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Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil
Journal of Chromatography A, 1214 (2008) 90–99
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil Manuel Suárez, Alba Macià, Maria-Paz Romero, Maria-José Motilva ∗ Food Technology Department, CeRTA-TPV, Escuela Técnica Superior de Ingeniería Agraria, Universidad de Lleida, Av/Alcalde Rovira Roure 191, 25198 Lleida, Spain
a r t i c l e
i n f o
Article history: Received 5 November 2007 Received in revised form 13 October 2008 Accepted 15 October 2008 Available online 6 November 2008 Keywords: Phenolic compounds HPLC UPLC MS/MS Olive oil
a b s t r a c t An improved liquid chromatography (LC) tandem mass spectrometry (MS/MS) method has been developed for the determination of phenolic compounds (phenyl alcohols, phenyl acids, secoiridoid derivatives, lignans and flavonoids) in virgin olive oil. The used LC technique was ultra-performance LC with columns packed with 1.7 m particles. The obtained results (retention times, linearity, reproducibility, detection limits (LODs) and quantification limits (LOQs) for the analysis of 14 phenolic compounds in standard solutions were compared with those obtained by high-performance LC (HPLC)–fluorescence and UPLC–diode array detection (DAD). When the 1.7 m column was used, the retention times were decreased three times with respect to conventional HPLC (5 m). The reproducibility of these methods, expressed as relative standard deviation (RSD) in terms of concentration ranged from 0.4–5.0%. In general, the LODs and LOQs were lower in UPLC–MS/MS than the other two methodologies for all the analytes, with the exception of vanillic acid and pinoresinol which values of LODs and LOQs by HPLC–fluorescence were similar to the values obtained by UPLC–MS/MS. Afterwards, the improved UPLC–MS/MS methodology was used to determine the studied compounds in spiked refined olive oil (ROO) by combining a liquid–liquid extraction (LLE) as a sample pre-treatment technique. The recoveries of the analytes were higher than 70%, with the exception of pinoresinol and 3,4-DHPEA-EDA, which were 61% and 67%, respectively. The LODs and the LOQs ranged from 0.44–127.78 g/kg, and from 1.11–427.78 g/kg, respectively for all the analytes. The reproducibility of the method was lower than 3.2%. The LLE–UPLC–MS/MS was successfully applied to analyze phenolic compounds in a virgin olive oil sample within 18 min. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Polyphenols are an important group of natural compounds, which are produced in the secondary metabolism of many plants in nature. The importance of these compounds resides in their antioxidant activity (demonstrated in in vivo and in vitro experiments) [1], their possible effects against degeneration illness, and some pharmaceutical effects, such as anti-carcinogenic, anti-atherogenic and anti-microbial properties [2–4]. The most important phenolic compounds that have been identified in olive oil are phenolic alcohols (such as hydroxytyrosol and tyrosol) secoiridoid derivates (such as the dialdehydic form of elenolic acid linked to tyrosol (p-HPEA-EDA), the aldehydic form of elenolic acid linked to tyrosol (p-HPEA-EA), the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), 4(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC), oleuropein aglycone (3,4-DHPEA-EA) and its methylated form (methyl 3,4-
∗ Corresponding author. Tel.: +34 973 702817; fax: +34 973 702596. E-mail address: [email protected] (M.-J. Motilva). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.098
DHPEA-EA)), phenolic acids and derivates (such as vanillic acid and vanillin respectively), lignans (pinoresinol and acetoxypinoresinol) and flavonoids (including luteolin and apigenin) [5]. The qualitative and quantitative determination of these phenolic compounds in oil samples is very important and several analytical methodologies have been reported. In early years, nonspecific analytical methods, such as thin layer chromatography (TLC) [6] and UV spectroscopy (Folin) [7,8], were applied for the analysis of polyphenols with limited success. Afterwards, these traditional methods were replaced by other more specific ones, such as high performance liquid chromatography (HPLC) [9–17] and gas chromatography (GC) [18,19], and later, capillary electrophoresis (CE) [20–23] given the need to profile and identify the individual phenolic compounds in olive oil samples. The results obtained by GC are very reliable and interesting, but the use of this technique is less common because a derivatization step is necessary. Carrasco-Pancorbo et al. [21–23] developed different methodologies to determine phenolic compounds in olive oil samples by CE using both UV and mass spectrometry (MS) as the detector system. The results were very attractive, with short analysis time and high efficiency peak separation, but the downside of this technique, as
M. Suárez et al. / J. Chromatogr. A 1214 (2008) 90–99
is known, is the low concentration sensitivity. On the other hand, HPLC methods with UV [12–14], fluorescence [15], MS [16,17] and tandem MS (MS/MS) [9,10] detectors have also been reported to be highly efficient. All of them, MS and MS/MS have the greatest potential for the analysis of phenolic compounds in olive oil, since these compounds are found at trace levels in this complex sample matrix. Additionally, apart of its high sensitivity, this detection system has the capability to both determine the molecular weight and provide structural information. Recently, ultra-performance liquid chromatography (UPLC) has been developed as a result of the improvement in the packing materials used for the chromatographic separation. This technique is based in the van Deemter equation which shows that, as the particle sizes decreases to less than 2.5 m, there is a significant gain in efficiency (in this case the particle size is 1.7 m) and it does not diminish at increased flow rates [24]. The principle advantages reported for UPLC are the increase in the signal-to-noise ratio (S/N) (narrower peaks), a reduction in the analysis time and an enhancement in peak resolution [25,26]. As well as the analytical separation technique, olive oil samples have to be pre-treated in order to clean up the sample matrix and preconcentrate the polyphenols, since these are found at trace levels in the oil. The most common technique to accomplish this objective has been to perform either a liquid–liquid extraction (LLE) [7,27,28] or a solid-phase extraction (SPE) [12,29]. Although SPE is an attractive isolation technique in terms of speed, low cost and solvent consumption, Hrncirik et al. [30] concluded that it was problematic because of the selectivity towards the individual phenolic compounds, particularly the aglycone type ones. The aim of this study was to develop a rapid and sensitive method based on UPLC–MS/MS for the determination of phenolic compounds in olive oil. Previously, the obtained results (retention time and the quality parameters) in standard solutions were compared with those obtained for the analysis of 14 phenolic compounds by HPLC–fluorescence and UPLC–DAD. Afterwards, UPLC–MS/MS with an LLE as a sample pretreatment technique was used to determine the studied compounds spiked in refined olive oil (ROO). To our knowledge, for the first time the phenolic compounds present in the olive oil were analysed and quantified by UPLC–MS/MS. In addition, the quality parameters (linearity, reproducibility, LOD, LOQ, and matrix effect) of the developed method were studied for the analysis of these compounds in spiked olive oil. Finally, the developed method (LLE–UPLC–MS/MS) was applied to determine the compounds under study in a virgin olive oil sample. A tentative quantification was also performed for the determination of some secoiridoid derivates (3,4-DHPEA-AC, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EA and ligstroside and oleuropein derivates) in this sample. 2. Experimentation 2.1. Chemicals and reagents Apigenin, apigenin 7-O-glucoside, luteolin, luteolin 7-Oglucoside, oleuropein, hydroxytyrosol, tyrosol, and vanillin were purchased from Extrasynthese (Genay, France). Caffeic and vanillic acids were purchased from Fluka Co. (Buchs, Switzerland) and (+)-pinoresinol was acquired from Arbo Nova (Turku, Finland). The secoiridoid derivatives (3,4-DHPEA-AC, 3,4-DHPEA-EDA, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EDA and p-HPEAEA) and the lignan acetoxypinoresinol were not available commercially and were isolated from virgin olive by semipreparative HPLC [31].
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Standard stock solutions of each compound were prepared in methanol. All the solutions were stored in a dark flask at 4 ◦ C. A standard stock mixture of the phenolic compounds was prepared weekly at a concentration of 50 mg/l. Fig. 1 shows the chemical structure of the studied phenolic compounds. Methanol (HPLC grade), acetonitrile (HPLC grade), n-hexane and acetic acid were all provided by Scharlau Chemie (Barcelona, Spain). Water was of Milli-Q quality (Millipore Corp, Bedford, MA, USA). 2.2. Instrumentation 2.2.1. HPLC HPLC analyses were performed in a Waters system (Milford, USA) consisted of an in-line degasser (with high-purity helium), a pump (Waters 600 E), a Waters 717 Plus autosampler with a 20 l loop injector, a multi lambda fluorescence detector Waters 2475. The column was an Inertsil ODS-3 column (5 m, 15 cm × 4.6 mm) (GL Sciences Inc.) equipped with a Spherisorb S5 ODS-2 precolumn (5 m, 1 cm × 4.6 mm) (Technokroma, Barcelona). The software used to manage the chromatographic separation was Empower (Milford, USA). The mobile phase was MilliQ water/acetic acid (100/0.2, v/v) as eluent A and acetonitrile as eluent B. The elution started at 5% of eluent B for 2 min, then was linearly increased 25% of eluent B in 8 min, further increased to 40% B in 10 min, an additional increased to 50% B in 10 min, and more increased to 100% B in 10 min. Then, it was kept isocratic for 5 min and back to initial conditions for 5 min. The reequilibration time was 5 min. The flow-rate was 1.5 ml/min, the injected volume was 20 l and all the samples were filtered through 0.45 m before the chromatographic analyses. The fluorescence characteristics were 339 nm emission and 278 nm excitation. All the analyses were carried out at room temperature. 2.2.2. UPLC The UPLC system consisted of an AcQuityTM UPLC equipped with a binary pump system Waters (Milford, MA, USA) using an AcQuity UPLCTM BEH C18 column (1.7 m, 100 mm × 2.1 mm i.d.) equipped with a VanGuardTM Pre-Column AcQuity UPLCTM BEH C18 (2.1 mm × 5 mm, 1.7 m) also from Waters. During the analysis, the column was kept at 30 ◦ C and the flow-rate was 0.4 ml/min. The mobile phase was eluent A, MilliQ water/acetic acid (100/0.2, v/v) and eluent B, acetonitrile. The elution started at 5% of eluent B for 5 min, then was linearly increased 40% of eluent B in 20 min, further increased to 100% of eluent B in 0.1 min and kept isocratic for 1.9 min. Then, back to initial conditions in 0.1 min, and the reequilibration time was 1.9 min. The injection volume was 2.5 l, and all the samples were filtered through 0.22 m before the chromatographic analyses. The UPLC was coupled to a PDA detector AcQuity UPLCTM and a TQDTM mass spectrometer (Waters, Milford, MA, USA). The software used was MassLynx 4.1. The wavelengths in the PDA detector were set at 278 and 339 nm. Ionization was achieved using electrospray (ESI) interface operating in the negative mode [M–H]− and the data were collected in the selected reaction monitoring (SRM). The ionization source parameters were capillary voltage 3 kV, source temperature 150 ◦ C and desolvation gas temperature 400 ◦ C, with a flow-rate of 800 l/h. Nitrogen (99.99% purity, N2 LCMS nitrogen generator, Claind, Como, Italy) and argon (≥99.99% purity, Aphagaz, Madrid, Spain) were used as cone and collision gases respectively. The SRM transitions and the individual cone voltage and collision energy for each phenolic compound were evaluated by infusing 10 mg/l of each compound in order to obtain the best instrumental conditions. Two SRM transitions were studied in order to find the most abundant product ions, selecting the most sensitive transition for quantification and a
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Fig. 1. Chemical structures of the studied phenolic compounds.
Table 1 Optimized SRM conditions for the analyses of the studied phenolic compounds by UPLC–MS/MS. Phenolic group
Compound
Precursor ion (m/z)
Quantitation (SRM1 )
Confirmation (SRM2 )
Product ion
Cone voltage (V)
Collision energy (eV)
Product ion
Cone voltage (V)
Collision energy (eV)
Ion ratio
Hydroxytyrosol Tyrosol
153 137
123 106
35 40
10 15
95 119
35 40
25 15
35 1.9
Phenyl acids and derivates
Vanillic acid Caffeic acid Vanillin
167 179 151
123 135 136
30 35 20
10 15 10
152 117 92
30 35 20
15 20 15
1.0 25 6.5
Flavonoids
Luteolin-7-O-Glucoside Apigenin-7-O-Glucoside Luteolin Apigenin
447 431 285 269
285 269 133 123
50 85 55 30
25 20 25 10
256 311 151 152
50 85 55 30
40 25 25 15
23 2.5 1.9 1.6
Secoiridoid
Oleuropein
539
377
35
15
275
35
20
1.0
Secoiridoid derivates
3,4-DHPEA-AC 3,4-DHPEA-EDA Ligstroside derivate p-HPEA-EA p-HPEA-EDA Ligstroside derivate Methyl 3,4-DHPEA-EA Oleuropein derivate 3,4-DHPEA-EA
195 319 335 361 303 393 409 365 377
135 195 199 291 285 317 377 229 275
30 40 40 30 30 40 30 35 35
10 5 10 10 5 15 5 10 10
107 183 155 259 179 257 275 185 307
30 40 40 30 30 40 30 35 35
20 10 15 10 5 15 10 15 10
3.0 1.7 5.0 2.1 1.1 4.1 3.5 2.8 1.1
Lignans
Pinoresinol Acetoxypinoresinol
357 415
151 235
40 45
30 15
136 151
40 45
30 15
1.0 3.0
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Phenyl alcohols
Ion ratio: abundance SRM1 /abundance SRM2.
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second one for confirmation purposes. For the positive identification of the analytes in the sample, the chromatographic retention time of the analyte in the sample would not vary more than 2% compared to that of a standard, and the relative abundance of the two SRM transitions monitored had to be within 15% of the ratios obtained for the standards. The results are shown in Table 1. 2.3. Samples Refined olive oil (ROO), which was obtained from an industrial process, was used as a blank for recovery and quality parameters studies. To test the applicability of the developed method, virgin olive oil samples were analyzed. This oil was obtained from an olive oil mill in Catalonia (Spain) during the harvest season and was from the Arbequina variety of olive fruit. An LLE was used to isolate the phenolic fraction of the oil. The LLE protocol was similar to described by Morelló et al. [31–33] but with some modifications. Briefly, 20 ml of methanol:water (80:20, v/v) were added to 45 g of virgin olive oil and homogenized for 2 min with a Polytron (Lutau, Switzerland). After that, two phases were separated by centrifugation at 637 × g for 10 min and the hydroalcoholic phase was transferred to a balloon. This step was repeated twice and the extracts were combined in the balloon. Then, the hydroalcoholic extracts were rotatory evaporated up to a syrupy consistency at 31 ◦ C and were dissolved in 5 ml of acetonitrile. Afterwards, the extract was washed three times with 10 ml of n-hexane and the rejected n-hexane was treated with 5 ml of acetonitrile. The acetonitrile solution was finally rotatory evaporated to dryness and then re-dissolved in 5 ml of methanol and maintained at −40 ◦ C before the chromatographic analysis. 2.4. Quality parameters The instrumental quality parameters, such as linearity, calibration curve, reproducibility, LODs and LOQs were determined firstly for methanol spiked with known concentrations of polyphenols, and secondly for ROO spiked with known concentrations of phenolic compounds and extracted according to the procedure described in Section 2.3. 3. Results and discussion 3.1. Analysis of phenolic compounds in standard solutions The initial conditions for the analysis of the studied compounds were those reported in our previous studies by HPLC [31–33]. Then, in order to convert the HPLC gradient to UPLC conditions, the AcQuity UPLCTM Columns calculator (Waters, Milford, USA) was used. Then, the gradient was slightly modified in order to improve the separation of the studied compounds. The flow-rate was 0.4 ml/min. In our previous reports, methanol was used as eluent B, but in the present study this organic solvent was modified by acetonitrile because when methanol was used at a flow-rate of 0.4 ml/min, in some percentages of this organic solvent in the mobile phase, the pressure was higher than 15000 psi. The SRM transitions and the corresponding acquisition parameters (cone voltage and collision energy) were selected according to the results obtained from infusing 10 mg/l of a standard solution of each compound into the mobile phase in the negative ESI mode. The selected parameters were those which had a better response in each case and the results are shown in Table 1. As can be seen in this table, two product ions were studied in SRM: one was used to allow the quantification of the phenolic compound and the other
was used as a confirmation ion. This is necessary when complex samples, such as virgin olive oil, are analyzed. The product ions obtained were in agreement with those reported in the literature [17]. The SRM ratio of each compound was used to confirm the identification of the analyte. All the SRM ratio calculated were within 15% of the ratio calculated upon standards. Related with the secoiridoid derivatives it was possible to obtain by semipreparative HPLC enough quantity for all the compounds to optimize SRM conditions (by the infusion of a solution of each individual compound into the mobile phase in the negative ESI mode). However the quantity of 3,4-DHPEA-EDA and p-HPEA-EDA obtained was only enough to study the parameters of the validation assay. The lower concentration of the other secoiridoid derivatives in the phenolic extract of virgin olive oil did not permit to obtain enough quantity of these compounds to carry out the complete validation procedure. In consequence these compounds were tentatively quantified in reference of 3,4-DHPEA-EDA and p-HPEA-EDA as standards. Fig. 2 shows the total ion chromatogram (TIC) obtained for the analysis of 14 selected phenolic compounds (hydroxytyrosol, tyrosol, vanillic acid, caffeic acid, vanillin, luteolin-7-G, apigenin-7G, 3,4-DHPEA-EDA, oleuropein, luteolin, pinoresinol, p-HPEA-EDA, acetoxypinoresinol and apigenin) in the negative mode. The 14 analytes were resolved in within 18 min. The concentration of these compounds was 5 mg/l, except to luteolin 7-O-glucoside, apigenin 7-O-glucoside, luteolin and apigenin which was 1 mg/l, tyrosol which was 2 mg/l and p-HPEA-EDA which was 15 mg/l. As it can been seen in Fig. 2 3,4-DHPE-EDA and p-HPEA-EDA were broad peaks. This fact could be attributed to the presence of isomeric forms of these compounds resulting of the hydrolysis of oleuropein and ligstroside, the main phenols of olive fruit, during the olive oil extraction process. Phenolic molecules are reactive chemical species, vulnerable to hydrolysis, oxidation, conjugation, polymerization and complexation. During crushing and malaxation, steps that break cell walls and expose the olive paste to enzymes, oxygen and mild heat, many chemical and enzymatic processes take place that could explain the presence in the virgin olive oil of the different secoiridoids and their isomers resulting of the hydrolysis of oleuropein and ligstroside. In order to show the purity of 3,4-DHPEA-EDA and p-HPEA-EDA and the other phenols that were isolated by semipreparative HPLC, an analysis in full-scan mode was performed and the MS spectrum at the front, at the middle and at the tail were compared. As example, Fig. 3 shows the MS chromatogram in full-scan mode of 3,4-DHPEA-EDA at the three zones. The figure shows that the MS spectrum were similar at the three zones, obtaining in all the cases the precursor ion m/z 319 and the products ions 195 and 183 which were the quantitation ion and the confirmation ion respectively. Therefore, this fact confirmed the purity of the peak and demonstrated that the width of this peak could be due to the presence of the isomeric forms. Analogous behavior was obtained in the analysis of the other phenolic compounds. The chromatograms obtained with this methodology, UPLC–DAD and UPLC–MS/MS, were compared to that obtained for the analysis of these compounds by HPLC–fluorescence (chromatograms not shown). When the studied compounds were analysed by HPLC–fluorescence, only eight of them (hydroxytyrosol, tyrosol, vanillic acid, oleuropein, 3,4-DHPEA-EDA, pinoresinol, acetoxypinoresinol, and p-HPEA-EDA) were detected, since the use of this detector is limited to a reduced group of phenolic compounds. As it can be seen in Table 2, the retention times in UPLC methodologies were shorter than the retention times in HPLC method, as it was expected. In our study, an average decrease in retention time about three times was observed by using the 1.7 m column.
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Fig. 2. Total ion chromatograms (TIC) in SRM acquisition obtained from the analysis of the 14 phenolic compounds in standard solution. Peak designation and its concentration was: (1) hidroxytyrosol, 5 mg/l; (2) tyrosol, 2 mg/l; (3) vanillic acid, 5 mg/l; (4) caffeic acid, 5 mg/l; (5) vanillin, 5 mg/l; (6) luteolin 7-O-G, 1 mg/l; (7) apigenin 7-O-G, 1 mg/l; (8) 3,4-DHPEA-EDA, 5 mg/l; (9) oleuropein, 5 mg/l; (10) luteolin, 1 mg/l; (11) pinoresinol, 5 mg/l; (12) p-HPEA-EDA, 15 mg/l; (13) acetoxypinoresinol, 5 mg/l; and (14) apigenin, 1 mg/l.
3.2. Quality parameters Standards solutions were analyzed by UPLC–MS/MS in order to determine the linearity range, reproducibility, LODs and LOQs. Then, the obtained results were compared with to those obtained for the analysis of these compounds by UPLC–DAD and HPLC–fluorescence. Table 2 show the quality parameters obtained for the analysis of these 14 compounds by HPLC–fluorescence, UPLC–DAD and UPLC–MS/MS. The linearity range of the analytical procedure was performed by serial dilution of a stock solution of the studied compounds. All the calibrations curves (obtained based on the integrated peak area) were linear over the range of study and were calculated by using seven points at different concentrations. Furthermore, each concentration was injected three times. The determination coefficients (R2 ) were higher than 0.996 for all analytes both for HPLC and UPLC.
The reproducibility, as the relative standard deviation (RSD%), in terms of concentration, was determined at two concentration levels, 50 mg/l and the lowest value of the range of study (data not shown) on three different days. As can be seen in Table 2, good RSDs were achieved in the three methodologies, and these ranged from 0.4 to 4.0% for all the analytes, except to p-HPEA-EDA, which was 5% in HPLC–fluorescence. The LODs and LOQs, calculated using the signal-to-noise ratio criterion of 3 and 10, respectively, were between low g/l and hundreds g/l, respectively for the analysis of these compounds by UPLC–MS/MS. These values were lower than the obtained by HPLC–fluorescence and UPLC–DAD. In contrast, for some compounds such as tyrosol, vanillic acid, and pinoresinol, these values were similar than the obtained by HPLC–fluorescence. By comparing these results with to those reported in the literature, these were similar or lower [15,29].
Fig. 3. MS chromatogram and MS spectrum obtained at the front (A), at the middle (B) and at the tail (C) of the peak 3,4-DHPEA-EDA.
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Fig. 4. Extracted ion chromatograms of the identified phenolic compounds in a virgin olive oil sample by LLE–UPLC–MS/MS.
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Table 2 Retention time, linearity, reproducibility (RSD%), LODs and LOQs for the analysis of the 14 phenolic compounds by HPLC–fluorescence, UPLC–DAD, and UPLC–MS/MS in standard solutions. Compound
Retention time (min)
HPLC–fluorescence Hydroxytyrosol Tyrosol Vanillic acid Oleuropein 3,4-DHPEA-EDA Pinoresinol Acetoxypinoresinol p-HPEA-EDA
8.2 11.1 12.7 16.8 18.9 20.5 20.8 22.8
UPLC–DAD Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-glucoside Apigenin-7-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin UPLC–MS/MS Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-glucoside Apigenin-7-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin a b c d e f g
Linearity (mg/l)
RSD% (n = 3), 50 mg/l
LOD (mg/l)
0.45–50 0.014–50 0.014–50 0.27–30 2.5–50 0.006–50 0.2–18 5–50
1.6 2.1 1.3 1.8a 0.7 1.3 2.2b 5.0
0.21 0.009 0.006 0.14 1.5 0.002 0.1 2.0
2.5 4.05 5.28 5.66 7.52 11.39 12.42 13.25 13.69 15.06 15.65 16.02 16.41 17.21
0.3–50 0.4–50 0.2–50 0.2–50 0.1–50 0.15–50 0.08–36 6–50 0.5–50 0.09–50 0.2–50 7–50 3–50 0.08–50
0.4 0.9 0.5 1.4 0.7 1.3 0.6c 0.5 1.5 2.6 0.4 0.7 0.5 1.4
0.17 0.22 0.07 0.12 0.04 0.08 0.04 3.00 0.20 0.06 0.1 0.7 0.5 0.05
2.5 4.05 5.28 5.66 7.52 11.39 12.42 13.25 13.69 15.06 15.65 16.02 16.41 17.21
0.06–42 0.02–50 0.03–50 0.03–50 0.03–50 0.01–50 0.025–36 0.6–10 0.04–30 0.004–50 0.03–50 0.6–15 0.3–5 0.003–5
1.8d 1.6 0.9 1.1 0.4 0.9 0.9c 1.3e 1.2a 1.4 2.1 3.0f 4.0g 2.1g
0.03 0.01 0.02 0.02 0.008 0.004 0.006 0.4 0.02 0.002 0.01 0.3 0.1 0.001
LOQ (mg/l) 0.69 0.031 0.021 0.47 5.0 0.007 0.4 6.5 0.60 0.70 0.20 0.40 0.12 0.30 0.10 15.00 0.60 0.20 0.50 9.00 5.00 0.10 0.08 0.06 0.06 0.06 0.03 0.01 0.02 1.4 0.06 0.007 0.08 1.0 0.8 0.005
30 mg/l. 18 mg/l. 36 mg/l. 42 mg/l. 10 mg/l 15 mg/l. 5 mg/l.
3.3. Analysis of oil sample The developed method, UPLC–MS/MS, was validated for the analysis of olive oil. In these samples, a pre-treatment is necessary before the analytical separation technique to get rid of matrix components and to enrich the analytes, usually performed by LLE. Firstly, the extraction process was investigated with 22 and 45 g of ROO in order to test differences in recoveries. These extraction recoveries were estimated using this oil spiked with the analytes at a concentration of 5 mg/l. No difference between recoveries was observed when the studied phenolic compounds were extracted in 22 and 45 g of ROO and the recoveries were acceptable. Therefore, we chose 45 g as the optimum amount since higher amounts can give lower LODs. Table 3 shows the extraction recoveries (R%) and these were above 70%, except with pinoresinol and 3,4-DHPEA-EDA, whose recoveries were about 61% and 67%, respectively. These values were in agreement with the literature for the extraction of these compounds both for LLE [30,34] and SPE [29,30].
Then, the method was used to analyze ROO spiked with the analytes at different concentrations in order to determine the linear range, the calibration curves, the RSDs, the LODs and the LOQs. The calibrations curves were linear over the range of study with determination coefficients higher than 0.990 for all the analytes. Reproducibility, expressed as RSD% and in terms of concentration, was obtained by analyzing three replicates of 45 g spiked analytes at 1111 g/kg. The RSDs were lower than 3.2% for all the compounds. The LODs and the LOQs, calculated using a signal-to-noise ratio of 3 and 10 respectively, were from 0.44 to 127, and from 1.11 to 428 g/kg, respectively. Despite the increasing success of LC coupled to an MS detector, matrix effects have limited the ESI applications. Matrix effects result from co-eluting matrix components that compete for ionization capacity. This competition produces significant errors in the accuracy and precision of the analytical method. Matrix effect is observed by a decrease or increase of the analyte signal present in a matrix extract with the same analyte present in organic solvent, respectively [35].
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Table 3 Recovery (R%), linearity, reproducibility (RSD), LOD and LOQ for the analysis of 14phenolic compounds in spiked ROO UPLC–MS/MS. Compound Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-O-glucoside Apigenin-7-O-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin a
R (%) 76 84 87 75 73 84 82 67 89 104 61 71 80 89
Linearity (g/kg)
RSD% (n = 3) (1111 g/kg)
LOD (g/kg)
LOQ (g/kg)
67–5555 6–5555 2.7–1111 3.3–5555 2.7–5555 3.3–2222 6–555 167–1333 2.2–5555 0.7–1111 6–5555 333–3333 33–1222 1.1–222
1.8 1.9 0.3 1.8 0.8 2.3 1.3a 3.0 2.7 2.2 1.8 3.1 3.2 1.3
3.33 7.78 3.33 1.11 2.22 1.11 2.22 72.22 5.56 0.44 1.11 127.78 16.67 0.67
55.52 72.22 35.56 11.11 16.67 11.11 16.67 244.44 55.56 1.11 11.11 427.78 611.11 2.22
555.56 g/kg.
In our study an evaluation of signal suppression was conducted in order to assess its effect on phenolic compounds quantification. This evaluation was done by comparing the detector response of the phenolic compounds spiked in organic solvent (methanol) with those from ROO extracts, at different concentration levels. It was observed either a positive or negative effect, lower than 15% (data not shown). In order to minimize this effect, the extract was diluted 1:2 to prevent it but this dilution produced an increase of LODs, then no dilution of the sample was done because this effect could be considered small. These results are consistent with other studies previously reported in the literature which concluded that MS detection proceeded by an efficient UPLC separation is less susceptible to matrix effects, when this is only produced for the coelution of two different compounds, and may contribute to a reduction in ion suppression [36,37]. 3.4. Application of the developed method to virgin olive oil In order to show the applicability of the developed method, a commercial virgin olive oil was analyzed. Fig. 4 shows the extracted ion chromatograms of the selected 14 compounds included in the validation procedure, and the other secoiridoids (3,4-DHPEA-AC, 3,4-DHPEA-EA, p-HPEA-EA, methyl 3,4-DHPEA-EA), and oleuropein and ligstroside derivates present in the phenolic fraction of virgin olive oil. As it has been previously explained, the broad of the peaks belonging to 3,4-DHPEA-EDA and p-HPEA-EDA could be due to their isomeric forms. Table 4 presents the quantitative results obtained for the analysis of virgin olive oil by LLE-UPLC-MS/MS. Analyte concentrations were quantified by calibration curves for the phenolic compounds spiked in ROO. As it has been commented before, the SRM ratio of each compound was used to confirm the identity of the phenolic compound. All the SRM ratio calculated were within 15% of the ratio calculated upon standards. Some of secoiridoid derivates identified in the virgin olive oil (3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, 3,4-DHPEA-AC, p-HPEAEA, and oleuropein and ligstroside derivates) were tentatively quantified. We attempted to quantify these compounds using 3,4DHPEA-EDA and p-HPEA-EDA as standards in the basis of the similarity in chemical properties of each compound with the standard chosen for its quantification. In this way 3,4-DHPEA-AC, 3,4-DHPEA-EA and methyl 3,4-DHPEA-EA were tentatively quantified with the calibration curve of 3,4-DHPEA-EDA; p-HPEA-EA with the calibration curve of p-HPEA-EDA; and the ligstroside and oleuropein derivatives with the calibration curve of oleuropein. The amount of the phenolic compounds in the olive oil varies from traces to relatively high levels, as it is known, and there-
Table 4 Identified phenolic compounds in virgin olive oil and their quantification. Compound
Concentration (mg/kg olive oil)
Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-O-G Apigenin-7-O-G 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin 3,4-DHPEA-ACa 3,4-DHPEA-EAa Methyl 3,4-DHPEA-EAa p-HPEA-EAb Ligstroside derivatec Oleuropein derivatea
2.5 3.0 0.9 n.d. 0.5 n.q. n.d. 152 n.d. 4.1 2.3 20 0.9 1.5 1.6 68 5.0 42 25 1.4
n.q.: not quantified, n.d.: not detected. a Quantified with the calibration curve of 3,4-DHPEA-EDA. b Quantified with the calibration curve of p-DHPEA-EDA. c Quantified with the calibration curve of oleuropein.
fore virgin olive oil contained low amounts of phenyl acids and phenyl alcohols and high concentrations of secoiridoid derivates. In our study, the most abundant phenolic compounds were 3,4DHPEA-EDA, 3,4-DHPEA-EA, p-HPEA-EA and their concentrations were 152, 68 and 42 mg/kg, respectively, although 3,4-DHPEA-EA and p-HPEA-EA were tentatively quantified. In order to quantify 3,4-DHPEA-EDA, luteolin, pinoresinol, pHPEA-EDA, 3,4-DHPEA-AC, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EA and ligstroside derivate it was necessary to dilute the sample because the concentrations of these compounds were outwith the linear range of the corresponding calibration curve. Furthermore, caffeic acid, apigenin 7-O-glucoside and oleuropein were not detected and luteolin 7-O-glucoside was not quantified because its concentration was between its detection and quantification limits. These results were in agreement with those reported in the literature for the analysis of these compounds in virgin olive oil from the Arbequina cultivar [29,38]. 4. Conclusions This paper develops a rapid, efficient and sensitive method for the determination of phenolic compounds in virgin olive oil
M. Suárez et al. / J. Chromatogr. A 1214 (2008) 90–99
by UPLC–MS/MS. The developed methodology was compared in terms of speed, sensitivity and reproducibility with the results obtained by HPLC–fluorescence and UPLC–DAD in standard solutions. The use of columns packed with 1.7 m particles allowed analysing the 14 phenolic compounds in within 18 min, which represented three-fold reduction in the analysis time in comparison to conventional HPLC. Generally, the LODs and LOQs by UPLC–MS/MS were lower than the obtained by UPLC–DAD and HPLC–fluorescence, but for some analytes these values in UPLC–MS/MS and HPLC–fluorescence were similar. UPLC–MS/MS in combination with a LLE, as a sample pretreatment technique, was developed by spiking the ROO sample with the studied phenolic compounds. The extraction recoveries ranged between 73–104%, except for 3,4-DHPEA-EDA and pinoresinol which were 67% and 61%, respectively. The reproducibility was lower than 3.2% and the LODs and LOQs were between low g/kg and hundred g/kg. Finally, the developed methodology was successfully applied to a broad range of phenolic compounds, such as phenyl acids, phenyl alcohols, lignans, flavonoids and secoiridoid derivates in a commercial virgin olive oil from the Arbequina cultivar. Acknowledgements This work was supported by the Spanish Ministry of Education and Science (project AGL2005-07881-C02-01/ALI) and the grant received by Manuel Suarez (BES-2006-14136). References [1] F. Visioli, C. Galli, Crit. Rev. Food Sci. Nutr. 42 (2002) 209. [2] D. Ryan, M. Antolovich, P. Prenzler, K. Robards, S. Lavee, Sci. Hort. 92 (2002) 147. [3] M. Patumi, R. D’Andria, V. Marsilio, G. Fontanazza, G. Morelli, B. Lanza, Food Chem. 77 (2002) 27. [4] L. Bravo, Nutr. Rev. 56 (1998) 317. [5] L.S. Artajo, M.P. Romero, M. Suárez, M.J. Motilva, Eur. Food Res. Technol. 225 (2007) 617. [6] R. Capasso, A. Evidente, F. Scognamiglio, Phytochem. Anal. 3 (1992) 270. [7] G. Montedoro, M. Servili, M. Baldioli, E. Miniati, J. Agric. Food Chem. 40 (1992) 1571. [8] M.J. Tovar, M.P. Romero, J. Girona, M.J. Motilva, J. Sci. Food Agric. 82 (2002) 892.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil Manuel Suárez, Alba Macià, Maria-Paz Romero, Maria-José Motilva ∗ Food Technology Department, CeRTA-TPV, Escuela Técnica Superior de Ingeniería Agraria, Universidad de Lleida, Av/Alcalde Rovira Roure 191, 25198 Lleida, Spain
a r t i c l e
i n f o
Article history: Received 5 November 2007 Received in revised form 13 October 2008 Accepted 15 October 2008 Available online 6 November 2008 Keywords: Phenolic compounds HPLC UPLC MS/MS Olive oil
a b s t r a c t An improved liquid chromatography (LC) tandem mass spectrometry (MS/MS) method has been developed for the determination of phenolic compounds (phenyl alcohols, phenyl acids, secoiridoid derivatives, lignans and flavonoids) in virgin olive oil. The used LC technique was ultra-performance LC with columns packed with 1.7 m particles. The obtained results (retention times, linearity, reproducibility, detection limits (LODs) and quantification limits (LOQs) for the analysis of 14 phenolic compounds in standard solutions were compared with those obtained by high-performance LC (HPLC)–fluorescence and UPLC–diode array detection (DAD). When the 1.7 m column was used, the retention times were decreased three times with respect to conventional HPLC (5 m). The reproducibility of these methods, expressed as relative standard deviation (RSD) in terms of concentration ranged from 0.4–5.0%. In general, the LODs and LOQs were lower in UPLC–MS/MS than the other two methodologies for all the analytes, with the exception of vanillic acid and pinoresinol which values of LODs and LOQs by HPLC–fluorescence were similar to the values obtained by UPLC–MS/MS. Afterwards, the improved UPLC–MS/MS methodology was used to determine the studied compounds in spiked refined olive oil (ROO) by combining a liquid–liquid extraction (LLE) as a sample pre-treatment technique. The recoveries of the analytes were higher than 70%, with the exception of pinoresinol and 3,4-DHPEA-EDA, which were 61% and 67%, respectively. The LODs and the LOQs ranged from 0.44–127.78 g/kg, and from 1.11–427.78 g/kg, respectively for all the analytes. The reproducibility of the method was lower than 3.2%. The LLE–UPLC–MS/MS was successfully applied to analyze phenolic compounds in a virgin olive oil sample within 18 min. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Polyphenols are an important group of natural compounds, which are produced in the secondary metabolism of many plants in nature. The importance of these compounds resides in their antioxidant activity (demonstrated in in vivo and in vitro experiments) [1], their possible effects against degeneration illness, and some pharmaceutical effects, such as anti-carcinogenic, anti-atherogenic and anti-microbial properties [2–4]. The most important phenolic compounds that have been identified in olive oil are phenolic alcohols (such as hydroxytyrosol and tyrosol) secoiridoid derivates (such as the dialdehydic form of elenolic acid linked to tyrosol (p-HPEA-EDA), the aldehydic form of elenolic acid linked to tyrosol (p-HPEA-EA), the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), 4(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC), oleuropein aglycone (3,4-DHPEA-EA) and its methylated form (methyl 3,4-
∗ Corresponding author. Tel.: +34 973 702817; fax: +34 973 702596. E-mail address: [email protected] (M.-J. Motilva). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.10.098
DHPEA-EA)), phenolic acids and derivates (such as vanillic acid and vanillin respectively), lignans (pinoresinol and acetoxypinoresinol) and flavonoids (including luteolin and apigenin) [5]. The qualitative and quantitative determination of these phenolic compounds in oil samples is very important and several analytical methodologies have been reported. In early years, nonspecific analytical methods, such as thin layer chromatography (TLC) [6] and UV spectroscopy (Folin) [7,8], were applied for the analysis of polyphenols with limited success. Afterwards, these traditional methods were replaced by other more specific ones, such as high performance liquid chromatography (HPLC) [9–17] and gas chromatography (GC) [18,19], and later, capillary electrophoresis (CE) [20–23] given the need to profile and identify the individual phenolic compounds in olive oil samples. The results obtained by GC are very reliable and interesting, but the use of this technique is less common because a derivatization step is necessary. Carrasco-Pancorbo et al. [21–23] developed different methodologies to determine phenolic compounds in olive oil samples by CE using both UV and mass spectrometry (MS) as the detector system. The results were very attractive, with short analysis time and high efficiency peak separation, but the downside of this technique, as
M. Suárez et al. / J. Chromatogr. A 1214 (2008) 90–99
is known, is the low concentration sensitivity. On the other hand, HPLC methods with UV [12–14], fluorescence [15], MS [16,17] and tandem MS (MS/MS) [9,10] detectors have also been reported to be highly efficient. All of them, MS and MS/MS have the greatest potential for the analysis of phenolic compounds in olive oil, since these compounds are found at trace levels in this complex sample matrix. Additionally, apart of its high sensitivity, this detection system has the capability to both determine the molecular weight and provide structural information. Recently, ultra-performance liquid chromatography (UPLC) has been developed as a result of the improvement in the packing materials used for the chromatographic separation. This technique is based in the van Deemter equation which shows that, as the particle sizes decreases to less than 2.5 m, there is a significant gain in efficiency (in this case the particle size is 1.7 m) and it does not diminish at increased flow rates [24]. The principle advantages reported for UPLC are the increase in the signal-to-noise ratio (S/N) (narrower peaks), a reduction in the analysis time and an enhancement in peak resolution [25,26]. As well as the analytical separation technique, olive oil samples have to be pre-treated in order to clean up the sample matrix and preconcentrate the polyphenols, since these are found at trace levels in the oil. The most common technique to accomplish this objective has been to perform either a liquid–liquid extraction (LLE) [7,27,28] or a solid-phase extraction (SPE) [12,29]. Although SPE is an attractive isolation technique in terms of speed, low cost and solvent consumption, Hrncirik et al. [30] concluded that it was problematic because of the selectivity towards the individual phenolic compounds, particularly the aglycone type ones. The aim of this study was to develop a rapid and sensitive method based on UPLC–MS/MS for the determination of phenolic compounds in olive oil. Previously, the obtained results (retention time and the quality parameters) in standard solutions were compared with those obtained for the analysis of 14 phenolic compounds by HPLC–fluorescence and UPLC–DAD. Afterwards, UPLC–MS/MS with an LLE as a sample pretreatment technique was used to determine the studied compounds spiked in refined olive oil (ROO). To our knowledge, for the first time the phenolic compounds present in the olive oil were analysed and quantified by UPLC–MS/MS. In addition, the quality parameters (linearity, reproducibility, LOD, LOQ, and matrix effect) of the developed method were studied for the analysis of these compounds in spiked olive oil. Finally, the developed method (LLE–UPLC–MS/MS) was applied to determine the compounds under study in a virgin olive oil sample. A tentative quantification was also performed for the determination of some secoiridoid derivates (3,4-DHPEA-AC, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EA and ligstroside and oleuropein derivates) in this sample. 2. Experimentation 2.1. Chemicals and reagents Apigenin, apigenin 7-O-glucoside, luteolin, luteolin 7-Oglucoside, oleuropein, hydroxytyrosol, tyrosol, and vanillin were purchased from Extrasynthese (Genay, France). Caffeic and vanillic acids were purchased from Fluka Co. (Buchs, Switzerland) and (+)-pinoresinol was acquired from Arbo Nova (Turku, Finland). The secoiridoid derivatives (3,4-DHPEA-AC, 3,4-DHPEA-EDA, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EDA and p-HPEAEA) and the lignan acetoxypinoresinol were not available commercially and were isolated from virgin olive by semipreparative HPLC [31].
91
Standard stock solutions of each compound were prepared in methanol. All the solutions were stored in a dark flask at 4 ◦ C. A standard stock mixture of the phenolic compounds was prepared weekly at a concentration of 50 mg/l. Fig. 1 shows the chemical structure of the studied phenolic compounds. Methanol (HPLC grade), acetonitrile (HPLC grade), n-hexane and acetic acid were all provided by Scharlau Chemie (Barcelona, Spain). Water was of Milli-Q quality (Millipore Corp, Bedford, MA, USA). 2.2. Instrumentation 2.2.1. HPLC HPLC analyses were performed in a Waters system (Milford, USA) consisted of an in-line degasser (with high-purity helium), a pump (Waters 600 E), a Waters 717 Plus autosampler with a 20 l loop injector, a multi lambda fluorescence detector Waters 2475. The column was an Inertsil ODS-3 column (5 m, 15 cm × 4.6 mm) (GL Sciences Inc.) equipped with a Spherisorb S5 ODS-2 precolumn (5 m, 1 cm × 4.6 mm) (Technokroma, Barcelona). The software used to manage the chromatographic separation was Empower (Milford, USA). The mobile phase was MilliQ water/acetic acid (100/0.2, v/v) as eluent A and acetonitrile as eluent B. The elution started at 5% of eluent B for 2 min, then was linearly increased 25% of eluent B in 8 min, further increased to 40% B in 10 min, an additional increased to 50% B in 10 min, and more increased to 100% B in 10 min. Then, it was kept isocratic for 5 min and back to initial conditions for 5 min. The reequilibration time was 5 min. The flow-rate was 1.5 ml/min, the injected volume was 20 l and all the samples were filtered through 0.45 m before the chromatographic analyses. The fluorescence characteristics were 339 nm emission and 278 nm excitation. All the analyses were carried out at room temperature. 2.2.2. UPLC The UPLC system consisted of an AcQuityTM UPLC equipped with a binary pump system Waters (Milford, MA, USA) using an AcQuity UPLCTM BEH C18 column (1.7 m, 100 mm × 2.1 mm i.d.) equipped with a VanGuardTM Pre-Column AcQuity UPLCTM BEH C18 (2.1 mm × 5 mm, 1.7 m) also from Waters. During the analysis, the column was kept at 30 ◦ C and the flow-rate was 0.4 ml/min. The mobile phase was eluent A, MilliQ water/acetic acid (100/0.2, v/v) and eluent B, acetonitrile. The elution started at 5% of eluent B for 5 min, then was linearly increased 40% of eluent B in 20 min, further increased to 100% of eluent B in 0.1 min and kept isocratic for 1.9 min. Then, back to initial conditions in 0.1 min, and the reequilibration time was 1.9 min. The injection volume was 2.5 l, and all the samples were filtered through 0.22 m before the chromatographic analyses. The UPLC was coupled to a PDA detector AcQuity UPLCTM and a TQDTM mass spectrometer (Waters, Milford, MA, USA). The software used was MassLynx 4.1. The wavelengths in the PDA detector were set at 278 and 339 nm. Ionization was achieved using electrospray (ESI) interface operating in the negative mode [M–H]− and the data were collected in the selected reaction monitoring (SRM). The ionization source parameters were capillary voltage 3 kV, source temperature 150 ◦ C and desolvation gas temperature 400 ◦ C, with a flow-rate of 800 l/h. Nitrogen (99.99% purity, N2 LCMS nitrogen generator, Claind, Como, Italy) and argon (≥99.99% purity, Aphagaz, Madrid, Spain) were used as cone and collision gases respectively. The SRM transitions and the individual cone voltage and collision energy for each phenolic compound were evaluated by infusing 10 mg/l of each compound in order to obtain the best instrumental conditions. Two SRM transitions were studied in order to find the most abundant product ions, selecting the most sensitive transition for quantification and a
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Fig. 1. Chemical structures of the studied phenolic compounds.
Table 1 Optimized SRM conditions for the analyses of the studied phenolic compounds by UPLC–MS/MS. Phenolic group
Compound
Precursor ion (m/z)
Quantitation (SRM1 )
Confirmation (SRM2 )
Product ion
Cone voltage (V)
Collision energy (eV)
Product ion
Cone voltage (V)
Collision energy (eV)
Ion ratio
Hydroxytyrosol Tyrosol
153 137
123 106
35 40
10 15
95 119
35 40
25 15
35 1.9
Phenyl acids and derivates
Vanillic acid Caffeic acid Vanillin
167 179 151
123 135 136
30 35 20
10 15 10
152 117 92
30 35 20
15 20 15
1.0 25 6.5
Flavonoids
Luteolin-7-O-Glucoside Apigenin-7-O-Glucoside Luteolin Apigenin
447 431 285 269
285 269 133 123
50 85 55 30
25 20 25 10
256 311 151 152
50 85 55 30
40 25 25 15
23 2.5 1.9 1.6
Secoiridoid
Oleuropein
539
377
35
15
275
35
20
1.0
Secoiridoid derivates
3,4-DHPEA-AC 3,4-DHPEA-EDA Ligstroside derivate p-HPEA-EA p-HPEA-EDA Ligstroside derivate Methyl 3,4-DHPEA-EA Oleuropein derivate 3,4-DHPEA-EA
195 319 335 361 303 393 409 365 377
135 195 199 291 285 317 377 229 275
30 40 40 30 30 40 30 35 35
10 5 10 10 5 15 5 10 10
107 183 155 259 179 257 275 185 307
30 40 40 30 30 40 30 35 35
20 10 15 10 5 15 10 15 10
3.0 1.7 5.0 2.1 1.1 4.1 3.5 2.8 1.1
Lignans
Pinoresinol Acetoxypinoresinol
357 415
151 235
40 45
30 15
136 151
40 45
30 15
1.0 3.0
M. Suárez et al. / J. Chromatogr. A 1214 (2008) 90–99
Phenyl alcohols
Ion ratio: abundance SRM1 /abundance SRM2.
93
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second one for confirmation purposes. For the positive identification of the analytes in the sample, the chromatographic retention time of the analyte in the sample would not vary more than 2% compared to that of a standard, and the relative abundance of the two SRM transitions monitored had to be within 15% of the ratios obtained for the standards. The results are shown in Table 1. 2.3. Samples Refined olive oil (ROO), which was obtained from an industrial process, was used as a blank for recovery and quality parameters studies. To test the applicability of the developed method, virgin olive oil samples were analyzed. This oil was obtained from an olive oil mill in Catalonia (Spain) during the harvest season and was from the Arbequina variety of olive fruit. An LLE was used to isolate the phenolic fraction of the oil. The LLE protocol was similar to described by Morelló et al. [31–33] but with some modifications. Briefly, 20 ml of methanol:water (80:20, v/v) were added to 45 g of virgin olive oil and homogenized for 2 min with a Polytron (Lutau, Switzerland). After that, two phases were separated by centrifugation at 637 × g for 10 min and the hydroalcoholic phase was transferred to a balloon. This step was repeated twice and the extracts were combined in the balloon. Then, the hydroalcoholic extracts were rotatory evaporated up to a syrupy consistency at 31 ◦ C and were dissolved in 5 ml of acetonitrile. Afterwards, the extract was washed three times with 10 ml of n-hexane and the rejected n-hexane was treated with 5 ml of acetonitrile. The acetonitrile solution was finally rotatory evaporated to dryness and then re-dissolved in 5 ml of methanol and maintained at −40 ◦ C before the chromatographic analysis. 2.4. Quality parameters The instrumental quality parameters, such as linearity, calibration curve, reproducibility, LODs and LOQs were determined firstly for methanol spiked with known concentrations of polyphenols, and secondly for ROO spiked with known concentrations of phenolic compounds and extracted according to the procedure described in Section 2.3. 3. Results and discussion 3.1. Analysis of phenolic compounds in standard solutions The initial conditions for the analysis of the studied compounds were those reported in our previous studies by HPLC [31–33]. Then, in order to convert the HPLC gradient to UPLC conditions, the AcQuity UPLCTM Columns calculator (Waters, Milford, USA) was used. Then, the gradient was slightly modified in order to improve the separation of the studied compounds. The flow-rate was 0.4 ml/min. In our previous reports, methanol was used as eluent B, but in the present study this organic solvent was modified by acetonitrile because when methanol was used at a flow-rate of 0.4 ml/min, in some percentages of this organic solvent in the mobile phase, the pressure was higher than 15000 psi. The SRM transitions and the corresponding acquisition parameters (cone voltage and collision energy) were selected according to the results obtained from infusing 10 mg/l of a standard solution of each compound into the mobile phase in the negative ESI mode. The selected parameters were those which had a better response in each case and the results are shown in Table 1. As can be seen in this table, two product ions were studied in SRM: one was used to allow the quantification of the phenolic compound and the other
was used as a confirmation ion. This is necessary when complex samples, such as virgin olive oil, are analyzed. The product ions obtained were in agreement with those reported in the literature [17]. The SRM ratio of each compound was used to confirm the identification of the analyte. All the SRM ratio calculated were within 15% of the ratio calculated upon standards. Related with the secoiridoid derivatives it was possible to obtain by semipreparative HPLC enough quantity for all the compounds to optimize SRM conditions (by the infusion of a solution of each individual compound into the mobile phase in the negative ESI mode). However the quantity of 3,4-DHPEA-EDA and p-HPEA-EDA obtained was only enough to study the parameters of the validation assay. The lower concentration of the other secoiridoid derivatives in the phenolic extract of virgin olive oil did not permit to obtain enough quantity of these compounds to carry out the complete validation procedure. In consequence these compounds were tentatively quantified in reference of 3,4-DHPEA-EDA and p-HPEA-EDA as standards. Fig. 2 shows the total ion chromatogram (TIC) obtained for the analysis of 14 selected phenolic compounds (hydroxytyrosol, tyrosol, vanillic acid, caffeic acid, vanillin, luteolin-7-G, apigenin-7G, 3,4-DHPEA-EDA, oleuropein, luteolin, pinoresinol, p-HPEA-EDA, acetoxypinoresinol and apigenin) in the negative mode. The 14 analytes were resolved in within 18 min. The concentration of these compounds was 5 mg/l, except to luteolin 7-O-glucoside, apigenin 7-O-glucoside, luteolin and apigenin which was 1 mg/l, tyrosol which was 2 mg/l and p-HPEA-EDA which was 15 mg/l. As it can been seen in Fig. 2 3,4-DHPE-EDA and p-HPEA-EDA were broad peaks. This fact could be attributed to the presence of isomeric forms of these compounds resulting of the hydrolysis of oleuropein and ligstroside, the main phenols of olive fruit, during the olive oil extraction process. Phenolic molecules are reactive chemical species, vulnerable to hydrolysis, oxidation, conjugation, polymerization and complexation. During crushing and malaxation, steps that break cell walls and expose the olive paste to enzymes, oxygen and mild heat, many chemical and enzymatic processes take place that could explain the presence in the virgin olive oil of the different secoiridoids and their isomers resulting of the hydrolysis of oleuropein and ligstroside. In order to show the purity of 3,4-DHPEA-EDA and p-HPEA-EDA and the other phenols that were isolated by semipreparative HPLC, an analysis in full-scan mode was performed and the MS spectrum at the front, at the middle and at the tail were compared. As example, Fig. 3 shows the MS chromatogram in full-scan mode of 3,4-DHPEA-EDA at the three zones. The figure shows that the MS spectrum were similar at the three zones, obtaining in all the cases the precursor ion m/z 319 and the products ions 195 and 183 which were the quantitation ion and the confirmation ion respectively. Therefore, this fact confirmed the purity of the peak and demonstrated that the width of this peak could be due to the presence of the isomeric forms. Analogous behavior was obtained in the analysis of the other phenolic compounds. The chromatograms obtained with this methodology, UPLC–DAD and UPLC–MS/MS, were compared to that obtained for the analysis of these compounds by HPLC–fluorescence (chromatograms not shown). When the studied compounds were analysed by HPLC–fluorescence, only eight of them (hydroxytyrosol, tyrosol, vanillic acid, oleuropein, 3,4-DHPEA-EDA, pinoresinol, acetoxypinoresinol, and p-HPEA-EDA) were detected, since the use of this detector is limited to a reduced group of phenolic compounds. As it can be seen in Table 2, the retention times in UPLC methodologies were shorter than the retention times in HPLC method, as it was expected. In our study, an average decrease in retention time about three times was observed by using the 1.7 m column.
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Fig. 2. Total ion chromatograms (TIC) in SRM acquisition obtained from the analysis of the 14 phenolic compounds in standard solution. Peak designation and its concentration was: (1) hidroxytyrosol, 5 mg/l; (2) tyrosol, 2 mg/l; (3) vanillic acid, 5 mg/l; (4) caffeic acid, 5 mg/l; (5) vanillin, 5 mg/l; (6) luteolin 7-O-G, 1 mg/l; (7) apigenin 7-O-G, 1 mg/l; (8) 3,4-DHPEA-EDA, 5 mg/l; (9) oleuropein, 5 mg/l; (10) luteolin, 1 mg/l; (11) pinoresinol, 5 mg/l; (12) p-HPEA-EDA, 15 mg/l; (13) acetoxypinoresinol, 5 mg/l; and (14) apigenin, 1 mg/l.
3.2. Quality parameters Standards solutions were analyzed by UPLC–MS/MS in order to determine the linearity range, reproducibility, LODs and LOQs. Then, the obtained results were compared with to those obtained for the analysis of these compounds by UPLC–DAD and HPLC–fluorescence. Table 2 show the quality parameters obtained for the analysis of these 14 compounds by HPLC–fluorescence, UPLC–DAD and UPLC–MS/MS. The linearity range of the analytical procedure was performed by serial dilution of a stock solution of the studied compounds. All the calibrations curves (obtained based on the integrated peak area) were linear over the range of study and were calculated by using seven points at different concentrations. Furthermore, each concentration was injected three times. The determination coefficients (R2 ) were higher than 0.996 for all analytes both for HPLC and UPLC.
The reproducibility, as the relative standard deviation (RSD%), in terms of concentration, was determined at two concentration levels, 50 mg/l and the lowest value of the range of study (data not shown) on three different days. As can be seen in Table 2, good RSDs were achieved in the three methodologies, and these ranged from 0.4 to 4.0% for all the analytes, except to p-HPEA-EDA, which was 5% in HPLC–fluorescence. The LODs and LOQs, calculated using the signal-to-noise ratio criterion of 3 and 10, respectively, were between low g/l and hundreds g/l, respectively for the analysis of these compounds by UPLC–MS/MS. These values were lower than the obtained by HPLC–fluorescence and UPLC–DAD. In contrast, for some compounds such as tyrosol, vanillic acid, and pinoresinol, these values were similar than the obtained by HPLC–fluorescence. By comparing these results with to those reported in the literature, these were similar or lower [15,29].
Fig. 3. MS chromatogram and MS spectrum obtained at the front (A), at the middle (B) and at the tail (C) of the peak 3,4-DHPEA-EDA.
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Fig. 4. Extracted ion chromatograms of the identified phenolic compounds in a virgin olive oil sample by LLE–UPLC–MS/MS.
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Table 2 Retention time, linearity, reproducibility (RSD%), LODs and LOQs for the analysis of the 14 phenolic compounds by HPLC–fluorescence, UPLC–DAD, and UPLC–MS/MS in standard solutions. Compound
Retention time (min)
HPLC–fluorescence Hydroxytyrosol Tyrosol Vanillic acid Oleuropein 3,4-DHPEA-EDA Pinoresinol Acetoxypinoresinol p-HPEA-EDA
8.2 11.1 12.7 16.8 18.9 20.5 20.8 22.8
UPLC–DAD Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-glucoside Apigenin-7-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin UPLC–MS/MS Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-glucoside Apigenin-7-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin a b c d e f g
Linearity (mg/l)
RSD% (n = 3), 50 mg/l
LOD (mg/l)
0.45–50 0.014–50 0.014–50 0.27–30 2.5–50 0.006–50 0.2–18 5–50
1.6 2.1 1.3 1.8a 0.7 1.3 2.2b 5.0
0.21 0.009 0.006 0.14 1.5 0.002 0.1 2.0
2.5 4.05 5.28 5.66 7.52 11.39 12.42 13.25 13.69 15.06 15.65 16.02 16.41 17.21
0.3–50 0.4–50 0.2–50 0.2–50 0.1–50 0.15–50 0.08–36 6–50 0.5–50 0.09–50 0.2–50 7–50 3–50 0.08–50
0.4 0.9 0.5 1.4 0.7 1.3 0.6c 0.5 1.5 2.6 0.4 0.7 0.5 1.4
0.17 0.22 0.07 0.12 0.04 0.08 0.04 3.00 0.20 0.06 0.1 0.7 0.5 0.05
2.5 4.05 5.28 5.66 7.52 11.39 12.42 13.25 13.69 15.06 15.65 16.02 16.41 17.21
0.06–42 0.02–50 0.03–50 0.03–50 0.03–50 0.01–50 0.025–36 0.6–10 0.04–30 0.004–50 0.03–50 0.6–15 0.3–5 0.003–5
1.8d 1.6 0.9 1.1 0.4 0.9 0.9c 1.3e 1.2a 1.4 2.1 3.0f 4.0g 2.1g
0.03 0.01 0.02 0.02 0.008 0.004 0.006 0.4 0.02 0.002 0.01 0.3 0.1 0.001
LOQ (mg/l) 0.69 0.031 0.021 0.47 5.0 0.007 0.4 6.5 0.60 0.70 0.20 0.40 0.12 0.30 0.10 15.00 0.60 0.20 0.50 9.00 5.00 0.10 0.08 0.06 0.06 0.06 0.03 0.01 0.02 1.4 0.06 0.007 0.08 1.0 0.8 0.005
30 mg/l. 18 mg/l. 36 mg/l. 42 mg/l. 10 mg/l 15 mg/l. 5 mg/l.
3.3. Analysis of oil sample The developed method, UPLC–MS/MS, was validated for the analysis of olive oil. In these samples, a pre-treatment is necessary before the analytical separation technique to get rid of matrix components and to enrich the analytes, usually performed by LLE. Firstly, the extraction process was investigated with 22 and 45 g of ROO in order to test differences in recoveries. These extraction recoveries were estimated using this oil spiked with the analytes at a concentration of 5 mg/l. No difference between recoveries was observed when the studied phenolic compounds were extracted in 22 and 45 g of ROO and the recoveries were acceptable. Therefore, we chose 45 g as the optimum amount since higher amounts can give lower LODs. Table 3 shows the extraction recoveries (R%) and these were above 70%, except with pinoresinol and 3,4-DHPEA-EDA, whose recoveries were about 61% and 67%, respectively. These values were in agreement with the literature for the extraction of these compounds both for LLE [30,34] and SPE [29,30].
Then, the method was used to analyze ROO spiked with the analytes at different concentrations in order to determine the linear range, the calibration curves, the RSDs, the LODs and the LOQs. The calibrations curves were linear over the range of study with determination coefficients higher than 0.990 for all the analytes. Reproducibility, expressed as RSD% and in terms of concentration, was obtained by analyzing three replicates of 45 g spiked analytes at 1111 g/kg. The RSDs were lower than 3.2% for all the compounds. The LODs and the LOQs, calculated using a signal-to-noise ratio of 3 and 10 respectively, were from 0.44 to 127, and from 1.11 to 428 g/kg, respectively. Despite the increasing success of LC coupled to an MS detector, matrix effects have limited the ESI applications. Matrix effects result from co-eluting matrix components that compete for ionization capacity. This competition produces significant errors in the accuracy and precision of the analytical method. Matrix effect is observed by a decrease or increase of the analyte signal present in a matrix extract with the same analyte present in organic solvent, respectively [35].
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Table 3 Recovery (R%), linearity, reproducibility (RSD), LOD and LOQ for the analysis of 14phenolic compounds in spiked ROO UPLC–MS/MS. Compound Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-O-glucoside Apigenin-7-O-glucoside 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin a
R (%) 76 84 87 75 73 84 82 67 89 104 61 71 80 89
Linearity (g/kg)
RSD% (n = 3) (1111 g/kg)
LOD (g/kg)
LOQ (g/kg)
67–5555 6–5555 2.7–1111 3.3–5555 2.7–5555 3.3–2222 6–555 167–1333 2.2–5555 0.7–1111 6–5555 333–3333 33–1222 1.1–222
1.8 1.9 0.3 1.8 0.8 2.3 1.3a 3.0 2.7 2.2 1.8 3.1 3.2 1.3
3.33 7.78 3.33 1.11 2.22 1.11 2.22 72.22 5.56 0.44 1.11 127.78 16.67 0.67
55.52 72.22 35.56 11.11 16.67 11.11 16.67 244.44 55.56 1.11 11.11 427.78 611.11 2.22
555.56 g/kg.
In our study an evaluation of signal suppression was conducted in order to assess its effect on phenolic compounds quantification. This evaluation was done by comparing the detector response of the phenolic compounds spiked in organic solvent (methanol) with those from ROO extracts, at different concentration levels. It was observed either a positive or negative effect, lower than 15% (data not shown). In order to minimize this effect, the extract was diluted 1:2 to prevent it but this dilution produced an increase of LODs, then no dilution of the sample was done because this effect could be considered small. These results are consistent with other studies previously reported in the literature which concluded that MS detection proceeded by an efficient UPLC separation is less susceptible to matrix effects, when this is only produced for the coelution of two different compounds, and may contribute to a reduction in ion suppression [36,37]. 3.4. Application of the developed method to virgin olive oil In order to show the applicability of the developed method, a commercial virgin olive oil was analyzed. Fig. 4 shows the extracted ion chromatograms of the selected 14 compounds included in the validation procedure, and the other secoiridoids (3,4-DHPEA-AC, 3,4-DHPEA-EA, p-HPEA-EA, methyl 3,4-DHPEA-EA), and oleuropein and ligstroside derivates present in the phenolic fraction of virgin olive oil. As it has been previously explained, the broad of the peaks belonging to 3,4-DHPEA-EDA and p-HPEA-EDA could be due to their isomeric forms. Table 4 presents the quantitative results obtained for the analysis of virgin olive oil by LLE-UPLC-MS/MS. Analyte concentrations were quantified by calibration curves for the phenolic compounds spiked in ROO. As it has been commented before, the SRM ratio of each compound was used to confirm the identity of the phenolic compound. All the SRM ratio calculated were within 15% of the ratio calculated upon standards. Some of secoiridoid derivates identified in the virgin olive oil (3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, 3,4-DHPEA-AC, p-HPEAEA, and oleuropein and ligstroside derivates) were tentatively quantified. We attempted to quantify these compounds using 3,4DHPEA-EDA and p-HPEA-EDA as standards in the basis of the similarity in chemical properties of each compound with the standard chosen for its quantification. In this way 3,4-DHPEA-AC, 3,4-DHPEA-EA and methyl 3,4-DHPEA-EA were tentatively quantified with the calibration curve of 3,4-DHPEA-EDA; p-HPEA-EA with the calibration curve of p-HPEA-EDA; and the ligstroside and oleuropein derivatives with the calibration curve of oleuropein. The amount of the phenolic compounds in the olive oil varies from traces to relatively high levels, as it is known, and there-
Table 4 Identified phenolic compounds in virgin olive oil and their quantification. Compound
Concentration (mg/kg olive oil)
Hydroxytyrosol Tyrosol Vanillic acid Caffeic acid Vanillin Luteolin-7-O-G Apigenin-7-O-G 3,4-DHPEA-EDA Oleuropein Luteolin Pinoresinol p-HPEA-EDA Acetoxypinoresinol Apigenin 3,4-DHPEA-ACa 3,4-DHPEA-EAa Methyl 3,4-DHPEA-EAa p-HPEA-EAb Ligstroside derivatec Oleuropein derivatea
2.5 3.0 0.9 n.d. 0.5 n.q. n.d. 152 n.d. 4.1 2.3 20 0.9 1.5 1.6 68 5.0 42 25 1.4
n.q.: not quantified, n.d.: not detected. a Quantified with the calibration curve of 3,4-DHPEA-EDA. b Quantified with the calibration curve of p-DHPEA-EDA. c Quantified with the calibration curve of oleuropein.
fore virgin olive oil contained low amounts of phenyl acids and phenyl alcohols and high concentrations of secoiridoid derivates. In our study, the most abundant phenolic compounds were 3,4DHPEA-EDA, 3,4-DHPEA-EA, p-HPEA-EA and their concentrations were 152, 68 and 42 mg/kg, respectively, although 3,4-DHPEA-EA and p-HPEA-EA were tentatively quantified. In order to quantify 3,4-DHPEA-EDA, luteolin, pinoresinol, pHPEA-EDA, 3,4-DHPEA-AC, 3,4-DHPEA-EA, methyl 3,4-DHPEA-EA, p-HPEA-EA and ligstroside derivate it was necessary to dilute the sample because the concentrations of these compounds were outwith the linear range of the corresponding calibration curve. Furthermore, caffeic acid, apigenin 7-O-glucoside and oleuropein were not detected and luteolin 7-O-glucoside was not quantified because its concentration was between its detection and quantification limits. These results were in agreement with those reported in the literature for the analysis of these compounds in virgin olive oil from the Arbequina cultivar [29,38]. 4. Conclusions This paper develops a rapid, efficient and sensitive method for the determination of phenolic compounds in virgin olive oil
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by UPLC–MS/MS. The developed methodology was compared in terms of speed, sensitivity and reproducibility with the results obtained by HPLC–fluorescence and UPLC–DAD in standard solutions. The use of columns packed with 1.7 m particles allowed analysing the 14 phenolic compounds in within 18 min, which represented three-fold reduction in the analysis time in comparison to conventional HPLC. Generally, the LODs and LOQs by UPLC–MS/MS were lower than the obtained by UPLC–DAD and HPLC–fluorescence, but for some analytes these values in UPLC–MS/MS and HPLC–fluorescence were similar. UPLC–MS/MS in combination with a LLE, as a sample pretreatment technique, was developed by spiking the ROO sample with the studied phenolic compounds. The extraction recoveries ranged between 73–104%, except for 3,4-DHPEA-EDA and pinoresinol which were 67% and 61%, respectively. The reproducibility was lower than 3.2% and the LODs and LOQs were between low g/kg and hundred g/kg. Finally, the developed methodology was successfully applied to a broad range of phenolic compounds, such as phenyl acids, phenyl alcohols, lignans, flavonoids and secoiridoid derivates in a commercial virgin olive oil from the Arbequina cultivar. Acknowledgements This work was supported by the Spanish Ministry of Education and Science (project AGL2005-07881-C02-01/ALI) and the grant received by Manuel Suarez (BES-2006-14136). References [1] F. Visioli, C. Galli, Crit. Rev. Food Sci. Nutr. 42 (2002) 209. [2] D. Ryan, M. Antolovich, P. Prenzler, K. Robards, S. Lavee, Sci. Hort. 92 (2002) 147. [3] M. Patumi, R. D’Andria, V. Marsilio, G. Fontanazza, G. Morelli, B. Lanza, Food Chem. 77 (2002) 27. [4] L. Bravo, Nutr. Rev. 56 (1998) 317. [5] L.S. Artajo, M.P. Romero, M. Suárez, M.J. Motilva, Eur. Food Res. Technol. 225 (2007) 617. [6] R. Capasso, A. Evidente, F. Scognamiglio, Phytochem. Anal. 3 (1992) 270. [7] G. Montedoro, M. Servili, M. Baldioli, E. Miniati, J. Agric. Food Chem. 40 (1992) 1571. [8] M.J. Tovar, M.P. Romero, J. Girona, M.J. Motilva, J. Sci. Food Agric. 82 (2002) 892.
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