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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Journal of Agricultural and Food Chemistry

 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

297

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

298

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

299

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

303

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

304

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

311

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

312

obtained in +bbCID spectra.

313

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

315

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

316

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

317

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

319

was successfully working in the rapid method.

320

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

322

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

326

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

327

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

328

unknown molecules using HR-QTOF.30, 31

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

330

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

331

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

332

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

333

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

334

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,

337

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

339

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

340

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