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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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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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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Journal of Agricultural and Food Chemistry

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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Journal of Agricultural and Food Chemistry

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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