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UHPLC-QQQ MS/MS Quantitation of Polyphenols and Secoiridoids in California-style Black Ripe Olives and Dry-Salt Cured Olives Eleni Melliou, Jerry A Zweigenbaum, and Alyson Elayne Mitchell J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506367e • Publication Date (Web): 10 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

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UHPLC-QQQ MS/MS Quantitation of Polyphenols and Secoiridoids in

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California-style Black Ripe Olives and Dry-Salt Cured Olives

4 Eleni Melliou†,‡,*, Jerry A. Zweigenbaum§ and Alyson E. Mithcell‡

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†

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University of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece;

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‡

Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,

Department of Food Science and Technology, University of California, Davis, One Shields

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Avenue, Davis, CA 95616, USA;

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§

Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808, USA

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* To whom correspondence should be addressed. Tel: +30210 7274052. E-mail:

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[email protected]

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1 ACS Paragon Plus Environment


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Abstract

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The chemical composition of finishedtable olive products isinfluenced by the olive variety and

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the processing method used to debitter or cure table olives. Herein a rapid UHPLC-QqQ MS/MS

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method, utilizing dynamic MRM, was developed for the quantitation of 12 predominant phenolic

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and secoiridoid compounds in olive fruit including: hydroxytyrosol, oleuropein, hydroxytyrosol-

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4-O-glucoside, luteolin-7-O-glucoside, rutin, verbascoside, oleoside-11-methyl ester, 2,6-

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dimethoxy-p-benzoquinone, phenolic acids (chlorogenic acid, o-coumaric acid), oleuropein

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aglycone and ligstroside aglycone. Levels of these compounds were measured in fresh and

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California-style black ripe processed Manzanilla olives, and in two dry-salt cured olive varieties

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(Mission from California and Throuba Thassos from Greece).Results indicate that the variety

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and debittering processing method have strong impact on the profile of phenolic and secoiridoid

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compoundsin table olives. The dry-salt cured olives contained higher amounts of most

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compounds studied and especially oleuropein (1459.5±100.1µg/g),whereas California-style black

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ripe olives had a significant reduction or loss of these bioactive compounds (e.goleuropein level

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at 36.7±3.1µg/g).

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Keywords: table olives, polyphenols, secoiridoids, California-style black ripe, dry-salt, UHPLC-

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QqQ MS/MS

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INTRODUCTION

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Table olives from Olea europaea L. are a traditional product and an important

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component of the Mediterranean diet.World consumption of table olives is assessed at 2,521,500

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tons in 2013.1 There are three main categories of table olives based upon the trade preparation

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methods including: green (or Spanish style), natural black (or Greek style), and California-style

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black ripe olives.2 The California-style black ripe olives are the most widely consumed olive

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among American consumers. Olive fruit is a rich source of compounds with health protecting

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activities and include: hydroxytyrosol, oleuropein, and many other related biophenols and

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secoiridoid derivatives.3 The complement of bioactives in the final olive products is influenced

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by the olive variety and the debittering method used in preparing the olives.4Debittering methods

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are of interest, asprevious studies indicate they can lead to olives with increased or significantly

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reduced healthy properties.5

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A native to the Mediterranean, historically olives were first consumed as tree-ripened

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fruit. Olives lose bitterness through natural ripening processes (in Greece known as throuba or

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stafidolia).6 Inhabitants of the Mediterranean area, who relied on olives as an essential part of

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their diet, developed various methods to preserve olives after harvesting. For many centuries, the

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basic ingredient usedto preserve olives was salt. Additional ingredients used in olive preservation

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included: honey, olive oil, vinegar and vine juice.6 The major bitter component of olives is

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oleuropein and its derivatives.7 Oleuropein is removed during dry-salt curing and can also be

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removed through other processes including lye-assisted hydrolysis of oleuropein.7-9 In Greece

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olives are now produced primarily using brine (known as Greek style), dry-salt, and lye (i.e. a 1-

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2% sodium hydroxide solution). In other countries such as Spain, Italy and the United States,

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most producers rely on lye to assist in debittering olives (Spanish-style and California-style black

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ripe olives).

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Throuba Thassos olives (also called Stafidolies, twisted, or wrinkled olives) are a



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culturally important edible olive in Greece. Traditionally, this fruit isover-ripened on the tree

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giving it a wrinkled appearance and requires no additional processing to reduce bitterness.

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However, for economic reasons Throuba Thassos olives are now produced almost exclusively

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using dry-salt processing to give a similar wrinkled appearance as tree ripened olives. Despite

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the similar appearance, the two different types of processing lead to olives with different

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chemical profiles. In contrast to the naturally overripe olives, which contain very low amounts of

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oleuropein,9 dry-salt processing leads to a higher concentration of oleuropein5 highlighting the

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critical role of processing in the chemical composition of finished olives.4 California-style black

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ripe olive processing methods involve harvesting olives before complete maturity (green). The

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raw olives are either directly treated with lye to remove bitterness or preserved in an acidic brine

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solution (0.2−0.4% calcium chloride, pH < 4.0) until they can be lye treated and processed. A

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mild fermentation may occur during the initial brine storage.This style of processing results in

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olives with the lowest levels of oleuropein and bitterness.10

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Herein, we describe a rapid ultrahigh-pressureliquid chromatography (UHPLC) triple

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quadrupole MS/MS (QqQ MS/MS) method utilizing dynamic multiple reaction monitoring

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(dMRM) for the measurement ofa range of keybitter and bioactive constituents in olives. The

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method was applied for the simultaneous quantitation of hydroxytyrosol-4-O-glucoside, 1,

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hydroxytyrosol, 2, 2,6-dimethoxy-p-benzoquinone, 3, chlorogenic acid, 4, oleoside-11-methyl

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ester, 5, rutin, 6, verbascoside, 7, luteolin-7-O-glucoside, 8, o-coumaric acid, 9, oleuropein, 10,

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oleuropein aglycone monoaldehydic form, 11 and ligstroside aglycone monoaldehydic form, 12

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(Figure 1) in three different varietiesof fresh and processed olives cured using either dry-salt or

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California-style black ripe processing methods.

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MATERIALS AND METHODS

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Chemicals. HPLC grade formic acid, acetonitrile, hexane and methanol were obtained from

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Fisher Scientific (Fair Lawn, NJ).

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Samples. Dry-salt cured Throuba Thassos olives were purchased from a local market in Davis,

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CA and the producer was contacted for processing conditions. The Throuba Thassos olives were

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treated with 40% dry-salt for 2 mo. After curing, the olives were washed with water and dried at

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ambient temperature for 2-3 days before packaging. Dry-salt Mission olives were purchased

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directly from the grower/processor at the local Farmers Market in Davis, CA. According to the

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producer, the olives were treated with dry-salt 1:1 w/w for 3 mo. After curing, the olives were

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washed with water and sun dried. California-style black ripe olives were prepared using fresh

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Manzanilla olives collected in early October 2011 in Davis, CA. The fresh samples were

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analyzed on the day they were collected. California-style black ripe olives were produced using

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typical commercial processing.11 Briefly, the olives were prepared by immersion in a 1− 2%

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sodium hydroxide solution over three consecutive days until the lye penetrated the cuticle layers,

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1-2 mm of the pulp and to the pit. During the intervals between lye treatments, the fruit was

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suspended in water in which air is bubbled for 24 h. The oxygen is required for the black color

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formation, an oxidation reaction which results from the oxidation and polymerization of o-

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diphenols, mainly hydroxytyrosol and caffeic acid. A ferrous gluconate solution (0.1% w/v) was

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added for 24 h to fix the developed black color. Olives were then rinsed with water to neutralize

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the solution. Olives were packed in a 3% sodium chloride solution and sterilized at 121 ° C for

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15 min.

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Reference Compounds. Hydroxytyrosol, oleuropein, rutin, chlorogenic acid, o-coumaric acid

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were purchased from Extrasynthese (Genay, France) and their purity as stated by the supplier

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was >98%. Hydroxytyrosol-4-O-glucoside, luteolin-7-O-glucoside, verbascoside, oleoside-11-



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methyl ester, 2,6-dimethoxy-p-benzoquinone, oleuropein and ligstroside aglycones were isolated

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after chromatographic purification from olives. The identity and purity of the isolated

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compounds was established using NMR spectroscopy and in all cases was>95%.

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Extraction and Sample Preparation. A random sampling of 20-30 olives were separated into

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three sub-samples. Each subsample was homogenized individually. A 10 g (wet weight) sample

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of olive pulp was removed and extracted immediately with 25 mL of MeOH:H2O (v/v 4:1) in an

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ultrasonic bath for 45 min at 25 oC. A 25-mL aliquot of hexane was added for oil removal and

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the mixture was shaken for 30 s. The supernatant was separated with centrifugation at 4000 rpm

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for 3 min. The hexane phase was removed and a 1-mL aliquot of the MeOH:H2O phase was

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filtered through a 0.45-µm filter (EMD Millipore, Billerica, MA), diluted with H2O (1:50) and

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injected into the LC-MS.

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UHPLC-MS/MS Analysis. Analysis of the 12 phenolic and secoiridoid compounds was

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performed on an 1290 Infinity ultrahigh-pressure liquid chromatography system (UHPLC)

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interfaced to an 6460 triple-quadrupole mass spectrometer (QqQ MS/MS) with electrospray

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ionization (ESI) via Jet Stream Technology (Agilent Technologies, Santa Clara, CA). The

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UHPLC was equipped with a binary pump with an integrated vacuum degasser (G4220A), an

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autosampler (G4226A) with thermostat (G1330B), and a thermostated column compartment

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(G1316C). The column used was a 150 mm x 2.1 mm i.d., 2.7 µm, Poroshell 120 EC-C18

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(Agilent Technologies, Santa Clara, CA). The mobile phase consisted of 0.1% formic acid in

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water (A) and acetonitrile (B) with the following gradient program: 0-2.5 min 10% B; 2.5-3.0

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min 10-25% B; 2.5-3.0 25% B; 3.0-6.0min 25% B; 6.0-7.5 min 25-40% B; 7.0-8.5 min 40-95%

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B; 8.5-9.5 min 95% B. The flow rate was 0.4 mL/min, and the injection volume was 1.0 µL.


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Negative and positive ESI mode was used. The drying gas temperatures and flow rate were

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250°C and 8 L/min, respectively. The sheath gas temperature and flow rate were 350°C and 11

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L/min, respectively and the nebulizer gas pressure was 45 psi. The capillary voltage was 3.5 kV

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for positive and negative modes and the nozzle voltage was 0 kV and 1.5 kV for positive and

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negative mode respectively.

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Dynamic multiple reaction monitoring (dMRM) mode was used to analyze olive phenolic and

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secoiridoid compounds. A cycle time of 500 ms was used. Dwell times were automatically set

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using cycle time and peak overlap. Precursor ions and product ions were identified and

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optimized using MassHunter Optimizer (Agilent Technologies). A summary of the precursor

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ions and product ions used for quantitation is given in Table 1.

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Calibration Curves. Standard stock solutions of 1.0 mg/mL were prepared in methanol. Stock

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solutions were diluted in MeOH:H2O (4:1, v/v) to give concentration ranging from 0.001 µg/mL

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to 50 µg/mL. Stock solutions were used to identify the linear range for quantitation (LRQ), the

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limit of detection (LOD; S/N > 3) and limit of quantitation(LOQ; S/N > 10) for each analyte.

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Olive samples were spiked to give concentrations of analytes at 0.002, 0.02, 0.2, 1, 2, 5 mg/g

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olive by diluting appropriate volumes of the stock standard solutions with MeOH:H2O (4:1 v/v)

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and mixing with 10.0 g of olive flesh (cv. Manzanilla) after California-style black ripe

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processing. This olive matrix was selected because it presented the lowest concentrations of the

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analytes making easier the construction of the calibrations curves. The area of each analyte was

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measured in a blank matrix standard and then again after the addition of a known amount of

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standard analyte to the matrix. The difference in area measured for each analyte peak, before and

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after spiking, was directly correlated with the spiked amount to construct the calibration curve.

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Method Validation.The method was checked for the linearity, precision [calculated as the

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relative standard deviation % (RSD %)], accuracy [evaluated as the relative percentage error %

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(Er%)], and sensitivity [defined by the LOD and LOQ].

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RESULTS AND DISCUSSION

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A UHPLC-QqQ MS/MS method utilizing dynamic MRM was developed to

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quantitate a range of bitter and biologically active phenolic and secoiridoid compounds in table

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olives. This method offers improved sensitivity, selectivity and throughput (< 10 min/ per run) in

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comparison to previously reported methods as reviewed by Segura-Carretero et al.12 The

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usefulness of the new method was demonstrated in the simultaneous quantitation of a broad

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range of phenolic and secoiridoid compounds in fresh and finished olive products generated by

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dry-salt or California-style debittering methods. Rapid reliable methods for the quantitation of

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hydroxytyrosol, oleuropein and their derivatives is of special interest since these compounds

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have been recently recognized by the EU as agents protecting low density lipoprotein (LDL)

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oxidation and thus offering cardiovascular protection.13 Currently this health claim is recognized

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only for olive oil. To date several GC/MS, HPLC-UV or LC/MS methods are available for

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monitoring phenolic and secoiridoid compounds in olive products (e.g. olive oil, olive leaves,

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olive fruits).12 The GC/MS methods require derivatization and long analysis times (30-120

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min).14 The HPLC-UV methods have low sensitivity (e.g LOQ = 1 µg/mL for hydroxytyrosol5)

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and are limited by long chromatographic separation times (20-100 mins).12 Additionally in some

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cases, HPLC-UV methods are unable to resolve overlapped peaks15 making difficult the

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simultaneous quantitation of numerous compounds, especially in short separation times. Rapid

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LC/MS methods have been applied to unprocessed olives but are restricted to the quantitation of

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oleuropein and hydroxytyrosol.16,17 An HPLC-Orbitrap MS/MS method for quantitation of nine

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compounds in fresh olives was recently reported.18 The HPLC-Orbitrap and the current UHPLC 8 ACS Paragon Plus Environment

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QqQ methods present similar LODs and LOQs for oleuropein, hydroxytyrosol, oleuropein

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aglycone and ligstroside aglycone, the only four common compounds studied using the two

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methods, however the Orbitrap method presented longer analysis time (31 min) and the

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quantitation range was restricted to higher concentrations (e.g 1000-50000 ng/mL as compared

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to 100-12500 ng/mL for oleuropein aglycone and 500-20000 ng/mL vs 50-6250 ng/mL for

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hydroxytyrosol). Moreover, the HPLC-Orbitrap method relies on calibration curves that were

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constructed by dilution of standard compounds in a different solvent (acetonitrile) than the

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solvent used for the dilution of the real samples (methanol and water). Extraction or dilution of

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oleocanthal and oleacein using methanol can have a negative impact on their levels as recently

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reported19,20 and has the potential to impact other compounds (e.g. oleuropein aglycone and

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ligstroside aglycone) as these two compounds easily isomerize20 and require well controlled

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conditions for accurate measurements.

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Recovery from the olive matrix varied. At high concentrations of added standards (1-5

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ppm) recoveries were 85-95% whereas at low concentrations (0.1-0.5 ppm) recoveries were 50-

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60%. For this reason it was necessary to use calibration curves constructed with addition of

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known amounts to olive matrix instead of calibration curves constructed with pure compounds in

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solution. The method was checked for linearity, precision, accuracy, LOD and LOQ and the

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results were satisfactory. These results show that the method can be efficiently used for the

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measurement of target analytes. Intra-day precision (repeatability) was in all cases

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