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Anal. Chem. 1980, 52,2430-2432
comitant with a rise in column back-pressure. This problem can be reduced or eliminated by periodically flushing the columns with deionized water followed by methanol. When this procedure is not convenient, the addition of a bactericide such as trichlorobutanol or phenol to the mobile phase may help alleviate this problem. Repacking the precolumn periodically is probably the best solution to this problem, however. As has been reported by other investigators utilizing LC with electrochemical detection ( I 3 ) ,the first few samples injected produced a low detector response. After the system was preconditioned, quantitation of subsequent injections was highly reproducible. Because of the labile nature of ascorbic acid, it is recommended that fresh tissues be analyzed whenever possible and that the tissue preparation time be minimized. Tissues frozen in liquid nitrogen and stored a t -60 "C were also analyzed. This treatment did not appear to significantly alter the ascorbic acid content of certain tissues if homogenization was conducted before the tissue thawed. The effect of prolonged storage on the ascorbic acid content of various tissues has not been thoroughly studied and should be evaluated if frozen tissues are to be analyzed. The importance of vitamin C in vertebrate systems is well documented. This new analytical method for ascorbic acid is being used in our laboratory to monitor the tissue free ascorbic acid concentrations of various invertebrates during environmental and pollutant-induced stress (14). The role of ascorbic acid in maintenance of homeostasis during stress
does not appear to be limited to vertebrates. It is hoped that tissue ascorbic acid levels may be used as an index of stress in monitoring the physiological and nutritional status of animals subjected to anthropogenic influences in nature.
LITERATURE CITED (1) Roe, J. H.; Kuether, C. A. J. Biol. Chem. 1943, 747, 399-407. (2) Sullivan, M. X.; Clarke, H. C. N. ASSOC.Off. Agric. Chem. 1955, 38, 514-5 18. (3) Day, B. R.; Williams, D. R.; Marsh, C. A. Clin. Biochem. (Amsterdam) 1979, 72, 22-26. (4) Summerwell, W. N.; Sealock, R. R. J. Biol. Chem. 1952, 796, 753-759. (5) Malckel, R. P. Anal. Biochem. 1960, 7 , 498-501. (6) Zannoni, V.; Lynch, M.; Goldstein, S.; &to, P. Biochem. Med. 1974, 7 7 , 41-48. (7) Horwitz, W., Ed. "Official Methods of Analysis of the Association of Official Analytical Chemists"; No. 43.051 Association of Official Analy?ical Chemists: Washington, DC, 1975; p 829. (8) Margarelli, P. C., Jr.; Cobin, L. B. Roc. Annu. Meet.- WWM Mafic. Soc. 1970. 9 , 235-241. (9) Allison, J. H.; Stewart, M. A. Anal. Biochem. 1971, 4 3 , 401-409. (10) Thrivckraman, K. V.; Refshauge, C.; Adams, R. N. Life Sci. 1974, 75, 1335-1342. (11) Pachla, L. A.; Kissinger, P. T. Methods Enzymol. 1979, 62, 15-24. (12) Pettibone, M. B u / l . - U . S . NaN. Mus. 1963, No. 227, part I. (13) Pachla, L. A.; Klssinger, P. T. Anal. Chem. 1976, 48, 364-367. (14) Thomas, P. T.; Carr, R. S.; Neff, J. M. "Marine Pollution and Physiology of Marine Organisms", In press.
RECEIVED for review May 27, 1980. Accepted September 5 , 1980. This study was supported in part by Grant No. OCE77-24551 from the National Science Foundation, International Decade of Ocean Exploration, Pollutant Responses in Marine Animals (PRIMA) Program.
Potassium Fluoride Assisted Derivatization of Carboxylic Acids to Phenacyl Esters for Determination by High-Performance Liquid Chromatography Jack M. Miller," Ian D. Brindle, Stephen R. Cater, and Kwok-Hung So Department of Chemistty, Brock University, St. Catharines, Ontario, Canada L2S 3A 1
James H. Clark Department of Chemistry, University of York, Heslington, York, England YO 1 5DD
Phenacyl esters of carboxylicacids have become increasingly important recently because of their sensitivity to detection in high-performance liquid chromatographs. Since traditional procedures have inherent problems such as slowness of reaction ( I ) and variable products (2), which have not lent themselves to exploitation, newer methods of derivatization have emerged. Thus Borch ( 3 )and Hullett and Eisenreich ( 4 ) used a nonaqueous system which achieved >90% yield after 8 h at room temperature or after 2 h at 50 "C. Durst (5,6) has developed a more rapid procedure which involves the use of crown ethers as catalysts, with reaction times of 10-20 min. Durst's derivatization and HPLC method have also been used recently in the analysis of C S 4 & fatty acids as part of a medical study (7) and in the determination of low molecular weight, volatile, fatty acids (8). This interest in phenacyl derivatives suggests that a simple procedure, for their preparation, using inexpensive reagents, is needed. We recently reported (9) an alternative method which involves the use of KF in a suitable solvent or, in the case of liquid acids, no solvent at all. We now report in detail the results of our macroscopic and microscopic derivatization of carboxylic acids using KF-assisted reactions in the formation 0003-2700/80/0352-2430$0 1.OO/O
of phenacyl and substituted phenacyl esters. Since this paper was submitted, Nagels et al. have published a paper ( I O ) which illustrates the analytical derivatization of quinic acid and derivatives using our K F method.
EXPERIMENTAL SECTION Equipment and Apparatus. Infrared spectra were obtained from either KBr disks or mulls on KBr plates by using a PE 237 grating spectrophotometer. Proton nuclear magnetic resonance spectra were obtained on either a Varian A-60 or Bruker WP-60 spectrometer. High-performance liquid chromatography data were obtained by using a Perkin-Elmer Series 3 HPLC with Rheodyne injector and the PE LC55 visible/UV detector operating at 254 nm. Separation was achieved by using 10 cm X '/4 in. 0.d. stainless steel reverse-phase columns with 10-pm C18 ODS packing with a solvent mix programmed from 10% acetonitrile/gO% water to 90% acetonitrile/lO%water over a 10-min period, or alternatively unprogrammed using 25% acetonitrile/75% water. Macroscopic Esterification. A typical reaction involved stirring together KF (1.28 g, 0.022 mol) and a-bromoacetophenone (phenacyl bromide) (1.99 g, 0.01 mol) for 1 min in 10 g of anhydrous DMF, after which the acid, e.g., acetic acid (0.6 g, 0.01 mol), was added and the mixture stirred at room temperature for 30 min. Ether (50 mL) was then added and the DMF extracted by shaking with three 50-mL aliquots of water. The ethereal 0 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
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Table I. Phenacyl Ester Yields
acid acetic trimethylacetic palmitica benzoic 2,4,6-trimethylbenzoic chloroacetic dichloroacetic acrylic 4-nitrobenzoic 3,S-dinitrobenzoic 3-methyl- Z-nitrobenzoic
p-bromophenacyl esters ester yield, % time, h 95.3 87.7 88.2 90 94.7 98 94.2 94.6
96.1 90.9 91 98 95 98
phenacyl esters ester yield, % time, h
1.0 0.25 1.0 1.5 0.5
98.2 95 96 98
0.5 0.2 0.2 2
96
0.3
0.5
96.5 87 90 94.7 90 95.2 81.7 89.8 95.2
0.5 0.25 0.7 5
0.5 0.25 1.0
1.0 0.5
p-phenylphenacyl esters ester yield, % time, h 98 94.1
93.8 98 92.3 82 85.6 82.5 91 97.2 68 76.1 94.8 83.6
1 0.5 0.5 0.5 0.5 0.5
0.5 0.25 0.5 :t.o
0.5 0.5 0.5 'I.O
1.0
-1.o
0.5
2-hydroxy benzoic 0.5 0.5 4-hy droxy benzoic 0.5 0.5 2-amino benzoicb 0.5 0.5 a Heated to 100 "C. This system is being investigated with respect to possible formation of the diester under some reaction conditions. solutions were dried over anhydrous MgSO, and evaporated to give a light yellow crystalline solid, mp 49-50 "C, compared with a literature value of 51-52 "C (II), yield 98.2%. Only in cases where the product ester was insoluble in ether was the procedure modified. As the ether/water step appeared to be necessary for effective removal of DMF for the ether insoluble esters, chloroform was added to the ether after the water washings. With higher molecular weight acids, the p-phenylphenacyl esters were also chloroform insoluble and these were worked up by filtration and water washing. Pure esters were then obtained from the crude by crystallization from ethanol, 1-propanol, ethyl acetate/ethanol, or chloroform/ethanol. Most recrystallized from ethanol in greater than 80% yield. The products were characterized by IR, NMR, and melting point data; phenacyl esters have a characteristic 'H NMR signal at 6 5.5 ppm for the O=C-CH2-Oprotons and a strong IR band at 1235 cm-' for a C - 0 - C stretching frequency. Separate C=O stretching frequencies were observed for the ester and phenacyl carbonyls. Microderivatization. Microscale derivatizations were carried out on DMF solutions of acetic acid and 4-hydroxybenzoic acids M. Equimolar over concentration ranges from lo-' to quantities of acid and a,p-dibromoacetophenone were used along with an excess of KF. Typical reactions involved efficiently stirring 3-9 mL of acid/phenacyl bromide solution with excess KF for 30 min at room temperature. After the solution was filtered, 1-5 pL of the DMF solution was injected into the HPLC. The peaks due to unreacted bromide and unreacted acid in the case of the aromatic acid (acetic acid cannot be detected by the UV HPLC detector) were small compared with the ester. The sensitivity of the detector was such that, without the procedure being modified, it should be possible to work down to lo-" M acid, Le., with subpicogram detectability.
RESULTS AND DISCUSSION In Table I, we summarize respectively the data on phenacyl, p-bromophenacyl, and p-phenylphenacyl esters, of a series of representative aliphatic and aromatic carboxylic acids, both substituted and unsubstituted. Unless otherwise stated these reactions were carried out a t room temperature and in DMF solution. in most cases, the phenacyl derivatives appear to have the highest yields and form fastest, while for aliphatic acids the yields of the p-bromophenacyl derivatives are the lowest. In the hydroxybenzoic acid cases, the lack of phenol alkylation is probably due to the preferential attack of F- on the carbonyl moiety. Any slight preference for more rapid formation of the phenacyl derivative is probably steric. The resulting order of reaction rates is a-bromoacetophenone > apdibromoacetophenone > a-bromo-p-phenylacetophenone. These rapid, inexpensive reactions have been readily adapted to the laboratory derivatization of carboxylic acids as part of their characterization. This led to the suggestion
03
0
io
20
MIN
Flgure 1. Liquid chromatogram of M acetic acid derivatized with p-bromophenacyl bromide: 1 pL injection; sensitivity 0.02 AUFS (absorbancy units full scale); chart speed 0.25 cm/min; (a) p-brome phenacyl acetate, (b) p-bromophenacyl bromide, (c) N,Ndimethylformamide.
i
A 10
20
MIN
Flgure 2. Liquid chromatogram of lo-' M 4-hydroxybenzoic acid derivatized with p-bromophenacyl bromide: 5 pL injection;sensitivity 0.05 AUFS: (a) 4-hydroxybenzoic acid, (b) p-bromophenacyl 4hydroxybenzoate, (c) p-bromophenacyl bromide, (d) N,Ndimethylformamide. that good yields might also be obtained on the micro scale. Thus when we looked at the micro derivatization, the resulting method was far simpler than those used by other authors (3-8) and we used cheaper reagents, Le., KF instead of crown ether. Figures 1 and 2 show HPLC traces for the M acetic acid and lo4 M 4-hydroxybenzoic acid solutions after reaction with p-bromophenacyl bromide. Sensitivity is excellent and the technique may obviously be pushed to much lower concentrations. For the acetate, 'jsoth of the concentration would still have been detectable with a 1-pL injection, and if a 10-pL
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Anal. Chem. 1980, 52, 2432-2435
injection were used, concentrations of acid on the order of 2 X M are measurable. From Figure 2, noting the recorder sensitivity etc., it is clear that the 4-hydroxybenzoic acid could easily be detected in 10-l' M solution as its p-bromophenacyl derivative. Also, there is no reason the quantities of solutions used in the reaction could not be reduced below 1 mL by use of small vials and appropriate stirrers. Jordi (12)has indicated that the level of detectability for p-bromophenacyl derivatives lies in the range of mol but clearly, in this work, limits of detectability lie in the range of mol. This is perhaps a reflection of the improvements in instrumentation over the last few years and is consistent with the results obtained by Barcelona e t al. (8). Morozowich and Douglas (13) have also reported a fast derivatization method for p-nitrophenacyl esters of prostaglandins using N,N-diisopropylethylamine.The authors in this paper indicate a limit of detection of 1 ng which, again, may be a reflection of the state of the art in 1975. p-Nitrophenacyl bromide is also much less readily available than the reagent used in this work. T h u s we have a derivatization technique which, when combined with a modern HPLC and the intense phenacyl chromophore, permits subpicogram detection and analysis of carboxylic acids.
ACKNOWLEDGMENT T h e authors thank H. L. Holland and T. R. B. Jones for their assistance. LITERATURE CITED (1) Shriner, R. L.; Fuson, R. C.; Curtin, D. Y. "The Systematic Identification of Organic Compounds", 5th ed.; Wiley: New York, 1964; p 235. (2) Pokras. H. H.; Bernstein, H. I. J . Am. Chem. SOC. 1943, 65, 2096. (3) Borch, R. F. Anal. Chem. 1975, 4 7 , 2437. (4) Hullett, D. A.; Eisenreich, S. J. Anal. Chem. 1979, 51, 1953. (5) Durst. H. D. Tetrahedron Lett. 1974, 2421. (6) Durst, H. D.; Milano, M.; Kikta, E. J.; Connelly, S. A,; Grushka. E. Anal. Chem. 1975, 47, 1797. (7) Takagama, K.; Qureshi, N.; Jordi, H. C.; Schnoes, H. K. J . Liq. Chromatogr. 1979, 2 , 861. (8) Barcelona, M. J.; Liljestrand, H. M.; Morgan, J. J. Anal. Chern. 1980, 52,321. (9) Clark, J. H.;Miller, J. M. Tetrahedron Leff. 1977, 599. (10) Nagels, L.; De Beuf, C.; Esmans, E. J . Chromatogr. 1980, 190, 41 1. (11) GiraMi. P. N. f a r m c o , Ed. S d . 1959, 14, 90; Chem. Abstr. 1960, 54, 3299. (12) Jordi, H. C. J . Liq. Chromatogr. 1978, 1 , 215. (13) Morozowich, W.; Douglas, S. L. Prostaglandins 1975, 10, 19.
RECEIVED for review April 7,1980. Accepted August 18, 1980. The authors thank Imperial Oil and the National Science and Engineering Research Council of Canada for the award of research grants to J. M. Miller and NATO for a travel grant to J. M. Miller and J. H. Clark.
Determination of Colchicine and Colchiceine in Microbial Cultures by High-Performance Liquid Chromatogra phy Allan E. Klein and Patrick J. Davis* Division of Pharmaceutical Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 787 12
Colchicine (1))the major alkaloid of Colchicum species, has traditionally been employed for the treatment of gout (1). The antineoplastic activity of colchicine and its derivatives have recently increased interest in their potential use as chemotherapeutic agents (2). While colchicine itself may prove too toxic for use in this regard ( 3 , 4 ) ,there is ample evidence to indicate that derivatives of colchicine exhibit a higher therapeutic index and, hence, hold more promise for clinical application (5, 6). For this reason, we are examining the use of microorganisms to metabolically prepare derivatives of colchicine and related alkaloids (7). Colchiceine (2) is a reported mammalian metabolite of colchicine (8) and is a potential microbial metabolite as well. The suggestion has been made (9) that the 0-dealkylated product of colchicine with Streptomyces griseus as reported by Velluz et al ( 1 0 , l l )may, in fact, be colchiceine, although this still appears to be open to question. A problem with such studies is that the analytical techniques reported thus far for colchicine have lacked specificity for the determination of the parent compound in the presence of its various metabolites or derivatives. Thin-layer chromatography has been successfully used for the qualitative evaluation of some colchicine derivatives (12, 13). Normalphase (14)as well as reverse-phase (15-1 7) high-performance liquid chromatographic (HPLC) systems have been employed for colchicine derivatives, and we recently reported the successful separation of six colchicine derivatives on a reversephase system (18). Colchiceine (2) has not been previously determined in the presence of colchicine and related compounds. Thin-layer chromatography has been described for this acidic metabolite (13). However, we have observed poor mobility in diethylamine-containing solvent systems. The compound typically is strongly adsorbed by silica gel and shows little or no mobility in a variety of solvent systems. In
NHCOCH3 OR
0 3 , R=CH2CH3
7 , R=CH3
addition, the chromatographic analysis is complicated by the vinylagous acid functionality which favors the formation of
0003-2700/80/0352-2432$01.00/00 1980 American Chemical Society
