Article pubs.acs.org/JAFC
Effect of the Solvent and the Sample Preparation on the Determination of Triterpene Compounds in Two-Phase Olive-MillWaste Samples Antonia Fernández-Hernández,*,† Antonio Martinez,‡ Francisco Rivas,‡ Jose A. García-Mesa,† and Andres Parra‡ †
Centro “Venta del Llano” del Instituto Andaluz de Investigación y Formación Agraria, Pesquera, Agroalimentaria, y de la Producción Ecológica (IFAPA), Mengíbar, 23620 Jaén, Spain ‡ Departamento de Quimica Organica, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, ES-18071 Granada, Spain ABSTRACT: A simple and rapid extraction method has been employed to determine several value-added compounds, mainly triterpenes, in two-phase olive-mill-waste samples. The compounds were extracted with methanol or ethyl acetate, and the initial fresh samples were treated for classic techniques such as drying, drying and oil extraction, and drying and sifting of the olive stones. For the identification and quantitation of the compounds, an ultra performance liquid chromatography−mass spectrometry method was employed. The best results of the triterpenic compound content were achieved by extraction with methanol from the fresh sample for the oleanolic and ursolic acids, and erythrodiol and uvaol; and from the dried-extracted sample for the maslinic acid. Conversely, the best results for the linoleic acid content were reached by extraction with ethyl acetate from the dried-sifted sample. These are remarkable processes that make the solid wastes from the olive-oil industry reach a high added value. KEYWORDS: two-phase olive-mill-waste alperujo, maslinic acid, oleanolic acid, ursolic acid, triterpene diols, linoleic acid
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INTRODUCTION In Spain, the world’s leading producer of olive oil, the modern system of two-phase centrifugation for extracting olive oil has quickly replaced the three-phase system used in the early 1990s. This olive oil extraction system has produced a new solid waste called two-phase olive-mill-waste (TPOMW) or alperujo, which is generated in large quantities during a short period of time. Approximately six-million tons of TPOMW are produced annually in Spain,1 causing serious management problems due to its phytotoxicity and its semisolid texture.2,3 Different technologies have been proposed for the TPOMW treatment. However, this byproduct is a promising source of potentially useful compounds such as phenolic compounds,4−6 oligosaccharides,7,8 mannitol,7,9,10 and triterpene compounds.11,12 This natural starting material is a significant source of triterpenes that can be used to produce chiral intermediates and may have many applications such as flavorings, fragrances, pharmaceuticals, or biocontrol agents.13 Among them, the main acids are oleanolic (3β-hydroxyolean-12-en-28-oic acid, 1), maslinic (2α,3β-dihydroxyolean-12-en-28-oic acid, 2), and ursolic (3βhydroxyursan-12-en-28-oic acid, 3); meanwhile, the main alcohols are erythrodiol (olean-12-ene-3β,28-diol, 4) and uvaol (urs-12-ene-3β,28-diol, 5) (Figure 1). The studies published on these pentacyclic triterpenes have demonstrated that they display a wide variety of biological effects such as antiinflammatory, anti-HIV, analgesic, antimicrobial, hepatoprotective, and virostatic properties.14−20 The simultaneous determination of triterpene acids using chromatographic and spectroscopic methods has been thoroughly studied over recent years.21−28 Many extraction techniques to recover triterpene acids (Soxhlet, heat reflux, © 2015 American Chemical Society
Figure 1. Chemical structures of the main triterpene compounds found in TPOMW samples.
ultrasonic, and microwave) have been described from diverse plant tissues such as herbs, fruits, olive leaves, and olive-mill wastes.29 Romero et al.30 extracted oleanolic acid and maslinic acid from table olives, using an exhaustive solid−liquid extraction technique with a mixture of methanol:ethanol (1:1), and Guinda et al.31 also suggested ethanol as an appropriate solvent for extraction. In general, the extraction of pentacyclic terpene acids is usually performed with ethyl acetate,32 methanol, ethanol, or diethyl ether.33 The objective of the current study is to compare two different solvents, such as methanol or ethyl acetate, and the different sample-preparation processes, to evaluate their efficacy on the extraction of the triterpene content from TPOMW. On Received: Revised: Accepted: Published: 4269
December March 11, March 14, March 14,
19, 2014 2015 2015 2015 DOI: 10.1021/jf506171y J. Agric. Food Chem. 2015, 63, 4269−4275
Article
Journal of Agricultural and Food Chemistry Table 1. Optimized MRM Conditions for the Analyses of the Value-Added Compounds by UPLC−MS/MS quantitation (MRM1)
confirmation (MRM2)
compound
precursor ion (m/z)
product ion
cone voltage (V)
collision energy (eV)
product ion
cone voltage (V)
collision energy (eV)
oleanolic acid (1) maslinic acid (2) ursolic acid (3) erythrodiol + uvaol (4 + 5) linoleic acid (6) 18β-glycyrrhetinic acid (7)
455.54 471.48 455.49 265.31 279.37 469.47
407.65 393.36 407.45 97.05 261.38 425.45
90 80 85 50 45 90
40 40 40 20 17 40
389.71 405.21 166.47 216.56 58.95 355.37
90 80 85 50 45 90
45 50 45 20 17 50
an aliquot (100 μL in methanol or ethyl acetate) together with 100 μL of a solution of 18β-glycyrrhetinic acid (7, 5 mg/L), as an internal standard, was injected into the liquid chromatograph. UPLC−MS Analyses. The UPLC system consisted of an AcQuity ultra-performance liquid chromatographer equipped with a binary pump system, using an AcQuity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), all from Waters (Milford, MA, USA). The mobile phase consisted of (A) acetonitrile and (B) water, containing 0.1% formic acid, 75:25 (v/v), and was kept isocratic for 10 min with a flow rate of 0.4 mL/min. During the analysis, the column was kept at 30 °C, and the injection volume was 10 μL. The UPLC was coupled to a PDA detector AcQuity UPLC and a TQD mass spectrometer (Waters, Milford, MA, USA). The software used was Masslynx 4.1. The wavelength in the PDA detector was set at 205−215 nm. For ionization, an electrospray (ESI) interface was used, operating in the negative mode, and the data were collected in the selected reaction monitoring (MRM). The ionization source parameters were: capillary voltage 3 kV, source temperature 150 °C, and desolvation gas temperature 350 °C with a flow-rate of 650 L/h. Nitrogen (99.99% purity, N2 LC−MS nitrogen generator ZEFIRO 35, Strumenti Scientifici Cinel, Vigonza, Italy) and argon (>99.99% purity, Air−Liquide, Madrid, Spain) were used as cone and collision gases, respectively. The MRM transitions and the individual cone voltage and collision energy for each triterpene compound were evaluated by infusing 5 μg/mL to establish the best instrumental conditions. Two MRM transitions were studied to find the most abundant product ions, selecting the most sensitive transition for quantitation, and a second one for confirmation purposes. For the positive identification of the analytes in the sample, the chromatographic retention time of each analyte should not vary more than 2% compared to the standard, and the relative abundance of the two MRM transitions monitored had to be within 15% of the ratios calculated for the standards (Table 1). Standards oleanolic, maslinic, ursolic, and linoleic acids, as well as uvaol and erythrodiol, were used to identify peaks. Stock solutions containing 10 μg/mL were prepared by dissolving the standards in methanol and diluted to a series of appropriate concentrations to construct the calibration curve. Triterpene compounds were quantified using a TargetLynx (Waters Inc.) system. Statistical Analysis of Data. All data are presented as the means ± standard deviation from the independent experiments made in triplicates. The data were evaluated by one-way ANOVA with the Statistix 8 program for Windows, and the differences between means were assessed using the Fisher LSD test. Statistical significance was considered at p < 0.05.
the basis of the literature available, this is the first attempt to determine the extraction efficiency of different samples of TPOMW (several types of drying and degreasing procedures, including the olive stones or not) on their triterpene content. This study is expected to provide researchers information on an efficient extraction method of triterpene compounds from TPOMW for industrial application.
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MATERIALS AND METHODS
Materials and Reagents. Oleanolic (1) and linoleic (6) acid standards were purchased from Fluka (Steinheim, Germany) (Figure 1). Maslinic (2) and ursolic (3) acids standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Erythrodiol (4) and uvaol (5) were purchased from Extrasynthese (Genay, France). 18β-Glycyrrhetinic acid (7), also from Extrasynthese, was used as an internal standard. Methanolic-stock standard solutions in the range of 0.05−1.0 μg mL−1 were prepared for each analyte and stored at −20 °C. Standard solutions were analyzed by LC−MS daily. HPLC-grade ethyl acetate and methanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Analytical-grade hexane, from Panreac (Barcelona, Spain), was used for the oil extraction in samples with the Soxhlet method. Deionized water, acetonitrile, and methanol of LC−MS grade, from Waters (Barcelona, Spain), were used for the preparation of chromatographic mobile phases. Formic acid, from Waters (Barcelona, Spain), was used as an additive in the mobile phases to enhance ionization required for MS detection. Sample Preparation. All samples of TPOMW were obtained from virgin olive-oil extraction using an Abencor laboratory oil mill (MC2 Ingenieriá y Sistemas S.L. Sevilla, Spain), which kneaded the olive paste at 28 °C for 30 min. This system provided more homogeneous and representative samples with the control of two key variables, that is, temperature and extraction time, not an easy task in an industrial system. (A) Fresh sample (FS). Original wet sample from the TPOMW. (B) Dried sample (DS). The FS was dried in a forced-air oven at 105 °C until constant weight. (C) Dried-extracted sample (DES). DS was placed in celluloseextraction thimbles and extracted in a Soxhlet apparatus with 200 mL of hexane for 6−8 h. Then the solvent of the sample without oil was evaporated in a forced-air oven at 60 °C for 6 h. This type of oven has been used given the high stability of the compounds studied. (D) Dried-sifted sample (DSS). DS was sifted sequentially with a 0.5 mm and a 0.1 mm pore size to separate the olive stones. Extraction of Triterpene Compounds. The extraction procedure is similar to the method described by Guinda31 and Romero,30 with some modifications. That is, 1 g of each sample (FS, DS, DES, or DSS) was mixed, in a 15 mL centrifuge tube, with 10 mL of methanol or ethyl acetate. The mixture was stirred for 1 h and then centrifuged at 4000 rpm for 6 min. Finally, the extract was separated from the solid phase, and the solvent of this extract was removed under reduced pressure. The residue was dissolved in 2 mL of methanol or ethyl acetate and then was filtered through 0.45-μm pore size. This solution was diluted (1:20, v/v) in acetonitrile/water (75:25, v/v) (mobile phase). Finally, this solution was filtered through 0.2-μm pore size, and
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RESULTS AND DISCUSSION UPLC−MS/MS Method Validation. The development of the UPLC−MS/MS method was validated in terms of precision, accuracy, and linearity. The regression curve of the six compounds was drawn using the external-standard method. All of the correlation coefficient (r > 0.9950) values indicated appropriate correlations between the concentrations of the investigated compounds and their peak areas, within the test ranges. Assay precision was verified by using six independent tests. The accuracy of the assay method was evaluated in 4270
DOI: 10.1021/jf506171y J. Agric. Food Chem. 2015, 63, 4269−4275
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Table 2. Retention Time, Regression Equation (r2), Reproducibility (RSD, %), LOD, and LOQ, for the Analysis of the Triterpenic Compounds by UPLC−MS/MS, in Standard Solutions
a
compound
retention time (min)
oleanolic acid (1) maslinic acid (2) ursolic acid (3) erythrodiol + uvaol (4 + 5) linoleic acid (6) 18β-glycyrrhetinic acid (7)
4.54 1.78 4.28 3.69 6.42 2.10
regression equation y y y y y y
= = = = = =
0.0003x − 1.1388 0.0002x − 3.7261 0.0003x − 1.2226 0.0005x − 5.1192 0.0013x + 1.5205 0.00002x + 0.3189
r2a
RSDb (%)
LODc (mg/kg)
LOQd (mg/kg)
0.9999 0.9966 0.9998 0.9972 0.9999 0.9966
3.87 3.39 3.57 5.36 6.83 7.96
0.35 0.72 0.37 0.36 0.017 0.001
0.97 1.97 1.03 0.98 0.047 0.002
Correlation coefficient. bRSD (%) = (SD/mean) × 100. cLimit of detection (S/N = 3). dLimit of quantitation (S/N = 10).
triplicate using low, middle, and high concentration levels. The limit of detection (LOD) and limit of quantitation (LOQ) were determined by injecting a series of diluted solutions with known concentrations. LOD and LOQ were defined as the signal-to-noise ratio equal to 3 and 10, respectively. These results are shown in Table 2. An internal standard, 18βglycyrrhetinic acid (7), was added to the fresh TPOMW sample before any handling (extraction procedure, drying, and sifting) to gain information about the recovery of the compounds studied. This recovery was 96.3% for methanol and 99.5% for ethyl acetate. Comparison of Extraction with Different Solvents. The selection of the most suitable solvent for extracting the analytes of interest from the sample matrix is not only a fundamental step for the development of an extraction method, but also for the solubility of the analytes in the solvent. Methanol, ethanol, ethyl acetate, and diethyl ether are the most frequently solvents used to extract the triterpene acids from plant material.23,30−33 Ethanol and ethanol/water29,33 are poor extractors for triterpenes, while better results were achieved with methanol and diethyl ether. In this study, the solvents methanol and ethyl acetate, for their polarity, were selected for the extraction of the valueadded compounds from the TPOMW samples (Table 3). These value-added compounds extracted from TPOMW samples (FS, DS, DES, and DSS) were triterpene acids (oleanolic, maslinic, and ursolic) and diols (erythrodiol and uvaol) (Figure 1). Also, the extract showed a large content in linoleic acid, a fatty acid of the olive oil saponifiable fraction. The extraction results (Table 3) showed that methanol was the most efficient solvent in extracting the value-added compounds from the FS, in agreement with several authors who consider methanol the best solvent,23,29 probably because the sample contained water. By contrast, ethyl acetate, the best solvent for Romero et al.,30 proved to be the best extracting solvent with the DSS. With the other samples (DS and DES), the results varied depending on the type of compound analyzed. Thus, ethyl acetate gave the best extraction results with oleanolic and linoleic acids, the less polar compounds, whereas methanol was the best for the more polar products, maslinic acid, and diols (erythrodiol and uvaol). Finally, ursolic acid had the same results of extraction with both solvents. Effects of the Type of TPOMW Sample Preparation on Their Content of Each Compound. In this study, classic techniques such as drying (DS), drying and olive oil extraction with a Soxhlet (DES), and drying and sifting of the olive stones (DSS) were compared with the FS to analyze the influence of the type of sample on the content of each compound extracted from them. The average content in oleanolic acid (1) was highly varied for the diverse TPOMW samples (FS, DS, DES, and DSS;
Table 3. Average Content of Several Value-Added Compounds Isolated from Different Preparations of TwoPhase Olive-Mill-Waste Samplesa solvent extraction compound
sample
methanol
ethyl acetate
FSb oleanolic acid (g/kg) maslinic acid (g/kg) ursolic acid (mg/kg) erythrodiol + uvaol (mg/kg) linoleic acid (mg/kg)
2.03 25.5 287 632 542
± ± ± ± ±
0.25a 1.75a 24a 91a 94a
1.81 23.7 181 466 385
± ± ± ± ±
0.43a 1.2b 53b 61a 31a
0.74 15.6 133 523 403
± ± ± ± ±
0.13a 1.2 a 17a 122a 16a
0.82 11.7 131 400 449
± ± ± ± ±
0.15a 0.8b 14a 52a 22a
1.47 27.9 169 509 323
± ± ± ± ±
0.27b 1.20a 29a 40a 7a
1.89 25.2 169 378 324
± ± ± ± ±
0.15a 0.93b 22a 28b 8a
0.55 23.0 240 317 545
± ± ± ± ±
0.08b 0.64b 27a 28a 63a
0.74 25.4 242 348 626
± ± ± ± ±
0.08a 1.5a 39a 39a 33a
DSc oleanolic acid (g/kg) maslinic acid (g/kg) ursolic acid (mg/kg) erythrodiol + uvaol (mg/kg) linoleic acid (mg/kg) DESd oleanolic acid (g/kg) maslinic acid (g/kg) ursolic acid (mg/kg) erythrodiol + uvaol (mg/kg) linoleic acid (mg/kg) DSSe oleanolic acid (g/kg) maslinic acid (g/kg) ursolic acid (mg/kg) erythrodiol + uvaol (mg/kg) linoleic acid (mg/kg) a
The results are referenced to initial sample weight and expressed as mean ± standard deviation (n = 10). Results in a same row, not sharing a common letter, are significantly different (P < 0.05). bFS (fresh two-phase olive-mill-waste sample). cDS (dried two-phase olivemill-waste sample). dDES (dried and extracted two-phase olive-millwaste sample). eDSS (dried two-phase olive-mill-waste sample without olive stones).
Figure 2). The best results of extraction (Table 3) were achieved using the FS with both solvents (2.03 g kg−1 in methanol, and 1.81 g kg−1 in ethyl acetate) while using the DES with ethyl acetate (1.89 g kg−1). Conversely, with the DS and DSS samples, the extraction of this compound (1) was clearly lower (between 0.82 and 0.55 g kg−1). Maslinic acid (2) is a more abundant compound in these olive-mill wastes (Figure 2). The average content of this triterpenic acid in the extraction of the diverse TPOMW samples was more regular (between 27.9 and 23.0 g kg−1; Table 3), except for the DS, for which the extraction was considerably lower (15.6 g kg−1 in methanol, and 11.7 g kg−1 in ethyl acetate). 4271
DOI: 10.1021/jf506171y J. Agric. Food Chem. 2015, 63, 4269−4275
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Journal of Agricultural and Food Chemistry
Figure 2. Content of triterpene compounds found using methanol (purple) or ethyl acetate (green) as solvent extraction from the different samples of TPOMW. The results are referenced to the sample weight and expressed as mean ± standard deviation (n = 10). Vertical bars with different capital letters (methanol) or small letters (ethyl acetate) indicate significant differences between the sample preparation process according to the Fisher LSD test at a P < 0.05 level of probability.
techniques (DSS) hinders the extraction of the more polar fraction (triterpene diols). The best results of extraction of linoleic acid (6) were achieved from the DSS with both solvents (626 mg kg−1 in ethyl acetate, and 545 mg kg−1 in methanol) and from the FS with methanol (542 mg kg−1) (Figure 2). In this case, the removal of the olive stones from the DS seemed to help the extraction of the lipid fraction (linoleic acid, 6) contrary to what happened with the triterpene diols (4 and 5). In conclusion, the analysis of the average content of the different value-added compounds for all the TPOMW samples (FS, DS, DES, and DSS) indicated that for compounds 1, 3, 4, and 5, the best results were achieved by extraction with methanol from the FS. Furthermore, for compound 2, the best results were reached also with methanol but from the DES. Finally, for linoleic acid (6), the best solvent was ethyl acetate on the DSS.
Ursolic acid (3) was the less abundant triterpene acid in these olive-mill wastes (Figure 2). The best extraction result (Table 3) was reached using the FS with methanol (287 mg kg−1), which was clearly higher than with ethyl acetate (181 mg kg−1). In the other TPOMW samples (DS, DES, and DSS), no differences were found between the two solvents, achieving the best extraction results (around 240 mg kg−1) using the DSS. In summary, the comparison of the average contents of the three triterpene acids (1−3) of the dried sample (DS) with those of the dried sample extracted in a Soxhlet (DES) suggests that the oil presence in the first sample seems to hinder the extraction of these triterpene acids (Table 3). The average content of erythrodiol (4) and uvaol (5) of the diverse TPOMW samples (Figure 2) was higher when the corresponding sample was extracted with methanol (632−509 mg kg−1) than with ethyl acetate (466−378 mg kg−1), except for the DSS sample (Table 3). Therefore, it seems that the elimination of the olive stones from the DS by sifting 4272
DOI: 10.1021/jf506171y J. Agric. Food Chem. 2015, 63, 4269−4275
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Journal of Agricultural and Food Chemistry Study of the Type of TPOMW Sample on the Triterpenic Compound Contents by PCA. When the results of the extraction with methanol of the compounds with higher content (1, 2, 4, and 5) on the TPOMW samples (FS, DS, DES, and DSS) were submitted to “principal component analysis” (PCA), we could see that maslinic acid (2) explained 98% of the data variance (PC1), whereas oleanolic acid (1) and the triterpene diols (4 and 5) accounted for only 1% (PC2). The visualization of the four sample types, as well as their distribution into groups according to principalcomponent scores, is displayed in Figure 3. The groups of
Figure 5. PCA plots for TPOMW samples using oleanolic acid, maslinic acid, and erythrodiol + uvaol content with their ethyl acetate responses. FS (original fresh sample), DS (dried sample), DES (driedextracted sample) and DSS (dried-sifted sample).
Figure 3. PCA plots for TPOMW samples using the oleanolic acid, maslinic acid, and erythrodiol + uvaol content with their methanol responses. FS (original fresh sample), DS (dried sample), DES (driedextracted sample), and DSS (dried-sifted sample).
TPOMW samples established were as follows: Group I (FS), very high content of the triterpene compounds analyzed; Group II (DS), high content of erythrodiol + uvaol and low content of maslinic and oleanolic acids; Group III (DES): very high content of maslinic acids and intermediate content of oleanolic acid and erythrodiol + uvaol; and Group IV (DSS), high content of maslinic acid and low content of oleanolic acid and erythrodiol-uvaol. Loading plot of array is displayed in Figure 4. Maslinic acid presented the highest value for the first principal component
Figure 6. PCA loading plot for TPOMW samples using 1, 2, and 3−4 content with their ethyl acetate responses.
oleanolic acid had the highest one for PC2, while the triterpene diols (erythrodiol and uvaol) showed lower values for both components (PC1 and PC2). The groups of TPOMW samples established were as follows: Group I (FS and DES), very high content of maslinic and oleanolic acids; Group II (DS), high content of erythrodiol + uvaol and low content of maslinic and oleanolic acids; and Group III (DSS), very high content of maslinic acid and low content of oleanolic acid and erythrodiol + uvaol. In summary, we describe and validate a UPLC−MS method for separating and quantitating five value-added compounds in several sample-preparation processes of TPOMW. Moreover, these extraction processes offer greater advantage for future applications due to the simplicity and relatively good extraction efficiency. For an industrial DES TPOMW sample, methanol should be used as the solvent for the highest extraction of maslinic acid, and ethyl acetate for oleanolic acid. For the industrial extraction of the highest content of triterpene diols (erythrodiol and uvaol), methanol should be used with a FS TPOMW sample. For the selective extraction of linoleic acid, industry should start from a DSS TPOMW sample and use ethyl acetate as solvent extraction. In conclusion, the use of these processes for extracting the solid wastes of the olive-oil industry can reduce the environmental impact of these residues, since several bioactive compounds with high value-added can be isolated. These results may contribute to the revaluation of the TPOMW in view of the promising bioactivity properties of the natural triterpene acids.
Figure 4. PCA loading plot for TPOMW samples using compounds 1, 2, and 3−4 content with their methanol responses.
(PC1), and the oleanolic acid had the highest one for PC2, while the triterpene diols (erythrodiol and uvaol) showed lower values for both components (PC1 and PC2). When the extraction solvent was ethyl acetate and these data were submitted to the PCA (Figure 5), we found that maslinic acid (2) explained 99% of the data variance (PC1), while oleanolic acid (1) and triterpene diols (4 and 5) accounted for only 1% (PC2). Loading plot of array is displayed in Figure 6. Maslinic acid presented the highest value for PC1, and 4273
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AUTHOR INFORMATION
Corresponding Author
*Phone: +34671532209. Fax: +34953366380. E-mail: antonia.
[email protected]. Funding
A.F.-H. deeply appreciates the contract grant funded by the Agricultural Research Institute of Spain (INIA) and the European Social Fund. This work was also financially supported by grants from the “Consejeriá de Innovación, Ciencia y ́ (FQM−7372), and the Empresa” of the “Junta de Andalucia” “Plan Propio” of the University of Granada. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank David Nesbitt for reviewing the English in the manuscript. ABBREVIATIONS USED TPOMW, two-phase olive-mill-waste; UPLC−MS, ultraperformance liquid chromatography tandem mass spectrometry; FS, fresh sample; DS, dried sample; DES, dried-extracted sample; DSS, dried-sifted sample; PDA, photodiode array detector; TQD, tandem quadrupole detector; ESI, electrospray ionization; MRM, multiple reaction monitoring; LSD, least significant difference; LOD, limit of detection; LOQ, limit of quantitation; PCA, principal component analysis
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