Previous PageTable Of ContentsNext Page

MEASUREMENT OF AROMATIC CHOLINE ESTERS IN RAPESEED BY HPLC

Nicolas Mabon, Jean-Paul Wathelet and Michel Marlier

FacultÚ universitaire des Sciences agronomiques, UnitÚ de chimie gÚnÚrale et organique
Passage des DÚportÚs, n░2, B-5030 Gembloux, Belgium
e-mail: wathelet.jp@fsagx.ac.be

ABSTRACT

Aromatic choline esters are natural compounds occurring in Brassicaceae especially in rapeseed among whom the most important is called sinapine (choline ester of the 3,5-dimethoxy-4-hydroxycinnamic acid). Choline esters are easily hydrolysed by entero bacteria in digestive tractus giving the correspondent acid and a choline molecule. Choline could then be broken down, by a deaminase, into trimethylamine, compound inducing a negative flavour ("fishy odour") in the milk, the meat or in yolk. So, the method improved in our laboratory is a power tool for breeders and other scientists who would like to evaluate individual aromatic choline esters in seeds or meals. Seeds (10 g) are first ground in a coffee mill (20 sec). 200 mg of ground seeds are weighed in a test tube and then placed in a water bath (75░C). Then 10 ml of boiling methanol/acetic acid (0.05M) mixture (70/30) are added plus 0.5 ml of internal standard solution (3,5-dimethoxybenzoic choline ester, 10 Ámol/ml).The heterogeneous content is stirred with a magnetic stirrer for 10 min. Only one extraction step is necessary to obtain good results. Then, 1 ml of crude extract is purified with a cation exchange column (CM Sephadex C25-120). Aromatic choline esters are eluted with acetic acid 1N. Separation and quantification of individual choline esters are further realised by high performance liquid chromatography with an Inertsil 5 ODS-2 (3 x 250 mm, 5 Ám) using a ternary solvent gradient (water-acetonitrile-phosphate buffer: NaH2PO4, 20 mM at pH 2 with o-phosphoric acid). In these optimised chromatographic conditions, the choline esters are separated and quantified. The retention times and the response factors have been determinated for the 36 different choline esters (benzoic or cinnamic structures with hydroxy or methoxy groups in ortho, para or meta position) synthesised in our laboratory.

KEYWORDS Brassicaceae, rapeseed, flavour, sinapine, trimethylamine

INTRODUCTION

Aromatic choline esters, of which sinapine is the best known, are present in appreciable amounts in Brassicaceae especially in rapeseed among whom the most important is called sinapine (choline ester of the 3,5-dimÚthoxy-4-hydroxycinnamic acid). These quaternary ammonium compounds are easily hydrolysed by a deaminase into trimethylamine. Trimethylamine restricts utilisation of high quality rapeseed protein as food and feed. This compound produce a "fishy" odour in eggs (Fenwick et al., 1981), a disagreeable taste in the meat of calves (Anderson et al., 1984) and in the milk of cows (Andersen and Andersen, 1982). That is the reason why measurement of these choline esters is important. Aromatic choline esters can be separated by thin layer chromatography, ion exchange chromatography, high performance liquid chromatography (Clausen et al., 1983; Clossais-Besnard and Bouchereau, 1994; Pl÷eger et al., 1985) or micellar electrokinetic capillary chromatography (Bjergegaard et al., 1993, 1994) where micelles are formed with sodium dodecyl sulfate. In this experimentation, the HPLC method published by Clausen et al. (1983) has been improved for measuring easily choline esters. The method developed in our laboratory is a power tool for breeders and other scientists who would like to evaluate individual aromatic choline esters in seeds or meals.

REAGENTS and MATERIAL

Reagents

All chemicals were analytical grade obtained from commercial way.

Material

Hewlett Packard (serie 1050) is the chromatograph (HPLC) used for separation of choline esters.

RESULTS and DISCUSSION

The method used by Clausen et al. (1983) has been improved at different steps of the protocol.

Sampling and extraction

Seeds (10 g) are first ground in a coffee mill (20 sec). 200 mg of ground seeds are weighed in a test tube placed in a water bath (75░C). Then 10 ml of boiling methanol/acetic acid (0.05M) mixture (70/30) are added plus 0.5 ml of internal standard solution (3,5-dimethoxybenzoic choline ester, 10 Ámol/ml). The heterogeneous content is stirred with a magnetic stirrer for 10 min and the upper phase is collected. Ultra turrax is not used avoiding contamination's. A single extraction step is sufficient to obtain good results while Clausen et al. (1983) recommended 2 extractions.

Purification

Then, 1 ml of the crude extract is purified with a cation exchange column (CM Sephadex C25-120, 50 mg). Aromatic choline esters are eluted with acetic acid 1N (10 ml). Regeneration of the resin is made with HCl 1 N (5 ml). Another resin has also been tested (SP-Sephadex C25-120) with HCl 1N as eluent, but results were not improved.

HPLC analysis

Separation and quantification of individual choline esters are further realised by high performance liquid chromatography with an Inertsil 5 ODS-2 (3 x 250 mm, 5 Ám) using a ternary solvent gradient (A water, B acetonitrile, C phosphate buffer: NaH2PO4, 20 mM at pH 2 with o-phosphoric acid). Elution gradient is the following: time 0 min (A 40%, B 10% C 50%), time 30 min (A 15%, B 35%, C 50%). Compounds are detected at 210 nm or 280 nm and 335 nm. In these optimised chromatographic conditions, all the choline esters tested are well separated. Retention times and response factors have been determinated in these chromatographic conditions for the 36 different choline esters (benzoic or cinnamic structures with hydroxy or methoxy substituants in ortho, para or meta position) synthesised in our laboratory (Table 1). As you can see (Figure 3) the u.v. spectra of choline ester and their corresponding aromatic acid are very close.

u.v. library

The u.v. spectrum of each choline ester has been stored.

Figure 1: Separation of aromatic choline esters from rapeseed (SINERGY) (Inertsil 5 ODS-2 column)

Figure 2: structure of sinapine

Figure 3: u.v. spectra of choline ester and his corresponding acid

Table 1: Structure and retention time of the 36 choline esters synthesised

RT

Acidic part

R 2

R 3

R 4

R 5

R 6

NAME

Formula

5.51

Benzoic

 

OH

OH

OH

 

gallic choline ester

C12NH18O5

5.65

Benzoic

 

OH

OH

   

protocatechic choline ester

C12NH18O4

5.65

Benzoic

 

OH

 

OH

 

resorcylic choline ester

C12NH18O4

6,58

Benzoic

OH

OH

       

C12NH18O4

7.32

Benzoic

   

OH

     

C12NH18O3

8.29

Benzoic

 

OMe

OH

   

vanillic choline ester

C13NH20O4

9.00

Benzoic

 

OH

       

C12NH18O3

9.00

Benzoic

OH

 

OH

   

β resorcylic choline ester

C12NH18O4

9.00

Benzoic

 

OH

OMe

   

isovanillic choline ester

C13NH20O4

9.00

Benzoic

 

OMe

OH

OMe

 

syringic choline ester

C14NH22O5

9.30

Cinnamic

 

OH

OH

   

cafeic choline ester

C14NH20O4

10.04

Benzoic

OH

   

OH

 

gentisic choline ester

C12NH18O4

12.23

Benzoic

OH

     

OH

 

C12NH18O4

12.44

Benzoic

OH

OMe

       

C13NH20O4

13.59

Benzoic

 

OMe

OMe

   

hesperaline

C14NH22O4

13.59

Cinnamic

   

OH

   

p-coumaric choline ester

C14NH20O3

13.88

Benzoic

OMe

       

o-anisic choline ester

C13NH20O3

14.34

Cinnamic

 

OMe

OH

OMe

 

sinapine

C16NH24O5

14.50

Benzoic

OH

       

salicylic choline ester

C12NH18O3

15.10

Cinnamic

 

OMe

OH

   

ferulic choline ester

C15NH22O4

15.30

Cinnamic

 

OH

     

m-coumaric choline ester

C14NH20O3

15.80

Cinnamic

 

OH

OMe

   

isoferulic choline ester

C15NH22O4

16.76

Benzoic

   

OMe

   

p-anisic choline ester

C13NH20O3

17.32

Benzoic

 

OMe

     

m-anisic choline ester

C13NH20O3

17.61

Benzoic

OH

   

OMe

   

C13NH20O4

18.27

Cinnamic

OH

       

o-coumaric choline ester

C14NH20O3

18.99

Benzoic

OH

 

OMe

     

C13NH20O4

20.50

Cinnamic

 

OMe

OMe

   

C16NH24O4

21.42

Benzoic

 

OMe

 

OMe

   

C14NH22O4

23.04

Cinnamic

           

C14NH20O2

24.45

Benzoic

           

C12NH18O2

24.94

Benzoic

 

OMe

OMe

OMe

   

C15NH24O5

25.12

Cinnamic

 

OMe

OMe

OMe

   

C17NH26O5

26.24

Cinnamic

OMe

OMe

       

C16NH24O4

27.50

Cinnamic

OMe

   

OMe

   

C16NH24O4

28.83

Cinnamic

OMe

 

OMe

     

C16NH24O4

CONCLUSIONS

Aromatic choline esters present in Brassicaceae are easily quantify by HPLC with the improved method. 36 aromatic choline esters can be identified by their retention time and their u.v. spectrum.

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

1. Andersen H. and Andersen P. (1982): Anvendelse af raps i foderblandiner til kvaeg. Production, 22, 23-26, in Danish

2. Andersen H, Varnum P., Andersen P, Klastrup S., S÷rensen S., S÷rensen H. and Olsen O. (1984): Dobbeltlav rapsskra i kraftfoderblander til kalve og ungtyre. Meddelelse Statens husdyrbrugsforsog. 4 pp, in Danish

3. Bjergegaard C., Ingvardsen L. and S÷rensen H. (1993): Determination of aromatic choline esters by micellar electrokinetic capillary chromatography, Journal of Chromatography, 653, 99-108

4. Bjergegaard C., Ingvardsen L. and S÷rensen H. (1994): Determination of aromatic choline esters accumulated in cruciferous seeds and associated to dietary fibres. G.C.I.R.C. Bulletin, 183-190

5. Clausen S., Olsen O. and S÷rensen H. (1983): Separation of aromatic choline esters by high performance liquid chromatography. Journal of Chromatography, 260, 193-199

6. Clossais-Besnard and Bouchereau (1994): Analyse des facteurs antinutritionnels du colza. Glucosinolates et esters de choline. G.C.I.R.C. Bulletin, 115-116

7. Fenwick G. (1981): Trimethylamine taint in eggs. In: Quality of eggs. Proc. Eur. Symp. 1st Apeldoorn, 18-23 May, 144-152

8. Pl÷eger A., Larsen L., Olsen O., Clausen S., M÷eller P., Rasmussen K., Nielsen J. and S÷rensen H. (1985): Aromatic choline esters, aromatic choline esterase, glucosinolates and myrosinases in oilseed rape and other crucifers. Royal Veterinary and Agricultural University, Chemistry Department, Copenhagen, Denmark, thesis, 1-121

Previous PageTop Of PageNext Page