A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

1. Preparation of Solutions Needed for the Glucosinolate Extraction

1. Prepare 500 mL of 70% methanol (MeOH) in water (ultrapure) in a glass bottle. For 100 samples and 5 standards, 420 mL of 70% MeOH is needed.
2. Prepare 20 mM sodium acetate (NaOAc) (pH = 5.5) by adding 0.82 g of NaOAc or 1.36 g of NaOAc x 3 H₂O in 500 mL of water; adjust the pH with hydrogen chloride (HCl). Store the NaOAc in the refrigerator. For 100 samples and 5 standards, 370 mL of 20 mM NaOAc (pH = 5.5) in water is needed.
3. To prepare the column material, mix 10 g of cross-linked dextran gel (type G-25) with 125 mL of ultrapure water. Store the resulting mixture in a refrigerator (4 °C).
4. Preparation of the sulfatase solution
   1. Dissolve 10,000 units of aryl sulfatase (type H-1 from Helix pomatia) in 30 mL of ultrapure water and add 30 mL of absolute ethanol (EtOH). Mix the solution well and transfer it to one 250-mL centrifuge tube or multiple smaller tubes, depending on availability.
   2. Centrifuge the mixture at 2,650 x g for 20 min at room temperature (RT). Transfer the supernatant(s) to a beaker; add 90 mL of EtOH and mix it. Centrifuge the mixture in one or more centrifuge tubes at 1,030 x g for 15 min at RT.
   3. Discard the supernatants. Dissolve and combine the pellets in a total of 25 mL of ultrapure water. Vortex well, dispense in 1-mL tubes, and store in a freezer at -20 °C; they will keep for at least one year.
5. Preparation of the sinigrin reference solution
   1. Weigh in around 9 mg of sinigrin monohydrate with 1-µg accuracy (e.g., 8.769 mg). Transfer it into a 10-mL volumetric flask and dissolve the sinigrin monohydrate in 10.0 mL of ultrapure water.
   2. Calculate the molarity of the stock solution and prepare five sinigrin references between 50-750 µM by diluting of the stock solution. For a detailed example, see Supplementary File S1.
   3. Dispense the different sinigrin references in 1.5-mL reaction tubes and freeze them at 20 °C. Include a series of five sinigrin references in each extraction batch. Always use the correct molarity values for the currently used sinigrin references when calculating the concentrations.

2. Preparation of the Extraction Setup

1. Prepare a column rack for the Pasteur pipette columns (for the preparation of columns, see step 3.1). Label the rack or the columns to keep track of the sample numbers (see Figure 1).
2. Prepare a block holding the same number of labelled 2-mL microcentrifuge tubes (see step 3.3 and Figure 1)

Figure 1: Rack for glucosinolate extraction. Front view (left) and top view (right) of a self-made column rack and block for the glucosinolate extraction. Height: 100 mm, distance between shifted holes (to hold the Pasteur pipette columns): 30 mm (x-axis) x 15 mm (y-axis). Please click here to view a larger version of this figure.

3. Preparation of the Columns and Microcentrifuge Tubes

1. Prepare one column per sample and five extra columns for the five sinigrin reference samples.
   1. In each glass pipette, place a piece of glass wool approximately 1 cm x 1 cm. Use a wooden or glass stick to push the glass wool down to the point where the pipette narrows. The glass wool should prevent the column material from running out, but do not use too much, as it will slow the elution of the columns. Wear gloves when working with the glass wool.
2. Place the columns on the column rack (see Figure 1). Place the column rack in a lab tray (waste tray) to catch the eluents used for the consecutive column washes.
3. Cut the first 5 mm off the tip of a plastic 1-mL pipette tip and pipette 0.5 mL of the prepared column material (G-25 gel in water, see step 1.3) into each column. Shake the bottle containing the column material well before pipetting. Discard leaking columns (G-25 material running out through the glass wool layer) and replace with new ones.
   1. After pipetting the column material into the columns, add 1 mL of ultrapure water to flush down the material.
4. Prepare one 2-mL reaction tube per sample and five tubes for the five sinigrin reference samples; label them. Use a dissecting needle to make holes in the caps for later freeze-drying and place the prepared tubes into the tube block in the exact same pattern as the columns (step 2.2, see Figure 1).

4. Extraction of Glucosinolates

1. Weigh freeze-dried and finely-ground plant material (usually 50.0-100.0 mg of dry weight; the final glucosinolate concentrations in the extract should be in the range of the reference curve) to the nearest 0.1 mg in 2-mL, labeled, round-bottom reaction tubes. Add two small metal balls (3 mm in diameter) as boiling retardants to each tube.
   NOTE: The protocol can also be applied to fresh, flash-frozen materials, that have been ground under liquid nitrogen and kept frozen until extraction. Increase the amount of material weighed for extraction and the percentage of MeOH in the extraction liquid to 85% to compensate for dilution by the water in the materials¹⁹.
2. Pipette 1 mL of 70% MeOH into each tube and vortex briefly. Close the tubes and seal them with safety caps before placing them as quickly as possible into a hot water bath (90-92 °C) for a few minutes (~5 min), until the 70% MeOH just boils. Caution: Wear safety goggles during this step!
3. Place the sample tubes in an ultrasonic bath for 15 min. Meanwhile, take the sulfatase and the five sinigrin reference samples out of the freezer to thaw them at RT.
4. After ultra-sonication, centrifuge the sample tubes at 2,700 x g in a benchtop centrifuge for 10 min at RT; a pellet should form in each tube. Add the supernatants to the labelled columns and pipette the five reference samples onto separate columns.
   1. While pipetting the supernatants, keep the tip well over the pellet to avoid pipetting plant materials. Note that when dried samples are used, the supernatant volume will be less than 1.0 mL.
5. Add 1 mL of 70% MeOH to the remaining pellets in the sample tubes and vortex the tubes before placing them in an ultrasonic bath for 15 min. Centrifuge the tubes again, as in step 4.4, and add the supernatants to the respective columns; due to the properties of the column material, the negatively charged sulfate group of the glucosinolates will be specifically retained on the column.
6. Wash the columns with the extracts in three sequential steps.
   1. Pipette 2 x 1 mL of 70% MeOH onto each column. Wait for the column to run dry before adding the next 1 mL; this will remove more apolar compounds from the extracts (e.g., chlorophyll).
   2. Flush out the MeOH by adding 1 mL of ultrapure water to each column.
   3. Pipette 2 x 1 mL of 20 mM NaOAc buffer to each column to create the optimal conditions for the sulfatase reaction.
7. Take the rack with the columns out of the waste tray and dry the feet of the rack with a tissue. Place the rack over the block with vials and labeled tubes. Make sure that each column tip is in the corresponding, labeled, 2-mL tube (see Figure 1).
8. Add 20 µL of sulfatase solution to the columns. Ensure that the sulfatase reaches the surface of the column material. Pipette 50 µL of NaOAc buffer onto each column to flush down the sulfatase. Cover the columns with aluminum foil and let stand overnight.
   NOTE: Due to the activities of the sulfatase, the sulfate group will be removed, releasing the desulfoglucosinolates from the column so that they can elute with the water. For a detailed description, see Crocoll et al.¹⁹.
9. The next day, elute the desulfoglucosinolates by pipetting 2 x 0.75 mL of ultrapure water onto each column. When all columns have run dry, lift the column rack and remove it from the reaction tubes.
   1. Cap the tubes (make sure that there are holes in the caps) and freeze them in liquid nitrogen or in a -80 °C freezer for 30 min. Freeze-dry the samples for 12-24 h (depending on the number of samples and the capacity of the freeze-drier) to remove all water.
10. After freeze-drying, re-dissolve the residue in an exact volume (usually 1.0 mL) of ultrapure water. Transfer the samples and the five sinigrin references to labeled HPLC vials. Keep the samples in a refrigerator (4 °C) for up to two weeks or a freezer (-20 °C) for up to one year before analyzing them with HPLC.
11. Let the glass columns dry under the hood overnight and dispose them when dry. Recover the metal balls from the sample tubes used in step 4.5 for reuse and put the tubes in the waste disposal bin.

5. HPLC Measurements of Extracted Samples

1. Separate the glucosinolates on a reversed-phase C₁₈ column (4.6 x 150 mm, 3 µm, 300 Å) with an acetonitrile-water gradient (see Table 1) and a flow of 0.75 mL/min and a column temperature of 40 °C. Perform detection and quantification at 229 nm.
2. Measure the sinigrin references on the same column but with an adapted gradient method to reduce solvent use (see Table 2). Start with injecting the five sinigrin references, beginning with the reference with the lowest concentration. Then inject the samples, including a methanol injection (blank) after every tenth sample to clean the column and to prevent the carryover of peaks.

Table 1: Acetonitrile-water gradient for glucosinolate separation and analysis on reversed-phase HPLC.

Table 2: Shortened acetonitrile-water gradient for quantification of the five sinigrin references used for the quantification of glucosinolates.

6. Glucosinolate Identification and Quantification

1. Identification
   1. Compare the UV spectra and retention times of the peaks in the samples with commercially available, pure glucosinolate references. See Figure 2 for examples of UV spectra from the various glucosinolate classes.
   2. Identify unknown glucosinolates with reference standards or on LC-MS^18,19, if possible.
   3. Identify unknown glucosinolates with nuclear magnetic resonance (NMR), if possible.
   4. Compare the UV spectra and retention times of the peaks with those in Figure 2 and Table 3, databases, or literature references.

Figure 2. UV spectra of the most common glucosinolate classes. UV absorption spectra (200-350 nm) of six desulfoglucosinolates (GSL), based on solutions made of commercially available reference compounds extracted as described here, representing the most common structural classes. The common name, structural name (between brackets), and structural class are given. (A) Sinigrin (2-propenyl GSL), alkenyl; (B) glucoerucin (4-methylthiobutyl GSL), thioalkenyl; (C) glucoraphanin (4-methylsulfinlybutyl GSL), sulfinyl; (D) glucobrassicin (indol-3-ylmethyl GSL), indole; (E) gluconasturtiin (2-phenylethyl GSL), aromatic; (F) sinalbin (4-hydroxybenzyl GSL), aromatic. Please click here to view a larger version of this figure.

2. Quantification
   1. Integrate the area under the peaks of the five sinigrin references and the samples.
   2. Calculate a calibration curve of the five measured sinigrin references based on the integrated area. Use the equation of the regression line (see Equation 1) to calculate the interpolated amount of glucosinolates (see Equation 2).
   3. To calculate the concentration of the different glucosinolates, multiply the interpolated amount (x) by the response factor (M) for detection at 229 nm from EC (1990)¹⁶, Buchner (1987)¹⁵, or Brown et al. (1993)¹⁷; the consensus response factors of the most commonly detected desulphoglucosinolates are given in Table 3.
      NOTE: Response factors may vary between systems-this may be the reason that there are different values given by different references (see References 15-17). Nevertheless, the values are mostly quite close and in a similar range with structural classes. Following the convention, set the response factor of unidentified glucosinolates to 1, although for unknown indole glucosinolates with a UV spectrum similar to that of glucobrassicin and other indole glucosinolates (Figure 2), it would be advisable to use 0.25 as a response factor to obtain a realistic estimate of the concentration (Table 3).
   4. To calculate the final amount of glucosinolates (x_t) in the sample, take the dilution factor (D) and the mass of sample (w) used for the analysis into account (see Equation 3).
      (1)
      (2)
      (3)
      y = Peak area
      m = slope of the reference 5-point regression curve
      c = the intercept of the regression line with the y-axis
      x = amount of glucosinolates in the extract
      x_t = concentration of glucosinolates in the plant sample
      D = dilution factor
      M = response factor for the detection at 229 nm from Buchner (1987) or Brown et al. (1993)
      w = mass of sample used for extraction