4-Bromomethyl-7-methoxycoumarin as a new fluorescence label for

Yinfeng Xie , Guoliang Li , Jinmao You , Xinwei Bai , Chengyan Wang , Lin Zhang , Fuhua Zhao , Xiaoyun Wu , Zhongyin Ji , Zhiwei Sun. Chromatographia ...
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(46) J. H. Beynon, R. G. Cooks, J. W. Amy, W. E. Baitinger, and T. y.Ridley, Anal. Chern., 45, 1023A (1973). (47) J. F. Litton, PhD Thesis, Purdue University, Chap. 3 (1976). (48) T. L. Kruger, J. F. Litton, and R. G. Cooks, Anal. Lett., 9, 533 (1976). (49) T. L. Kruger, J. F. Litton, R. W. Kondrat, and R. G. Cooks, Anal. Chem., 48, 21 13 (1976). (50) K. Levsen and H. D. Beckey, Org. Mass Spectrom., 9, 570 (1974). (51) K. Levsen and H.-R. Shuiten, Biorned. Mass Spectrorn., in press. (52) D. H. Smith, C. Djerassi, K. H. Maurer, and U. Rapp, J. Am. Chern. Soc., 96, 3482 (1974). (53) E. Soitero-Rigau,M.S. Thesis, Purdue University (1976).

(54) J. H. Beynon (1976).

and R. G. Cooks, h t . J. Mass Spectrom. /on fhys., 9, 107

RECEIVEDfor review September 28, 1976, ~ ~N ~ -~ vember 19, 1976. This work was supported by the National Science Foundation. T.L.K. was supported in part by the Special Leave with Pay Program at Ball State University, Muncie, Ind.

4-Bromomethyl-7-methoxycoumarin as a New Fluorescence Label for Fatty Acids Wolfgang Dunges Pharmakologisches lnstitut der Universitat Mainz, 0-6500Mainz, West Germany

Reaction with 4-bromomethyl-7-methoxycoumarin (Br-Mmc) allows the fluorescence labeling of fatty acids. With the aid of microliter reflux techniques and thin-layer chromatography, an extenslve screening program was performed. All unsubstituted and most substituted aliphatic monocarboxylic acids gave Mmc derivatives with strong blue fluorescence. Mmc esters of different fatty acids were prepared in millimole amounts. Their structure was confirmed by mass spectrometry and elementary analysis. Thin-layer chromatographic separation and in situ fluorescence measurement of their Mmc esters allows the determination of picomole amounts of fatty acids.

Fluorescence labeling of nonfluorescent compounds facilitates their determination by thin-layer chromatography or high pressure liquid chromatography. Detection sensitivity is improved and the range of linearity between signal and substance amount is expanded over several orders of magnitude ( I ) . 5-Dimethylamino-1-napthalenesulfonylchloride (DNS-chloride) ( Z ) , fluorescamine (3),and several other reagents have been used for this purpose. Fluorescence labels have mainly been attached to amino groups and to phenolic and alcoholic OH groups. About 8% of all known organic compounds contain a carboxyl group (4).Nevertheless fluorescence labeling of organic acids has not been attempted ( I ) . Only the formation of ultraviolet (uv) absorbing derivatives of fatty acids has been used in combination with high pressure liquid chromatography ( 5 , 6 )although fluorescence detection units are commercially available. Through the development of new apparatus (7,8)thin-layer chromatography has attracted new interest. Whereas separations are improved by these methods, detection sensitivity for fatty acids on thin-layer chromatograms is still poor compared to gas chromatography. Nonderivatized volatile fatty acids cannot be submitted to thin-layer chromatography. For the purpose of fluorescence labeling of fatty acids 4bromomethyl-7-methoxycoumarin(Br-Mmc) appeared to be useful. Reactions of Br-Mmc with OH, "2, and NHR groups have been investigated in aqueous media, but with little success (9, I O ) . In a nonaqueous medium, palmitic acid (among others) is quantitatively methylated with methyl iodide (11). This method of derivative formation for gas chromatography is based on Claisen's' synthesis of phenylallyl ethers (12). Similar reaction conditions were used for the formation of 442

ANALYTICAL CHEMISTRY, VOL. 49, NO. 3, MARCH 1977

derivatives of Br-Mmc. The purpose of this report is to demonstrate the applicability and scope of Br-Mmc labeling in the trace analysis of fatty acids.

MATERIALS AND METHODS Reagents and Chemicals. All reagents and chemicals were of A grade. They were purchased from E. Merck, Darmstadt, Germany. 4-Bromomethyl-7-methoxycoumarin (Br-Mmc) was either purchased from Regis Chemical Company, Morton Grove, Ill., or it was prepared according to Secrist et al. (10).Fatty acids were a gift of Henkel and Cie., Dusseldorf, Germany. Their purity was checked by thin-layer chromatography. Screening Experiments for the Reactivity of Br-Mmc. Each compound was reacted with Br-Mmc in a molar ratio of 1:3. All compounds were dissolved in acetone or (in some cases) in ethyl acetate. The final volume was 25 pl. After the addition of 2 mg of finely powdered KzC03, the solutions were refluxed for 1h using a microrefluxer ( 1 3 ) .Twelve solutions were refluxed at the same time (one blank with Br-Mmc). After cooling, the microvessels were closed with PTFE caps. The reaction mixtures could be stored at 4 "C for several months without losses of liquid. For the preparation of greater quantities of derivatives, e.g., in order to test reaction conditions, up to 9 reactions in volumes of 400 pl or 6 reactions in volumes of 5 ml could be carried out simultaneously using the ml-refluxer (14). The microrefluxer and the ml-refluxer are available in the United States from Regis Chemical Company, Morton Grove, Ill., and in Europe from Forschungsinstitut Berghof, D-7400 Tubingen, W. Germany. Synthesis of Reference Compounds. 4-Methyl-7-methoxycoumarin derivatives ("Mmc-derivatives") of representative fatty acids were prepared by refluxing for 1 h solutions of 1 mmol of the compound and 3 mmol of Br-Mmc in 800 ml of acetone together with 5 g of solid KzC03. The solutions were filtered and the solvent removed in vacuo. The residues were crystallized repeatedly from appropriate solvents in order to remove Br-Mmc. Preparative yields of the pure esters were between 20 and 50%. The purity of the Mmc derivatives was checked by thin-layer chromatography. The structures of the new compounds were confirmed by mass spectrometry and by elementary analysis. In addition purity and structure of one compound was verified using 'H NMR. For this purpose it was dissolved in deuterated dimethyl sulfoxide a t 60 "C. Thin-Layer Chromatography. Two p1 of the reaction mixtures and of solutions of the free acids were applied directly to silica gel plates with inorganic fluorescence indicator (SIL-A-25-UV,Macherey and Nagel, Duren, Germany) with the aid of a micropipet according to Kaiser ( 1 5 ) .The chromatograms were developed in vapor phase saturated flat chambers according to Seiler (16).The thin-layer plates were divided into two 10 X 20 cm areas. A support with two contact zones on opposite edges was used. Since the chromatographic path length was only 8 cm, the diffusion of the spots was minimal. The spots had a diameter of 1-2 mm as they were supplied with a small bore micropipet. Thus both sides of the plates could be used and a total of 38 samples could be separated in one run. Benzene/ethyl acetate (17/1)was the standard solvent system, alternatively benzene/ethyl

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T a b l e I. R e a c t i o n of !Aliphatic Carboxylic Acids w i t h B r - M m c A. F a t t y acids Straight chain

Substituted straight chain

Branched

Cyclic

Formic acida,c,d Acetic acid