MEASUREMENT OF AROMATIC CHOLINE ESTERS IN RAPESEED BY HPLC

Nicolas Mabon, Jean-Paul Wathelet 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

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 µmol/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 µm) 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-diméthoxy-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; Plöeger 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 µmol/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 µm) 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			b 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

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

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

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

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

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

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

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

Plöeger A., Larsen L., Olsen O., Clausen S., Möeller P., Rasmussen K., Nielsen J. and Sörensen 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