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Analysis of tryptamine at the femtomole level in tissue using negative ion chemical ionization gas chromatography-mass spectrometry
Journal of’ Chromatography, 440 (1988) 253-259 Elsevier Science Publishers B.V., Amsterdam -
CHROM.
Printed
in The Netherlands
20 299
ANALYSIS OF TRYPTAMINE AT THE FEMTOMOLE USING NEGATIVE ION CHEMICAL IONIZATION GRAPHY-MASS SPECTROMETRY
DAVID
A. DURDEN
LEVEL IN TISSUE GAS CHROMATO-
and A. A. BOULTON*
Neuropsychiatric Research Unit, Cancer and Medical Research Building, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO (Canada)
SUMMARY
An ultra sensitive method for the detection of tryptamine, an endogenous amine in mammalian neuronal systems, at the femtomole level has been developed using negative chemical ionization gas chromatography-mass spectrometry (NCIGC-MS). The amine is converted into a perfluorinated spirocyclic derivative, e.g. l-pentafluoro-2-methylenepyrrolidine-3-spiro-3’-(3~-indole) which is detected using selected-ion monitoring of the (M - 2HF) ions of the endogenous and deuterated internal standard compounds. Two mass spectrometers were compared; they gave minimum detectable quantities from tissue samples of 40 pg (VG-7070F) and 0.9 pg (VG-70s) respectively. These detection levels are approximately 5-200 times lower than have been obtained by previous MS methods.
INTRODUCTION
Tryptamine is a naturally occuring trace amine present in the brain of all animals including humans1v2. It is a pharmacologically active amine3 which readily crosses the blood-brain barrier4, influences both human and animal behaviour5-9 and may be implicated in some neurological and psychiatric disorderslO-i2. Tryptamine is formed by decarboxylation of L-tryptophan13-is and is catabolized by monoamine oxidase’+lg. Its metabolite, indole-3-acetic acid, has been detected in human cerebrospiral fluid20 and in rat brain21,22. It has a rapid turnover approaching that of 5-hydroxytryptamine’ 5,23~24 and a very short half life after intracerebral injections25,26. Although tryptamine is known to be distributed within the cell and located in synaptosomes2’, only recently has there been a concerted search for its brain binding siteszs-34. Tryptamine concentrations in the brain are very low even in the striatum1’24 where it is maximally concentrated and although lesions of the mid-brain raphe do not affect striatal levels35, niagrostriatal lesions do36 suggesting an involvement with dopamine pathways3’. Finally, iontophoretically applied tryptamine inhibits cortical cell firing3s,39 independently of 5-hydroxytryptamine and in its presence modulates it. All of the above evidence indicates that tryptamine is a neuromodulator and may 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
D. A. DURDEN,
254
A. A. BOULTON
be a neurotransmitter. In order to study this substance further more detailed information on its location, subcellular distribution and the effect of lesions is required. Such investigations will require the development of super sensitive assays for its detection. The original method developed in this laboratory for the analysis of tryptamine1JJ4 using direct probe electron impact ionization-mass spectrometry (EI-MS) of the 5-dimethylamino-1-napthalenesulfonyl (dansyl) derivative is still one of the most sensitive available with a sensitivity (minimum detectable amount) of about 1 pmol (100 pg). Gas chromatography-mass spectrometry (GC-MS) methods using packed columns22,40 were unable to detect tryptamine at this level and thus workers using this procedure were only able to put upper limits on the endogenous concentrations of tryptamine in whole rat brain. A somewhat more sensitive procedure using high-performance liquid chromatography (HPLC) with fluorescence detection41 has been developed. The most sensitive procedure to date is the GC-MS procedure of Beck and Flodberg4* in which the heptafluorobutyrate derivative was isolated by capillary column GC-MS. We report here an ultra sensitive GC-MS procedure which obtains its high sensitivity from the use of negative chemical ionization (NCI) of a highly electrophilic derivative of tryptamine. MATERIALS
AND METHODS
All solvents were of HPLC-grade and water was purified by reverse osmosis and then by ion exchange to a resistance of 18 MS2 (Barnstead, Nanopure II). Tryptamine hydrochloride was obtained from Sigma (St. Louis, MO, U.S.A.) and a,@$-tetradeutero-tryptamine . HCl (tryptamine-d4) was synthesised in this laboratory by B. A. Davis. Acetic anhydride (Fisher) was redistilled in glass and stored at -20°C before use. Pierce fluorinated acyl anhydrides, pentafluoropropionic anhydride (PFPA), heptafluorobutyric anhydride (HFBA), and trifluoroacetic anhydride (TFAA) were purchased from Chromatographic Specialties (Brockville, Canada). PFPA was redistilled from phosphorus pentoxide and stored in 1 ml ampoules at -20°C. Brain tissue was obtained from 200 g Wistar rats (Charles River Labs., Quebec, Canada). The animals were killed by decapitation, their brains removed, and the hypothalami and striata dissected out and frozen on dry ice. Tissue weights were about 30 mg and 100 mg respectively per animal. Tissues were homogenized in 1 ml 0.1 M perchloric acid containing l-2 ng of tryptamine-d4 and then centrifuged (Fisher micro-centrifuge Model 235B at 12 000 g) for 15 min. To the supernatants were added 0.2 ml acetic anhydride and approx. 0.1 g sodium bicarbonate. The solution was mixed (Vortex Genie) and additional sodium bicarbonate added over a period of 45-60 min until all bubbling ceased. The acetylated tryptamine was then extracted into ethyl acetate (2 x 3 ml). The organic phase was transferred to l-ml Reactivials (Pierce) and taken to dryness at 40°C under a stream of nitrogen. Dichloromethane (100 ~1) was added and again taken to dryness to remove azeotropically any remaining water. PFPA (200 ~1) was then added, the vials sealed with PTFE-faced seals and heated at 80°C for 1 h. The vials were cooled and about 0.4 ml hexane added and the reactants washed with 200 ~1 of phosphate buffer (pH 6). The organic phase was transferred to 200-~1 Reactivials and reduced to a volume of 5-10 ~1 from which an
NCI-GC-MS
OF TRYPTAMINE
255
aliquot (1 ~1) was injected. Blanks containing only the tryptamine-d4 and check samples containing 2.0 ng each of tryptamine and tryptamine-d4 were carried through the entire procedure. For sensitivity checks larger quantities (i.e. 40 pg of each amine) were derivatized as described above. The perfluoroacyl reaction was also repeated for the larger quantities using TFAA and HFBA. MS analysis was carried out using a VG Analytical 7070F double focussing mass spectrometer with a Hewlett-Packard Model 5700 gas chromatograph containing a capillary column connected directly to the ion source. Initial experiments were carried out using for the capillary column an SGE BP5 50 m x 0.32 mm I.D. l-pm bonded phase (thick film capillary column) and for later experiments (including tissues) a J & W DBl 60 m x 0.32 mm I.D. 0.25~pm bonded phase (thin film capillary column). These columns were operated with a helium flow of 3340 cm/s and a temperature program of 170°C isothermal, 2 min, lO”C/min to 290°C and then isothermal for 4 min. Injection was on column (at 200°C) into a short length (15 cm) of megabore column (J & W DBl 0.52 mm I.D. 1.5-pm bonded phase) which was then coupled to the main capillary column with a Supelco union and Vespel graphite seals. In either case the capillary was pushed into the megabore column so that the sample did not pass over the seals. Spectra were recorded under EI conditions at 70 eV, 200 PA collector current and ion source temperature of 170°C and under NC1 conditions, 50 or 100 eV, 2000 PA emission and ion source temperature 140-150°C using either methane or ammonia CI gas. A megabore column (J & W DBl 15 m x 0.52 mm I.D. 1.5-pm bonded phase) with the carrier (5 ml/min) and the make-up gas (25 ml/min) or a packed column 6 x 2 mm I.D., 3% SP2100 on Supelcoport SO/l00 was also connected via the jet separated and used for some experiments, For methane, the source housing pressure was 5 . lo-’ mbar and for ammonia 2 . lop5 mbar to give maximum signals. One ion source was kept for NC1 use and modifications were made to maximize the NC1 signal. The CI aperture of the sliding slit was widened from the original 0.07 mm x 5 mm to 0.3 1 mm x 5 mm. Compared to a second source with a CI slit of 0.11 mm x 5 mm the signal gain was at least a factor of 25. The polarity of the electron beam magnets was also chosen for optimum signal. The beam focussing controls were initially set with perfluorokerosene (PFK) (1 .O ~1 into the septum inlet) present in the source. This was then pumped away and the beam refocussed to maximize the signal on the residual PFK peak at m/z 281 or on the GC column bleed peaks. For quantitation, selected-ion monitoring (SIM) was performed on the (M - 2HF) peak of the spirocyclic derivatives of tryptamine m/z 290.0667 and tryptamine-d4 (M - 2HF) m/z 293.0855 using m/z 280.9824 of PFK as a reference and a resolution of 2000. Although the absolute signal height was reduced by a factor of 2 at this resolution, the signals due to the column bleed ions were reduced much more and hence the background signal was lowered dramatically. SIM was performed using the VG Digital multiple ion detector (DIGMTD) unit to control the accelerating and electric sector potentials and a magnetic field selector43 to control the magnet and the choice of reference mass. The output of the DIGMID unit was filtered using Bessel filters44. A few analyses were performed using a much more sensitive mass spectrometer, the VG 70s. This instrument was equipped with a Hewlett-Packard 5890A gas chromatograph, a universal split/splitless injector and a DBl capillary column (30 m x 0.25 mm I.D. with 0.25~pm bonded film) also connected directly to the ion source.
256
BOULTON
loo-
(M-ZHF) 290
ic. E t 9! ,c
310
128 0, 50
100
I, 150
200
250
L
I
I 300
I
Mass
Fig. 1. NC1 spectrum of the tryptamine derivative l-pentafluoropropionyI-2-methylenepyrrolidine-3-spiro-3’-(3H-indole) (MW = 330), obtained using the 7070F mass spectrometer with methane reagent
gas.
Temperature program was 160°C; 2 min, lO”C/min to 250°C with the spirocyclic derivative eluting at about 210°C. NC1 was undertaken using methane reagent gas and the pressure optimized at about 2 + 10m5-5 . 1O-5 mbar. This instrument possesses a three-position ion source exit slit and the mid-sized EI/CI slit was used. This instrument was also operated at 2000 resolution and SIM profiles of m/z 281, 290 and 293 collected using the VG 1 l-250 J data system and VG SIR software45 using m/z 281 and 331 of PFK as lock masses. The sensitivity was optimized with PFK present in the ion source. RESULTS
AND
DISCUSSION
The reaction of N-acetyltryptamine with PFPA is expected to produce a 3,3spirocyclic compound, l-pentafluoropropionyl-2-methylenepyrrolidine-3-spiro-3’(3H-indole), similar to that from melatonin 46*47. The NC1 spectrum (Fig. 1) supports this structure as does the EI spectrum with a molecular ion of m/z 330. This compound is amazingly electrophilic and a much greater sensitivity (approx. 50-100 times) was obtained in the NC1 mode compared to the EI mode when the spectra were recorded. There was some evidence for the presence of a non-cyclized structure, i.e. N-acetyl-N,N’-dipentafluoropropionyltryptamine43 but it appeared to decompose or cyclize on the column and with the thick film BP5 column (1 pm film) only one product was observed (eluting at 280-290°C). On the thin film DBl column, the spirocyclic structure eluted at a lower temperature and was preceded by a small peak due either to the non-cyclized tryptamine derivative or perhaps a cyclized p-carboline (1 -methyl-pentafluoro-/Lcarboline). The structure and process are under investigation. Even though this small peak was present using the 0.25~,um film DBl column, the sensitivity was not compromised as the majority of the product was as the spirocyclic compound and the GC resolution was much improved over the heavier loaded BP5 column. With a clean ion source and using the packed column, we have been able to detect as little as 160 fg of tryptamine on column4*. This value was obtained with a signal-to-noise (S/N) value of 3 and the output of the digital MID unit heavily filtered (10 s time constant) with the Bessel filter44. Fig. 2 shows the detection of tryptamine from a single caudate nucleus using the 7070F and Fig. 3, a similar tissue
NC&CC-MS
257
OF TRYPTAMINE
10;; ”
---lJ
m/z 293.0955
5 00
515
530
5 45
6:00
6 30
6 15
Fig. 2. SIM analysis of tryptamine with 1.28 ng of tryptamine-d4 internal standard from a single caudate nucleus. Column, DBI, 60 m x 0.32 mm, 0.25 pm. Temperature program, 200°C 2 min; lO”C/min to 290°C. VG 7070F mass spectrometer. Fig. 3. SIM analysis of tryptamine with 1.95 ng tryptamine-d4 internal standard from a single caudate nucleus. Column, DBI, 30 m x 0.25 mm, 0.25 pm. Temperature program, 160°C 2 min; 10”C/min to 250°C. VG 70s mass spectrometer. Only the portion of chromatogram from 5:0 to 6:30 min is shown.
on the 70s instrument. In both cases only about 15-20% of the final solution was injected into the gas chromatograph. Table I shows the results of analysis of tryptamine in the two tissues (using the 7070F) compared to previous values obtained in this laboratory using the dansyl technique’. It is clear that due to the much higher sensitivity of the NCI-GC-MS procedure, the variability is much reduced, i.e. from 39% to 3% for the caudate values and from 23% to 12% for the hypothalamus values. It must be noted that in both cases we are comparing results from single tissues in the case of the NC1 technique and pooled tissues in the case of the dansyl technique. A minimum detectable level from tissues can be calculated using data from Figs. 2 and 3 (shown in Table TABLE
I
TRYPTAMINE
CONCENTRATIONS
Values are means f standard column). 1.28 ng tryptamine-d,
Caudate nucleus Hypothalamus l
standard
IN RAT BRAIN
REGIONS
error of the mean. NCI-GC-MS of the tryptamine internal standard. Tissue from one rat.
PFP derivative
(DBI
Tryptamine
Weight
Concentration
Previous values*
fpg)
Img)
(nglgl
IngIg)
219 f 24 60 f 11
68.3 f 14.1 f
3.20 f 0.10 4.28 f 0.51
2.93 f 0.94 f
7.9 1.6
From ref. 1. Direct probe EI-MS of the tryptamine with tissues from 10 rats pooled.
dansyl derivative.
1.14 0.22
25 pg tryptamine-d,
internal
258
D. A. DURDEN,
TABLE
A. A. BOULTON
I1
COMPARISON OF SIGNAL-TO-NOISE RATIOS OF TWO MASS SPECTROMETERS MUM DETECTABLE QUANTITIES FROM TISSUES
AND MINI-
The signal values from the two instruments
directly
Identity
are in different
707oF* Signal
units and cannot
be compared
7os** Noise
SIN
Min. der.
Signal
Noise
SIN
(Pgi m/z 290 m/z 293
400 1930***
20 20
DBl 60 m x 0.32 mm * DBl 30 m x 0.25 mm l ** Corresponds to 1.28 pg g Corresponds to 1.95 pg l
l
2O:l 96:1
40 40
Min. det. (PS)
320 1822$
0.28 0.28
1143:l 6507: 1
0.5 0.9
I.D. column, ammonia reagent gas. I.D. column, methane reagent gas. tryptamine-d., added to the homogenate. tryptamine-da added to the homogenate.
II) using a minimum S/N ratio of 3. For the 7070F, the minimum detectable level from the tissue used would be about 40 pg whereas a similar tissue on the 70s the minimum detectable level appears to be 500-900 fg. When the Bessel filter is not used on the 7070F the S/N value reduces (to 6:l for m/z 290 in this example) and the minimum detectable level is about 120 pg. This method demonstrates the extremely high sensitivity of NC1 with an appropriate derivative and that with appropriate MS, low femtomol quantities of amines can be detected. ACKNOWLEDGEMENTS
We wish to thank the Medical Research Council of Canada and Saskatchewan Health for continuing financial support, Dr. B. A. Davis for synthesis of the deuterated compounds and E. Zarycki, R. Janzen and N. Pidskalny for assistance with tissue preparation, derivatization and MS analysis respectively. We wish especially to thank Dr. Paul Brooks of the Institute for Sedimentary Petroleum Geology, Calgary, Alberta, for the analysis using the 70s mass spectrometer. REFERENCES 1 2 3 4 5 6 7 8 9
S. R. Philips, D. A. Durden and A. A. Boulton, Can. J. Biochem., 52 (1974) 447-451. S. R. Philips, 9. Rozdilsky and A. A. Boulton, Biol. Psychiat., 13 (1978) 51-57. R. S. G. Jones, Frog. Neurobiol., 19 (1982) 117-139. W. H. Oldendorf, Am. J. Physiol., 21 (1971) 1629-1639. W. G. Dewhurst, Nature (London), 218 (1968) 1130-1133. A. Foldes and E. Costa, Biochem. Pharmacol., 24 (1975) 1617-1621. C. A. Marsden and G. Curson, Psychopharmacology (Berlin), 57 (1978) 71-76. 9. Cox, A. Davis, V. Juton, T.F. Lee and D. Martin, J. Physiol. (London), 337 (1983) 441450. A. Delini-Stula and E. Radeke, in A. A. Boulton, P. R. Bieck, L. Maitre and P. Riederer (Editors), Neuropsychopharmacology of the Trace Amines: Experimental and Clinical Aspects, Humana Press, Clifton, NJ, 1985. pp. 125-140.
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OF TRYPTAMINE
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10 P. H. Yu, B. A. Davis, R. D. Bowen, S. Wormith, D. Addington and A. A. Boulton, in A. A. Boulton, G. B. Baker, W. G. Dewhurst and M. Sandler (Editors), Neurobiology ofthe Trace Amines: Analytical, Physiological, Pharmacological, Behavioural and Clinical Aspects, Humana Press, Clifton, NJ, 1984, pp. 4755486. 11 E. Kienzl, P. Riederer, K. Jellinger and H. Noller, in A. A. Boulton, G. B. Baker, W. G. Dewhurst and M. Sandler (Editors), Neurobiology of the Trace Amines: Analytical, Physiological, Pharmacological, Behavioural and Clinical Aspects, Humana Press, Clifton, NJ, 1984, pp. 571-579. 12 J. Montplaisir, J. de Champlain, S. N. Young, K. Missala, T. L. Sourkes, J. Walsh and G. Remillard, Neurology, 32 (1982) 129991302. 13 J. M. Saavedra and J. Axelrod, J. Pharm. Exp. Ther., 185 (1973) 523-529. 14 H. Weil-Malherbe, J. Neurochem., 27 (1976) 8299834. 15 J_ J. Warsh, D. V. Coscina, D. D. Godse and P. W. Chan, J. Neurochem., 22 (1979) 1191-1196. 16 H. Weissback, W. Lovenberg, B. G. Redfield and S. Udenfriend, J. Pharm. Exp. Ther., 131 (1961) 2632. 17 P. H. Wu and A. A. Boulton, Can. J. Biochem., 51 (1973) 1104-1112. 18 N. H. Neff and H.-Y. T. Yang, @e Ski., 14 (1974) 2001-2074. 19 P. H. Yu, in A.A. Boulton, P. R. Bieck, L. Maitre and P. Riederer (Editors), Neuropsychopharmucology of the Trace Amines: Experimental and Clinical Aspects, Humana Press, Clifton, NJ, 1985, pp. 301308. 20 L. Bertilsson and L. Palmer, Science (Washington, DC), 177 (1972) 7476. 21 J. J. Warsh, P. W. Chan, D. D. Godse, D. V. Coscina and H. C. Stancer, J. Neurochem., 29 (1977) 955-958. 22 F. Artigas and E. Gelpi, Anal. Biochem., 92 (1979) 233-242. 23 D. A. Durden and S. R. Philips, J. Neurochem., 34 (1980) 1725-1732. 24 A. V. Juorio and D. A. Durden, Neurochem. Res., 9 (1984) 128331293. 25 D. A. Durden, T.-V. Nguyen and A. A. Boulton, Neurosci Res., (1987) in press. 26 J. L. Meek, A. R. Krall and M. A. Lipton, J. Neurochem., 17 (1970) 162771635. 27 A. A. Boulton and G. B. Baker, J. Neurochem., 25 (1975) 477481. 28 K. J. Kellar and C. S. Cascio, Europ. J. Pharmacol., 78 (1982) 475478. 29 C. A. Altar, A. M. Wasley and I. L. Martin, Neuroscience, 17 (1980) 263-273. 30 G. Bruning and H. Rommelspacher, Life Sci., 34 (1984) 144-1446. 31 P. L. Wood, C. Pitapil, F. Lafaille, N. P. V. Nair and R. A. Glennon, Arch. Znt. Pharmacodyn., 268 (1984) 194-201. 32 D. C. Perry, J. Pharm. Exp. Ther., 236 (1986) 548-559. 33 J. K. McCormack, A. J. Beitz and A. A. Larson, J. Neurosci., 6 (1980) 94-101. 34 D. Graham and S. Z. Langer, Neuropharmacol.. 26 (1987) 109331097. 35 A. V. Juorid and A. J. Greenshaw, J. Neurochem., 45 (1985) 422426. 36 A. V. Juorio and A. J. Greenshaw, Brain Res., 371 (1986) 3855389. 37 A. V. Juorio, A. J. Greenshaw and T. V. Nguyen, J. Neurochem., 48 (1987) 13461350. 38 R. S. G. Jones and A. A. Boulton, Life Sci., 27 (1980) 1849-1856. 39 R. S. G. Jones, Br. J. Pharmacol., 73 (1981) 4855493. 40 J. J. Warsh, D. D. Godse, H. C. Stancer, P. W. Chan and D. D. Coscina, Biochem. Med., 18 (1977) 10-20. 41 A. A. Larson and N. L. Dalo, J. Chromatogr., 375 (1986) 3747. 42 0. Beck and G. Flodberg, Biomed. Mass Specfrom., 11 (1984) 1555158. 43 D. A. Durden, unpublished reseults. 44 D. A. Durden and B. A. Bailey, J. Chromatogr., 368 (1986) 499.58. 45 J. C. Bill, B. W. Gray, B. N. Green and J. A. Yoffe, presented at the 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, May 24-29, 1987. 46 K. Blau, G. S. King and M. Sandler, Biomed. Mass Specfrom., 4 (1977) 232-236. 47 A. J. Lewy and S. Markey, Science (Washington, DC), 201 (1978) 741-743. 48 D. A. Durden, presented at the 34th ASMS Conference on Mass Spectrometry and ANied Topics. Cincinnati, OH, June 8-13. 1986.
CHROM.
Printed
in The Netherlands
20 299
ANALYSIS OF TRYPTAMINE AT THE FEMTOMOLE USING NEGATIVE ION CHEMICAL IONIZATION GRAPHY-MASS SPECTROMETRY
DAVID
A. DURDEN
LEVEL IN TISSUE GAS CHROMATO-
and A. A. BOULTON*
Neuropsychiatric Research Unit, Cancer and Medical Research Building, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO (Canada)
SUMMARY
An ultra sensitive method for the detection of tryptamine, an endogenous amine in mammalian neuronal systems, at the femtomole level has been developed using negative chemical ionization gas chromatography-mass spectrometry (NCIGC-MS). The amine is converted into a perfluorinated spirocyclic derivative, e.g. l-pentafluoro-2-methylenepyrrolidine-3-spiro-3’-(3~-indole) which is detected using selected-ion monitoring of the (M - 2HF) ions of the endogenous and deuterated internal standard compounds. Two mass spectrometers were compared; they gave minimum detectable quantities from tissue samples of 40 pg (VG-7070F) and 0.9 pg (VG-70s) respectively. These detection levels are approximately 5-200 times lower than have been obtained by previous MS methods.
INTRODUCTION
Tryptamine is a naturally occuring trace amine present in the brain of all animals including humans1v2. It is a pharmacologically active amine3 which readily crosses the blood-brain barrier4, influences both human and animal behaviour5-9 and may be implicated in some neurological and psychiatric disorderslO-i2. Tryptamine is formed by decarboxylation of L-tryptophan13-is and is catabolized by monoamine oxidase’+lg. Its metabolite, indole-3-acetic acid, has been detected in human cerebrospiral fluid20 and in rat brain21,22. It has a rapid turnover approaching that of 5-hydroxytryptamine’ 5,23~24 and a very short half life after intracerebral injections25,26. Although tryptamine is known to be distributed within the cell and located in synaptosomes2’, only recently has there been a concerted search for its brain binding siteszs-34. Tryptamine concentrations in the brain are very low even in the striatum1’24 where it is maximally concentrated and although lesions of the mid-brain raphe do not affect striatal levels35, niagrostriatal lesions do36 suggesting an involvement with dopamine pathways3’. Finally, iontophoretically applied tryptamine inhibits cortical cell firing3s,39 independently of 5-hydroxytryptamine and in its presence modulates it. All of the above evidence indicates that tryptamine is a neuromodulator and may 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
D. A. DURDEN,
254
A. A. BOULTON
be a neurotransmitter. In order to study this substance further more detailed information on its location, subcellular distribution and the effect of lesions is required. Such investigations will require the development of super sensitive assays for its detection. The original method developed in this laboratory for the analysis of tryptamine1JJ4 using direct probe electron impact ionization-mass spectrometry (EI-MS) of the 5-dimethylamino-1-napthalenesulfonyl (dansyl) derivative is still one of the most sensitive available with a sensitivity (minimum detectable amount) of about 1 pmol (100 pg). Gas chromatography-mass spectrometry (GC-MS) methods using packed columns22,40 were unable to detect tryptamine at this level and thus workers using this procedure were only able to put upper limits on the endogenous concentrations of tryptamine in whole rat brain. A somewhat more sensitive procedure using high-performance liquid chromatography (HPLC) with fluorescence detection41 has been developed. The most sensitive procedure to date is the GC-MS procedure of Beck and Flodberg4* in which the heptafluorobutyrate derivative was isolated by capillary column GC-MS. We report here an ultra sensitive GC-MS procedure which obtains its high sensitivity from the use of negative chemical ionization (NCI) of a highly electrophilic derivative of tryptamine. MATERIALS
AND METHODS
All solvents were of HPLC-grade and water was purified by reverse osmosis and then by ion exchange to a resistance of 18 MS2 (Barnstead, Nanopure II). Tryptamine hydrochloride was obtained from Sigma (St. Louis, MO, U.S.A.) and a,@$-tetradeutero-tryptamine . HCl (tryptamine-d4) was synthesised in this laboratory by B. A. Davis. Acetic anhydride (Fisher) was redistilled in glass and stored at -20°C before use. Pierce fluorinated acyl anhydrides, pentafluoropropionic anhydride (PFPA), heptafluorobutyric anhydride (HFBA), and trifluoroacetic anhydride (TFAA) were purchased from Chromatographic Specialties (Brockville, Canada). PFPA was redistilled from phosphorus pentoxide and stored in 1 ml ampoules at -20°C. Brain tissue was obtained from 200 g Wistar rats (Charles River Labs., Quebec, Canada). The animals were killed by decapitation, their brains removed, and the hypothalami and striata dissected out and frozen on dry ice. Tissue weights were about 30 mg and 100 mg respectively per animal. Tissues were homogenized in 1 ml 0.1 M perchloric acid containing l-2 ng of tryptamine-d4 and then centrifuged (Fisher micro-centrifuge Model 235B at 12 000 g) for 15 min. To the supernatants were added 0.2 ml acetic anhydride and approx. 0.1 g sodium bicarbonate. The solution was mixed (Vortex Genie) and additional sodium bicarbonate added over a period of 45-60 min until all bubbling ceased. The acetylated tryptamine was then extracted into ethyl acetate (2 x 3 ml). The organic phase was transferred to l-ml Reactivials (Pierce) and taken to dryness at 40°C under a stream of nitrogen. Dichloromethane (100 ~1) was added and again taken to dryness to remove azeotropically any remaining water. PFPA (200 ~1) was then added, the vials sealed with PTFE-faced seals and heated at 80°C for 1 h. The vials were cooled and about 0.4 ml hexane added and the reactants washed with 200 ~1 of phosphate buffer (pH 6). The organic phase was transferred to 200-~1 Reactivials and reduced to a volume of 5-10 ~1 from which an
NCI-GC-MS
OF TRYPTAMINE
255
aliquot (1 ~1) was injected. Blanks containing only the tryptamine-d4 and check samples containing 2.0 ng each of tryptamine and tryptamine-d4 were carried through the entire procedure. For sensitivity checks larger quantities (i.e. 40 pg of each amine) were derivatized as described above. The perfluoroacyl reaction was also repeated for the larger quantities using TFAA and HFBA. MS analysis was carried out using a VG Analytical 7070F double focussing mass spectrometer with a Hewlett-Packard Model 5700 gas chromatograph containing a capillary column connected directly to the ion source. Initial experiments were carried out using for the capillary column an SGE BP5 50 m x 0.32 mm I.D. l-pm bonded phase (thick film capillary column) and for later experiments (including tissues) a J & W DBl 60 m x 0.32 mm I.D. 0.25~pm bonded phase (thin film capillary column). These columns were operated with a helium flow of 3340 cm/s and a temperature program of 170°C isothermal, 2 min, lO”C/min to 290°C and then isothermal for 4 min. Injection was on column (at 200°C) into a short length (15 cm) of megabore column (J & W DBl 0.52 mm I.D. 1.5-pm bonded phase) which was then coupled to the main capillary column with a Supelco union and Vespel graphite seals. In either case the capillary was pushed into the megabore column so that the sample did not pass over the seals. Spectra were recorded under EI conditions at 70 eV, 200 PA collector current and ion source temperature of 170°C and under NC1 conditions, 50 or 100 eV, 2000 PA emission and ion source temperature 140-150°C using either methane or ammonia CI gas. A megabore column (J & W DBl 15 m x 0.52 mm I.D. 1.5-pm bonded phase) with the carrier (5 ml/min) and the make-up gas (25 ml/min) or a packed column 6 x 2 mm I.D., 3% SP2100 on Supelcoport SO/l00 was also connected via the jet separated and used for some experiments, For methane, the source housing pressure was 5 . lo-’ mbar and for ammonia 2 . lop5 mbar to give maximum signals. One ion source was kept for NC1 use and modifications were made to maximize the NC1 signal. The CI aperture of the sliding slit was widened from the original 0.07 mm x 5 mm to 0.3 1 mm x 5 mm. Compared to a second source with a CI slit of 0.11 mm x 5 mm the signal gain was at least a factor of 25. The polarity of the electron beam magnets was also chosen for optimum signal. The beam focussing controls were initially set with perfluorokerosene (PFK) (1 .O ~1 into the septum inlet) present in the source. This was then pumped away and the beam refocussed to maximize the signal on the residual PFK peak at m/z 281 or on the GC column bleed peaks. For quantitation, selected-ion monitoring (SIM) was performed on the (M - 2HF) peak of the spirocyclic derivatives of tryptamine m/z 290.0667 and tryptamine-d4 (M - 2HF) m/z 293.0855 using m/z 280.9824 of PFK as a reference and a resolution of 2000. Although the absolute signal height was reduced by a factor of 2 at this resolution, the signals due to the column bleed ions were reduced much more and hence the background signal was lowered dramatically. SIM was performed using the VG Digital multiple ion detector (DIGMTD) unit to control the accelerating and electric sector potentials and a magnetic field selector43 to control the magnet and the choice of reference mass. The output of the DIGMID unit was filtered using Bessel filters44. A few analyses were performed using a much more sensitive mass spectrometer, the VG 70s. This instrument was equipped with a Hewlett-Packard 5890A gas chromatograph, a universal split/splitless injector and a DBl capillary column (30 m x 0.25 mm I.D. with 0.25~pm bonded film) also connected directly to the ion source.
256
BOULTON
loo-
(M-ZHF) 290
ic. E t 9! ,c
310
128 0, 50
100
I, 150
200
250
L
I
I 300
I
Mass
Fig. 1. NC1 spectrum of the tryptamine derivative l-pentafluoropropionyI-2-methylenepyrrolidine-3-spiro-3’-(3H-indole) (MW = 330), obtained using the 7070F mass spectrometer with methane reagent
gas.
Temperature program was 160°C; 2 min, lO”C/min to 250°C with the spirocyclic derivative eluting at about 210°C. NC1 was undertaken using methane reagent gas and the pressure optimized at about 2 + 10m5-5 . 1O-5 mbar. This instrument possesses a three-position ion source exit slit and the mid-sized EI/CI slit was used. This instrument was also operated at 2000 resolution and SIM profiles of m/z 281, 290 and 293 collected using the VG 1 l-250 J data system and VG SIR software45 using m/z 281 and 331 of PFK as lock masses. The sensitivity was optimized with PFK present in the ion source. RESULTS
AND
DISCUSSION
The reaction of N-acetyltryptamine with PFPA is expected to produce a 3,3spirocyclic compound, l-pentafluoropropionyl-2-methylenepyrrolidine-3-spiro-3’(3H-indole), similar to that from melatonin 46*47. The NC1 spectrum (Fig. 1) supports this structure as does the EI spectrum with a molecular ion of m/z 330. This compound is amazingly electrophilic and a much greater sensitivity (approx. 50-100 times) was obtained in the NC1 mode compared to the EI mode when the spectra were recorded. There was some evidence for the presence of a non-cyclized structure, i.e. N-acetyl-N,N’-dipentafluoropropionyltryptamine43 but it appeared to decompose or cyclize on the column and with the thick film BP5 column (1 pm film) only one product was observed (eluting at 280-290°C). On the thin film DBl column, the spirocyclic structure eluted at a lower temperature and was preceded by a small peak due either to the non-cyclized tryptamine derivative or perhaps a cyclized p-carboline (1 -methyl-pentafluoro-/Lcarboline). The structure and process are under investigation. Even though this small peak was present using the 0.25~,um film DBl column, the sensitivity was not compromised as the majority of the product was as the spirocyclic compound and the GC resolution was much improved over the heavier loaded BP5 column. With a clean ion source and using the packed column, we have been able to detect as little as 160 fg of tryptamine on column4*. This value was obtained with a signal-to-noise (S/N) value of 3 and the output of the digital MID unit heavily filtered (10 s time constant) with the Bessel filter44. Fig. 2 shows the detection of tryptamine from a single caudate nucleus using the 7070F and Fig. 3, a similar tissue
NC&CC-MS
257
OF TRYPTAMINE
10;; ”
---lJ
m/z 293.0955
5 00
515
530
5 45
6:00
6 30
6 15
Fig. 2. SIM analysis of tryptamine with 1.28 ng of tryptamine-d4 internal standard from a single caudate nucleus. Column, DBI, 60 m x 0.32 mm, 0.25 pm. Temperature program, 200°C 2 min; lO”C/min to 290°C. VG 7070F mass spectrometer. Fig. 3. SIM analysis of tryptamine with 1.95 ng tryptamine-d4 internal standard from a single caudate nucleus. Column, DBI, 30 m x 0.25 mm, 0.25 pm. Temperature program, 160°C 2 min; 10”C/min to 250°C. VG 70s mass spectrometer. Only the portion of chromatogram from 5:0 to 6:30 min is shown.
on the 70s instrument. In both cases only about 15-20% of the final solution was injected into the gas chromatograph. Table I shows the results of analysis of tryptamine in the two tissues (using the 7070F) compared to previous values obtained in this laboratory using the dansyl technique’. It is clear that due to the much higher sensitivity of the NCI-GC-MS procedure, the variability is much reduced, i.e. from 39% to 3% for the caudate values and from 23% to 12% for the hypothalamus values. It must be noted that in both cases we are comparing results from single tissues in the case of the NC1 technique and pooled tissues in the case of the dansyl technique. A minimum detectable level from tissues can be calculated using data from Figs. 2 and 3 (shown in Table TABLE
I
TRYPTAMINE
CONCENTRATIONS
Values are means f standard column). 1.28 ng tryptamine-d,
Caudate nucleus Hypothalamus l
standard
IN RAT BRAIN
REGIONS
error of the mean. NCI-GC-MS of the tryptamine internal standard. Tissue from one rat.
PFP derivative
(DBI
Tryptamine
Weight
Concentration
Previous values*
fpg)
Img)
(nglgl
IngIg)
219 f 24 60 f 11
68.3 f 14.1 f
3.20 f 0.10 4.28 f 0.51
2.93 f 0.94 f
7.9 1.6
From ref. 1. Direct probe EI-MS of the tryptamine with tissues from 10 rats pooled.
dansyl derivative.
1.14 0.22
25 pg tryptamine-d,
internal
258
D. A. DURDEN,
TABLE
A. A. BOULTON
I1
COMPARISON OF SIGNAL-TO-NOISE RATIOS OF TWO MASS SPECTROMETERS MUM DETECTABLE QUANTITIES FROM TISSUES
AND MINI-
The signal values from the two instruments
directly
Identity
are in different
707oF* Signal
units and cannot
be compared
7os** Noise
SIN
Min. der.
Signal
Noise
SIN
(Pgi m/z 290 m/z 293
400 1930***
20 20
DBl 60 m x 0.32 mm * DBl 30 m x 0.25 mm l ** Corresponds to 1.28 pg g Corresponds to 1.95 pg l
l
2O:l 96:1
40 40
Min. det. (PS)
320 1822$
0.28 0.28
1143:l 6507: 1
0.5 0.9
I.D. column, ammonia reagent gas. I.D. column, methane reagent gas. tryptamine-d., added to the homogenate. tryptamine-da added to the homogenate.
II) using a minimum S/N ratio of 3. For the 7070F, the minimum detectable level from the tissue used would be about 40 pg whereas a similar tissue on the 70s the minimum detectable level appears to be 500-900 fg. When the Bessel filter is not used on the 7070F the S/N value reduces (to 6:l for m/z 290 in this example) and the minimum detectable level is about 120 pg. This method demonstrates the extremely high sensitivity of NC1 with an appropriate derivative and that with appropriate MS, low femtomol quantities of amines can be detected. ACKNOWLEDGEMENTS
We wish to thank the Medical Research Council of Canada and Saskatchewan Health for continuing financial support, Dr. B. A. Davis for synthesis of the deuterated compounds and E. Zarycki, R. Janzen and N. Pidskalny for assistance with tissue preparation, derivatization and MS analysis respectively. We wish especially to thank Dr. Paul Brooks of the Institute for Sedimentary Petroleum Geology, Calgary, Alberta, for the analysis using the 70s mass spectrometer. REFERENCES 1 2 3 4 5 6 7 8 9
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