ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
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Fluorometric Assay of a-Methylene Carbonyl Compounds with N'-Methylnicotinamide Chloride Hiroshi Nakamura" and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo 1 13, Japan
Scheme I
A spectrophotofluorometric method has been developed for the determination of a-methylene carbonyl compounds by using N'-methylnicotinamide chloride (NMN). When reacted at 0 O C for 10 min with NMN in the presence of alkali and heated at 92 O C for 3 min after the addition of formic acid solution, only compounds having an a-methylene carbonyl group (-CH,-CO-) gave intense violet to green fluorescence. By measuring the fluorescence, as little as 5 pmol of acetophenone, 1-indanone and a-tetralon could be determined. The relative standard deviations of 1.00% and 1.61% are observed for the analyses of 1 X lo-' mol and 1 X lo-'' mol of acetophenone, respectively. The mechanism associated with the reaction is also discussed.
&-CARBINOL
0
H-ADDUCT
Spectrophotometric determination of a-methylene carbonyl compounds has been performed by using reactions of carbonyl groups or those of active methylene groups adjacent to carbonyl groups. Various carbonyl reagent such as salicyloylhydrazide ( I , 2 ) , 2-diphenylacetyl-1,3-indandione-lhydrazone (3-7) and dansyl hydrazine (8-20) have been proposed as a tool for the fluorometric assay; however, their applicability is often limited by the fact that the reagents themselves are fluorescent and their fluorescence characteristics are not too different from their reaction products. Kramer et al. (11) reported the insensitive fluorescent reaction using o-nitrobenzaldehyde for the detection of methyl ketones. On the other hand, there has scarcely been fluorometric assay based upon t h e reactivity of active methylenes except the procedure described by Bartos (12) using 1,2-naphthoquinone-4-sulfonic acid. T h e same reagent allows the fluorometric estimation of primary and secondary amines under the same condition (13). Various ketones such as acetone (14-17), methyl ethyl ketone (18, 19) and acetophenone (20) have been used as reagents for fluorometric determination of quarternary N'alkylpyridinium derivatives of nicotinamide such as N'methylnicotinamide, NAD+, NADP', and nicotinamide mononucleotide. T h e procedure involves the reaction of N'dkylpyridinium compounds with ketones in alkaline media (first step) and the successive heating with excess acids to produce fluorophores (second step). Huff (14)had tentatively identified the fluorophore produced from A'l-methylnicotinamide and acetone as 1,7-dimethyl-5-oxo-(l,s-dihydro-1,6naphthyridine) and proposed a probable pathway of the reaction. If this is correct, generally carbonyl compounds having an a-methylene carbonyl group (R-CH,-CO-R') seem to produce similar fluorescent products with the pyridinium compounds according to the following steps (Scheme I). However, the application of the fluorogenic reaction to the fluorometric assay of carbonyl compounds has not been reported. In the present investigation, N'-methylnicotinamide chloride (NMN, R"=CH3 in Scheme I) was proved to be an excellent reagent for some carbonyl compounds having an a-methylene carbonyl group, which led to the development of a sensitive and specific fluorometric determination of them. 0003-2700/78/0350-2047S0 1. O O / O
CYCLIZED &-ADDUCT ( hONFLUORESCENT)
F-UOROPHORE
EXPERIMENTAL Apparatus. The following were used: a Hitachi MPF-2A grating spectrophotofluoronieter equipped with a xenon lamp and I-cm quartz cells. a water bath circulator (model BT-35, Yamato Scientific, Tokyo), and a Toa HM-5 pH meter (Toa Denpa Kogyo, Tokyo, Japan). Materials. N'-Methylnicotinamide chloride (NMN) (Tokyo Kasei, Tokyo): formic acid ( 8 8 7 ~GR), ~ and sodium hydroxide pellets (Kanto Chemical, Tokyo) were used as received. Other chemicals and solvents used were of the highest purity commercially available. Preparation of Stock Solutions of Carbonyl Con~pounds. A stock solution of 10 mM of each carbonyl compound was prepared with distilled water whenever possible or ethanol. Each 10-pL aliquot of the 10 mM solutions was added with 9.99 mL of distilled water to make a 0.01 mM stock solution. Distilled water was used to prepare dilute solutions. Solutions of a-keto acids were prepared just before use. Recommended Assay Procedure. To 100 1 L of aqueous sample solution is added 100 pL of 6 M NaOH. The mixture is then mixed with 100 pL of 50 mM NMN in 10 M HC1 under vigorous mixing on a vortex type mixer. After 10 min or a certain time, the alkaline mixture is acidified by ,adding 1500 pL of 18.66 M formic acid. All the above procedures are performed in an ice bath (0 "C). The acidic solution is heated at 92 "C for 3 min and cooled to 0 "C in the ice bath. The fluorescence intensity is measured at respective excitation and emission wavelengths against a reagent blank containing no carbonyl compound. RESULTS Preliminary Examination of the Reaction of NMN with Carbonyl Compounds. In order to mark latently reactive compounds, a preliminary screening test was performed by using ca. 20 mg of materials. When test compounds were mixed with 0.1 mL of 1.5 M NaOH and mixed with 0.1 m L of 10 m M NMN in M HC1, intense violet, blue, or green colors of fluorescence were observed immediately under a long-wave UV light only when some carbonyl compounds were employed. A reagent blank gave faint bluish green fluorescence but it was almost lost by the addition of acids (e.g., 0.5 mL of 88% formic acid) to the alkaline reaction mixture, in contrast the fluorescence of the reactive compounds remained unchanged or intensified with slight changes
'P 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
I
0°C
l
30°C W
m 0 LL W
0
3
LL
I "
':I
Figure 1. Effect of NaOH concentration on the first step. 0.1 mL each of varying concentrations of NaOH, 0.01 mM a-methylene carbonyl M HCI were mixed in this order compound and 10 mM NMN in at a certain temperature and incubated for 10 min. The samples were added with 1.5 mL of 88% formic acid, heated at 92 "C for 3 min and cooled in an ice bath. The fluorescence was measured at optimal excitation and emission wavelengths (acetophenone, 378/430; cyclohexanone, 375/457; phenylacetaldehyde, 3741488, in nm). Values are corrected with a reagent blank
in colors of fluorescence. Brief heating of the acidic solutions caused marked increase in fluorescence intensity. T h e final fluorescence obtained by the heating was observed only with carbonyl compounds containing an a-methylene carbonyl group (-CH,-CO-) such as: methyl ketones (CH,-CO-), n-methylene ketones (-CH2-CO-), cyclic a-methylene ketones (-CH,-CO-), a-methylene aldehydes (-CH,-CHO), 1,3-diketones (-CO-CH,CO-), 1,4-diketones (-CO-CH,-CH,-CO-), a-keto acids (-CH2-CO-COOH), P-keto acid (-CO-CH,-COOH), y-keto acid (-CO-CH2-CH2-COOH), keto sugars, and keto steroids. Some acid esters (-CH2-COOR) and dialkyl malonates (CHJCOOR),) fluoresced in the test. As the exceptions, carboxylic acids, their derivatives, and those which are rigid in structure or have bulky substituents such as camphor, dimedone, isoketopinic acid and pinacolone yielded no or very weak fluorescence. Other carbonyl compounds having less than two hydrogen atoms a t a-carbon or having no a-carbon showed no or very weak fluorescence. Active methylenes such as pyrrole, indole, resorcinol, orcinol, and phloroglucinol behaved negatively. The present test was also negative with following classes of compounds: amines, polyols, steroids, alcohols, ethers, purines, pyrimidines, nitriles, thiols, disulfides, sulfides, sulfonic acids, and nitro compounds. Therefore, these results indicate that the a-methylene carbonyl group (--CH,-CO-) is essential for compounds to be reactive in the present fluorogenic reaction using NMN, though the steric factor should not be negligible in the final fluorescence intensity (see supplementary material). Some analytical factors have been examined below for the fluorometric determination of a-methylene carbonyl compounds by taking acetophenone as a main model. Effect of NaOH Concentration on the First Step of the Reaction. T h e rection of carbonyl compounds with N M N did not take place in acidic and neutral media. The formation of fluorescent products was affected by the NaOH concentration both a t 0 "C and 30 "C (Figure 1). In the cases of acetophenone and cyclohexanone, even 6 M NaOH was not sufficient to obtain maximal fluorescence. In contrast, amethylene aldehydes such as phenylacetaldehyde gave maximal fluorescence a t 0 "C with 0.5 M NaOH and stronger alkali reduced the final fluorescence a t 30 "C. The reaction a t higher temperature resulted in the increased fluorescence yield with cyclohexanone but led to the opposite result with acetophenone. As shown in Figure 2 , the formation of fluorescent products at 0 "C proceeded slowly in all cases with time depending on the NaOH concentration, while when reacted at 30 "C t h e fluorescence originated from acetophenone and phenylacetaldehyde (data not shown) maximized within 1 min and decreased rapidly with time. Such a de-
Figure 2. Development of fluorescence dependent on NaOH concentration and reaction time at 0 "C and 30 "C. Conditions are the same as in Figure 1
'
TEV?ERATLRE(~NC!
1
.
'5'C
'4 L NU E s
Figure 3. Effect of temperature on the first step. (a) Conditions are the same as in Figure 1 except that the incubations were performed by adding 0.1 mL of 6 M NaOH for 1 min at various temperatures. (b) An aliquot (0.1 mL) of 0.01 mM acetophenone was added with 0.1 mL each of 6 M NaOH and 10 mM NMN and incubated at various temperatures. After an appropriate time, the samples were added with 1.5 mL of 88% formic acid and similarly treated as described in Figure 1
creased fluorescence observed with acetophenone at 30 "C seemed to be primarily due to the degradation of the a-adduct. Effect of Temperature on the First Step. When reacted for 1 min with 6 M NaOH, reactions of carbonyl compounds with N M N were dependent on the temperature, showing maxima at around 15 "C with acetophenone and at 40 "C with cyclohexanone (Figure 3a). However, when the period of reaction of acetophenone with N M N was prolonged, the fluorescence decreased with time a t higher temperature than 5 "C (Figure 3b), indicating lability of the a-adduct of acetophenone t o temperature. Similar phenomenon was also observed with propiophenone. In contrast, apparent degradation of the intermediate was not observed up to 40 "C in the cases of cyclohexanone, acetone, and pinacolone. Effect of NMN Concentration on the First Step. The fluorescence yields obtained by reacting acetophenone a t 0 "C and cyclohexanone a t 40 "C with varying concentration of N M N are illustrated in Figure 4. In both cases, generally the fluorescence increased with increasing concentration of NMN. Judging from the signal to noise ( S / N ) ratio, the optimal N M N concentrations were determined as 50 mM for acetophenone and 30 mM for cyclohexanone, respectively. T h e condition determined for cyclohexanone (reaction a t 40 "C with 30 m M N M N ) necessarily surpassed that for acetophenone (reaction a t 0 "C with 50 m M N M N ) in the production of fluorophore from cyclohexanone. The S / N ratio a t 40 "C was less than half of that a t 0 "C because the fluorescence increased markedly by heating in the blank. Therefore, the condition determined for acetophenone was employed for the first step in the following experiments.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
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w
0 Z W
0 U-I
W
a
0
3 LL
hYNI n Y :
Figure 4. Effect of NMN concentration on the first step. 0.1 mL of 0.01 mM a-methylene carbonyl compound was added with 0.1 mL each M of 6 M NaOH and various concentrations of NMN dissolved in HCI and incubated at 0 OC for acetophenone or 40 OC for cyclohexanone for a given time. The broken lines indicate the fluorescence of blanks. Other conditions are the same as in Figure 1
I
I
0
1
I
1
-
2 3 4 FINAL pti
5
Figure 6. Effect of acids on the second step. A 0.1-mL aliquot of 0.01 mM acetophenone (1 nmol) was treated by the procedure established in the text except that 18.66 M formic acid was replaced by various acids with varying concentrations
Pli Figure 5. Dependence on pH of fluorescence intensity of the fluorophore from acetophenone. A 0.2-mL aliquot of the neutralized reaction mixture containing the fluorophore from acetophenone, equivalent to 1 nmol, was mixed with 2 mL of 1 M HCI, 1 M CH,COONa-1 M HCI buffer (pH 0.5 M KCI)-0.5 M Na,CO, buffer (pH 0.65-5.2), (0.5 M H,BO, 7.4-11.0) or 0.1 M NaOH and the fluorescence intensity corrected for the blank was plotted vs. the actual pH values measured
+
Effect of pH on the Second Step of the Reaction. When the reaction mixture of the first step was heated with varying p H of buffers a t 92 "Cfor 3 min and adjusted final p H to ca. 0.5 with formic acid, the maximal fluorescence was observed at, around p H 3, which indicates t h e p H dependence of the conversion of t h e a-adduct to the fluorophore. Effect of pH on the Fluorescence Intensity of Fluorophore. When the fluorophore derived from acetophenone was investigated for fluorescence intensity in buffers, three types of fluorescence characteristics were observed (Figure 5). T h e main fluorescence was in t h e acidic region with excitation maximum (Ex) a t 378 nm and emission maximum (Em) a t 430 n m (violet fluorescence), and strongest at p H 0.5 and 2. T h e second was light blue fluorescence with Ex 378 n m and E m 475 n m being maximal aroung pH 6. At the alkaline region above p H 10, greenish fluorescence with E x 414 n m and E m 482 n m was predominant. Assuming the acetophenone fluorophore was single, two apparent pK, values: 4.5 and 8.7, were obtained from the curves. The fluorescence should be measured in acidic media for analytical purpose because both the intensity and the S / N ratio in acidic region were greater than those in neutral and alkaline regions. Selection of Acid Both for the Second Step and for the Solvent for Fluorescence Measurement. Although it was
proved above that the conversion o f the intermediate of acetophenone to its fluorophore was maximal near p H 3 and the fluorophore fluoresced most intensely between p H 0.5 and pH 2, it was not practical to employ the above conditions in view of reproducibility, simplicity of the procedure, and probable contamination with reactive carbonyl compounds in buffers. T o overcome these shortcomings, various acids in place of buffers were examined. There were optimal concentrations for every acid used in the second step. T h e conversion of the intermediate into the fluorophore was apparently most effective when it was performed a t p H 0-1 except for the cases with acetic and propionic acids (Figure 6). All of the acids used gave a product with identical fluorescence properties. T h e fluorescence was stable a t 0 "C for 48 h in formic, nitric, phosphoric, hydrochloric, and sulfuric acids while it decreased in acetic and propionic acids. These results indicated that formic acid was the choice of solvent in regard to the fluorescence intensity and stability. When the fluorescence intensity induced by formic acid was compared with that obtained by a procedure involving double adjustments of p H (3.0 and 0.5), both gave almost the same intensity. As an acid source for the second step of the reaction, formic acid was superior to the combination of buffers because of lower blank. Conversion of the Intermediate to Fluorophore with Formic Acid. T h e formation of acetophenone fluorophore in formic acid was slow a t 0 "C. However, complete conversion of the intermediate into the fluorophcre was achieved within 5 min a t 50 "C and a few minutes a t 92 "C. Allowing the reaction mixture of the first step to stand a t 0 "C for 30 min in the formic acid solution did not cause any side-reaction except the gradual transformation to the fluorophore, which is of convenience in procedure because the final fluorescence is independent of the time between the addition of formic acid and the beginning of the heat treatment. Determination of a-Methylene Carbonyl Compounds. Based on the above findings, a procedure was established based on the conditions for acetophenone. When analyzed by t h e procedure, as little as 5 pmol of acetophenone, 1indanone, and a-tetralon could be determined. T h e working curves were linear a t least up to 1 nmol. T h e precision of the
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 14, DECEMBER 1978
Table I. Fluorescence Properties of Various Carbonyl Compounds in the Present Procedure optimal wave- relative length fluorexcita- emiss- escence tion ion at optimaxi- maximal mum, mum, wavecompound nma .ma length acetophenone 378 430 1o o b 400 445 86 1-indanone a-tetralon 45 413 458 propiophenone 377 450 43 370 439 10 Na a-ketoglutarate 362 420 7 Na pyruvate 7 phenylace taldehy de 374 488 cyclopentanone 6 380 460 375 457 5 cyclohexanone 365 478 5 oxalacetic acidC 4 methyl ethyl ketone 370 457 p-phenylpropionaldehyde 1 380 435 propionaldehyde 367 451 0. 7 0.4 n-butyraldehyde 366 449 acetone 356 432 0.4 testosterone 0.4 418 471 progesterone 419 472 0.3 377 457 0.1 androsterone a The excitation and emission maxima are not Acetophenone is arbitrarily taken as 100 corrected. Cis form. method at two levels was evaluated by analyzing standard acetophenone solution ten times. T h e standard deviations mol for 1 x were 0.010X mol of acetophenone and 0.016 X lo-'' mol for 1 X mol of acetophenone. Table I summarizes the fluorescence characteristics and relative fluorescence intensity of some positive compounds. Q u a l i t a t i v e Tests on Reaction Mechanism. When the test tube containing 0.1 mL each of 50 m M N M N in M HC1, 100 m M acetophenone, and 6 M NaOH was tightly covered with filter paper impregnated with Nessler solution (21)and incubated a t 0 "C for 15 min, no detectable amount of ammonia was found. However, after heating the tube at 95 "C for a few minutes, the paper was stained brown with almost the same degree of intensity as the case of the blank containing no acetophenone. T h e result indicated that the intermediate produced in the first step contained an amide group. The alkaline reaction mixture of the first step similarly prepared by using excess amount of N M N (200 mM) to acetophenone (50 mM) showed a positive response to the test for active methylenes using 2,4-dinitrochlorobenzene (22), giving a reddish brown color with the same degree of intensity as t h a t of the corresponding amount of acetophenone. T h e result indicated t h a t the active methylene group of acetophenone remained in the structure of the intermediate. Acidification of the reaction mixture of the first step, which was prepared from 100 m M each of N M N and acetophenone, by the addition of 1.5 m L of 18.66 M formic acid followed by incubation at 0 "C for 5 min changed the intermediate into a nonamide compound which did not liberate ammonia when heated a t 95 "C and p H 13 for 10 min.
DISCUSSION By reacting with N M N in the presence of alkali and by successive heating in acid, only a-methylene carbonyl compounds having a general formula R-CH,-CO-R gave intense fluorescence. The finding obtained in the present investigation supports our initial assumption that the fluorescent reaction between N M N and acetone presented by Huff (14) would be applicable to other a-methylene carbonyl compounds. According to Huff (14), the a-adduct of acetone first undergoes
Scheme I1 A
h20
' R CH3 DEPY3RCGENATE3 ci -ADDUCT
Scheme I11
cyclization and then dehydrogenation by losing two hydrogens between C8 and C9 to form the fluorophore. However, as to the route from the wadduct to the fluorophore, an alternative which involves dehydrogenation of a-adduct and successive dehydration may be considered (Scheme 11). Huff (14)did not establish whether the cyclized cu-adduct formed in alkaline media or in acidified ones. Therefore, the possibility of three intermediates exists, the a-adduct, cyclized cu-adduct, and dehydrogenated a-adduct, in the alkaline reaction mixture. T h e acidification of the alkaline solution did not result in the rapid development of fluorescence but it required additional heating to obtain maximal fluorescence. Therefore, it is clear that the final fluorophore does not form in the first step. Qualitative tests reveal that the intermediate (R = H, in Scheme 11) formed from acetophenone and N M N contain both an amide and an active methylene group. T h e findings deny the presence of cyclized or dehydrogenated a-adducts but suggest the a-adduct is the predominant intermediate in the first step. The a-adduct is rapidly converted by acid a t 0 "C into the second intermediate having no amide group. Therefore, the formation of the fluorophore from the a-adduct is considered to take place via the cyclized a-adduct as the proximate fluorophore, and not via the dehydrogenated a-adduct. The dehydrogenation of the cyclized a-adduct was accelerated by the addition of excess amount of N M N to the acidified solution when the a-adduct was prepared with limited amounts of NMN. Therefore, N M N which is a sole organic proton acceptor in the assay system may serve in the dehydrogenation of the cyclized 0-adduct. T h e overall pathways of the present reactions are shown in Scheme 111, though there remains a possibility of another structure for the fluorophore derived from the y-adduct a t C4 of N M N . T h e a-adducts of acetophenone and propiophenone were temperature-sensitive in alkaline media while those of cy-
ANALYTICAL CHEMISTRY, VOL. 50,
clohexanone, acetone, and pinacolone seemed to be apparently stable under the condition. Although the acid amide group of the a-adduct was stable a t 0 "C in the alkaline reaction mixture as stated above, alkaline hydrolysis of the amide group was observed a t higher temperature generating ammonia. +
CH3
NaOH-
+NH3
R
In all compounds tested, the final fluorescence was in inverse proportion to the amounts of ammonia liberated which was semiquantitatively determined with the Nessler reagent-filter paper. The greenish fluorescence observed with time in the alkaline reaction mixture seems to be due to a-carbinol of N M N (14) which is weakly fluorescent and almost diminished by heating in acid to liberate NMN. On the other hand, prolonged incubation or heating of the alkaline reaction mixture caused strong bluish fluorescence which cannot be quenched by acid any more. This irreversible fluorescence is considered due to side reaction of the aldehyde produced by opening of the pyridine ring of a-carbinol of N M N between N1 and C2. T h e high specificity of the present reaction is due to simultaneous requirement of two different functional groups, active methylene and adjoining carbonyl groups, for the formation of fluorophores. Therefore, indole and pyrrole, which can be detected with colorimetric reagents for active methylenes such as sodium nitroprusside (23, 24). 1,2naphthoquinone-4-sulfonic acid (25, 26) and p-dimethylaminobenzaldehyde (25), are not detectable in the present fluorescent reaction. Moreover, some other typical active methylenes such as creatinine, resorcinol, orcinol, and phloroglucinol which may produce a cyclic a-methylene carbonyl group and show positive response to the colorimetric reagents or some cyclic a-methylene carbonyls such as dimedone, camphor, and isoketopinic acid gave no fluorescence, probably because of t h e steric hindrance around the amethylene carbonyl groups. Of the expected positive compounds 1,3-diketones were nonfluorescent or weakly fluorescent in the present reaction. This may be due to the isomerization (27) of the compounds into stable enol forms. T h e application of N M N as a fluorogenic labeling reagent for carbonyl compounds in high performance liquid chromatography has been successfully performed with both
NO. 14, DECEMBER 1978
2051
pre-column and post-column derivatization methods (28). Supplementary Material Available: A table (6 pages) containing fluorescence data for various classes of carbonyl compounds after reaction with NMN followed by heating in acid will appear following these pages in the microfilm edition of this volume of the journal. Photocopies of the supplementary material from this paper or microfiche (105 X 148 mm, 24X reduction, negatives) may be obtained from Business Operations, Books and Journals Division, Americal Chemical Society, 1155 16th St., N.W., Washington, D.C. 20036. Full bibliographic citation (journal, title of article, author) and prepayment, check or money order for $5.50 for photocopy ($7.00 foreign) or $3.00 for microfiche (S4.00 foreign) are required.
LITERATURE CITED B. Camber, Nature (London), 174, 1107 (1954). P. S. Chen. Jr., Anal. Chem.. 31, 296 (1959). R. Brandt and N. D. Cheronis, Microchem. J . , 5 , 110 (1961). R. Brandt, J. C. Kouines, and N. D. Cheronis. Microchem. J . , 6, 519 (1962). R. Brandt and N. D. Cheronis, Microchin?.Acta. 3, 467 (1963). R. Brandt, J. C. Kouines, and N. D. Cheronis, J . Chromatogr., 12, 380 (1963). D. J. Pietrzyk and E. P. Chan, Anal. Chem., 42, 37 (1970). V. Graef, Z . Klin. Chem. Klin. Biochem., 8, 320 (1970). R. Chayen, R. Dvir, S. Gould, and A. Harell, Isr. J . Chem., 8, 157p (1970). R. Chayen, R. Dvir, S . Gould, and A. Harell, Anal. Blochem., 42, 283 (1971). D. N. Kramer, L. U. Tolentino, and E. B. Ha'ckley,Anal. Chem., 44, 2243 ( 1972). J. Bartos, Ann. Pharm. Fr., 27, 691 (1969) M. Pesez and J. Bartos, Ann. Pharm. F r . , 27, 161 (1969). J. W. Huff, J . Blol. Chem., 167, 151 (1947). J. W. Huff and W. A. Perlzweig, J . Biol. Chem., 167, 157 (1947). N. Levitas, J. Robinson, F. Rosen, J. W. Huff, and W. A Perlzweig, J . Biol. Chem., 167, 169 (1947). J. Robinson, N. Levitas, F. Rosen. and W. A. Perlzweig, J . Biol. Chem., 170, 653 (1947). K. J. Carpenter and E. Kodicek, Biochem. J . , 46, 421 (1950). H. B. Burch, C. A. Storvick, R. L. Bicknell, H. C. Kung, L. G. Alejo, W. A. Everhart, 0. H. Lowry, C. G. King, and 0.A. Bessey, J . Biol. Chem., 212, 897 (1955). B. R. Chrk, R. M. Halpern, and R. A. Smith, Anal. Biochem.,68, 54 (1975). M. J. Johnson, J . Biol. Chem., 137, 575 (1941). M. Ishidate and T. Sakaguchi, J . Pharm. SOC.Jpn., 70, 444 (1950). E. Legal, Jahresber. forfschr. Chem., 1648 (1883). B. Bitto, Ann. Chem., 267, 372 (1883). P. Ehrlich and C. A. Herter, Z . Physiol. Chem., 41, 329 (1904). F. Feigl and 0. Frehden, Microchemie, 16, 79 (1934). M. Ishidate and Y. Yamane. Chem. pharm. Bull. (Tokyo), 8, 1116 (1960). H. Nakamura and 2. Tamura, J , Chromatogr., in press.
RECEIVED for review July 5, 1978. Accepted September 5, 1978. Presented in part a t the 98th Annual Meeting of the Pharmaceutical Society of Japan, Okayama, April 3-5,1978. T h e authors are grateful to the Pharmacological Research Foundation, for partial support of this work.
FIuorimet ric Determination of Pyridine after Hydroxylation with the Hamilton Hydroxylation System Michelle P. Wong and Kenneth A. Connors" School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
Pyridine and some substituted pyridines were subjected to the Hamilton hydroxylation system, consisting of hydrogen peroxide, catechol, and ferric ion. The resulting hydroxylated pyridines were measured fluorimetrically. The method is applicable to lo-' to M aqueous solutions of several pyridine compounds; linear fluorescence concentration plots were obtained.
T h e ability of the Hamilton hydroxylation system (1-3), 0003-2700/78/0350-2051$01.00/0
consisting of catechol, ferric perchlorate, and hydrogen peroxide, to introduce a hydroxy group on an aromatic ring is potentially useful analytically. This hydroxylation system was previously used ( 4 , 5 ) as a reagent for the analysis of aqueous solutions of aromatic compounds by their conversion t o phenols, which were subsequently determined colorimetrically. The purpose of the present research was to carry out the hydroxylation step and then to measure the resulting hydroxylated product fluorimetrically, because many hydroxy compounds fluoresce strongly. Attention was restricted to @ 1978 American Chemical Society
