Anal. Chem. 1982, 54, 2587-2590
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Fluorometric: Determination of Citric and Aconitic Acids with DicyclohexyIcarbodiimide Sheng-Chlh Chen School of Pharmacy, China Medical College, 9 1 Hsueh Shih Road, Taichung, Taiwan, Republic of China
When citric acid and cl's- and trans-accnnltic acids reacted wlth dlcyciohexylcarbodllmide (DCC) In the presence of acetic acld, they readily gave fluorescent species with excitation wavelength at 4102 nm and emisalon wavelengths at 490 nm (citric acld) anid 485 nm (aconitic acids). By application of this reactlon, new fluorometric methods have been developed for determlnatlon of these aclds. Various reaction conditions have been standardized. The cailbratlon curves were linear from 5 to 90 nmoi for citric add and from 0.2 to 90 nmoi for aconitic acids. The relative standard devlatlons obtained wlth 50 nmol of citric, cls-aconltlc, and transaconltic acids were 5.50, 6.75, and 5.26%, respectively. The speclflclty and mechanlwm associated wlth the reaction are also described.
Citric and aconitic acids, which are mlembers of the tricarboxylic acid cycle, are biochemically and clinically interesting substances present in urine and blood. The former in used as an acidulant in 'beverages, confectionery, and pharmaceutical syrups and is a component of anticoagulant citrate solution; the latter, as a plasticizer for buna rubber and plastics, is also utilized in the manufacturing industry. For the determination of these compounds in such complicated samples, a highly specific method is required. Fluorometric determination of citric acid has been performed by using the reaction of the acid with pyridine in the presence of acetic anhydride (1). Another method which was developed for the deterrnination of polycarboxylic acids (2) by using resorcinol and sulfuric acid as reagents has also been recommended for the same acid (3). Moreover, Hori et al. (4) have introduced o-aminsthiophenol as a fluorogenic reagent, for citric acid. Although tlhe acetic anhydride-pyridine method is relatively amenable to routine use, these methods either are of low degree of specificity or are time-consuming. In the case of aconitic acid, except for two reactions ( 5 , 6 ) which were described for thin-layer chromatography (TLC), few fluorometric methods have been reported. In the previous paper (7), the author has established a new detection method for some di- and tricarboxylic acids on TLC plates and papers with dicyclohexylcarbodiimide (DCC) as a spray reagent. In this paper, the author, applying the same reagent, describes a new fluorometric method for specific determination of citric and aconitic acids. EXPERIMENTAL SECTION Apparatus. A Shimadzu RF-520 dual beam difference spectrofluorophotometer equipped with a xenon lamp and 1-cm quartz cells was used for the determination of fluorescence intensity. The excitation and emission bandwidths were set at 10 nm. Wavelengths indicated are uncorrected. Materials and Reagenta. DCC and solvents were purchased from Merck (Darmstadt, West Germany) and used as received. Other materials were the same as described previously (7). Procedure A for the Determination of Citric Acid. To 100 pL of the sample solution, which contained 5-90 nmol of citric 0003-2700/82/0354-2587$01.25/0
acid in 0.3 M butanolic acetic acid, 100 pL of 0.4 M DCC in 1-butanol was added and mixed with the aid of a Vortex type mixer. The mixture was then heated immediately at 60 "C for 10 min. After the mixture had been cooled in an ice bath (ca. 5 s) and then diluted with 2.5 mL of methanol, the fluorescence intensity was measured with excitation wavelength at 402 nm and emission wavelength at 490 nm against a reagent blank. Procedure B for the Determination of cis- or transAconitic Acid. The procedure for the determination of cis- or trans-aconitic acid was similar to that for citric acid described above. But, the sample solution containing 0.2-90 nmol per 100 pL was prepared with 1.0 M methanolic acetic acid and reacted with 100 p L of 0.8 M DCC in methanol. Finally, after dilution with 2.5 mL of methanol the fluorescence intensity was measured with excitation wavelength at 402 nm and emission wavelength at 485 nm. Thin-LayerChromatographic Examination of the Fluorophore Formed in the Reaction Mixture. Precoated TLC aluminum sheets (silica gel 60, 0.2 mm layer) from Merck (Darmstadt, West Germany) were used to detect the fluorophore in the reaction mixture with ethyl acetate-methanol (8:2, v/v) as a solvent system at room temperature (ca. 20 "C). RESULTS Effect of Solvent on the Fluorogenic Reaction. As indicated by Khorana (8) and Mikolajczyk and Kielbasinski (9),solvents exert a remarkable effect on the reaction rate and the reaction products of carbodiimides with acids. In order to obtain a proper solvent for the determination of the acids, various solvents were investigated. In this study, a 100-pL aliquot of 5 mM acid solution was used to react with 400 PL of 0.8 M DCC, and the relative fluorescence intensity was measured. The result, as shown in Table I, demonstrates that each acid reacted in different solvents gave quite different fluorescence intensity, especially in the case of citric and trans-aconitic acids, and therefore that the solvents used affected the fluorogenic reaction of DCC with these acids. From the table, 1-butanol was chosen as a solvent for the reaction of citric acid, and methanol for that of aconitic acids, since they gave relatively strong fluorescence. Accelerator for the Fluorogenic Reaction. Acetic acid, acetic anhydride, formic acid, aluminum chloride, cupric chloride, and pyridine were separately investigated for the fluorogenic reaction of DCC with the acids. Among these, acetic acid accelerated the reactions of both citric and aconitic acids, whereas pyridine accelerated that of aconitic acids but had no effect on the reaction of citric acid with DCC. The others, namely, formic acid, aluminum chloride, and cupric chloride, caused fluorogenic reaction failure. Figures 1 and 2 show respectively the results of the investigations on the effects of acetic acid and pyridine on the fluorogenic reaction. They illustrate that 0.5 M and 0.15 M of acetic acid, and 50450% of pyridine in the reaction mixtures gave maximum fluorescence intensity to aconitic, citric, and aconitic acids, respectively. However, excess amounts of both acetic acid and pyridine made the fluorescence intensity decrease. It is also noticeable that when acetic acid was absent, cis-aconitic acid gave stronger fluorescence than the trans isomer did, which also can be found in Table I, indicating that 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
Table I. Relative Fluorescence Intensities of the Reaction Mixtures of Acids and DCC in Various Solvents citric acid optimal RFI at wavelength optimal (Ex/Em, nm)" wavelength
solvent methanol ethanol 1-propanol 2-propanol 1-butanol 2-methyl-1-propanol 2-methyl-2-propanol 1-pentanol dioxane ethyl acetate acetone dime th ylf ormamide acetonitrile pyridine diethylamine
3971488 4001488 4051484 4001488 4021490 40 514 90 3851474 4001488 3971485 3961480 3971480 3851474 39 514 76 3951475 3651442
25 63
7 86 100 34 29 97 67 56 22 34 48 29
7
trans-aconitic acid optimal RFI at wavelength optimal (Ex/Em, nm)" wavelength 4101490 4131492 4121501 4161505 4151501 4131503 4181508 4 1 51498 4101484 4061486 4201500 4051483 4601816 40 21490 3971472
cis-aconitic acid optimal RFI at wavelength optimal (Ex/Em, nm)" wavelengthb
168 84 101
79 137 110 60 146 165 56 82 147 124
538c
7
4121492 413149 2 4121494 4 1 21496 4121496
280 23 8 222 224 278
4121496 4121496 ' 4101485 4101488 4121490 4051488 4191507 3921472 3981473
19 5 24 8 2 59 366 193 221 332 572c
15
a The excitation and emission maxima are uncorrected. One-tenth milliliter of 5 mM acid solution and 0.4 mL of 0.8 M DCC were mixed and then treated with the standard procedure described in the Experimental Section. In the case of aconitic acids, the mixture was allowed to stand for 1 min at 28 "C, rather than being heated, before dilution. The fluorescence intensity was increasing while the solution was measured or allowed to stand.
32 c4 06 aB ACETIC A C I D ( M )
0
Y
0
02
0'
reaction. Fifty nanomoies of the acid (m, citric acid; A,cis-aconitic acid; 0 , trans-aconitic acid) reacted with 0.4 M DCC in 200 pL of alcoholic solution (1-butanol for citric acid; methanol for aconitic acids) containing various amounts of acetic acid at 60 OC for 10 min. The subsequent experiment was performed with the standard procedures.
0
M
4
0
6
0
8
0
1
0
03
DCC (
Flgure 1. Effect of the concentration of acetic acid on the fiuorogenic
05
04
M)
Figure 3. Effect of DCC concentration on the fiuorogenic reaction. One hundred nanomoies of the acid citric acid; A,cis-aconitic acid; 0 , trans-aconitic acid) reacted with various amounts of DCC in 200 pL of reaction mixture by the method described in the Experimental Section. Broken line is for the reaction in 50% pyridine performed with a procedure as indicated in Figure 2.
m,
3
PYRlDINE (V0v i v )
Flgure 2. Effect of pyridine concentration on the fiuorogenic reaction.
Fifty nanomoies of aconitic acid (0,fransaconitic acid; A,cis-aconitic acid) reacted with 0.4 M DCC in 200 pL of reaction mixture containing various amounts of pyridine at 60 OC for 10 min. After dilution with 2.5 mL of methanol, the fluorescence intensity was measured with excitation wavelength at 402 nm and emission wavelength at 490 nm.
the cis isomer was more active. However, it was reversed when acetic acid was added into the reaction mixture. Effect of the Concentration of DCC on the Fluorogenic Reaction. Figure 3 depicts the relation between the fluorescence intensity and the concentration of DCC. In the case of reaction in 50% pyridine, only less than 0.05 M DCC was required for aconitic acids to give maximum fluorescence intensity. However, in acetic acid solution at least 0.1 M or 0.3 M DCC was required for citric or aconitic acids, respectively. For increased the reproducibility, 0.1 M, 0.4 M, and 0.8 M DCC were chosen for standard procedures and reacted with an equal volume of the sample solution of aconitic acids in
0
5
73
15
20
MINUTES
Flgure 4. Time courses of the fiuorogenic reactions of citric acid (a),
trans-aconitic acid (b), and cis-aconitic acid (c) with DCC. The sample solution and DCC solution were the same as that of the standard procedures and reacted at 40 OC (0,W), 50 OC (A, A)and 60 OC (0, 0).separateiy. Dotted lines are for those reacted in 50% pyridine with 0.1 M DCC.
pyridine, citric acid in 1-butanol, and aconitic acids in methanol with acetic acid as an accelerator, respectively. DCC
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
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Table 11. Relative Fluorescence Intensities of Various Acids in the Present Methods procedure A optimal RFI at wavelength optimal (Ex/Em, nm) a wavelength trans-aconitic acid cis-aconitic acid citric acid isocitric acid hippuric acid leucine-hy drochloride a
4131493 4131493 4021490 4021477 3491415
0
The excitation and emission maxima are uncorrected.
L.
0
5
10
15
31 5 266 100 4 3
-_
20
procedure B optimal RFI at wavelength optimal (ExIEm, nm) a wavelength 4021485 4021485 4021490 4021477 3321398 3761456
100
85 28 1 1
0.1
Citric acid and trans-aconitic acid are separately taken as 100. shows that, except those given in Table 11, salts of these acids and cis-aconitic anhydride, all were negative to DCC in acetic acid solution. The relative fluorescence intensities of the acids listed in Table 11were determined on the molar basis. The table shows that the present methods are highly specific for citric acid and cis- and trans-aconitic acids. In addition, since aconitic acids have a double bond on their structures, it is reasonable to expect that they can easily be distinguished from citric acid by hydrogenation.
WATER (%,v/v)
Flgure 5. Effect of water on the fluorogenic reaction. One hundred microliters of the sample solution containing 50 nmol of trans-aconitic acki and various amounts of water reacted with 100 pL of 0.8 M DCC as described in the Experimental Section.
in these solutions could retain its activity even at 60 "C for at least 1 h. Optimal Temperature and Time for the Fluorogenic Reaction. With the optimal solvent and concentration of DCC and the accelerators, temperature and time for the reaction had been investigated for each acid. Figure 4 shows separately the results that all of these acids gave maximum fluorescence intensity when they readed in acetic acid solution at 60 "C for 10 min. However, in 50% pyridine, the optimal temperature and time for cis- and trans-aconitic acids were 50 "C, 10 min and 50 "C, 20 min, respectively. Effect of Water on the Fluorogenic Reaction. A preliminary test indicated that the fluorescence arising from the reaction of DCC with the acids disappeared after the addition of water. However it has been described by Kasai et al. (10) that water suppresses the coupling reaction of DCC with hydroxylamine-hydrocliloride. In this study, the effect of water on the reaction of DCC with trans-aconitic acid was investigated. The result (Figure 5) shows that water affected the fluorogenic reaction. Determination of Citric and cis-and trans-Aconitic Acids. With the standardized conditions, three procedures had been established. However, because the procedure with pyridine as the accelerator gave larger relative standard deviation (about two times of the others) and bad linearity at low concentration of the acids, only two are given in the Experimental Section. With the procedures, 5-90 nmol of citric acid or 0.2-90 nmol of aconitic acids can be determined with a linear calibration curve against a practically nonfluorescent blank. The relative standard deviations obtained with 50 nmol of citric and cisand trans-aconitic acids (n = 5) were 5.50, 6.75, and 5.26%) respectively. Specificity of the Present Methods. Although it has been investigated on TIE plates in previous paper (7))the specificity of the reactions of DCC with acids in acetic acid solution was reinvestigated. With the standard procedures 50 compounds with one to three carboxyl and related groups, as used in the previous paper (7), were examined at the 10 pmol level. The result
DISCUSSION DCC has been proved to be an excellent reagent for synthesis of peptides (11-13)) ester (14-16)) and also for dehydration of @-hydroxyketone (17,18) and @-hydroxyester (19) to give a,@-unsaturatedcompounds. In the present methods, DCC is also used presumably as a dehydrating agent to convert citric acid to aconitic acid, aconitic anhydride, and then fluorescent species. Among the tested compounds in the specificity investigation, dicarboxylic acids such as malic, fumaric, maleic, itaconic, and citraconic acids, a tricarboxylic acid such as tricarballylic acid, and trimethyl esters of citric and trans-aconitic acids cannot produce fluorescence species when they reacted with DCC in the presence of acetic acid. However, sodium citrate, cis-aconitic anhydride, and cis-aconitic, trans-aconitic, citric, and isocitric acids are positive to this reagent, indicating that three carboxyl groups (or anhydride, but not ester) with a double bond, e.g., aconitic acids, or with a @-hydroxygroup, e.g., citric and isocitric acids, were essential for the reaction with DCC. Therefore, it is possible to predict that dehydration of the @-hydroxygroup of citric acid, as well as that of @hydroxy ketone and @-hydroxy ester, and formation of a resonating carbanion intermediate (20, 21), through which dehydroxylation, trans to cis isomerization, or anhydride formation arises, would take place. In addition, since cisaconitic anhydride can give the same fluorescence species and carboxylic acid can be dehydrated to anhydride by DCC (8, 9),it is likely that DCC converts cis-aconitic acid to aconitic anhydride and then fluorescence species. This reaction pathway is similar to that supposed by Pesez and Bartos (22). They have reported a fluorometric method for tertiary aliphatic amines with aconitic acid in the presence of acetic anhydride and supposed that this reaction converts aconitic acid with acetic anhydride to aconitic anhydride and then with tertiary amine gives a fluorescence species with excitation wavelength at 405 nm and emission wavelength at 485 nm. In the present methods, therefore, DCC may behave as a dehydrating agent and source of tertiary amine, since the addition of methanol or acetic acid to DCC can lead to 0methyl-N,N'-dicyclohexylisourea (23) or 0-acetyl-N,N'-dicyclohexylisourea (8,9) in the presence of acetic acid. Other evidence of the requirement of an amine for the preseik reaction can be found in Figures 2 and 3, that pyridine content vs. fluorescence intensity was linear over the range of 10 to
Anal. Chsm. l Q 8 2 , 54, 2590-2591
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50% and pyridine affected the amount of DCC required for the reaction, respectively. The present reaction needs only one reagent and proceeds under mild conditions, and so it is more simple than the two-reagent system of the pyridine-acetic anhydride method or o-aminothiophenol method, in which 125 OC and 15 h of heating are required. Moreover, in view of specificity, the present methods are excellent for citric and aconitic acids, because some polycarboxylic acids (especially,such as malonic acid) can fluoresce with tertiary amines in the presence of acetic anhydride (24, 25). Therefore, despite the similar sensitivity to that of pyridine-acetic anhydride method, the established methods are more amenable to specific routine work or automatic analysis. ACKNOWLEDGMENT The author thanks Zenzo Tamura and Hiroshi Nakamura, Faculty of Pharmaceutical Sciences, University of Tokyo, for their critical review of this manuscript and valuable suggestions. LITERATURE CITED Pellet, M. V.; Seigner, Ch.; Cohen, H. Pathol. Biol. 1989, 77, 909-914. Frohman, C. E.;Orthen, J. M. J . Biol. Chem. 1953, 205, 717-723. Yamamoto, D.; Kawamura, T. Me!//Dalgaku Ncgakubu Kenkyu Hokoku 1971, 26. 1-13. Hori, M.; Kometani, T.; Ueno, H.; Morimoto, H. Blochem. Med. 1974, 7 1 , 49-59.
(5) Massa, V.; Suspiugas, P.; Salabert, J. Trav. SOC.Pharm. Montpellier. 1974, 3 4 , 71-77. (6) Lang, H.; Lang, E. J . Chromatogr. 1972, 73, 290-291. (7) Chen, S . 4 . J . Chromatog. 1982, 238, 480-482. (8) Khorana, H. G. Chem. Rev. 1953, 5 3 , 145-166. (9) Mikolajczyk, M.; Kielbasinski, P. Tetrahedron 1981, 3 7 , 233-284. (10) Kasai, Y.; Tanimura, T.; Tamura, 2 . Anal. Chem. 1975, 47, 34-37. (11) Sheehan, J. C.; Hess, G. P. J . Am. Chem. SOC. 1955, 7 7 , 1067-1068. (12) Biout, E. R.; DesRoches, M. E. J . Am. Chem. SOC. 1959, 87, 370-372. (13) Albertson, N. F. "Organic Reactions"; Wlley: New York, 1962; Vol. 12, pp 205-213. (14) Khorana, H. G.; Todd, A. R. J . Chem. SOC. 1953, 2257-2260. (15) Khorana, H. G. J . Am. Chem. SOC. 1954, 7 6 , 3517-3522. (16) Decker, C. A.; Khorana, H. G. J . Am. Chem. SOC. 1954, 78, 3522-3527. (17) Corey, E. J.; Andersen, N. H.; Carbon, R. M.; Paust, J.; VedeJs, E.; Viattas, I.; Winter, R. E. K. J . Am. Chem. SOC. 1988, 9 0 , 3245-3247. (18) Aiexandre, C.; Rouessac, F. Bull. SOC.Chim. Fr. 1971, 1637-1840. (19) Alexandre, C.; Rouessac, F. C.R . Hebd. Seances Acad. Sci., Ser. C 1972, 274, 1585-1588. (20) Kllnman, J. P.; Rose, I . A. Biochemistry 1971, IO, 2259-2266. (21) Ambler, J. A.; Roberts, E. J. J . Org. Chem. 1948, 13, 399-402. (22) Pesez, M.; Bartos, J. Talanta 1989, 78, 331-336. (23) Moffatt, J. G.; Khorana, H. 0. J . Am. Chem. SOC. 1957, 79, 3741-3746. (24) Roeder, G. J . Am. Pharm. Assoc. 1941, 3 0 , 74-78. (25) Thomas, A. D. Talanta 1975, 2 2 , 865-889.
RECEIVED for review April 6, 1982. Accepted September 10, 1982. Presented at the Annual Meeting of the Pharmaceutical Society of the Republic of China, Taipei, Dec 1981.
CORRESPONDENCE Effect of Analog-to-Digital Converter Resolution on Absorbance Measurements Sir: The wide use of digital data acquisition in chemical instrumentation has led to an appreciation of the effects of amplitude quantization of signal-to-noise ratio. It has been shown theoretically that, under most experimental conditions, the effect of quantization is to add white or uncorrelated noise to the total signal noise and that the root mean square magnitude of the quantization is q/121/2, where q is the quantization interval (I,2). The derivation of this expression is given in ref 2 and will not be repeated here. It is of interest to inquire whether these theoretical predictions can be verified experimentally under practical laboratory conditions. We report here a method for measuring quantitatively the effect of quantization noise in experimental signals and demonstrate its application to absorbance measurements made with a continuum-source atomic absorption spectrometer. The method requires the acceptance of four basic assumptions. First, one must assume that noises add quadratically P=Q2+N2
(') where T is the total noise, Q is the quantization noise, and N represents all other noise sources such as photon noise, flicker noise, etc. Second, quantization noise must be assumed to be directly proportional to the Size of the quantization interval Qs = 4Q10 =
16Q12
(2)
where Qs, Ql0, and QI2are the quantization noises for the 8-,
lo-, and 12-bit resolutions, respectively. Third, we must assume that all other noises are not a function of resolution. Fourth, it is necessary to assume that the quantization noise is smaller than other noise sources. On the basis of these four assumptions one can write equations for the total noise T with 8-bit and 12-bit quantization Ta2= Qa2 IF' = (16Q12)2 N2 (3)
+
+
T122= QlZ2+ IF' (4) where Taand T12represent the total noise for an 8- and 12-bit quantization, respectively. Both Ta and T12can be measured from one set of 12-bit data by masking the four least significant bits to get 8-bit data. Solving eq 3 and 4 simultaneously for Qlz yields
Values of Q for 10- and 8-bit quantization may be obtained from eq 2. EXPERIMENTAL SECTION Measurements were made on a continuum-source atomic absorption spectrometer (3) which utilizes a 300-w sc source, a wavelength modulation spectrometer with a photomultiplier detector, and a 12-bit analog-to-digital(ADC) converter interfaced to a minicomputer. Intensities are measured directly by the ADC and are converted into absorbances by the minicomputer. Two types of noise measurements were performed:
0003-2700/82/0354-2590$01.25/00 1982 American Chemlcai Society
