elution with absolute ethyl alcohol demonstrate that spectral differences and displacements were not due to chemical alteration of the hydrazones by the adsorbents. Three studies reveal that bathochromic displacements in going from transmittance to reflectance do take place and are more pronounced, in the case of the two hydrazones studied, near the visible region of the spectrum. The displacements are fairly constant, attesting to the reproducibility of the reflectance technique and presumably to the similar nature of the factor(s) responsible. The effect of particle size in spectral reflectance and its role in causing spectral displacements have been considered briefly by Johnson and Studer (IO) and Lermond and Rogers ( I S ) . Accessory evidence supports the view that the spectral displacements are not inherent in the reflectance technique but are primarily due to or arise from a state of subdivision. Shibata (18) in his work on suspensions of carotene crystals in water found that the three absorption bands in the transmittance spectra of these suspensions were shifted toward shorter wave lengths when compared with the transmittance spectra of the benzene extracts of the carotenes. The displacements detected in the transmittance spectra of the suspensions are undoubtedly due to the state of subdivision of the carotene crystals in the suspensions relative to those in true solution. The findings of Naughton and
coworkers (15) can be explained similarly because no displacements were noted when the reflection spectra of the heme pigments in tuna meat were compared with the corresponding transmittance spectra in solution. These n-orkers may have been comparing substances of approximately similar particle size if the heme pigments, mainly oxy- and metmyoglobin, being macromolecules, were not in true solution but were present in both instances in the colloidal state. Finally, the most direct evidence is given by the results of the comparison of the maxima of the transmittance and reflection spectra of the two hydrazones measured directly on filter paper under a variety of conditions which show clearly that they are similar (Table I). These findings are considered significant because direct comparison of the transmittance and reflection spectra of substances of identical particle size revealed no displacements. LITERATURE CITED
(1) Borisov, M. D., Izvest. Akad. Nauk, S.S.S.R. Ser. Fiz. 17, 689 (1953). (2) Bradfield, A. E., Flood, A. E., J. Chem. Soc. 1952, 4740. 3) Brady, 0. L., Ibid., 1931, 756. 4) Braude, E. A., Jones, E. R. H., Ibid., 1945, 498. (5) Buyske, D. A, Owen, L. H., Wilder, P.. Hobbes. AI. E.. ANAL.CHEM.28. 910 (1956).' (6) Dirscherl, W.,Nahm, H., Ber. 73B, 448 (1940). (7) Elvidge, J. A,, Whalley, % Chem. I., & Ind. (London) 1955, 589.
I
(8) Fisher, R. B., Vratny, F., Anal. Chim. Acta 13, 588 (1955). (9) Guilmart, T., Bull. SOC. chim. [5], 5, 1209 (1938). (10) Johnson, P. D., Studer, F. J., J. Opt. SOC.Am. 40, 121 (1950). (11) Jones, L. A., Holmes, J. C., Seligman, R. B., ANAL.CHEW28, 190 (1956). (12) Lautsch, V. W., Kurth, G., Broser, W. J.. 2.Naturforsch. 8B. 640 (1953). (13) Lekmond, A . , Rbgers,' L. 'B., ANAL.CHEM.27,340 (1955). (14) Lynn, W. S., Steele, L. A., Staple, E.,Zbid.,28,132(1956). (15) >aughton, J. J., Frodyma, M. M., Zeitlin. H.. Science 125. 121 (1957). (16) Pippen,'E. L., Eyring, E. J,, Nonaka, Masahide, ASAL. CHEM. 29, 1305 (1957). (17) Pruckner, F., Schuienburn, hf., Schwuttke. G.. Naturwissenschaften38, 45 (1951).' ' (18) Shibata, K. , Biochim. Biophys. Acta 22,398 (1956). (19) Shibata, K., J. Biochem. (Japan) 45, 599 (1958). (20) Shibata, K., private communication. (21) Shihata, K., Benson, A. A., Calvin, M.. Biochim. BioDhvs. Acta 15. 461 (1954). (22) Smith, J. H. C., Shibata, K., Hart, R. W.,Arch. Biochem. Biophys. 72, 457 (1957). (23) Timmons, C. J., J . Chem. SOC.1957, 2613. (24) Yamaguchi, K., Fujii, S., Tahata, T., Kato, S., Yakugaku Zasshi. 74, 1322 (1954). (25) Ibid., p. 1327. (26) Zeitlin, H., Niimoto, A., Nature 181, 1616 (1958).
e.
I
"
RECEIVED for review August 11, 1958. Accepted hlarch 16, 1959. Work supported by a Frederick Gardner Cottrell grant from The Research Corp. of New York.
Spectrofluorometric Estimation of Adrenochrome in Human Plasma A. N. PAYZA and M. E. MAHON Psychiatric Research Unit, University Hospital, Saskatoon, Canada
b A determination of adrenochrome (2,3 -dihydro - 3 -hydroxy- N-methylindole-5,6-quinone) and similar compounds in plasma is based on the formation of a fluorescent compound in the presence of zinc acetate. The Farrand spectrofluorometer is used to measure fluorescence. Adrenochrome concentrations of 0.1 to 1 y yield a linear fluorescent curve. Adrenolutin (3,5,6-trihydroxy-N-methylindole) is the end product of the reaction. Other compounds tested-adrenaline, noradrenaline, tryptophan, tryptamine, 5-hydroxytryptophan, serotonin, 3-indofeacetic acid, 5,6-dihydroxy-N methylindole, 3,4-dihydroxyphenylalanine, dopachrome, epinine, epinochrome, and adrenoxyl-do not give 1170
ANALYTICAL CHEMISTRY
increased fluorescence after addition of zinc salts in acetone. 2-lodoadrenochrome, 5,6-dihydroxynorephedrinechrome, and noradrenochrome yield fluorescent substances with zinc salts to a lesser degree than adrenochrome,
follow the excretion of adrenochrome into urine in laboratory animals (2). It has also been used to develop an assay for small quantities of adrenochrome in plasma and cerebrospinal fluid.
A
Crystalline adrenochrome was prepared as described (6). Other compounds tested (reagent grade) were: DLtryptophan, serotonin, 3,4dihydroxyphenylalanine (Nutritional Biochemicals Corp.), noradrenaline hydrochloride (Delta Chemical Works, Inc.), 3,4dihydroxynorephedrine hydrochloride (Mann Research Laboratories Inc.), 3-indoleacetic acid, epinephrine, tryptamine hydrochloride (Eastman Kodak Co.), (epinine) 3,4-dihydroxyphenyl-
MATERIALS A N D METHOD
was an intermediate stage in the oxidation of epinephrine by a mammalian catechol oxidase in human plasma (6, 7 ) . Although this indicates adrenochrome may be present in human plasma, no assay method has been developed to test this possibility. Zinc and aluminum salts change adrenochrome to a fluorescent compound ( I , 4). This reaction was used to DRENOCHROME
ethylmethylamine hydrochloride, crystalline 2-iodoadrenochrome, and 5,6d i h y d r o x y-N-m e t h y 1i n d o 1e. 5Hydroxytryptophan was obtained from hIerck & Co., Inc. Noradrenochrome, 5,6-dihydroxynorephedrine-chrome, dopachrome, and epinochrome were prepared by oxidation of the corresponding parent compounds with silver oxide in dilute aqueous solutions. Each aminochrome solution was adjusted to the same absorbance a t 480 mp maximum, as that of a known solution to compare fluorescence before and after zinc acetate addition. Adrenolutin was prepared according to HarleyMason and recrystallized before use (3). Preparation of Reagents. 1. Zinc Acetate Solution 1.511.1. Zinc acetate (32.9 grams, British Drug Houses, Analar) was dissolved in distilled water (75 ml.) by gentle heat; the solution was cooled and made u p to 100 ml. with distilled water. Turbid solutions should be discarded because they interfere with the estimation of fluorescence. The reagent should be stored a t room temperature and renewed every 2 days. 2 . Ascorbic Acid Solution. Ascorbic acid (1 gram, British Drug Houses, U.S.P.) was dissolved in distilled water (100 ml.) and used within 4 hours. Older solutions mere discarded. 3. Zinc Acetate-Ascorbic Acid Reagent. Reagent l (30 ml.) was added to reagent 2 (0.9 ml.) and the mixture diluted to 45 ml. with distilled water. This gave a solution oi 1M zinc acetate containing 20 mg. yo of ascorbic acid. This was prepared just before use and was discarded after 30 minutes. 4. Acetone (Merck & Co., Inc., reagent grade). A mixture of 2 ml. of reagent 3 plus 1 of distilled water and 4 of acetone must remain clear; if turbid, it should be discarded. 5. Acetone-Ascorbic Acid Reagent. Reagent 2 (2 ml.) was added to reagent 4 (9'3 m1.1.~ 6. Adrenochrome. Stock solution, crystalline adrenochrome (10 mg.) freshly prepared in distilled water (100 ml.) daily. Dilute solution, 1 ml. of stock solution (100 y) diluted to 20 ml. with distilled water (5 y per ml.). 7 . Glass double-distilled water was used. Spectrofluorometer (Farrand Optical Go., Inc.), Model 104293 (5-ml. quartz cuvette), was used. Procedure. All glassware was rinsed first with glass double-distilled water and then with reagent grade acetone. PREPARATION OF UNKNOWN SAMPLE. Heparinized blood (10 ml.) was centrifuged and the plasma removed. The plasma (1 ml.) mas placed in 25-ml. Erlenmeyer flask and reagent 3 (2 ml.) was added and allowed to stand for 30 seconds. Reagent 5 (4 ml.) was added with swirling and allowed to stand for 5 minutes a t room temperature to precipitate the plasma proteins and convert adrenochrome into adrenolutin. The precipitate was filtered using H. Reeve Angel & Co. filter paper No. 812 and a clear filtrate was obtained. Some paper contains fluorescent impurities.
Table 1.
Effect of Concentration of Zinc Acetate-Ascorbic Acid on Fluorescence
Test Tube NO.
1
2 3 4
5 6 7
8 9
Zinc Acetate Solution, hfl. 0 1
2
4 5
Fluorescence, Arbitrary JLliK- -
Acetone Ascorbic Acid, Mg. %
Blank A
Sample Bb
Zinc acetate concentration variable 10 23 10 36 10 38 10 42 45 ~.
10
_.
10
6
8 10 10
48
55
10 10 0
58 22
25 1300 1600 2200
2600 2700 2600 2600 2400
B-A Ditf .
7
12ti4 1562
3158 2555
2652
2545 25 12
238
Ascorbic acid concentration variable 34 1000 966 0 2 34 1800 1766 10 3 38 2000 1962 20 4 2100 2012 50 38 2100 2055 5 100 45 5 2200 2138 6 200 62 5 7 34 0 0 200 34 a 495-mp excitation, 500-mp emission. Final volume in each tube was 16 ml. with distilled water. Quartz cuvette ( 5 ml.) used for reading fluorescence. Above results from one experiment. * Contained 0.75 ml. of dilute adrenochrome solution. 1
If preferred, the plasma is centrifuged in a Serval1 Laboratory Equipment centrifuge a t 9000 r.p.m. for 2 minutes. PLASM.4 BLANK. This is prepared using plasma (1 ml.) and reagent 5 (6 ml.). INTERNAL STANDARD.Plasma (1 ml.) plus 0.1 ml. of dilute adrenochrome solution (0.5 y) was treated the same as the unknown sample. READINGOF FLUORESCENCE. Fluorescence was read 10 minutes after mixing reagents a t 405-mp excitation and 500-mp emission on the microammeter of the spectrofluorometer. These are the excitation and fluorescence maxima of 3,5,6-trihydroxy-N-methylindole (Figure 1).
EXPERIMENTAL
EFFECT OF ASCORBICACID CONCENTRATION IN PRESENCE OF ZIKC ACETATEON FORMATION OF FLUORESCENCE FROM ADRENOCHROME. The following reagents were used: zinc acetate (21.9 grams) in distilled water (100 ml.), ascorbic acid in distilled water, 1%, diluted to the following concentrations : 10, 20, 50, 100,and 200 mg. %, in distilled mater, and adrenochrome (10 mg. %) in distilled water. This stock solution was diluted 10 times.
Calculation. Reading of unknown sample reading of plasma blank Reading of internal standard reading of unknown sample
-
X
1000 2
gave micrograms of adrenochrome per liter of plasma. The difference between a reagent blank prcpared from reagent 3 (2 ml.), distilled m t e r (1 ml.) and reagent 5 (4 nil,)j and a solution consisting of distilled water (1 ml.) plus reagent 5 (6 ml.) should be subtracted from the reading of the unknown sample, in the numerator of the above equation. Stability of Instrument. The instrument was calibrated against a standard quinine solution (8) and two known concentrations of adrenochrome solution were read dailv before the unknowns were analyzed." A new xenon arc lamp gave higher readings of samples and reagent blanks. It is essential t o provide a steady light source in the spectrofluorometer by using a voltage stabilizer delivering 115 volts.
IB I
350
450
550
W A V E LENGTH, mu
Figure 1
.
Fluorescence characteristics
of adrenochrome (Zinc acetate-ascorbic acid) and adrenolutin in acetone A. Excitation and emission of adrenolutin ( 1 pmole/lO ml.) in acetone and in presence of zinc acetate-ascorbic acid 6. Excitation and emissionmaxima of adrenochrome ( 1 pmole/lO ml.) plus zinc acetateascorbic acid in acetone
VOL. 31, NO. 7,JULY 1959
1171
Table 11.
Comparison of Fluorescence Formation from 1 y of Adrenochrome by Different Reagents Fluorescence, Maxima Arbitrary Excitation Emission Units Sodium hydroxide (1M) 405 520 55.4 Aluminum sulfate (1M) 390 460 14.0 Zinc acetate (1M) 405 500 194.0 Reagents. Adrenochrome (1 y ) in distilled water (8 ml.). Each reagent listed (lml.). Respective blank values subtracted from sample readings.
Table 111. Adrenochrome Concn,, r/Liter 500
Stability of Fluorescence
Fluorescence, Time, Arbitrary Units" Min. Sample Blank Diff. 10 1500 35 1465 20 1500 65 1435 60 1100 18 900 10 580 78 502 20 600 94 506 60 600 86 514
The effect on fluorescence because of increasing quantities of ascorbic acid (20 to 200 mg. %) is shown in Table I. Concentrations between 20 and 200 mg. % do not markedly affect fluorescence. Therefore, 20 mg. % was selected as the working concentration.
than either aluminum sulfate or sodium hydroxide, all a t the same molarity as shown in Table 11. ZINCACETATECONCENTRATION. The effect of zinc acetate concentration on development of fluorescence from adrenochrome was tested using the following reagents: zinc acetate (21.9 grams) and ascorbic acid (20 mg.) in distilled water (100 ml.), ascorbic acid (20 mg.) in acetone (100 ml.) , and adrenochrome (10, mg.) in distilled water (100 ml,). This stock solution was diluted 10 times for this experiment. Table I shows that 1 part of zinc acetate (1M) containing 20 mg. of ascorbic acid plus 2 parts of acetone containing 20 mg. of ascorbic acid yields more fluorescence than other ratios.
Between p H 3 and 7 , the formation of fluorescence from adrenochrome by Other compounds tested, including zinc acetate-ascorbic acid was not dihydromaleic acid, urea, ethylenealtered. 200 diamine, hydrazine, thiourea, oxalic Other zinc salts (fluoride, chloride, acid, and dihydroxyfurnaric acid, did bromide, iodide, sulfate, nitrate, fluoronot affect the production of fluorescence silicate, salicylate, formate, phenolby zinc acetate. These, therefore, cansulfonate, thiocyanate, lactate, tartrate, not replace ascorbic acid. and borate) did not produce any fluoresCOMPARISONOF ZINC ACETATETO cence from adrenochrome a t any conOTHER COMPOUNDS WHICH PRODUCE centration of salt. 405 mp excitation, 500-mp emission. Results of one experiment. FLUORESCENCE FROM ADRENOCHROME. I n another experiment, the concenZinc acetate yields more fluorescence tration of zinc acetate was changed while the molarity of total salts was kept constant by the addition of 1M sodium chloride. Zinc acetate (1M) was necesTable IV. Calibration Curve of Adrenochrome and Fluorescence in Presence and sary and could not be replaced by Absence of Plasma sodium chloride. Fluorescence4, Arbitrary Unite STABILITYOF FLUORESCENCE. The Adrenochrome, Plasma Plasma y/Sample Sample Blank DiiT. sample blank Diff. stability of the fluorescence of adrenochrome-zinc acetate-ascorbic acid in 0.1 130 30 100 270 180 acetone was measured using three con150 30 120 270 180 90 centrations of adrenochrome (500, 200, 150 30 110 260 180 90 130 30 100 and 50 y per liter) a t three time inter260 180 80 140 30 110 270 180 90 vals (10, 20, and 60 minutes). The procedure described was used. Results 0.2 270 30 240 3.50 170 .. _. __. -18n -_ -. (Table 111) indicate that fluorescence 250 30 220 360 170 190 260 was stable up to 1 hour a t lower con30 230 350 170 180 250 30 220 350 170 180 centration of adrenochrome and then 260 30 230 340 170 170 declined. 0.5 530 40 490 610 180 430 .~ ~. . ~ - . 520 40 480 600 180 420 RELATIONSHIPOF ADRENOCHROW 540 30 510 580 180 400 CONCENTRATIONTO FLUORESCENCE 180 410 520 30 490 590 FORMATION BY ZINCACETATE-ASCORBIC 480 510 30 ACID IN ACETONE. Adrenochrome, 0.1 1.0 980 30 950 1000 180 820 to 1.0 y per sample, produced fluores970 30 940 1000 180 820 cence directly proportional to the 960 30 930 1000 820 ... __.. -180 __ _- 960 30 930 980 180 800 quantity used (Table IV). Experi980 30 950 mental conditions, as described, were used, replacing the plasma with disa 405-mpexcitation, 500-mp emission. One experiment out of six. tilled water. Each concentration was replicated five times. To ble V. Adrenolutin Fluorescence Maxima and Intensity in Various Solvents A similar experiment was run six Fluorescence, times in the presence of plasma. ReMaximum, Mp Arbitrary sults are shown in Table IV. Solvent Emission Excitation Units The presence of plasma proteins had Methanol 490 405 2000 a quenching effect on the fluorescence Distilled water 505 410 1400 formation from adrenochrome by zinc Sodium hydroxide (0.01N) 515 405 2400 acetate. Therefore, an internal standAcetic acid (0.01N) 500 405 1000 Acetone 490 405 3400 ard should be prepared for each plasma. Determinations were made on fresh Adrenolutin (10 y/ml.) in various solvents. plasma immediately after centrifugaFluorescence readings made at specific excitation and emission maxima. tion. Samples stored for some time ~
1175
ANALYTICAL CHEMISTRY
yielded lower values. The plasma Table VI. Comparison of Fluorescence of Compounds Before and After Treatment blank established the quantity of with Zinc Acetate fluorescing compounds already present in the plasma which do not react with Fluorescence Readings zinc acetate. These readings were subBefore Zinc After Zinc tracted from the measurements. FluoresFluorescence, A calibration curve of adrenochrome Concn. cence, arbitrary arbitrary in 10-Mi. added to plasma is shown in Figure 2. units Ex. Em. units Compound Sample Solvent Ex. Em. IDESTITY OF ADREXOCHROME-ZINC 35 405 500 D.W. , . . , . . 0 Adrenochrome 0.005 ACETATE-ASCORBIC 4CID AND ADRENO168 405 500 ... ... 0 0.025 D.W. LUTIN. The excitation (405 mp) and 37 405 500 0 0.005 Acetone . . . . . . emission (480 m,u) maxima for adreno180 405 500 0 0.025 Acetone . . . . . . lutin are shifted in the presence of zinc 34 395 495 395 495 0 5,6Dihydroxy0.005 D.W. acetate (1M)to 405-mh excitation and 118 395 495 395 495 0 norephedrine0.025 D.W. 32) 405 500 500-mp emission. Adrenochrome-zinc (405 500 0 chrome 0.005 D.W. 168) 405 500 (405 500 0 0.025 D.W. acetate-ascorbic acid had the same 46 395 495 0 0.005 Acetone 395 495 maxima as adrenolutin in the presence 190 395 495 0 0.025 Acetone 395 495 of zinc acetate as shown in Figure 1. 405 500 0 0.005 Acetone (405 500 45 1 405 500 EFFECTOF SOLVENTS ON ADRENO180) 0 0.025 Acetone (405 500 IUTIN FLUORESCENCE. The fluorescence 4.4 405 500 . . . ... 0 2-lodoadreno0.005 D.W. maxima and the intensity of fluores22.8 405 500 ... . . . 0 chrome 0,025 D.W. 2.0 405 500 0 0.005 Acetone . . . , . . cence of adrenolutin (10 y per ml. of 13.0 405 500 0 0.025 Acetone . . . . . . solvent) are shown in Table V. Ace390 495 10.0 0 tone was the most suitable solvent. 390 495 66 0 0 FLUORESCENCE OBTAINED FROM (405 500 11.8) 0 PLASMA. The fluorescence maxima of 62.6) (405 500 0 the plasma blank, plasma plus zinc 15.0 380 460 0 0.005 Acetone . . . . . . 71.0 380 460 0 0.025 Acetone . . . . . . acetate-ascorbic acid in acetone (un(405 500 14.6) 0 0.005 Acetone . . . . . . known plasma as under method), and (405 500 71.6) 0 0.025 Acetone . . . . . . the plasma containing adrenochrome Adrenolutin 0.005 D.W. 405 480 40 405 500 40 (0.5 y) are shown in Figure 3. Cere405 500 170 0.025 D.W. 405 480 170 brospinal fluid was similarly analyzed. 0.005 Acetone 405 480 45 405 500 45 The identity of curves 2 and 3 maxima 405 500 190 0.025 Acetone 405 480 190 indicated the presence of adrenochrome Adrenochrome excitation and emission maxima after zinc acetate treatment were 405 in plasma and cerebrospinal fluid. mp ex. and 500 mr em. To compare the fluorescence of the other compounds, measureSPECIFICITY OF REACTION. Direct ments were made at these maxima (parentheses) and at maxima for each compound. Epinine, ephedrine, 5,6-dihydroxynorephedrine, and epinochrome did not fluoresce with or fluorescence readings of some comwithout zinc acetate in distilled water or acetone. pounds tested alone and after addition of zinc acetate are shown in Table VI. Only adrenochrome ( I ) , 2-iodoadrenochrome (11),5,6-dihydroxynorephedrinefound in urine and will be reported chrome (HI), and noradrenochrome elsewhere. (IV) gave an intense fluorescence. Compounds I and I1 in the presence of DISCUSSION zinc acetate have the same fluorescence maxima as adrenolutin; noradrencOf the compounds tested, adrenochrome yields a slightly loner intensity chrome, 2-iodoadrenochrome, 5,6-diand different maxima. hydroxynorephedrine-chrome and norEpinephrine, 3,4-dihydroxyphenyl40 adrenochrome yielded fluorescence, with alanine (DOPA), 3,4-dihydroxyphenylzinc acetate-ascorbic acid in acetone. li alanine+hrome, 3-indoleacetic acid, 5,6The second and third compounds have 8 n dihydroxy-Ar-methylindole, noradrenalu' not been shown in plasma. Norepinephine, serotonin, tryptamine, tryptophan, rine is not oxidized into noradrenoand 5-hydroxytryptophan have no fluochrome in the body (6). Therefore, rescence in acetone with or without zinc the method is specific for adrenochrome. d acetate. All fluoresce in distilled water The other compounds listed under with various excitation and emission x 20 specificity show a decrease in fluoresmaxima and the addition of zinc acetate cence after zinc acetate addition in decreases the intensity. distilled water and when acetone was APPLICATIONO F METHOD. xormal used as solvent, there was no fluoresLevels. Forty-five normal subjects IO cence before or after the addition of zinc tested for adrenochrome levels had an acetate. Therefore, these compounds average value of 71 y per liter with a ' if present, would not interfere with the maximum of 106 and a minimum of 21 determination of adrenochrome. 0 7 per liter. Cerebrospinal fluids have The structural requirements of fluo0 I about the same values, averaging 75 y 2 3 rescence formation with zinc acetate MICROGRAMS OF ADRENOCHROME per liter in seven normal subjects. appear t o be a 5,6-o-quinone and a The administration of adrenochrome Figure 2. Fluorescence formation hydroxyl in c-3 position, because corn(10 ml.) by intravenous injection or of adrenochrome after reaction with pounds without a hydroxyl in (3-3 zinc acetate LSD-26 orally (100 y) increases plasma position such as dopachronie and epinoadrenochrome several times above the chrome do not show an increase of 1. Adrenochrome alone initial levels. Parallel values were 2. Adrenochrome in presence of plasma fluorescence with zinc acetate. The (I
501
8
I
VOL. 31, NO. 7, JULY 1959
1173
o-quinone structure on the benzene ring is not sufficient to produce fluorescence n-ith zinc acetate-0-benzoquinone with zinc acetate gave a nonfluorescerit green color. Iodine in the C-2 position decreased the production of fluorescence, although the presence of methyl had no effect. Because neither adrenochrome or any of its derivatives have yet been isolated from blood or urine, there is no absolute proof that this method measures adrenochrome. However, the method is accurate when adrenochrome is added to plasma or injected intravenously into blood. Plasma contains fluorescent factors which behave as if they n-ere adrenochrome. Therefore it is a working assumption that adrenochrome is being measured. However, proof must await isolation studies. After this manuscript was submitted, Szara, Axelrod, and Perlin (9) reported a sensitive specific method for measuring adrenochrome in plasma. They found less than 20 y equivalent of fluorescence which had no specific fluorescence peak. The two methods have been compared for sensitivity and are equally specific. Ten milligrams of authentic adrenochrome was injected intravenously into a subject. Ten minutes later blood was drawn from the other arm and placed in a flask containing heparin. This method showed 400 y per liter in the plasma. The Szara method yielded 76 y per liter. With this method, 1 y of authentic adrenochrome yielded 0.25 fluorescence unit compared to an increase of approximately 0.0335 unit (same scale) or one eighth the increase. Thus the method of Szara et al. is not sufficiently sensitive to settle the question as to whether or not adrenochrome is present in plasma. ACKNOWLEDGMENT
The authors thank R. Heacock for
S.F
Figure 3. Fluorescence spectra of plasma and spinal fluid acetone extracts before and after reaction with zinc acetate Plasma P. and spinal fluid S.F. acetone extracts Fluorescence measured a t 405-mp excitation 1. Before zinc acetate reaction 2. After zinc acetate reaction 3. After zinc acetate reaction with addition of 1 y of adrenochrome 4. Nonspecific light scattering peak
VI Y w P
3
300
400
500
600
&/ 3
300
400 500 W A V E LENGTH, Mp
the preparation of crystalline 2-iodoadrenochrome and 5,6-dihydroxy-Nmethylindole and Burroughs Wellcome & Co. who supplied the 3,4-dihydroxyphenylethylmethylene hydrochloride (epinine). LITERATURE CITED
(1) Fischer, P., Derouaux, G., Lambot, H., Lecomte, J., Bull. soc. chim. Belges 59,72 (1950). (2) Fischer, P., Lecomte, J., Ibid., 33, 569 (1951). (3) Harley-Mason, J., J. Chem. SOC. 1950,1276.
600
(4) Harley-Mason, J., Bu'Lock, J. D., Nature 166, 1037 (1950). (5) Heacock, R. A,, Nerenberg, C., Payza, A. N., Can. J . Chem. 36, 853 (1958). (6) Payza, A. N., Hoffer, A., to be published. ((7!,F&\ter, 7 ) Richter, D., Biochem. J . 3'1, 2022
(1937). \1aClr ). (8) Sprince, H., Rowley, G. R., Science 125,25 (1957). (9) Szara, S., Axelrod, J., Perlin, S., Am. J . Psychiat. 115, 162 (1958). RECEIVEDfor review June 23, 1958. Accepted February 3, 1959. Research supported by National Health Grants,
Ottawa, The Rockefeller Foundation, New York, and The Saskatchewan Committee on Schizophrenia Research.
Spectroscopic Techniques for Identification of Organosilicon Compounds A. LEE SMITH and J. A. McHARD Dow Corning Corp., Midland, Mich. ,Silicones may b e identified with the aid of chemical and Physical tests, but in many cases, simple spectroscopic examination, particularly by infrared, will satisfactorily characterize the material with a minimum of time and effort. Techniques for examining both organosilicon monomers and polymers are discussed. Some infrared
1174
ANALYTICAL CHEMISTRY
spectra of typical chlorosilanes and silicone polymers are shown, and the spectra are interpreted in terms of group frequencies. The applicability of ultraviolet, Raman# emission, and nuclear magnetic resonance spectroscopy to identification of silicones is reviewed.
A
of chemical and physical techniques may be applied to the problem of identifying organosilicon compounds (32). Although reliable chemical analyses are useful in characterizing these materials, supplementary structural information is often required which involves spectroscopic examination. I n many cases, too, satisfactory YARIETY
