1398
ANALYTICAL CHEMISTRY, VOL. 50,
NO. 9,
AUGUST 1978
Fluorescence Labeling of Dicarboxylic Acids for High Performance Liquid Chromatographic Separation Sir: Easy and efficient methods are needed for the separation and detection of dicarboxylic acids in urine or in blood serum. Present methods frequently involve the derivatization of the acids for separation and identification by GC or GC-MS. Jellum ( I ) has recently reviewed the use of GC-MS in the analysis of organic acid in body fluids. T h e analysis of dicarboxylic acids and a-keto acids by H P L C is not yet widespread. One of the major reasons for t h a t fact lies in the lack of a suitably sensitive detector for all but the aromatic acids. Some attempts have been made to use the quinoxalone derivatives of a-keto acid (viz., 2 ) in HPLC. T h e quinoxalone reagent is highly selective toward @-ketoacids, and the derivative can be detected by UV or fluorescence. Phenacyl derivatives of dicarboxylic acids have been used in H P L C in conjunction with a UV detector (3). Recently Dunges ( 4 ) has reported the use of 4-bromomethyl-7-methoxycoumarin (Br-Mmc) as a fluorescence labeling reagent in the TLC analysis of fatty acids. Dunges et al. (5)have reported an improved procedure using crown ether. Lam and Grushka further improved the methodology by using a crown ether catalyst (6) and they have separated t h e derivatives using reversed phase HPLC. Dunges, in his work ( 4 , 5 ) ,reported t h a t t h e dicarboxylic acids could not be derivatized with Br-Mmc. Fluorescence labeling in conjunction with HPLC is attractive since the detection limits are quite low. T o the best of our knowledge, however, the preparation and separation of fluorescing derivatives of dicarboxylic acid have not been attempted. This is somewhat surprising in view of the physiological importance of these compounds. T h e present paper described the procedure by which the Mmc derivatives of dicarboxylic acids and a-keto acids can be formed. Use is being made of the fact t h a t the potassium salts of the acids can be transferred t o a n aprotic solvent with crown ethers. T h e carboxylate ions in an aprotic solvent are rather reactive, thus facilitating alkylation reactions. T h e derivatives of the acids can then be separated by H P L C and detected with a fluorometer. EXPERIMENTAL Apparatus. The liquid chromatograph consisted of a model
396 Milton Roy Minipump (LDC, Riveria Beach, Fla.), and a Gilson (Middletown, Wis.) Spectra/Glo fluorometer. The excitation filter had a maximum transmittance at 360 nm and the emission filter had a cut-off valve of 400 nm. The samples were introduced via a Rheodyne injection valve model 70-10 purchased from Altex Scientific Co. (Berkeley, Calif.). The stainless steel column, 22.3 cm X 4.2 mm id., was packed by the conventional slurry technique with CI8 bonded Partisil 10. Regents. Partisill0 was obtained from Whatman Co. (Clifton: N.J.). The dicarboxylic acids, 18-crown-6, DNS (5-dimethylaminel-naphthalenesulfonyl chloride), and solvents were obtained from various sources and used without further purification. 4-Bromomethyl-7-methoxycoumarin (Br-Mmc) was prepared according to Secrist et al. ( 7 ) . The a-keto acids were obtained from Sigma Chemical Co. (St. Louis, Mo.) and were used without further purification. The mobile phase consisted of various ratios of reagent grade methanol and distilled, deionized water. Procedure. The derivatives were prepared by adding 4-6 mg samples of the acids, 3-4 mg of K2C03,and 1 - 2 mg of crown ether to a 50-mL round bottom flask, and dissolving the mixture in 10 mL of a previously prepared solution of 4-bromomethyl-7methoxycoumarin in acetone (0.15 g in 100 mL acetone). The reaction flask was placed in a preheated oil bath at 8G90 “C where it was allowed to reflux for 1 h. Samples for injection were 0003-2700/78/0350-1398$01 O O / O
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Flgure 2. Relationship between the detector response and the amount of acid (azelaic) used in the reaction obtained directly from the product solution without further treatment. The injection was made with a 25-pL syringe. The chromatograms are given in terms of relative intensity. Capacity ratios for the a-keto acids were measured relative to DNS. All the chromatographic runs were made at ambient temperatures. RESULTS A N D DISCUSSION The excitation and emission spectra of the Mmc derivatives are described elsewhere ( 5 , 6). Lam and Grushka (6) have studied various derivatization schemes of mono acids with Br-Mmc. They have shown that the use of K 2 C 0 3to form the salt prior to t h e phase transfer yields an excellent compromise between the ease of the procedure and the rate of derivative formation. Using crown ether, the reaction time was reduced drastically from that reported by Dunges ( 4 ) . As indicated earlier, Dunges could not derivatize dicarboxylic acids. I t was felt that with the crown ether transferring the dicarboxylate to an organic solvent, the derivatization reaction with Br-Mmc can be accelerated. Figure 1 shows the result of a reaction rate study. T h e amount of suberic acid-Mmc ester is plotted vs. t h e reaction time. T h e amount of ester formed is proportional to the peak height ratios, Le., the height of the suberic ester relative to that of the anthracene internal standard. The figure shows that derivatization can take place and that the reaction is essentially completed in 1h. However, sufficient acid is derivatized in a 15- to 30-minute period to give reproducible response with t h e fluorescence detector. T h e products of the derivatization procedure should be proportional to the amount of the starting acids. Figure 2 shows a typical result. A plot of t h e peak height of azelaic acid-Mmc derivative relative to anthracene vs. t h e amount of the acid introduced to the reaction varied yields a straight line. Each experimental point was obtained after a reaction 6 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
1399
Table I. k' Capacity Ratios (Relative to DNS) of Some &-KetoAcidsa cy-keto acids
k'
glyoxalic pyruvic a-ketobutyric ox alace tic a-ketoglutaric
4.26 4.72 7.01 5.97 1.99
a C,,-Partisil 1 0 column, 50% waterimethanol mobile phase, Flowrate: 0 . 7 1 m l i m i n .
.................... I
1
MIN Figure 3. Separation of dicarboxylic acid derivatives on a C18column. Detector: fluorometer. Mobile phase: 55 % water/methanol;flow rate: 2.33 mL/min. (1) malonic, (2) succinic, (3) glutaric, (4) adipic
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20
MIN Figure 4. Separation of dicarboxylic acid derivatives on a C,, column. Detector: fluorometer. Mobile phase: 50 % water/methanol; flow rate: 3.30 mL/min. (1) pimelic, (2) suberic, (3) azelaic, (4) sebacic
period of 1 h. T h e straight line indicates that the procedure is suitable for quantitative purposes. Figures 3 and 4 show some chromatographic separations of several dicarboxylic acids. Although no attempts were made to optimize t h e resolution and maximize the number of acid separated, it is clear from the figures that a methanol gradient can be used t o separate t h e C2 to Clo dicarboxylic acids with little difficulty. Oxalic acid, not shown in the figure, can be derivatized rather easily. T o ascertain the detection limits, two different samples of azelaic acid were derivatized. T h e resultant derivative so-
lutions were diluted successively and injected into t h e chromatograph until the signal was twice the noise level. The detection limit was found to be about 2 X lo-'' mol. a-Keto acid can also be derivatized using t h e above procedure. Table I shows the capacity ratios, k', of several such acids. D N S was used as the inert peak in the calculations of the k'values. The large differences in the k'values indicates t h a t the acids show in Table I can be resolved easily. T h e retention order of oxalacetic acid and a-ketoglutaric acid should be compared with that of succinic and glutaric acid. It is not clear to us why the shorter oxalacetic acid should elute before t h e longer a-ketoglutaric acid. Note t h a t t h e keto monoacids elute according to the chain length: the longer the chain, the larger t h e k'value. It is clear from the results shown above that a crown ether can be used to catalyze the formation of Mmc derivatives of dicarboxylic acids as well as a-keto acids. T h e derivatization reaction is simple, straightforward, and it can be used in quantitation studies. T h e reagent Br-Mmc is attractive for the following reason. In the analysis scheme used here, the reagent itself does not fluoresce, t h u s eliminating possible interferences from unreacted Br-Mmc. T h e disadvantages of t h e method are minor: (1) the Mmc derivatives slowly hydrolyze in t h e presence of water; ( 2 ) the reagent and the derivatives decompose when exposed t o light over a long period of time. Consequently, d solutions should be prepared within a short time before analysis. These minor disadvantages are compensated for by the ease of preparation and sensitive detection.
LITERATURE CITED (1) E. Jellurn, J . Chromatogr., 143, 427 (1977). (2) J. C. Liao, N. E. Hoffman, J. J. Barboriak, and D. A. Roth, Clin. Chem. (Winston-Salem, N.C.), 23, 802 (1977). (3) E. Grushka, H. D. Durst, and E. J. Kikta, Jr., J . Chromatogr., 112, 673 (1975). (4) W. Dunges, Anal. Chem., 49, 442-445 (1977). (5) W. Dunges, A. Meyer, K. H. Muller, M. Muller, R. Pietschmann. C. Rachetta, R. Sehr, and H. Tuss, Fresenius' Z . Anal. Chem., 288, 361 (1977). (6) S. Lam and E. Grushka, J . Chromatogr., accepted for publication. (7) J. A. Secrist 111, J. R. Barrio, and N. J. Leonard, Biochem. Biophys. Res. Commun., 45, 1267 (1971).
Eli Grushka* Stanley Lam John Chassin Department of Chemistry State University of New York a t Buffalo Buffalo, New York 14214
RECEIVED for review March 20, 1978. Accepted May 26, 1978.
