Resonance Raman Spectra of the Aminochromes of Some Biochemically Important Catecholamines Michael D. Morris Department of Chemistry, University of Michigan, Ann Arbor, Mich. 48 104
Aminochromes (I) are formed by oxidation of catechol-(
I
R" I amines with air, triiodide, ferricyanide, or other common inorganic oxidants ( I ) . These red compounds (A, = 480-500 nm) are the first products in the common trihydroxyindole fluorimetric procedures for catecholamine determination (2). A continuing flood (3) of publications on this topic attests to the importance of catecholamine determinations in biochemistry and clinical analysis and to the inadequacy of existing techniques. Recently, we have suggested that resonance Raman spectra of the aminochromes of noradrenaline and adrenaline can be used for the determination of those compounds ( 4 ) . We report here the major bands of the aminochromes of adrenaline, noradrenaline, isoproterenol, dopamine, and epinine and present tentative band assignments. The resonance enhanced bands can all be assigned to vibrations of the immonium p-quinone moiety. We report them as vc=c, etc., although this is a clear oversimplification.
EXPERIMENTAL Reagents. Adrenaline, noradrenaline, and isoproterenol were obtained as the bitartrates (Sigma Chemical Co.) and used as received. Dopamine (Sigma) and epinine (K and K) were obtained as the hydrochlorides and used as received. All other chemicals were reagent grade. Distilled water was used to prepare all solutions. Procedure. Aminochromes were prepared by reaction of the appropriate catecholamine (approx. 2 X 10-4M) with excess triiodide in a 1:l acetic acid-sodium acetate buffer (approx. 2 X 10-*M in each, p H 4.8). After 2-3 minutes, excess triiodide was destroyed with potassium thiosulfate solution and diluted to volume so that the final nominal aminochrome concentration was about 1 X 10-4M, based on complete reaction. Raman Spectra. A Spex 1401 double monochromator equipped with a cooled RCA C31034 photomultiplier and both dc and photon counting detection systems was used for these experiments. All three slits were set for 200 microns, a resolution of about 4.5 cm-l. The excitation source was a Coherent Radiation CR-5 argon ion laser. Laser power was restricted to approximately 200 milliwatts a t the sample, but was less than that for some minor lines. Except as noted, spectra were obtained using 488.0-nm excitation. Samples were contained in conventional 2-mm melting point capillaries.
1 I700
I
I
I600 3lJ,
3 433
8530
cm
Figure 1. Resonance Raman spectrum of 1 X 10-4M adrenochrome, pH 4.8. he, = 488.0 nm. Spectrometer bandpass, 4.5 cm-'
RESULTS AND DISCUSSION The adrenochrome (derived from adrenaline) resonance Raman spectrum is shown as Figure 1. The principal bands of the spectra of the aminochrome resonance Raman spectra of the aminochromes investigated and their tentative assignments are shown as Table I. Depolarization ratios are included for adrenochrome. Table I also includes the absorption maxima for the blue-green region of the spectrum. The values listed here agree quite well with earlier measurements on these aminochromes ( 1 ) . In addition to the bands tabulated in Table I, weaker bands are observed around 1170,650, and 460 cm-l. These bands are not useful for trace analysis and will not be discussed further here. The 1665-1680 cm-l band is tentatively assigned to carbonyl stretching. This band is in the quinone carbonyl region, Moreover, the band is not particularly sensitive to the effects of substitution on the five-membered ring, derived from the ethyl amine tail of the parent catecholamine. The band in the 1605-1635 cm-l region we assign primarily to C=C stretching. The frequency region is correct. There is little dependence on substitution at the 3-position (the indole numbering system is used in this communication), although nitrogen substitution influences the position of the band somewhat. One would expect a t least two bands with large C=C stretching contributions because of the presence of the oxy-
Table I. Aminochrome Resonance Raman Spectra0 Parent compound
Adrenaline
Avc=o
(1665-1680 cm-')
1679 (0.29)b
Avc=c
(1605-1635 cm-')
1628 (0.20)b
Noradrenaline 1675 1632 (1585, sh) Isoproterenol 1680 1605 Dopamine 1665 1635 Epinine 1672 1628 a Av in cm-I, A,, = Ar+ 4 8 8 . 0 n m . bDepolarization ratio.
1474 (0.39)b 1454 (0.27)b 1425 1463 1445 1435 1415 1463 1420
490 485 492 475 475
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
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ol
I
460
I
I 480
1
I
500
I
l \ l l 520
1, nm
Figure 2. Excitation profile of adrenochrome. ( a ) Absorption spectrum. (b)1679 cm-'. (c) 1628 cm-'. (41474 cm-'
gen substituent at C-6. In noradrenochrome, there is a weak band at 1585 cm-' visible as a shoulder on the water band. It is quite possible that C=C bands of the other aminochromes are obscured by water scattering, on which these bands are superimposed. The most useful bands of the spectra are the substituent sensitive pairs of bands in the 1415-1480 cm-l region. We propose that these have a large contribution from C=N+ or C=N+-C stretching. The bands occur a t considerably lower frequencies than C=N or C=N+ stretches normally do, ca. 1600-1700 cm-l. However, the strong dependence on details of the substitution pattern on and near to the nitrogen means that these vibrations must involve nitrogen. Since the r-system is implicated in the electronic absorption near 490 nm, the C=N+ band must be involved. Despite the general occurrence of C=N stretches a t or above 1600 cm-l, the infrared spectra of phenoxazines and phenoxazones have strong absorptions in the 1493-1516 cm-I region, which have been assigned to C=N stretching on the basis of N15 substitution ( 5 ) .Thus, our assignment is reasonable. The excitation profiles and the relevant portion of the absorption spectrum of adrenochrome are presented as Figure 2. The corrected integrated band intensities roughly
parallel the absorption spectrum. The 1455-cm-' band overlaps the more intense 1475-cm-' band too much to allow accurate estimation of its area, and it is omitted. As expected, the relative intensitites peak near the maximum of the absorption spectrum, 485 nm. We have attempted pH dependence measurements to probe the effects of oxy-anion protonation and immonium ion hydroxylation on the spectra. However, aminochromes are unstable in basic solution or in strongly acid solution. Over the pH range where all samples are quite stable, pH 4-7, frequencies and relative intensities are not pH dependent. Although catecholamines themselves are weakly fluorescent, the adrenochromes are nonfluorescent. As such, they are suitable derivatives for resonance Raman spectrometry. As we have shown ( 4 ) , the detection limits for adrenaline and noradrenaline by aminochrome resonance Raman spectrometry are about 2 X 10-6M. The other aminochromes give signals of similar intensities, so that similar detection limits can be expected for these compounds as well. Such detection limits are higher than those attainable by fluorimetric procedures. However, catecholamine analysis usually involves adsorption of the catecholamines on alumina to concentrate them and remove interferences prior to the actual reaction and measurement sequence. Such preconcentration can bring catecholamines within the range of resonance Raman spectrometry and allow the simultaneous determination of several catecholamines in a single sample. This possibility is being investigated in our laboratories.
LITERATURE CITED R. A. Heacock. Adv. Heterocycl. Chem., 5,205-290 (1965). H. Weil-Malherbe, in D. Glick, ed., Metbods Biocbem. Anal., Suppl. Vol,, Analysis of Biogenic Amines and their Related Enzymes, Wlley-lnterscience, New York, 1971, pp 119-192. (3) N. Gochman and C. S. Young, Anal. Cbem., 47, 16R (1975). (4) M. S. Rahaman and M. D. Morris, Talanta, in press. (5) H. Musso. K. Spauke, and K. K. Walter, Chem. Ber., 100, 1436 (1967).
RECEIVEDfor review July 18, 1975. Accepted September 9, 1975.
Cadmium, Copper, Mercury, and Zinc Ions as Inorganic Probes in Phosphorimetric Analysis of Nucleosides G. D. Boutilier," J. R. Andrew, C. M. O'Donnell,2and 1.N. Solie3 Department of Chemistry, Colorado State University, Fort Collins, Colo. 80523
The luminescence technique of phosphorimetry has been shown to be highly sensitive for the analysis of selected drugs (1-4) and vitamins (5, 6). The use of mixed aqueous solvents (7) and spinning sample cells (8) has solved two major problems in adapting phosphorimetry to routine biochemical and pharmacological analysis. The problem of overlapping phosphorescence spectra remains, because phosphorescence spectra are bands which usually extend over several tens of nanometers. In contrast with atomic fluorescence spectrometry where narrow line spectra proPresent address, Department of Chemistry, University of Florida, Gainesville, Fla. 32611. * Present address, Bio-Science Laboratories, 7600 Tyrone Avenue, Van Nuys, Calif. 91405. Author to whom reprint requests should be sent. 2454
vide an efficient means for resolving mixtures, the use of high resolution monochromators is not as important or as useful in phosphorimetry. However, phosphorescence lifeto 10 sec, depending upon the nature times range from of the emitting triplet state, and it has been suggested that the resolution of mixtures of phosphors can be accomplished by time-resolved phosphorimetry (9). The feasibility of this approach has been demonstrated in the analysis of halogenated biphenyls (IO)and selected drugs ( 4 ) using pulsed source-time resolved phosphorimetry (11, 12). Recent instrumental improvements in phosphorimetry are reviewed by O'Donnell and Winefordner (13). The use of alkali halide salts, such as sodium iodide, for external heavy atom perturbations of the phosphor ( 1 4 ) provides a method of increasing the sensitivity of phospho-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
