’1064
ANALYTICAL CHEMISTRY LITERATURE CITED
dried over fresh barium oxide. The melting point was 89.0” to 92.0” C. Upon recrystallization from ethyl alcohol the melting point was redetermined as 92.5’ to 93.8” C. A mixed melting point with the bis-(O,O-diisopropyl dithiophosphoryl) disulfide prepared previously by direct oxidation with iodine produced no change in melting point. It) would appear t,hat the reaction a t the micro platinum electrode is: 2-SP(S)(OR)* -+ (RO),P(S)SS[S]P(OR)2 2eThe El;?us. saturated calomel electrode values for the dialkyl dithiophosphates become increasingly negative as the molecular weight increases. At 0.5 X 10-3111 t’he Eli2 values determined were for isopropyl, -0.104 volt; n-propyl, -0.131 volt; secbutyl, -0.203 volt; isobutyl, -0.200 volt; and n-butyl -0.204 volt. This increase would be expected if insoluble precipitat,es of increasingly lower solubilities were being formed (15). At’ concentrations up to 1 X 10-3M the i d values for all the butyl salts studies are nearly identical, with deviation becoming apparent only at concentrstions above 1 X Results are shown in Table 111.
American Cyanamid Co., Brit. Patent 588,090 (May 14, 1947). Buchanan, G. H., Mining a n d Met., 11, 567 (1930). Buchanan, G. H., U. S. Patent 1,868,192 (July 17, 1933). Carius, L., Ann., 112, 190 (1859). Edsberg, R. L., AXAL.CHEST., 26, 724-6 (1954). Gaines, J. C., J . Econ. Entomol., 46, 896-9 (1948). Gaudin, A. M., “Flotation,” IIcGraw-Hill, New York, 1932. Kabachuik, RI. I., and Rlastruykova, T. A , Bull. Acnd. Sci. C.S.S.R., NO. 4, 727-35 (1952). Kolthoff, I. h l . , and Barnum, C., J . Am.Chem. Soc., 62, 3061 (1940). Lingane, J. J., Ibid., 67, 1917 (1945). Lingane, J. J., and Laitinen, H. A.. IND. ExG., Cmnr., ASAL. ED., 11, 504 (1939). Mastin, T. W., Norman, G. R., and Weilmuenster, E. -L,,J. Am. Chem. Soc., 67, 1662 (1945). Muller, 0. H., Ibid., 69, 2992 (1947). Muller, 0. H., “Polarographic Method of Analysis,” 2nd ed., p. 69, Chemical Education Publishing Co., Easton, Pa., 1951. Revenda, J., Collection Czechosloa. Chem. C o m n ~ u n s . ,6, 453 (1934). Romieus, C. J., and Wohnsieddter, H. P., U. S. Patent 1,748,619 (Feb. 25, 1930). Sargent and Co., E. H., Chicago, Ill., “AIanual of Instructions for Sargent Model XI1 High Speed Photographic Polarograph.”
+
ACKNOWLEDGMENT
The authors wish t,o express appreciation to Dale McCowen and Gary Babcock for repeztirig part of the experimental work described.
RECEIVED for review September 13, 1954.
Accepted hlarch 7, 1955
Polarographic Measurement of I-Noradrenaline and I-Adrenaline JOANNE HENDERSON’ and A. STONE FREEDBERG Department o f Medicine, Harvard M e d i c a l School, M e d i c a l Research Department, Yaminr Research Laboratories, and M e d i c a l Service, Beth Israel Hospital, Boston, Mass.
This work was initiated to meet the need for an accurate and simple method for the measurement of Z-adrenaline and Z-noradrenaline. The compounds were converted by iodate oxidation to their derivatives iodoadrenochrome or iodonoradrenochrome. Aliquots of the reaction solutions were measured polarographically. Half-wave potentials for iodoadrenochrome and iodonoradrenochrome were found to be Eli2 = +0.03 volt and El!?= $0.02 volt, respectively, in 0.1M acetic acidacetate buffer pH 4.52, 0.01% in gelatin. A linear relation between concentration and diffusion current was found over the investigated range of the equivalent of 1 to 50 y of adrenaline or noradrenaline. Hapid and accurate routine analysis of these amines can be carried out by the described method.
S
TLTDIES of adrenal medullary activity in man have been handicapped by the lack of an accurate and reasonablv simple method for the measurement of adrenaline (epinephrine) and noradrenaline (Arterenol). The bioassay method developed b y von Euler ( 5 ) is carried out on two isolated or intact animal organs. The quantity of adrenaline and noradrenaline in an unknown mixture is calculated from the difference in response of the hen’s rectal cecum or rat uterus and the cat’s blood pressure, as compared with the response to known amounts of the two amines. The chemical methods now available have not been entirely satisfactory. Early emphasis on the colorimetric measurement of various derivatives of the amines ( 1 , 5 , 13) has been superseded by more sensitive fluorimetric techniques (9, 11). During the course of these studies, the publications of Keil-lfalherbe and Bone ( 1 4 ) and Manger and others (12) have come to the authors’ attention. They have not had the opportunity to examine this 1
Present address, Arthur D. Little Co., Inc., Cambridge, Mass.
method. Although the sensitivity has thus been increased, it has not been conclusively established that the fluorescence measured is entirely that of the adrenaline or noradrenaline derivatives.
M,
H
A
B Figure 1.
C
D
Structure of amines
A.
Adrenochrome E . Noradrenochrome C . Iodoadrenochrome D. Iodonoradrenochrome
The present study is concerned with the application of the polarographic method t o the measurement of adrenaline and noradrenaline. The catechol nucleus of adrenaline is oxidized at a potential so positive that its measurement a t the dropping mercury electrode would not be practical. However, p - and oquinones are easily reduced and produce well defined, reversible polarographic waves which bear a linear relationship between concentration and diffusion current. Adrenochrome, the oquinone of adrenaline, although reversibly reduced over a wide range of pH, as shown by Wiesner (15),is unstable and eventually precipitates as melanin. Under suitable conditions, adrenaline is converted quantitatively to a stable derivative, iodoadrenochrome (Figure 1). Since this compound has the same o-keto structure as adrenochrome, it was expected that this derivative would produce a polarographic wave a t a similar potential. The present study presents a modification of the iodate oxidation described by Bouvrt (3) for the conversion of adrenaline to iodoadrenorhrome. The method has been extended to the prep-
1065
V O L U M E 27, NO. 7, J U L Y 1 9 5 5
- - 0.4 .'ui - 0.9
of the amine derivatives. The prepurified gas was passed through two bottles containing Fieser's sclution (81, through a solution of lead acetate, and finally through distilled water before being admitted to the test solution. All polarographic curves were recorded on a Leeds and iicrthrup Electrochemograph, Type E. A 0.1N potassium nitrate agar bridge was connected to the reaction vessel with a saturated calomel electrode. The resistance of the external circuit was assumed to be negligible for the small currents recorded. The capillary constant, K,was equal to 6.87. Measurements were made a t a sensitivity chosen to give a 5to 3 0 - m i . diffusion current with anodic-cathodic polarization ( 1 0 . 2 to -1.0 volt us. S.C.E.). The solutions were rebubbled with nitrogen and a second curve n-as recorded to confirm the complete absence of oxygen.
0
0
d + 0.2 ::
a b
a
d
r
GALV. 0
Figure 2.
Typical polarographic curves
0~1.V HAC-Ac buffer, pH 4.52, 0.01% in gelatin
Iodoadrenochrome
Iodonoradrenochrome d
h
Concn. T sec. d n s , PA. H (mni).
D
2.27 X-lO-4X
4.52 X-10-4.1I
3 8 5 17 3 3
3 8 5 17 3 3
3.45 X'10-4.1f 4 2 5 If3 8 3
6.88 X-IO-4-U 3 9 5 16 6 3
aration of the iodonoradrenochrome. Both compounds can easily be prepared and aliquots of the reaction solutions can be polarographed without purification or separation from excess reagents. Diffusion currents increase linearly with concentration over the range investigated of 1 to 50 y in 5 ml. of total volume. PREPARATIOIV OF IODOADRENOCHROME
lpproximately 10-2 to 10-3,1f stock solutions were prepared from crystalline adrenaline bitartrate or free adrenaline in 2% acetic acid. Two tenths of a milliliter of hydrochloric acid and methanol were added to dissclve the less soluble free adrenaline in the preparation of the 10-23f solutions. These stock solutions remained colorless for several days under refrigeration. T o an adrenaline aliquot containing approximately millimole, 2% acetic acid was added to give a total reaction volume of approximately 5 ml. The exact total volume was found to be not critical if the solution was well buffered. Six millimoles of 0.LV potassium iodate per millimole of adrenaline were added and the solutions were mechanically stirred for exactly 4 minutes. During stirring the solution changes from colorless to a final color ranging from salmon pink to red-orange or deep red, depending on the initial concentration of adrenaline present. After stirring exactly 4 minutes, 2N sulfuric acid (1 ml.) was added, which decreased the pH to 1. The color of the solution changes to mauve or deep purple, indicating the substitution of an iodine atom in the adrenochrome indole ring. An amount of 0.2% potassium iodide (the stock solution was acidified with 0.5 ml. of 2N sulfuric acid) equivalent to 1.7 millimoles per millimole of adrenaline was added as soon as the mauve or purple color cf iodoadrenochrome appeared (less than 0.5 minute). Ali uots of this solution were then pipetted into the polarograp%ic cell which contained 0.1 ml. of 0.5'% gelatin and sufficient 0.1M acetic acid-acetate buffer (of pH 4.52) to give a final total volume of 5 ml. After the addition of the iodoadrenochrome aliquot, oxygen \vas removed from the buffer solution by bubbling a stream of prepurified nitrogen through the cell for 10 to 15 minutes. Complete removal of oxygen is required since it gives a wave in the vicinity
Table I. Titration of Excess Iodine with 0.1N Thiosulfate Equivalent of Adrenaline,
Conversion t o Iodoadrenochrome,
Mg.
%
21 4 18.2
88 110
18 2 ~.
09 .. 80 93 112
~
18.2 18.2 13.66
Av. Equivalent of Noradrenaline, hlg.
97
Conversion t o Iodonoradrenochrome
% 94 100 103
33,42 33,42 33.42 AV.
99
~
PREPARATION OF IODONORADREXOCHROME
Stock solutions were prepared from crystalline noradrenaline bitartrate monohydrate to contain approximately 10-2 to 10-3 millimole per milliliter. I n contrast to adrenaline, noradrenaline solutions diluted to volume with 0.2N sodium acetate were stable for about 4 to 8 hours. Solutions diluted with distilled water showed a color change only after several days. The oxidation was carried out with the same proportions of reagents used for adrenaline, hut 0 . 2 r sodium acetate was substituted for the 2 % acetic acid to give a less acid medium. The oxidation time TTas increased to exactly 6 minutes. The remainder of the procedure was carried out ex40 actly as for adrenaline. Aliquots were analyzed in ';. the same acetic acida c e t a t e buffer of p H L 1.52. 2 30
;; Y
\
LL Y 4 0 Y
RESULTS
Aliquots of the iodoadrenochrome or iodonoradrenochrome solutions containing the equivalent of 1 y or more of adrenaline or noradrenaline produced well defined and re01 02 producible polarographic c u r v e s (Figure 2). I n POLAROGRAPHIC D I F F U S I O N CURRENT I101 0 . l M acetic acid-acetate Fieure 3. Relation of Dolarographic diffusion current buffer of pH 4.52, 0.01% to the concentration of adin gelatin, at 2 5 ~ the renaline observed half-wave potentials were: E, 2 = 0.03 volt for iodoadrenochrome, and Eitz = 4- 0.02 volt for iodonoradrenochrome. Standard curves obtained from aliquots of iodoadrenochrome or iodonoradrenochrome solutions containing the equivalent of 1 to 50 y of adrenaline or noradrenaline (Figures 3 and 4) showed a linear relation between diffusion current and concentration for both compounds, The polarographic data are reproducible for duplicate or varying amounts of amine contained in aliquots of solutions of iodoadrenochrome or iodonoradrenochrome. S o difference was observed in conversion of free adrenaline and the bitartrate salt. To check the conversion of adrenaline and noradrenaline to their iodo derivatives by an independent method, the amines were oxidized following the above procedure and the excess iodine was titrated immediately with 0.1N sodium thiosulfate in the presence of 2 ml. of starch solution. Crystals of iodoadrenochrome and iodonoradrenochrome formed in these more concentrated solutions (containing the equivalent of 13 t o 33 mg. of amine) did not interfere with the titrations. Calculations were based on the published total uptake of 6 atoms of iodine per molecule of amine following Bouvet ( 3 ) . The titrations of five iodoadreno-
c,,
+
ANALYTICAL CHEMISTRY
1066 chrome solutions averaged 96.9%; those of three determinations of iodonoradrenochrome solutions, 99.0% (Table I). The reproducibility of conversion of each amine to its derivative and its subsequent measurement was established by a series of analyses carried out on known amounts of crystalline adrenaline bitartrate and noradrenaline bitartrate monohydrate. Stock solutions were used only on the day of preparation. From tTvo t o six aliquots containing the equivalent of 0.158 to 0.738 mg. of adrenaline were converted to the iodo compound according to the procedure given above. I n the case of noradrenaline, however, the amounts of each compound were treated as “unknown” -that is, to each solution was added an excess of 0.1N potassium iodate (0.4 ml.) and 0.2y0potassium iodide (0.8 ml.), sufficient t o convert as much as 6.6 X 10-3 millimole (1.1 mg.) of noradrenaline. Polarographic measurements were made in duplicate on aliquots of the iodoadrenochrome solutions containing the equivalent of about 20 y of amine. From the diffusion current obtained, the weight of adrenaline or noradrenaline contained in the aliquot was calculated from the slopes of the standard curves (Figures 3 and 4). The total amounts of compound were then found from the known volumes of the reaction solutions. The data are tabulated in Table 11. The per cent error of the method is 5 to 10%. The conversion of either adrenaline or noradrenaline is quantitative and reproducible under rigorously controlled conditions in both concentrated and dilute solutions. The theoretical minimum amounts required for complete conversion of the amines as calculated from Bouvet ( 3 )are 6 millimoles of iodate per millimole of adrenaline (or noradrenaline) and 1.7 millimoles of potassium iodide per millimole of adrenaline. Under these conditions, reproducibility of conversion of the amines to their derivatives depends on precise timing of the oxidation reaction and adequate stirring of the solution. Although the wave height is independent of variation in iodate added (Table I), the ratio of iodate to iodide must be kept constant to avoid either a large excess of iodate (greater than about twelvefold) which enhances t h e iodoadrenochrome wave, or e6 iodide, which itself p r o d u c e s a masking a n o d i c wave. f eoThe a p p l i c a b i l i t y of t h i s E method to bioz s logic s o l u t i o n s IsI s raises certain 0 questions. AdrenIOa l i n e a n d noradrenaline cannot 0 be measured indiI viduallp in the z c 82 6 same s o l u t i o n , since the differen8 tiating m e t h y l group, lying in 0 I I I I I I I I the indole ring, 0 0.02 0.04 0.06 0 0 6 0 10 0.12 0.14 does not signifiPOLAROQRAPHIC DIFFUSION CURRENT (,MI) cantly affect the Figure 4. Relation of polarographic diffusion current to the concentratials for the retion of noradrenaline spective reactions. I n contrast to other methods for the measurement of catechol amines, however, colored or fluorescent substances normally found troublesome do not interfere. Previous workers ( 2 , 4, 6, I O , 11) have separated noradrenaline and adrenaline by partition chromatography. Utilizing a paper
~
p
s
chromatographic method for the separation of noradrenaline and adrenaline prior to polarographic analysis, preliminary studies in the authors’ laboratory indicate the feasibility of collecting eluates containing these amines, converting to the iodo derivatives in the same test tubes and analyzing an aliquot of each. A single conversion and analysis can be done in 20 minutes. Less time is required for a group of samples done in series. Table 11. Accuracy of Measurement of Adrenaline and Noradrenaline after Conversion to Corresponding Iodonor- or Iodoadrenochrome Adrenaline No. of Conversions (Each RIeasured in Duplicate)
No. of Conversions (Not Measured in Duplicate) 2
Polarographic measurement, (a\. ), mg. 0 522 0 678 0 353 0 386 0 467 0 333 0 148 0 234 Millimole KlOa Added 0.025 0.02 0.005 0.02 0,005 0 02 0,009 0.02 0.01 0.01 0.04 0.01 0.04
Theoretical, mg.
0 0 0 0 0 0 0 0
499 738 394 362 527 316 158 246 Nonadrenaline Polarographic Theomeasurement retical, (av.), mg. mg. 0.487 0.441 0.245 0.220 0.234 0.239 0,267 0.239 0.252 0.241 0.241 0.229 0.260 0.237 0,240 0.237 0.416 0.403 0.368 0.395 0.318 0.365 0.334 0 344 0.365 0.426
Error,
%
+ 5 - 8 - 10
t i -11 + 5 - 6 - 5
Error,
70
+IO +11 - 2 +I1
+- 2 +IO
t
1
f 3
- 8 - 13 - 3 - 4
The polarographic method has the advantage of being rapid, specific, and sensitive. It should be particularly valuable for studies of the output of adrenaline and noradrenaline in urine. Levels of the catechols excreted by normal young males per 24 hours are of the order of 11.5 y of adrenaline and 29.0 y of noradrenaline (’7). This is within the optimum working range of the polarographic method. ACKNOWLEDGMENT
The technical assistance of Mary Jane Vetter is acknowledged. The authors wish to thank Sheppard Crane of Rinthrop-Steams, Inc., for his generosity in supplying the adrenaline and noradrenaline salts and J. B. PIIcCarthy of Parke-Davis, Inc., for adrenaline in the free form. LITERATURE CITED
Auerbach, 11.E., and Angell, E., Science, 109, 537 (1949). (2) Bergstrom, S., Euler, U. S. von, and Hamberg, U., Acta Chem.
(1)
Scand., 3 (1949). (3) Bouvet, P., Ann. pharm., 7, 721 (1949). (4) Crawford, T. B. B., and Outschoorn, 8. S., Brit. J . Pharmacol., 6,8 (1951). f5) Euler. U. S. von. Pharmacol. Reps.. 3. 247 (1951). i6j Euler, U. S. von, Hamberg, U., and Hellner, S., Biochem. J., 49, 655 (1951). (7) Euler, E. S. yon, and Hellner, S., Acta Physiol. Scand., 22, 161 (1951). (8) Fieser, L. F., “Experiments in Organic Chemistry,” 2nd ed., p. 395, Heath, Boston, 1941. (9) Heller, J. H., Setlow, R. B., and Mylon, E., Am. J . Physiol., 161, 268 (1950). (10) James, W. O., S a t u r e , 161, 851 (1948). (11) Lund, 4.,Acta Pharmacol. Toricol., 5, 231 (1949). (12) Manger, 13’. AI., Baldes, E. J., Flock, E. V.,Bollman, J. L.,
Berkson, J., and Jacobs, >I., Proc. Staff Meetings Mayo
Clinic, 28, 526-31 (1953). (13) Shaw, F. H., Biochem. J . , 32, 19 (1938). (14) Weil-llalherbe, H., and Bone, A. D., I h i d . , 51, 311 (1952). (15) Wiesner, K., Biochem. Z., 313, 48 (1942). RECEIVED for review June 1, 1954. Accepted February 10, 1955. This investigation was supported b y a grant-in-aid (H-1509) from the United States Public Health Service.
