Di

AJa

b

a

ARAA

KPPTU

1

m(ectehrots

oismi

1d

Journal of Chromatography B, 877 (2009) 1169–1176

Contents lists available at ScienceDirect

Journal of Chromatography B

journa l homepage: www.e lsev ier .com/ locate /chromb

etermination of procyanidins and their metabolites in plasma samples bymproved liquid chromatography–tandem mass spectrometry

ida Serraa, Alba Maciàa, Maria-Paz Romeroa, Maria-Josepa Salvadób, Mario Bustosb,uan Fernández-Larreab, Maria-José Motilvaa,∗

Department of Food Technology, Escola Tècnica Superior d’Enginyeria Agrària, Universitat de Lleida, Avda/Alcalde Rovira Roure 191, 25198 Lleida, SpainDepartment of Biochemistry and Biotechnology, Campus Sescelades, Universitat Rovira i Virgili, c/ Marcel·li Domingo s/n, 43007 Tarragona, Spain

r t i c l e i n f o

rticle history:eceived 28 November 2008ccepted 4 March 2009vailable online 11 March 2009

eywords:lasma

a b s t r a c t

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography–tandem massspectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, cat-echin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analyticalmethod, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrixeffect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was65%. The LODs and LOQs were lower than 0.003 and 0.01 �M, respectively, for all the procyanidins studied,

rocyanidinsandem mass spectrometryPLC

except for the trimers, which were 0.8 and 0.98 �M, respectively. This methodology was then applied forthe analysis of rat plasma obtained 2 h after ingestion of grape seed phenolic extract. Monomers (catechinand epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples,and their concentration ranged from 0.85 to 8.55 �M. Moreover, several metabolites, such as catechinand epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechinmethyl-sulphate were identified. These conjugated forms were quantified, in reference to the respectiveunconjugated form, showing concentrations between 0.06 and 23.90 �M.

. Introduction

Flavanols are found in a wide range of food sources as bothonomers and oligomeric procyanidins. Flavanol monomers are

−)-epicatechin and (+)-catechin, and procyanidins are oligomers ofpicatechin and catechin. The main food sources of flavanols includeocoa, red wine, green tea, red grapes, berries and apples [1]. Overhe past decade, different studies have reported on the health ben-fits, mainly cardio-protective effects, of flavanols. These findingsave been attributed in part to their antioxidant properties. Thus,esearch into the bioavailability, metabolism and pharmacokineticsf ingested flavanols in the diets of volunteers is of great impor-ance as it may in turn reflect the antioxidant status of the subjectstudied.

Biomarkers are biological molecules found in blood, body tissuesr physiological fluids. These biological molecules are useful tools

n nutritional epidemiology to determine food or nutrient expo-ure because they combine the measurement of bioavailability andetabolism. The main advantage of biomarkers is that they are an

mportant alternative to the more traditional dietary assessment

∗ Corresponding author. Tel.: +34 973 702817; fax: +34 973 702596.E-mail address: motilva@tecal.udl.cat (M.-J. Motilva).

570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jchromb.2009.03.005

© 2009 Elsevier B.V. All rights reserved.

tools, such as diet histories, food frequency questionnaire or dietdiaries [2] with significant variability related to food processing andstorage conditions, and dietetic estimations.

Several studies in humans have demonstrated the relationshipbetween the phenols present in biological fluids, such as bloodor urine, and dietary intake [3,4]. Dietary polyphenols undergometabolic conversion in the liver, including methylation of ahydroxyl group and reduction of a carbonyl group, as well as conju-gation leading to glucurono and sulphoconjugates. In order to usethese specific metabolites as biomarkers, it is essential that the ana-lytical technique for determining them in biological samples to besensitive, selective, reliable and robust.

Different analytical methodologies have been reported for theanalysis of procyanidins in biological samples, such as plasma[5–20], urine [9,11,13,18,19], and body tissues [13,15,19]. Thesemethods include high performance liquid chromatography (HPLC)coupled to mass spectrometry (MS) [15,16,18], tandem MS [8,13,14],fluorimetric [6,17], diode array detection (DAD) [11], and electro-chemical detector (ECD) [7–9,12], gas chromatography (GC) coupled

to MS [5,19], although this technique often requires derivatization,and capillary electrophoresis (CE) coupled to DAD [20].

Recent advances in analytical techniques have improved theireffectiveness and could expand their potential for analyzing andidentifying biomarkers. The use of MS and tandem MS (MS/MS) as

1 togr. B

ats

aaStock

2

2

dMltstctw

Rvoptfi

(gfM

2

nnt1

buaB(Hnidate4Tcr

170 A. Serra et al. / J. Chroma

detector is a useful tool for determining analytes at low concentra-ions and for the positive identification of compounds in complexamples, such as biological ones.

The aim of this study is to develop and validate a rapid, sensiblend reliable method for the determination of monomers, catechinnd epicatechin, dimers and trimers in plasma samples by off-linePE-UPLC-MS/MS. After validation, the method was used to iden-ify and quantify procyanidins and their metabolites in rat plasmabtained 2 h after administration, by intragastric gavage, of a doseorresponding to 1 g of grape seed procyanidin extract (GSPE) perilogram of body weight.

. Experimental

.1. Chemicals and reagents

Standards of (−)-epicatechin, (+)-catechin and the internal stan-ard (IS) catechol were purchased from Sigma–Aldrich (St. Louis,O, USA); and procyanidin B2 from Fluka Co. (Buchs, Switzer-

and). Individual stock standard solutions of 2000 mg/l, except tohe dimer which was 500 mg/l, were dissolved in methanol andtored in a dark-glass flask at −18 ◦C. A stock standard mixed solu-ion of catechin, epicatechin and dimer was prepared weekly at aoncentration of 100 mg/l in methanol. The working standard solu-ions were prepared daily by diluting the stock standard solutionsith the solution of acetone:water:acetic acid (70:29.5:0.5, v:v:v).

The GSPE, whose commercial name is Vitaflavan® (Les Dérivesésiniques et Terpéniques, Dax, France) was obtained from vitisinifera grape seed from southwest France. The procyanidin contentf the extract was >75% (monomers 22%, dimers 20% and trimers toentamers 56%). The cocoa nib samples corresponded to the Foras-ero variety from Ghana (West Africa). Cocoa nibs were obtainedrom roasted cocoa beans separated from their husks and brokennto small pieces.

Acetonitrile (HPLC-grade), methanol (HPLC-grade), acetoneHPLC-grade) and glacial acetic acid (≥99.8%) were of analyticalrade (Scharlab, Barcelona, Spain). Ultrapure water was obtainedrom a Milli-Q water purification system (Millipore Corp., Bedford,

A, USA).

.2. Isolation of the trimer as standard

The trimer was isolated by using semi-preparative HPLC inormal phase from a phenolic extract obtained from cocoaibs [21]. The cocoa nib phenolic extract was dissolved in ace-one:water:acetic acid (70:29.5:0.5, v:v:v) at a concentration of00 mg/ml before the chromatographic fractionation.

The semi-preparative HPLC system includes a Waters 1525EFinary HPLC pump, a Waters Flexinject, an Inertsil ODS-3 col-mn (5 mm, 25 cm × 10 mm i.d., GL Sciences Inc.) equipped withSpherisorb S5 ODS-2 (5 mm, 10 cm × 10 mm i.d., Technokroma,

arcelona, Spain) precolumn, a Waters 2487 � absorbance detector280 nm) and a Waters Fraction Collector II. The semi-preparativePLC system was operated using Brezze software. 50 �l of phe-olic extract of cocoa nibs was injected manually into the

njector module (1 ml sample loop). The mobile phase wasichloromethane:methanol:water:acetic acid (84:14:2:2, v:v:v:v)s the eluent A, and methanol:water:acetic acid (96:2:2, v:v:v) ashe eluent B. The elution started with 0% of eluent B, then was lin-

arly increased to 17.6% of eluent B in 30 min, further increased to5% of B in 15 min, and an additional increase to 50% B in 5 min.hen, it was kept isocratic for 10 min and returned to the initialonditions for 10 min. The reequilibration time was 10 min. The flowate was 1 ml/min.

877 (2009) 1169–1176

The fractions corresponded to trimer were collected manuallyby observing the detector output on the recorder and accordingto their retention time [22,23]. After that, the organic solvent wasremoved by rotary evaporation (Buchi, Labortechnick AG, Switzer-land) under a partial vacuum at 30 ◦C. Finally, the water extract wasfreeze-dried in Lyobeta 15 lyophilizer (ImaTelstar, Spain) and thenthe fractions were stored in an N2 atmosphere at −40 ◦C. An aliquotof the lyophilized fraction was exactly weighted and dissolved inacetone:water:acetic acid (70:29.5:0.5, v:v:v) at final concentrationof 5.8 �M. Then, this solution was spiked to the plasma matrix inorder to study the quality parameters of the developed method.

2.3. Plasma samples

Rat plasma samples were obtained from male Wistar rats (three-month-old). These rats weighed between 370 and 420 g and werepurchased from Charles River Laboratories (Barcelona, Spain). TheAnimal Ethics Committee of the Rovira i Virgili University approvedall the procedures. The rats were maintained in air-conditionedquarters at 22 ◦C under 12 h dark/12 h light cycles (light from 9 a.m.to 9 p.m.). All the animals were fed a standard diet of PanLab A04(Panlab, Barcelona, Spain) and water.

Procyanidin-rich extract (Vitaflavan®) was administered to therats by intragastric gavage, after the animals had been in fastingconditions between 16 and 17 h with only access to tap water. Theprocyanidin extract was administered to the rats at a dose of 1 gGSPE per kilogram of body weight. 2 h after the treatment the ratswere anaesthetized with ketamine–xylazine and blood sampleswere collected from abdominal aorta. The plasma samples wereobtained by centrifugation (2000 × g, 30 min at 4 ◦C) and stored at−40 ◦C until chromatographic analysis.

2.4. Plasma phenols extraction

Prior to the chromatographic analysis, the rat plasma sampleswere pretreated by off-line SPE. OASIS HLB (60 mg, Waters Corp.,Milford, MA) cartridges were used. These were conditioned sequen-tially with 5 ml of methanol and 5 ml of Milli-Q water/acetic acid(99.8:0.2, v:v). 20 �l of phosphoric acid 85% and 50 �l of catechol(IS) at a concentration of 20 mg/l were added to 1 ml of plasmasample, and then this solution was loaded into the cartridge. Theloaded cartridges were washed with 3 ml of Milli-Q water and 5 mlof Milli-Q water/acetic acid (99.8:0.2, v:v). Then, the retained pro-cyanidins were eluted with 4 ml of solution acetone:water:aceticacid (70:29.5:0.5, v:v:v). The elution solvent was evaporated todryness under a nitrogen stream in an evaporating unit at 30 ◦C(PIERCE Model 18780, IL, USA) and reconstituted with 100 �l ofelution solution. Finally, the extract was filtered through 0.22 �mnylon syringe filter (Teknokroma, Barcelona, Spain) and transferredto the autosampler vial before the chromatographic analysis. Theinjection volume was 2.5 �l.

In order to obtain a plasma matrix free from procyanidins, poolplasma from basal conditions was dephenolized. To dephenoliza-tion, the plasma was loaded through the cartridge (OASIS HLB200 mg, Waters Corp., Milford, MA) and the elution of the loadedplasma was collected. This sample matrix was used as the blankplasma to validate the procedure.

2.5. UPLC-ESI-MS/MS

The UPLC analysis of procyanindin extracts was performed using

a Waters Acquity Ultra-PerformanceTM liquid chromatography sys-tem (Waters, Milford, MA, USA), equipped with a binary pumpsystem (Waters, Milford, MA, USA). The separations were achievedusing an Acquity HSS T3 column (100 mm × 2.1 mm i.d., 1.8 �mparticle size) from Waters (Milford, MA, USA). The analytes were

A. Serra et al. / J. Chromatogr. B 877 (2009) 1169–1176 1171

Table 1Optimized SRM conditions for analyzing catechol (IS), catechin, epicatechin, dimer B2, and trimer by UPLC-MS/MS in negative mode.

Compound SRM1 (quantification) Cone voltage (V) Collision energy (eV) SRM2 (confirmation) Cone voltage (V) Collision energy (eV)

Catechol (IS) 109 > 91 40 15 – – –CEDT

s(ow0c

seaTva4act(actctssfcpfcts

2

tdl(

sdactTft

dsddmtp

atechin 289 > 245 45 10picatechin 289 > 245 45 10imer B2 577 > 289 45 20rimer 865 > 577 60 20

eparated with a mobile phase consisting of water:acetic acid99.8:0.2, v/v) (eluent A) and acetonitrile (eluent B) at a flow ratef 0.4 ml/min. The gradient elution was reported in our previousork [21] and the total analysis time was 12.5 min. Briefly, it was:–10 min, 5–35%B; 10–10.10 min, 35–80%B; 10.10–11 min, 80%B iso-ratic; 11–11.10 min, 80–5%B; 11.10–12.50 min, 5%B isocratic.

The tandem MS analyses were carried out on a TQD masspectrometer (Waters, Milford, MA, USA) equipped with a Z-spraylectrospray interface. The analyses were done in negative mode,nd the data was acquired in selected reaction monitoring (SRM).he ionization source working conditions were as follows: capillaryoltage, 3 kV; source temperature, 150 ◦C; cone gas flow rate, 80 l/hnd desolvation gas flow rate, 800 l/h; desolvation temperature,00 ◦C. Nitrogen (>99% purity) and argon (99% purity) were useds nebulizing and collision gases, respectively. Cone voltages andollision energies were optimized by infusion of a standard solu-ion of 10 mg/l of each standard in a mixture of acetonitrile:water50:50, v/v) at a flow rate of 10 �l/min. First, full-scan spectra werecquired in order to select the most abundant m/z value and theone voltage was optimized. In all cases, [M−H]− ions were foundo be the most abundant. These ions were selected as the pre-ursor ions, and afterwards, the collision energies were studiedo find the most abundant product ions. Therefore, the most sen-itive transition was selected for quantification purposes and theecond one for confirmation. Table 1 shows the MS/MS transitionsor quantification and confirmation as well as cone voltage andollision energy values optimized for each of the standard com-ounds. For catechol, the product ion 91 m/z was the only oneormed and hence no second MS/MS transition was available foronfirmation purposes. The dwell time established for each transi-ion was 30 ms. Data acquisition was carried out by MassLynx v 4.1oftware.

.6. Validation procedure

The method developed was validated using a serial dilution ofhe blank plasma spiked with the standard catechin, epicatechin,imer B2, and trimer. The parameters considered were: recovery,

inearity, calibration curves, precision, accuracy, detection limitLOD), quantification limit (LOQ), and the study of the matrix effect.

The linearity of the method was evaluated using blank plasmapiked with the analytes. Calibration curves (based on peak abun-ance) were plotted using y = a + bx, where y is the (analyte/IS) peakbundance ratio and x is the (analyte/IS) concentration ratio. Con-entrations of the procyanidins were calculated by interpolatingheir (analyte/IS) peak abundance ratios on the calibration curve.he calibration curves were obtained by analyzing five points at dif-erent concentration levels and each standard solution was injectedhree times.

The precision of the method, expressed by relative standardeviations (% RSDs) of the concentration and peak abundance, wastudied at two concentration levels, 0.2 and 1.4 �M, except for the

imer and trimer, which were 0.07 and 0.6 �M, on three differentays (inter-day) and one injection each day. The accuracy of theethodology was evaluated by sample quantification at a concen-

ration level of 2 �M for the trimer, and 0.2 �M for the rest of therocyanidins. The accuracy was calculated from the ratio between

289 > 205 45 15289 > 179 45 15577 > 425 45 15865 > 695 60 25

the concentrations of the procyanidin found compared to the spikedconcentration. This quotient was then multiplied by 100.

The LODs and LOQs were calculated using the signal-to-noisecriterion of 3 and 10, respectively. The extraction recoveries of theanalytes were determined using blank plasma spiked with the ana-lytes at a concentration of 3 �M, except for the dimer and the trimer,which were 1.5 and 5.8 �M, respectively. To determine extractionrecoveries, the responses of the analytes spiked in plasma matricesbefore and after extraction were compared.

The matrix effect was evaluated by comparing peak abundancesobtained from blank plasma samples spiked after sample pre-treatment with those obtained from standard solutions.

3. Results and discussion

3.1. Trimer isolation

Trimer procyanidin is not commercially available, and so for thisstudy, the trimer was obtained from an extract rich in procyani-dins by semi-preparative HPLC-DAD. Two extracts were tested forthis purpose, these being the GSPE extract (Vitaflavan®), and thephenolic cocoa nib extract. Semi-preparative HPLC in the normalphase was used because various authors have reported that thismode separates the procyanidins by their degree of polymerizationbetter than in the reverse mode [22,23].

The amount of procyanidins in the GSPE extract was higher thanin the cocoa extracts. However, the chromatographic peaks corre-sponding to different procyanidins in the GSPE extract were broaderthan the peaks from the cocoa extracts. This could be related tothe greater presence of the isomeric forms in the structure of theprocyanidins (dimer, trimer, etc.) in the GSPE extract than in theprocyanidins from the cocoa extract. The major isomerization levelof the procyanidins in the GSPE extract made it difficult to isolatethe trimer by semi-preparative HPLC.

Fig. 1 shows the extracted ion chromatograms of the trimer pro-cyanidin isolated from the (A) GSPE extract, and the (B) cocoa nibphenolic extract by HPLC-MS/MS in the normal phase. As Fig. 1Bshows, the trimer procyanidin isolated from the cocoa extract isvery simple, and only two isomeric forms were observed. By con-trast, the trimer isolated from the GSPE extract shows a greatnumber of isomeric forms (see Fig. 1A). In consequence, the cocoaextract was selected to purify the trimer because it showed a higherpeak efficiency than that obtained by the GSPE extract and thismade it easy to study the validation procedure.

3.2. SPE procyanidins extraction

Off-line SPE was used as the sample pretreatment techniqueto clean-up the plasma sample matrix and determine the pro-cyanidins at low concentration levels. The main objective of theclean-up step is to remove proteins and endogenous substancespresent in the biological fluids that could interfere with the

chromatographic analysis of the procyanidins. A critical aspectof the clean-up is the optimization of the recovery of the ana-lytes of interest during the elution steps to obtain the maximumrecoveries. For this purpose, the commercial standards of the pro-cyanidins (catechin, epicatechin and dimer B2) and the purified

1172 A. Serra et al. / J. Chromatogr. B 877 (2009) 1169–1176

Fig. 1. Extracted ion chromatograms of trimer procyanidin isolated by semi-preparative HPLC and analyzed by HPLC-MS/MS from (A) GSPE extract, and (B)pte

tPp[

itffta

moeacasmw

Fig. 2. Extracted ion chromatograms from blank plasma sample spiked with cate-

TRp

C

CEDT

urified cocoa nib extract. The concentration of each extract was 10 mg/ml, andhis was dissolved in the extraction solution. See Experimental Section 2.2 for thexperimental conditions.

rimer were spiked in blank plasma at different concentrations.lasma sample acidification was taken into account to disrupthenol–protein binding and to enhance recovery of the analytes24].

Different volumes (1–5 ml) of water and acidic water were stud-ed in the clean-up step to remove any possible interference fromhe plasma matrix without eluting the retained procyanidins. Dif-erent volumes were evaluated, and finally we selected 3 ml of waterollowing by 5 ml of acid water (0.2% acetic acid) to clean-up the car-ridge, removing the interference substances without elution of thenalytes of interest.

Afterwards, the procyanidins elution step was studied. To deter-ine the appropriate composition of the elution solvent, different

rganic solvents or solutions and different volumes of these werevaluated. The elution solvents studied were acetone:water:aceticcid (70:29.5:0.5, v:v:v), and methanol/acetone at different per-entages, from 0% to 100%. The recoveries were higher when

cetone:water:acetic acid (70:29.5:0.5, v:v:v) was used as elutionolvent. When 1 ml of this solvent was used, almost 100% of theonomers catechin and epicatechin were eluted. However, 4 mlas necessary to elute the procyanidins, dimer and trimer. To

able 2etention time (RT), recovery (%R), linearity, calibration curves, reproducibility, accuracy, Llasma samples.

ompound RT (min) %R Linearity (�M) Calibration curve

atechin 3.68 102 0.02–3 y = 6.913x + 0.252picatechin 4.55 96 0.02–3 y = 5.830x + 0.270imer B2 4.09 84 0.008–0.8 y = 5.524x + 0.030rimer 4.93 65 0.9–5.8 y = 0.045x − 0.085x

a RSD% was calculated as concentration.b 0.6 �M.c 0.07 �M.

chin, epicatechin, dimer B2 and trimer. The peak designation and its concentrationwas (1) catechol, 9 �M; (2) catechin, 1.5 �M; (3) epicatechin, 1.5 �M; (4) dimer B2,0.4 �M; and (5) trimer, 3.4 �M.

increase the pre-concentration factor, evaporation to dryness wascarried out in an N2 stream in an evaporating unit at 30 ◦C as hasbeen described in Experimental Section 2.4, and reconstituted with100 �l of elution solution. Then, the extract was re-dissolved inthe minimum volume of solvent in order to obtain a high pre-concentration factor. A volume of 100 �l of two different solvents,acetone:water:acetic acid (70:29.5:05, v:v:v) and acetonitrile, weretested to re-dissolve the dried procyanidin plasma extract. Whenacetonitrile was used, a peak distortion was obtained duringthe chromatographic analysis. Therefore, acetone:water:acetic acid(70:29.5:05, v:v:v) was selected and the obtained recoveries werehigher than 84% for all the procyanidins, except for the trimer, whichwas 65% (see Table 2).

As mentioned above, pool plasma from basal conditions wasdephenolized in order to obtain a plasma matrix free from procyani-dins for the validation procedure. When this plasma sample waspre-treated and pre-concentrated by off-line SPE, no interference

peaks were observed. Fig. 2 shows the extracted ion chromatogramsof a blank plasma sample spiked with 1.5 �M of catechin and epi-catechin, 0.4 �M of dimer B2, and 3.4 �M of trimer after the SPEextraction.

ODs and LOQs for the analysis of the studied compounds by UPLC-MS/MS in spiked

RSD% (n = 3)a Accuracy LOD (�M) LOQ (�M)

1.4 �M 0.2 �M

4.3 8.9 104 0.004 0.0133.9 8.6 99.8 0.004 0.0153.5b 8.5c 98 0.003 0.0104.2b 8.8c 102 0.800 0.980

A. Serra et al. / J. Chromatogr. B 877 (2009) 1169–1176 1173

F rocyanc s of th

3

mapd(toitbop

3

we

rttwtd

ig. 3. Off-line SPE-UPLC-DAD chromatogram of (A) control rat plasma, and (B) patechin and epicatechin, (E) dimer B2, and (F) trimer. See the text for the condition

.3. Matrix effect

When ESI is used as the ionization technique in MS, one of theain problems is the signal suppression or enhancement of the

nalyte response due to the other components present in the sam-le matrix (matrix effect) [25]. To evaluate this matrix effect, theetector responses of the procyanidins spiked in elution solventacetone:water:acetic acid (70:29.5:0.5, v:v:v)) were compared tohose spiked in plasma at different concentrations. Either a positiver negative effect was observed, lower than 17%, which meant anncrease or decrease in the detector response, respectively. Never-heless, this effect could be considered small. To reduce inaccuraciesy matrix, as well as the clean-up step of the plasma sample byff-line SPE, the preparation of the calibration curves with spikedlasma was considered more appropriate.

.4. Validation of the analytical procedure

Blank plasma spiked with different procyanidin concentrationsas analyzed by off-line SPE and UPLC-MS/MS to determine the lin-

arity, calibration curves, reproducibility, accuracy, LODs and LOQs.Linearity was tested following the procedure developed in the

ange from 0.02 to 5.8 �M. All the compounds showed R2 higher

han 0.998 (Table 2). The precision of the method, calculated ashe relative standard deviation (% RSD), in terms of concentration,as calculated at two concentration levels, 0.07 and 0.6 �M for the

rimer and dimer, and 0.2 and 1.4 �M for the rest of the procyani-ins, and these gave results lower than 8.9% and 4.3%, respectively.

idin rat plasma at 280 nm. Extracted ion chromatograms of (C) catechol (IS), (D)e experiment.

The accuracy of the method was between 98% and 104%. TheLODs and LOQs were lower than 0.003 and 0.01 �M, respectively, forall the procyanidins studied, except for the trimers, which were 0.8and 0.98 �M, respectively. These values were similar to and lowerthan the results reported in the literature [5,6,11,16,17,19,20]. One ofthe greatest advantages of the UPLC method developed is the possi-bility of quantifying the procyanidins studied at low concentrationswithin 5 min. This analysis time is between 2 and 5-times shorterthan the methods reported in the literature for the analysis of thesecompounds by HPLC [5–11,13–19].

Additionally, the UPLC system allowed the volume of the sampleinjected to be between 4 and 40-fold lower than the other methodsreported in the literature [14–17].

3.5. Application of the method for determining procyanidins inplasma samples

In order to show the applicability of the method developed, ratplasma was analyzed. Procyanidin-rich plasma was obtained fromthe abdominal aorta of the rats 2 h after administration, via directstomach intubation, of a dose of GSPE extract corresponding to1 g/kg of weight. Rat plasma extracted before ingestion of the GSPEextract was used as a control. The UPLC-DAD chromatograms of

the procyanidins in the control and procyanidin-rich plasmas areshown in Fig. 3A and B, respectively. Important differences in thechromatographic profile of the two plasma samples were observed.Thus, chromatographic peaks corresponding to monomers (cate-chin and epicatechin, peaks 2 and 3, respectively), dimers (peak 4)

1174 A. Serra et al. / J. Chromatogr. B

Table 3Procyanidin concentration detected in rat plasma sample by off-line SPE-UPLC-MS/MS.

Compound Concentration (�M)

CEDT

atect(

rtswmse

Fg

atechin 0.85picatechin 1.28imer 2.40rimer 8.55

nd trimers (peak 5) were only identified in plasma obtained afterhe ingestion of the GSPE extract (Fig. 3B). Fig. 3C–F also shows thextracted ion chromatograms of the catechol (IS) and the free pro-yanidins. The quantification of the free procyanidins (monomerso trimers) showed concentrations ranging from 0.85 to 8.55 �MTable 3).

The detection and quantification of dimers and trimers in theat plasma are relevant, because their presence could suggesthat these oligomeric forms are absorbed and metabolized in the

ame way as the monomeric forms. The results obtained agreedith those in the literature, where some authors also detectedonomers [10,14], dimers [14,26,27], and trimers [14] in plasma

amples after intake of procyanidin-rich extracts, such as grape seedxtract [27], cocoa [6–8,16,28], apple [14] and tea [16].

ig. 4. Extracted ion chromatograms and its MS spectra of the generated metabolites:lucuronide, (M4) epicatechin methyl-glucuronide, (M5) catechin methyl-sulphate, and (M

877 (2009) 1169–1176

In MS, it is very important to quantify each analyte with therespective calibration curve because the ionization can vary in func-tion of the molecule structure. This was observed in the presentstudy with the quantification of the trimer. When the trimer wasquantified using its calibration curve obtained with the purifiedstandard from cocoa phenol extract (see Experimental Section 2.2),the concentration of this oligomer in the plasma was 8.55 �M. Incontrast, the quantification of the trimer using the calibration curveof catechin, epicatechin or dimer showed concentrations between0.05 and 0.06 �M.

During digestion and intestinal absorption, polyphenols are sub-jected to three main types of conjugation: methylation, sulphation,and glucuronidation [29]. In consequence, apart of the free pro-cyanidins, different conjugated metabolites could be present in ratplasma after the ingestion of GSPE extract. As well as applyingthe validation method to quantify the free forms of the procyani-dins in rat plasma, the identification of the potential generatedmetabolites was considered. For this purpose, analyses in MS

(full-scan mode) and MS/MS (based on neutral loss scan and prod-uct ion scan mode) were performed. These techniques (full-scanand product ion scan) are excellent tools for verifying structuralinformation about the compounds when standards are not avail-able.

(M1) catechin glucuronide, (M2) epicatechin glucuronide, (M3) catechin methyl-6) epicatechin methyl-sulphate. See the text for the conditions of the experiment.

togr. B 877 (2009) 1169–1176 1175

tWmhaTsedts

bmlamootta

ma3i

alm

cwemct

wcccssttb

TP

M

MM

M

M

M

M

Table 5Quantification of procyanidin metabolites by off-line SPE-UPLC-MS/MS.

Compound SRM (quan-tification)

Conevoltage (V)

E collision(eV)

Concentration(�M)

Catechinglucuronidea

465 > 289 40 20 23.90

Epicatechinglucuronideb

465 > 289 40 20 20.57

Catechin methyl-glucuronidea

479 > 303 40 25 13.75

Epicatechinmethyl-glucuronideb

479 > 303 40 25 9.06

Catechinmethyl-sulphatea

383 > 303 40 15 1.05

Epicatechinmethyl-

383 > 303 40 15 1.30

A. Serra et al. / J. Chroma

First, analyses were carried out in the full-scan mode (from 80o 1200 m/z) by applying different cone voltages from 20 to 60 V.

hen low cone voltages were applied, the MS spectrum gave infor-ation about the precursor ion or the [M−H]−. In contrast, when

igh cone voltages were applied, specific fragment ions were gener-ted and the MS spectrum gave information about their structure.he structural information was also verified by using product ioncan and neutral loss scan in the MS/MS mode. In product ion scanxperiments, the product ions are produced by collision-activatedissociation of the selected precursor ion in the collision cell. Neu-ral loss scan of 80 and 176 units were used to characterize theulphate and glucuronide forms, respectively.

In the analysis of rat plasma, six procyanidin metabolites coulde identified by the fact that their product ions produced ions at/z 289 that matched that of catechin/epicatechin. The metabo-

ites identified were catechin and epicatechin glucuronide, catechinnd epicatechin methyl-glucuronide, and catechin and epicatechinethyl-sulphate, as shown in Table 4. Considering the UPLC elution

rder of the catechin and epicatechin (Fig. 2) and the elution orderf the metabolites (Fig. 4), together with the MS fragmentation pat-ern, the earlier eluting M1, M3 and M5 peaks have been assignedo catechin metabolites, while the M2, M4 and M6 peaks have beenssigned to epicatechin metabolites.

Two ions gave a precursor ion of m/z 479 and product ions of/z 303 and m/z 289. These ions could be identified as catechin

nd epicatechin methyl-glucuronide, because the product ion m/z03 is due to the loss of glucuronide molecule (176 units) and the

on m/z 289 to the loss of methyl group (14 units).The two ions with precursor ion of m/z 465 could be identified

s catechin and epicatechin glucuronide, respectively, due to theoss of 176 units (m/z 289) that could correspond to glucuronide

olecule.Then, the last procyanidin metabolite identified could be cate-

hin and epicatechin methyl-sulphate (precursor ion of 383 m/z)hose product ions were m/z 303 and m/z 289. These could be

xplained by the loss of a sulphate molecule (80 units) and theethyl group (14 units), respectively. Fig. 4 shows the extracted ion

hromatograms of the metabolites generated and their MS spec-rum.

The metabolites detected in rat plasma in the present studyere in agreement with those of other authors, who detected

atechin and epicatechin glucuronide [13,30], catechin and epi-atechin methyl-glucuronide [13]. However, in others studies, theonjugated forms of procyanidins were not identified in plasmaamples like these because previous enzymatic treatments of the

ample using �-glucuronidase and sulphatase were applied beforehe chromatographic analysis [10,11,17,20]. The recovery and quan-ification of these metabolites in biological samples are complexecause of the lack of standards, those justify the enzymatic treat-

able 4rocyanidin metabolites identified in rat plasma sample by off-line SPE-UPLC-MS.

etabolitea Compound RT (min) [M−H]−

(m/z)MS2 ions (m/z)

1 Catechin glucuronide 2.59 465 289, 1752 Epicatechin

glucuronide3.03 465 289, 175

3 Catechinmethyl-glucuronide

3.79 479 303, 289

4 Epicatechinmethyl-glucuronide

4.16 479 303, 289

5 Catechinmethyl-sulphate

4.52 383 303, 289, 245

6 Epicatechinmethyl-sulphate

5.01 383 303, 289, 245

a See Fig. 4.

sulphateb

a Quantified with the calibration curve of catechin.b Quantified with the calibration curve of epicatechin.

ment of the samples to their tentative quantification in referenceto the respective free forms. However the enzymatic treatmentcould be more an additional confirmation in the identification ofthe conjugate forms of procyanidins than an accurate quantifica-tion. The time and temperature conditions during the enzymatictreatment could result in a loss of the free procyanidins, leading tounder-quantification. In this study, the enzymatic treatment wasnot considered previously to quantification, and the procyanidinmetabolites were quantified in catechin and epicatechin equiva-lents by UPLC-MS/MS and two SRM transitions were selected forthis. Table 5 shows the quantification of these metabolites in plasmasamples using the calibration curves of the respective free forms,and the concentrations are expressed as catechin or epicatechinequivalents. The results confirmed an important metabolism of theprocyanidins after the ingestion of the GSPE extract, mainly glu-curonoconjugates of catechin and epicatechin.

4. Conclusions

The present study describes a rapid, simple and sensitivemethod for determining procyanidins and their metabolites inplasma samples. The developed method, off-line SPE-UPLC-MS/MS,allowed procyanidins to be determined at low �M concentrationlevels in 5 min, and the metabolites generated after the diges-tion and intestinal absorption to be identified and tentativelyquantified. The detected metabolites were catechin and epicate-chin glucuronide, methyl-glucuronide, and methyl-sulphate. Themethod developed could thus be used successfully for pharma-cokinetic and bioavailability studies in humans and can serve asan advantageous alternative to those previously reported due to itsspeed, sensitivity, selectivity and low sample amount.

Acknowledgement

This work was supported by the CENIT program from the SpanishMinister of Industry through Shirota Functional Foods, S.L.

References

[1] R.M. Hackman, J.A. Polagruto, Q.Y. Zhu, B. Sun, H. Fujii, C.L. Keen, Phytochem.Rev. 7 (2008) 195.

[2] A.R. Kristal, U. Peters, J.D. Potter, Cancer Epidemiol. Biomarkers Prev. 14 (2005)2826.

[3] H. Ito, M. Gonthier, C. Manach, C. Morand, L. Mennen, C. Remesy, Br. J. Nutr. 94(2005) 500.

[4] J. Linseisen, S. Rohrmann, Eur. J. Nutr. 47 (2008) 60.[5] D.L. Luthria, A.D. Jones, J.L. Donovan, A.L. Waterhouse, J. High Resol. Chromatogr.

20 (1997) 621.[6] M. Richelle, I. Tavazzi, M. Enslen, E.A. Offord, Eur. J. Clin. Nutr. 53 (1999) 22.

1 togr. B

[[

[[

[

[

[

[[

[[

[

[

[

[[[

176 A. Serra et al. / J. Chroma

[7] D. Rein, S. Lotito, R.R. Holt, C.L. Keen, H.H. Schmitz, C.G. Fraga, J. Nutr. 130 (2000)2109S.

[8] R.R. Holt, S.A. Lazarus, M. Cameron Sullards, Q.Y. Zhu, D.D. Schramm, J.F. Ham-merstone, Am. J. Clin. Nutr. 76 (2002) 798.

[9] J.L. Donovan, C. Manach, L. Rios, C. Morand, A. Scalbert, C. Remesy, Br. J. Nutr.87 (2002) 299.

10] A. Kotani, N. Miyashita, F. Kusu, J. Chromatogr. B 788 (2003) 269.11] K.O. Chu, C.C. Wang, M.S. Rogers, K.W. Choy, C.P. Pang, Anal. Chim. Acta 510

(2004) 69.12] A. Bolarinwa, J. Linseisen, J. Chromatogr. B 823 (2005) 143.13] C. Tsang, C. Auger, W. Mullen, A. Bornet, J.-M. Rouanet, A. Crozier, Br. J. Nutr. 94

(2005) 170.14] T. Shoji, S. Masumoto, N. Moriichi, H. Akiyama, T. Kanda, Y. Ohtake, J. Agric. Food

Chem. 54 (2006) 884.15] B. García-Ramírez, J. Fernández-Larrea, M.J. Salvadó, A. Ardèvol, L. Arola, C.

Bladé, J. Agric. Food Chem. 54 (2006) 2543.16] Y. Masukawa, Y. Matsui, N. Shimizu, N. Kondou, H. Endou, M. Kuzukawa, J.

Chromatogr. B 834 (2006) 26.17] D. Gossai, C.A. Lau-Cam, J. Liq. Chromatogr. Relat. Technol. 29 (2006) 2571.18] S. Baba, N. Osakabe, Y. Kato, M. Natsume, A. Yasuda, T. Kido, Am. J. Clin. Nutr. 85

(2007) 709.

[

[

[

877 (2009) 1169–1176

[19] C.H. Grün, F.A. van Dorsten, D.M. Jacobs, M. Le Belleguic, E.J.J. van Velzen, M.O.Bingham, J. Chromatogr. B 871 (2008) 212.

20] D.A. El-Hady, N.A. El-Maali, Talanta 76 (2008) 138.21] N. Ortega, M.P. Romero, A. Macià, J. Reguant, N. Anglès, J.R. Morelló, J. Agric.

Food Chem. 56 (2008) 9621.22] J.F. Hammerstone, S.A. Lazarus, A.E. Mitchell, R. Rucker, H.H. Schmitz, J. Agric.

Food Chem. 47 (1999) 490.23] M.A. Kelm, J.C. Johnson, R.J. Robbins, J.F. Hammerstone, H.H. Schmitz, J. Agric.

Food Chem. 54 (2006) 1571.24] V. Ruiz-Gutierrez, M.E. Juan, A. Cert, J.M. Planas, Anal. Chem. 72 (2000)

4458.25] B.K. Choi, D.M. Hercules, A.I. Gusev, J. Chromatogr. A 907 (2001) 337.26] S. Baba, N. Osakabe, M. Natsume, J. Terao, Free Radic. Biol. Med. 33 (2002) 142.27] A. Sano, J. Yamakoshi, S. Tokutake, K. Tobe, Y. Kubota, M. Kikuchi, Biosci. Biotech-

nol. Biochem. 67 (2003) 1140.

28] P.R. Poudel, H. Tamura, I. Kataoka, R. Mochioka, J. Food Compos. Anal. 21 (2008)

622.29] C. Manach, A. Scalbert, C. Morand, C. Remesy, L. Jimenez, Am. J. Clin. Nutr. 79

(2004) 727.30] F.A. Tomas-Barberan, E. Cienfuegos-Jovellanos, A. Marin, B. Muguerza, A. Gil-

Izquierdo, B. Cerdá, J. Agric. Food Chem. 55 (2007) 3926.