Evaluation of dialdehydic anti-inflammatory active principles in extra-virgin olive oil by reactive paper spray mass spectrometry

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Contents lists available at ScienceDirect

International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms

Evaluation of dialdehydic anti-inflammatory active principles in extra-virgin olive oil by reactive paper spray mass spectrometry

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Fabio Mazzotti ∗ , Leonardo Di Donna, Domenico Taverna, Monica Nardi, Donatella Aiello, Anna Napoli, Giovanni Sindona

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Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Via P. Bucci Cubo 12/C, I-87036 Arcavacata di Rende (CS), Italy

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a r t i c l e

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Article history: Received 25 June 2013 Received in revised form 23 July 2013 Accepted 23 July 2013 Available online xxx

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Keywords: Paper spray Olive oil, Nonsteroidal Antiinflammatory Drugs (NSAIDs) Mass spectrometry Precursor ion scan

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1. Introduction

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A novel approach for the identification and evaluation of Nonsteroidal Antiinflammatory Drugs (NSAIDs), found in olive oil is presented. The method is based on the exploitation of paper spray (PS) tandem mass spectrometry (MS/MS), which allows a high throughput screening of the active principles characterized by a dialdehyde moiety after an in situ derivatization with methoxyamine that leads to stable alkyloxime derivatives. The quantification analysis were performed in the precursor ion scan mode by comparing the signal intensity of the reference compound in the standard solution vs the signal intensity of the analytes obtained by the experiments performed on the olive oil. To verify the reliability of the proposed approach, a sample of seed oil spiked with a known amount of hydroxyoleocanthal has been analyzed leading to accuracy values in the range from 92% to 107%. The method here presented allows the direct analysis of olive oil samples in order to quickly verify its quality, providing molecular information on the presence of such anti-inflammatory molecules in a rapid and convenient way. © 2013 Published by Elsevier B.V.

Mass spectrometry is an analytical technique that can provide both qualitative and quantitative information on molecular species after their conversion into ions. It is widely applied in the agrofood sector for the analysis of proteins and metabolites [1,2]. Many major difficulties in taking full advantages of mass spectrometry have been overcome by the ability to produce gas phase ions from aqueous solution. Electrospray ionization mass spectrometry (ESIMS) is the most frequently used ion source for a variety of important analytical applications; it provides a sensitive, robust, and reliable tool for studying nonvolatile and thermally labile biomolecules. In combination with HPLC, ESI-MS has become a very powerful technique capable of analyzing both small and large molecules of various polarities in a complex matrixes [3–5]. Mass spectrometry has been used also to evaluate the food authenticity and origin [6,7]. In the last years, a new family of MS sources have been developed in order to produce ions under ambient conditions [8–12]. Ambient mass spectrometry possesses the ability to record mass spectra of ordinary samples, in their native environment, without sample preparation or pre-separation by creating ions outside the instrument [13]. Paper spray (PS) ionization has been

∗ Corresponding author. Tel.: +39 0984 493317; fax: +39 0984 493307. E-mail address: [email protected] (F. Mazzotti).

developed as a direct, fast and low-cost sampling and ionization method for qualitative and quantitative mass spectrometric (MS) analysis of complex mixtures [14–17]. For instance, paper spray mass spectrometry has been used to detect pesticides from fruit surface; similar approaches have been used to analyze agrochemicals and contaminants directly from food matrixes [18–20]. Qualitative and semi-quantitative analysis performed using ambient MS experiments typically requires only few seconds. The present study reports a paper spray mass spectrometry (PS/MS) method for the estimation of olive oil phenolic dialdehydes by in situ derivatization reaction. Olive oil is a food product obtained from the fruit of olive trees; it is one of the most important constituents of the Mediterranean diet; its constant intake may provide potential health benefits to humans, lowering the incidence of cardiovascular disease [21–25]. It contains antioxidant and anti-inflammatory active principles such as E vitamin, carotenoids and phenolic compounds which also may protect against cardiovascular diseases. The main phenolic compounds are hydroxytyrosol, tyrosol and the phenolic dialdehydes, the most known of which is oleocanthal [26–29]. The quality of virgin olive oil is associated with the presence of microcomponents whose healing effects have been documented [30]. The main goal of the method here presented is the direct evaluation of the anti inflammatory dialdehydes of olive oil (Scheme 1), after in situ derivatization into stable alkyloxime derivates, by paper spray mass spectrometry (PS/MS).

1387-3806/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijms.2013.07.012

Please cite this article in press as: F. Mazzotti, et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.07.012

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Scheme 1. 1 R = COOCH3 ; R = OH; MW 378; oleuropein aglycon; I A mono-derivatized protonated species m/z 408; I B bis-derivatized protonated species m/z 437. 2 R = COOCH3 ; R = H; MW 362; ligstroside aglycon; II A mono-derivatized protonated species m/z 392; II B bis-derivatized protonated species m/z 421. 3 R = H; R = OH; MW 320; hydroxyoleocanthal, III B bis-derivatized protonated species m/z 379. 4 R = R = H; MW 304; oleocanthal, IV B bis-derivatized protonated species m/z 363.

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d0 and d3 -methoxyamine provides a fast and quantitative derivatization of those active principles. The developed method was tested with four samples olive oils purchased at local store. The measurements were carried out with low-energy collision dissociation in a tandem mass spectrometer (CID-MS/MS) using the precursor ion scan method.

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2. Experimental

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Chemicals. Solvents and reagents were commercially available (Sigma-Aldrich, St. Luis, MO). Standards. Hydroxyoleocanthal standard was obtained as reported [27]. Oil samples. Four extra virgin olive oils were stored in amber glass bottles at room temperature until analysis. S1, S2, S3, and S4 were purchased at a local store. Seed oil was used as blank matrix for analytical parameters calculations.

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2.1. Sample preparation

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500 mg of olive oil were added of a 50 ␮L solution of benzaldehyde (100 ppm) and then vortexed for 3 min. The obtained mixture was then preloaded (15 ␮L) onto a paper triangle and dried at room temperature for 2 min before mass spectrometry analysis.

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2.2. In situ derivatization

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Chemical derivatization reaction has been carried out using 15 ␮L of 1.5 M methoxyamine hydrochloride methanol/water (1:1, v/v) solution; the latter was spotted onto a paper triangle preloaded with 15 ␮L volume of olive oil. After drying, the paper triangle was positioned in front of the ion transfer tube of the mass spectrometer and the high voltage was applied. Then, 3× 15 ␮L volume of

the methoxyamine solution were spotted onto the paper to allow 2 min acquisition for the precursor ion scan mode experiment. 2.3. Mass spectrometry and paper spray ionization The MS analyses were carried out with a TSQ Quantum Vantage (Thermo Fischer Scientific, San José, CA) triple-stage quadrupole mass spectrometer implemented for PS/MS application. 10 ␮L of sample was spotted on triangular shaped paper matrix and, once dried, the same volume of methanol was added to allow the spray desorption. All the instrument parameters were controlled by Xcalibur software, version 2.0.0 (Thermo Fisher Scientific). The homemade paper spray source was operating in positive ion mode. The following working conditions were applied: spray voltage, 5.0 kV; capillary temperature 290 ◦ C. The collision gas was argon used at a pressure in the collision cell (Q2) of 0.8 mTorr, and the mass resolution at the first (Q1) and third (Q3) quadrupoles was set at 0.7 u at full width at half-maximum (fwhm). The scan time was set at 0.4 s while the number of micro scans at 2. The collision energy (CE) was optimized individually per compound and then set at 25 eV (averaged value). The precursor ion scan mode was used to quantify the analytes; in particular, the ions at m/z 121 and 137 were selected for two scan events within the same experiment. Further, a third scan event was set for the ion at m/z 109 (related to the derivatized benzaldehyde, used as internal standard). Instrument control and data processing were carried out by means of Xcalibur software. The total acquisition time was of 120 s, allowing 40 s per scan event. 3. Results and discussion Chemical derivatization of the analytes is often used to enhance the detection sensitivity in mass spectrometry and in particular

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m/z Fig. 1. Paper spray full scan MS spectrum of olive oil obtained, spotting the sample adsorbed on the paper, with CH3 OH (A), a solution of NH2 OCH3 in methanol (B) and a solution of NH2 OCD3 in methanol (C).

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in ESI-MS [31]; in the presented methodology, the derivatization reaction of the investigated molecules has been performed with NH2 OCH3 . The derivatization of the aldehydic moiety of the active principles is needed to overcome the isomerization of the analytes which takes place in solution [27]. Fig. 1 shows the full scan paper spray spectra obtained sampling a sample of olive oil spotted with methanol (Fig. 1A) and the same sample spotted with a solution of NH2 OCH3 /CD3 in methanol

(Fig. 1B and C). The spectrum in Fig. 1A displays the ions at m/z 379, 363, 321 and 305 which may be easily ascribed to the protonated species of the dialdehydes 1–4 under investigation; Fig. 1B shows the ions generated by the in situ derivatization: in particular the species at m/z 437, 421, 379, 363 (I B–IV B, Scheme 1) correspond to the bis-methoxy-pentanedialdehydes obtained from 1 to 4, while those at m/z 408 and 392 (I A–II A, Scheme 1) may be attributed to the derivatization of a single aldehydic group present in 1 and 2.

Table 1 Amount of analytes founds in virgin olive oils and spiked samples, repeatability value (RSD%)a and calculated accuracy. Analytes amount (mg/kg) Samples

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RSD%

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85 ± 12 92 ± 15 131 ± 19 75 ± 9 68 ± 6

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44 ± 6 25 ± 5 35 ± 7 28 ± 6 31 ± 7

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Spiked 100 mg/kg Spiked 200 mg/kg

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72 ± 11 120 ± 15 135 ± 18 65 ± 10 95 ± 13

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92 ± 12 213 ± 22

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Accuracy value 92% 107%

± ± ± ± ±

The repeatability of the measurements was determined by analyzing five times each sample.

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Fig. 2. Paper spray MS/MS spectrum of derivatized hydroxyoleocanthal (A) and oleocanthal (B).

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In addition, the MS full scan has been acquired by spotting a solution of NH2 OCD3 in methanol (Fig. 1C); the spectrum shows the same ions recorded in the spectrum of Fig. 1B increased of six or three u. it should be noted that in all the spectra acquired using d0 or d3 methoxyamine, were not found any signal of the underivatized dialdehydes ions, confirming that the in situ derivatization is quantitative. The MS/MS spectra of the derivatized molecules display the tyrosol/hydroxytyrosol moiety as base peaks (ion at m/z 121 for derivatized 2 and 4 and m/z 137 for derivatized 1 and 3). In Fig. 2 the MS/MS spectra of the species at m/z 379 (III B) and m/z 363 (IV B) are reported as an example; those species corresponds to the bis-derivatized species from 3 and 4 (Scheme 1). The precursor ion scan methodology has been chosen to perform the semi quantitative assay. The samples were acquired together with a solution of benzaldehyde, which was added as internal standard to minimize the errors due to the variable position of triangle paper. The assay was, hence, carried out by monitoring the precursor ion current of three different ions: (a) the fragment ion at m/z 121 (4-hydroxy styrenium ion) for the assay of 2 and 4, (b) the fragment ion at m/z 137 (3,4-dihydroxy styrenium ion) for the assay of 1 and 3 and (c) the fragment ion at m/z 91 which was obtained by the fragmentation of the benzaldehyde molecule. The semi-quantitative information have been obtained comparing the signal intensity of the ion III B (m/z 379) obtained from the standard solution vs the signal intensity of the ions I B-IV B and I A–II A (Scheme 1) obtained by the experiments performed on the olive oil. Table 1 shows the amount of the four dialdehydes present in different olive oils obtained from the local market; the amount of 1 and 2 has been evaluated adding the two different contribution of both mono and bis derivatized molecules. The experiment has been performed directly on the olive oil matrix without any extraction step. The reproducibility values were evaluated after five repeated injections of each samples. To demonstrate the reliability of the proposed approach, a sample of seed oil spiked with a known amount of hydroxyoleocanthal has been submitted to analysis: the accuracy data reported in Table 1 shows

that the agreement of the observed vs the theoretical concentration value is near 100%. 4. Conclusions The paper spray mass spectrometry can be used for the direct analysis of extra virgin olive oil. The anti-inflammatory dialdehydes principles can be readily analyzed from untreated oil samples without sample preparation. The semi-quantitative analysis is adequate to verify the quality of virgin olive oil. The method provides molecular information on the active molecules in a rapid and convenient way and has potential application to rapid screening in other kind of vegetable and biological matrices. Moreover, a significant advantage of paper spray is to generate ions for MS analysis directly from raw samples loaded onto the paper substrate. Finally, the paper spray is not time consuming, very cheap and also “green”, because it allows for mass spec determination and semi-quantification, bypassing chromatography and using just few microliters of samples spotted onto a paper triangle. Acknowledgments University of Calabria and QUASIORA project of the Calabria APQ-RAC network and Italian National Project, MIUR-PRIN 2009XZ7XAB 003 are gratefully acknowledged. References [1] A. Napoli, D. Aiello, L. Di Donna, P. Moschidis, G. Sindona, Vegetable proteomics: the detection of Ole e 1 isoallergens by peptide matching of MALDI MS/MS spectra of underivatized and dansylated glycopeptides, Journal of Proteome Research 7 (2008) 2723–2732. [2] L. Di Donna, F. Mazzotti, A. Napoli, R. Salerno, A. Sajjad, G. Sindona, Secondary metabolism of olive secoiridoids. New microcomponents detected in drupes by electrospray ionization and high-resolution tandem mass spectrometry, Rapid Communications in Mass Spectrometry 21 (2007) 273–278. [3] L. Di Donna, F. Mazzotti, H. Benabdelkamel, B. Gabriele, P. Plastina, G. Sindona, Effect of H/D isotopomerization in the assay of resveratrol by tandem mass

Please cite this article in press as: F. Mazzotti, et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.07.012

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208 209 210

[4]

211 212 213 214

[5]

215 216 217 218

[6]

219 220 221 222

[7]

223 224 225 226

[8]

227 228 229

[9]

230 231 232

[10]

233 234 235

[11]

236 237 238 239

[12]

240 241

[13]

242 243 244

[14]

245 246 247

[15]

248 249 250

[16]

251 252 253

[17]

spectrometry and isotope dilution method, Analytical Chemistry 81 (2009) 8603–8609. F. Mazzotti, L. Di Donna, B. Macchione, L. Maiuolo, E. Perri, G. Sindona, Screening of dimethoate in food by isotope dilution and electrospray ionization tandem mass spectrometry, Rapid Communications in Mass Spectrometry 23 (2009) 1515–1518. L. Di Donna, D. Taverna, F. Mazzotti, H. Benabdelkamel, M. Attya, A. Napoli, G. Sindona, Comprehensive assay of flavanones in citrus juices and beverages by UHPLC-ESI-MS/MS and derivatization chemistry, Food Chemistry 141 (2013) 2328–2333. H. Benabdelkamel, L. Di Donna, F. Mazzotti, A. Naccarato, G. Sindona, A. Tagarelli, D. Taverna, Authenticity of PGI “clementine of Calabria” by multielement fingerprint, Journal of Agricultural and Food Chemistry 60 (2012) 3717–3726. L. Di Donna, F. Mazzotti, A. Naccarato, R. Salerno, A. Tagarelli, D. Taverna, G. Sindona, Secondary metabolites of Olea europaea leaves as markers for the discrimination of cultivars and cultivation zones by multivariate analysis, Food Chemistry 121 (2010) 492–496. Z. Takats, J.M. Wiseman, B. Gologan, R.G. Cooks, Mass spectrometry sampling under ambient conditions with desorption electrospray ionization, Science 306 (2004) 471–473. Z. Takats, I. Cotte-Rodriguez, N. Talaty, H. Chen, R.G. Cooks, Direct, trace level detection of explosives on ambient surfaces by desorption electrospray ionization mass spectrometry, Chemical Communications (2005) 1950–1952. H. Chen, S. Yang, A. Wortmann, R. Zenobi, Neutral desorption sampling of living objects for rapid analysis by extractive electrospray ionization mass spectrometry, Angewandte Chemie International Edition 46 (2007) 7591–7594. J.F. García-Reyes, F. Mazzotti, J.D. Harper, N.A. Charipar, S. Oradu, Z. Ouyang, G. Sindona, R.G. Cooks, Direct olive oil analysis by low-temperature plasma (LTP) ambient ionization mass spectrometry, Rapid Communications in Mass Spectrometry 19 (2009) 3057–3062. R.G. Cooks, Z. Ouyang, Z. Takats, J.M. Wiseman, Ambient mass spectrometry, Science 311 (2006) 1566–1570. M.E. Monge, G.A. Harris, P. Dwivedi, F.M. Fernández, Mass Spectrometry, Recent advances in direct open air surface sampling/ionization, Chemical Reviews 113 (2013) 2269–2308. H. Wang, J. Liu, R.G. Cooks, Z. Ouyang, Paper spray for direct analysis of complex mixtures using mass spectrometry, Angewandte Chemie International Edition 49 (2010) 877–880. J. Liu, H. Wang, N.E. Manicke, J.M. Lin, R.G. Cooks, Z. Ouyang, Development, characterization, and application of paper spray ionization, Analytical Chemistry 82 (2010) 2463–2471. H. Wang, N.E. Manicke, Q.A. Yang, L.X. Zheng, R.Y. Shi, R.G. Cooks, Z. Ouyang, Direct analysis of biological tissue by paper spray mass spectrometry, Analytical Chemistry 83 (2011) 1197–1201. N.E. Manicke, P. Abu-Rabie, N. Spooner, Z. Ouyang, R.G. Cooks, Quantitative analysis of therapeutic drugs in dried blood spot samples by paper spray mass

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30] [31]

5

spectrometry: an avenue to therapeutic drug monitoring, Journal of the American Society for Mass Spectrometry 9 (2011) 1501–1507. J. Liu, H. Wang, R.G. Cooks, Z. Ouyang, Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry, Analytical Chemistry 83 (2011) 7608–7613. D. Taverna, L. Di Donna, F. Mazzotti, B. Policicchio, G. Sindona, High-throughput determination of sudan azo-dyes within powdered chili pepper by paper spray mass spectrometry, Journal of Mass Spectrometry 48 (2013) 544–547. S.A. Oradu, R.G. Cooks, Multistep mass spectrometry methodology for direct characterization of polar lipids in green microalgae using paper spray ionization, Analytical Chemistry 84 (2012) 10576–10585. W.C. Willett, F. Sacks, A. Trichopoulou, G. Drescher, A. Ferro-Luzzi, E. Helsing, D. Trichopoulos, Mediterranean diet pyramid: a cultural model for healthy eating, The American Journal of Clinical Nutrition 61 (1995) 1402–1406. R. Estruch, M.A. Martnez-Gonzalez, D. Corella, J. Salas-Salvado, J. RuizGutierrez, M.I. Covas, M. Fiol, E. Gomez-Gracia, V.E. Lopez-Sabater, F. Aros, M. Conde, C. Lahoz, J. Lapetra, G. Saez, E. Ros, Effects of a Mediterranean-style diet on cardiovascular risk factors, Annals of Internal Medicine 145 (2006) 1–11. A. Iacono, R. Gomez, J. Sperry, J. Conde, B. Bianco, R. Meli, J.J. Gomez-Reino, A.B. Smith III, O. Gualillo, Effect of oleocanthal and its derivatives on inflammatory response induced by LPS in chondrocyte cell line, Arthritis & Rheumatism 62 (2010) 1675–1682. W. Li, B.J. Sperry, A. Crowe, J.Q. Trojanowski, A.B. Smith III, Y.V.M. Lee, Inhibition of tau fibrillization by oleocanthal via reaction with amino groups of tau, Journal of Neurochemistry 110 (2009) 1339–1351. F. Visioli, A. Poli, C. Galli, Antioxidant and other biological activities of phenols from olives and olive oil, Medicinal Research Reviews 65 (2002) 65–75. G.K. Beauchamp, R.S. Keast, D. Morel, J. Lin, J. Pika, Q. Han, C.H. Lee, A.B. Smith, P.A. Breslin, Phytochemistry: ibuprofen-like activity in extra-virgin olive oil, Nature 45 (2005) 45–46. L. Di Donna, H. Benabdelkamel, F. Mazzotti, A. Napoli, M. Nardi, G. Sindona, High-throughput assay of oleopentanedialdehydes in extra virgin olive oil by the UHPLC-ESI-MS/MS and isotope dilution methods, Analytical Chemistry 83 (2011) 1990–1995. F. Mazzotti, H. Benabdelkamel, L. Di Donna, L. Maiuolo, A. Napoli, G. Sindona, Assay of tyrosol and hydroxytyrosol in olive oil by tandem mass spectrometry and isotope dilution method, Food Chemistry 135 (2012) 1006–1010. A. De Nino, F. Mazzotti, E. Perri, A. Procopio, A. Raffaelli, G. Sindona, Virtual freezing of the hemiacetal–aldehyde equilibrium of the aglycones of oleuropein and ligstroside present in olive oils from Carolea and Coratina cultivars by ionspray ionization tandem mass spectrometry, Journal of Mass Spectrometry 3 (2000) 461–467. F. Visioli, C. Galli, Olive oil phenols and their potential effects on human health, Journal of Agricultural and Food Chemistry 46 (1998) 4292–4296. F. Mazzotti, H. Benabdelkamel, L. Di Donna, C.M. Athanassopoulos, A. Napoli, G. Sindona, Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages, Journal of Mass Spectrometry 7 (2012) 932–939.

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