Materials and Reagents

Horseradish tissue (hypogeous and epigeous portion) (see Note 1)
Methanol (MeOH) (LC/MS grade) (Carlo Erba Reagents, catalog number: 414831 )
Acetonitrile (ACN) (LC/MS grade) (Carlo Erba Reagents, catalog number: 412342 )
Ultrapure Milli-Q water
Liquid nitrogen
Sinigrin hydrate from horseradish (99%) (Sigma-Aldrich, catalog number: S1647 )
Rapeseed ERM certified Reference Material containing gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin and gluconaturtiin (Sigma-Aldrich, catalog number: ERMBC367 )
Glucoiberin (C2 Bioengineering, catalog number: 10-JS 12-05-02 )
Glucobarbarin (C2 Bioengineering, catalog number: 18-DM 19-10-99 )
Glucotrapeolin (C2 Bioengineering, catalog number: 16-PM 19-10-99 )
70% MeOH (see Recipes)

In addition only for intact GLS determination

Formic acid (gradient grade) (Sigma-Aldrich, catalog number: F0507 )
0.1% Formic acid (HCOOH) (see Recipes)

In addition only for desulfo-method

DEAE-Sephadex A-25 (formiate form) obtained by using DEAE-Sephadex A-25 (chloride form) (Sigma-Aldrich, catalog number: A25120 ) and imidazole (Sigma-Aldrich, catalog number: 56750 )
Sulfatase type H-1 (Sigma-Aldrich, catalog number: S-9626 )
Sulfatase type H-1 (1 to 2.5) (see Recipes)
DEAE-Sephadex A-25 (formiate form) (see Recipes)

Equipment

Freeze Dry Systems (e.g. Labconco, model: Freezone 4.5 )
Laboratory mill
Disposable 50 ml and 15 ml polypropylene tubes
2 ml sample vials
Water bath: beaker filled with water and placed on a heating device (electric hotplate or similar device).
Thermometer
Vortex mixer
Refrigerated centrifuge (50 ml tubes) (e.g. Heraeus, model: Varifuge F )
Glass Pasteur pipettes
0.22 µm nylon filter (Whatman)
HPLC system with a photodiode array detector (e.g. Agilent, model: Agilent 1200 HPLC Liquid Chromatrography System)
HPLC 2 ml glass vials (Phenomenex, model: AR1-3910-12 ) with caps (Phenomenex, model: AR0-8959-13-B )
Liquid chromatography (LC) coupled with electrospray ionization (ESI) and high resolution mass spectrometry (MS) (e.g. Thermo Fisher Scientific, model: LC-ESI-FTICR MS )
Intact glucosinolates Discovery C₁₈ column, 250 x 4.6 mm, 5 µm particle size (pore size, 180Å) (Sigma-Aldrich, catalog number: 504971 ), with a Discovery C₁₈ 20 x 4 mm security guard cartridge (Sigma-Aldrich, catalog number: 505129 )
Desulfoglucosinolates (Nucleodur C₁₈ column, 125 mm x 3 mm) (MACHEREY-NAGEL, catalog number: MN760051.30 )

Procedure

Intact GLS

GLS extraction
1. Clean plant material with distilled water and dry with paper towels. Separate roots from epigeous portion, weight and immediately freeze at -80 °C to inhibit myrosinase activity.
2. Lyophilize the frozen tissues and grind to a fine powder using a laboratory mill. Chill roots in liquid nitrogen before the lyophilization to allow the crushing.
3. Weigh 200 mg of frozen dry material in 50 ml polypropylene tubes and place in a water bath heated with an electric hotplate at 70-80 °C for 10 min. During this process, carry out the following steps: After 1 min add 2 ml of 70% methanol solvent and after 5 min mix with vortex for 20 sec.
4. At the end of 10 min remove the polypropylene tube from the water bath and mix again with vortex for 20 sec.
5. Centrifuge at 4 °C for 10 min at 2,400 x g.
6. Collect the supernatant in a disposable 15 ml polypropylene tube by using glass Pasteur pipettes.
7. Extract again the remaining pellet with 2 ml of 10% methanol in water bath heated with an electric hotplate at 70-80 °C for 10 min; after 5 min mix with vortex for 20 sec and the follow the same procedure from step A4-6.
8. Combine the supernatants and vortex the extract for 20 sec.
9. Filter through 0.22 µm nylon filter and transfer into 2 ml sample vials.

GLS detection by LC MS

Inject 25 µl of each sample in LC-ESI-FTICR MS system equipped with Discovery C₁₈ column, 250 x 4.6 mm, 5 µm particle size (pore size, 180 Å), with a Discovery C₁₈ 20 x 4 mm security guard cartridge.
Settings:
Flow rate: 1 ml/min
Column temperature: 25 °C
Solvent gradient for chromatographic separation:

Time (min)
% Solvent A
0.1% HCOOH water
% Solvent B
ACN
0
90
10
10
76
24
12
40
60
15
90
10
20
90
10

Connect the LC system to the mass spectrometer by a laboratory-made splitter with a split ratio of 1:4 after the analytical column to allow 200 µl/min to enter the ESI source.

Perform full-scan experiments in both the linear trap and the ICR cell. Collect data in profile mode in the range of m/z 50−1,000 by setting:
Ionization mode: negative
Resolution: 100.000 (FWHM) at m/z 400
ESI needle voltage: -4.60 kV
Capillary voltage: -22 V
Temperature of the heated capillary: 350 °C
Sheath gas (N₂) ﬂow rate: 80 (arbitrary units)
In these conditions GLS elute with a retention time and molecular exact mass to charge ratio as reported in the following table (Agneta et al., 2012; Agneta et al., 2014):

Chemical name	Molecular formulae	Monoisotopic exact value as [M-H]^- (m/z)	t_R
3-(Methylsulfinyl)propyl-GLS	C₁₁H₂₁NO₁₀S₃	422.02549	4.3
2-Propenyl-GLS	C₁₀H₁₇NO₉S₂	358.02720	4.4
2-Methylsulfonyl-oxo-ethyl-GLS	C₁₀H₁₇NO₁₂S₃	437.98402	4.6
3-Butenyl-GLS	C₁₁H₁₉NO₉S₂	372.04285	5.5
1-Methylpropyl-GLS	C₁₁H₂₁NO₉S₂	374.05850	6.2
2-Methylpropyl-GLS	C₁₁H₂₁NO₉S₂	374.05850	6.4
4-Mercaptobuthyl-GLS	C₁₁H₂₁NO₉S₃	406.03057	6.5
7-Methylsulfinylheptyl-GLS	C₁₅H₂₉NO₁₀S₃	478.08808	7.3
4-Hydroxyindol-3-ylmethyl-GLS	C₁₆H₂₀N₂O₁₀S₂	463.04866	7.4
Unidentified isomer of 4-hydroxyindol-3-ylmethyl-GLS	C₁₆H₂₀N₂O₁₀S₂	463.04866	7.5
2(S)-Hydroxy-2-phenylethyl-GLS and/or 2(R)-Hydroxy-2-phenylethyl-GLS	C₁₅H₂₁NO₇S	438.05341	7.8
4-Pentenyl-GLS	C₁₂H₂₁NO₉SO₂	386.05850	7.8
Benzyl-GLS	C₁₄H₁₉NO₉S₂	408.04285	8.5
Indol-3-ylmethyl-GLS	C₁₆H₂₀N₂O₉S₂	447.05375	10.3
2-Phenylethyl-GLS	C₁₅H₂₁NO₉S₂	422.05850	12.1
4-Methoxyindol-3-ylmethyl-GLS	C₁₇H₂₂N₂O₁₀S₂	477.06431	12.7
7-Methylthioheptyl-GLS	C₁₅H₂₉NO₉S₃	462.09317	16.7

Note: It is possible to use the extracts to quantify GLS by preparing a calibration curve for each GLS using a series of dilution and calculating the area under the peak of each compound.

Figure 1. Example of eXtracted Ion Chromatograms (XICs) using high resolution LC-ESI-FTICR MS acquired in negative ion mode of root crude extract of A. rusticana. The ions monitored are displayed in each trace (plots A-O) and correspond to the most abundant deprotonated molecules, [M−H]⁻, using a restricted window of ±0.0010 m/z unit centered around each selected ion. Peak numbers in bold correspond to: (1) 3-(methylsulfinyl)propyl-GLS; (2) 2-propenyl-GLS; (3)* 2-methylsulfonyl-oxo-ethyl-GLS; (4) 3-butenyl-GLS; (5)* 1-methylpropyl-GLS; (6)* 2-methylpropyl-GLS; (7)* 4-mercaptobuthyl-GLS; (8)* 7-methylsulfinylheptyl-GLS; (9) 4-hydroxyindol-3-ylmethyl-GLS; (10) unidentified isomer of 4-hydroxyglucobrassicin; (11) 2(S)-hydroxy-2-phenylethyl-GLS and/or 2(R)-hydroxy-2-phenylethyl-GLS; (12) 4-pentenyl-GLS; (13) benzyl-GLS; (14) indol-3-ylmethyl-GLS; (15) 2-phenethyl-GLS; (16) 4-methoxyindol-3-ylmethyl-GLS; (17)* 7‐methylthioheptyl-GLS (peaks with asterisk are tentatively assigned). The XIC signals of some peaks were acquired using a sample extract diluted 1:20 with the mobile phase. In bold are indicated also the molecular formula and the range of monoisotopic value as [M-H]^– ion (Δ m/z) (Agneta et al., 2014).

Desulfo GLS

GLS extraction
1. Follow the procedure of intact GLS step A1-8.
2. Transfer 500 µl of the extract on the top of a 20 mg of Sephadex DEAE-A 25 (shortened Pasteur pipet) in the formiate form.
3. Wash the column twice with 1 ml of deionized water
4. Add 100 µl of sulfatase type H-1 diluted 1 to 2.5 with water to achieve the desulfation and incubate overnight at 39 °C (minimum 16 h) (Möllers et al., 1999)
5. Elute desulfatated GLS with 3 x 500 µl deionized water and collect in 15 ml polypropylene tubes.
6. Vortex the sample for 20 sec.
7. Filter through 0.22 µm nylon filter and transfer into 2 ml sample vials.

GLS detection by HPLC

Inject 25 µl of each sample in HPLC system equipped with Nucleodur C₁₈ column, 125 mm x 3 mm.
Settings:
Flow rate: 0.6 ml/min
Column temperature: 35 °C
UV detection: 229
Solvent gradient for chromatographic separation:

Time(min)	% Solvent A Water	% Solvent B ACN
0	99	1
20	80	20
25	80	20
22	99	1

In these conditions GLS elute with a retention time as reported in the following table:

Chemical name	t_R	Relative response factor^a
3-(Methylsulfinyl)propyl-GLS	2.8	1.07
2-Propenyl-GLS	4.3	1.00
3-Butenyl-GLS	7.3	1.11
1-Methylpropyl-GLS and/or	8.9	1.00^b
2-Methylpropyl-GLS	8.9	1.00^b
2(S)-Hydroxy-2-phenylethyl-GLS and/or 2(R)-Hydroxy-2-phenylethyl-GLS	10.1	0.98^c
4-Pentenyl-GLS	10.9	1.15
Benzyl-GLS	11.8	0.95
Indol-3-ylmethyl-GLS	13.9	0.29
2-Phenylethyl-GLS	16.1	0.95
4-Methoxyindol-3-ylmethyl-GLS	16.5	0.25

^aThe response factors values used for the desulfated glucosinolates quantification as reported in EN ISO 9167-1 (1992), with except of following desulfated molecules: 1-methylpropyl-GLS, 2-methylpropyl-GLS and 2(S)-hydroxy-2-phenylethyl-GLS and/or 2(R)-Hydroxy-2-phenylethyl-GLS
^bResponse factor of 2-propenyl-GLS as desulfated molecules used for the relative quantification
^cExperimental response factor determinate by using the 2(S)-hydroxy-2-phenylethyl-GLS in comparison to 2-propenyl-GLS as desulfated molecules (see Note 2)

Notes

Plant tissues were collected from a field collection of the Institute of Plant Genetics, National Research Council, Thematic Centre for the Preservation of Mediterranean Biodiversity, located in Policoro (MT) (40° 17′ 30″ N, 16° 65′ 16″ E), where many accessions of horseradish, previously collected from various villages of the internal areas of the Basilicata region, are maintained and vegetatively propagated (for details see Sarli et al., 2012).
It is possible to use the extracts to quantify desulfo GLS by preparing a calibration curve for each GLS by using a series of dilution and calculating the area under the peak of each compound.
It is also possible to quantify desulfo GLS by using the response factors reported in the table above, if you add an internal standard after step A3 (Procedure A), and by applying the following formula:
µmol Area_dg x RF_dg x n
------- = ------------------------
g Area_st x RF_st X m
Wherein:
Area_dg is the peak area of the desulfoglucosinolate, Area_st is the peak area of internal standard, RF_dg is the response factor of the corresponding desulfoglucosinolate, RF_st is the response factor of the internal standard, n is the quantity, in µmol, of the internal standard added to the sample, m is the mass, in g, of the sample.

Recipes

70% MeOH
70 ml of MeOH
30 ml of Milli-Q water
0.1% Formic acid (HCOOH)
1 ml of Formic acid
999 ml of Milli-Q water
Sulfatase type H-1 (1 to 2.5)
1 ml of sulfatase type H-1
1.5 ml of Milli-Q water
DEAE-Sephadex A-25 (formiate form)
Convert the chloride form to the formiate form by adding for each column 500 µl of 6 mM imidazole formiate (dissolve 40 g imidazole in 100 ml 30% formic acid) and then rinsing the column with water.

References

Agneta, R., Lelario, F., De Maria, S., Möllers, C., Bufo S. A. and Rivelli A. R. (2014). Glucosinolate profile and distribution among plant tissues and phenological stages of field-grown horseradish. Phytochem 106: 178-187.
Agneta, R., Rivelli, A. R., Ventrella, E., Lelario, F., Sarli, G. and Bufo, S. A. (2012). Investigation of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (Armoracia rusticana) using LC-ESI-hybrid linear ion trap with Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation. J Agric Food Chem 60(30): 7474-7482.
EN ISO 9167-1 (1992). Rapeseed-Determination of glucosinolates content-Part 1: Method using high-performance liquid chromatography.
Möllers, C., Nehlin, L., Glimelius, K. and Iqbal M. C. M. (1999). Inﬂuence of in vitro culture conditions on glucosinolate composition of microspore-derived embryos of Brassica napus. Physiol Plantarum 107(4): 441-446.
Sarli, G., De Lisi, A., Agneta, R., Grieco, S., Ierardi, G., Montemurro, F., Negro, D., Montesano, V. (2012). Collecting horseradish (Armoracia rusticana, Brassicaceae): local uses and morphological characterization in Basilicata (Southern Italy). Genet Resour Crop Ev 59(5): 889-899.