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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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.
Page 1 of 21
Journal of Agricultural and Food Chemistry
1 2
UHPLC-QQQ MS/MS Quantitation of Polyphenols and Secoiridoids in
3
California-style Black Ripe Olives and Dry-Salt Cured Olives
4 Eleni Melliou†,‡,*, Jerry A. Zweigenbaum§ and Alyson E. Mithcell‡
5 6 7
†
8
University of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece;
9
‡
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,
Department of Food Science and Technology, University of California, Davis, One Shields
10
Avenue, Davis, CA 95616, USA;
11
§
Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808, USA
12 13 14
* To whom correspondence should be addressed. Tel: +30210 7274052. E-mail:
15
[email protected]
16 17 18 19
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 21
Abstract
20 21 22
The chemical composition of finishedtable olive products isinfluenced by the olive variety and
23
the processing method used to debitter or cure table olives. Herein a rapid UHPLC-QqQ MS/MS
24
method, utilizing dynamic MRM, was developed for the quantitation of 12 predominant phenolic
25
and secoiridoid compounds in olive fruit including: hydroxytyrosol, oleuropein, hydroxytyrosol-
26
4-O-glucoside, luteolin-7-O-glucoside, rutin, verbascoside, oleoside-11-methyl ester, 2,6-
27
dimethoxy-p-benzoquinone, phenolic acids (chlorogenic acid, o-coumaric acid), oleuropein
28
aglycone and ligstroside aglycone. Levels of these compounds were measured in fresh and
29
California-style black ripe processed Manzanilla olives, and in two dry-salt cured olive varieties
30
(Mission from California and Throuba Thassos from Greece).Results indicate that the variety
31
and debittering processing method have strong impact on the profile of phenolic and secoiridoid
32
compoundsin table olives. The dry-salt cured olives contained higher amounts of most
33
compounds studied and especially oleuropein (1459.5±100.1µg/g),whereas California-style black
34
ripe olives had a significant reduction or loss of these bioactive compounds (e.goleuropein level
35
at 36.7±3.1µg/g).
36 37 38
Keywords: table olives, polyphenols, secoiridoids, California-style black ripe, dry-salt, UHPLC-
39
QqQ MS/MS
40
2 ACS Paragon Plus Environment
Page 3 of 21
41
Journal of Agricultural and Food Chemistry
INTRODUCTION
42
Table olives from Olea europaea L. are a traditional product and an important
43
component of the Mediterranean diet.World consumption of table olives is assessed at 2,521,500
44
tons in 2013.1 There are three main categories of table olives based upon the trade preparation
45
methods including: green (or Spanish style), natural black (or Greek style), and California-style
46
black ripe olives.2 The California-style black ripe olives are the most widely consumed olive
47
among American consumers. Olive fruit is a rich source of compounds with health protecting
48
activities and include: hydroxytyrosol, oleuropein, and many other related biophenols and
49
secoiridoid derivatives.3 The complement of bioactives in the final olive products is influenced
50
by the olive variety and the debittering method used in preparing the olives.4Debittering methods
51
are of interest, asprevious studies indicate they can lead to olives with increased or significantly
52
reduced healthy properties.5
53
A native to the Mediterranean, historically olives were first consumed as tree-ripened
54
fruit. Olives lose bitterness through natural ripening processes (in Greece known as throuba or
55
stafidolia).6 Inhabitants of the Mediterranean area, who relied on olives as an essential part of
56
their diet, developed various methods to preserve olives after harvesting. For many centuries, the
57
basic ingredient usedto preserve olives was salt. Additional ingredients used in olive preservation
58
included: honey, olive oil, vinegar and vine juice.6 The major bitter component of olives is
59
oleuropein and its derivatives.7 Oleuropein is removed during dry-salt curing and can also be
60
removed through other processes including lye-assisted hydrolysis of oleuropein.7-9 In Greece
61
olives are now produced primarily using brine (known as Greek style), dry-salt, and lye (i.e. a 1-
62
2% sodium hydroxide solution). In other countries such as Spain, Italy and the United States,
63
most producers rely on lye to assist in debittering olives (Spanish-style and California-style black
64
ripe olives).
65
Throuba Thassos olives (also called Stafidolies, twisted, or wrinkled olives) are a
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 21
66
culturally important edible olive in Greece. Traditionally, this fruit isover-ripened on the tree
67
giving it a wrinkled appearance and requires no additional processing to reduce bitterness.
68
However, for economic reasons Throuba Thassos olives are now produced almost exclusively
69
using dry-salt processing to give a similar wrinkled appearance as tree ripened olives. Despite
70
the similar appearance, the two different types of processing lead to olives with different
71
chemical profiles. In contrast to the naturally overripe olives, which contain very low amounts of
72
oleuropein,9 dry-salt processing leads to a higher concentration of oleuropein5 highlighting the
73
critical role of processing in the chemical composition of finished olives.4 California-style black
74
ripe olive processing methods involve harvesting olives before complete maturity (green). The
75
raw olives are either directly treated with lye to remove bitterness or preserved in an acidic brine
76
solution (0.2−0.4% calcium chloride, pH < 4.0) until they can be lye treated and processed. A
77
mild fermentation may occur during the initial brine storage.This style of processing results in
78
olives with the lowest levels of oleuropein and bitterness.10
79
Herein, we describe a rapid ultrahigh-pressureliquid chromatography (UHPLC) triple
80
quadrupole MS/MS (QqQ MS/MS) method utilizing dynamic multiple reaction monitoring
81
(dMRM) for the measurement ofa range of keybitter and bioactive constituents in olives. The
82
method was applied for the simultaneous quantitation of hydroxytyrosol-4-O-glucoside, 1,
83
hydroxytyrosol, 2, 2,6-dimethoxy-p-benzoquinone, 3, chlorogenic acid, 4, oleoside-11-methyl
84
ester, 5, rutin, 6, verbascoside, 7, luteolin-7-O-glucoside, 8, o-coumaric acid, 9, oleuropein, 10,
85
oleuropein aglycone monoaldehydic form, 11 and ligstroside aglycone monoaldehydic form, 12
86
(Figure 1) in three different varietiesof fresh and processed olives cured using either dry-salt or
87
California-style black ripe processing methods.
88 89
MATERIALS AND METHODS
90
4 ACS Paragon Plus Environment
Page 5 of 21
Journal of Agricultural and Food Chemistry
91
Chemicals. HPLC grade formic acid, acetonitrile, hexane and methanol were obtained from
92
Fisher Scientific (Fair Lawn, NJ).
93 94
Samples. Dry-salt cured Throuba Thassos olives were purchased from a local market in Davis,
95
CA and the producer was contacted for processing conditions. The Throuba Thassos olives were
96
treated with 40% dry-salt for 2 mo. After curing, the olives were washed with water and dried at
97
ambient temperature for 2-3 days before packaging. Dry-salt Mission olives were purchased
98
directly from the grower/processor at the local Farmers Market in Davis, CA. According to the
99
producer, the olives were treated with dry-salt 1:1 w/w for 3 mo. After curing, the olives were
100
washed with water and sun dried. California-style black ripe olives were prepared using fresh
101
Manzanilla olives collected in early October 2011 in Davis, CA. The fresh samples were
102
analyzed on the day they were collected. California-style black ripe olives were produced using
103
typical commercial processing.11 Briefly, the olives were prepared by immersion in a 1− 2%
104
sodium hydroxide solution over three consecutive days until the lye penetrated the cuticle layers,
105
1-2 mm of the pulp and to the pit. During the intervals between lye treatments, the fruit was
106
suspended in water in which air is bubbled for 24 h. The oxygen is required for the black color
107
formation, an oxidation reaction which results from the oxidation and polymerization of o-
108
diphenols, mainly hydroxytyrosol and caffeic acid. A ferrous gluconate solution (0.1% w/v) was
109
added for 24 h to fix the developed black color. Olives were then rinsed with water to neutralize
110
the solution. Olives were packed in a 3% sodium chloride solution and sterilized at 121 ° C for
111
15 min.
112 113
Reference Compounds. Hydroxytyrosol, oleuropein, rutin, chlorogenic acid, o-coumaric acid
114
were purchased from Extrasynthese (Genay, France) and their purity as stated by the supplier
115
was >98%. Hydroxytyrosol-4-O-glucoside, luteolin-7-O-glucoside, verbascoside, oleoside-11-
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 21
116
methyl ester, 2,6-dimethoxy-p-benzoquinone, oleuropein and ligstroside aglycones were isolated
117
after chromatographic purification from olives. The identity and purity of the isolated
118
compounds was established using NMR spectroscopy and in all cases was>95%.
119 120
Extraction and Sample Preparation. A random sampling of 20-30 olives were separated into
121
three sub-samples. Each subsample was homogenized individually. A 10 g (wet weight) sample
122
of olive pulp was removed and extracted immediately with 25 mL of MeOH:H2O (v/v 4:1) in an
123
ultrasonic bath for 45 min at 25 oC. A 25-mL aliquot of hexane was added for oil removal and
124
the mixture was shaken for 30 s. The supernatant was separated with centrifugation at 4000 rpm
125
for 3 min. The hexane phase was removed and a 1-mL aliquot of the MeOH:H2O phase was
126
filtered through a 0.45-µm filter (EMD Millipore, Billerica, MA), diluted with H2O (1:50) and
127
injected into the LC-MS.
128 129
UHPLC-MS/MS Analysis. Analysis of the 12 phenolic and secoiridoid compounds was
130
performed on an 1290 Infinity ultrahigh-pressure liquid chromatography system (UHPLC)
131
interfaced to an 6460 triple-quadrupole mass spectrometer (QqQ MS/MS) with electrospray
132
ionization (ESI) via Jet Stream Technology (Agilent Technologies, Santa Clara, CA). The
133
UHPLC was equipped with a binary pump with an integrated vacuum degasser (G4220A), an
134
autosampler (G4226A) with thermostat (G1330B), and a thermostated column compartment
135
(G1316C). The column used was a 150 mm x 2.1 mm i.d., 2.7 µm, Poroshell 120 EC-C18
136
(Agilent Technologies, Santa Clara, CA). The mobile phase consisted of 0.1% formic acid in
137
water (A) and acetonitrile (B) with the following gradient program: 0-2.5 min 10% B; 2.5-3.0
138
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%
139
B; 8.5-9.5 min 95% B. The flow rate was 0.4 mL/min, and the injection volume was 1.0 µL.
6 ACS Paragon Plus Environment
Page 7 of 21
Journal of Agricultural and Food Chemistry
140
Negative and positive ESI mode was used. The drying gas temperatures and flow rate were
141
250°C and 8 L/min, respectively. The sheath gas temperature and flow rate were 350°C and 11
142
L/min, respectively and the nebulizer gas pressure was 45 psi. The capillary voltage was 3.5 kV
143
for positive and negative modes and the nozzle voltage was 0 kV and 1.5 kV for positive and
144
negative mode respectively.
145
Dynamic multiple reaction monitoring (dMRM) mode was used to analyze olive phenolic and
146
secoiridoid compounds. A cycle time of 500 ms was used. Dwell times were automatically set
147
using cycle time and peak overlap. Precursor ions and product ions were identified and
148
optimized using MassHunter Optimizer (Agilent Technologies). A summary of the precursor
149
ions and product ions used for quantitation is given in Table 1.
150 151
Calibration Curves. Standard stock solutions of 1.0 mg/mL were prepared in methanol. Stock
152
solutions were diluted in MeOH:H2O (4:1, v/v) to give concentration ranging from 0.001 µg/mL
153
to 50 µg/mL. Stock solutions were used to identify the linear range for quantitation (LRQ), the
154
limit of detection (LOD; S/N > 3) and limit of quantitation(LOQ; S/N > 10) for each analyte.
155
Olive samples were spiked to give concentrations of analytes at 0.002, 0.02, 0.2, 1, 2, 5 mg/g
156
olive by diluting appropriate volumes of the stock standard solutions with MeOH:H2O (4:1 v/v)
157
and mixing with 10.0 g of olive flesh (cv. Manzanilla) after California-style black ripe
158
processing. This olive matrix was selected because it presented the lowest concentrations of the
159
analytes making easier the construction of the calibrations curves. The area of each analyte was
160
measured in a blank matrix standard and then again after the addition of a known amount of
161
standard analyte to the matrix. The difference in area measured for each analyte peak, before and
162
after spiking, was directly correlated with the spiked amount to construct the calibration curve.
163
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 21
164
Method Validation.The method was checked for the linearity, precision [calculated as the
165
relative standard deviation % (RSD %)], accuracy [evaluated as the relative percentage error %
166
(Er%)], and sensitivity [defined by the LOD and LOQ].
167 168
RESULTS AND DISCUSSION
169
A UHPLC-QqQ MS/MS method utilizing dynamic MRM was developed to
170
quantitate a range of bitter and biologically active phenolic and secoiridoid compounds in table
171
olives. This method offers improved sensitivity, selectivity and throughput (< 10 min/ per run) in
172
comparison to previously reported methods as reviewed by Segura-Carretero et al.12 The
173
usefulness of the new method was demonstrated in the simultaneous quantitation of a broad
174
range of phenolic and secoiridoid compounds in fresh and finished olive products generated by
175
dry-salt or California-style debittering methods. Rapid reliable methods for the quantitation of
176
hydroxytyrosol, oleuropein and their derivatives is of special interest since these compounds
177
have been recently recognized by the EU as agents protecting low density lipoprotein (LDL)
178
oxidation and thus offering cardiovascular protection.13 Currently this health claim is recognized
179
only for olive oil. To date several GC/MS, HPLC-UV or LC/MS methods are available for
180
monitoring phenolic and secoiridoid compounds in olive products (e.g. olive oil, olive leaves,
181
olive fruits).12 The GC/MS methods require derivatization and long analysis times (30-120
182
min).14 The HPLC-UV methods have low sensitivity (e.g LOQ = 1 µg/mL for hydroxytyrosol5)
183
and are limited by long chromatographic separation times (20-100 mins).12 Additionally in some
184
cases, HPLC-UV methods are unable to resolve overlapped peaks15 making difficult the
185
simultaneous quantitation of numerous compounds, especially in short separation times. Rapid
186
LC/MS methods have been applied to unprocessed olives but are restricted to the quantitation of
187
oleuropein and hydroxytyrosol.16,17 An HPLC-Orbitrap MS/MS method for quantitation of nine
188
compounds in fresh olives was recently reported.18 The HPLC-Orbitrap and the current UHPLC 8 ACS Paragon Plus Environment
Page 9 of 21
Journal of Agricultural and Food Chemistry
189
QqQ methods present similar LODs and LOQs for oleuropein, hydroxytyrosol, oleuropein
190
aglycone and ligstroside aglycone, the only four common compounds studied using the two
191
methods, however the Orbitrap method presented longer analysis time (31 min) and the
192
quantitation range was restricted to higher concentrations (e.g 1000-50000 ng/mL as compared
193
to 100-12500 ng/mL for oleuropein aglycone and 500-20000 ng/mL vs 50-6250 ng/mL for
194
hydroxytyrosol). Moreover, the HPLC-Orbitrap method relies on calibration curves that were
195
constructed by dilution of standard compounds in a different solvent (acetonitrile) than the
196
solvent used for the dilution of the real samples (methanol and water). Extraction or dilution of
197
oleocanthal and oleacein using methanol can have a negative impact on their levels as recently
198
reported19,20 and has the potential to impact other compounds (e.g. oleuropein aglycone and
199
ligstroside aglycone) as these two compounds easily isomerize20 and require well controlled
200
conditions for accurate measurements.
201
Recovery from the olive matrix varied. At high concentrations of added standards (1-5
202
ppm) recoveries were 85-95% whereas at low concentrations (0.1-0.5 ppm) recoveries were 50-
203
60%. For this reason it was necessary to use calibration curves constructed with addition of
204
known amounts to olive matrix instead of calibration curves constructed with pure compounds in
205
solution. The method was checked for linearity, precision, accuracy, LOD and LOQ and the
206
results were satisfactory. These results show that the method can be efficiently used for the
207
measurement of target analytes. Intra-day precision (repeatability) was in all cases
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.
Page 1 of 21
Journal of Agricultural and Food Chemistry
1 2
UHPLC-QQQ MS/MS Quantitation of Polyphenols and Secoiridoids in
3
California-style Black Ripe Olives and Dry-Salt Cured Olives
4 Eleni Melliou†,‡,*, Jerry A. Zweigenbaum§ and Alyson E. Mithcell‡
5 6 7
†
8
University of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece;
9
‡
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,
Department of Food Science and Technology, University of California, Davis, One Shields
10
Avenue, Davis, CA 95616, USA;
11
§
Agilent Technologies, 2850 Centerville Road, Wilmington, DE 19808, USA
12 13 14
* To whom correspondence should be addressed. Tel: +30210 7274052. E-mail:
15
[email protected]
16 17 18 19
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 21
Abstract
20 21 22
The chemical composition of finishedtable olive products isinfluenced by the olive variety and
23
the processing method used to debitter or cure table olives. Herein a rapid UHPLC-QqQ MS/MS
24
method, utilizing dynamic MRM, was developed for the quantitation of 12 predominant phenolic
25
and secoiridoid compounds in olive fruit including: hydroxytyrosol, oleuropein, hydroxytyrosol-
26
4-O-glucoside, luteolin-7-O-glucoside, rutin, verbascoside, oleoside-11-methyl ester, 2,6-
27
dimethoxy-p-benzoquinone, phenolic acids (chlorogenic acid, o-coumaric acid), oleuropein
28
aglycone and ligstroside aglycone. Levels of these compounds were measured in fresh and
29
California-style black ripe processed Manzanilla olives, and in two dry-salt cured olive varieties
30
(Mission from California and Throuba Thassos from Greece).Results indicate that the variety
31
and debittering processing method have strong impact on the profile of phenolic and secoiridoid
32
compoundsin table olives. The dry-salt cured olives contained higher amounts of most
33
compounds studied and especially oleuropein (1459.5±100.1µg/g),whereas California-style black
34
ripe olives had a significant reduction or loss of these bioactive compounds (e.goleuropein level
35
at 36.7±3.1µg/g).
36 37 38
Keywords: table olives, polyphenols, secoiridoids, California-style black ripe, dry-salt, UHPLC-
39
QqQ MS/MS
40
2 ACS Paragon Plus Environment
Page 3 of 21
41
Journal of Agricultural and Food Chemistry
INTRODUCTION
42
Table olives from Olea europaea L. are a traditional product and an important
43
component of the Mediterranean diet.World consumption of table olives is assessed at 2,521,500
44
tons in 2013.1 There are three main categories of table olives based upon the trade preparation
45
methods including: green (or Spanish style), natural black (or Greek style), and California-style
46
black ripe olives.2 The California-style black ripe olives are the most widely consumed olive
47
among American consumers. Olive fruit is a rich source of compounds with health protecting
48
activities and include: hydroxytyrosol, oleuropein, and many other related biophenols and
49
secoiridoid derivatives.3 The complement of bioactives in the final olive products is influenced
50
by the olive variety and the debittering method used in preparing the olives.4Debittering methods
51
are of interest, asprevious studies indicate they can lead to olives with increased or significantly
52
reduced healthy properties.5
53
A native to the Mediterranean, historically olives were first consumed as tree-ripened
54
fruit. Olives lose bitterness through natural ripening processes (in Greece known as throuba or
55
stafidolia).6 Inhabitants of the Mediterranean area, who relied on olives as an essential part of
56
their diet, developed various methods to preserve olives after harvesting. For many centuries, the
57
basic ingredient usedto preserve olives was salt. Additional ingredients used in olive preservation
58
included: honey, olive oil, vinegar and vine juice.6 The major bitter component of olives is
59
oleuropein and its derivatives.7 Oleuropein is removed during dry-salt curing and can also be
60
removed through other processes including lye-assisted hydrolysis of oleuropein.7-9 In Greece
61
olives are now produced primarily using brine (known as Greek style), dry-salt, and lye (i.e. a 1-
62
2% sodium hydroxide solution). In other countries such as Spain, Italy and the United States,
63
most producers rely on lye to assist in debittering olives (Spanish-style and California-style black
64
ripe olives).
65
Throuba Thassos olives (also called Stafidolies, twisted, or wrinkled olives) are a
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 21
66
culturally important edible olive in Greece. Traditionally, this fruit isover-ripened on the tree
67
giving it a wrinkled appearance and requires no additional processing to reduce bitterness.
68
However, for economic reasons Throuba Thassos olives are now produced almost exclusively
69
using dry-salt processing to give a similar wrinkled appearance as tree ripened olives. Despite
70
the similar appearance, the two different types of processing lead to olives with different
71
chemical profiles. In contrast to the naturally overripe olives, which contain very low amounts of
72
oleuropein,9 dry-salt processing leads to a higher concentration of oleuropein5 highlighting the
73
critical role of processing in the chemical composition of finished olives.4 California-style black
74
ripe olive processing methods involve harvesting olives before complete maturity (green). The
75
raw olives are either directly treated with lye to remove bitterness or preserved in an acidic brine
76
solution (0.2−0.4% calcium chloride, pH < 4.0) until they can be lye treated and processed. A
77
mild fermentation may occur during the initial brine storage.This style of processing results in
78
olives with the lowest levels of oleuropein and bitterness.10
79
Herein, we describe a rapid ultrahigh-pressureliquid chromatography (UHPLC) triple
80
quadrupole MS/MS (QqQ MS/MS) method utilizing dynamic multiple reaction monitoring
81
(dMRM) for the measurement ofa range of keybitter and bioactive constituents in olives. The
82
method was applied for the simultaneous quantitation of hydroxytyrosol-4-O-glucoside, 1,
83
hydroxytyrosol, 2, 2,6-dimethoxy-p-benzoquinone, 3, chlorogenic acid, 4, oleoside-11-methyl
84
ester, 5, rutin, 6, verbascoside, 7, luteolin-7-O-glucoside, 8, o-coumaric acid, 9, oleuropein, 10,
85
oleuropein aglycone monoaldehydic form, 11 and ligstroside aglycone monoaldehydic form, 12
86
(Figure 1) in three different varietiesof fresh and processed olives cured using either dry-salt or
87
California-style black ripe processing methods.
88 89
MATERIALS AND METHODS
90
4 ACS Paragon Plus Environment
Page 5 of 21
Journal of Agricultural and Food Chemistry
91
Chemicals. HPLC grade formic acid, acetonitrile, hexane and methanol were obtained from
92
Fisher Scientific (Fair Lawn, NJ).
93 94
Samples. Dry-salt cured Throuba Thassos olives were purchased from a local market in Davis,
95
CA and the producer was contacted for processing conditions. The Throuba Thassos olives were
96
treated with 40% dry-salt for 2 mo. After curing, the olives were washed with water and dried at
97
ambient temperature for 2-3 days before packaging. Dry-salt Mission olives were purchased
98
directly from the grower/processor at the local Farmers Market in Davis, CA. According to the
99
producer, the olives were treated with dry-salt 1:1 w/w for 3 mo. After curing, the olives were
100
washed with water and sun dried. California-style black ripe olives were prepared using fresh
101
Manzanilla olives collected in early October 2011 in Davis, CA. The fresh samples were
102
analyzed on the day they were collected. California-style black ripe olives were produced using
103
typical commercial processing.11 Briefly, the olives were prepared by immersion in a 1− 2%
104
sodium hydroxide solution over three consecutive days until the lye penetrated the cuticle layers,
105
1-2 mm of the pulp and to the pit. During the intervals between lye treatments, the fruit was
106
suspended in water in which air is bubbled for 24 h. The oxygen is required for the black color
107
formation, an oxidation reaction which results from the oxidation and polymerization of o-
108
diphenols, mainly hydroxytyrosol and caffeic acid. A ferrous gluconate solution (0.1% w/v) was
109
added for 24 h to fix the developed black color. Olives were then rinsed with water to neutralize
110
the solution. Olives were packed in a 3% sodium chloride solution and sterilized at 121 ° C for
111
15 min.
112 113
Reference Compounds. Hydroxytyrosol, oleuropein, rutin, chlorogenic acid, o-coumaric acid
114
were purchased from Extrasynthese (Genay, France) and their purity as stated by the supplier
115
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
117
after chromatographic purification from olives. The identity and purity of the isolated
118
compounds was established using NMR spectroscopy and in all cases was>95%.
119 120
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
122
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
125
for 3 min. The hexane phase was removed and a 1-mL aliquot of the MeOH:H2O phase was
126
filtered through a 0.45-µm filter (EMD Millipore, Billerica, MA), diluted with H2O (1:50) and
127
injected into the LC-MS.
128 129
UHPLC-MS/MS Analysis. Analysis of the 12 phenolic and secoiridoid compounds was
130
performed on an 1290 Infinity ultrahigh-pressure liquid chromatography system (UHPLC)
131
interfaced to an 6460 triple-quadrupole mass spectrometer (QqQ MS/MS) with electrospray
132
ionization (ESI) via Jet Stream Technology (Agilent Technologies, Santa Clara, CA). The
133
UHPLC was equipped with a binary pump with an integrated vacuum degasser (G4220A), an
134
autosampler (G4226A) with thermostat (G1330B), and a thermostated column compartment
135
(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
137
water (A) and acetonitrile (B) with the following gradient program: 0-2.5 min 10% B; 2.5-3.0
138
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%
139
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
142
L/min, respectively and the nebulizer gas pressure was 45 psi. The capillary voltage was 3.5 kV
143
for positive and negative modes and the nozzle voltage was 0 kV and 1.5 kV for positive and
144
negative mode respectively.
145
Dynamic multiple reaction monitoring (dMRM) mode was used to analyze olive phenolic and
146
secoiridoid compounds. A cycle time of 500 ms was used. Dwell times were automatically set
147
using cycle time and peak overlap. Precursor ions and product ions were identified and
148
optimized using MassHunter Optimizer (Agilent Technologies). A summary of the precursor
149
ions and product ions used for quantitation is given in Table 1.
150 151
Calibration Curves. Standard stock solutions of 1.0 mg/mL were prepared in methanol. Stock
152
solutions were diluted in MeOH:H2O (4:1, v/v) to give concentration ranging from 0.001 µg/mL
153
to 50 µg/mL. Stock solutions were used to identify the linear range for quantitation (LRQ), the
154
limit of detection (LOD; S/N > 3) and limit of quantitation(LOQ; S/N > 10) for each analyte.
155
Olive samples were spiked to give concentrations of analytes at 0.002, 0.02, 0.2, 1, 2, 5 mg/g
156
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
158
processing. This olive matrix was selected because it presented the lowest concentrations of the
159
analytes making easier the construction of the calibrations curves. The area of each analyte was
160
measured in a blank matrix standard and then again after the addition of a known amount of
161
standard analyte to the matrix. The difference in area measured for each analyte peak, before and
162
after spiking, was directly correlated with the spiked amount to construct the calibration curve.
163
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Method Validation.The method was checked for the linearity, precision [calculated as the
165
relative standard deviation % (RSD %)], accuracy [evaluated as the relative percentage error %
166
(Er%)], and sensitivity [defined by the LOD and LOQ].
167 168
RESULTS AND DISCUSSION
169
A UHPLC-QqQ MS/MS method utilizing dynamic MRM was developed to
170
quantitate a range of bitter and biologically active phenolic and secoiridoid compounds in table
171
olives. This method offers improved sensitivity, selectivity and throughput (< 10 min/ per run) in
172
comparison to previously reported methods as reviewed by Segura-Carretero et al.12 The
173
usefulness of the new method was demonstrated in the simultaneous quantitation of a broad
174
range of phenolic and secoiridoid compounds in fresh and finished olive products generated by
175
dry-salt or California-style debittering methods. Rapid reliable methods for the quantitation of
176
hydroxytyrosol, oleuropein and their derivatives is of special interest since these compounds
177
have been recently recognized by the EU as agents protecting low density lipoprotein (LDL)
178
oxidation and thus offering cardiovascular protection.13 Currently this health claim is recognized
179
only for olive oil. To date several GC/MS, HPLC-UV or LC/MS methods are available for
180
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
182
min).14 The HPLC-UV methods have low sensitivity (e.g LOQ = 1 µg/mL for hydroxytyrosol5)
183
and are limited by long chromatographic separation times (20-100 mins).12 Additionally in some
184
cases, HPLC-UV methods are unable to resolve overlapped peaks15 making difficult the
185
simultaneous quantitation of numerous compounds, especially in short separation times. Rapid
186
LC/MS methods have been applied to unprocessed olives but are restricted to the quantitation of
187
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
190
aglycone and ligstroside aglycone, the only four common compounds studied using the two
191
methods, however the Orbitrap method presented longer analysis time (31 min) and the
192
quantitation range was restricted to higher concentrations (e.g 1000-50000 ng/mL as compared
193
to 100-12500 ng/mL for oleuropein aglycone and 500-20000 ng/mL vs 50-6250 ng/mL for
194
hydroxytyrosol). Moreover, the HPLC-Orbitrap method relies on calibration curves that were
195
constructed by dilution of standard compounds in a different solvent (acetonitrile) than the
196
solvent used for the dilution of the real samples (methanol and water). Extraction or dilution of
197
oleocanthal and oleacein using methanol can have a negative impact on their levels as recently
198
reported19,20 and has the potential to impact other compounds (e.g. oleuropein aglycone and
199
ligstroside aglycone) as these two compounds easily isomerize20 and require well controlled
200
conditions for accurate measurements.
201
Recovery from the olive matrix varied. At high concentrations of added standards (1-5
202
ppm) recoveries were 85-95% whereas at low concentrations (0.1-0.5 ppm) recoveries were 50-
203
60%. For this reason it was necessary to use calibration curves constructed with addition of
204
known amounts to olive matrix instead of calibration curves constructed with pure compounds in
205
solution. The method was checked for linearity, precision, accuracy, LOD and LOQ and the
206
results were satisfactory. These results show that the method can be efficiently used for the
207
measurement of target analytes. Intra-day precision (repeatability) was in all cases
