at 410 and 430 m p Tvere utilized to construct calibration graphs for each wave length. The graphs were straight lines, but a t concentrations greater than 12 p.p.m., nonlinearity was encountered with each compound. Procedure. Two solutions lvere prepared for each unknown. One solution was made u p t o p H 5.5 b y dilution with acetate buffer, and t h e other was made u p t o pH 12.0 b y addition of 2 M sodium hydroxide. The fluorescence intensities of the two solutions were read a t 410 and 430 mp under 314-mp excitation. The readings on the solution a t p H 5.5 yield the concentration of salicylic acid directly from the calibration graphs. The readings on the solution at p H 12.0 are then utilized to obtain the intensity of the m-hydroxybenzoic acid fluorescence b y deducting the corre-
sponding intensity of the solution with a p H of 5.5. These intensities and the calibration graphs for nz-hydroxybenzoic acid yield the concentration of this compound present. If the first determination indicates a total concentration of the two compounds greater than 12 p.p.ni., a suitable dilution should be made and the determination repeated. RESULTS
Five test mixtures were prepared and analyzed b y the procedure outlined. The results (Table I) indicate that, in mixtures of 0- and m-hydroxybenzoic acids, both components can be determined viith a n error of less than 5%. p-Hydroxybenzoic acid is not fluorescent when exposed to 314-mp radiation and should have no effect on the method. This was tested by preparing
mixtures of the three isomers and applying the procedure. The results shorn in Table I1 indicate that the para isomer has no detrimental effect on the determination of the ortho and nieta compounds, LITERATURE CITED
(1) Boweri, E. J., Wokes, F., “Fluorescence of Solutions,” p. 42, Long-
mans, Green, London, 1953.
( 2 ) Collat, J. IT., Rogers, L. B., A N ~ L . CHEU.27, 961 (1955).
RECEIVEDfor review August 19, 1957. t\ccepted April 11, 1958. Division of Analytical Chemistry, 131st Meeting, dCS, Miami, Fla., April 1957. Work supported by a fellowship grant from Eastman Kodnk Co. Abstracted from thesis resented to Michigan State University y G. A. Thommes in partial fulfillment of requirements for the degree of doctor of philosophy.
E
Fluorometric Determination of Adrenalin and Noradrenalin in Aqueous Solution SIDNEY ROSTON’ Department o f Biochemistry,
U. S.
Army Medical Research laboratory, Fort Knox, Ky.
A method for determining adrenalin and noradrenalin in aqueous solution has been developed which depends on differential transformation of adrenochrome and noradrenochrome to the corresponding fluorescent substances. This technique results in values for adrenalin much more independent of the noradrenalin present than obtained by previous methods, and vice versa, and has high specificity and sensitivity.
T
HAS BEES conbiderable interest recently in the chemical deterniination of adrenalin and noradrenalin. The production of fluorescent compounds from adrenalin and noradrrnalin has brought the sensitivity of tlieir chemical determination within lmlogical range. One fluorescence ilietliod involves condensation of the c:itrchol amines n-ith ethylenediamine, \ u t is so nonspecific that catechol and niany other related conipound~ produce approximately the Fame fluorescwce as adrenalin and noradrenalin ( 4 ) . Another technique depends upon the frill dei-elopment of both adrenochrome aiid noradrenochrome at pH 5 to 6 and of only adrenochrome a t p H 3 to 4 ;
HERE
Present address, 2403 Clarendon Xve., 1,ouisville 5, Ky.
this results from the action of various oxidation agents, including manganese dioxide and potassium ferricyanide (1, 2 ) . A difficulty arises from the fact that the determination of a small amount of noradrenalin in the presence of a large amount of adrenalin depends upon the small difference between two relatively large values of fluorescence. The present method of determining adrenalin and noradrenalin does not depend upon differential development, but upon differential transformation of adrenochronie and noradrenochrome. Values for adrenalin are more independent of the amount of noradrenalin present than in previous methods, and vice versa. The specificity of the determination of adrenalin aiid noradrenalin through the adrenochrome and noradrenochrome stage is probably very high, no other biological substances of +dar structure having been found to react appreciably in a similar fashion
(0. EXPERIMENTAL
Materials.
T h e Farrand Optical
Co. fluorometer, Model A, n-as used t o measure fluorescence. T h e primary filter was Fisher KO.365 with maximum transmittance a t 365 nip, and the secondary filter \vas K r a t t e n KO. 57, green in color. All cheniicals were of ACS reagent grade. The
adrenalin and noradrenalin were in the form of the bitartrates, from Kinthrop-Strarns Co., S e w York. Solutions of 10 y of these amines per nil. of 0.01S hydrochloric acid were stable for a t least several months if refrigerated; aliquots were taken for daily use. Other reagcnts included 5‘Y sodium hydroxide ; 0.25y0 aqueous potassium ferricyanide solution; 1V sodium ncetate buffer, p H 5.5; and a solution containing 8 nig. of ascorbic acid per nil. of water, which was prepared each day. Dietilled water was used. Methods. From t h e aqueous solution containing t h e unknown amount of catechol amines. 2-ml. portions are pipetted into two separate test tubes. T o each tube is added 1 ml. of t h e acetate buffer and 0.1 ml. of the ferricyanide solution. T h e reaction is then prrniitted to proceed a t 25’ C. for 5 minutes. bIean\Thile, three test tubes are prepared: One contains 0.i5 ml. of the 5-1- sodium hydroxide and 5.9 nil. of n-ater, a second contains 0.25 nil. of the ascorbic acid solution, aiid a third contains 4 nil. of the ascorbic acid solution, 0.75 1111. of 5-1- sodium hydroside, and 2.15 nil. of water. I n the preferential determination of noradrenalin-a technique n-hich will be designated the noradrenalin method -the contents of one of the two tubes containing the oxidized catechol amines are intermixed sei-era1 times rapidly TI ith the contents of the tube containVOL. 30, NO. 8, AUGUST 1958
1363
ing the sodium hydroxide only. After 30 seconds 0.25 ml. of ascorbic acid solution is added and mixed thoroughly. To determine adrenalin preferentially -the adrenalin method-the contents of the second tube containing the oxidized catechol amines are added to the sodium hydroxide and 4 ml. of ascorbic acid and mixed thoroughly. For either method. the fluorescence is read after 5 minutes. The fluorescence remains unchanged for a period of a t least 0.5 hour. It is essential to use only one cuvette compartment and one fluorometer tube and to have the tube lined u p in identical fashion for each reading, as a single sample may give significantly different readings when the tubes and compartments used as well as the tube positions are varied. Several standards of different concentrations should be run simultaneously with the unknon-n (adrenalin in the adrenalin method and noradrenalin in the noradrenalin method). I n case several amplification settings must be used to determine the optimal fluorescence readings of the samples, a standard n-ill be available for each scale of amplification. The blanks of the adrenalin and noradrenalin methods are prepared by adding either 2 mg. or 32 mg. of ascorbic acid first to 2-ml. aliquots of the unknown solution, followed by the addition of the ferricyanide and the other reagents of the tests. Five minutes after mixing of the reagents, the fluorescence reading of the blank in the noradrenalin method is 1.3 times and in the adrenalin method, 1.4 times the reading of distilled water. The net fluorescence of the unknown solutions and the standards is determined by subtracting the readings of fluorescence of the appropriate blanks from the readings of the samples and standards. The calculation of the amount of adrenalin and noradrenalin originally present involves the solution of two linear simultaneous equations: KA
+ 0.22KN
0.043KA
=
X
+ 0.43KN = Y
ANALYTICAL CHEMISTRY
1. lu’oradrenalin, 1 7 ; water reading, 7 3. Adrenalin, 1 y; water reading, 4
noradrenalin in the noradrenalin niet hod. 9 and Y may each be determined in that part of the range of amplification where the determination is most accurate, but not necessarily a t the same amplification. It is necessary in each case to have available a standard whose reading falls appropriately near the reading of the unknown. I n case different amplifications are used, the values of K in Equations 1 and 2 above will be different. The solutions of Equations 1 and 2 are
d
=
(X - O.XY)/0.98K
and N = (Y
- 0.04X)/0.42K
RESULTS AND DISCUSSION
Noradrenalin Method. T h e basis for t h e preferential determination of noradrenalin is that, while adrenochrome is transformed almost in-
(1)
(2)
where K is the net reading of 1 y of adrenalin in the adrenalin method as determined from the measurements of the standards; A is the amount of adrenalin and AV is the amount of noradrenalin; X is the net fluorescence reading found for the unknown in the adrenalin method and Y , that found in the noradrenalin method. The coefficients of Equation 1 result from the fact that noradrenalin yields 22% of the fluorescenee derived from adrenalin in the adrenalin method. The coefficient of noradrenalin in Equation 2 is derived from the fact that the fluorescence of 1 y of noradrenalin in the noradrenalin method is 43y0that of 1 y of adrenalin in the adrenalin method, Finally, the coefficient of the adrenalin in Equation 2 results from the fact that adrenalin yields only 10% of the fluorescence of the same amount of 1364
Figure 1. Fluorescence vs. time of contact with 0.5N sodium hydroxide before addition of ascorbic acid
.r‘ N O R A D R E N A L I N
Figure 2. Fluorescence vs. amount of noradrenalin with varying amounts of adrenalin present, obtained according to noradrenalin method 1. aidrenalin, 0 y ; water reading, 6 2 . ildrenalin, 1 7 ; water reading, 6
3. Adrenalin, 5
y;
water reading, 6
stantaneously into adrenolutine, the formation of noradrenolutine from noradrenochrome proceeds, in exponential fashion, essentially t o completion in 3 minutes (1). If an alkaline environment is permitted to act upon adrenochrome and noradrenochrome for various periods of time before the addition of 2 mg. of ascorbic acid, the results shown in Figure 1 are obtained. I n the absence of ascorbic acid, the different patterns of oxidation of adrenochrome and noradrenochrome result from the differences in the rate of formation of adrenolutine and noradrenolutine, as the rates of breakdown of adrenolutine and noradrenolutine are approximately the same (9,3’). The ascorbic acid prevents further destruction of the fluorescent substances while not preventing their formation. It is evident from Figure 1 that the amount of residual fluorescence is considerably less for adrenochrome than for noradrenochrome. Amounts of sodium hydroxide between 0.65 and l ml. yield optimal results with the least loss of noradrenolutine fluorescence relative to the residual adrenolutine fluorescence. For amounts of sodium hydroxide from 0.65 to 0.05 ml., the residual noradrenolutine fluorescence increases but the residual adrenolutine fluorescence is relatively greater. Thirty seconds of contact n i t h 0.75 ml. of sodium hydroxide before addition of ascorbic acid gives the best results-Le., retention of 70% of the noradrenolutine fluorescence but only 4% of the adrenolutine fluorescence. Bubbling of oxygen through the solution during the 30second period does not change the results. The results of the noradrenalin procedure as outlined aboi-e are shown in Figure 2, both for noradrenalin separately and in mixtures with adrenalin. The results for adrenalin alone are strictly linear up to at least 10 y. For 5 y of adrenalin the resultant fluorescence is less than the sum of the fluorescences derived separately from the adrenalin and noradrenalin present, as the line for adrenalin plus noradrenalin is not parallel to that for noradrenalin alone. For 1 y of adrenalin, this divergency of lines is less marked. Even for 2 y of adrenalin, because of the lo^ residual fluoresceiice of adrenolutine relative to noradrenolutine, very little error 11-ill be introduced as a reqult of the nonadditivity. This nonadditivity is more marked a t 60 secoiids’ reaction with 0.75 ml. of sodium hydroxide. I n the presence of up to 2 y of adrenalin, noradrenalin on the a i erage yields a fluorescence equal to that of 10 times as much adrenalin. The results of four sets of readings, each consisting of six simultaneous determinations by the noradrenalin
y 90
4
5 BO
301 20
i
2
LL
j
l
0
I
5
,
IO
i
I
I
I
I
I
I
15 20 25 30 35 40 45 MG. ASCORBIC ACID
]
o
0
50
Figure 3. Fluorescence from adrenalin and noradrenalin obtained according to adrenalin method vs. amount of ascorbic acid
Final total volume maintained at 10 nil. by adjusting water added at last stage of reaction 1. Koradrenalin, 1 r ; water reading, 7 2. Adrenalin, 0.5 -1; water reading, 8
method, are presented in Table I ; calculations have been made, where necessary, in terms of the amount of noradrenalin which would ,produce the same readings. The standard deviations fall \\-ell xithin 10% of the corresponding results. Adrenalin Method. T h e method for t h e preferential determination of adrenalin depends upon t h e finding that, in a n alkaline solution containing 2 t o 48 mg. of ascorbic acid, there is a much greater loss of noradrenolutine t h a n of adrenolutine fluorescence. As increasing amounts of ascorbic acid are added t o t h e noradrenochrome solution simultaneously with 0.75 nil. of 5.V sodium hydroxide, there is a lessening of the final plateau values to 11-hich the noradrenolutine fluorescence rises (Figure 3). As compared with the results for 0.75 nil. of sodium hydroxide and 32 mg. of ascorbic acid, there is a 30y0 increase in the noradrenolutine fluorescence upon increasing the sodium hydroxide to 1 ml.. and a 2OY0 decrease upon decreasing it to 0.5 nil. The adrenolutine fluorescence remains unchanged betn-een 0.5 and 1 nil. of 5 s sodium hydroxide. The optimal value is 0.75 ml. rather than 0.5 ml. because, in potential applications of this technique to blood, tissues. and urine, the higher alkalinity nil1 be less significantly affected by any buffering agents iyhich may be present in biological preparations. K i t h 32 nig. of ascorbic acid, also the optimal amount for the effects desired, the loss of adrenolutine fluorescence is only loyo, but that of the noradrenolutine fluorescence is 64%. Curves of adrenolutine and noradrenolutine fluorescence, individually or to-
, .I
I .2
I
I
.3 .4
I
I I .5 -6 .7 ?' A D R E N A L I N
.8
l
l
.9 .IO
Figure 4. Fluorescence vs. amount of adrenalin with varying amounts of noradrenalin present, obtained according to adrenalin method 1. 2.
Xoradrenalin, 0 Noradrenalin, 2
y; -?;
x-ater reading, 3 water reading, 3
gether, are linear, and parallelism of lines exists where expected (Figure 4). Thus, there is no evidence of nonadditivity of the fluorescence derived from mixtures of adrenalin and noradrenalin in this method. On the average, adrenalin yields the same net fluorescence as 4.5 times as much nqradrenalin. The results of four sets of readings, each consisting of six simultaneous determinations in the adrenalin method,
Table I.
Determination of Adrenalin and Noradrenalin
(Values given in micrograms) Equivalent WorAmount adren- Adren- Pres- Std. a h alin ent Found Dev. Noradrenalin Method Xoradrenalin 0.50 ... . . . 0.48 0.02
Present
0.10 0.20 0:70 0.10 0.40
0.093 0.006 0 : 2 i 0 0.255 0.014 0,140 0.147 0.007
Adrenalin Method Adrenalin
. . . 0.10 0.20 0.20 0:OkO 0.30 0.10
0 , 0 9 8 0,004 0:044 0.046 0.002 0.094 0.098 0,005 0.166 0.160 0.006
Table II. Determination of Adrenalin and Noradrenalin in Mixtures ~
Present, y XoradrenAdrenalin alin 0.70 0.80 0.20 0.03 0.15 0.15
0.40 0.15 0.65 0.02 0.25 0.06
Found, y XorAdrenadrenalin 0.62 0.76 0.31 0 025 0.17 0.15
alin 0.39 0.16 0.60 0,023 0.26 0.066
2
20 4 0 60 80 0 100 120 140 160 180 L
y.
SECONDS
0 I0
Figure 5. Fluorescence vs. time of exposure of adrenochrome and noradrenochrome to ascorbic acid a t pH
5.5 Action of ascorbic acid terminated by adding 0.75 ml. of sodium hydroxide and sufficient water to make 10-ml. final volume 1. Ascorbic acid, 32 mg.; noradrenalin, 1 -/; water reading, 9 2 . Aiscorbicacid, 16 mg.; noradrenalin, 1 -/; water reading, 9 3. Ascorbic acid, 32 mg.; adrenalin, 0.5 y ; water reading, 10 4. -1scorbic acid, 16 mg.; adrenalin, 0.5 -/; water reading, 10 expressed, where necessary, in terms of the equivalent amounts of adrenalin, are presented in Table I. Again, the standard deviations fall well within 10% of the corresponding results. Ascorbic acid, in amounts from 2 to 50 nig., has no significant destructive effect upon formed adrenolutine and noradrenolutine. A possible explanation for the mode of action of the ascorbic acid in these reactions may be deduced from the results shown in Figure 5 , Bt p H 5.5 the interaction of the ascorbic acid and the adrenochrome and noradrenochrome causes a loss of adrenolutine and noradrenolutine fluorescence. Cnder these conditions even as little as 2 mg. of ascorbic acid produces a 35% loss of noradrenalin fluorescence in 5 minutes and a 62% loss in 10 minutes; for adrenalin the corresponding losses are 18 and 2670, respectively. If it is assumed that a similar interaction of these oxidation products of the catechol amines and ascorbic acid occurs in 0.5S sodium hydroxide. tn o simultaneous pathways of reaction would be available t o noradrenochrome and adrenochrome-Le. the interaction with ascorbic acid and the forniation of adrenolutine and noradrenolutine. The almost instantaneous formation of adrenolutine from adrenochrome would prevent any significant interaction with the ascorbic acid. Horvever, the slower rate of formation of noradrenolutine from noradrenochrome ivould allow time for some interaction with ascorbic acid, VOL. 30, NO. 8, AUGUST 1958
1365
and there would be a resultant loss of noradrenolutine fluorescence. Determination in Mixtures. T h e results of t h e determination of both the noradrenalin and adrenalin in a series of six aqueous solutions containing known amounts of these amines are presented in Table 11. For 0.03 to 1 y of adrenalin and 0.05 to 1 y of noradrenalin the recovery of the catechol amines is within lOyo of
the amount added. The lower limits of the method, for 20% accuracy, are Wproximately 0.01 y of adrenalin and 0.02 y of noradrenalin. ACKNOWLEDGMENT
The author would like to express his appreciation t o Hans Jeiisen and Ulrich Westphal for their interest in his work.
LITERATURE CITED
(1) Euler, U. S. v., Floding, I., Acta Physiol. Scand. 33, Suppl. 118, 45 (1955). (2) Lund, A., Acta Pharmacol. Tozicol. 6 , 137 (1950). (3) Mylon, E., Roston, S., Am. J . Physiol. 172, 612 (1953). (4) 11-eil-hlalherbe, H., Bone, -4. D., Bzochem. J . 51, 311 (1952).
RECEIVEDfor review, ~~l~ 31, 1957. Accepted RIarch 6, 1958.
Complexometric Titration of Calcium in the Presence of Magnesium ALEXANDER D. KENNY and VICTOR H. COHN Department o f Pharmacology, Harvard Medical School, and Biological Research laboratories, Harvard School o f Dental Medicine, Boston 7 5, Mass.
b By modifying the (ethylenedinitril0)tetraacetate complexometric titration method of Munson and his associates for the determination of calcium (20to 50-7aliquots), it has been possible to raise the limit of magnesium interference, arbitrarily taken as greater than 24/,, from a magnesium to calcium weight ratio of 3 to above 16. The modification entailed ensuring that the p H of the titration solution was 12.4 to 12.5. At lower pH values calcium was overestimated, and a t higher pH values it was underestimated.
A
and precise (ethylenedinitri1o)tetraacetate (EDTA) coniplexometric titration method for determining calcium has been developed ( 5 ) . Kenny and Toverud ( 4 ) indicated the noninterference of phosphate in a similar method in the presence of phosphorus to calcium ratios as high as 8. The degree of magnesium interference was studied so that the possibilities of the application of the method to the analysis of calcium. particularly in biological tissues, could be more clearly defined. K i t h the usual procedure, there was interference a t ratios of magnesium to calcium as lovi as 3; with the modified method, no important interference occurred with ratios as high as 16. RAPID
REAGENTS AND EQUIPMENT
Coleman junior spectropliotometer, using Evelyn colorimeter tubes (No. 4635, Rubicon Co., Philadelphia, Pa.) in a Coleman cuvette adapter (No. 6-102, Coleman Co., Inc., Los Angeles, Calif.). The adapter was turned on a lathe to enlarge the internal diameter for use with the Evelyn tubes. 1366
ANALYTICAL CHEMISTRY
A Beckman Rlodel G pH meter was used for p H determinations with the Beckman Type E glass electrode. EDTA solution, 0.18 gram of disodium dihydrogen (ethylenedinitril0)tetrancetate p e j liter of distilled water. Standard calcium solution. 10 nig. per 100 ml. Anhydrous calciuni carbonate, standard luminescent grade (llallinckrodt Chemical Korks, St. Louis, No.), was dried for 4 hours a t 130" C., and 0.2497 gram of t h r dried salt was dissolved in 25 ml. of water containing 2 ml. of concentrated hydrochloric acid and diluted to 1 liter with water. This was assumed to be a primary standard ( 1 ) . Stock magnesium solution, 100 mg. per 100 ml. Reagent grade magnesium sulfate heptahydrate m s used. \Torking magnesium solution, 50 and 5 mg. per 100 nil. Ammonium purpurate solution. The ammonium purpurate, 50 mg. (Hagan Corp., Pittsburgh, Pa.), was shaken n-ith 25 ml. of water sild filtered into a broivn bottle. Fresh solution was prepared after 2 days. 2-Octanol, 20% in 9.5% ethyl alcohol. All water used ]vas double-distilled. PROCEDURE
The E D T A complexometric titration method developed by hIunson and his associates ( 5 ) n a s used with a slight modification. The calcium solution to be analyzed vas unbuffered and a t a neutral pH to avoid the necessity of finding the precise amount of sodium hydroxide needed for a final pH of 12.4 to 12.5. The solution for titration was prepared in an Evelyn colorimeter tube by mixing a calcium aliquot of 20 to 50 y, varying amounts of working niagnesiuni solutions, water to n total volume of 9.0 nil., 2.0 nil. of 0 . 2 5 sodium hydroxide, 1 drop of 2070 2octanol, and 0.3 nil. of ainnioniuni purpurate solution. The final p H was
12.4 to 12.5. The colorimeter tube was immediately placed in the special cuvette adapter in the spectrophotonieter. A bubbling device and a buret with extended tip were lowered into the colorimeter tube. The solution was titrated in the spectrophotometer a t 520 nib. Nitrogen or air was bubbled in to mix the contents during each addition of 0.2 ml. of the EDTA solution. Readings of transmittance were taken after each addition. By plotting the points on seiiiilogarithmic graph paper, the end point n a s determined by the point of intersection of the two straight lines. There Ivere three or four replicates a t each ratio of magneqiuni to calcium. All values of calcium found were expressed as a percentage of the theoretical calcium contents follon ed by the standard deviation. The values which received suhsequcnt statistical treatment n-ere determined on random samples, the analyst being unanare of the code. RESULTS
The original procedure (6) rvas follo\ved in which the pH was not rigidIy controlled; 4 drops of 9 N sodium hydroxide n'ere addfd to the calciuni aliquot in a total volume of 10 nil. As the ratio of the neight of magnesium to calcium was increased from 0 to 10, the degree of interference Ivas greater. From the data of an experiment in which the aliquot of calcium \vas 50 y. the regression line, represented by the equation I' = 99.99 - 0.721X, was calculated. I t is plotted in Figure 1, experiment 1 together with the observed points. Extrapolation of the regression line, represented by the dotted line, is justifiable; in other experiments conducted under similar conditions. a magnesium to calcium
