Rapid determination of the free and total hydroxytyrosol and tyrosol content in extra virgin olive oil by stable isotope dilution analysis and paper spray tandem mass spectrometry

Journal Pre-proof Rapid determination of the free and total hydroxytyrosol and tyrosol content in extra virgin olive oil by stable isotope dilution analysis and paper spray tandem mass spectrometry Lucia Bartella, Fabio Mazzotti, Giovanni Sindona, Anna Napoli, Leonardo Di Donna PII:

S0278-6915(19)30900-7

DOI:

https://doi.org/10.1016/j.fct.2019.111110

Reference:

FCT 111110

To appear in:

Food and Chemical Toxicology

Received Date: 26 September 2019 Revised Date:

27 December 2019

Accepted Date: 29 December 2019

Please cite this article as: Bartella, L., Mazzotti, F., Sindona, G., Napoli, A., Di Donna, L., Rapid determination of the free and total hydroxytyrosol and tyrosol content in extra virgin olive oil by stable isotope dilution analysis and paper spray tandem mass spectrometry, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2019.111110. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Credit author statement Lucia Bartella: Visualization, Validation, Investigation, Writing - Review & Editing. Fabio Mazzotti: Formal analysis, Investigation. Anna Napoli: Investigation. Giovanni Sindona: Data Curation. Leonardo Di Donna: Conceptualization, Methodology, Resources, Writing - Original Draft, Writing - Review & Editing, Supervision, Funding acquisition.

Rapid determination of the free and total hydroxytyrosol and tyrosol content in extra virgin olive oil by stable isotope dilution analysis and paper spray tandem mass spectrometry Lucia Bartella, Fabio Mazzotti, Giovanni Sindona, Anna Napoli and Leonardo Di Donna* Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, via P. Bucci, cubo 12/D, I-87030 Rende (CS) – Italy [email protected] [email protected] [email protected] [email protected] *

Correspondence to:

Leonardo Di Donna, Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, via P. Bucci, cubo 12/D, I-87030 Rende (CS) - Italy; phone: +39-0984-492857; E-mail address: [email protected];

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Abstract A rapid analytical method for the determination of phenylethanoids content in extra virgin olive oil has been developed. The method intends to address the European regulation EU 432/2012 on health claims, which allows to report on the front label of olive oil, the positive health effects due to the consumption of this food. The innovative method is based on paper spray tandem mass spectrometry using deuterated standards. It relies on a two-step analysis, needed to assess the free form of tyrosol and hydroxytyrosol and their ester conjugates after hydrolysis treatment. Different olive oil samples have been analyzed and the classical analytical parameters such as accuracy, LOQ and LOD were calculated from fortified samples. The good values of the latters show the reliability of the new approach, that limits the time of analysis and sample preparation to few minutes. Keywords Olive oil; stable isotope dilution analysis; Hydroxytyrosol; Tyrosol; Paper Spray Mass Spectrometry 1. Introduction Olive oil is a natural oil obtained by pressing the whole fruit of olive trees: it is used in cooking as food dressing or in frying. There are different categories of olive oil, depending on its quality level: the most appreciated is, of course, the extra virgin olive oil (EVOO) which must respect certain requirements in term of molecular content, manufacturing, aroma and taste sensing. Its molecular composition is quite unique among edible fats and provides different beneficial health effects (Estruch, 2010; Estruch et al., 2013). For example the presence of particular monounsaturated fatty acid such as oleic acid seems to be associated with reduced risk of cardiovascular mortality, cardiovascular events and stroke (Schwingshackl and Hoffmann, 2014); in addition, the presence of lipophilic antioxidants, i.e. vitamin E complex (tocopherols), and polar antioxidants (tyrosol, hydroxytyrosol and their derivatives) may prevent atherogenesis (Kaliora et al., 2006). On the other 2

hand, the pharmacological activity of both vitamin E and phenylethanoids has been deeply analysed by government food agencies (EFSA Panel, 2010; EFSA Panel, 2011). Based on these reports, the European Union has issued a regulation (EC, 2012) which allows to report on the packaging of the olive oil important health claims. For example, regarding the phenylethanoids, the olive oil, which contains “at least 5 mg of hydroxytyrosol and its derivatives (e.g. oleuropein complex and tyrosol) per 20 g…”, may report on the label that “Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress”. It should be pointed out that the phenylethanoids present in olive oil consist in a family of molecules comprising the simple alcohols hydroxytyrosol (1, Htyr) and tyrosol (2, Tyr) and their ester derivatives (3-6) (Figure 1). However, the analysis of these compounds should be conducted by analytical tools, which allow the highest level of selectivity and accuracy. Even using the latter methodologies, the experiments may result very tricky and time consuming for several reasons: the lack of suitable authentic standards and the interferences of analytical conditions on their chemical stability. For example, the ester derivatives (3-6 figure 1) are dialdehydes, which readily interconvert in acetals and hemiacetals in presence of acidic methanol. Insert figure 1 It is worth to note that EU regulation does not report any analytical method to assay these molecules, therefore several papers have been published to address this task (table 1), the most of which are based on simple HPLC/UV detection (Ricciutelli et al., 2017; Tsimidou et al., 2019), while others rely on GC-MS or HPLC/UPLC analysis coupled to mass spectrometry (Purcaro et al., 2014; Bartella et al., 2018). The lack of suitable standards has been wisely overcome by the use of hydrolytic reactions prior the analyses, which convert the ester derivatives to simple phenylethanols (Mastralexi et al., 2014; Bartella et al., 2018; Bellumori et al., 2019). Some authors attempt to eliminate the problem of the formation of acetals by using different elution solvents, but in general none of these methods pull out the limitation above exposed, and many of them remain quite time

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consuming in the sample preparation step excluding few exceptions which use fast hydrolysis processes (Bartella et al., 2018). Insert table 1 Ambient mass spectrometry is a relatively new field in mass spectrometry: the ion sources which exploit this technique take advantages on the fact that ions are generated in their native state. Paper spray mass spectrometry is the simplest ambient ionization technique: the main advantage of this source is the relatively easy of analysis set-up, and also the nonnecessity of tedious pre-treatments of the sample to be analyzed. Paper spray, as all the ambient ionization sources, is really useful to get molecular profiles of complex matrixes: coupled with chemometrics, it is capable to discover differences between samples of different origin or type (Vaclavik et al., 2009). Paper spray may also be used for quantitative purposes even if there are some issues that limit its use for this target: in particular, the MS spectra are not quite repeatable for what concerns the absolute intensity of the peaks, although the relative intensity of the signals within the spectrum does not fluctuate. Furthermore, using a single stage mass spectrometer (e.g. a single quadrupole) the technique does not possess good sensitivity, and this is due for the most to the background noise typical of this source. To overcome this problem, hence, it is mandatory the use of suitable internal standards and the presence of a multistage and sensitive mass analyzer such as a triple quadrupole (Taverna et al., 2016; Di Donna et al. 2017; Bartella et al., 2019). Here we present an innovative and very time saving method to analyze tyrosol and hydroxytyrosol derivatives by means of paper spray tandem mass spectrometry (PS-MS/MS) and microwave assisted acid hydrolysis. The accuracy of the method is assured by the use of deuterated internal standards, which together with tandem mass spectrometry represent the unsurpassed analytical methodology to determine small molecules in complex matrixes such as olive oil (Taverna et al., 2016; Di Donna et al. 2017; Bartella et al., 2019). 2. Materials and Method 4

2.1 Chemicals Solvents (HPLC grade) and chemicals were commercially available (Sigma–Aldrich, St. Louis, MO). Hydroxytyrosol, tyrosol and oleuropein standards were purchased at Extrasynthese (Genay Cedex, France). Labeled internal standards, d2-Hydroxytyrosol and d2-Tyrosol were synthesized as reported by literature with slight modifications [Kuwajima et al., 1998]. 2.2 Oil samples Ten extra virgin olive oils were kindly furnished by C.R.A. (Centro di Ricerca per l’Olivicoltura e l’Industria Olearia, Rende, Italy). The samples were stored in amber glass bottles at 4 °C until the analysis. Corn oil was purchased from a local market and used as blank sample for the analytical parameters evaluation. 2.3 Standard solutions Standard solutions of unlabeled and labeled standards were prepared at concentration of 2000 mg/L by dissolving the pure standards in MeOH/H2O 80/20 v/v. The calibration curves were obtained by analyzing five solutions of hydroxytyrosol and tyrosol at different concentration (2.5, 5, 7, 15, 20 mg/L), and internal standards at the fixed concentration (7 mg/L). 2.4 Evaluation of free Hydroxytyrosol and Tyrosol 28 µl of a stock solution containing d2-hydroxytyrosol and d2-tyrosol at 500 mg/L were added to 1 g of each extra virgin olive oil sample and homogenized by vortex for 3 min. The mixture was dissolved in 5 ml of n-hexane and loaded into a normal-phase SPE cartridge (Sep-Pak Silica Plus Long Cartridge, 690 mg, 55-105 µm particle size, 125Å, Waters). The cartridge was washed with nhexane (3 × 5 ml), and finally eluted with 2 ml of MeOH. The methanol fraction was directly analyzed by Paper Spray Mass Spectrometry (PS-MS/MS). 2.5 Evaluation of total Hydroxytyrosol and Tyrosol 5

Each olive oil sample was submitted to extraction and microwave-assisted hydrolysis, according to our previous work, with some modifications (Bartella et al., 2018). Briefly, 56 µl of a d2Hydroxytyrosol and 56 µl of a d2-Tyrosol, both from stock solution at 2000 mg/L were added to 2g of each sample; the mixture was homogenized for 3 min by vortex and then extracted with 4 ml of EtOH/H2O (0,1% HCOOH) 7:3 v/v under vigorous stir for 3 min. After centrifugation, 500 µl of the extract were mixed with 500 µl of 2M HCl in a suitable microwave vial and submitted to hydrolysis for 4 min, in a microwave Anton Paar Multiwave 3000 setting the maximum power at 1400W and the temperature at 140°C. The hydrolysis mixture was diluted in water (5 ml) and loaded onto a reversed phase SPE cartridge (Strata C18-E, 500 µm particle size, 70Å, 500 mg/6ml, Phenomenex), which was first washed with water (3 × 5ml) and then eluted with 2 ml of methanol. The obtained solution was submitted to PS-MS/MS analysis. 2.6 Mass Spectrometric analysis The MS analyses were carried out using a LC 320 triple-stage quadrupole mass spectrometer (Varian Inc., Palo Alto, CA, USA), in-house implemented for PS-MS applications. A volume of 15 µL for each sample was spotted onto a triangular paper and left to dry for 1 min. The same volume of methanol was spotted to the paper during the acquisition every 30 s (total acquisition time of 2 min) in order to allow the ion generation. The analyses were performed in negative ionization mode with the following optimized MS parameters: needle voltage -5000 V; shield -600 V; housing temperature 35 °C and the detector fixed at 1400 V. Argon was used as collision gas at a pressure inside the collision cell (Q2) of 2 mTorr, the mass resolution at the first (Q1) and third (Q3) quadrupole was set at 0.7 u at full width at half-maximum (FWHM). Scan time was set at 0.060 s. The multiple reaction monitoring (MRM) scan mode was used for the quantification, following the fragmentation channels typical of tyrosol and hydroxytyrosol (table S1). Collision energy (CE) and capillary were optimized for each compound. The ion current of each monitored transition was averaged over the total acquisition time and used for the quantitative analysis. 6

2.7 HPLC-UV analysis The HPLC-UV analyses, needed to calculate the recovery of the extraction process, were performed using the FractionLynx system from Waters (Milford, MA) working in analytical mode, equipped with a 2535 quaternary pump and a 2989 UV/visible detector. The chromatographic separation was carried out using a C18 reversed-phase column, Luna (250 × 4.6 mm, 5µm, Phenomenex) and an elution gradient built with 0.1% HCOOH/H2O (solvent A) and ACN (solvent B), composed by the following step: linear gradient from 100% A to 85% A in 5 min; from 85% A to 70% A in 10 min; isocratic elution 70% A for 7 min; linear gradient from 95% A to 60% A in 33 min, linear gradient from 60% A to 40% A in 5 min, linear gradient from 70% A to 5% A in 8 min; isocratic elution 5% A for 5 min; linear gradient from 5% A to 100% A in 5 min; equilibration of the column for 10 min. The run time was 50 min, at flow rate of 1 ml/min, injecting a volume of 20 µl. The chromatograms were recorded at 280 nm wavelength. The concentration of hydroxytyrosol and tyrosol was obtained by a calibration curve gathered analyzing five standard solutions of analytes, with a concentration range from 2,5 mg/L to 50 mg/L. 2.8 Limit of quantitation (LOQ) and limit of detection (LOD) The analytical parameters, the LOQ and the LOD, were determined according to the directives of IUPAC and American Chemical Society’s Committee on Environmental Analytical Chemistry: SLOD = SRB + 3σRB SLOQ = SRB + 10σΡΒ where SLOD is the signal at the limit of detection, SLOQ is the signal at the limit of quantitation, SRB is the averaged ratio between the signals of the analyte transition and that of the internal standard from a blank sample (corn oil), and σRB is its standard deviation. 3. Results and Discussion 7

3.1 PS-MS/MS experiment and sample preparation procedure In this paper, we report a methodology able to determine the amount of phenylethanoids in a complex food, such as olive oil, by means of paper spray tandem mass spectrometry with the aid of deuterated internal standards. It is worth to remember that the phenylethanoid family (figure 1) is composed mainly by two alcohols (tyrosol (1) and hydroxytyrosol (2)) and their four esters: oleocanthal (3), oleaecin (4), ligstroside aglycon (5) and oleuropein aglycone (6). The method for the quantitative determination is, therefore, divided in two steps: in the first step, free Tyr and Htyr are analyzed and quantified; in the second step, the esters are hydrolyzed to simple alcohols, which are then analyzed by MS/MS. This two-step procedure is necessary to gather information on the amount of esterified compounds in order to address the EU regulation, which requires to assess the amount of each molecule. The first step is quite easy to perform: in practice, the olive oil is mixed with known amount of deuterated standard, homogenized and diluted in n-hexane; thereafter, a purification step on a normal phase cartridge is necessary to avoid interferences in the ionization step due to the excess of triacylglycerols. The pre-purified sample is then spotted onto a triangular piece of paper and allowed to ionize in the electric field of the source. For what concerns the second step of quantification, the olive oil sample enriched with deuterated standards is extracted in a polar solvent mixture and then submitted to a fast microwave hydrolytic step (Bartella et al., 2018). Even in this case, the reaction product is purified by a SPE cartridge step (reverse phase in this case) and then analyzed by PS-MS/MS. The analysis is performed in multiple reaction monitoring (MRM) mode by following the main fragmentation of tyrosol, hydroxytyrosol and their corresponding deuterated standards (table S1). It is worth to note that in both steps the mass spectrometric analysis is accomplished in 2 minutes acquisition time. Figure 2 represents the typical ion chromatogram of a PS-MS/MS experiment performed on an olive oil sample: the figure 2A reports the ion traces of Htyr and d2-Htyr, while figure 2B shows the ion chromatograms of Tyr and d2-Tyr. Insert figure 2 8

The ratio of the averaged signal intensities derived from the analysis is interpolated to a calibration curve obtained in a suitable concentration range (see experimental). 3.2 Extra virgin olive oil samples Table 2 shows the result of the analyses performed on ten extra virgin olive oil samples. In particular, the third column of the table refers to the amount of free Tyr and Htyr, while the fifth column shows the total amount after hydrolysis. Insert table 2 The reproducibility of the experiments is quite good considering that the each sample has been submitted to the whole process three times over three weeks. Table 3 shows the value of phenylethanoid as requested by the EU regulation. In particular, each value has been obtained by the expression reported in the footnote of the table the takes into account the molecular weight of the esters of Tyr and Htyr. The values are expressed as mg per 20 g as specified by the regulation. Insert table 3 The most of the oils are above the threshold that allows to report the health claim on the front food label. 3.3 Method validation The consistency of the has been demonstrated by evaluating the main analytical parameters. 3.3.1 Limit of detection (LOD), limit of quantification (LOQ) and linearity. The LOD and LOQ were evaluated for both steps of the procedure. The values were determined by analyzing corn oil sample spiked with the internal standards (see section 2.8). Table 4 shows the values of LOD and LOQ calculated both for solution, after the sample preparation procedure, and for oil. The LOQ values obtained in solution, are below the lowest calibration level, which means 9

that the PS-MS/MS method provides good sensitivity for the determination of the analytes in oil matrix. The linearity of the instrumental response was assessed by means of a calibration curves obtained from the analysis of five solutions at known concentration of Htyr and Tyr in the range 2.5 - 20 mg/L, mixed with the internal standards at a fixed concentration of 7 mg/L. The standard solutions were analyzed in triplicate as described in section 2.6. The method response was linear in the selected range of concentration providing a correlation coefficient value higher than 0.998. Further evidence of the linearity was provided by adding the LOQ analysis responses to the calibration curve; even in this case the correlation coefficient stands above 0.98. Insert table 4 3.3.2 Accuracy and precision (repeatability and reproducibility) The accuracy of the methodology was evaluated at two level of concentration for both steps of procedure, according with our previous work (Bartella et al. 2018). The accuracy values, related to the analysis of free Htyr and Tyr, were calculated using two fortified blank samples (corn oil naturally lacking of phenylethanoids), arranged with low and high phenolic content. In the case of the experiment performed on total Htyr and Tyr, oleuropein too was added to corn oil in order to mimic the presence of esters. In both cases, the accuracy values remain in the acceptable range 90110%. Spiked samples were also used to evaluate the precision in terms of repeatability (expressed as the percentage relative standard deviation, RSD) calculated by performing three instrumental analyses for each fortified samples: in all cases, RSD values were below 15%, providing good repeatability. Finally, the method reproducibility was tested by preparing and analyzing the spiked samples three times over a period of three weeks: even in this case the RSD values were satisfactory. The results are summarized in table 5. Insert table 5

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3.3.4 Specificity and recovery. The specificity of the method is assured by the use of multiple reaction monitoring analyzing scan in a triple quadrupole mass spectrometer. In particular, table S1 reports two fragmentation channels per analyte, the first used for the assay, and the second needed for confirmatory purposes. Since the samples were purified by a SPE cartridge step prior the analysis by PS-MS/MS, it has been necessary to calculate the recovery values; this experiment has been carried out by HPLC-UV analysis. The value of recovery is in all cases above 85% (table 4). 3.3.5 Robustness of the method In order to demonstrate the suitability of the developed approach, its robustness has been evaluated by comparing the results with those obtained by a different approach. In particular, the same extra virgin olive oil samples were also analyzed by a procedure based on HPLC-UV analysis; the results are quite similar to those obtained by the PS-MS/MS methodology (table 6) providing values completely superimposable. Insert table 6 4. Conclusion A fast and reliable method to determine the amount of phenylethanoids in olive oil has been developed. The method intends to address the European regulation on health claims which may greatly increase the value of the product EVOO. The developed method, based on the use of paper spray mass spectrometry and isotopomers as internal standards, is very rapid having a MS acquisition time of only two minutes per step. The whole time of the two-steps experiment comprising the sample pretreatment and microwave hydrolysis is limited to no more than ten minutes. 5. Competing interests.

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6. Acknowledgments The authors wish to thank Calabria Regional institution for its financial support through the POR FESR-FSE Calabria 2014/2020, Action 1.5.1 grant. LB wishes to thank Italian Ministry of Education, University and Research for its grant n. AIM1899391 – 1 in the framework of the project “Azione I.2, Mobilità dei Ricercatori, PON R&I 2014-2020”

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Legends to figures Figure 1. Phenylethanols and ester conjugates present in extra virgin olive oil. Figure 2. Ion signal obtained from a sample of olive oil: (A) Htyr and d2-Htyr transitions, (B) Tyr and d2-Tyr transitions.

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References Bartella, L., Mazzotti, F., Napoli, A., Sindona, G., Di Donna, L., 2018. A comprehensive evaluation of tyrosol and hydroxytyrosol derivatives in extra virgin olive oil by microwave-assisted hydrolysis and HPLC-MS/MS. Anal. Bioanal. Chem., 410(8), 2193-2201. Bartella, L., Di Donna, L., Napoli, A., Sindona, G., Mazzotti, F. 2019. High-throughput determination of vitamin E in extra virgin olive oil by paper spray tandem mass spectrometry. Anal. Bioanal. Chem., 411(13), 2885-2890. Bellumori, M., Cecchi, L., Innocenti, M., Clodoveo, M.L., Corbo, F., Mulinacci, N., 2019. The EFSA health claim on olive oil polyphenols: Acid hydrolysis validation and total hydroxytyrosol and tyrosol determination in italian virgin olive oils. Molecules, 24(11), 2179-2195. Di Donna, L., Taverna, D., Indelicato, S., Napoli, A., Sindona, G., Mazzotti, F., 2017. Rapid assay of resveratrol in red wine by paper spray tandem mass spectrometry and isotope dilution. Food Chem., 229, 354-357. EC, 2012. Commission Regulation (EU) No 432/2012 of 16 May 2012 establishing a list of permitted health claims made on foods, other than those referring to the reduction of disease risk and to children’s development and health. Off. J. Eur Comm. No L136/1. https://eurlex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32012R0432&from=IT. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010. Scientific Opinion on the substantiation of health claims related to vitamin C and reduction of tiredness and fatigue (ID 139, 2622), contribution to normal psychological functions (ID 140), regeneration of the reduced form of vitamin E (ID 202), contribution to normal energyffyielding metabolism (ID 2334, 3196), maintenance of the normal function of the immune system (ID 4321) and protection of DNA, proteins and lipids from oxidative damage (ID 3331) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 8, 1815. 14

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2011. Scientific Opinion on the substantiation of health claims related to polyphenols in olive and protection of LDL particles from oxidative damage (ID 1333, 1638, 1639, 1696, 2865), maintenance of normal blood HDL cholesterol concentrations (ID 1639), maintenance of normal blood pressure (ID 3781), “antiinflammatory properties” (ID 1882), “contributes to the upper respiratory tract health” (ID 3468), “can help to maintain a normal function of gastrointestinal tract” (3779), and “contributes to body defences against external agents” (ID 3467) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 9, 2033. Estruch, R., 2010. Anti-inflammatory effects of the Mediterranean diet: the experience of the PREDIMED study. Proc. Nutr. Soc. 69, 333-340. Estruch, R., Ros, E., Salas-Salvadó, J., Covas, M.I., Corella, D., Arós, F., Gómez-Gracia, E., RuizGutiérrez, V., Fiol, M., Lapetra, J., Lamuela-Raventos, R.M., Serra-Majem, L., Pintó, X., Basora, J., Muñoz, M.A., Sorlí, J.V., Martínez, J.A., Martínez-González, M.A., 2013. PREDIMED Study Investigators: Primary prevention of cardiovascular disease with a Mediterranean diet. N. Engl. J. Med. 368, 1279-1290. Kaliora, A.C., Dedoussis, G.V.Z., Schmidt, H., 2006. Dietary antioxidants in preventing atherogenesis. Atherosclerosis, 187(1), 1-17. Kuwajima, H., Takai, Y., Takaishi, K., Inoue, K., 1998. Chem. Pharm. Bull. 46(4), 581–586. Mastralexi, A., Nenadis, N., & Tsimidou, M.Z., 2014. Addressing analytical requirements to support health claims on "olive oil polyphenols" (EC regulation 432/2012). J. Agr. Food Chem., 62(12), 2459-2461. Purcaro, G., Codony, R., Pizzale, L., Mariani, C., Conte, L., 2014. Evaluation of total hydroxytyrosol and tyrosol in extra virgin olive oils. Eur. J. Lipid Sci. Tech., 116(7), 805-811.

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Ricciutelli, M., Marconi, S., Boarelli, M.C., Caprioli, G., Sagratini, G., Ballini, R., Fiorini, D., 2017. Olive oil polyphenols: A quantitative method by high-performance liquid-chromatographydiode-array detection for their determination and the assessment of the related health claim. J. Chromatog. A, 1481, 53-63. Schwingshackl, L., Hoffmann, G., 2014. Monounsaturated fatty acids, olive oil and health status: A systematic review and meta-analysis of cohort studies. Lipids Health Dis. 13, 154-169. Taverna, D., Di Donna, L., Bartella, L., Napoli, A., Sindona, G., Mazzotti, F., 2016. Fast analysis of caffeine in beverages and drugs by paper spray tandem mass spectrometry. Anal. Bioanal. Chem., 408(14), 3783-3787. Tsimidou, M.Z., Sotiroglou, M., Mastralexi, A., Nenadis, N., García-González, D.L., Toschi, T.G., 2019. In house validated UHPLC protocol for the determination of the total hydroxytyrosol and tyrosol content in virgin olive oil fit for the purpose of the health claim introduced by the EC regulation 432/2012 for “olive oil polyphenols”. Molecules, 24(6), 1044-1060. Vaclavik, L., Cajka, T., Hrbek, V., Hajslova, J., 2009. Ambient mass spectrometry employing direct analysis in real time (DART) ion source for olive oil quality and authenticity assessment. Analytica Chimica Acta, 645(1-2), 56-63.

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Table 1. Comparison between the methodologies used for the detection of tyrosol and hydroxytyrosol derivatives. Method

plus

drawbacks

Reference

Acid hydrolysis, HPLC-UV

Detector commonly utilized

Slow sample preparation step and lengthy UV analysis

Ricciutelli et al, 2019

Acid hydrolysis, HPLC-UV

Detector commonly utilized

Slow sample preparation step

Tsimidou et al., 2017

Acid hydrolysis, derivatization, GC-MS

Specificity of the technique

Slow sample preparation step and lengthy GC analysis

Purcaro et al., 2014

Microwave acid hydrolysis, HPLC-MS/MS

Specificity of the technique, rapid sample preparation

Use of deuterated standard

Bartella et al., 2018

Acid hydrolysis, HPLC-UV

Detector commonly utilized

Slow sample preparation step and lengthy GC analysis

Bellumori et al., 2019

Table 2. Hydroxytyrosol and tyrosol free and total content in selected extra virgin olive oil samples. Olive Oil Sample

Free Tyr-Htyr (mg/kg)

RSD* (%)

Total Tyr-Htyr (mg/kg)

RSD* (%)

CA

Tyr Htyr

14,1 ± 1,1 N.D.

7,8 ----

71 ± 5 N.D.

7,0 ---

CO

Tyr Htyr

10,7 ± 0,9 5,4 ± 0,7

8,4 13,0

215 ± 8 121 ± 4

3,7 3,4

DD

Tyr Htyr

11,4 ± 0,9 5,4 ± 0,5

7,9 9,2

84 ± 5 37 ± 4

5,9 10,8

DR

Tyr Htyr

N.D. N.D.

-----

84 ± 7 46 ± 1

8,3 2,2

PE

Tyr Htyr

N.D. 4,2 ± 0,2

--4,8

86 ± 7 102 ± 3

8,1 2,9

LU

Tyr Htyr

5,8 ± 0,2 3,0 ± 0,1

3,4 3,3

199 ± 6 212 ± 7

3,0 3,3

PI

Tyr Htyr

13,6 ± 0,4 11,2 ± 0,5

2,9 4,5

133 ± 6 94 ± 2

4,5 2,1

CS

Tyr Htyr

5,6 ± 0,4 N.D.

7,3 ---

80 ± 8 41 ± 1

10,0 2,4

OT

Tyr Htyr

7,5 ± 0,6 N.D.

7,8 ---

167 ± 8 147 ± 4

6,1 2,7

NO

Tyr Htyr

6,1 ± 0,5 N.D.

8,2 ---

63 ± 1 53 ± 1

1,6 3,8

N.D. not detected

* The reproducibility of the measurements was achieved by repeating three times (seven for sample CA) the experiment for each sample every seven days.

Table 3. Amount of phenylethanols and their derivatives in selected olive oils expressed as mg/20 g according to 432/2012 EU Regulation. Olive Oil Sample

(mg/20 g)a,b

RSD (%)

CA

3,0 ± 0,3

10,0

CO

15,4 ± 0,7

4,5

DD

5,3 ± 0,5

9,4

DR

6,1 ± 0,4

5,1

PE

8,5 ± 0,5

5,9

LU

19,0 ± 0,6

3,1

PI

10,0 ± 0,4

4,0

CS

5,6 ± 0,5

8,9

OT

14,5 ± 0,6

4,1

NO

5,3 ± 0,2

3,8

a The sum of the concentrations of each analyte X is calculated using the formula (Xt-Xf)×cf+Xf, where Xt is the total concentration of the species, (e.g. tyrosol or hydroxytyrosol and their esters), Xf is the free concentration of the species and cf is the ratio between the average molecular weight of the ester conjugate (3,4 and 5,6) and the molecular weight of X. b

Total amount of species reported as indicated by the EU directive 432/2012 i.e. sum of total amount of Htyr and Tyr derivatives expressed as mg per 20 g (EU, 2012).

Table 4. Recovery, LOD and LOQ values gathered on fortified corn oils for the determination of free and total Htyr/Tyr content. LOD (mg/L)

LOD (mg/kg)

LOQ (mg/L)

LOQ (mg/kg)

Recovery (%)

Solution

Oil sample

Solution

Oil sample

Free Tyr

1.6

3,1

2.6

5,2

Quantitative

Free Htyr

0.5

1,1

1.6

3,1

86,4

Total Tyr

0.3

2,1

3.4

27,4

90,5

Total Htyr

1.8

14,1

3.8

30,2

90,6

Table 5. Accuracy, reproducibility and repeatability values gathered on fortified corn oils for the determination of free and total Htyr and Tyr content. Fortified sample

Spiking concentration (mg/kg)

Found concentration (mg/kg)

Mean accuracy (%)

RSDa (%)

RSDb (%)

Free Htyr and Tyr Low content corn oil High content corn oil

Tyr

8

8,8 ± 0,8

110

9,1

10,3

Htyr

8

8,8 ± 0,4

110

4,5

6,1

Tyr

16

15,6 ± 0,3

97

1,9

2,8

Htyr

16

15,3 ± 0,9

96

5,9

5,7

Tyr

50

45 ± 5

90

11,1

12,5

Htyr

30

38 ± 3

Oleuropein

30

---

101

7,9

7,5

Tyr

150

157 ± 22

105

14,0

13,2

Htyr

100

131 ± 6

Oleuropein

100

---

102

4,6

5,9

Total Htyr and Tyr Low content corn oil

High content corn oil a b

Reproducibility Repeatability

Table 6. Concentration of free and total Tyr and Htyr obtained by different approaches. Olive Oil Sample

Free Tyr-Htyr (mg/kg)

Free Tyr-Htyr (mg/kg)

Total TyrHtyr (mg/kg)

Total Tyr-Htyr (mg/kg)

PS-MS/MS

HPLC-UV

PS-MS/MS

HPLC-UV

CA

Tyr Htyr

14,1 ± 1,1 N.D.

16,2 ± 2,0 2,0 ± 0,2

71 ± 5 N.D.

67 ± 3 13,3 ± 1,2

CO

Tyr Htyr

10,7 ± 0,9 5,4 ± 0,7

9,3 ± 0,4 7,1 ± 1,1

215 ± 8 121 ± 4

209 ± 8 130 ± 6

DD

Tyr Htyr

11,4 ± 0,9 5,4 ± 0,5

10,8 ± 1,4 6,3 ± 0,8

84 ± 5 37 ± 4

78 ± 7 39± 4

DR

Tyr Htyr

N.D. N.D.

2,0 ± 0,3 N.D.

84 ± 7 46 ± 1

90 ± 7 48 ± 3

PE

Tyr Htyr

N.D. 4,2 ± 0,2

N.D. 4,1 ± 0,6

86 ± 7 102 ± 3

81± 5 108 ± 6

LU

Tyr Htyr

5,8 ± 0,2 3,0 ± 0,1

7,3 ± 0,5 5,0 ± 0,3

199 ± 6 212 ± 7

185 ± 8 220 ± 10

PI

Tyr Htyr

13,6 ± 0,4 11,2 ± 0,5

13,0 ± 0,3 11,5 ± 0,6

133 ± 6 94 ± 2

124 ± 5 102 ± 3

CS

Tyr Htyr

5,6 ± 0,4 N.D.

8,5 ± 0,9 N.D.

80 ± 8 41 ± 1

87 ± 5 43 ± 2

OT

Tyr Htyr

7,5 ± 0,6 N.D.

6,1 ± 0,7 3,0 ± 0,2

167 ± 8 147 ± 4

157 ± 9 163 ± 6

NO

Tyr Htyr

6,1 ± 0,5 N.D.

5,4 ± 0,8 N.D.

63 ± 1 53 ± 2

58 ± 5 51 ± 4

Highlights •

A rapid determination of phenylethanoids in olive oil has been set up.

•

The whole experiment lasts few minutes.

•

The analysis utilizes labeled standards and paper spray tandem mass spectrometry.

•

The method was tested to extra virgin olive oil and fortified oil samples.

•

The proposed method addresses the European regulation 432/2012 on health claims

1

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: