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The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography
Journal of Chromatography A, 1120 (2006) 205–210
The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography Simone Rochfort a,∗ , Domenico Caridi b , Melanie Stinton b , V. Craige Trenerry a , Rod Jones c b
a PIRVic DPI-Werribee, 621 Sneydes Road, Werribee, Vic. 3030, Australia School of Molecular Sciences, Victoria University, Werribee, Vic. 3030, Australia c PIRVic DPI-Knoxfield, 621 Burwood Highway, Knoxfield, Vic. 3152, Australia
Available online 2 February 2006
Abstract Plant foods contain not only essential nutrients, e.g. protein, amino acids, vitamins and minerals, but also phytochemicals that have added health benefits. One such class of phytochemicals are the glucosinolates. Glucosinolates, particularly glucoraphanin, are predominant in plants of the Brassica genus, most notably in vegetables such as broccoli. There is a growing interest in the role glucoraphanin plays in chemoprotection and as a result there is a requirement to accurately determine the levels of glucoraphanin in vegetable products. Reverse phase ion pair high performance liquid chromatography (HPLC) is the method of choice; however, this work has been hindered by the lack of available standard reference materials. Broccoli seeds, which are particularly rich in glucoraphanin (20–50 mg/g), have proved to be ideal for the isolation of glucoraphanin on the preparative scale. A novel preparative scale HPLC method with simple compound recovery has been developed to meet the need for a glucoraphanin standard. © 2006 Elsevier B.V. All rights reserved. Keywords: Glucoraphanin; Preparative HPLC; SPE; Glucosinolates; Brassica sp.
1. Introduction Many cancer prevention approaches recommend the need for lifestyle changes accentuating exercise, the cessation of smoking and the increased consumption of plant materials such as fruits and vegetables. The consumption of plant materials is especially important because there are various substances in fruits and vegetables that are important for maintaining health. This has been substantially demonstrated in various studies [1]. Secondary plant metabolites fall into many chemical classes, some of which are beneficial for health. These beneficial phytochemicals are biologically active non-nutrients that have been associated with the protection of human health [2]. Glucosinolates, which are present in the genus Brassica and the family Crucifereae, have generated particular interest with respect to cancer prevention [3]. Glucoraphanin (4-methylsulfinylbutyl
∗
Corresponding author. Tel.: +61 3 97428704. E-mail address: [email protected] (S. Rochfort).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.01.046
glucosinolate) (Fig. 1) is the most predominant glucosinolate in broccoli [4,5] and is hydrolysed by the enzyme myrosinase to sulforaphane when it is released from the plant vacuoles after mechanical stress, e.g. during cutting or chewing. Sulforaphane is the active component that inhibits Phase 1 enzymes and induces Phase II enzymes which is thought to result in certain cancer protection [6–8]. There is a requirement to accurately determine the levels of glucosinolates in various vegetables due to the growing awareness by scientists and consumers of the role glucosinolates play in cancer prevention. This work has been hampered by the lack of suitable quantities of pure glucosinolate reference materials, in particular glucoraphanin. A number of approaches to isolate glucoraphanin from plant material have been documented. These include high-speed column chromatography, counter current chromatography [9] and preparative HPLC [10,11]. Fahey et al. [9] isolated large (20 g) quantities of glucoraphanin using counter current chromatography; however, this technique is not commonly available and requires specialist attention. Preparative HPLC is an attractive alternative to counter current chromatography and has been used
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Fig. 1. Glucoraphanin R (CH2 )4 SOCH3 .
to prepare smaller quantities of glucoraphanin from white cabbage [10]. Broccoli seeds, which are particularly rich in glucoraphanin (20–50 mg/g) [4,5,12], appear to be a good substrate for preparative HPLC. (Note: glucoraphanin is only predominant in broccoli; cauli, cabbage and Brussels sprouts have mainly sinigrin, progoitrin or glucobrassicin, with only a small amount of glucoraphanin being present.) This paper reports a rapid method for the isolation and purification of glucoraphanin from broccoli seeds using solid phase extraction (SPE) and preparative HPLC based on a method developed in our laboratories for the quantification of glucoraphanin in broccoli seeds and broccoli florets [13]. 2. Experimental 2.1. Chemicals Glucoraphanin, potassium salt was obtained from the Royal Veterinary and Agricultural University (Copenhagen, Denmark). Tetramethyl ammonium bromide (TMAB) was obtained from Sigma Chemical Company, Sydney, Australia. All other chemicals and reagents were AR or HPLC grade. C18 (1 g) and amino propyl (1 g) cartridges were obtained from Varian Inc., USA. Stock and working standards of glucoraphanin were prepared by dissolving glucoraphanin potassium salt in deionised water. The solutions were stable for at least 3 months when stored at 4 ◦ C. 2.2. Samples and extraction procedure 2.2.1. Samples Local seed producers in Victoria, Australia, provided samples of broccoli seeds. The seeds were stored in a sealed container at 4 ◦ C until used. 2.2.2. Extraction procedure To approximately 3 g of broccoli seeds 30 ml of boiling water was added and the mixture boiled for 5 min. The bulk of the water was decanted and the seeds transferred to a mortar with 5 ml of water. The seeds were ground to a paste. The resultant slurry was transferred to a 100 ml volumetric flask with deionised water, made to the mark and sonicated for 5 min. The extract was filtered under vacuum through Whatman No 4 filter paper. Mega Bond Elut C18 cartridges (1 g) were activated with methanol and washed with water. Mega Bond Elut NH2 car-
tridges (1 g) were activated with methanol and equilibrated with 1% acetic acid in water. The C18 and NH3 + cartridges were connected in series and 10 ml of the extract loaded onto the C18 cartridge. The cartridges were washed with 6 ml of deionised water, the C18 cartridge discarded and the NH3 + cartridge washed with 6 ml of methanol. The glucosinolate fraction was removed from the NH3 + cartridge with 10 ml of freshly prepared 2% solution of concentrated NH4 OH solution in methanol. The solvents were evaporated to dryness under a stream of nitrogen at room temperature. The entire 100 ml was processed in this manner and the residues were combined and dissolved in 1 ml of deionised water. A 100 l aliquot was removed and diluted to 1 ml for analytical HPLC analysis. The remaining material was kept for preparative HPLC analysis. 2.3. Instrumentation 2.3.1. Analytical HPLC The extraction procedure was monitored with either of the following sets of conditions: 2.3.1.1. Analytical Method 1. The analyses were performed with an Agilent series 1100 HPLC equipped with a quaternary gradient pump, autosampler and diode array detector using a 10 m C18 300 mm × 3.9 mm stainless steel Bondapak HPLC column (Waters, Milford, MA, USA) fitted with a C18 guard column. The mobile phase consisted of 0.005 M TMAB dissolved in 2% v/v methanol/water. The flow rate was 1.5 ml per min and the chromatograms were monitored concurrently at 230 and 270 nm. Chemstation software was used to process the chromatographic data. The column was flushed with 20% v/v methanol/water after the end of each series of analyses. 2.3.1.2. Analytical Method 2. The analyses were performed with a Varian 9012 Solvent Delivery System equipped with a Varian 9100 Autosampler and a Varian 9050 Variable Wavelength UV–vis detector set at 234 nm using a 5 m C18 250 mm × 4.6 mm stainless steel C18 Prevail column and guard column (Alltech Assoc., Baulkham Hills, NSW, Australia). The mobile phase consisted of 0.05 M sodium di-hydrogen orthophosphate dissolved in 2% v/v methanol/water at a flow rate of 1 ml per min. Varian Star Version 5.5 software was used to process the chromatographic data. The column was flushed with 20% v/v methanol water after the end of each series of analyses. 2.3.1.3. Analytical Method 3. The analyses were performed with the HPLC equipment described in (1) above but using a 5 m 250 mm × 4.6 mm stainless steel C18 Prevail column and guard column (Alltech Assoc., Baulkham Hills, NSW, Australia). A mobile phase consisting of 1% v/v acetonitrile/water (0.1% v/v formic acid in both solvents) was used with a flow rate of 1 ml per min. The column was flushed with 20% v/v acetonitrile/water after the end of each series of analyses.
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2.3.2. Preparative HPLC 2.3.2.1. Mobile phases as utilised in Analytical Methods 1 and 2 (Section 2.3.1). Preparative HPLC was performed with a 5 m C18 250 mm × 21.4 mm stainless steel Varian Dynamax (Varian Inc., CA, USA) preparative column fitted to a Varian Prostar 215 Solvent Delivery Module and a Varian Prostar 320 UV–vis detector. The sample solution was loaded onto the column through a 1 ml sample injection loop. The flow rate was 22 ml/min. Varian Star software was used to monitor the chromatography. The glucoraphanin peak was collected manually. 2.3.2.2. Mobile phase as utilised in Analytical Method 3 (Section 2.3.1). Preparative HPLC was performed with a 5 m C18 250 mm × 21.4 mm stainless steel Varian (Varian Inc., CA, USA) preparative column fitted to a Waters 600E HPLC system equipped with a Waters HPLC autosampler (1 ml injection loop) and a Waters 440 variable wavelength UV detector set at 230 nm. The flow rate was 10 ml/min. The HPLC equipment was controlled by Waters Empower software (Waters, Milford, MA, USA). The glucoraphanin peak was collected manually. 2.3.3. Mass spectrometry Mass spectra were obtained by infusing a dilute aqueous solution of the extract and a pure glucoraphanin standard into the electrospray source of a ThermoElectron LTQ ion trap mass spectrometer (Thermo Electron Corporation CA, USA) operating in the Negative Ion Mode. The sample was introduced by infusion at 5 ul/min to the IonMax ESI probe without the use of auxiliary gases. The source voltage was 4 kV, the capillary voltage −44 V and the heated capillary was maintained at 250 ◦ C. The structure was confirmed by comparing the MS2 fragmentation pattern of the molecular ion from the sample with the MS2 fragmentation pattern of pure glucoraphanin. MS2 fragmentation was generated using helium as a collision gas and carried out under the following conditions: MS2 isolation width: 2.00; MS2 normalized coll. energy: 35.0; MS2 activation Q: 0.250 and MS2 activation time: 30.000 ms. 2.3.4. NMR spectroscopy Proton nuclear magnetic resonance (NMR) spectroscopy was performed in D2 O using a Bruker high resolution DPX300 NMR Spectrometer at 300 mHz (Bruker Biospin Corporation, USA). The DEPT experiment was acquired on the same instrument at 75 MHz. The DEPT experiment revealed all protonated carbons in the molecule and allowed the assignment of carbon multiplicity, providing additional evidence for the identification of glucoraphanin. 3. Results and discussion Glucoraphanin has been isolated in limited quantities by preparative scale HPLC [10]. Previous attempts to isolate pure glucosinolates by preparative HPLC have indicated that this is difficult to achieve due to the complex chromatograms and
Fig. 2. Crude seed extract after combined C18-amino SPE clean-up in de-ionised water, 98% sodium di-hydrogen orthophosphate with 2% methanol, 234 nm, ambient temperature, 1.0 ml/min.
that preparative HPLC only yields small amounts of compounds [10,14]. We recently reported the isolation and determination of glucoraphanin in broccoli seeds using SPE as a means of isolating and concentrating the glucoraphanin prior to analysis by either Micellar Electrokinetic Capillary Chromatography (MECC) or HPLC [13]. HPLC using a Bondapak C18 column and a mobile phase consisting of 0.005 M TMAB in 2% v/v methanol/water was used to determine the levels of glucoraphanin in the seeds. Similar separations were achieved with a number of different mobile phases. A typical chromatogram demonstrating the separation of glucoraphanin from other compounds is shown in Fig. 2. Hot aqueous extraction followed by SPE seems an efficient way to prepare a concentrated glucosinolate solution suitable for preparative HPLC [13]. Treatment of the seed with boiling water to deactivate myrosinase followed by crushing and extraction with hot water gave a glucosinolate fraction rich in glucoraphanin. The seed extract (100 ml), which was too dilute to use for preparative HPLC, also contained other plant material that could potentially damage the preparative HPLC column. A crude glucosinolate concentrate was readily obtained from the seed extract by passing the solution through a C18 SPE cartridge and a protonated amino propyl anion exchange SPE cartridge in series. Unwanted organic material was trapped on the C18 cartridge whilst the charged glucosinolates passed straight through and were trapped on the protonated amino propyl column. The C18 cartridge was removed, the amino propyl cartridge washed with methanol and the glucosinolates removed by washing with 2% ammonia solution in methanol. The solvent was removed in vacuo leaving a residue rich in glucoraphanin (98 mg) (see Fig. 2). This procedure offered a viable alternative to removal of the water in vacuo as it also allowed for the removal of unwanted plant material that might potentially interfere with the separation or block the preparative HPLC column. HPLC and MECC [13] analysis of the residue showed that glucoraphanin was quantitatively recovered from the SPE clean up. Glucoraphanin was identified from its retention time and UV spectral information from the photodiode array detector of the HPLC system. This residue was dissolved
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S. Rochfort et al. / J. Chromatogr. A 1120 (2006) 205–210 Table 1 Comparison of techniques employed in the preparative HPLC isolation of glucoraphanin Preparative HPLC mobile phase
Isolation of pure glucoraphanin
Reference
2% v/v CH3 OH, 98% aqueous 0.05 M TMAB
1. Add NH4 Cl
[11]
2% v/v CH3 OH, 98% aqueous 0.05 M NaH2 PO4 , pH 3.2 Fig. 3. Analysis of glucoraphanin prepared using prep mobile phase 1 (TMAB). Analytical conditions: 98% 50 mM sodium di-hydrogen orthophosphate with 2% v/v methanol, 234 nm, ambient temperature, 1.0 ml/min.
in a minimum amount of water and subject to preparative HPLC. 3.1. Preparative HPLC Fig. 3 shows the purity of the glucoraphanin collected by preparative HPLC with mobile phase as in Analytical Method 1 as described in Section 2. Similar results were obtained with mobile phase as in Analytical Method 2. When fractions are collected from the preparative HPLC, it is then necessary to remove the mobile phase in order to purify the glucosinolate. There are different ways to remove the mobile phase from the sample. In many cases, the removal of the solvent is the primary step, followed by the removal of the mobile phase salt. Initially, preparative HPLC was attempted based on literature analytical procedures. The preparative HPLC chromatograms mirrored the analytical procedures allowing for a pure fraction to be collected. The purity of the extract was confirmed by analytical HPLC (see Fig. 4) and mass spectral analysis; however, NMR revealed surprisingly little product, given the mass recovered. The difficulty lay in separating the glucoraphanin from residual salts remaining from the preparative LC mobile phases (sodium phosphate and tetramethyl ammonium bromide) (see Table 1). This led to the development of an alternative mobile phase without the use of inorganic salts. Formic acid was chosen as the pH modifier as it is reasonably volatile and should be readily
1% v/v CH3 CN (0.1% formic acid), 99% aqueous 0.1% formic acid
2. CHCl3 extraction, 3. Removal of solvent in vaccuo 1. Removal of mobile phase in vaccuo 2. Precipitation of NaH2 PO4 with CH3 OH 3. Filter to remove NaH2 PO4 4. Remove CH3 OH in vaccuo 1. Removal of mobile phase in vaccuo (T < 45 ◦ C)
[10]
New method
removed with the other components of the mobile phase under reduced pressure. Glucoraphanin was well separated from the other components in both the analytical and preparative HPLC using the
Fig. 5. 1 H NMR (D2 O, 300 MHz).
Fig. 4. Preparative HPLC collection, 1% aqueous acetonitrile with 0.1% formic acid, 234 nm, ambient temperature, 10.0 ml/min. NB: the trace shown here is for a 5 mg injection of material. Good seperation was still achieved with 50 mg though the UV–vis trace is off scale.
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209
Fig. 6. DEPT (D2 O, 75 MHz).
mobile phase as in Analytical Method 3 described in Sections 2.3.1 and 2.3.2 (see Fig. 4). Removal of the mobile phase under reduced pressure at room temperature gave a viscous, colourless liquid (18 mg) which was shown to be glucoraphanin (acid) from a comparison of the 1 H NMR (Fig. 5), DEPT NMR (Fig. 6) and mass spectra (Fig. 7) of the sample with those of the pure reference compound [10]. Fig. 8 identifies the MS2 mass spectra of the purified glucoraphanin. Here, the molecular ions are fragmented with an inert gas in the ion trap of the mass spectrometer. The glucoraphanin standard, purified material and fractions obtained throughout the purification procedure produced spectra that were all very sim-
Fig. 8. MS2 spectrum acquired on LTQ mass spectrometer by infusion.
ilar. The fragment patterns in all three spectra displayed peaks at 436, 372 and 259 amu and the MS2 fragment patterns are at similar relative ratios providing additional conclusive proof that the purified preparative collections contain glucoraphanin. 4. Conclusion Pure glucoraphanin was obtained in 0.6% (6 mg/g) overall yield from an aqueous extraction of broccoli seeds followed by SPE and preparative HPLC using a mobile phase consisting of 1% v/v acetonitrile/water (0.1% v/v formic acid in both solvents). The glucoraphanin (17.6 mg) was purified from 98 mg of crude material obtained from 3 g of broccoli seed. The purity was assessed by HPLC, MS, NMR and UV spectroscopy. The described method is rapid (less than 1 day), robust and suitable for preparing useable quantities of pure glucoraphanin suitable for the quantitation of glucoraphanin in Brassica species. Acknowledgments
Fig. 7. MS spectrum acquired on LTQ mass spectrometer by infusion.
The authors wish to acknowledge the assistance of Ms. JaneWhitford (DPI cadet) for her technical support and Dr. Andrew Smallridge (Victoria University) for his assistance in NMR data acquisition.
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References [1] M. Nestle, Nutr. Rev. 56 (1998) 127. [2] A. Milner, J. Nutr. 129 (1999) 1295. [3] J.W. Fahey, Y. Zhang, P. Talalay, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 10367. [4] N. Rangkadilok, M.E. Nicolas, R.N. Bennett, R.R. Premier, D.R. Eagling, P.W.J. Taylor, Sci. Hortic. 96 (2002) 27. [5] L.G. West, K.A. Meyer, B.A. Balch, F.J. Rossi, M.R. Schultz, G.W. Haas, J. Agric. Food Chem. 52 (2004) 916. [6] D. Bertelli, M. Plessi, D. Braghiroli, A. Monzani, Food Chem. 63 (1998) 417. [7] G. Kiddle, R.N. Bennett, N.P. Botting, N.E. Davidson, A.A.B. Robertson, R.M. Wallsgrove, Phytochem. Anal. 12 (2001) 226.
[8] F.M.V. Pereira, E. Rosa, J.W. Fahey, K.K. Stephenson, R. Carvalho, A. Aires, J. Agric. Food Chem. 50 (2002) 6239. [9] J.W. Fahey, K.L. Wade, K.K. Stephenson, F.E. Chou, J. Chromatogr. A 996 (2003) 85. [10] L.G. Arguello, D.K. Sensharma, F. Qiu, A. Nurtaeva, Z. El Rassi, J. AOAC Int. 82 (1999) 115. [11] T. Prestera, J.W. Fahey, W.D. Holtzclaw, C. Abeygunawardana, J.L. Kachinski, P. Talalay, Anal. Biochem. 239 (1996) 168. [12] L. West, I. Tsui, G. Haas, J. Chromatogr. A 996 (2002) 227. [13] V.C. Trenerry, D. Caridi, A. Elkins, O. Donkor, R. Jones, Food Chem. 98 (2006) 179–187. [14] J.K. Troyer, K.K. Stephenson, J.W. Fahey, J. Chromatogr. A 919 (2001) 299–304.
The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography Simone Rochfort a,∗ , Domenico Caridi b , Melanie Stinton b , V. Craige Trenerry a , Rod Jones c b
a PIRVic DPI-Werribee, 621 Sneydes Road, Werribee, Vic. 3030, Australia School of Molecular Sciences, Victoria University, Werribee, Vic. 3030, Australia c PIRVic DPI-Knoxfield, 621 Burwood Highway, Knoxfield, Vic. 3152, Australia
Available online 2 February 2006
Abstract Plant foods contain not only essential nutrients, e.g. protein, amino acids, vitamins and minerals, but also phytochemicals that have added health benefits. One such class of phytochemicals are the glucosinolates. Glucosinolates, particularly glucoraphanin, are predominant in plants of the Brassica genus, most notably in vegetables such as broccoli. There is a growing interest in the role glucoraphanin plays in chemoprotection and as a result there is a requirement to accurately determine the levels of glucoraphanin in vegetable products. Reverse phase ion pair high performance liquid chromatography (HPLC) is the method of choice; however, this work has been hindered by the lack of available standard reference materials. Broccoli seeds, which are particularly rich in glucoraphanin (20–50 mg/g), have proved to be ideal for the isolation of glucoraphanin on the preparative scale. A novel preparative scale HPLC method with simple compound recovery has been developed to meet the need for a glucoraphanin standard. © 2006 Elsevier B.V. All rights reserved. Keywords: Glucoraphanin; Preparative HPLC; SPE; Glucosinolates; Brassica sp.
1. Introduction Many cancer prevention approaches recommend the need for lifestyle changes accentuating exercise, the cessation of smoking and the increased consumption of plant materials such as fruits and vegetables. The consumption of plant materials is especially important because there are various substances in fruits and vegetables that are important for maintaining health. This has been substantially demonstrated in various studies [1]. Secondary plant metabolites fall into many chemical classes, some of which are beneficial for health. These beneficial phytochemicals are biologically active non-nutrients that have been associated with the protection of human health [2]. Glucosinolates, which are present in the genus Brassica and the family Crucifereae, have generated particular interest with respect to cancer prevention [3]. Glucoraphanin (4-methylsulfinylbutyl
∗
Corresponding author. Tel.: +61 3 97428704. E-mail address: [email protected] (S. Rochfort).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.01.046
glucosinolate) (Fig. 1) is the most predominant glucosinolate in broccoli [4,5] and is hydrolysed by the enzyme myrosinase to sulforaphane when it is released from the plant vacuoles after mechanical stress, e.g. during cutting or chewing. Sulforaphane is the active component that inhibits Phase 1 enzymes and induces Phase II enzymes which is thought to result in certain cancer protection [6–8]. There is a requirement to accurately determine the levels of glucosinolates in various vegetables due to the growing awareness by scientists and consumers of the role glucosinolates play in cancer prevention. This work has been hampered by the lack of suitable quantities of pure glucosinolate reference materials, in particular glucoraphanin. A number of approaches to isolate glucoraphanin from plant material have been documented. These include high-speed column chromatography, counter current chromatography [9] and preparative HPLC [10,11]. Fahey et al. [9] isolated large (20 g) quantities of glucoraphanin using counter current chromatography; however, this technique is not commonly available and requires specialist attention. Preparative HPLC is an attractive alternative to counter current chromatography and has been used
206
S. Rochfort et al. / J. Chromatogr. A 1120 (2006) 205–210
Fig. 1. Glucoraphanin R (CH2 )4 SOCH3 .
to prepare smaller quantities of glucoraphanin from white cabbage [10]. Broccoli seeds, which are particularly rich in glucoraphanin (20–50 mg/g) [4,5,12], appear to be a good substrate for preparative HPLC. (Note: glucoraphanin is only predominant in broccoli; cauli, cabbage and Brussels sprouts have mainly sinigrin, progoitrin or glucobrassicin, with only a small amount of glucoraphanin being present.) This paper reports a rapid method for the isolation and purification of glucoraphanin from broccoli seeds using solid phase extraction (SPE) and preparative HPLC based on a method developed in our laboratories for the quantification of glucoraphanin in broccoli seeds and broccoli florets [13]. 2. Experimental 2.1. Chemicals Glucoraphanin, potassium salt was obtained from the Royal Veterinary and Agricultural University (Copenhagen, Denmark). Tetramethyl ammonium bromide (TMAB) was obtained from Sigma Chemical Company, Sydney, Australia. All other chemicals and reagents were AR or HPLC grade. C18 (1 g) and amino propyl (1 g) cartridges were obtained from Varian Inc., USA. Stock and working standards of glucoraphanin were prepared by dissolving glucoraphanin potassium salt in deionised water. The solutions were stable for at least 3 months when stored at 4 ◦ C. 2.2. Samples and extraction procedure 2.2.1. Samples Local seed producers in Victoria, Australia, provided samples of broccoli seeds. The seeds were stored in a sealed container at 4 ◦ C until used. 2.2.2. Extraction procedure To approximately 3 g of broccoli seeds 30 ml of boiling water was added and the mixture boiled for 5 min. The bulk of the water was decanted and the seeds transferred to a mortar with 5 ml of water. The seeds were ground to a paste. The resultant slurry was transferred to a 100 ml volumetric flask with deionised water, made to the mark and sonicated for 5 min. The extract was filtered under vacuum through Whatman No 4 filter paper. Mega Bond Elut C18 cartridges (1 g) were activated with methanol and washed with water. Mega Bond Elut NH2 car-
tridges (1 g) were activated with methanol and equilibrated with 1% acetic acid in water. The C18 and NH3 + cartridges were connected in series and 10 ml of the extract loaded onto the C18 cartridge. The cartridges were washed with 6 ml of deionised water, the C18 cartridge discarded and the NH3 + cartridge washed with 6 ml of methanol. The glucosinolate fraction was removed from the NH3 + cartridge with 10 ml of freshly prepared 2% solution of concentrated NH4 OH solution in methanol. The solvents were evaporated to dryness under a stream of nitrogen at room temperature. The entire 100 ml was processed in this manner and the residues were combined and dissolved in 1 ml of deionised water. A 100 l aliquot was removed and diluted to 1 ml for analytical HPLC analysis. The remaining material was kept for preparative HPLC analysis. 2.3. Instrumentation 2.3.1. Analytical HPLC The extraction procedure was monitored with either of the following sets of conditions: 2.3.1.1. Analytical Method 1. The analyses were performed with an Agilent series 1100 HPLC equipped with a quaternary gradient pump, autosampler and diode array detector using a 10 m C18 300 mm × 3.9 mm stainless steel Bondapak HPLC column (Waters, Milford, MA, USA) fitted with a C18 guard column. The mobile phase consisted of 0.005 M TMAB dissolved in 2% v/v methanol/water. The flow rate was 1.5 ml per min and the chromatograms were monitored concurrently at 230 and 270 nm. Chemstation software was used to process the chromatographic data. The column was flushed with 20% v/v methanol/water after the end of each series of analyses. 2.3.1.2. Analytical Method 2. The analyses were performed with a Varian 9012 Solvent Delivery System equipped with a Varian 9100 Autosampler and a Varian 9050 Variable Wavelength UV–vis detector set at 234 nm using a 5 m C18 250 mm × 4.6 mm stainless steel C18 Prevail column and guard column (Alltech Assoc., Baulkham Hills, NSW, Australia). The mobile phase consisted of 0.05 M sodium di-hydrogen orthophosphate dissolved in 2% v/v methanol/water at a flow rate of 1 ml per min. Varian Star Version 5.5 software was used to process the chromatographic data. The column was flushed with 20% v/v methanol water after the end of each series of analyses. 2.3.1.3. Analytical Method 3. The analyses were performed with the HPLC equipment described in (1) above but using a 5 m 250 mm × 4.6 mm stainless steel C18 Prevail column and guard column (Alltech Assoc., Baulkham Hills, NSW, Australia). A mobile phase consisting of 1% v/v acetonitrile/water (0.1% v/v formic acid in both solvents) was used with a flow rate of 1 ml per min. The column was flushed with 20% v/v acetonitrile/water after the end of each series of analyses.
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2.3.2. Preparative HPLC 2.3.2.1. Mobile phases as utilised in Analytical Methods 1 and 2 (Section 2.3.1). Preparative HPLC was performed with a 5 m C18 250 mm × 21.4 mm stainless steel Varian Dynamax (Varian Inc., CA, USA) preparative column fitted to a Varian Prostar 215 Solvent Delivery Module and a Varian Prostar 320 UV–vis detector. The sample solution was loaded onto the column through a 1 ml sample injection loop. The flow rate was 22 ml/min. Varian Star software was used to monitor the chromatography. The glucoraphanin peak was collected manually. 2.3.2.2. Mobile phase as utilised in Analytical Method 3 (Section 2.3.1). Preparative HPLC was performed with a 5 m C18 250 mm × 21.4 mm stainless steel Varian (Varian Inc., CA, USA) preparative column fitted to a Waters 600E HPLC system equipped with a Waters HPLC autosampler (1 ml injection loop) and a Waters 440 variable wavelength UV detector set at 230 nm. The flow rate was 10 ml/min. The HPLC equipment was controlled by Waters Empower software (Waters, Milford, MA, USA). The glucoraphanin peak was collected manually. 2.3.3. Mass spectrometry Mass spectra were obtained by infusing a dilute aqueous solution of the extract and a pure glucoraphanin standard into the electrospray source of a ThermoElectron LTQ ion trap mass spectrometer (Thermo Electron Corporation CA, USA) operating in the Negative Ion Mode. The sample was introduced by infusion at 5 ul/min to the IonMax ESI probe without the use of auxiliary gases. The source voltage was 4 kV, the capillary voltage −44 V and the heated capillary was maintained at 250 ◦ C. The structure was confirmed by comparing the MS2 fragmentation pattern of the molecular ion from the sample with the MS2 fragmentation pattern of pure glucoraphanin. MS2 fragmentation was generated using helium as a collision gas and carried out under the following conditions: MS2 isolation width: 2.00; MS2 normalized coll. energy: 35.0; MS2 activation Q: 0.250 and MS2 activation time: 30.000 ms. 2.3.4. NMR spectroscopy Proton nuclear magnetic resonance (NMR) spectroscopy was performed in D2 O using a Bruker high resolution DPX300 NMR Spectrometer at 300 mHz (Bruker Biospin Corporation, USA). The DEPT experiment was acquired on the same instrument at 75 MHz. The DEPT experiment revealed all protonated carbons in the molecule and allowed the assignment of carbon multiplicity, providing additional evidence for the identification of glucoraphanin. 3. Results and discussion Glucoraphanin has been isolated in limited quantities by preparative scale HPLC [10]. Previous attempts to isolate pure glucosinolates by preparative HPLC have indicated that this is difficult to achieve due to the complex chromatograms and
Fig. 2. Crude seed extract after combined C18-amino SPE clean-up in de-ionised water, 98% sodium di-hydrogen orthophosphate with 2% methanol, 234 nm, ambient temperature, 1.0 ml/min.
that preparative HPLC only yields small amounts of compounds [10,14]. We recently reported the isolation and determination of glucoraphanin in broccoli seeds using SPE as a means of isolating and concentrating the glucoraphanin prior to analysis by either Micellar Electrokinetic Capillary Chromatography (MECC) or HPLC [13]. HPLC using a Bondapak C18 column and a mobile phase consisting of 0.005 M TMAB in 2% v/v methanol/water was used to determine the levels of glucoraphanin in the seeds. Similar separations were achieved with a number of different mobile phases. A typical chromatogram demonstrating the separation of glucoraphanin from other compounds is shown in Fig. 2. Hot aqueous extraction followed by SPE seems an efficient way to prepare a concentrated glucosinolate solution suitable for preparative HPLC [13]. Treatment of the seed with boiling water to deactivate myrosinase followed by crushing and extraction with hot water gave a glucosinolate fraction rich in glucoraphanin. The seed extract (100 ml), which was too dilute to use for preparative HPLC, also contained other plant material that could potentially damage the preparative HPLC column. A crude glucosinolate concentrate was readily obtained from the seed extract by passing the solution through a C18 SPE cartridge and a protonated amino propyl anion exchange SPE cartridge in series. Unwanted organic material was trapped on the C18 cartridge whilst the charged glucosinolates passed straight through and were trapped on the protonated amino propyl column. The C18 cartridge was removed, the amino propyl cartridge washed with methanol and the glucosinolates removed by washing with 2% ammonia solution in methanol. The solvent was removed in vacuo leaving a residue rich in glucoraphanin (98 mg) (see Fig. 2). This procedure offered a viable alternative to removal of the water in vacuo as it also allowed for the removal of unwanted plant material that might potentially interfere with the separation or block the preparative HPLC column. HPLC and MECC [13] analysis of the residue showed that glucoraphanin was quantitatively recovered from the SPE clean up. Glucoraphanin was identified from its retention time and UV spectral information from the photodiode array detector of the HPLC system. This residue was dissolved
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S. Rochfort et al. / J. Chromatogr. A 1120 (2006) 205–210 Table 1 Comparison of techniques employed in the preparative HPLC isolation of glucoraphanin Preparative HPLC mobile phase
Isolation of pure glucoraphanin
Reference
2% v/v CH3 OH, 98% aqueous 0.05 M TMAB
1. Add NH4 Cl
[11]
2% v/v CH3 OH, 98% aqueous 0.05 M NaH2 PO4 , pH 3.2 Fig. 3. Analysis of glucoraphanin prepared using prep mobile phase 1 (TMAB). Analytical conditions: 98% 50 mM sodium di-hydrogen orthophosphate with 2% v/v methanol, 234 nm, ambient temperature, 1.0 ml/min.
in a minimum amount of water and subject to preparative HPLC. 3.1. Preparative HPLC Fig. 3 shows the purity of the glucoraphanin collected by preparative HPLC with mobile phase as in Analytical Method 1 as described in Section 2. Similar results were obtained with mobile phase as in Analytical Method 2. When fractions are collected from the preparative HPLC, it is then necessary to remove the mobile phase in order to purify the glucosinolate. There are different ways to remove the mobile phase from the sample. In many cases, the removal of the solvent is the primary step, followed by the removal of the mobile phase salt. Initially, preparative HPLC was attempted based on literature analytical procedures. The preparative HPLC chromatograms mirrored the analytical procedures allowing for a pure fraction to be collected. The purity of the extract was confirmed by analytical HPLC (see Fig. 4) and mass spectral analysis; however, NMR revealed surprisingly little product, given the mass recovered. The difficulty lay in separating the glucoraphanin from residual salts remaining from the preparative LC mobile phases (sodium phosphate and tetramethyl ammonium bromide) (see Table 1). This led to the development of an alternative mobile phase without the use of inorganic salts. Formic acid was chosen as the pH modifier as it is reasonably volatile and should be readily
1% v/v CH3 CN (0.1% formic acid), 99% aqueous 0.1% formic acid
2. CHCl3 extraction, 3. Removal of solvent in vaccuo 1. Removal of mobile phase in vaccuo 2. Precipitation of NaH2 PO4 with CH3 OH 3. Filter to remove NaH2 PO4 4. Remove CH3 OH in vaccuo 1. Removal of mobile phase in vaccuo (T < 45 ◦ C)
[10]
New method
removed with the other components of the mobile phase under reduced pressure. Glucoraphanin was well separated from the other components in both the analytical and preparative HPLC using the
Fig. 5. 1 H NMR (D2 O, 300 MHz).
Fig. 4. Preparative HPLC collection, 1% aqueous acetonitrile with 0.1% formic acid, 234 nm, ambient temperature, 10.0 ml/min. NB: the trace shown here is for a 5 mg injection of material. Good seperation was still achieved with 50 mg though the UV–vis trace is off scale.
S. Rochfort et al. / J. Chromatogr. A 1120 (2006) 205–210
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Fig. 6. DEPT (D2 O, 75 MHz).
mobile phase as in Analytical Method 3 described in Sections 2.3.1 and 2.3.2 (see Fig. 4). Removal of the mobile phase under reduced pressure at room temperature gave a viscous, colourless liquid (18 mg) which was shown to be glucoraphanin (acid) from a comparison of the 1 H NMR (Fig. 5), DEPT NMR (Fig. 6) and mass spectra (Fig. 7) of the sample with those of the pure reference compound [10]. Fig. 8 identifies the MS2 mass spectra of the purified glucoraphanin. Here, the molecular ions are fragmented with an inert gas in the ion trap of the mass spectrometer. The glucoraphanin standard, purified material and fractions obtained throughout the purification procedure produced spectra that were all very sim-
Fig. 8. MS2 spectrum acquired on LTQ mass spectrometer by infusion.
ilar. The fragment patterns in all three spectra displayed peaks at 436, 372 and 259 amu and the MS2 fragment patterns are at similar relative ratios providing additional conclusive proof that the purified preparative collections contain glucoraphanin. 4. Conclusion Pure glucoraphanin was obtained in 0.6% (6 mg/g) overall yield from an aqueous extraction of broccoli seeds followed by SPE and preparative HPLC using a mobile phase consisting of 1% v/v acetonitrile/water (0.1% v/v formic acid in both solvents). The glucoraphanin (17.6 mg) was purified from 98 mg of crude material obtained from 3 g of broccoli seed. The purity was assessed by HPLC, MS, NMR and UV spectroscopy. The described method is rapid (less than 1 day), robust and suitable for preparing useable quantities of pure glucoraphanin suitable for the quantitation of glucoraphanin in Brassica species. Acknowledgments
Fig. 7. MS spectrum acquired on LTQ mass spectrometer by infusion.
The authors wish to acknowledge the assistance of Ms. JaneWhitford (DPI cadet) for her technical support and Dr. Andrew Smallridge (Victoria University) for his assistance in NMR data acquisition.
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