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Taian ta, Vol. 23, pp. 65-66. Pergamon Press. 1976. Printed in Great Britain

DETE RMINATION OF ADRENALINE AND NORADRENALINE BYRESONANCE RAMAN SPECTROMETRYMOHAMMEDS. RAHAMAN* and MICHAEL D. MORRIS

Department of Chemistry, University of Michigan, Ann Arbor, MI 48104, U.S.A.

(Received 25 February 1975. Accepted 25 Ma rch 1975)

Conventionally, determination of adrenaline and norad-renaline in urine and other matrices is carried out by fluor-imetry after conversion of the catecholamines into the tri-hydroxyindoles. is2 The method suffers from certain disad-vantages. First, as with other fluorimetric methods, it issusceptible to interference from a variety of quenchingagents. Second, many pharmaceuticals give fluorescentproducts, which may interfere. Finally, adrenaline and nor-adrenaline are not differentiated by the procedure. How-ever, adrenaline alone will undergo the initial oxidationin acidic solution, so noradrenaline can be determinedby difference.In the past few years, resonance Raman spectrometryhas emerged as a sensitive and selective probe for manymolecules of biological interest.3 Most workers have con-fined their studies to molecules which themselves can showresonance-enhanced Raman spectra. A few attempts to pre-pare derivatives suitable for resonance Raman spectro-metry have been reported.4*5Moreover, the potential of resonance Raman appearsto have been largely overlooked by analytical chemists,although the technique is simple, offers high resolution andfreedom from the quenching effects which plague fluores-cence.6 The principal drawback of resonance Raman spec-trometry is the fact that conventional spectrometers cannotdistinguish between Raman scattering and fluorescence.With the advent of convenient instrumentation for fluores-cence-rejecting techniques such as coherent anti-Stokesscattering, 3s inverse Raman spectrometry,g and time-resolved Raman spectrometry~ the fluorescence prob-lem appears tractable.In the present communication we report the applicationof resonance Raman spectrometry to the determination ofadrenaline and noradrenaline as the aminochromes. Aerial.oxidation of these molecules, catalysed by copper( iswell known. The technique is simple, applicable to con-centrations as low as 1 x 10m6M and allows determinationof adrenaline and noradrenaline on a single sample.

EXPERIMENTALApparatusA Spex 1401 double monochromator, a cooled RCAC31034A photomultiplier and both d.c. and photon-count-ing detection systems were used for these experiments. Slit-widths of 4OOpm (9cm-) were employed. Scan-rates of2&50 cm- i/min were used. The excitation source was aCoherent Radiation CR-5 argon-ion laser. Melting pointcapillaries were used as sample containers, with conven-tional transverse excitation and observation at 90 to theincident radiation. Absorption spectra were obtained witha Cary 14 spectrophotometer.

* Present address: Department of Chem. & Chem. Eng.Michigan Technological University, Houghton, Michigan49931.

ReagentsAdrenaline hydrogen tartrate and noradrenaline hydro-

gen tartrate were obtained from the Sigma Chemical Com-pany and used as received. Stock solutions of these rea-gents were stored under refrigeration and replaced at fre-quent intervals. All other reagents were of ACS reagentgrade.Procedure

The aminochromes were prepared by addition to thecatecholamines of stock copper(I1) sulphate, ammoniumchloride and potassium nitrate solutions to make the solu-tions O.OlM in cupric ion, 0.04M in ammonium ion, and005M in nitrate. The pH of the resulting solution wasadjusted to 5 with ammonia. Raman scattering intensitywas measured at 1480 cm- (adrenaline) and 1430 cm-(noradrenaline), the nitrate 1050~cm- line being used asan internal standard. Ar+ 4965-nm excitation is optimum,but Ar+ 488.0nm may be used.

RESULTS AND DISCUSSIONAbsorption spectra of the aminochromes formed areshown in Fig. 1. The compounds have similar spectra, butthe adrenaline product shows higher absorption. In theabsence of ammonium ion similar spectra were observed,but with lower intensities, implying incomplete oxidation.The resonance Raman spectra of the adrenaline and nor-adrenaline aminochromes are presented in Fig. 2. In addi-tion to these bands, weaker bands occur around 1680,1625, 1100 and 500 cm- . The two bands of the adrenalinespecies at 1480 and 1465 cm-i and the single band ofnoradrenaline at 1430 cm-i allow ready differentiation ofthese compounds. Interpretation of these spectra is in pro-gress and will be reported at a later date.

400 600

Fig. 1. Absorption spectra of aminochrome systems,pH = 5. l-l x 10e4M adrenaline; 2-l x 10e4Mnoradrenaline.

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Fig. 2. Resonance Raman spectra of aminochromes, 1, =4965nm. l-l x 10w5M adrenaline; 2-l x lo-Mnoradrenaline.Table 1 shows analytical data for these solutions. Theworking curve is linear over the range 5 x 10v6-2 x10-4A4. Above the IO-M level, the precision of themeasurements is about f 3%. Although signals are detect-able down to about the 10m6M level, the noise in oursystem is too great to allow analytical use of the signalsbelow about 5 x 10-6M. More sophisticated signal condi-tioning could probably lower the detection limits and thelimit of the useful concentration range.Our current studies have been limited to synthetic solu-tions. We have carried out some preliminary experimentson urine matrices. Prior concentration of the catechol-

amines by the conventional technique of adsorption on alu-mina and elution with acid is necessary to bring the con-centrations to a usable level. This step also serves toremove fluorescent substances from the sample. Our results

Table 1. Concentration dependence of aminochromeresonance Raman scatteringAdrenaline Noradrenaline

C, IO- 5M &mqd~NO; c> 10-5M &-,ple/~NO;0.45 0.032 0.55 0.025I 05 0.082 1.00 0.0451.60 0.122 4.0 0.1402.03 0.158 6.0 0.2053.00 0.230 7.5 0.2606.0 0.47, 9.0 0.30,7.0 0.56, 10.0 0.35,8.0 0.64,10.0 0.80,

show that adrenaline and noradrenaline from urine sam-ples are detectable by this technique. Studies of variousclinical specimens are now in progress to determine therelative merits of the resonance Raman and fluorimetricapproaches to catecholamine determination.

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REFERENCESP. L. Wolf, D. Williams, T. Tsudaka and L. Acosta,Methods and Techniques in Clinical Chemistry, pp. 107-113. Wiley-Interscience, New York, 1972.J. A. Lorraine and E. T. Bell, Hormone Assays andTheir Clinical Application, 3rd Ed., pp. 251-287. Liv-ingstone, Edinburgh, 1971.T. G. Spiro, Acct. Chem. Res., 1974, I. 339.P. R. Carey, A. Froese and H. Schneider, Biochemistry,1973, 12. 2198.P. R. Carey and H. Schneider, Biochem. Biophys.Research. Com mun., 1974, 57. 831.C.-W. Tsai and M. D. Morris, Anal. Chim. Acta, inthe press.R. F. Begley, A. B. Harvey, R. L. Byer and B. S. Hud-son, J. Chem. Phys., 1974, 61. 2466.R. F. Begley, A. B. Harvey and R. L. Byer, Appl. Phys.Lett., 1974, 25. 387.E. Yeung, J. Mol. Spectry., 1974, 53. 319.R. P. VanDuyne, D. L. Jeanmaire and D. F. Shriver,Anal. Chem., 1974, 46. 213.F. E. Lytle and M. S. Kelsey, ibid., 1974, 46. 855.R. A. Heacock, Advan. Heterocyclic Chem., 1965, 5.205.

Summary-Aminochromes of adrenaline and noradrenaline show resonance Raman scattering at 1480and 1430 cm- 1 respectively, with Ar excitation. Scattering intensity is a linear function of con-centration. Detection limits are 1 x 10m6M. Both catecholamines can be determined in a single measure-ment.