Crotonic acid and methyl crotonic acid do not fall on the line of the D us. u plot (Figure 2) but require more polar solvents to obtain the reactivity predicted from the substituent constants. Hine and Bailey (10) found that crotonic acid reacts much more slowly with diphenyldiazomethane than m-ould be expect,ed from the polar substituent constant originally obtained from ionization studies. They explained this on the basis that some of the resonance due to conjugation of the carbonyl group with the olefinic grouli was lost in going to the transition state. In the present study, this explanation also applies that the effect should he more pronounced because there is a complete loss of conjugation in the bromination reaction and therefore a complete loss of such resonance. In summary, some generalizations concerning the selection of a solvent for the bromination of specific compounds, especially as applied to rate studies, can be made. For conjugated unsaturated carboxylic acids, a solvent of dielectric constant about 55, for exa,mple 50Y0 methanol, should be used, but’ for acids in which the carbonyl group is not conjugated with the unsaturated linkage, nonpolar solvents should be used. For olefinic compounds substituted with an amide or nitrile group, 707, concentration or higher in methanol up to pure methanol are appropriate. For esters,
solvents of lower polarity range, acetic acid to methanol are indicated. Because triply bonded compounds have more electrophilic character than olefinic compounds, their rates of bromination are relatively slower and, for this reason, water was a good solvent for butynediol. In contrast, olefinic compounds containing hydroxyl groups require the most nonpolar solvents and other conditions such as lowered temperatures are necessary. Compounds containing only the electrondonating alkyl substituents also require special conditions to slow the reactions sufficiently. For unsaturated compounds, mono-, di-, and tri-substituted with halogens, the corresponding solvents indicated are the least polar ones-pure methanol and about 60% methanol, respectively. The reactions of tetra-substituted halogen compounds are too slow even in the most polar solvents and these compounds require special conditions such as elevated temperatures. In each of these cases, additional suLstituents including alkyl groups in the same molecule contribute to the electronic character of the unsaturated bond and will alter the total u value. LITERATURE CITED
(1) Albright, P. S., Gosting, L. J., J . Am. Chem. SOC.68, 1061 (1946).
( 2 ) Anantakrishnan, S. V.)Ingold, C. K., J . Chem. SOC.1935, pp. 984, 1396.
(3) Anantakrishnan, S. Y.j Venkataraman, R., Ibad., 1939, p. 224. (4! Charton, L l . , J . Org. Chem. 26, 735 (1961). (5) Charton, M.,Sfeislich, H., J . A m , (‘hem. SOC.80, 5940 (1958). (6) Frost, A. A., Pearson, R. G., “Kinetics and Mechanism,” p. 130, %‘]ley, Sew York, 1953. ( 7 ) Hammett, L. P., Chem. Revs. 17, 125 119351. (8) Hammett, L. P., Trans. Faraday SOC.34, 156 (1938). (9) Hine, J., “Physical Organic Chemistrv,” D. 215.- McGraw-Hill. New Yock, 1$62. (10) Hine, J., Bailey, W. C., Jr., J . Am. (‘hem. SOC.81, 2075 (1959). (11) Ingold, C. K., “Structure and Mechanism in Organic Chemistry,” p. 658, Cornel1 Cniversity Press, Ithaca, 1953. (12) JaffB, H. H., Chem. Revs. 53, 191 (1953).
(13) kirkwood, J. G., J . Chem. Phys. 2, 351 (1934). (14) Imdler, K. J., Eyring, H., Ann. ‘Y. 1’. Acnd. Sci. 39, 303 (1940). (15) LIcDaniel, 1). H., Brown, H. C., J . Org. (’hem. 23, 420 (1958). (16) Price, \$’. C., (’hem. Revs. 41, 257 (1947). (17) Robertson, P. W., de la Mare, P. B. D., Johnston, W. T. G., J . Chem. SOC. 1943, p. 276. (18) Siggia, S., Hanna, J. G., Serencha, 5 . XI.. ANAL.CHEM.35. 362 (19631. (19) Walker, I. K., Robertson, P. ’W., J . Chem. SOC.1939,p . 1515. RECEIVEDfor review April 13, 1964. Resubmitted October 28, 1964. Accepted February 18, 1965.
Single Extraction Method for the Simultaneous Fluorometric Determination of Serotonin, Dopamine, and Norepinephrine in Brain ROBERT M. FLEMING and WILLIAM G. CLARK Psychopharmacology Research 1aboratories, Veterans Administration Hospital, Sepulveda, Calif., and Department of Biological Chemistry, University of California Center for Health Sciences, 10s Angeles, Calif. ERIC D. FENSTER’ and JACK C. TOWNE Veterans Administration Hospital (Research), and Department of Biochemistry, Northwestern University Medical School, Chicago, 111.
A method has been developed for a single solvent extraction and the simultaneous determination of serotonin (5HT), dopamine (DA), and norepinephrine (NE), in less than 1 gram of brain tissue in the presence and in the absence of relatively large amounts of their precursor amino acids and some related analogs. Essentially the method involves acetone extraction of the amines from brain homogenates; removal of the acetone by evaporation in vacuo;
692
ANALYTICAL CHEMISTRY
S
extraction of the residue with butanol saturated with 0.01 N HCI; addition of heptane to return the amines to an aqueous phase; passage of the aqueous phase through an ion exchange column which retains the amines but not the amino acids; and elution and fluorometric analysis of the amines. The endogenous amine content (pg.1 gram fresh tissue f std. dev.) found in 1 1 mouse brain pairs was: 5HT, 0.55 f 0.03; DA, 0.70 5 0.06;
to the roles of t h e biogenic amines 5HT, D-4, and NE in brain have been made possible by increasingly improved and sensitive means of determination. Present methods are capable of efficient extraction of just one or two amines by agents such as butanol (6, I ? , fg), acetone (1, 2, 9. l o ) , perchloric acid ( 5 ) , or trichloroacetic acid (Wf). However,
NE, 0.48
Ill.
f
0.03.
TUDIES RELATED
1 Present address, Department of Biochemistry, rniversity of Chicago, Chicago,
none of these cited methods are suitable for the relatively raliid and simultaneous determination of the three major monaminer in the same sinal1 sample of brain or other tissue. Recent,ly two of us, E. F. and J . T. (22, 25), report,ed in abstract form an acetone extraction method for t’he fluorometric analysis of brain 5 H T and S E . This made possible better comparison of bioassay and fluorometric results. We have now extended this work to permit quantitative, simultaneous extraction and determination of 5HT, NE, and D h in less than 1 gram of brain tissue, even in the presence of large amounts of the precursor amino acids 5-hydroxytryptophan (5HTP), 3,4-dihydroxyphenylalanine (DOPA), and its analog 3-(3,4dihydroxyphenyl)-2-methylalanine (CYmethyldopa). Application of this method a t its full sensitivity requires great care in the preparation of reagents. All solutions of mineral acids, bases, and buffers are prepared from reagent grade chemicals with water that has been double-distilled from glass. EXPERIMENTAL
Preparation of Solvents and Reagents. ACETONE. Reagent grade acetone (J. T. Baker Chemical Co., Phillipsburg, X . J.), is distilled from potassium permanganate (KMnO,, C.S.P.), 1.5 grams of K M n 0 4 per liter of acetone, and stored in the refrigerator. Experience has shown t h a t the same results are obtained after several weeks storage as with freshly distilled acetone. Just prior to use, it is saturated with potassium metabisulfite (K2S20L,, reagent grade, Rlerck & Co., Inc., Rahway, 9.J.), centrifuged, decanted, and 1 S formic acid (Mallinckrodt Chemical Works, S. Y.) added (formic :acetone, 1 :20
v.iv.). BUTANOL.
Reagent grade l-butanol (Fisher Scientific Co., Philadelphia, Pa.) is purified by successive washings once each with an equal volume of 1,V KaOH, 1N HC1, and three times or more with glass-distilled water, following which the pH of the butanol is above 3, as specified by Shore and Olin (19). The butanol is then equilibrated with 0.01N HC1 by mixing it twice with the acid (200 ml. of O.OILV HClAiter butanol). After the second mixing, the organic and aqueous phases are left in contact with each other. This can be stored at, room temperature for a t least a month. WHEPTANE. Reagent grade n-heptane (Phillips Petroleum Co., Bartlesville, Okla., 99 mole % minimum), is washed successively with a n equal volume of LV S a O H , 1A‘ HC1, and twice with glass-distilled water. I t can be stored a t room teniperature a t least a month. IOI)II\JE REAGENT(0.02,V). Reagent grade 11 (Mallinckrodt Chemical Works, N. Y.), 253.8 mg.] is dissolved in 100 ml. of absolute ethanol. This is prepared every month and stored in a
brown bottle at room temperature in the dark. SOIIIUMSULFITE(0.02s). Reagent grade anhydrous YazS03 (Mallinckrodt), 25.2 nig..ml. 4 . 5 s S a O H , is made every month and stored a t room temperature in a polyethylene bottle. SODIUM THIOSULFATE (0.025N). Reagent grade NaZS203. 5 H 2 0 (Mallinckrodt), 620 nig./lOO ml. water, is made every month and stored a t room temperature. ASCORBICACID (0.011.Y) FOR DX OXIDATION.Reagent grade (Fisher), 2.0 mg./ml. 5.V HCI, is made just prior to use. ASCORBIC.ACID (0.0115) FOR XE OXIDATION.Same, but 2 mg./:ml. of 5 S KaOH, is made just prior to use. .ASCORBIC .ACID (0.0112V) IN HzO FOR S E TISSUE BLASK. Same, but, 2 mg./ml. of H 2 0 , is made just prior to use. STANDARI) .A~IINI: SOLUTIONS.Solutions of the standard amines calculated as free base (5HT, D h , S E ) are made up to concentrations of 40 pg./ml. in 0 . 0 1 S HC1. The solutions are stable in the refrigerator for several weeks. Working standards are prepared daily by appropriate dilution with 0.01:4’ HCI. The amines used are 5HT, 5-hydroxytryptamine creatinine sulfate (“13”-grade, Calbiochem, Los Angeles); DX, Dopamine, 3hydroxytyramine HCI (“A”-grade, Calbiochem) ; NE) L-norepinephrine bitart’rate (Winthrop Laboratories,
s.Y.).
Preparation of Ion Exchange Resin. Amberlite CG-50, type 2, 200- to 400-mesh (Rohrn and Haas Co., Philadelphia, Pa.) is washed and cycled through the sodium and acid forms according to Hirs, Moore, and Stein ( 1 3 ) , and buffered to pH 6.1 u-ith 0.2.V NaH2PO4-K2HPO4 buffer, p H 6.1. The buffered resin can be stored for several weeks a t room temperature. Extraction of Amines from Brain Homogenates. ])rains, from 18 to 22 grams male, Swiss albino mice sacrificed by decapitation, were rapidly removed, rinsed in ice cold isotonic saline, blotted, weighed on a torsion balance, and homogenized with the special acetone for 30 seconds in a WrTis high speed homogenizer (VirTis Co., Inc., Gardiner, N. Y.) a t 40,000 r.p.m. Each tissue sample comprised two brains with a total weight of 0.75 to 0.90 gram, although the method can be adapted for much smaller samples, using smaller volumes and microcuvettes. The acetone: tissue ratio was 20: 1, Amine extraction was facilitated by allowing the homogenate to stand for 30 minutes with occasional manual swirling, and was then centrifuged for 5 minutes a t 380 x G and the supernatant fluid decanted into 1.5 X 25 em. screw-cap, roundbottom glass centrifuge t’ubes. This first extraction removed 1 0 0 ~ o of the estractahle 5 H T and 95% each of D h and S E from the tissue, making it unnecessary to re-extract the residue. The tubes were placed in a I3uchler Rotary “Evapo-Mix” (Iluchler Instru~
7
.
1
~
.
ment>s,Inc., S . Y.) and the extract was evaporated to dryness in vacuo (10-1 mm. Hg) at, room temperature. Foaming was avoided by the rapid rotational movement before and during the application of the vacuum. Eight ml. of the 0.01S HCI-equilibrated butanol was then added and the tubes were agitated for 1 to 2 minutes on a Vortex Jr. Mixer (Scientific Industries, Inc., Queen’s Village, N. Y.) to expedite solubilization of the residue. One ml. of 0.0LV HCI and 16 ml. of heptane were added and the amines were driven into the aqueous phase using agitation on the Vortex mixer for about 1 minute. After centrifuging a t 380 X G for 5 minutes to separate the two phases, all but traces of the organic phase were carefully aspirated off. hliquots (described below) of the 1.8-ml. aqueous phase, resulting from the added HC1 and t’he 0.8 ml. dissolved in the butanol phase, were analyzed fluorometrically for the amines with the Xminco-Rowman spectrophotofluorometer (American Instrument Co., Silver Spring, Md.) using an Osram xenon arc, a 1P21 photomultiplier tube, slit arrangement No. 5 and fused quartz cuvettes (4.25 ml. total volume). I n experiments in which amino acid precursors or their analogs were present, the aqueous phase was first passed through a CG-50 resin column and the eluate was analyzed for the amines. I n this case the 1 ml. of 0.01N NC1 is omitted and addition of heptane results in an. 0.8-ml. aqueous phase which is then buffered to pH 6.0 with 0.7 ml. of the 0.2M sodium-potassium phosphate buffer p H 6.1 and run through a 0.6 X 1-cm. bed of the resin at atmospheric pressure. Traces of organic phase remaining were carefully aspirated off prior to running the sample through the column. The resin bed was contained in a 0.6 X 15-cm. glass chromatograph tube, previously washed with 10 ml. of 0.02144 ?ITaH2P04-K2HP04buffer a t pH 6.1. The sample tube was rinsed with 0.5 ml. of 0.02M phosphate buffer and the rinse was also placed on the column. When the level of the liquid had dropped just to the top of the resin, 1 ml. more of the 0.02M phosphate buffer was run through to wash through the amino acids. The amines were retained on the resin and were eluted with 4 ml. of 1 J i sodium acetate-acetic acid buffer, p H 5.2, a t atmospheric pressure. Fluorometric Analysis of Amines. 5 H T was determined by its inherent fluorescence in 3hr HC1 a t 300-mp excitation and 550-mp fluorescence wavelengths (6). A 0.6-ml. aliquot of the aqueous phase or 1.0 ml. of the column eluate was used for analysis. An equivalent volume of 6.ON HC1 was added to the aliquots and the samples were read in the spectrophotofluorometer. All mp values reported are uncorrected. .A reagent blank run through the entire procedure and read a t 3001550 m p was used as the 5HT blank. This reading was subtracted from that of the samlile to obtain the 5H‘T fluorescenw-contribution of the unknown or standard. VOL. 37, NO. 6, M A Y 1965
693
6-
Light scatter peak 2
;i 3 I: g ;;
6-
5-
3 31
5-
li 1;
I :i
RESULTS A N D DISCUSSION
4-
'
For all three amines the procedure has been studied regarding recovery, specificity, and sensitivity. h number of investigators using bioasbay procedures have alluded to the inability of acetone to permit a stable recover). of serotonin from biological tissues (24). Using the fluorometric techniques, we found that variable and poor recoveries uere obtained from reagent grade acetone: however, uuon distillation from K M n 0 4 and the addition of formic acid, the losses could be obviated. Metabisulfite is required for protection of norepinephrine but not for serotonin. The volatile acidic-reducing solvent (the weuared acetone) urovides a stable medium for the catechols which are unstable in an alkaline solution. When authentic standards were introduced into the prepared acetone, the acetone evaporated off and the residue containing the amines dissolved in 0.01S HC1, analysis of an aliquot of the resulting solution by the fluorometric procedure showed no loss of DA or S E but a loss Of 30 to 60% Was Observed in 5HT. h similar loss occurred when 5HT standard TS as merely introduced into a tube from which acetone had been evaporated. However, in the presence of tissue no such loss in 5HT occurred. The extraction recovery of each amine investigated was reliably uniformly 65% n hen compared to external standards, that is, authentic amines which had not been subjected to extrac-
luorescent peak i;
I I I
:; :;
3-
I I I
; ;
I I
!
2II
I
1
1
/
1
. A.
I
1
,
200 300 400 500 600 200 300 400 5 0 550 $00 Wavelength ( U p ) Figure 1 . Excitation spectra and light scatter (left) and fluorescence spectra (right) of aqueous phase 5HT Authentic standard, 0.5 p g . (-1; a p p a r e n t from 0 . 7 5 - g r a m brain ('"'~~',",~''); authentic, 0.5 p g . plus W h e n the excitotion wavelength w a s r e a g e n t blank (-,-I. a p p a r e n t from 0 . 7 5 - g r a m brain (---I; varied, the fluorescent wavelength w a s set a t 550 mp. W h e n the fluorescent wavelength was varied, the ex&tion wavelength w a s s i t a t 300 m p
DA was determined by its oxidation to the highly fluorescent 5,g-dihydroxyindole. A 0.4-1n1. aliquot of the aqueous phase or a 1.0-ml. aliquot of the column eluate was used. The 0.4ml. aliquot was brought to 1.0 ml. and pH 5.2 by adding 1-11 sodium acetate-acetic acid buffer a t pH 5.2. The aqueous phase and column eluate samples were both treated as follows: 0.2 ml. of 0.025 Iz added and mixed. Waited 3 minutes; 0.2 ml. of alkaline Na2S03 solution added and mixed. Waited 3 minutes; 0.4 ml. of 5A' HCIascorbic acid solution added and mixed. Waited 45 minutes; read at 330/385 mp. The tissue blanks were treated as follows: 0.4 ml. of L Y HC1-ascorbic acid solution added and mixed. Waited 3 minutes; 0.2 i d . of Xa2S03solution added and .mixed. Waited 3 minutes; 0.2 ml. of O.O2&Y I2 added an Waited 45 minutes; read a t mp and subtracted the blank va the sample reading to obtain D;l fluorescence contribution. KE was determined by its conversion to the highly fluorescent 3.5,6-trihydrosyindole. As in the determination of IIA, a 0.4-ml. aliquot of the aqueous phase was brought to 1.0 ml. and pH 5.2 or 1.0 i d . of the column eluate was used. The samples were treated as follows: 0.2 ml. of 0.02.Y Is added and mixed. Waited 6 minutes; 0.2 ml. 0.025S Xa2S203added and mixed. S o wait; 0.4 ml. 5>Y XaOHascorbic acid solution added and mixed. Waited 45 minutes; read at 410'520 nip. The tissue blanks were treated as follows: 0.2 ml. 0.025.Y SanSrOz added and mixed. m'aited 6 minutes; 0.2 i d . 0.02.\- I2 added and mixed. X o wait: 0.4 nil. ascorbic a(-id-water reagent added and niised. Waited 45 minutes. Read at 410 520 nip and 694
ANALYTICAL CHEMISTRY
subtracted the blank values from the sample reading to obtain NE fluorescence contribution. The ascorbic acidwater reagent was used in lieu of alkaline ascorbic acid because the former gives a smaller blank reading. Standards, recovery samples, and reagent blanks were carried through the entire procedure in the same way.
12 -
12
l0-
IO
8-
8
6-
6
8 4-
4
CI
.= c v)
=> Q)
.? c
0
-a Q)
Q)
u c
v)
2
= 0
LL
2
2I
200
I
300 330
400 200 Wavelength (Mp)
300
385400
Figure 2. Excitation spectra and light scatter (left) and fluorescence spectra and light scatter (right) of the aqueous phase fluorophore of DA
1; authentic, 0 . 5 pg. plus Authentic standard, 0 . 5 pg. (--); a p p a r e n t from 0 . 7 5 - g r a m brain ( a p p a r e n t from 0 . 7 5 - g r a m brain (- - -); tissue blanks have b e e n subtracted. W h e n the excitation wavelength wos voried, the fluorescent wavelength was set o t 385 mp. W h e n the fluorescent w a v e length was voried, the excitation wavelength was set a t 330 m p
Recovery of Authentic Amines Added to 0.75 Gram of Mouse Brain Tissue
Table I.
0.4 p g . added
5HT Sam97, ple Keanalyzed Found. covery 93 Aqueous 0.37 0.37 93 phase 0.37 93 0.40 100 Column 95 eluate 0 . 3 8 0.38
a
95
0.8
SE
11.4
c;
%c
Found 0 41 0 0 0 0 0 0
Recovery 102
42
105
39 38
98
Found 0 40 0 0 0 0 0
95
42
105
42 38
105 95
40
40 43
42 39 0 37
pg.
5HT
B
Recovery 100
Recovery
Found
100 100
107 10,i
added
DA
0 79 0 82
101
0 79 0 82 0 76
99 103 95
99
97
c70
Found 0 0 0 0 0
75
74 73 81 70
0 74
0 83
93
NE
-_ Recovery 94 93 94 101 99 93
104
%
Found 0 80 0.79
Recovery 100 99 105
0.84 0 81 0 74 0 74 0 86
101 93 93
107
Net, after subtracting endogenous values.
12 -
3
:I
{I
a 3 a
IO -
Light scatter peak
:I
iI
:I
5
il :I
:I
cb
:I
il :I
a
8-
64-
2-
-
I I
I I
1
300 400 500 520 600
200
Wavelength ( M p ) Figure 3. Excitation spectra and light scatter (left) and fluorescence spectra and light scatter (right) of the aqueous phase fluorophore of NE Authentic standard, 0.4 p g . (-I; a p p a r e n t from 0 . 7 5 - g r a m brain ( """"""'); authentic, 0.4 p g . plus a p p a r e n t from 0 . 7 5 - g r a m brain (- - -1. Tissue blanks h a v e b e e n subtracted. W h e n the excitation wavelength w a s varied, the nuarescent wavelength w a s set a t 5 2 0 m p . W h e n the fluorescent w a v e length w a s varied, the excitation wavelength was set a t 410 mL.r
tion by organic solvents. These recovery values are comparable to other extraction methods (4,19). Authentic amines in amounts approximating endogenous levels, which had been added to 0.75 gram brain tissue and carried through the entire procedure, were recovered with excellent precision. Recoveries were similar for both aqueous phase and column eluate samples (Table I). The excitation and fluorescence spectra of the apparent 5HT, D-1: and S E extracted from mouse brain were found to have the Fame general characteristics as the aut,hentic amines. Addition of each standard to tissue increased the fluorescence by that expected from the standard curve (Figure 1-3). There were no differences in the peak positions of aqueous phase, column eluate samples, or authentic amines.
Table II.
Endogenous Amine Concentration in Whole Mouse Brain
Reference Fleming et a2 Fleming et al
Extraction method Acetone (no amino acid) Acetone ( + amino
Albrecht et al. ( 1 )
Acetone
Wiegand and Perry
Butanol
acid)^
(Z5)
Leroy (15)
Butanol
Amine concentration 5HT DA
Carlsson and Lindqvist (8) Smith (20)
Perchloric acid
Smith ( 2 0 )
Trichloroacetic acid
pg
/grama
NE
0 55 i 0 03 0 70 f 0 06 0 48 i 0 03
iridium @), and gold (6) has been reported. In this scheme the 1)rccious metals are collected in molten tin when the sample material is fused at 1200' to 1250' C. with a flus containing stannic oside, sodium carbonate, silica, boras, and powdered coke. The resulting tin alloy is then treated by relatively sinil~le wet-chemical techniques to isolate and separate (8)the individual metal> prim to their determination by a1)l)rol)riatc spectrophotometric methods. The tin-collection schenit has tiern
applied successfully, in this laboratory, to the deterinination of one or more of the platinum metals and,,or gold in such diverse materials as silicate rocks, coppei~-nickel ores and concentrates, copper-nickel matte, meteorites of both the iron and stony types: and the minerals magnetite, chromite, and osmiridium. Some platiniferous materials such as certain of those listed above, also contain appreciable amounts of ruthenium and osmium. The deterinination of these elements is sometimes required in geochemical and mineralogical studies as n-ell a. in laboratoriec ahsociated n-it11 the production and refining of the
