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Determination of calystegines in several tomato varieties based on GC-Q-Orbitrap analysis and their classification by ANOVA Ana Romera-Torres, Francisco Javier Arrebola, José Luis Martínez Vidal, and Antonia Garrido Frenich J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06952 • Publication Date (Web): 13 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Journal of Agricultural and Food Chemistry

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Determination of Calystegines in Several Tomato Varieties Based on GC-Q-

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Orbitrap Analysis and Their Classification by ANOVA

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Ana Romera-Torres, Javier Arrebola-Liébanas, José Luis Martínez Vidal, Antonia

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Garrido Frenich*

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Department of Chemistry and Physics, Research Centre for Agricultural and Food

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Biotechnology (BITAL), University of Almería, Agrifood Campus of International

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Excellence, ceiA3, Carretera de Sacramento s/n, E-04120 Almería, Spain.

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*Corresponding

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[email protected])

author (Tel. +34 950015985; fax: +34 950015008. E-mail:

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ACS Paragon Plus Environment


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Abstract

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In this study, several calystegines (A3, A5, B1, B2, B3, B4 and C1) have been determined

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in tomato. A simple extraction followed by a derivatization step with silylating agents has

19

been performed prior to their analysis by gas chromatography coupled to high resolution

20

mass spectrometry (GC-HRMS-Q-Orbitrap), which allowed the monitoring of several

21

ions at accurate mass. The validation of the method has provided suitable values of

22

linearity, trueness (73.7-120.0%) and precision (≤ 20.0%, except for calystegines B3 and

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B4 at 0.5 mg/kg). The limit of quantitation (LOQ) was set at 0.5 mg/kg for all analytes.

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The validated method was successfully applied to the analysis of nine different tomato

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varieties, and calystegines A3, A5, B2 and C1 were found at concentrations ranging

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between 0.65 mg/kg (C1) and 12.47 mg/kg (B2). Tomato varieties were classified

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according to their calystegines content applying an analysis of variance (ANOVA).

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Keywords: Calystegines, Solanum lycopersicum, tomato varieties, GC-MS analysis, Q-

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Orbitrap, ANOVA.

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Introduction

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Calystegines are a group of alkaloids discovered in 1988 during a determination of

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opines by paper electrophoresis. They were initially discovered as positives spots in

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transformed root cultures of Calystegia sepium and further screening of intact Atropa

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belladonna roots also revealed their presence.1 Their chemical structure contains a

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nortropane ring system with 3, 4 or 5 hydroxyl groups (calystegines A, B or C,

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respectively) located at various positions and with differing stereochemistry, and also

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having an aminoketal functionality.2 Other group of calystegines, named calystegines N,

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are characterized by a bridgehead amino group and also contains a methylated nitrogen.3

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As iminosugars, they usually act as competitive glycosidase inhibitors and are considered

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for treatment of lysosomal storage diseases, e.g. Gaucher disease and Fabry disease.4

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Although it has not been established that they are the main agents, calystegines are

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presented in Solanum dimidiatum and Solanum kwebense, which causes crazy cow

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syndrome and maldronksiekte in South Africa.5

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Because of their closely related structure to tropane alkaloids, calystegines occurrence

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were investigated in several genera, such as Atropa, Datura, Duboisia, Hyoscyamus, and

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Scopolia belonging to Solanaceae family,6 and plant families such as Convolvulaceae,7

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Erythroxylaceae8 and Brassicaceae,9 which are well-known for the presence of tropane

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alkaloids. Solanaceae and Convolvulaceae families have many edible fruits and

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vegetables rich in calystegines. Tomatoes (Solanum lycopersicum L.) are the most

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popular vegetables in the world and around 150.000 million kg were produced in 2017

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according to data from the United Nations Food and Agriculture Organization (FAO).10

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Recently, analytical methods focused on the determination of calystegines have been

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reviewed.11 These polyhydroxylated alkaloids have a high water solubility and are usually

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extracted using different proportions of methanol/water, such as 50:50 (v/v),7,8,12–17 80:20,



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v/v5,18 or 20:80, v/v.14 They have also been extracted with ethanol/water (50:50, v/v)19 or

58

with 0.2% of formic acid in acetonitrile/water (50:50, v/v).20 Then, extracts were passed

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through cation and/or anion exchange resins columns in order to purify them and after

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that, calystegines were usually silylated by a derivatization process. Finally, although

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calystegines have been analyzed by thin layer chromatography21 or capillary

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electrophoresis using an indirect UV detector,22 they are usually analyzed by gas

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chromatography coupled to mass spectrometry with a single quadrupole analyzer (GC-

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Q-MS).7,12,15,18,19 In contrast, neither purification nor derivatization are widely reported

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for liquid chromatography-MS (LC-MS) analysis. In these cases, triple quadrupole

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(QqQ),16,20 Orbitrap17 and a hybrid quadrupole coupled to Orbitrap (Q-Orbitrap)20

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analyzer have been employed.

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High resolution MS (HRMS) instruments, e.g. time of flight (TOF) or Orbitrap

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instruments, measure ions at a high resolution power, and provide full-spectrum data with

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a high mass accuracy. Moreover, the capacity to determine the molecular formula of

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analytes from accurate-mass measurements has become the most important feature of

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HRMS,23 allowing screening of targeted and untargeted compounds. While several

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studies have employed HRMS to analyze similar compounds such as tropane

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alkaloids,24,25 until now, only two LC-HRMS methods have been applied to the

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determination of calystegines.17,20

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In general, studies have been focused on the content of calystegines in several potato

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varieties,5,13,14,26 but so far, little information about calystegines occurrence in tomato is

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available17,27,28 and only four calystegines have been studied. Although one study

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analyzed seven calystegines in tomato and tomato-based product using a 250 x 4.6 mm,

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3 µm, ACE HILIC-A column (Advanced Chromatography Technologies LTD, Aberdeen,

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Scotland),17 a thorough investigation of several varieties has not been carried out. The


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aim of this work was to evaluate the variation of calystegines (A3, A5, B1, B2, B3, B4 and

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C1) content within and between several tomato varieties obtained under the same

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agronomic and environmental conditions. A GC coupled to a hybrid quadrupole-Orbitrap

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analyzer (GC-Q-Orbitrap) was used for first time allowing a highly reliable identification

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of the compounds.

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Material and methods

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Chemicals, reagents and equipment

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Calystegine standards (A3, A5, B1, B2, B3, B4 and C1), structures from 1-7 shown in

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Figure 1, were kindly provided by Professor Naoki Asano of Hokuriku University

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(Kanazawa, Japan) and they were used as reference standards for GC analysis.

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LC-MS grade methanol (purity ≥99.9%) was supplied by Merck-Sigma (St. Louis,

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MO). LC-MS grade water was obtained from Scharlab (Barcelona, Spain), formic acid

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(Optima LC-MS) was acquired from Fisher Scientific (Erembodegem, Belgium), and

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HPLC grade n-hexane (purity 99.9%) was provided by VWR Chemicals (Radnor, PA).

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Anhydrous pyridine (purity 99.8%), hexamethyldisilazane (HMDS) (reagent grade, ≥

98

99%) and chlorotrimethylsilane (TMCS) for GC derivatization (purity ≥ 99.0%) were

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provided by Merck-Sigma (St. Louis, MO).

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A Trace 1300 GC (Thermo Fisher Scientific, Bremen, Germany) was used for the

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chromatographic analysis. It was equipped with a TriPlus RSH autosampler (Thermo

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Fisher Scientific), and the column used was a 30 m x 0.25 mm i.d., 0.25 μm, VF-5ms

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(Agilent, Santa Clara, CA). As MS detector, a Q-Exactive Orbitrap mass analyzer

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(Thermo Fisher Scientific) was used.

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Samples collection



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Samples of nine tomato varieties were supplied by a greenhouse farmer from Almería

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(southeast Spain). All they were grown under the same climatic and agronomic conditions

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and into the same greenhouse. Three different samples of each var. were selected in order

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to evaluate potential differences between varieties and within varieties. The tomato types

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were: one oxheart, var. “Monterosa”; one black tomato, var. “Kumato”; two cherry, (one

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common cherry, var. “Zoraida”, and another cherry oval, var. “Satyplum”); two saladette,

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var. “Bernal” (A and B); and three cluster or tomato on the vine (TOV), varieties

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“Ateneo”, “Guanche” and “Motto Fi”.

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Sample extraction

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Each sample (≥1 kg) was ground and homogenized. An aliquot of 1 g was weighed

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and mixed with 10 mL of methanol/water (50:50, v/v). The mixture was shaken for 1 min

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with a vortex. Afterwards, the sample was centrifuged for 10 min at 5000 rpm (4136 g).

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The supernatant was filtered using a 0.22 μm Nylon filter vials (Agilent, Santa Clara,

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CA), and 1 mL was collected for the derivatization step.

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Derivatization

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One mL of the extract was evaporated under a nitrogen stream, frozen and then,

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lyophilized for 20 h. Next, 250 µL of pyridine, 40 µL of HMDS and 10 µL of TMCS

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were added and the mixture was kept at 70 ºC for 30 min. After that, 700 µL of n-hexane

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were added and homogenized increasing the total volume up to 1 mL, ready to be injected

128

into the GC-Q-Orbitrap system.

129 130

GC-Q-Orbitrap analysis


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One µL of sample was injected at the injector at 250 ºC using splitless mode for 2 min.

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Helium (with electronically controlled constant flow of 1 mL/min) was used as carrier

133

gas. For the separation of the calystegines, the GC oven was programmed as follows:

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initially, the temperature was set at 100 ºC and held for 2 min, then, it was increased to

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240 ºC at 5 ºC/min and finally, it was held at 240 ºC for 10 min, with a total running time

136

of 40 min.

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The positive electron ionization (EI) source was operated at 70 eV at a temperature of

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250 ºC, and the transfer line temperature was set at 250 ºC. The full scan-MS acquisition

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mode was performed at a resolution power of 60000 full width at half maximum (FWHM)

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in a scan range of m/z 60-600 with 1 µscan. Lock masses were used for column bleed at

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m/z 73.04680 for C3H9Si+, m/z 133.01356 for C3H9O2Si2+, m/z

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C5H15O3Si3+, m/z 281.05114 for C7H21O4Si4+ and m/z 355.06990 for C9H27O5Si5+.

207.03235 for

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The chromatogram was processed using Xcalibur version 4.1, with Quanbrowser and

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Qualbrowser, and the data was evaluated using TraceFinder 4.1 software (Thermo Fisher

145

Scientific, Les Ulis, France). Statistical analysis (analysis of variance, ANOVA) was

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carried out with IBM SPSS Statistics v23 (Armonk, NY).

147 148

Method validation

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A validation protocol was carried out in order to ensure an adequate identification and

150

quantitation of the target compounds. The performance characteristics of the method were

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established evaluating linearity, trueness, precision and lower limits, following the

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criteria established in SANTE/11813/2017.29 As many calystegines are natural

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compounds which occur in several Solanaceae plants, there is not available a blank matrix

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of tomato for all the target compounds. Therefore, and because the sample used for

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method validation purposes was not a standard reference material, all the calystegines



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that were not naturally present in this sample (B1, B3, B4 and C1) were evaluated for the

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validation of the method.

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Standard calibration solutions prepared in methanol at concentrations that ranged from

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10 to 1000 µg/L were subjected to the sample pretreatment (evaporation, lyophilization

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and derivatization) and analyzed. Linearity was studied from the least-squares regression

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of peak area versus concentration and determination coefficients (R2) were calculated.

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Intra-day precision, expressed as relative standard deviation (% RSD), was estimated

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by analyzing three aliquots of tomato sample at three different spiked concentrations: 0.5,

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1 and 5 mg/kg. Additionally, three aliquots of non-spiked samples were also extracted

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and repeatability of the method for those calystegines (A3, A5 and B2) naturally present

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in the sample was calculated. Trueness was determined through the recoveries of

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calystegines B1, B3, B4 and C1 at three different spiking levels (0.5, 1 and 5 mg/kg).

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Bearing in mind that no blank matrix was available, LOD and LOQ were established

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from the solvent calibration curve. LOD was considered as the lowest concentration that

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provided a mass spectrum with a characteristic ion measured with a mass error lower than

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5 ppm. LOQ was established as the lowest concentration providing a mass spectrum with

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a fragment ion having a mass error lower than 5 ppm, at the same retention time as the

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characteristic ion and with the same chromatographic profile. Additionally, the LOQ

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should present good linearity, precision and trueness.

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Results and discussion

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Optimization of GC-Q-Orbitrap conditions

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A derivatized standard solution of each calystegine was injected into the GC-Q-

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Orbitrap system. A full-MS was acquired for each calystegine in EI mode in order to

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select the characteristic ions.


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A predicted GC-MS spectrum was used as reference for each calystegine.30 Some

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possible characteristic ions were identified and their masses were extracted from the total

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ion chromatogram (TIC). Calystegines that belong to the same group (A, B or C) are

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positional isomers, so they present the same molecular formula. It was observed that

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calystegines within the same group provided the same ions, although slight differences

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between relative abundances of these ions were observed.

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Extracting the masses of the possible characteristic ions from the full-MS spectra, the

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retention time, the theoretical mass and the chemical formula were determined from the

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experimental mass. Several ions were confirmed for each calystegine as well as some

190

identified as common ions. The selected GC-Q-Orbitrap parameters are presented in

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Table 1.

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Figure 2 shows an example of the full-MS spectra including the proposed mechanism

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for calystegines A3, B4 and C1. As observed, the loss of a methyl group (m/z 15.02293)

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and trimethylsilanol (OH-TMS m/z 90.04954) are the most common fragmentations. it

195

should be mentioned that none of the molecular ions were detected, [M-CH3]·+ ion being

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the highest intensity ion detected.

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The described chromatographic method, based on a previously published work,15 was

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used and proved that reliable identification and quantitation of the compounds was

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possible due to goof chromatographic separation of the calystegine isomers studied.

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Figure 3 shows the obtained TIC using the optimized experimental conditions.

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Optimization of sample pretreatment

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Sample pretreatment was divided into two phases: sample extraction and

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derivatization. On the one hand, for the optimization of the sample extraction and

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considering the highly polarity of the studied compounds, two different extractive options



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were tested. Three aliquots of 10 g of tomato were spiked at 1 mg/kg and extracted using

207

methanol with 1% of formic acid (QuPPe-Method)31 and methanol/water (50:50, v/v),

208

which was frequently employed for their extraction.8,13,21 The QuPPe-Method had been

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developed for the extraction of highly polar pesticides and because of the sample

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pretreatment comprises an evaporation step, a non-aqueous solvent was thought to be

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appropriate and tested by analyzing three aliquots of tomato spiked as described above.

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However, recoveries ranging from 26-106% were obtained when 1% formic acid in

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methanol was used (Figure 4A), while methanol/water (50:50, v/v) provided better

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recoveries (64-118%) and higher sensitivity (Figure 4B). Bearing in mind that a marked

215

matrix effect was observed, the sample amount was reduced from 10 to 1 g. Three aliquots

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of 1 g of sample spiked at 1 mg/kg were extracted. A diminution of the matrix effect was

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observed and a clearer TIC were obtained with recoveries similar to those seen when 10

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g of sample were tested (Figure 4C). Thus, for further experiments, this last extraction

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procedure based on the use of only 1 g of sample was used.

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The derivatization step was also submitted to optimization (Figure 4). Different

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volumes of pyridine (30, 130 or 250 µL), HMDS (40 or 80 µL) and TMCS (10 or 20 µL)

222

were tested and the best results were obtained employing 250 µL pyridine, 40 µL HMDS

223

and 10 µL TMCS (Figure 4D). Although pyridine does not interfere with the reaction, the

224

entire solid residue obtained from the lyophilization step should be covered in order to be

225

completely dissolved and derivatized. Additionally, the use of a final clean-up step was

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also evaluated, mixing the final extract with water (50:50, v/v), but improvements in the

227

results were not observed and this additional clean-up step was finally discarded.

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Method validation


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The performance characteristics of the optimized method are shown in Table 2.

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Linearity was evaluated through the obtained determination coefficients (R2) which were

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always higher than 0.99 for all the compounds, except calystegines A3 and B4 (Table 2).

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The standard addition methodology was used for quantitation purposes due to a high

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matrix effect observed (not reported). LOD was established at 0.25 (calystegine A5, B1,

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B3 and C1) and 0.5 mg/kg (calystegine A3, B2 and B4), whereas LOQ was set at 0.5 mg/kg

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for all calystegines.

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As shown in Table 2, adequate intra-day precision (≤ 20.0%) was obtained at all the

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concentrations studied and for all the compounds, but slightly higher values were

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obtained for calystegine B3 at 0.5 mg/kg (22.4%) and calystegine B4 at 0.5 mg/kg (21.4%).

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Recoveries ranged from 73.7 (calystegine B4) to 120.0% (calystegine B3) at 0.5 mg/kg;

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from 89.5 (calystegine B4) to 118.8% (calystegine B1) at 1 mg/kg and from 95.8

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(calystegine B3) to 100.7% (calystegine B4) at 5 mg/kg. For calculating recovery rates to

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those calystegines natively present in the spiked samples, the concentrations found for

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those compounds in the blank analysis were subtracted.

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Very few reports determine calystegine in tomato, and only Romera-Torres et al.17

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include validation parameters; the sensitivity is similar to that presented herein, although

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slightly higher for some calystegines (LOQ 0.10 mg/kg for calystegine B4, 0.25 mg/kg

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for calystegine B3 and 0.50 mg/kg for calystegines A3, A5, B1, B2 and C1); similar results

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were observed for trueness and precision. However, comparing to other studies of

250

calystegines in other matrices, the proposed GC-Q-Orbitrap method is more sensitive and

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also lower than alternative GC methodologies that provided LOQ from 3-10 mg/L or 2-

252

20 mg/kg,5,7–9,13–15,18,19and than LC methods, with LOQ from 0.4 to 2.5 mg/kg.16,20

253 254

Analysis of samples



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It can be observed (Table 3) that ‘Zoraida’ and ‘Satyplum’ varieties presented the

256

highest concentrations of calystegines A3, A5, B2 and C1, while the concentration of the

257

analytes in ‘Monterosa’, ‘Bernal B’, ‘Ateneo’ and ‘Motto Fi’ varieties were below the

258

LOQ of the proposed analytical method. The concentrations calculated for the

259

calystegines found in the studied samples ranged from 0.65 mg/kg of C1 (‘Bernal A’) to

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12.47 mg/kg of B2 (‘Zoraida’). Figure 5 shows the chromatograms and full-MS spectra

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of calystegine A3 and calystegine B2 in two of the studied tomato samples.

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Previous studies found concentrations in tomato samples of calystegine A3 (0.2-21

263

mg/kg), calystegine A5 (0.9-7 mg/kg) and calystegine B2 (0.4-21 mg/kg).27 These results

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are in accordance to the results presented herein, although the wide variability in the

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concentrations of natural compounds must be taken into account, which is highlighted

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when observing the extensive range of concentrations found for instance in potato,

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ranging from 0.2 mg/kg of calystegine A318 to 10145 mg/kg of calystegine B2.5

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Finally, with the aim of checking whether the difference observed between the content

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of calystegines depends on the var., an ANOVA study was performed. The results

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obtained for calystegine A5 show that no significance difference was observed between

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varieties (‘Kumato’, ‘Satyplum’, ‘Zoraida’, ‘Bernal A’ and ‘Guanche’) (p value>10)

272

(Table 3). However, significant differences were observed between the concentrations

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found for calystegine C1 between varieties that in ‘Zoraida’ was significantly higher than

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in ‘Kumato’, ‘Satyplum’, ‘Bernal A’ and ‘Guanche’ (p value

1 Determination of Calystegines in Several Tomato ... - ACS Publications

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