Determination of calystegines in several tomato varieties based on GC

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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

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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

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

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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

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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, ≥

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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

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into the GC-Q-Orbitrap system.

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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

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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

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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

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

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

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Method validation

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

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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

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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

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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

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

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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

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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)

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

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

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

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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

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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

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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

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Analysis of samples



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

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

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

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

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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

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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)

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(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

Determination of calystegines in several tomato varieties based on GC

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