and one of kidney, were relyophilized for an additional 24 hr. No discernible losses of mercury were observed during the second cycle. Although it must be emphasized that these results are for the three forms of mercury stated, or their metabolites, it is obvious that the losses of mercury from blood and/or animal tissues during lyophilization are negligible. It has been stated (6) that the most likely form of mercury in fish tissues is methylmercury; therefore, for the animal tissues normally analyzed, these experiments should be directly applicable. Since the loss of inorganic mercury is generally accepted to be insignificant, the possibility of hydrolysis of the organomercurials used must be considered. The startling difference in organ-organ and organ-blood ratios indicates that the forms of mercury behave significantly differently; for example the liver-blood ratio for methylmercury fed rats (12 days after the last feeding of tagged compounds) was 0.4 and 16 for phenylmercuric acetate. The kidneyblood ratios were 2 and 3000, the liver-muscle ratios were 2 and 18, and the kidney-brain ratios were 20 and 1700, respectively. Blood activities for inorganic mercury were very low; after five days essentially all of the 203Hg activity is found in the kidney, with the liver being the only other organ containing enough activity to be measured reliably. The relatively high 203Hg activity in the brains of animals fed methylmercury chloride (at least 100 times greater than either phenylmercuric acetate or inorganic mercury) is also indicative of the known passage of methylmercury chloride across the "bloodbrain barrier." (6) G. Westoo. VarFoeda, 7, 137 (1969)
Since binding through sulfhydryl groups in proteins may be the binding mechanism in animals, it must be emphasized that for samples in which such binding groups are much less available, as, for example, in sediment or urine samples, significant losses may occur. Indeed, freeze drying aqueous solutions (5mM Na2C03 for methylmercuric chloride, 0.01M acetic acid for phenylmercuric acetate) of the tagged mercury compounds resulted in losses of up to 90% for the organomercurials and 10% for inorganic mercury (in 1M HCl). However, when tagged methylmercury chloride was pipetted directly into 3 ml of heparinized whole blood or a solution of 250 mg of l-cysteine in 3 ml of water, freeze-dry losses were less than 4% for blood and 7% for cysteine. In addition, the loss of mercury from the feces of rats fed phenylmercuric acetate was approximately 10%. When these limitations are kept in mind, the data presented here indicate that freeze drying is a practical approach to preparing tissue samples for mercury determination. ACKNOWLEDGMENTS The kind assistance of E. Van Loon and E. Miller of the U.S. Food and Drug Administration who provided the rats used in these experiments and of J. Held of the National Institutes of Health who provided the guinea pigs is gratefully acknowledged. The many helpful discussions and the encouragement of J. T. Tanner and D. N. Lincoln of the U.S. Food and Drug Administration are also acknowledged with thanks. Received for review August 10, 1972. Accepted February 6, 1973.
Fluorescent Detection of Tryptamine in the Nanogram Range James M. Cisy and B. C. Gerstein Ames Laboratory-USAEC and Department of Chemistry, lowa State University, Ames. lows
The determination of tryptamine has been of vast biological interest over the past two decades. In the past few years, studies of urine samples of schizophrenic patients have shown that preceding and during the period of schizophrenic behavior, increased excretions of indole metabolites occurred (1, 2). Improvement in the methods for detecting tryptamine have taken many routes: TLC ( 3 ) , native fluorescence (4, and norharman fluorescence (5, 6). Even so, quantitative detection limits for tryptamine have not reached the nanogram level. Recently Maickel and Miller (7) have found that trypt(1) G. Brune and H. E. Himwich, "Effects of Reserpine on Urinary Tryptamine and Indole-3-Acetic Acid Excretion in Mental Deficiency, Schizophrenia and Phenylpyruvic Oligophrenia," read before the Acta of the International Meeting on the Techniques for the Study of Psychotropic Drugs, Bologna, Italy, June 26-27, 1960. (2) J. Spaide, H. Tanimukai, J. R. Bueno, and H. E. Himwich, Arch. Gen. Psychiat, 18, 658 (1968). (3) J. N. Elbeand R . M. Brooker. Experientia, 18, 524 (1962) (4) E. D. Duggan, R . L. Bowman, E. E. Brodie, and S. Udenfriend. Arch. Biochem. Biophys., 68, 1 (1957). (5) S. M. Hess and S. Udenfriend, J . Pharmacol. Exp. Ther.. 127, 125 (1959). (6) D . Eccleston, G . W. Ashcroft, T. 6. 6 . Crawford. and R . Loose, J. Neurochem.. 9 , 113 (1962). (7) R. P. Maickei and F. D . Miller, Anal. Chem., 38, 1937 (1966).
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amine, after condensation with o-phthalaldehyde (OPT), does not fluoresce as well as serotonin (5-hydroxytryptamine) due to the lack of a hydroxyl group a t the 5 position of the indole ring. It is interesting to note that with native fluorescence, tryptamine has a lower detection limit than does serotonin ( 4 ) . Also some of their findings concerning the relationship between substituents a t the 3 and 5 positions of the indole ring have been challenged by the findings of Hakanson and Sundler (8) using formaldehyde as a condensation agent. For these reasons, we chose to investigate reagents which might make possible the quantitative fluorescent detection of tryptamine in the nanogram range. EXPERIMENTAL T r y p t a m i n e HC1 was o b t a i n e d f r o m K u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n of Cleveland, O h i o . D i s t i l l e d w a t e r a n d reagent-grade h y d r o c h l o r i c acid, benzaldehyde, s o d i u m hydroxide, chloroform, p e t r o l e u m ether (bp 60-110 "C), a n d d i e t h y l ether a n d spectrop h o t o m e t r i c - g r a d e m e t h a n o l were used in a l l r e a c t i o n m i x t u r e s a n d procedures. A l l compounds were used w i t h o u t f u r t h e r purification. (8) R . Hakanson and (1971),
F. Sundler. Biochem. Pharmacoi. 20. 3223
l-Phenyl-l,2,3,4-tetrahydro-/3-carboline was prepared by the following reaction sequence 4.0 3.5
* TRYPTAMINE
BENZALDEHYDE
BENZYLIDENETRYPTAMINE
I IkPHENYL - I . 2 , 3 . 4 - T E T R A H Y U R O - 8 - C 4 R B O L I N E
L m
3.0
k
z
2.5 t (L
d
k m a a
2.0
9 1.5
z Specifically 0.3007 g of tryptamine HC1 was dissolved in 50 ml of distilled water. Benzaldehyde (0.2451 g) was dissolved in 20 ml of methanol and refluxed for 1.0 hr. Dilute aqueous NaOH was added dropwise until the solution was made basic. Pale crystals were filtered and dried under vacuum. Benzylidenetryptamine (0.3684 g, 97%) was identified by NMR and mass spectral data: mp 121.5 "C (lit. (9) 120-121 "C) 1-Phenyl-1,2,3,4-tetrahydro-P-carboline was prepared following Jackson and Smith ( I O ) . Dry HC1 was bubbled through a solution of benzylidenetryptamine (0.1000 g) in 5.0 ml of dry ethyl ether. A yellow solid formed immediately. The ether was boiled off and the remaining solid was dissolved in 20 ml of distilled water. Dilute aqueous NaOH was added dropwise until the solution was made basic. The white solid was filtered and recrystallized from petroleum ether as tiny prisms. l-Phenyl-1,2,3,4-tetrahydro-P-carboline(0.0903 g, 90%) was identified from NMR and mass spectral data: m p 168 "C (lit. (10) 168 "C). A stock solution of l-phenyl-1,2,3,4-tetrahydro-P-carboline was prepared of concentration 1 X g/ml. From this, other solutions were prepared ranging in concentration from 1 X 10-6 to 1 x 10 -lo g/ml. Spectrophotometric-grade methanol was used in the stock solutions and in the subsequent dilutions. Fluorescence was measured in an Aminco-Bowman spectrophotofluorometer a t uncorrected wavelengths of 285 mp (excitation) and 360 f 5 mp (emission) with slits of 3116 in. (excitation and emission) and 1.0 mm (1P28 phototube). Reagent blanks consisted of absolute methanol. All fluorescent values have been corrected for the blank.
RESULTS AND DISCUSSION As shown in Figure 1 there is a linear relationship between fluorescence and concentration over a range from to g/ml. The detection limit for l-phenyl1,2,3,4-tetrahydro-P-carboline is 5 X 10-10 g/ml. The reactions required to generate the fluorescent product are essentially quantitative giving a more precise determination of tryptamine. With slightly purer solvents and better instrumentation, it should be possible to lower the detection limit. Care must be taken with the solvent to guard against contamination which will increase the background noise of the blank and distort the fluorescent values especially a t the lower concentrations. (9) T. Hoshino and V. Kotake. Justus Liebigs Ann. Chem., 516, 76
(1935). Jackson and A. E. Smith, Tetrahedron. 24, 403 (1968)
(10) A . H .
LL
1.0
(3
Q,
0.5 0.0 -6
I
-7
I
-8
I
h
"
I
-9
-10
-11
I
-12
I
-13
LOG CONCENTRATION, g/ml
Figure 1. Plot of t h e log of f l u o r e s c e n c e in arbitrary units vs. t h e log of c o n c e n t r a t i o n of 1-phenyl-l,2,3,4-tetrahydro-P-carboline
It should be noted that neither an aromatic 1,2-dialdehyde nor an unsaturated P-carboline ( e . g . , norharman) are needed for enhanced fluorescence and detection of tryptamine. Also the reactions and procedures are relatively simple and straightforward. The method does not require drastic conditions such as 10N HC1 as in the OPT method, (7) and it does not unde_rgoside reactions which, we have observed, occur in the OPT method. In attempting to find a method for determining serotonin in an analogous manner, we have found that the condensation product of serotonin and benzaldehyde does not precipitate when the solution is made basic. Therefore, serotonin will not interfere with the analysis for tryptamine if it is present in the solution. If any interference arises from other amines Hanson (11) has compiled many methods for isolating tryptamine from amine mixtures. More recently Narasimhachari (22) has developed a new method of separating tryptamine from other related amines. The use of these methods should remove any interference, if it is present. Received for review October 5, 1972. Accepted January 26, 1973. Work supported in part by the Ames Laboratory, USAEC, Contract No. W-7405-eng-82 and the Iowa State University Alumni Achievement Fund. (1 1 ) A. Hanson, "Handbook of Experimental Pharmacology," Vol. 19, Sprinaer-Verlaa. Berlin. 1966. Chaoter 2. (12) N. Narasirnhachari. J. M. Plant, and K. Y . Leiner, Biochem. Med.. 43, 304 (1971).
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