Rapid and Efficient Desulfonation Method for the Analysis of

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A Rapid and Efficient Desulfonation Method for the Analysis of Glucosinolates by High Resolution Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Jashbir Singh, Guddadarangavvanahal Jayaprakasha, and Bhimanagouda S. Patil J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04662 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Journal of Agricultural and Food Chemistry

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A Rapid and Efficient Desulfonation Method for the Analysis of

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Glucosinolates by High Resolution Liquid Chromatography Coupled with

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Quadrupole Time-of-Flight Mass Spectrometry

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Jashbir Singh, Guddadarangavvanahally K. Jayaprakasha,* Bhimanagouda S. Patil*

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Vegetable and Fruit Improvement Center, Department of Horticultural Sciences,

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Texas A&M University, 1500 Research Parkway, Suite A120, College Station, TX 77845

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8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

*Corresponding authors Tel.: +1 979 458 8090; fax: +1 979 862 4522 e-mail: [email protected] Tel.: +1 979 845 3864; fax: +1 979 862 4522 e-mail: [email protected]

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______________________________________________________________________________

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ABSTRACT: The goal of our present research was to develop a simple and rapid method for the

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quantitation of desulfoglucosinolates (desulfoGLS) without using column chromatography. The

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proposed method involves extraction, concentration, incubation of glucosinolates with sulfatase

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enzyme, and HPLC analysis. Identification of desulfoGLS in green kohlrabi was performed by

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LC-HR-ESI-QTOF-MS in positive ionization mode. A total 11 desulfoGLS were identified with

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neoglucobrassicin (3.32±0.05 µmol/g DW) as the predominant indolyl, whereas progoitrin and

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sinigrin were the major aliphatic desulfoGLS. The levels of aliphatic desulfoGLS such as

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glucoiberin, progoitrin, and glucoerucin were found to be 3.6, 1.9 and 1.6 fold higher than the

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conventional method, respectively, at 7 h. This technique was successfully applied to identify

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desulfoGLS from cabbage. The developed method has fewer unit operations, with maximum

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recovery and is reproducible to determine desulfoGLS in a large number of Brassicaceae

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samples in a short time.

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KEYWORDS: Kohlrabi, cabbage, desulfoGLS, LC-HR-ESI-QTOF-MS

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______________________________________________________________________________

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INTRODUCTION

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Glucosinolates (GLS) are biosynthetically derived from amino acids1 and they occur in all plants

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of the Capparales order.2, 3 GLS have a well-defined structure with a side chain (R-group) and D-

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glucopyranose as β-thioglucoside attached to (Z)-N-hydroximine sulfate esters.4,

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generally precursors of isothiocyanates, which are responsible for the pungent taste as well as

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many health beneficial properties6, 7 of the Brassica species, such as prevention of cardiovascular

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diseases, cancers, neurodegenerative, and chronic diseases.8 For example, Michaud et al.9

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reported a significant correlation between cruciferous vegetable consumption and reduced

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incidence of bladder cancer. Moreover GLS breakdown products such as sulphoraphane and

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indole-3-carbinol are released by breakdown of glucoraphanin, 4 and glucobrassicin, 8, and are

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inducers of phase I and II enzymes.10, 11

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GLS are

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Due to their high polarity of thioglucosides and relatively lower retention in the stationary

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phase, the HPLC separation of intact glucosinolates is a challenge.12 HPLC analysis of GLS in

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desulfonated form is well accepted and considered an effective method of quantification due to

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improved resolution.13 The desulfonation involves the binding of intact GLS on a weak anion

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exchange resin and the removal of the SO3- group from the parent moiety by the addition of aryl

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sulfatase enzyme by column chromatography.14, 15 After 18-24 h of incubation, the desulfoGLS

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are eluted with milli-Q water12, 16 and analyzed by HPLC. Open columns are not reproducible

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and give low yields due their differences in partition coefficients, adsorption and desorption

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properties.17, 18 Lee et al.19 reported the desulfonation of glucosinolates from turnip after 24 h

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incubation with aryl sulfatase on a Sephadex column and identified by LC-QTOF-MS with a 55

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min run time. Recently, the effect of storage, temperature and radiation treatments on GLS levels

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of broccoli was reported by a study using a Sephadex column followed by a 45 min LC

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method.20 Similarly, Park et al.21 reported the glucosinolate content in kohlrabi using a mini 3 ACS Paragon Plus Environment


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column containing DEAE-Sephadex A-25. The desulfonation was achieved by overnight

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incubation with aryl sulfatase, followed by elution and identification by LC-MS. Kohlrabi

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desulfoGLS identification has been performed using HPLC-PDA, typical UV spectra and LC-

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ESI-MS system.16, 21, 22

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A Scifinder search identified more than 190 papers that used column chromatography for the

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removal of the sulphate group for the analysis of desulfonated glucosinolates. This method takes

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>18-24 h incubation, followed by elution, concentration, and quantitation/identification. To

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facilitate rapid analysis of GLS in a large number of samples, it is critical to improve the sample

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preparation technique with fewer unit operations to minimize the recovery loss. The present

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study focused on developing a rapid method to analyze GLS from green kohlrabi in their

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desulfonated form without using column chromatography. In addition, LC-HR-ESI-QTOF-MS

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was used to characterize desulfoGLS by accurate mass and the possible fragmentation pattern.

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The developed method was successfully applied to validate the desulfonation process in GLS

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from cabbage. This is the first report on the identification of desulfoGLS using high resolution

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accurate mass spectrometry from green kohlrabi.

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MATERIALS AND METHODS

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Plant Materials. Green kohlrabi (Brassica oleracea var. gongylodes) and green cabbage

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(Brassica oleracea var. capitata) were obtained from the local supermarket (College station,

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TX), chopped, frozen at -80 °C for 24 h, lyophilized (Labconco FreeZone, Kansas City, MO),

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ground to obtain 60 to 80 mesh size powder and stored in -80 °C until further analysis.

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Chemicals. Aryl sulfatase from Helix pomatia (Type H-1, 23050 U/g solid), DEAE

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Sephadex A-25, HPLC grade acetonitrile and analytical grade phosphoric acid were purchased 4 ACS Paragon Plus Environment

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from Sigma-Aldrich (St. Louis, MO). Progoitrin, 2, sinigrin, 3, glucoraphanin, 4, gluconaphin, 5,

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glucoerucin, 7, and gluconasturtiin, 10, were purchased from Chromadex (Irvine, CA). HPLC

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grade water (resistivity 18.2 mΩcm) was obtained from Nanopure water purification system

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(Barnstead, Dubuque, IA). Sodium acetate purchased from EMD chemicals (Gibbstown, NJ) and

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acetic acid was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ).

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Sample Preparation. Stock sample was prepared by extracting 7 g of lyophilized green

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kohlrabi with 15 mL of boiling methanol/water (70:30, v/v) in water bath at 80 °C for 10 min to

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deactivate the endogenous myrosinase enzyme. The extract was cooled in an ice bath and

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vortexed for 2 min then sonicated for 30 min, centrifuged at 4480 g for 15 min and passed

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through Whatman filter paper No. 1. The residue was re-extracted twice with boiling

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methanol/water (70:30) (2 × 5 mL) to ensure the complete extraction of glucosinolates. All three

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extracts were pooled and concentrated to dryness under vacuum at 40 °C. The residue was

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dissolved in 25 mL of 0.1 M sodium acetate buffer (pH 5), passed through 0.45 micron filters

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and distributed into different tubes before being stored at -20 °C prior to optimization of the

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desulfonation method.

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Optimization of Rapid Desulfonation Method. Effect of Incubation Time. Five

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hundred microliters of sample was transferred into individual screwed capped vials and treated

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with 80 µL (1.84 units) of aryl sulfatase enzyme solution (1 mg/mL in buffer). The sample

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containing vials were vortexed for 30 s and incubated for 1, 4, 7, 10, 13, 16, 19 and 22 h for the

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conversion of intact GLS to desulfoGLS. After each incubation period, samples were passed

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through 0.45 µm Teflon PTFE syringe filters and subjected to HPLC analysis.



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Effect of Enzyme Volume. Briefly, 500 µL of extracted samples were treated with 10,

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20, 40, 80 and 160 µL (equivalent to 0.23, 0.46, 0.92, 1.84 and 3.68 units) of sulfatase enzyme

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and the total volume was adjusted to 1 mL with buffer. The resultant mixtures were vortexed for

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30 s then incubated for 1 h and 7 h at 25 °C with constant shaking. After the incubation period,

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filtered samples were injected to HPLC. The conversion of the intact GLS to desulfoGLS was

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analyzed by HPLC.

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Separation and Quantification of DesulfoGLS by HPLC. The above optimized

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conditions were subsequently used to analyze desulfoGLS in green kohlrabi. Briefly, 500 µL of

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samples were treated with 80 µL sulfatase enzyme and the total volume adjusted to 1 mL with

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buffer, vortexed for 30 s followed by incubation at 25 °C for 7 h with constant shaking. The

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resultant sample was filtered and subjected to HPLC analysis. Analysis of desulfoGLS was

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performed on a 1525 HPLC system (Waters, Milford, MA) equipped with a 2996 photodiode

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array detector and 717plus autosampler. Chromatographic separation was performed on a Zorbax

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Eclipse Plus C18 ODS (250 mm × 4.6 mm i.d., 5 µm) column (Agilent, Santa Clara, CA). A

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gradient mobile phase consisting of (A) 0.03 M phosphoric acid in water and (B)

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water/acetonitrile (70:30, v/v) at a flow rate of 0.8 mL/min. The separation was performed as

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isocratic 2% B (8 min), 2%−45% B (9 min), 45%−60% B (8 min), 60%−90% B (6 min), 90%

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−2% B (3 min), and 6 min isocratic at 2% B. The column was equilibrated 2 min before the next

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injection. A 20 µL sample was injected and the desulfoGLS were monitored at 227 nm.

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Empower-2 software was employed for processing the data.

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Standard DesulfoGLS Preparation and Calibration. Intact glucosinolates progoitrin,

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2, sinigrin, 3, glucoraphanin, 4, gluconaphin, 5, glucoerucin, 7, and gluconasturtiin, 10, were

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prepared at 1 mg/mL concentrations in 0.1 M acetate buffer (pH 5). All these individual 6 ACS Paragon Plus Environment

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standards were desulfonated by sulfatase enzyme (80 µL) as described above and confirmed by

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LC-MS. Further, all these compounds were subjected to serial dilutions (100, 50, 25, 12.5, 6.25,

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3.12, 1.56, and 0.78 µg/mL) and peak areas were used for preparing the calibration curves.

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Desulfonation by Conventional Method. To validate the levels of desulfoGLS obtained

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from the rapid method, conventional desulfonation was carried out according to Park et al.21.

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Briefly, the crude extract (500 µL) was loaded into a column (10 × 6 mm) containing 50 mg

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DEAE-Sephadex anion exchange resin, which was previously soaked in 0.1 M sodium acetate

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overnight. The column was washed twice with 1 mL of acetate buffer (pH 5). The desulfonation

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was carried out by adding sulfatase enzyme (80 µL) on a column and incubated for 18 h at 25

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°C. The column was eluted with nano-pure water to obtain desulfoGLS. The eluent was passed

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through 0.45 µm PTFE syringe filter and quantified by HPLC as mentioned above.

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Identification of Glucosinolates by LC-ESI-HR-QTOF-MS. The LC-MS identification

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was performed using HR-QTOF-MS on a maXis impact mass spectrometer (Bruker Daltonics,

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Billerica, MA) coupled to a 1290 HPLC (Agilent, Santa Clara, CA). The desulfoGLS were

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separated on a Zorbax Eclipse Plus C18 column (100 × 2.0 mm i.d., 3 µm) (Agilent, Santa Clara)

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at 65 °C with a flow rate of 100 µL/min using gradient mobile phase consisting of (A) 0.1%

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formic acid in water and (B) 0.1% formic acid in water/ acetonitrile (70:30, v/v). Elution of GLS

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was performed as follows, 0−2% B (10 min), 2%−45% B (12 min), 45%−60% B (5 min),

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60%−90% B (4 min), 90%−0% B (3 min) and 5 min isocratic A (100%). The column was

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equilibrated for 2 min before the next injection. MS and broadband collision induced dissociation

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(bbCID) data were acquired in the range of m/z 50–2000. Nitrogen was used for both nebuliser

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(gas pressure 2.1 bar) and drying gas (flow rate 8.0 L/min). The capillary ion voltage was 4,500

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V and temperature of drying gas was 200 °C. The transfer time of the source was 120.8 µs and 7 ACS Paragon Plus Environment


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the prepulse storage time 5 µs. The quadrupole MS and bbCID collision energies were set at 8

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and 30 eV, respectively. The spectrum acquisition rate was 1.4 Hz. External instrument

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calibration was performed with a sodium formate solution containing 1 mM sodium hydroxide in

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isopropanol/0.2% formic acid (1:1, v/v) according to the method used in our published work.23

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To identify the intact GLS, samples were run in electrospray negative ionization mode using

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same chromatographic conditions.

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Confirmation of DesulfoGLS. Certain desulfoGLS were not commercially available.

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To confirm the peak purity of identified desulfoGLS, HPLC peak fractions were collected

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manually and confirmed by high resolution mass spectrometry (HR-MS). For instance, six

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injections of 50 µL were injected in HPLC and chromatographic separation was carried out as

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described above. Each peak fraction was collected from the PDA detector outlet according to the

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expected retention time (tR) of desulfoGLS. Total 11 fractions were collected and immediately

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analyzed by the LC-MS. The desulfoGLS were separated on Zorbax Eclipse Plus C18 column

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(100 × 2.0 mm i.d., 3 µm) at 65 °C with a flow rate of 200 µL/min using a gradient mobile phase

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consists of (A) 0.1% formic acid in water and (B) 0.1% formic acid in water/acetonitrile (70:30,

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v/v). Elution of GLS was performed as follows, solvent A, 100−0% for 0−8 min, 0−100% A for

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8-11 min, then 2 min equilibrated in between the runs. The MS conditions were maintained as

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described above.

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Validation of Rapid Method. To validate the developed method for the desulfonation

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technique, lyophilized cabbage sample (500 mg) was extracted with 4 mL of 30% aqueous

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methanol at 80 °C for 10 min according to the protocol mentioned above. The extract (500 µL)

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was treated with sulfatase enzyme (80 µL), vortexed for 30 sec followed by incubation for 7 h at

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25 °C with constant shaking. The resultant sample was filtered and subjected to HPLC analysis. 8 ACS Paragon Plus Environment

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Statistical Analysis. Data was analyzed by one-way analysis of variance (ANOVA)

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using JMP Pro12 software (SAS, NC). Significance difference between means was observed by

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a Student’s t test at 5% probability level (P< 0.05).

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RESULTS AND DISCUSSION

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The present study reports the development of a simple, rapid and accurate method for the

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quantitation of desulfoGLS in kohlrabi and cabbage. The sample preparation and identification

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of desulfoGLS was performed without the tedious steps using DEAE-Sephadex column

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chromatography. The developed method involves fewer unit operations and requires 1-7 h for the

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desulfonation.

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HPLC Separation of Glucosinolates at Different Time Points. Figure 1 shows the

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chromatographic separation of eleven intact glucosinolates (GLS) and their desulfoGLS from

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kohlrabi at different time points. Intact glucosinolates glucoiberin, 1, progoitrin, 2, sinigrin, 3,

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glucoraphanin, 4, gluconaphin, 5, glucoibervirin, 6, glucoerucin, 7, glucobrassicin, 8, 4-methoxy

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glucobrassicin, 9, gluconasturtiin, 10 and neoglucobrassicin, 11, are denoted as GLS 1-11

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(Figure 1A), whereas desulfoGLS 1-11 represents their desulfonated forms at 7 h (Figure 1B).

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Peaks 8, 9, and 11 eluted at retention times (tR) of 31.9, 33.8, and 37.9 min, respectively, were

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tentatively identified as indolyl desulfoGLS. The peaks eluted at tR 10.6, 16.2, 18.2, 18.5, 22.9,

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24.4 and 27.8 min were identified as aliphatic desulfoGLS 1, 2, 3, 4, 5, 6 and 7, respectively

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(Figure 2). It was observed that for aromatic and indolyl peaks, 8-11, 70-90% were converted

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into their desulfonated form within 1 h. After 4 h, complete conversion had occurred. Aliphatic

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GLS peaks, 1-7, conversion was completed in 7 h. The desulfonation reaction was monitored for

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22 h to compare the results with the conventional method. 9 ACS Paragon Plus Environment


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Effect of Enzyme Volume on Desulfonation. For enzymatic assays, incubation time and

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enzyme volume are critical parameters to obtain end products.13, 24 A low volume may lead to

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incomplete desulfonation and high enzyme volume may cause a loss of desulfoGLS, which

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results in inaccurate quantitation.13 The effect of enzyme volume on desulfonation of GLS is

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shown in Figure 3. The results demonstrated that with an increase in enzyme volume, the rate of

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desulfonation increased. For instance, indolyl neoglucobrassicin, 11 and aliphatic progoitrin, 2,

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desulfoGLS levels increased more than two fold at 40 µL enzyme concentration as compared to

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10 µL. The rest of the compounds gradually increased with enzyme volume from 10-80 µL,

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while at higher enzyme volume (80-160 µL) did not affect yields significantly. Hennig et al.25

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reported that at higher enzymatic concentration, the peak intensities of indolyl GLS were

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reduced, while at lower concentration incomplete desulfonation of aliphatic GLS (glucoiberin, 1

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and glucoraphanin, 4 occurred. Similarly, our results showed lower content of indolyl derivatives

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with 160 µL enzyme than with 80 µL. On that basis, we concluded that 80 µL enzyme volume

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was sufficient for complete desulfonation of glucosinolates.

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Comparison of Conventional and Rapid Desulfonation Methods. The levels of

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individual desulfoGLS in kohlrabi were quantified by existing DEAE Sephadex column to

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compare the results with the rapid method. Figure 4 shows the levels of individual desulfoGLS

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obtained from the rapid optimized method at 7 h and conventional method at 18 h. Seven

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desulfoGLS, 1-7, showed a significantly higher value in the rapid method. The aliphatic

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glucosinolates, 3, 4 and 5 had slightly higher 11%, 4% and 21% respectively than the

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conventional method. For progoitrin, 2, and glucoerucin, 7, showed 1.9 and 1.6 fold higher

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levels, respectively. Similarly, glucoiberin, 1, was 3.8 fold higher than conventional method. The

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aromatic and indolyl desulfoGLS, 8-10, showed no significant differences in their levels. 10 ACS Paragon Plus Environment

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However, neoglucobrassicin, 11, had a significantly higher value 3.65±0.22 µmol/g DW in the

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conventional method as compare to the rapid method (3.32±0.06 µmol/g DW). The lower levels

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of desulfoGLS using the column may be due to incomplete elution from the resin or interaction

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between the anionic resin and the compounds. Thus, a robust and reproducible method is needed

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for rapid desulfonation and quantification of GLS.

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Characterization of Glucosinolates by LC-HR-ESI-QTOF-MS. LC-MS/MS analysis

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using quadrupole time of flight is considered a reliable and powerful analytical tool for the

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identification of GLS, especially when there is a lack of reference compounds.26 In LC-HR-ESI-

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QTOF-MS, target ions can be detected with sufficient sensitivity within a narrow retention time.

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DesulfoGLS were characterized by accurate high-resolution mass spectra obtained by

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electrospray positive ionization mode from green kohlrabi. Identification of eleven desulfoGLS

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was established based on the UV spectra and characteristic mass fragmentation / isotopic ratio

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patterns due to sulphur and nitrogen molecules. Table 1 presents the retention times, theoretical

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protonated accurate mass, and the key fragments of identified desulfoGLS (aliphatic, aromatic,

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indolyl derivatives) in green kohlrabi. Figure 5 represents the extracted ion chromatograms

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(EICs) of desulfoGLS identified in green kohlrabi. The tandem mass and +bbCID spectra of

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desulfoGLS of aliphatic, aromatic and indolyl derivatives obtained by positive ionization mode

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are shown in Figure 6. All mass spectra for the identified desulfoGLS were comprised of the

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precursor ion [M+H]+, the sodium adduct [M+Na]+ and the characteristic product ion due to the

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loss of the glucose moiety [M+H-162]+.

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Identification of Aliphatic Glucosinolates. A total of 7 aliphatic desulfoGLS were

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identified. The first peak was eluted at tR 5.2 min and exhibited an accurate mass at m/z 344.0823

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[M+H]+ (mass error +2.67 ppm) and at m/z 366.0650 [M+Na]+ which corresponded to the 11 ACS Paragon Plus Environment


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protonated form of desulfoGLS and its sodium adduct, respectively. It also showed a

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characteristic product ion at m/z 182.0301 [M+H-162]+ which was a result of the loss of a

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glucose moiety. The +bbCID spectra displayed a product ion at m/z 118.0318 [M+H-162-64]+

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which presumably originated by elimination of methyl sulfoxide. On the basis of mass data and

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UV spectra, the compound was identified as glucoiberin, 1. Similarly another peak eluted at tR

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6.8 min and exhibited an accurate mass value at m/z 310.0952 [M+H]+ (mass error +1.0 ppm)

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and its sodium adduct at m/z 332.0775 [M+Na]+. The +bbCID spectra also displayed a product

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ion at m/z 148.0427 [M+H-162]+ which appeared due to the loss of a glucose moiety. It also

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showed a less abundant product ion at m/z 130.0319 [M+H-162-18]+ due to loss of water from a

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hydroxyl-substituted side chain. It was thus inferred that the compound at tR 6.8 min was

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progoitrin, 2.

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Similar fragmentation patterns were observed for other aliphatic glucosinolates (tR 8.5,

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9.0, 18.6, 21.4, and 26.0 min) having an accurate mass [M+H]+ at m/z 280.0849 (mass error +0.1

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ppm), m/z 358.1005 (mass error -4.6 ppm), m/z 294.0963 (mass error +14.6 ppm), m/z 328.0850

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(mass error +10.1 ppm), and m/z 342.1033 (mass error +1.9 ppm) and their diagnostic ion

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fragment due to the loss of a glucose moiety [M+H-162]+ at m/z 118.0325, m/z 196.0471, m/z

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132.0462, m/z 166.0337 and m/z 180.0510 respectively. On the basis of the above fragmentation

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patterns the aliphatic glucosinolates were identified as sinigrin, 3, glucoraphanin, 4, gluconaphin,

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5, glucoibervirin, 6, and glucoerucin, 7. Interestingly, the +bbCID spectrum of 4 also displayed a

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minor product ion at m/z 132.0482 [M+H-162-64]+ due to the loss of methyl sulfoxide (CH4OS).

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Similarly the +bbCID spectra of 1 and 7 also exhibited a low abundant product ion at m/z

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118.0309 [M+H-162-CH3SH]+ and at m/z 132.0471 [M+H-162-CH3SH]+ respectively, which

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arose due to the loss of a methanethiol moiety. 12 ACS Paragon Plus Environment

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Identification of Aromatic and Indolyl Glucosinolates Derivatives. Aromatic

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desulfoGLS showed characteristic prominent product ion corresponding to tropylium ion.

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Gluconasturtiin, 10, at tR 31.6 min exhibited an accurate mass value at m/z 344.1155 [M+H]+

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with a mass error +2.14 ppm and its sodium adduct at m/z 366.0978 [M+Na]+. It also exhibited a

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prominent product ion at m/z 182.0633 [M+H-162]+ which corresponds to loss of a glucose

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moiety. DesulfoGLS 10 and 1 behaved as isobaric compounds due to same accurate mass in the

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positive ion MS, but the +bbCID spectra of both GLS displayed different prominent product ions

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due to their distinctive side chain (R-substituent). Gluconasturtiin, 10, showed a distinct product

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ion at m/z 91.0541 which corresponded to the abundance of tropylium ion while glucoiberin, 1,

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displayed a product ion at m/z 118.0318 [M+H-162-64]+.

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The three prominent peaks eluted at tR 27.8 min, 31.5 min and 36.4 min corresponded to

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the presence of indolic compounds and were identified as glucobrassicin, 8, 4-methoxy

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glucobrassicin, 9 and neoglucobrassicin, 11, respectively. Indolic glucosinolates were

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characterized by odd masses of aglycone fragments which indicated the presence of an even

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number of nitrogen atoms in their chemical structure.27 Desulfoglucobrassicin, 8, was putatively

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identified using the accurate mass value at m/z 369.1095 [M+H]+ (mass error +5.4 ppm) and its

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sodium adduct at m/z 391.0913 [M+Na]+. It also showed a dominant product ion at m/z 207.0580

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[M+H-162]+ due to loss of glucose moiety. Burke et al.28 reported the most important

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characteristic product ion of indolyl desulfoglucosinolates was abundance of [Rˊ]+. Similarly, the

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+bbCID spectrum of 8 showed a prominent characteristic product ion at m/z 130.0646 [Rˊ]+

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which corresponded to indole-3-methylene ion (C9H8N+). It also displayed a minor product ion at

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m/z 117.0565 [Rˊ-13]+. A peak eluted at tR 31.5 min showed an accurate mass value at m/z

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399.1205 [M+H]+ (mass error +3.9 ppm), its sodium adduct at m/z 421.1028 [M+Na]+ and 13 ACS Paragon Plus Environment


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diagnostic product ion at m/z 237.0692 [M+H-162]+. The +bbCID spectra also showed a

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prominent characteristic product ion at m/z 160.0750 [Rˊ]+ which corresponded to 4-methoxy

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indole-3-methylene (C10H10NO+). It showed further loss of a methoxy group followed by

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protonation to give minor fragment with small abundance at m/z 130.0650 [Rˊ-CH3O+H]+. On

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the basis of characteristic fragmentation pattern, the peak was identified as 4-

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methoxyglucobrassin, 9. Similarly, another prominent peak at tR 36.4 min was identified as

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neoglucobrassicin, 11, having an accurate mass value at m/z 399.1219 [M+H]+ (mass error

308

+0.37), its sodium adduct at m/z 421.1035 [M+Na]+, and a product ion peak at m/z 237.0689

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[M+H-162]+. The fragmentation pattern of 11 was similar to 9 but the +bbCID spectra of 11

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represented a characteristic intense product ion at m/z 130.0650 [Rˊ]+. Thus, methoxy containing

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isomers 9 and 11 were characterized by the difference in the intensities of their product ions

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obtained in +bbCID spectra.

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The extracted ion chromatograms (EICs) of intact GLS of green kohlrabi in negative

314

ionization mode at 0 h and the EICs at 7 h after incubation with sulfatase enzyme demonstrated

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that before the addition of enzyme (0 h), spectra showed an intact GLS peaks but after incubation

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with enzyme (7 h), no GLS precursor ions were observed in negative ionization mode. The EICs

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in positive ionization mode showed the presence of desulfoGLS at 7 h. The EICs obtained in

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positive and negative ionization modes provided strong evidence that the desulfonation process

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was successfully working in the rapid method.

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Confirmation of HPLC DesulfoGLS Fractions by HR-MS. Two different Eclipse Plus

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columns were used for analytical HPLC (250 mm × 4.6 mm i.d., 5 µm) and LC-MS (100 × 2.0

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mm i.d., 3 µm) separation. To confirm the peak identity from analytical HPLC, each peak

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fraction was collected manually and analyzed by HR-MS. The mass spectra of all the fractions 14 ACS Paragon Plus Environment

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were matched to their desulfoGLS accurate mass and their fragments (Table 1 and Figure 6).

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Thus, high resolution instruments are usually operated in an untargeted manner, which allows

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accurate mass information on every ion that reaches the detector.29 Moreover, the reliability,

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efficiency, sensitivity, and structural information will be more valid for identification of an

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unknown molecules using HR-QTOF.30, 31

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Quantification of Glucosinolates. After peak confirmation of desulfoGLS in HPLC and

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HR-MS, the same methodology was applied to analytical HPLC for the quantification of

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desulfoGLS. The quantification of 2, 3, 4, 5, 7 and 10, were achieved using the calibration curves

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of their respective desulfoGLS. The remaining aliphatic and indolyl glucosinolates were

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expressed relative to 4 and 10, respectively. All calibration curves showed excellent linearity

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with high correlation coefficient (R2) > 0.99. The limit of detection (LOD) and limit of

335

quantification (LOQ) were determined by injecting serial diluted standards solutions until the

336

signal-to-noise ratio (S/N) for each standard was 3 to 10, respectively.32,

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reaction, enzyme volume and incubation time are critical parameters.13, 24, 25 Figure 7A displays

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the formation of desulfoGLS at different time points using 80 µL of sulfatase enzyme from

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kohlrabi. A total 11 glucosinolates were quantified in green kohlrabi and ranged from

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0.16±0.007 to 3.32±0.02 µmol/g DW. DesulfoGLS 8, 9 and 10, showed significantly (P

Rapid and Efficient Desulfonation Method for the Analysis of

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