DETERMINATION OF GLUCOSINOLATES IN RAPESEED
IMPROVEMENT OF THE OFFICIAL HPLC ISO METHOD
(PRECISION AND SPEED)
Jean-Paul Wathelet, Nicolas Mabon and Michel Marlier
Faculté universitaire des Sciences agronomiques, Unité de chimie générale et organique
Passage des Déportés, n°2, B-5030 Gembloux, Belgium
e-mail: wathelet.jp@fsagx.ac.be
ABSTRACT
The official ISO protocol (similar to U.E. recommendations established in 1987 by six european laboratories) for determination of individual glucosinolate content in rapeseed using HPLC was published for the first time in 1992 (ISO 9167-1). Numerous laboratories, all over the world, use now this method, especially in order to control the 00 varieties. The goals of our research were to considerably reduce the analysis time without loosing precision. A single extraction is now suggested (200 mg of ground seeds stirred in 10 ml of 75°C methanol/water 70/30 with an internal standard for 10 min). Then 1-6 ml of the crude extract is directly put on DEAE A-25 resin prepared according to the ISO method. Glucosinolates are desulfated by addition of 100 µl of concentrated Helix pomatia sulfatase (H1, EC 3.1.6.1) quickly prepared by ethanol precipitation. The desulfatation process can be reduced to 1 hour without any problems with all kind of glucosinolate (alcenyl, benzyl, indolyl, methylthio, methylsulfinyl, methylsulfonyl...). Elution of the desulfo-glucosinolates is realised with 4 x 0.5 ml of distilled water. Desulfo-glucosinolates are separated by HPLC using an Inertsil 3 ODS-3 column (100 x 3 mm, 3 µm) with a water/acetonitrile gradient (from 2 to 25% in 35 min). Resolution is very nice and the limited flow (0.4 ml/min) reduce the solvent costs and the elimination of wastes. The suggested fast method has been compared with the official ISO method analysing the three certified reference materials prepared by BCR (now IRMM) and recommended by U.E. and ISO (CRMs 366, 190 and 367). The recovery of indolyl desulfo-glucosinolates, specially 4-OH glucobrassicin, is higher with the quicker method. Results obtained with the two methods are very close for other desulfo-glucosinolates.
KEYWORDS
Analysis, ISO, liquid chromatography, desulfo-glucosinolate, antinutritionnel, BCR
INTRODUCTION
Glucosinolates are a class of ca. 100 secondary plant compounds (Kjaer and Skrydstrup, 1987) generally found in Brassicaceae. These compounds have a similar structure characterised by a common part containing a beta thioglucose coupled with a sulfonated oxim group and by a variable organic side chain constituted by alkyl, alcenyl, hydroxy-alcenyl, aryl, indolyl, sulfinyl, sulfonyl or thio residues. Present in high or low level (00 varieties) in rapeseed and in rapeseed meals, these molecules are easily broken down during animal digestion, leading to compounds with detrimental and antinutritional characteristics. They are known to interfere with the thyroid and to damage vital organs (Fenwick et al., 1983). That is the reason why a group of six european laboratories developed since 1987 an European and, in a second time, an ISO (ISO 9167-1) method to quantify total and individual glucosinolates levels. These official protocols were mainly established on the basis of the work made by Spinks (Spinks et al., 1984) who separated, after desulfatation with Helix pomatia sulfatase, desulfo-glucosinolates with HPLC. Numerous laboratories, all over the world, use now this method, especially in order to control the 00 varieties and to obtain U.E. premium. The U.E. actively encourages production and use of rapeseed cake as a mean of reducing animal feed imports and of moving production away from crops grown in surplus. The goals of our research were to improve the official ISO method, to considerably reduce the analysis time without loosing precision and to validate the new method analysing BCR certified reference materials (Wathelet et al., 1991).
REAGENTS and MATERIAL
Reagents
All chemicals were analytical grade obtained from commercial sources. Helix pomatia (Type H1) sulfatase was a Sigma product.
Material
HPLC analysis were performed on a Hewlett Packard chromatograph (Type 1050) with a diode array detector.
RESULTS and DISCUSSION
Each step of the official ISO method (ISO 9167-1) has been checked and improved.
Sampling
In the ISO method it is first recommended to grind the seeds with a microgrinder and to weight 200 mg of ground seeds. The variability of total glucosinolate content depending on sample amount has been evaluated (Figure 1). A minimum of 200 mg of sample amount is recommended.
 |
Figure 1: Variability of the glucosinolate level according to the sample amount
Extraction
A single extraction (against 2 in the ISO method) is now suggested in a 10 ml test tube. 200 mg of ground seeds are stirred with 10 ml of 75°C methanol/water (70/30) with an internal standard. The mixture is permanently agitated with a magnetic stirrer for 10 min (Figure 2) and then centrifuged. Using this system it is possible to extract quickly a lot of samples.
 |
Figure 2: Kinetic of the glucosinolate extraction
Desulfatation and purification
1 to 6 ml of the crude extract depending on the glucosinolate content are directly put on a DEAE A‑25 resin (25 mg) prepared according to the ISO method. Glucosinolates are desulfated by addition of 100 µl of Helix pomatia sulfatase (H1, EC 3.1.6.1) quickly prepared by fractionated ethanol precipitation (75 mg in 40% ethanol, centrifugation, recover the upper phase and precipitation of sulfatase with 70% ethanol, take up the precipitate in 5 ml of distilled water and dilution 10 before use). The kinetic of desulfatation with diluted sulfatase (activity at pH 5.8 and 30°C: 0,05 u/ml; one activity unit corresponds to the desulfatation of 1 µmol of sinigrin per min) is represented figure 3. A minimum of 11 hours is necessary for complete desulfatation in this operating conditions. If necessary, the desulfatation time can be reduced to 2 hours with a ten times concentrated sulfatase solution (activity: 0,5 u/ml) without any problem with all kind of glucosinolates (alcenyl, benzyl, indolyl, methylthio, methylsulfinyl, methylsulfonyl...).
Figure 3: Kinetic of the glucosinolates desulfatation (sulfatase activity: 0,05 u/ml)
 |
Elution of the desulfo-glucosinolates is realised with 4 x 0,5 ml distilled water (Figure 4). The 4‑hydroxyglucobrassicin is eluted later than the other glucosinolates.
Figure 4: Profile of glucosinolates
elution
HPLC analysis
Desulfo-glucosinolates are separated by HPLC using an Inertsil 3 ODS-3 column (100 x 3 mm, 3 µm) with a water/acetonitrile gradient (from 2 to 25% in 35 min). Resolution is very nice (Figure 5) and the limited flow (0.4 ml/min) reduce simultaneously the costs of solvent and the elimination of wastes.
Figure 5: HPLC chromatogram of
rapeseed desulfo-glucosinolates
COMPARISON OF METHODS
The suggested fast method has been compared (Table 1) with the official ISO method analysing the three certified reference materials prepared by BCR (now IRMM) and recommended by U.E. and ISO (CRMs 366, 190 and 367).
Table 1: Glucosinolates level in different reference rapeseed materials using the official and the modified method (n=3)
|
Glucosinolate |
CRM 366 |
CRM 366 |
CRM 190 |
CRM 190 |
CRM 367 |
CRM 367 |
|
Method |
ISO |
Modified |
ISO |
Modified |
ISO |
Modified |
|
Progoitrin (PRO) Epiprogoitrin (EPRO) Gluconapoleiferin (GNL) Gluconapin (GNA) 4-OH glucobrassicin (4-OH) Glucobrassicanapin (GBN) Glucobrassicin (GBC) Gluconasturtiin (NAS) |
5.79 0.15 0.23 2.08 2.66 0.36 0.07 0.14 |
5.72 0.13 0.21 2.05 2.87 0.37 0.08 0.16 |
12.52 0.31 0.49 3.93 3.03 1.34 0.15 0.22 |
12.45 0.27 0.46 3.90 3.26 1.35 0.17 0.24 |
61.21 1.99 1.89 24.19 3.70 4.91 0.09 0.97 |
61.15 1.92 1.83 24.09 3.91 4.94 0.10 0.91 |
The recovery of indolyl desulfo-glucosinolates, specially 4-OH glucobrassicin, is higher with the quicker method. Results obtained for other desulfo-glucosinolates are very close.
CONCLUSION
The different steps of the official ISO method has been optimised in order to define an easier approach for glucosinolate determination. The suggested protocol to quantify individual or total glucosinolate content is faster (reduction of extraction number and desulfatation time) than the present ISO method without loosing precision (confirmation by analysing reference BCR materials: CRMs 190, 366 and 367). It is very useful for analysing a high number of samples. On an other hand, analysis costs are reduced (manpower, delay of desulfatation, HPLC solvents...). The new method will be tested in the future by other laboratories for validation before requesting to the International Standardisation Organisation a revision of the norm ISO 9167-1.
ACKNOWLEDGEMENTS
This work has been supported by the General Office of Research and Development of the Belgian Agricultural Ministery and by the General Direction of Technologies, Research and Energy of Ministery of "Region wallonne" in Belgium.
REFERENCES
Fenwick G., Heaney R., Mullin W. (1983): Glucosinolates and their breakdown products in food and food plants. CRC, Critical Review in Food Science and Nutrition 18:123-201
ISO norm (1992): Rapeseed- Determination of glucosinolate content-Part 1: Method using high-performance liquid chromatography. ISO 9167-1, 1-9
Kjaer A. and Skrystrup T. (1987): Selenoglucosinolates synthesis and enzymatic hydrolysis. Acta Chemistry Scandinavia, B41, 29-33
Spinks A., Sones K. and Fenwick G. (1984): The quantitative analysis of glucosinolates in cruciferous vegetables, oilseeds and forage crops using high performance liquid chromatography. Fette Seifen Anstrichmittel, 86, 228-231
Wathelet J-P, Wagstaffe P. and Boenke A. (1991): The certification of the total glucosinolate and sulphur contents of three rapeseeds (colza), CRMs 190, 366 and 367. Commission of the European Communities, report EUR 13339 EN, 1-75