- Email: [email protected]
Some specific fluorogenic reactions in pharmaceutical and environmental applications
trends in analytical chemistry, vol. 1, np. II, 1982
254
.
Some specific fluorogenic reactions in pharmaceutical and environmental applications Nonfluorescent compounds which cannot be treated with standard functional group derivatizing agents can sometimes be incorporated into specific fluorescent structures which are useful for analytical purposes. These reactions are intrinsically more selective because they are designed for specific applications and do not require a separation of excess fluorescent reagent.
David W. Fink Rahway,NJ, U.S.A. The usefulness of the derivatization of functional groups to generate fluorophores has been demonstrated by numerous analytical applications over the last 30 years. However, there are many cases in analytical chemistry in which the standard nonspecific functional group derivatizing reagents may not be applicable. Some of the more recently reported derivatizing agents are presented in Table I. An index such as this can often be used to select a suitable reaction. However, if the analyte does not contain any of these functional groups and if fluorescence measurement is judged to be a viable basis for the analysis, then it may be appropriate to design a specijk fluorogenic reaction based upon other structural features. This review describes some examples of specific solutions to such analytical problems which are probably more common, and certainly more challenging, than conventional functional group derivatization. The analytical methods used are based on standard fluorescence measurement as well as on fluorescence detection in liquid chromatography’. The approach to deriving a fluorophore from a nonluminescent substrate can be based upon a correlation of structural and photophysical properties*-+
following evaluation of the structure of the analyte. The principles involved in the following examples include the formation of conjugated polyene derivatives from saturated analytes, the extension of existing conjugation, and the introduction of molecular rigidity, which results in co-planarity of rings. These structural modifications are achieved via analytical reactions such as ring closing, dehydration, and other routes to aromatization. In some cases, the extension of these specific analytical reactions to a general compound class can be achieved after full characterization of the reaction mechanism, substrate properties, and reaction conditions. In addition, none of the reactions cited involves a fluorescent reagent (an excess ofwhich must be separated from the analytical fluorophore). The examples quoted are of nonfluorescent reagents which fluoresce only as a result of the analytical reaction. Compare this with some of the reagents in Table I, which are fluorescent ‘tags’ (e.g., dansyl derivatives, coumarins). The same fluorophore is present in both the reactant and product; thus, after the reaction, the excess reagent must be removed because it has the same fluorescence spectrum as the product.
Ring-closing reactions As pointed out by Parke+, the high fluorescence efficiency of many aromatic molecules (e.g., the xanthene dye fluorescein*psJ), often attributed to structural rigidity, is actually due to the conjugation of ring systems made easier by co-planarity. At any rate, ring-closing remains an especially useful technique for synthesizing luminescent compounds. There are many thiazole-containing compounds used in agricultural and pharmaceutical formulations. Studies of these agents in a variety of matrices (such as low concentration level formulations), bioavailability studies, and environmental samples often involve the determination of concentrations low enough to require the sensitivity of fluorescence analysis. However, thiazole per se is nonfluorescent6. A fluorometric method has been developed7 which is based upon a classical reaction originally used for semiconductor materials by Parker and Reess. In this treatment (Scheme I), thiazole is treated with zinc in 0 1982 Elsevier Scientific Publishing Company
255
trends in analytical chemistry, vol. 1, no. II, 1982
TABLE
I. Some fluorogenic
functional
agents Reference
Analyte
Reagent
group derivatizing
Fluorescamine Dansyl chloride Bansyl chloride I-Naphthalenemethylamine 1,2_Diaminonaphthalene I-Pyrenealdehyde 2-Fluorenealdehyde 4-Bromo-7-methoxycoumarin
primary amines amines, phenols amines isocyanates aldehydes primary amines
Weigele, M. et al. (1972) J. Am. Chem. Sot. 94,5927 Seiler, N. (1971) J. Chromatogr. 63,97 Seiler, N. et a(. (1973) J. Chromatogr. 84,95 Levine, S. P. et al. (1979) Anal. Chem. 51, 1106 Ohkura, Y. and Zaitsu, K. (1974) Talanta 2 1,547 Hwang, T. K. et al. (1978) Anal. Chim. Acta 99,305
fatty acids
Hydroxylation 2,2’-Dithiobis( I-aminonaphthalene) N-Methylnicotinamide Dimethoxyanthracene sulfonate
pyridines aromatic aldehydes a-CHs-carbonyls amines
Diinges, W. (1977) Anal. Chem. 49,442; Lam, S. and Grushka, E. (1978) J. Chromatogr. 158,207; Zelenski, S. G. and Huber, J. W., III (1978) Chromatographia 11,645 Wong, M. P. and Connors, K. A. (1978) Anal. Chem. 50,205l Ohkura, Y. et al. (1978) Anal. Chim. Acta 99,3 17 Nakamura, H. and Tamura, Z. (1978) Anal. Chem. 50,2047 Westerlund, D. and Borg, K. 0. (1976) Anal. Chim. Acta 67.89
an acidic solution to yield an intermediate - H&S, an alkyl sulfide or mercaptan. This intermediate then condenses with two moles ofp-phenylenediamine and, in a ring-closing reaction, the central ring is closed using ferric ion as the oxidizing agent to yield thionine for fluorescence measurementa. Thionine fluoresces with a red emission which has a maximum at 620 nm. A survey of model compounds was conducted to evaluate this technique as a general analytical method for this class of compounds. Saturation of the thiazole ring (thiazolidine) or removal of the nitrogen (thiophene) completely inhibits the reaction, as does fusing of the ring to form benzothiazole. In general, mono- or di-substitution at the 2- and 4-positions results in fluorescence which is of approximately equimolar (+25%) intensity with the parent unsubstituted thiazole. One specific example of an application of this reaction to a thiazole-containing compound is the peptide antibiotic thiopeptin, which is administered in Scheme I.
P Zn" H+
’
L- 1 -St
H2N
NH2
XY NH2
animal feed as a growth promoter and to improve feed efficiency.. It is used in the concentration range 10-20 g of drug per ton of feed. Thiopeptin has four thiazole rings, each similarly bonded to the C, H, N skeleton at both the 2- and 4-positionsta. Because the derivatization reaction (Scheme I) is not stoichiometric, the effects of zinc reducing agent, acidity of the reaction of analytical solution, heating time, and amount reagent are optimized. In the feed sample which is analyzed, thiopeptin is only a trace concentration level ingredient in a poorly defined matrix, requiring an efficient isolation scheme to separate the drug from any other thiazole rings. In addition to thiazole-containing extraneous material, a sulfur-containing protein can produce significant interference. This is important because these reactions have been used for the calorimetric determination of the mercaptan amino acid cysteinetr. Hence, the drug is extracted into ethyl acetate and introduced onto a gravity-fed adsorption column. After removing interferences, the drug is eluted with acetic acid in methyl alcohol, the eluate reduced to dryness, and the sample taken up in the HCl reaction medium. After the fluorogenic reaction, the fluorophore is partitioned into butanol for fluorescence measurement. This procedure yields an analytical method with an accuracy of 5% mean relative error over the concentration range 5-20 ppm thiopeptin in medicated feed. The drug amprolium is a quaternary compound which is structurally related to thiamine and which functions by a reversible thiamine inhibition mechanisml2. The fluorometric method applied to this drug is based upon the conversion of amprolium (I) to a fluorophore (II) by a reaction analogous to the oxidative production of thiochrome from thiamine, which was studied by Ohnesorge and Rogersrs. This is
-
“‘“‘$3Jc;Q
trends in analytical chemistry, vol. I, nb. II, 1982
256
another example of a ring-closing reactionl4. The reaction is sufficiently sensitive to be used for the analysis of plasma samples, and it is sufficiently rugged to have been adopted by the Association of Official Analytical Chemistsl5. Feed samples containing amprolium in the presence of a wide selection of other feed drugs have yielded analytical results ranging from 95% to 105% ofthe known medicated concentration of amprolium in the feed“+.
Dehydration reactions Compounds containing a hydroxyl group and a proton which are sterically positioned to allow facile elimination of water can often produce delocalized electronic systems. For instance, the avermectins are important new pharmaceutical products because they are a family of new anthelmintic compounds with a very .broad spectrum. The structures of these drugs have recently been described*6,17. They are fermentation products which exhibit a single electronic absorption band at h,,, 244 nm with shoulders on both the high- and low-energy sides. Their methanolic solutions are transparent at h > 310 nm and they are not fluorescent. Upon treatment with acetic anhydride in pyridine, two low-energy U.V. absorption bands appear and these are accompanied by the appearance of a visible fluorescence with a peak at 475 nm. The dihydrocyclohexene ring on this molecule (III) is fused to form a tetrahydrofuran ring and contains two hydroxyls with protons bonded trans to each. Heating with acetic anhydride in pyridine causes the elimination of two moles of water, producing an aromatic ring in conjugation with a diene system which is responsible for the fluorophore (IV) 18. The mechanism of this analytical reaction involves acetylation of the hydroxyl groups prior to dehydration. Recent work by Conners and co-workers has identified several nucleophilic catalysts for acylation by acetic anhydride which are superior to pyridine. These include 4-dimethylaminopyridine and some N-alkylated imidazoles’9-22. However, pyridine is retained in this analytical method because of the greater number of fluorescent interferences generated from endogenous plasma components when these more efficient catalysts were used. The derivative IV is the basis of a sensitive HPLC analytical method which uses fluorescence detection after preliminary sample isolationls. It has been applied to plasma samples obtained from animals dosed with the drug and which contain as little as 0.2 ng avermectin per milliliter of
PT
P
plasma in a 5-mL sample. A precision of 8% relative standard deviation was obtained at the 200-ng drug level and an accuracy of 5% mean relative error was achieved in a concentration range approaching 50 ppb of drug. The pharmaceutical applications of the method include the determination of pharmacokinetic parameters and comparisons of bioequivalence of experimental formulations. It has been known for many year&24 that oxytetracycline (V) can be heated in acidic solution to yield the more fluorescent anhydro-oxytetracycline (VI). This is a dehydration reaction. In alkaline solution, anhydro-oxytetracycline exhibits a fluorescence emission which is much more intense than that of the parent oxytetracycline. In practice a trace amount of thiopropionic acid is usually added to the analytical solution to retard oxidation of anhydro-oxytetracycline to nonfluorescent product+. Fig. 1 shows the fluorescence spectra obtained from drug-free bovine plasma and from plasma samples that have been supplemented with oxytetracycline and
9.1 ppm
3.6
porn
0.73 ppm
Blank Plasma
500
Wavelength
550
600
(nm)
Fig. 1. Fluorescence spectrum of the anhydro-oxytetracycline oxytetracvcline subblemented into drw- free bovine blasma.
derivative of
trends in analytical chemistry, vol. 1, no. 11,1982
treated to form the anhydro-oxytetracycline derivative. The concentration range is O-9 parts-per-million oxytetracycline. This fluorophore exhibits a fluorescence spectrum with a peak at 500 nm when excited at 390nm. The procedure has been used to evaluate a sustained-release pharmaceutical formulation of oxytetracycline. Fig. 2 presents the bioavailability results obtained after the drug was administered to cattle by intramuscular injection. The analyses resulting from the injection of a standard solution of oxytetracycline as a control in three daily doses exhibit three distinct peaks following each dose of the normal solution. The data obtained from the experimental microencapsulated oxytetracycline formulation after one dose clearly demonstrate sustained-release properties,
257
6
2 1 X
0.6
i
I-
20
Aromatization of an aliphatic analyte Aliphatic compounds can require major structural modification to yield fluorescent derivatives. Carboxymethyloxysuccinic acid (VII), which has been characterized as a complexing agent25, may find its way into environmental waters at low ppb concentration levels through its proposed use in synthetic detergents. Most environmental applications of fluorometric methods to air pollution and water pollution problems have been for polynuclear aromatic hydrocarbons, ofwhich many are suspected carcinogens. However, these are intrinsically fluorescent compounds, whereas VII is aliphatic and nonfluorescent. Although VII can be treated with any ofa number of carboxyl group derivatizing agents, a more selective method has been developed which involves incorporating its carbon atom framework in the fluorophore. This is another example of a ring-closing reaction, and is based on an historical spot test for malic acid26. In this method, the compound (following decarboxylation) is condensed with resorcinol, which adds to the ortho,paru positions of the two hydroxyls, eliminating two moles of water under dehydrating conditions with concentrated sulfuric acid to furnish 7-hydroxycoumarin (umbelliferone) (VIII) as the analytical fluorophore (Scheme II). Only those carboxylic acids with the basic structural features of malic acid interfere in this method. Because none of the acids (acetic, succinic, Scheme II.
40
60
80
100
Time (hr) Fig. 2. Pharmacokinetic application of fluorophore of Fig, I. Oxytetracycline concentration in plasma obtained from cattle following intramuscular injection of oxytetracycline formulations. (0) - Three (da+) injections of a control solution of ox~tetra~~line. (x) - One injection of a solutionformu~ated with microencapsutated o.+etracycline.
oxydiacetic), nor any of the aromatic carboxylic acids can form umbelliferone, they need not be separated from the environmental sample. With this fluorophore, concentrations as low as 0.5 parts-per-billion in a l-ml water sample can be determined. This coumarin fluorophore has also been used to make fluorescent derivatives of fatty acids for HPLC analysis; fluorometric coumarin substrates have been used in enzyme determinations by rate methods; and this compound is also used in dye lasers. The calibration curve is linear over a wide dynamic range (at least three decades of concentration); over the lo-6 to 10-7 M concentration range, the standard deviations from five replicate reactions, each at five different concentrations averaged cu. 9% relative to the mean fluorescence signal. This is a reasonable precision from an analytical reaction which is run as a melt. However, the fluorophore is unstable in the anionic form; the lactone ring splits in alkaline solution to yield its cinnamic acid product (IX). Umbelliferone, in addition, is photolabile, a process which is acceIerated under the excitation irradiation received in the fluorometer. Because this photoproduct fluoresces at a longer wavelength than the analytical fluorophore (500 nm vs 460 nm under the same excitation wavelength), its appearance can be monitored over a range of hydroxide ion concentrations*‘. The implication for the analytical method, however, is that suitable
AC” CH
“O
CH=CH--C&H
m -H,O
120
Ig:
7-OH-~ou~afin
2,4-diOH-Cinnamic
(Umbefliferonef
Aad
X exate :
370 nm
X excite:
370 nm
Xemit:
460 nm
Xemct.
500 nm
trends in analytical chemistry, vol. I, no. II, I982
258
conditions at near neutral pH can be found for thus allowing measurement analytical purposes, within a short enough time to avoid fluorophore degradation.
Conjugation of ring systems Thiabendazole (2-(4’-thiazolyl)benzimidazole) (X) is composed of a benzimidazole ring (XI) and a thiazole ring (XII) each bonded (Y to a nitrogen heteroatomz*. The use of thiabendazole formulations as agricultural fungicides has been approved for an ever-increasing variety of crops, including citrus fruits, bananas, tobacco, potatoes, soybeans, and sugar beets. This compound is of considerable environmental importance because of residues found in crops. For these residue studies, the analyte *of interest is the intact parent thiabendazole molecule (X) in the presence of its possible degradation products - the two stable heteroaromatic rings benzimidazole (XI) and thiazole (XII). Fig. 3 presents the U.V. spectra of thiabendazole (X) benzimidazole (XI), and thiazole (XII) in 0.1~ HCl. Thiabendazole is the only compound of these three which absorbs at h > 290 nm; both thiazole and benzimidazole are transparent at this wavelength. Hence, the absorption band at h,,, 302 nm in the figure reflects an electronic transition of a molecular orbital delocalized across both heteroaromatic rings, and is thus a characteristic physical property of intact thiabendazole. As a consequence, thiabendazole is intrinsically intensely fluorescent with an emission peak at 370 nm. Thiazole is not fluorescent and benzimidazole emits with a quantum yield of only 0.0629. Although this example is not a derivatization reaction, it is nevertheless relevant since it represents the formation of a low-energy U.V. fluorescent product from two nonfluorescent (in the near-u.v.) moieties. Direct measurement of fluorescence intensity under excitation at 300 nm, therefore, provides a sensitive
4
0.80 t\
@J$)-Q@j$)EQ N
X
XI
XII
assay which is specific for thiabendazole and which is widely used in agricultural applications for sensitive determinations of residues of the intact moleculeso.
Acknowledgement The author is indebted to John W. Tolan for collaborating on three of the derivatization projects cited in this review.
References Johnson,
E., Abu-Shumays,
A. and Abbott,
S. R. (1977),
J. Chromatogr. 107, 134
Wehry, E. L. (1967), in Fluorescence. Theory, Instrumentation, and Practice (G. G. Guilbault, ed.), Ch. 2, Marcel Dekker, New York Wehry, E. L. and Rogers, L. B. (1966) in Fluorescence and Phosphorescence Analysis (D. M. Hercules, ed.), Ch. 3, Interscience, New York Parker, C. A. (1968) Photoluminescence of Solutions, pp. 428-438, Elsevier, Amsterdam Fink, D. W. and Willis, C. R. (1970) J. Chem. Phys. 53, 4720 Metzger, J. V., Vincent, E.-J., Chouteau, J. and Mille, G. (1979) in The Chemistry of Heterocyclic Compounds (A. Weissberger and E. C. Taylor, eds), p. 51, Interscience, New York Shim, J.-S. K. , Tolan, J. W. and Fink, D. W. ( 1980) J. Pharm. Sci. 69, 275
Parker, C. A. and Rees, W. T. (1964) in Trace Analysis of (J. P. Cali, ed.), p. 243, Pergamon, Oxford 9 Hercules, D. M. (1966) Anal. Chem. 38, 29A 10 Hensens, 0. D. and Albers-Schonberg, G. (1978) Tetrahedron Semiconductor Materials
Lett. 3649
11 Vassel, B. (1941) J. Biol. Chem. 140, 323 12 Rogers, E. F. et al. (1960) J. Am. Chem. Sot. 82, 2974 13 Ohnesorge, W. E. and Rogers, L. B. (1956) Anal. Chem. 28, 1017 14 Kanora, J. and Szalkowski, C. R. (1964) J. Assoc. OfJic. Anal. Chem. 47, 209 15 Szalkowski, C. R. (1965) J. Assoc. Of3c. Anal. Chem. 48, 285 16 Albers-SchSnberg, G. et al. (1981) J. Am. Chem. Sot. 103, 4216
17 Springer, J. P., Arison, B. H., Hirschfield, J. M. and Hoopsteen, K. (1981)J. Am. Chem. SOL.103, 4221 18 Tolan, J. W., Eskola, P., Fink, D. W., Mrozik, H. and Zimmerman, L. A. (1980) J. Chromatogr. 190, 367 19 Pandit, N. K., Obaseki, A. 0. and Conners, K. A. (1980) Anal. Chem. 52, 1678 20 21 22 23 24 25 26
Wachowiak, R. and Conners, K. A. (1979) Anal. Chem. 51, 27 Conners, K. A. and Pandit, N. K. (1978) Anal. Chem. 50, 1542 Conners, K. A. and Albert, K. S. (1973) J. Pharm. Sci. 62,845 Hayes, J. E., Jr. and DuBuy, H. G. (1964) Anal. Biochem. 7,322 Scales, B. and Assinder, D. A. (1973) J. Phann. Sci. 62, 913 Rodriguez, C. E. and Devine, C. D. (1974) Talanta 21, 1313 Feigl, F. (1966), Spot Tests in Organic Analysis, 7th edn, pp. 352-353, Elsevier, Amsterdam 27 Fink, D. W. and Koehler, W. R. (1970) Anal. Chem. 42, 990 28 Brown, H. D. et al. (1961) J. Am. Chem. Sot. 83, 1764 29 Longworth, J. W., Rahn, R. 0. and Shulman, R. G. (1966)
A
0.20
-
0.10
-
J. Chem. Phys. 45, 2930 30 Ottender, H. and Hezel, U. (1975) J. Chromatogr. 109, 181 300 A,
350
nm
Fig. 3. Absorption spectra of thiabendaeole (X), benzimidazole (XI), and thiazole (XH) in O.lN HCI. Concentrations are3 X I@, I X 1k4, and2 X 1R4 M, respectively.
David W. Fink received his Ph.D. in analytical chemistry from Lehigh University in 1969. After Syears of analytical methods development at Lever Bras. Research and Development,. he joined the Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ, U.S.A., in 1971, where he is presently Assistant Director, Analytical, in Pharmaceutical Research and Development.
254
.
Some specific fluorogenic reactions in pharmaceutical and environmental applications Nonfluorescent compounds which cannot be treated with standard functional group derivatizing agents can sometimes be incorporated into specific fluorescent structures which are useful for analytical purposes. These reactions are intrinsically more selective because they are designed for specific applications and do not require a separation of excess fluorescent reagent.
David W. Fink Rahway,NJ, U.S.A. The usefulness of the derivatization of functional groups to generate fluorophores has been demonstrated by numerous analytical applications over the last 30 years. However, there are many cases in analytical chemistry in which the standard nonspecific functional group derivatizing reagents may not be applicable. Some of the more recently reported derivatizing agents are presented in Table I. An index such as this can often be used to select a suitable reaction. However, if the analyte does not contain any of these functional groups and if fluorescence measurement is judged to be a viable basis for the analysis, then it may be appropriate to design a specijk fluorogenic reaction based upon other structural features. This review describes some examples of specific solutions to such analytical problems which are probably more common, and certainly more challenging, than conventional functional group derivatization. The analytical methods used are based on standard fluorescence measurement as well as on fluorescence detection in liquid chromatography’. The approach to deriving a fluorophore from a nonluminescent substrate can be based upon a correlation of structural and photophysical properties*-+
following evaluation of the structure of the analyte. The principles involved in the following examples include the formation of conjugated polyene derivatives from saturated analytes, the extension of existing conjugation, and the introduction of molecular rigidity, which results in co-planarity of rings. These structural modifications are achieved via analytical reactions such as ring closing, dehydration, and other routes to aromatization. In some cases, the extension of these specific analytical reactions to a general compound class can be achieved after full characterization of the reaction mechanism, substrate properties, and reaction conditions. In addition, none of the reactions cited involves a fluorescent reagent (an excess ofwhich must be separated from the analytical fluorophore). The examples quoted are of nonfluorescent reagents which fluoresce only as a result of the analytical reaction. Compare this with some of the reagents in Table I, which are fluorescent ‘tags’ (e.g., dansyl derivatives, coumarins). The same fluorophore is present in both the reactant and product; thus, after the reaction, the excess reagent must be removed because it has the same fluorescence spectrum as the product.
Ring-closing reactions As pointed out by Parke+, the high fluorescence efficiency of many aromatic molecules (e.g., the xanthene dye fluorescein*psJ), often attributed to structural rigidity, is actually due to the conjugation of ring systems made easier by co-planarity. At any rate, ring-closing remains an especially useful technique for synthesizing luminescent compounds. There are many thiazole-containing compounds used in agricultural and pharmaceutical formulations. Studies of these agents in a variety of matrices (such as low concentration level formulations), bioavailability studies, and environmental samples often involve the determination of concentrations low enough to require the sensitivity of fluorescence analysis. However, thiazole per se is nonfluorescent6. A fluorometric method has been developed7 which is based upon a classical reaction originally used for semiconductor materials by Parker and Reess. In this treatment (Scheme I), thiazole is treated with zinc in 0 1982 Elsevier Scientific Publishing Company
255
trends in analytical chemistry, vol. 1, no. II, 1982
TABLE
I. Some fluorogenic
functional
agents Reference
Analyte
Reagent
group derivatizing
Fluorescamine Dansyl chloride Bansyl chloride I-Naphthalenemethylamine 1,2_Diaminonaphthalene I-Pyrenealdehyde 2-Fluorenealdehyde 4-Bromo-7-methoxycoumarin
primary amines amines, phenols amines isocyanates aldehydes primary amines
Weigele, M. et al. (1972) J. Am. Chem. Sot. 94,5927 Seiler, N. (1971) J. Chromatogr. 63,97 Seiler, N. et a(. (1973) J. Chromatogr. 84,95 Levine, S. P. et al. (1979) Anal. Chem. 51, 1106 Ohkura, Y. and Zaitsu, K. (1974) Talanta 2 1,547 Hwang, T. K. et al. (1978) Anal. Chim. Acta 99,305
fatty acids
Hydroxylation 2,2’-Dithiobis( I-aminonaphthalene) N-Methylnicotinamide Dimethoxyanthracene sulfonate
pyridines aromatic aldehydes a-CHs-carbonyls amines
Diinges, W. (1977) Anal. Chem. 49,442; Lam, S. and Grushka, E. (1978) J. Chromatogr. 158,207; Zelenski, S. G. and Huber, J. W., III (1978) Chromatographia 11,645 Wong, M. P. and Connors, K. A. (1978) Anal. Chem. 50,205l Ohkura, Y. et al. (1978) Anal. Chim. Acta 99,3 17 Nakamura, H. and Tamura, Z. (1978) Anal. Chem. 50,2047 Westerlund, D. and Borg, K. 0. (1976) Anal. Chim. Acta 67.89
an acidic solution to yield an intermediate - H&S, an alkyl sulfide or mercaptan. This intermediate then condenses with two moles ofp-phenylenediamine and, in a ring-closing reaction, the central ring is closed using ferric ion as the oxidizing agent to yield thionine for fluorescence measurementa. Thionine fluoresces with a red emission which has a maximum at 620 nm. A survey of model compounds was conducted to evaluate this technique as a general analytical method for this class of compounds. Saturation of the thiazole ring (thiazolidine) or removal of the nitrogen (thiophene) completely inhibits the reaction, as does fusing of the ring to form benzothiazole. In general, mono- or di-substitution at the 2- and 4-positions results in fluorescence which is of approximately equimolar (+25%) intensity with the parent unsubstituted thiazole. One specific example of an application of this reaction to a thiazole-containing compound is the peptide antibiotic thiopeptin, which is administered in Scheme I.
P Zn" H+
’
L- 1 -St
H2N
NH2
XY NH2
animal feed as a growth promoter and to improve feed efficiency.. It is used in the concentration range 10-20 g of drug per ton of feed. Thiopeptin has four thiazole rings, each similarly bonded to the C, H, N skeleton at both the 2- and 4-positionsta. Because the derivatization reaction (Scheme I) is not stoichiometric, the effects of zinc reducing agent, acidity of the reaction of analytical solution, heating time, and amount reagent are optimized. In the feed sample which is analyzed, thiopeptin is only a trace concentration level ingredient in a poorly defined matrix, requiring an efficient isolation scheme to separate the drug from any other thiazole rings. In addition to thiazole-containing extraneous material, a sulfur-containing protein can produce significant interference. This is important because these reactions have been used for the calorimetric determination of the mercaptan amino acid cysteinetr. Hence, the drug is extracted into ethyl acetate and introduced onto a gravity-fed adsorption column. After removing interferences, the drug is eluted with acetic acid in methyl alcohol, the eluate reduced to dryness, and the sample taken up in the HCl reaction medium. After the fluorogenic reaction, the fluorophore is partitioned into butanol for fluorescence measurement. This procedure yields an analytical method with an accuracy of 5% mean relative error over the concentration range 5-20 ppm thiopeptin in medicated feed. The drug amprolium is a quaternary compound which is structurally related to thiamine and which functions by a reversible thiamine inhibition mechanisml2. The fluorometric method applied to this drug is based upon the conversion of amprolium (I) to a fluorophore (II) by a reaction analogous to the oxidative production of thiochrome from thiamine, which was studied by Ohnesorge and Rogersrs. This is
-
“‘“‘$3Jc;Q
trends in analytical chemistry, vol. I, nb. II, 1982
256
another example of a ring-closing reactionl4. The reaction is sufficiently sensitive to be used for the analysis of plasma samples, and it is sufficiently rugged to have been adopted by the Association of Official Analytical Chemistsl5. Feed samples containing amprolium in the presence of a wide selection of other feed drugs have yielded analytical results ranging from 95% to 105% ofthe known medicated concentration of amprolium in the feed“+.
Dehydration reactions Compounds containing a hydroxyl group and a proton which are sterically positioned to allow facile elimination of water can often produce delocalized electronic systems. For instance, the avermectins are important new pharmaceutical products because they are a family of new anthelmintic compounds with a very .broad spectrum. The structures of these drugs have recently been described*6,17. They are fermentation products which exhibit a single electronic absorption band at h,,, 244 nm with shoulders on both the high- and low-energy sides. Their methanolic solutions are transparent at h > 310 nm and they are not fluorescent. Upon treatment with acetic anhydride in pyridine, two low-energy U.V. absorption bands appear and these are accompanied by the appearance of a visible fluorescence with a peak at 475 nm. The dihydrocyclohexene ring on this molecule (III) is fused to form a tetrahydrofuran ring and contains two hydroxyls with protons bonded trans to each. Heating with acetic anhydride in pyridine causes the elimination of two moles of water, producing an aromatic ring in conjugation with a diene system which is responsible for the fluorophore (IV) 18. The mechanism of this analytical reaction involves acetylation of the hydroxyl groups prior to dehydration. Recent work by Conners and co-workers has identified several nucleophilic catalysts for acylation by acetic anhydride which are superior to pyridine. These include 4-dimethylaminopyridine and some N-alkylated imidazoles’9-22. However, pyridine is retained in this analytical method because of the greater number of fluorescent interferences generated from endogenous plasma components when these more efficient catalysts were used. The derivative IV is the basis of a sensitive HPLC analytical method which uses fluorescence detection after preliminary sample isolationls. It has been applied to plasma samples obtained from animals dosed with the drug and which contain as little as 0.2 ng avermectin per milliliter of
PT
P
plasma in a 5-mL sample. A precision of 8% relative standard deviation was obtained at the 200-ng drug level and an accuracy of 5% mean relative error was achieved in a concentration range approaching 50 ppb of drug. The pharmaceutical applications of the method include the determination of pharmacokinetic parameters and comparisons of bioequivalence of experimental formulations. It has been known for many year&24 that oxytetracycline (V) can be heated in acidic solution to yield the more fluorescent anhydro-oxytetracycline (VI). This is a dehydration reaction. In alkaline solution, anhydro-oxytetracycline exhibits a fluorescence emission which is much more intense than that of the parent oxytetracycline. In practice a trace amount of thiopropionic acid is usually added to the analytical solution to retard oxidation of anhydro-oxytetracycline to nonfluorescent product+. Fig. 1 shows the fluorescence spectra obtained from drug-free bovine plasma and from plasma samples that have been supplemented with oxytetracycline and
9.1 ppm
3.6
porn
0.73 ppm
Blank Plasma
500
Wavelength
550
600
(nm)
Fig. 1. Fluorescence spectrum of the anhydro-oxytetracycline oxytetracvcline subblemented into drw- free bovine blasma.
derivative of
trends in analytical chemistry, vol. 1, no. 11,1982
treated to form the anhydro-oxytetracycline derivative. The concentration range is O-9 parts-per-million oxytetracycline. This fluorophore exhibits a fluorescence spectrum with a peak at 500 nm when excited at 390nm. The procedure has been used to evaluate a sustained-release pharmaceutical formulation of oxytetracycline. Fig. 2 presents the bioavailability results obtained after the drug was administered to cattle by intramuscular injection. The analyses resulting from the injection of a standard solution of oxytetracycline as a control in three daily doses exhibit three distinct peaks following each dose of the normal solution. The data obtained from the experimental microencapsulated oxytetracycline formulation after one dose clearly demonstrate sustained-release properties,
257
6
2 1 X
0.6
i
I-
20
Aromatization of an aliphatic analyte Aliphatic compounds can require major structural modification to yield fluorescent derivatives. Carboxymethyloxysuccinic acid (VII), which has been characterized as a complexing agent25, may find its way into environmental waters at low ppb concentration levels through its proposed use in synthetic detergents. Most environmental applications of fluorometric methods to air pollution and water pollution problems have been for polynuclear aromatic hydrocarbons, ofwhich many are suspected carcinogens. However, these are intrinsically fluorescent compounds, whereas VII is aliphatic and nonfluorescent. Although VII can be treated with any ofa number of carboxyl group derivatizing agents, a more selective method has been developed which involves incorporating its carbon atom framework in the fluorophore. This is another example of a ring-closing reaction, and is based on an historical spot test for malic acid26. In this method, the compound (following decarboxylation) is condensed with resorcinol, which adds to the ortho,paru positions of the two hydroxyls, eliminating two moles of water under dehydrating conditions with concentrated sulfuric acid to furnish 7-hydroxycoumarin (umbelliferone) (VIII) as the analytical fluorophore (Scheme II). Only those carboxylic acids with the basic structural features of malic acid interfere in this method. Because none of the acids (acetic, succinic, Scheme II.
40
60
80
100
Time (hr) Fig. 2. Pharmacokinetic application of fluorophore of Fig, I. Oxytetracycline concentration in plasma obtained from cattle following intramuscular injection of oxytetracycline formulations. (0) - Three (da+) injections of a control solution of ox~tetra~~line. (x) - One injection of a solutionformu~ated with microencapsutated o.+etracycline.
oxydiacetic), nor any of the aromatic carboxylic acids can form umbelliferone, they need not be separated from the environmental sample. With this fluorophore, concentrations as low as 0.5 parts-per-billion in a l-ml water sample can be determined. This coumarin fluorophore has also been used to make fluorescent derivatives of fatty acids for HPLC analysis; fluorometric coumarin substrates have been used in enzyme determinations by rate methods; and this compound is also used in dye lasers. The calibration curve is linear over a wide dynamic range (at least three decades of concentration); over the lo-6 to 10-7 M concentration range, the standard deviations from five replicate reactions, each at five different concentrations averaged cu. 9% relative to the mean fluorescence signal. This is a reasonable precision from an analytical reaction which is run as a melt. However, the fluorophore is unstable in the anionic form; the lactone ring splits in alkaline solution to yield its cinnamic acid product (IX). Umbelliferone, in addition, is photolabile, a process which is acceIerated under the excitation irradiation received in the fluorometer. Because this photoproduct fluoresces at a longer wavelength than the analytical fluorophore (500 nm vs 460 nm under the same excitation wavelength), its appearance can be monitored over a range of hydroxide ion concentrations*‘. The implication for the analytical method, however, is that suitable
AC” CH
“O
CH=CH--C&H
m -H,O
120
Ig:
7-OH-~ou~afin
2,4-diOH-Cinnamic
(Umbefliferonef
Aad
X exate :
370 nm
X excite:
370 nm
Xemit:
460 nm
Xemct.
500 nm
trends in analytical chemistry, vol. I, no. II, I982
258
conditions at near neutral pH can be found for thus allowing measurement analytical purposes, within a short enough time to avoid fluorophore degradation.
Conjugation of ring systems Thiabendazole (2-(4’-thiazolyl)benzimidazole) (X) is composed of a benzimidazole ring (XI) and a thiazole ring (XII) each bonded (Y to a nitrogen heteroatomz*. The use of thiabendazole formulations as agricultural fungicides has been approved for an ever-increasing variety of crops, including citrus fruits, bananas, tobacco, potatoes, soybeans, and sugar beets. This compound is of considerable environmental importance because of residues found in crops. For these residue studies, the analyte *of interest is the intact parent thiabendazole molecule (X) in the presence of its possible degradation products - the two stable heteroaromatic rings benzimidazole (XI) and thiazole (XII). Fig. 3 presents the U.V. spectra of thiabendazole (X) benzimidazole (XI), and thiazole (XII) in 0.1~ HCl. Thiabendazole is the only compound of these three which absorbs at h > 290 nm; both thiazole and benzimidazole are transparent at this wavelength. Hence, the absorption band at h,,, 302 nm in the figure reflects an electronic transition of a molecular orbital delocalized across both heteroaromatic rings, and is thus a characteristic physical property of intact thiabendazole. As a consequence, thiabendazole is intrinsically intensely fluorescent with an emission peak at 370 nm. Thiazole is not fluorescent and benzimidazole emits with a quantum yield of only 0.0629. Although this example is not a derivatization reaction, it is nevertheless relevant since it represents the formation of a low-energy U.V. fluorescent product from two nonfluorescent (in the near-u.v.) moieties. Direct measurement of fluorescence intensity under excitation at 300 nm, therefore, provides a sensitive
4
0.80 t\
@J$)-Q@j$)EQ N
X
XI
XII
assay which is specific for thiabendazole and which is widely used in agricultural applications for sensitive determinations of residues of the intact moleculeso.
Acknowledgement The author is indebted to John W. Tolan for collaborating on three of the derivatization projects cited in this review.
References Johnson,
E., Abu-Shumays,
A. and Abbott,
S. R. (1977),
J. Chromatogr. 107, 134
Wehry, E. L. (1967), in Fluorescence. Theory, Instrumentation, and Practice (G. G. Guilbault, ed.), Ch. 2, Marcel Dekker, New York Wehry, E. L. and Rogers, L. B. (1966) in Fluorescence and Phosphorescence Analysis (D. M. Hercules, ed.), Ch. 3, Interscience, New York Parker, C. A. (1968) Photoluminescence of Solutions, pp. 428-438, Elsevier, Amsterdam Fink, D. W. and Willis, C. R. (1970) J. Chem. Phys. 53, 4720 Metzger, J. V., Vincent, E.-J., Chouteau, J. and Mille, G. (1979) in The Chemistry of Heterocyclic Compounds (A. Weissberger and E. C. Taylor, eds), p. 51, Interscience, New York Shim, J.-S. K. , Tolan, J. W. and Fink, D. W. ( 1980) J. Pharm. Sci. 69, 275
Parker, C. A. and Rees, W. T. (1964) in Trace Analysis of (J. P. Cali, ed.), p. 243, Pergamon, Oxford 9 Hercules, D. M. (1966) Anal. Chem. 38, 29A 10 Hensens, 0. D. and Albers-Schonberg, G. (1978) Tetrahedron Semiconductor Materials
Lett. 3649
11 Vassel, B. (1941) J. Biol. Chem. 140, 323 12 Rogers, E. F. et al. (1960) J. Am. Chem. Sot. 82, 2974 13 Ohnesorge, W. E. and Rogers, L. B. (1956) Anal. Chem. 28, 1017 14 Kanora, J. and Szalkowski, C. R. (1964) J. Assoc. OfJic. Anal. Chem. 47, 209 15 Szalkowski, C. R. (1965) J. Assoc. Of3c. Anal. Chem. 48, 285 16 Albers-SchSnberg, G. et al. (1981) J. Am. Chem. Sot. 103, 4216
17 Springer, J. P., Arison, B. H., Hirschfield, J. M. and Hoopsteen, K. (1981)J. Am. Chem. SOL.103, 4221 18 Tolan, J. W., Eskola, P., Fink, D. W., Mrozik, H. and Zimmerman, L. A. (1980) J. Chromatogr. 190, 367 19 Pandit, N. K., Obaseki, A. 0. and Conners, K. A. (1980) Anal. Chem. 52, 1678 20 21 22 23 24 25 26
Wachowiak, R. and Conners, K. A. (1979) Anal. Chem. 51, 27 Conners, K. A. and Pandit, N. K. (1978) Anal. Chem. 50, 1542 Conners, K. A. and Albert, K. S. (1973) J. Pharm. Sci. 62,845 Hayes, J. E., Jr. and DuBuy, H. G. (1964) Anal. Biochem. 7,322 Scales, B. and Assinder, D. A. (1973) J. Phann. Sci. 62, 913 Rodriguez, C. E. and Devine, C. D. (1974) Talanta 21, 1313 Feigl, F. (1966), Spot Tests in Organic Analysis, 7th edn, pp. 352-353, Elsevier, Amsterdam 27 Fink, D. W. and Koehler, W. R. (1970) Anal. Chem. 42, 990 28 Brown, H. D. et al. (1961) J. Am. Chem. Sot. 83, 1764 29 Longworth, J. W., Rahn, R. 0. and Shulman, R. G. (1966)
A
0.20
-
0.10
-
J. Chem. Phys. 45, 2930 30 Ottender, H. and Hezel, U. (1975) J. Chromatogr. 109, 181 300 A,
350
nm
Fig. 3. Absorption spectra of thiabendaeole (X), benzimidazole (XI), and thiazole (XH) in O.lN HCI. Concentrations are3 X I@, I X 1k4, and2 X 1R4 M, respectively.
David W. Fink received his Ph.D. in analytical chemistry from Lehigh University in 1969. After Syears of analytical methods development at Lever Bras. Research and Development,. he joined the Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ, U.S.A., in 1971, where he is presently Assistant Director, Analytical, in Pharmaceutical Research and Development.