur, F. H.; Bhnger, M. I.; Khuhwr, M. Y.; J. Food Comp. Anl. 2006, 19, 698.

8 168 Method Vlidtion for Simultneous Determintion of Cholesterol nd Cholesterol Oxides in Milk J. Brz. Chem. Soc. 19. Dionisi, F.; Goly, P. A.; Aeschlimnn, J. M.; Fy, L. B.; J. Agric. Food Chem. 1998, 46, INMETRO - Instituto Ncionl de Metrologi, Normlizção e Qulidde Industril. Orientções sobre Vlidção de Ensios Químicos, DOQ-CGCRE-008; Brito, N. M.; Amrnte Júnior, O. P.; De Polese, L.; Ribeiro, M. L.; Rev. Ecotoxicol. Meio Ambiente 2003, 13, Bush, T. P.; Gross, H. B.; King, A. J.; J. Chromtogr. Sep. Tech. 2011, 2, Morles-Aizpúru, I. C.; Tenut-Filho, A.; Ciênc. Tecnol. Aliment. 2005, 25, Denobile, M.; Nscimento, S. E.; Brz. J. Phrm. Sci. 2004, 40, Stroher, G. L.; Rodrigues, A. C.; Dis, L. F.; Pedrão, M. R.; Pul, L. N.; Visentine, J. V.; de Souz, N. E.; Am. J. Anl. Chem. 2012, 3, Visentine, J. V.; Sldnh, T.; Brggnolo, N.; Frnco, M. R. B.; Ciênc. Tecnol. Aliment. 2005, 25, Mzlli, M. R.; Sldnh, T.; Brggnolo, N.; Rev. Inst. Adolfo Lutz 2003, 62, Piironen, V.; Toivo, J.; Lmpi, A. M.; J. Food Comp. Anl. 2002, 15, Mnzi, P.; Pizzoferrto, L.; Food Bioprocess Technol. 2010, 3, Orczewsk-Dudek, S.; Bedersk-Łojewsk, D.; Pieszk, M.; Pietrs, M. P.; Ann. Animl Sci. 2012, 12, 25. Submitted: October 24, 2013 Published online: November 29, 2013

9 Supplementry Informtion J. Brz. Chem. Soc., Vol. 25, No. 1, S1, Printed in Brzil Sociedde Brsileir de Químic $ SI UV-Vis Spectrometric Detection of Biodiesel/Diesel Blend Adultertions with Soyben Oil Dvid D. S. Fernndes, Adrino A. Gomes, Mrcelo M. de Fontes, b Gen B. d Cost, b Vlber E. de Almeid, c Mrio C. U. de Arújo, Roberto K. H. Glvão d nd Germno Vérs*,c Lbortório de Automção e Instrumentção em Químic Anlític/Quimiometri (LAQA), Deprtmento de Químic, Universidde Federl d Príb, Cix Postl 5093, João Pesso-PB, Brzil b Progrm de Pós-Grdução em Ciêncis Agráris nd c Deprtmento de Químic, CCT, Universidde Estdul d Príb, Cmpin Grnde-PB, Brzil d Divisão de Engenhri Eletrônic, Instituto Tecnológico de Aeronáutic, São José dos Cmpos-SP, Brzil Viscosity, refrctive index nd density were mesured for B5 biodiesel/diesel blends dulterted with soyben oil t the levels of 0.0 (i.e. without dultertion), 0.5, 1.0, 1.5, 2.0 nd 2.5% v/v (10 smples for ech level). Viscosity ws mesured using Cnnon-Fenske 200 viscosimeter (Vidrolbor). Prior to the viscosity mesurements, the smples were kept t 40 C during 20 minutes in thermosttic bth (Unique, USC-1800 model). Density ws mesured using Densito 30PX densimeter (Mettler Toledo). Refrctive index ws mesured using 2-wy ABBE refrctometer (Biobrix). The resulting verge vlues nd stndrd devitions re presented in Tble S1. Tble S1. Averge nd stndrd devition vlues of viscosity, density nd refrctive index of B5 blends dulterted with soyben oil (n = 10 smples for ech level of soyben oil) Soyben oil / % (v/v) Viscosity / (mm 2 s -1 ) Density / (g cm -3 ) Refrctive index ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± *e-mil: germno.vers@pq.cnpq.br