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Direct analysis of glucuronidated metabolites of main olive oil phenols in human urine after dietary consumption of virgin olive oil

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Direct analysis of glucuronidated metabolites of main olive oil phenols in human urine after dietary consumption of virgin olive oil
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  Analytical Methods Direct analysis of glucuronidated metabolites of main olive oil phenols inhuman urine after dietary consumption of virgin olive oil Olha Khymenets a,b,c , Magí Farré a,d , Mitona Pujadas a,b , Erika Ortiz a , Jesús Joglar e , Maria I. Covas b,f  ,Rafael de la Torre a,b,c, ⇑ a Human Pharmacology and Clinical Neurosciences Research Group, Institut Municipal d’Investigació Mèdica (IMIM-Hospital del Mar Research Institute), C/Dr. Aiguader 88,Barcelona 08003, Spain b CIBER de Fisiopatología de la Obesidad y Nutrición (CB06/03), CIBEROBN, Edificio D 1a planta, Hospital Clínico Universitario Santiago de Compostela, C/Choupana s/n,Santiago de Compostela 15706, Spain c Universitat Pompeu Fabra (CEXS-UPF), C/Doctor Aiguader, 80, Barcelona 08003, Spain d Universitat Autónoma de Barcelona (UDIMAS-UAB), C/Doctor Aiguader, 80, Barcelona 08003, Spain e Instituto de Química Avanzada de Cataluña, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), C/Jordi Girona 18-26, Barcelona 08034, Spain f  Cardiovascular Risk and Nutrition Research Group, IMIM-Hospital del Mar Research Institute, C/Dr. Aiguader 88, Barcelona 08003, Spain a r t i c l e i n f o  Article history: Received 21 May 2010Received in revised form 14 September2010Accepted 10 October 2010 Keywords: Olive oil phenolsMetabolismGlucuronidesUrinary excretion a b s t r a c t In the present study we report on a UPLC-MRM validated method for the simultaneous direct analysis of mainglucuronidatedmetabolitesofoliveoilphenols:tyrosol,hydroxytyrosolandits O -methylmetabolitehomovanillyl alcohol in human urine after dietary olive oil ingestion. The developed method was linearwithin the concentration range 20–2000ng/mL with adequate recovery of analytes (>86%). Intra- andinter-dayprecisionandaccuracywereaccordingtostandardrequirementsformethodvalidationcriteria.Usingthedevelopedmethod,urinaryconcentrationsandexcretionratesofglucuronidesofoliveoilphenolsweresuccessfullyestimatedinaninterventionstudywith11healthyvolunteerssupplementedwithadie-tarydoseofvirginoliveoil(VOO)(50mL).Therefore,about13%oftheconsumedoliveoilpolyphenolswererecoveredin24-hurine,where75%ofthemwereintheformofglucuronides(3 0 -and4 0 - O -hydroxytyrosolglucuronides, 4 0 - O -glucuronidesoftyrosol andhomovanillyl alcohol)and25%asfreecompounds.   2010 Elsevier Ltd. All rights reserved. 1. Introduction A number of epidemiological studies have provided evidence of the health benefits derived from the Mediterranean diet againstcancerandcardiovasculardiseases(LaVecchia,2009;Trichopoulou,Costacou, Bamia, & Trichopoulos, 2003). The main characteristicsof such a diet are its richness in natural vitamins and antioxidants,fromvegetablesandfruits, andahighcontentof monounsaturatedfatty acids, olive oil being the main source of fat (Trichopoulouet al., 2003). The biological benefits of olive oil consumption arenot only limited to its high content of monounsaturated fat, oliveoil minor components also display bioactive properties (Covaset al., 2006; Fitó et al., 2000). Phenolic compounds, the most stud-ied olive oil minor components, belong to the hydrosoluble frac-tion of olive oil. Some of the most representative phenoliccompounds in olive oil are hydroxytyrosol (HOTYR) and tyrosol(TYR) and their respective secoiridoid derivatives, oleuropein andligstroside (Servili & Montedoro, 2002). They have been shown toexhibit strong antioxidant properties that contribute to theprotection of olive oil against lipid rancidity. Recent interventionclinical trials have provided evidence that the phenolic content of an olive oil contributes to the protection in humans against lipidoxidative damage in a dose dependent manner (Covas et al.,2006; Weinbrenner et al., 2004).Oneofthefirststepsinlinkingthebiologicalactivitiesofphenolcompounds of dietary srcin to health benefits in humans is todemonstrate their bioavailability from diet. Several interventionstudies in human and animal models have reported that phenoliccompoundsarerapidlyabsorbedinadosedependentmannerwiththe phenolic content of the olive oil administered (Visioli et al.,2000; Weinbrenner et al., 2004). Olive oil phenolic compoundsare extensively metabolized in the gut and liver and, thus, in bio-logical fluids they are found mainly as phase II metabolites (e.g.glucuronides and sulphates) of HOTYR, TYR and 3- O -methylconju-gate of HOTYR (homovanillyl alcohol, HVAlc) (Miró-Casas et al.,2001,2003). InratsadministeredwithHOTYR, bothorallyorintra-venously, both glucuronide and sulphate conjugates of HOTYR andHVAlc were detected in different biological matrices and tissues(D’Angelo et al., 2001; Tuck, Hayball, & Stupans, 2002). Despite in-ter-species differences (Visioli et al., 2003), the first pass intestinaland hepatic metabolism plays an important role in the 0308-8146/$ - see front matter   2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2010.10.044 ⇑ Corresponding author at: Human Pharmacology and Clinical NeurosciencesResearch Group, IMIM-Hospital del Mar. C/Dr.Aiguader 88, Barcelona 08003, Spain.Tel.: +34 93 316 04 84; fax: +34 93 316 04 67. E-mail address:  rtorre@imim.es (R. de la Torre).Food Chemistry 126 (2011) 306–314 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem  bioavailability and disposition of olive oil phenolic compounds tosuch an extent that their free forms are present only at very lowconcentrations in biological fluids (Miró-Casas, Covas, Farre,et al., 2003; Miró-Casas et al., 2001).The biotransformation of HOTYR and TYR into their phase IImetabolites was predicted to negatively influence their activitiesas antioxidants (Nenadis, Wang, Tsimidou, & Zhang, 2005). PhaseII metabolites are generally considered to be pharmacologicallyinactive and targets for excretion. However, some phase II metab-olites (e.g. glucuronides) of food derived antioxidants, such as cat-echins, have been reported to be biologically active (Lu et al.,2003). Moreover, in a study in rats (Tuck et al., 2002) it was re- ported that the urinary excreted 3 0 - O -HOTYR-glucuronide, butnot its 3 0 - O -sulphate conjugate, was a more potent antioxidant(by DPPH test) than its parent compound HOTYR.TheprevalentpresenceofoliveoilphenolsintheformofphaseIImetabolites within the organism following olive oil phenolic com-poundsingestion(e.g.asoliveoil,oliveoilphenolextractsandpureolive oil phenols) was acknowledgedinmanystudies, (Miró-Casas,Covas, Farre, et al., 2003; Miró-Casas et al., 2003; Tuck, Freeman,Hayball,Stretch,&Stupans,2001;Visiolietal.,2003)howevernodi-rectmethodsfortheiridentificationandquantificationinbiologicalfluids were reported. The main limitation was the unavailability of corresponding standards. Therefore, olive oil phenol metaboliteshavenotbeenquantifiedaccuratelyuntilnowaswellastheirmet-abolicexcretionrateshaveneverbeenestimated.In the majority of the studies, the concentrations of phenoliccompounds tested in  in vitro  experiments to achieve biologicaleffects display a large disparity with those observed  in vivo  in thehumanbodyafterreal-lifedosesofphenolic-richfoods.Concentra-tions of total phenols inbiological fluids(after anenzymaticdiges-tionofsamples)havebeenusedasareferenceinmanyexperimentswithout taking into account the contribution of free phenolcompounds (presumed biologically active) and their metabolites(presumedbiologicallyinactive)tothisoverallquantitativeestima-tion. It is, therefore, mandatory to have a proper estimation of theexpected concentrations of phenols, and their main metabolites,inhumansafterphenolic-richfoodconsumption.Thesedataarere-quiredinordertoestablishtherangeofconcentrationstobetested in vitro  and  in vivo  in the evaluation of their biological activities.The availability of HOTYR and TYR metabolites, e.g. their glucu-ronides (Khymenets et al., 2006), can allow characterising qualita-tivelytheirmetabolicdispositionandestimatingquantitativelythecontribution of each metabolic pathway. These results should becombined with those obtained in studies designed at the evalua-tion of their biological activity. Their potential biological activityshould allow to review past clinical studies or to design new oneswhere the contribution of phenol compounds to biological effectsshould be revised. At this stage it is proposed that this evaluationshould be performed applying alternative experimental ap-proaches to those applied until now.Theaimof thepresentstudywastodevelopadirectandsimplemethod for detection and quantification of the main olive oil phe-nolic metabolites, glucuronides of TYR, HOTYR and its O-methyl-ated metabolite HVAlc, in human urine in order to estimate therole and the rates of glucuronidation of phenols derived afterreal-life doses of virgin olive oil. 2. Methods and materials  2.1. Reagents and chemicals Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol) (HOTYR)andtyrosol (2-(4-hydroxyphenyl)ethanol) (TYR) were purchased fromExtrasynthèse (Extrasynthèse, Lyon, France). Homovanillyl alcohol(HVAlc), 3-(4-hydroxyphenyl)propanol (HOPhPr) (used as internalstandard, I.S.2) were supplied by Sigma–Aldrich (Sigma–AldrichInc., St. Louis, MO). Synthetic urine UriSub  (CST TechnologiesInc., Great Neck, NY) was used for the UPLC-MRM method valida-tion. Methanol (MeOH) and acetonitrile (ACN) were of analyticalgrade from Scharlau (Scharlau Chemie, Barcelona, Spain). Mobilephase was filtered with 0.22 l m nylon filters (Whatman, Brent-ford, UK). Sodium bisulphite, acetic acid, ammonium hydroxide,hypochloric and phosphoric acid were supplied by Merck (LiChro-solv  , Barcelona, Spain). Ultrapure water was obtained using aMilli-Q purification system (Millipore, Molsheim, France).Glucuronides:4 0 - O -hydroxytyrosol(4 0 - O -Gluc-HOTYR)and3 0 - O -hydroxytyrysol (3 0 - O -Gluc-HOTYR) glucuronides, 4 0 - O -tyrosolglucuronide (4 0 - O -Gluc-TYR), 4 0 - O -homovanillyl alcohol (4 0 - O -Gluc-HVAlc), and 4 0 - O -hydroxyphenylpropanol (4 0 - O -Gluc-HOP-hPr) (used as internal standard, I.S.1) (Fig. 1) glucuronideswere synthesized according to a method previously described(Khymenetset al., 2006).  2.2. Subjects, diet, study design and sample collection Six healthy male (aged 22–28) and five female (aged 20–44)volunteers were recruited. The institutional ethics’ committee(CEIC-IMAS) approved the protocol and the participants signedan informed consent. All volunteers were healthy on the basis of a physical and medical examination and standard biochemicaland haematological tests.The VOO used in this investigation was of Spanish srcin andhad been utilised in former studies (Cicero et al., 2008). Theamount of total HOTYR, HVAlc, and TYR in VOO was determinedas previously described (Miró-Casas, Covas, Fitó, et al., 2003;Miró-Casas et al., 2001).Prior to the dietary intervention, volunteers followed a oneweek wash-out period in which sunflower oil was provided as asource of fat for all purposes. During the first four days of thewash-out period, participants were asked to follow an antioxi-dant-controlled diet consisting of no more than two pieces of fruit,twoservingsofvegetablesorlegumes,twocupsofteaorcoffeeperday, andthe total avoidanceof wine, beer, andolive oil. Duringthelast 3days of the wash-out period, and on the intervention day(food provided at the clinical trials unit), the volunteers followeda strict low-phenolic compound diet. Phenolic-rich foods (vegeta-bles, legumes, fruit, juice, wine, coffee, tea, caffeine-containingsoftdrinks, beer, cocoa, marmalade, olive oil, and olives) were totallyexcluded from the participants 0 diet. On the intervention day, atfasting state, 50mL (44g) of VOO were administered in a singledose with bread (200g).Spot urine was collected at 8a.m. at fasting state prior to VOOadministration and from0–6hand 6–24h after VOOconsumptionon the intervention day. Urine samples were preserved with so-dium bisulphite (1mM final concentration) at acidic conditions(0.24M HCl final concentration) and stored at  20  C prior to use.Blood samples were collected in EDTA-containing tubes imme-diately before (0h) and 1h, 6h, and 24h after VOO intervention.Plasma samples were obtained by centrifugation of whole blooddirectly after being drawn and were preserved at   20  C prior touse.  2.3. Analysis of free and glucuronoconjugated HOTYR, TYR, and HVAlc in urine samples by SPE-UPLC-MRM  Prior to analysis a mix of internal standards was added to eachtube (final concentration of 500ng/mL I.S.-1 and 1000ng/mL I.S.-2.) and dried under nitrogen (25  C, 10–15 psi, 1min). After thaw-ingatroomtemperature,aliquotsof1mLofurineweredistributedto glass tubes. Samples were diluted 1:1 with 4% H 3 PO 4  and O. Khymenets et al./Food Chemistry 126 (2011) 306–314  307  applied to pre-conditioned with 2mL of MeOH and equilibratedwith 2mL of water Oasis  HLB 3cc (60mg) cartridges (WatersCorporation, Ireland). Samples were washed with 2mL of water,extractedwith3mLofMeOHwhichwasevaporatedunderanitro-gen stream (25  C, 10–15psi), and the extracts were reconstitutedin 200 l L either of 0.5% acetic acid (for synthetic urine) or 1mMammonium acetate at pH 5 (real urine samples). This genericmethod of extraction demonstrated optimum recovery values forthe simultaneous extractionof glucuronides and their parent com-pounds in both synthetic and real urine samples (data not shown).Finally, samples were filtered using Spin-X  Centrifuge 2-mL poly-propylene tubes with 0.22 l m nylon filters (Corning  , CorningIncorporated, NY, USA) and analysed by UPLC–MS as describedbelow.Analysis was performed using a Waters Acquity Ultra Perfor-mance LC system (Waters, Milford, MA, USA) coupled to a triplequadrupole (Quattro Premier XE) mass spectrometer providedwith an orthogonal Z-spray–electrospray interface (ESI) (WatersAssociates, Milford, MA, USA). Gradient chromatographic separa-tion of HOTYR, TYR, HVAlc and their glucuronides was performedon an Acquity UPLC™ BEH C 18  column (100  2.1mm, i.d.,1.7 l m particle size) (Waters Corporation  , Ireland). For gradientelution, mobile phase [A] 1mM ammonium acetate at pH 5, andphase [B] 100% ACN, were applied. The following elution condi-tions were used: 0–2min linear gradient at 3% [B]; 2–2.2min gra-dient to 10% [B]; 2.2–3.0min linear; 3.0–3.2min gradient to 20%[B]; 3.2–4.8min linear; 4.8–5.2 gradient to 3% [B]; 5.2–6.5 linearat 3%[B] for columnequilibration. UPLCoperatingconditionswereas follows: column temperature, 40  C; flow rate, 0.4mL/min;injection volume, 10 l L.Ionisation was performed in the negative mode. The followinginlet conditions were applied: drying gas, nitrogen (1000L/h,400  C); capillary voltage, 3.0kV; cone voltage, 25.0V. All com-pounds were monitored in the multiple monitoring mode(MRM).Basedonthecharacteristicspectrumofcollisionenergyin-duced fragmentations identified using reference standards, thespecific MS transmission pairs (under optimum collision energy,eV) were chosen for their discriminatory identification (see Fig. 1.  Structures of parent compounds, their glucuronidated metabolites and corresponding internal standards (I.S.) used in the present study.308  O. Khymenets et al./Food Chemistry 126 (2011) 306–314  Table 1). All transitions were monitored within 0.04s dwell timewith 0.02s of delay in three consecutive MS segments: (1) 0–2.4min for all glucuronides and I.S.-1; (2) 2.4–3.2 for HOTYR only;and (3) 3.2–6.5 for TYR, HVAlc and I.S.-2.Analytes were quantified by comparison of their peak area ra-tios (analyte versus the corresponding I.S.) with calibration curvesin which the peak area ratios of spiked calibration standards hadbeen plotted versus their concentrations using a weighted (1/ v 2 )calibration model. Integrated peak area ratios of HOTYR, TYR, andHVAlc were compared with I.S.-2 (HOPhPr) and their glucuronideswith I.S.-1 (HOPhPrGluc4), respectively.  2.4. SPE-UPLC-MRM assay validation Within the framework of the development and validation of the analytical method, we should take into consideration thatall tested compounds were either endogenous (HOTYR and HVAlcare dopamine metabolites) or exogenous from unknown compo-nents of diet (HOTYR- and TYR-related compounds). As a result,there were no biological fluids (urine and plasma) completely freefrom these substances. For this reason synthetic urine (UriSub)was used as a surrogate matrix for method validation. Prior tothe application of the method to real urine samples, an intra-and inter-assay validation protocol was carried out in syntheticurine.Standardstocksolutionsof theanalytes(HOTYR,4 0 - O -Gluc-HO-TYR, 3 0 - O -Gluc-HOTYR, TYR, 4 0 - O -Gluc-TYR, HVAlc, and 4 0 - O -Gluc-HVAlc) and I.S.s (HOPhPr and 4 0 - O -Gluc-HOPhPr) were preparedin MeOH at a concentration of 1mg/mL each. Mixtures of the ana-lytes (glucuronides and parent compounds) at concentrations of 1,10, and 100 l g/mL and of the I.S.s containing 100 l g/mL of I.S.-2(HOPhPr) and 50 l g/mL of I.S.-1 (HOPhPr-Gluc4) were preparedby dilution/combination of the aforementioned stock solutions inmethanol for the UPLC–MS/MS analysis. All methanol solutionsof compounds were kept in dark-coloured, well-sealed vials at  20  C and were seen to be stable for more than a 1year period.Calibration curves were prepared in synthetic urine at concen-trationsof20, 100,500, 1000, and2000ng/mLforallanalytes,cov-ering a linear range within the expected in the urine matrix levelsof compounds (as free phenols and their glucuronides) before andafter dietary consumption of olive oil. Internal standards I.S.-1 andI.S.-2 were prepared at concentrations of 500 and 1000ng/mL.Control samples containing appropriate amounts of analytes atthree different concentrations: low (40ng/mL), medium (250ng/mL), and high (1500ng/mL) were used. Four replicate analyseswereperformedwithsynthetic urinesamples at all concentrationsof the calibrationcurve. Additionally, 3 and 10 standard deviations(SD) of the calculatedconcentrationsat the lowestcalibrationcon-centration (20ng/mL) were used in order to estimate the limits of detection (LODs) and quantification (LOQs), respectively. Precisionwascalculatedastherelativestandarddeviation(RSD)ofthequal-ity control sample concentrations (imprecision). Accuracy was ex-pressed as the relative error (ERR) of the calculated concentrations(bias). The intra-day precision and accuracy were evaluated byanalysing all three control samples (at low, mediumand high con-centration) three times during the same day and by analysing thesame control samples in three different days, respectively.Recoverywasdeterminedbycomparingtheabsolutepeakareasof standards and I.S spiked into synthetic urine and extracted bySPE with those spiked post extractions at three different concen-trations: 20, 500, and 2000ng/mL (the lowest, the medium, andthe highest calibrators). The extraction yield compared betweensynthetic and real urines was shown to be similar for all testedcompounds and I.S.s (data not shown). A standard curve preparedin 5 different basal urines (collected in the study) was used in thequantitative analysis of all tested compound in the urine samplesof the study, in order to minimise the influence of matrix effect(data not shown).  Table 1 Fragmentation pattern of studied compounds and MRM transitions selected for qualitative analysis performed in negative ionisation mode. Compounds (RT a , min) Molecularion [M  H] b ,  m/z   Main fragment ions Characteristic MS pair (optimum CE, eV) m/z   Fragment descriptionHOTYR (2.92) 153 123 [M  H  H 2 CO(30)] 153 ? 123 (15)4 0 - O -GlucHOTYR (1.06) 329 153 [M  H  Gluc(176)] 329 ? 153 (20)175 [Gluc  H] c 113 [Gluc  H  CO 2  H 2 O] d 3 0 - O -GlucHOTYR (1.22) 329 153 [M  H  Gluc(176)] 329 ? 153 (20)175 [Gluc  H] c 113 [Gluc  H  CO 2  H 2 O] d TYR (3.54) 137 106 [M  H  CH 3 O _ (31)] 137 ? 106 (15)93 [M  H  C 2 H 4 OH(44)]4 0 - O -GlucTYR (1.04) 313 137 [M  H  Gluc(176)] 313 ? 137 (25)175 [Gluc  H] c 113 [Gluc  H  CO 2  H 2 O] d HVAlc (3.94) 167 152 [M  H  CH 3 (15)] 167 ? 152 (15)122 [M  H  CH 3 (15)  H 2 CO(30)]4 0 - O -GlucHVAlc (1.36) 343 167 [M  H  Gluc(176)] 343 ? 167 (20)175 [Gluc  H] c 113 [Gluc  H  CO 2  H 2 O] d HOPhPr, I.S.-2 (4.27) 151 121 [M  H  H 2 CO(30)] 151 ? 106(15)106 [M  H  C 2 H 5 O(45)]4 0 - O -GlucHOPhPr I.S.-1 (1.50) 327 151 [M  H  Gluc(176)] 327 ? 151 (25)175 [Gluc  H] c 113 [Gluc  H  CO 2  H 2 O] da RT, retention time on chromatogram. b [M  H] – Deprotonated molecular ion. c [Gluc  H] – Anhydroglucuronic acid residue,  m/z   175. d [Gluc  H  CO 2  H 2 O] – Secondary fragment ion at  m/z   113 (loss of CO 2  and water from  m/z   175). O. Khymenets et al./Food Chemistry 126 (2011) 306–314  309   2.5. GC–MS plasma and urine sample analysis The urine and plasma samples collected in this study were ana-lysed in parallel by GC/MS for total and free HOTYR, TYR, andHVAlc as previously described (Miró-Casas, Covas, Farre, et al.,2003; Miró-Casas, Covas, Fitó, et al., 2003).  2.6. Data evaluation, quantification, and statistical analysis Normality of continuous variables was assessed by Kolmogo-rov–Levene test. For data comparison a paired Student  t  -test wasemployed. A least-squares (1/ v 2 ) regression analysis was used toobtain correlation coefficients and slopes. A linear regression testwas applied for correlation estimations.Statistical analyses were performed with SPSS for Windows(version12.0)andsignificancewasdefinedas P   <0.05.Dataareex-pressed as either Mean±SD, Mean (CV%), or Mean (CI 95% ) asspecified. 3. Results  3.1. UPLC-MRM method development and validation To understand the impact of glucuronidation on the metabolicpathway of olive oil phenols a direct SPE-UPLC-MRM method forthe simultaneous determination of TYR, HOTYR, and HVAlc andtheircorresponding O -glucuronidesinhumanurinewasdevelopedand validated.HOPhPr was used as an internal standard (I.S.-2) for parentcompounds (HOTYR, TYR and HVAlc) quantification since theyshare structural similarities (Fig. 1). It was also used in GC–MSsample analysis as previously reported (Miró-Casas, Covas, Farre,et al., 2003; Miró-Casas, Covas, Fitó, et al., 2003; Miró-Casaset al., 2001). With regard to glucuronides, the 4 0 - O -glucuronide of HOPhPr was predictedto be themost appropriate I.S. for suchtypeof metabolites and was, therefore, synthesized according to estab-lished methodology (Khymenets et al., 2006). Both I.S.s exhibitedanalogous extraction recoveries (94.8±7.1% for I.S.-2, 93.6±7.2%for I.S.-1), chromatographic behaviour (data not shown) and frag-mentationpattern (Table 1) in a similar manner to evaluated com-pounds (Table 2). The optimal resolution of the tested compounds,in particular the glucuronides of HOTYR, was obtained in syntheticurine samples under reported chromatographic conditions within4.5min (total analysis run time of 6.5min). Analysis of basal andpostprandial urine samples revealed that there were no co-elutingcompounds with the proposed I.S.s and that they were well sepa-rated from other peaks with similar MS fragmentation (seeFig. 2). Basal urine samples, despite being collected after a wash-out period, still contained visible traces of almost all tested com-pounds (Fig. 2).Good linearity for the developed assay ( r  2 >0.99) was foundwithintheinvestigatedcalibrationrangeof20–2000ng/mLinsyn-thetic urine, for all compounds. Recoveries within 89–94%and 92–97%wereobservedforglucuronidesandparentcompoundstested,respectively (Table 2). LODs and LOQs for all compounds were nothigher than the lower calibrator (20ng/mL), except for HOTYR. In-tra-andinter-dayprecisionandaccuracyresultswereaccordingtothe standard requirements for method validation criteria (RSD%and ERR% were 6 20% for low and 6 15% for mediumand high con-centration controls for both intra- and inter-day experiments(Hartmann, Smeyers-Verbeke, Massart, & McDowall, 1998). Allcompounds met these criteria except HOTYR particularly at thelowest concentration tested and marginally 4 0 - O -GlucTYR (Table 2).  3.2. HOTYR and TYR content in VOO and volunteers’ dietarycompliance TheconcentrationsofHOTYRandTYR,estimatedasthemixtureoffreeformsofHOTYRandTYRandthoseresultingfromtheacidichydrolysis of their secoiridoid derivatives present in olive oil, were67.6 l g/mL and 42.0 l g/mL, respectively. Therefore, the totalamounts of HOTYR and TYR consumed with 50mL of VOO were  Table 2 Summary for the UPLC-SPE-MRM assay pre-validation for simultaneous determination and quantification of free HOTYR, TYR and HVAlc and their glucuronidated conjugates insynthetic urine as surrogate matrix. Compound LOD (ng/mL) LOQ (ng/mL) Recovery (%) a Intra-day b Inter-day c RSD% d ERR% e RSD% d ERR% e 4 0 - O -GlucTYR 7.2 20 89.3 (13.6) 8.1 20.1 8.7 20.012.7 19.4 10.9 18.94.6 18.1 5.6 14.04 0 - O -GlucHOTYR 4.5 13.6 93.7 (11.5) 8.2 7.6 6.8 6.411.0 10.1 11.2 9.62.5 9.3 5.4 12.63 0 - O -GlucHOTYR 2.5 7.6 94.5 (11) 12.4 20.5 10.3 8.64.4 3.7 9.9 7.93.6 10.5 6.4 6.54 0 - O -GlucHVAlc 4.1 12.3 92.7 (11) 7.1 6.4 10.3 8.63.1 2.9 9.9 7.93.0 3.6 6.4 6.5HOTYR   24.7 75.06   92.8 (  25.2 )  57.9 59.8 68.4 50.6  23.9 24.9 21.5 19.7  12.7 9.4 9.7 7.4TYR 0.62 1.87 97.1 (13.7) 11.9 9.6 11.4 11.09.9 8.21 12.5 12.43.9 6.6 8.2 7.2HVAlc 2.87 8.7 94.1 (15.7) 6.9 9.0 14.9 12.89.7 7.4 9.4 6.90.5 10.6 8.7 9.7 a Mean (CV%),  n  =12 (quadruplicates per each of three concentrations: 20, 500 and 2000ng/mL). b Intra-day:  n  =3 per each control quality samples: low, medium, and high, respectively; c Inter-day:  n  =9, 3days per each control quality sample: low, medium, and high, respectively; d RSD: relative standard deviation of the control samples concentrations (imprecision). e ERR: relative error of the estimated concentrations for control samples (bias).310  O. Khymenets et al./Food Chemistry 126 (2011) 306–314
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