1461
Anal. Chem. 1985, 57, 1461-1464
were dispersed within each other with sufficient reproducibility that standard addition could be performed over a wide, controllable range of standard/analyte concentration ratios. I t is interesting to observe that the new variant of the gradient technique is partially based on the underlying concept of FIA titration ( I ) , where elements of fluid with identical D values are exploited, and partially on the concept of zone penetration ( 5 ) as designed for selectivity measurements, where elements of fluid with identical delay times t were used. Thus, another variation of the concept dispersed sample zone has been discovered and exploited. Its use is not limited to flame methods, as reaction based assays, like spectrophotometry, would equally benefit from the novel way of performing standard addition. Instruments equipped with photodiode arrays and appropriate fast data collection and storage facilities should increasingly find use in future applications. Registry No. Li, 7439-93-2; Na, 7440-23-5;K, 7440-09-7; Ca, 7440-10-2; water, 7732-18-5.
LITERATURE C I T E D Ruzicka, J.; Hansen, E. H. Anal. Chim Acta 1983, 145, 1. Janata, J.; Ruzicka, J. Anal. Chim. Acta 1982, 139, 105. Greenfield, S. Spectrochim. Acta, Part 8 1983, 388, 93. Black, C. A. "Methods of Soil Analysis, Part 2"; American Society of Agronomy, Inc.: Madison, W I , 1965; pp 894-895. (5) Zagatto, E. A. G.; Jacintho, A. 0.; Krug, F. J.; Reis, B. F.; Bruns, R . E.; Arujo, M. C. U. Anal. Chim. Acta 1983, 145, 169. (6) Tyson, J. F.; Appleton, J. M.; Idris, A. B. Anal. Cbim. Acta 1983. 745, 159. (7) Israel, Y.; Barnes, R. M. Anal. Chem. 1984, 56, 1188. (8) Hansen, E. H.; Ruzicka, J. Anal. Chim. Acta 1983, 148, 111. (9) Gine, M. F.; Reis, B. F.; Zagatto, E. A. G.; Krug, F. J.; Jacintho, A. 0. Anal. Chim. Acta 1983, 155, 131. (IO) Ramsing, A. U.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1981, 129, 1.
(1) (2) (3) (4)
RECEIVED for review August 10, 1984. Resubmitted March 11,1985. Accepted March 11,1985. The authors express their gratitude to the Danish National Council for Scientific and Industrial Research for financial assistance to Z. Fang and J. Harris and to the Academia Sinica for grant of leave to Z. Fang.
Dicyclohexylcarbodiimide as a Cleaving Agent for Colorimetric Determination of Pyridyl and Pyrimidinyl Compounds Sheng-Chih C h e n School of Pharmacy, China Medical College, 91 Hsueh Shih Road, Taichung, Taiwan, Republic of China
By use of dlcyclohexylcarbodAmIde (DCC) and dimethylbarblturic acid (DMBA) as reagents, a colorlmetric method for the deterrninatlon of pyrldyl and pyrlmldlnyl compounds has been established. DCC breaks the pyrldlne or pyrlmldine ring to afford giutaconaldehyde or maionaldehyde and then reacts wlth DMBA to produce chromophores. These heterocycles could be determined by measuring the chromophores. The reiatlve standard devlatlons obtained with different amounts of these compounds were In the range of 0.63 to 5.36% ( n = I O ) . The reactlon mechanism Is also discussed.
Carbodiimides are widely used as excellent coupling or dehydrating agents in synthesis (1-3) and analysis (3-10). Recently, Wilchek and co-workers (10,II) have demonstrated their cleaving activity on pyridine (eq 1) and established a colorimetric method for the determination of carbodiimides (IO). Since then the author, using the same reaction, has developed a fluorometric method for the determination of malonic acid (9). At present, the cleavage of pyridine to form glutaconaldehyde for colorimetry is carried out with cyanogen halide (12,13),pyridylpyridinium dichloride (14),or gem-polyhalogen compounds (15-18). However, few reagents have been reported for breaking the pyrimidine ring. In the present study the author, interested in the wide occurrence of nitrogen aromatic heterocycles and also in the cleaving activity on the pyridine ring as well as in the chromogenic reaction of the cleft product with dimethylbarbituric acid (DMBA), has investigated the action of dicyclohexylcarbodiimide (DCC) on some nitrogen aromatic heterocycles and developed a colorimetric
method for the determination of pyridyl and pyrimidinyl compounds. EXPERIMENTAL SECTION Apparatus. A Shimadzu UV-21OA double beam spectrophotometer and a Shimadzu RF-520 dual-beam difference spectrofluorometer equipped with a 150-W xenon lamp and 1-cm quartz cells were used for the determination of absorbance and fluorescence intensity, respectively. The wavelengths indicated were uncorrected. Mass spectrometry wm performed on a Hitachi M-52 mass spectrometer. The electron ionization energy was 20 eV. The final step of the purification of the chromophore was carried out on a Hewlett-Packard 1084B liquid chromatograph equipped with a Hewlett-Packard 79850B LC terminal and a reversed-phase RP-18 column (5 bm, 250 X 4.6 mm; E. Merck) with methanol as the eluent. Materials and Reagents. Nitrogen aromatic heterocycles were purchased from Tokyo Kasei (Tokyo, Japan; 2-aminopyrimidine, pyrimidine, pyrazole, phthalazine, pyridazine, imidazole, 2aminopyridine,and isoquinoline),Wako Pure Chemicals (Osaka, Japan; piperidine and quinoline), Kanto Chemical (Tokyo, Japan; sulfadiazine, sulfamerazine, and sulfamethazine), E. Merck (Darmstadt, West Germany; pyridine and pyrrole), Aldrich (Milwaukee, WI; 2-amino-4-methylpyrimidine and 2-amino-6methylpyridine), Sigma (St Louis, MO; pyridoxine HCl), and local drug stores (pharmacopial grade; sulfaphenazole, sulfadimethoxine, sulfisoxazole, sulfaguanidine, sulfamoxole, sulfisomidine, sulfamethoxazole, sulfamethoxypyridazine, chlorpheniramine maleate, niacin, isoniazid, and thiamine HCl). All of these compounds were used as received. The reagents, DCC and DMBA, were obtained from Tokyo Kasei and Fluka (Buchs, Switzerland), respectively, and were dissolved in methanol (E. Merck) without further purification. Thin-layer chromatography for the identification of the chromophore from sulfadiazine and malonaldehyde bis(di-
0003-2700/85/0357-1461$01.50/00 1985 American Chemical Society
1462
ANALYTICAL CHEMISTRY, VOL. 57,
NO. 7, JUNE 1985
Table I. Absorption and Fluorescence Characteristics of the Chromophores Derived from Pyridyl and Pyrimidinyl Compoundsa
compound pyridine pyrimidine pyridazine 2-aminopyrimidine 2-amino-4-methylpyrimidine sulfadiazine sulfamerazine thiamine HC1 chlorpheniramine maleate isoniazid niacin quinoline isoquinoline
Amaxtb nm
588 (588/605)
sensitivity,cpmol 5.74
490
0.244
489
f
linearity range, pmol
RSD,d 70
0->10 0->1.0e 0-0.8
2.01 (5.0) 3.03 (0.5)e 0.53 (0.5) 2.17 (0.5)
490
0.877
5119 490 511 415
0->1.0
0.025 0.301
0-0.1 0->1.0 0-8.0
472
400h 408 415 410
ca. 10 1.16
0.96 (0.05) 0.98 (0.5) 5.36 (5.0) 0.63 (2.5)
0->5.0
2.20 3.46
>10 >10
a The experiment was performed with the standardized procedure. *The parenthetical figures are excitation/emission wavelengths. The sensitivity refers to an amount required to produce an absorbance value of 0.3 in 3.0 mL of solution. dThe relative standard deviations were determined at the amounts (pmol) indicated in parentheses (n = 10). eThe data were obtained by fluorometry. fThe plateau of the calibration curve began at 1.0 pmol, where the absorbance value was 0.174. gThe color changed from yellowish green to pale brown after 10 min of reaction (10 pmol gave a 0.164 absorbance value at, ,A 511 nm). 'It was arbitrarily set at 400 nm because the visible absorption continued to the UV region.
methylacetal) (Tokyo Kasei) was performed on precoated aluminum TLC sheets (silica gel 60,0.2 mm layer; from E. Merck) with 1-butanol as a developer. Procedure for the Determination of Pyridyl and Pyrimidinyl Compounds. A 0.5-mL aliquot of the sample in methanol-water (9:l) was mixed successively with 0.25 mL of 3% methanolic DMBA and 0.25 mL of 60% methanolic DCC in a glass-stoppered test tube on a mixer. The mixture was warmed immediately at 45 "C for 40 min. After dilution with 2.0 mL of methanol the absorbance or fluorescence intensity was determined against a blank at the appropriate wavelength indicated in Table I.
RESULTS Because a preliminary test on the nitrogen aromatic heterocycles listed under the Materials and Reagents indicated that sulfadiazine afforded the strongest absorption intensity, the following studies were carried out with sulfadiazine as a model compound t o optimize the reaction conditions. Effect of Solvent and Water Content on the Reactions. In the previous study (9),the author used DCC and pyridine containing 15% water to produce glutaconaldehyde for the determination of malonic acid. Because the situation is not the same as that in the previous report where pyridine was used as a solvent, solvent content and water content for the present reactions were reinvestigated. Among the tested solvents that are water-miscible, methanol gave the largest absorbance and larger solubility to the reagents. Therefore, methanol was chosen as the solvent for the present reactions. On the other hand, the optimal amount of water required for the reactions was elucidated to be almost the same as that of the previous study (9) and hence 10% of water was added in the sample solution. Effect of the Concentrations of DCC and DMBA on the Reactions. The reagent solutions of DCC and DMBA were freshly and separately prepared in methanol since the mixed reagent solution was unstable on standing. The concentrations of DCC and DMBA solutions were set a t 60% and 3%, respectively, because the higher the concentrations of both solutions the larger the absorbance. Effect of Time and Temperature on the Reactions. Figure 1 is the time courses of the reactions at different temperatures, indicating that 45 "C was optimal for the reactions. Furthermore, the figure also indicates the reactions required more than 2 h to reach plateau a t 45 "C. However, in order to save time, 40 min was set despite a 10% decrease in absorbance.
60 " MINUTE
0;
"
120
"
Figure 1. Time courses of the reactions of sulfadiazine. Fifty nanomoles of sulfadiazine reacted with the standard procedure at 45 "C (0)and 5 1 OC (0).
Determination of Pyridyl and Pyrimidinyl Compounds. Solutions of 20 mM nitrogen aromatic heterocycles listed under the Materials and Reagents were prepared for the specificity test. Compounds that were positive to the present method are given with their absorption or fluorescence characteristics in Table I and their spectra in Figure 2. The table shows that pyridine, pyrimidine, %aminopyrimidine, sulfadiazine, sulfamerazine, thiamine hydrochloride, and chlorpheniramine maleate could be determined by the present method with high precision. Particularly, sulfadiazine showing the highest sensitivity and specificity could be easily determined in a complex mixture. Although the sensitivity was not as good as that of sulfadiazine, chlorpheniramine maleate, sulfamerazine, and thiamine hydrochloride were also specific and, hence, can be determined in their pharmaceutical preparations. Finally, it is also worthy of note that pyridine could be determined fluorometrically. DISCUSSION That carbodiimides acting as a cleaving agent on pyridine can afford glutaconaldehyde (I, eq 1) and then react with
I
II
DMBA to form a chromophore (11)has been chemically proved by Wilchek et al. (10). The reaction occurs through the electrophilic attack of the centric carbon of DCC on nitrogen where the negative pole of the resonating structure exists. Therefore, because pyrimidine and pyridazine have a similar negative pole a t the ring nitrogen, it is believed that they
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
1463
m/e
Figure 3. Mass spectra of the chromophore (A) and DMBA (B).
WAVELENGTH ( nm ) 1.2
B
L
L
we
346
( not observed)
0.9 Lu
1l
2
SI
0.6
9 0.3
-co
0.a
WAVELENGTH (nm)
Figure 2. Absorption spectra of the derivatives of ten selected compounds: (A) 1.0 pmol of (1) pyrimidine, (2) sulfamerazine, (3) 2aminopyrimidine,(4) chlorpheniramine maleate, and (5)pyridazine; (B) 0.1 pmol of (1) sulfadiazine and 5.0 pmol of (2) isoniazid, (3)niacin, (4) pyridine, and (5)thiamine hydrochloride were reacted following the standard procedure. behaved similarly as pyridine and gave similar chromophores in the present study. Moreover, because it is more polar, due to the presence of two electron-withdrawing nitrogens in a 1,3-relationship, pyrimidine showed more reactivity than pyridine. In contrast to the case of the six-membered aromatic heterocycles, pyrrole, imidazole, and pyrazole, having their positive pole of the dipolar resonance structures at nitrogen, were negative to the reactions. With respect to the amino derivatives of pyridine and pyrimidine, it was found that the amino group attached at the 2-position made the absorbance of pyrimidinyl derivatives decrease (e.g., 2-aminopyrimidine) or nullified the chromogenic reaction of pyridyl derivatives (e.g., 2-aminopyridine and 2-amino-6-methylpyridine). Although the phenomenon of the position 2 amino group may be partly a result of steric hindrance (19),tautomerism between the amino group and ring nitrogen (20)may also play an important role in the inhibition of the attack of DCC on the ring nitrogen. On the other hand, when the relation between the absorptivities and the structures of sulfadiazine, sulfamerazine, and sulfamethazine is compared, it can be found that the methyl group a t the 4 position of the pyrimidine ring led to the difficult production of chromophore and also the red shift of the A., Moreover, an additional methyl group, when introduced at the 6 position, made the formation of the aldehyde impossible and, consequently, abolished the chromogenic reaction of sulfamethazine. Following the same reason, sulfadimethoxine, sulfamethomidine
H
m/
*
;NCH, +CH=C, OH
m/e
99
71
Figure 4. Postulated fragmentation pattern of the chromophore.
and sulfisomidine gave no visible chromophore. From the above interpretation, it can be concluded that a cleaving or chromogenic reaction, similar to that of pyridine, might occur to pyrimidinyl derivatives. To prove both the supposition and the mechanism proposed by Wilchek et al. ( I O ) , a minute amount of the chromophore has been isolated from the reaction mixture by a lengthy and tedious procedure including silica gel column chromatography, reprecipitation from water, and finally HPLC. The chromophore appeared as red crystals with a melting point higher than 300 OC. It was soluble in water but not in sodium chloride saturated solution. The mass spectra of the chromophore and DMBA are shown in Figure 3. According to the mass spectrum of DMBA and the previous papers reported by Gilbert et al. and Skinner et al. (21,22),the postulated fragmentation pattern is given in Figure 4. The results indicate as expected that the chromophore was produced from the reaction of DMBA and malonaldehyde (111,a tautomer of 3-hydroxy-2-propenal; eq 2) which derived from the cleavage of the pyrimidine ring.
111
IV
To obtain further evidence, DMBA was reacted at 45 "C with malonaldehyde bis(dimethylaceta1) in the presence of hydrochloric acid. The chromophore resulting from the reaction had the same behavior on TLC plates and also the same absorption characteristics as that from sulfadiazine. Therefore, the reactions of sulfadiazine or other pyrimidinyl com-
1464
Anal. Chem. 1985, 57, 1464-1469
pounds with DCC and DMBA are proposed as eq 2.
(3) Williams, A.; Ibrahim, I. T. Cbem. Rev. 1981, 8 1 , 589-636. (4) Kasai, Y.; Tanimura, T.; Tamura, 2. Anal. Chem. 1975, 47, 34-37. ACKNOWLEDGMENT (5) Kasai, Y.; Tanimura, T.; Tamura, 2.; Ozawa, Y. Anal. Cbem. 1977, 49, 655-656. The author thanks Shu-Jen Chang and Chia-Li Wu, Tam(6) Chen, S.-C. J . Chromatogr. 1982, 238, 480-482. kang University, for the mass spectra measurements. (7) Chen, S.-C. Anal. Cbem. 1982, 5 4 , 2587-2590. (8) Chen, S.-C. Anal. Biocbem. 1983, 132, 272-275. Registry No. 11, 78902-42-8;111, 78902-50-8;DCC, 538-75-0; (9) Chen, S.-C. Anal. Biocbem. 1984, 140, 196-199. DMBA, 769-42-6;pyridine, 110-86-1;pyrimidine, 289-95-2;py(IO) Wilchek, M.; Miron, T.; Kohn, J. Anal. Biocbem. 1981, 114, 419-421. (11) Kohn, J.; Wilchek, M. Biocbem. Biopbys. Res. Commun. 1978, 84, ridazine, 289-80-5; 2-aminopyrimidine, 109-12-6; 2-amino-47-14. methylpyrimidine, 108-52-1; sulfadiazine,68-35-9; sulfamerazine, (12) Chaudhuri, D. K. Indian J . Med. Res. 1951, 39, 491-505. 127-79-7; thiamine HCl, 67-03-8; chlorpheniramine maleate, (13) Asmus, E.; Garschagen, H. 2.Anal. Chem. 1953, 139, 81-89. 113-92-8;isoniazid, 54-85-3;niacin, 59-67-6; quinoline, 91-22-5; (14) Anger, V.; Ofri, S. Talanta 1983, IO, 1302-1303. K. Sitzungsber. Naturforscb. Ges. Rostock 1916, 6 , 33; isoquinoline, 119-65-3;1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5- (15) Fujiwara, Cbem. Abstr. 1917, 1 1 , 3201. y1)-3-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylidene)methyl1(16) Friedman, P. J.; Cooper, J. R. Anal. Cbem. 1958, 30, 1674-1676. propene, 95798-81-5; 1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5- (17) Leibman, K. C.; Hindman, J. D. Anal. Cbem. 1964, 36, 348-351. (18) Uno, T.; Okumura, K.: Kuroda, Y. Chem. Pharm. Bull. 1982, 30, y1)-3-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylidene)-2-( 3-thia1876-1 879, zolyliummethyl)propenamine, 95798-82-6; [3,7-bis(1,3-di(19) Sasagi, T. "Heterocyclic Chemistry", 1st ed. (Japanese); Tokyo Kagamethyl-2,4,6-pyrimidinetrione-5-yl)heptyl] dimethylamine, ku Dojin: Tokyo, 1972; p 106. 95798-84-8; 1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-y1)-5-(di- (20) Barnes, R. A. "Pyridine and Its Derivatives, Part one", Interscience Publishers, Wiley: New York, 1960; pp 70-74. methyl-2,4,6-pyrimidinetrione-5-ylidene)-3-aminocarbamoyl-l,3J. N. T.; Millard, B. J.; Powell, J. W. J . Pharm. Pbarmac. 1970, pentadiene, 95841-20-6;54 1,3-dimethy1-2,4,6-pyrimidinetrione- (21) 2Gilbert, 2 , 897-901. 5-y1)-2-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-ylmethyl)penta- (22) Skinner, R. F.: Gallaher. E. G.; Predmore, D. B. Anal. Cbem. 1973, 45, 574-576. dienoic acid, 95798-86-0.
LITERATURE CITED (1) Khorana, H. G. Chem. Rev. 1953, 53, 145-166. (2) Mikolajczyk, M.; Kielbasinski, P. Tetrahedron 1981, 37, 233-264.
RECEIVED for review February 5 , 1985. Accepted February 26, 1985.
Reverse-Phase High-Performance Liquid Chromatography/Nuclear Magnetic Resonance Spectrometry Separations of Biomolecules with 1-1 Hard Pulse Solvent Suppression D. A. Laude, Jr., R. W.-K. Lee, and C. L. Wilkins*
Department of Chemistry, University of California, Riverside, California 92521
Recently developed solvent suppresslon methods that rely upon appllcatlon of rf excitation wlth zero spectral denslty at solvent resonances are demonstrated to provide slgnlflcant advantages for the reverse-phase LC/NMR experiment. I n particular, with mlnimal delays between scans and the abillty to suppress multiple solvent resonances, these suppresslon techniques are clearly superior to presaturation methods previously employed. I n the present work, parameters for the 1-1 hard pulse suppression technique are optimized for continuous-flow LC/NMR. Applicatlon of the method Is demonstrated wlth reverse-phase separations of amino acid, vltamln, and nucleoside mixtures.
Modern reverse-phase high-performance liquid chromatography (LC) has developed into the preeminent tool for biochemical analysis. Unfortunately, the coupling of reverse-phase methods with NMR detection is made significantly more difficult by solvent limitations; protonated HzO, acetonitrile, and methanol are not as readily substituted for as their normal phase solvent counterparts. Protonated solvents present two major difficulties for LC/NMR including the potential for spectral interference with the analyte, and constraints upon detection limits imposed by the dynamic
range limitations of the analog to digital converter (ADC). Attempts to solve these critical problems have included the use of larger ADCs (I),deuterated or halogenated solvents (2),or selective saturation solvent suppression pulse sequences ( I , 3 , 4 ) . Although all three approaches improve the dynamic range of LC/NMR, only the use of nonprotonated solvents effectively eliminates solvent spectral interferences. Especially for normal-phase methods, nonprotonated solvents such as Freon-113, carbon tetrachloride, and deuteriochloroform have been utilized. Although the application of deuterated solvents to reverse-phase separations on a preparative scale would be cost prohibitive, the development of analytical scale LC/NMR ( 1 , 5 )reduces solvent volumes with typical separations requiring 10 to 20 mL of solvent. At these levels, DzO becomes a viable solvent and several chromatographic applications (aqueous size exclusion and ion exchange) are amenable to LC/NMR analysis. Deuterated acetonitrile and methanol are several orders of magnitude more costly and, except for use as modifiers, are not feasible. The dynamic range of the ADC determines a ratio of the largest to smallest observable NMR signal equal to
R = 2b where b is the number of bits in the ADC. FT-NMR spectrometers are often equipped with 12-bit ADCs which limit
0003-2700/85/0357-1464$01.50/00 1985 American Chemical Society
