ppb, *5% a t 200 ppb, and *2% at 500 ppb. For selenium, the sensitivity is approximately 150 ng per gram of coal with precision values of &15% a t 250 ppb, *lo% at 500 ppb, and f5% a t 1ppm. Preservation and duplication of the counting geometry are two very important aspects of utilizing a low energy photon detector in that (1) the crystal itself is wafer thin, therefore, the sample must cover the surface area of the crystal, and (2) the soft photons or X-rays emitted from the sample are easily absorbed by the matrix of the sample itself; therefore, the sample and standard must be as thin as possible for maximum sensitivity. Using a point X-ray source of 57C0, a series of thirty separate counts were taken over the 201-mm2 area of the LEPD window, and the largest variation registered was *4.5%. This indicates that the counting geometry within this area is relatively uniform, thus providing accurate measurement of the mercury X-rays at any point in the coal sample. This is an especially important feature that enhances the ease of mercury analysis in coal using the LEPD. Use of the Low Energy Photon Detector, as applied to coal analysis can be optimized, sensitivity-wise, in several ways as follows.
1. An irradiation at a 1014 n/cmz sec flux could be used. 2. The detector cover cap could be designed such that the sample being counted would be only several millimeters from the beryllium window. 3. A flat plastic sample holder could be devised with a thin bottom which would reduce the 20% loss of mercury X-rays due to attenuation as was experienced with our sample holder. 4. The large diameter 25-mm Ortec LEPD could be used.
ACKNOWLEDGMENT The author wishes to acknowledge the assistance of M. 0. Schlesinger, U.S. Bureau of Mines, for supplying the coal samples used in this analysis. Received for review February 16, 1973. Accepted April 11, 1973. Presented at the 163rd National Meeting of the American Chemical Society in Boston, Mass., April 1972. This research was supported by the Source Sample and Fuels Analysis Branch of the Environmental Protection Agency.
Chemical Ionization Mass Spectrometry of Complex Molecules: Biogenic Amines G. W. A. Milne,
H. M. Fales, and R.
W. Colburn
National Heart and Lung Institute, National lnstitutes of Health and National lnstitute of Mental Health, Bethesda. Md. 20074
It is now well-known ( I ) that a group of compounds known as “biologically important amines” or “biogenic amines” is involved in the transmission of signals in the nervous system. The complex modes of action of these compounds are poorly understood and studies on this problem have been impeded by the lack of specific and sensitive analytical methods for the detection and quantitation of these compounds in biological preparations. These biologically important amines are widely distributed in many tissues but are present only at extremely low concentrations (nanograms per milliliter) (2). To be useful for their detection and quantitation, any analytical method, as well as being specific, must therefore be applicable at the nanogram-picogram range. Many otherwise useful analytical techniques are not adequately sensitive to deal with this problem and, in recent years, the two most useful methods to emerge have been fluorescence spectrophotometry (3-5) and mass spectrometry (6, 7). The majority of the compounds so far identified as putative transmitters are decarboxylation products of aromatic amino acids such as tyrosine, tryptophan, histidine, and 3,4-dihydroxyphenylalanine(dopa). These compounds (1) P. B. Molinoff and J. Axelrod. Ann. Rev. Biochem.. 40, 465 (1971). (2) L. Bertilsson and L. Palmer, Science. 177, 74 (1972). (3) A. Anton and D. Sayre. J. Pharmacol. Exp. Ther.. 138, 360 (1962). (4) E. G. McGeer and P L. McGeer, Can. J. Biochem. Physiol.. 40. 1141 (1962). (5) D. M. Shaw, A. Malleson, E. Eccleston, and D. Gundy. Brit. J. Psychiat.. 111, 993 (1965). (6) A. A. Boulton and J. R. Majer. J . Chromatogr., 48, 322 (1970). (7) L. Bertilsson. A. J. Atkinson, J. R. Althaus, A. Harfast, J. E. Lindgren, and E. Holmstedt. Anal. Chem., 44, 1434 (1972).
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are well-known (8) to be particularly labile at the carboncarbon bond of the side chain toward electron impact (EI). The reason for this is, of course, the fact that the odd-electron molecular ion can cleave to yield either of the very stable ions I or I1 and the corresponding radical 111 which is itself stabilized by resonance with the aromatic ring. In the case of histamine, cleavage of the side chain is so facile that the resulting ion gives rise to the base peak of the El mass spectrum at m / e 82 while the relative abundance of the molecular ion is under 5%. Similarly, the molecular ions of tryptamine, tyramine, and dopamine have very low relative intensities, the fragment ion at the appropriate m / e value being 5-30 times more intense.
\
III
+
I1
(8) K Biemann. “Mass Spectrometry,” McGraw-Hill. New York. N. Y . , 1962, p 90.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
Table I. lsobutane CI Mass Spectra of Biologically Important Aminesa Compound (mol wt) Ephedrine (165) Histamine (1 11)
Creatinine (1 13) Tryptamine ( 1 60) Tyramine (137) Dopamine (153) 3-Methoxy-4-hydroxy phenethylamine (167) Metanephrine (197) Normetanephrine (183) Octopamine (1 53) Norepinephrine (183) p-Chlorophenylalaninemethyl ester (213) 5-Hydroxyindole acetic acid (191) Tryptophan (204) @-Methyltyrosine(195)
Temp,OC 150 180 170 180 180 150 100 150 150 140 150 150 170 180 170
MH+-
MH+
100 100 100 100 100 100 100 100 66 94 100 100 50 100 100
MH--NH3
COOCH3
MH--HpO
MH*-
COOHp
%Xlb70
9 100 100 100 100 100 98.9 98.6 94.9 95.2
21
6 7 10
82 100 100 47
93.2 6 100 8 17
4
100 99.2 100 95.5
aAll intensities are expressed as a percentage of the base peak (100).bThe percentage of the total ion current above m/e 70 reported in the table. Ions containing isotopes such as 13C and 37CIare ignored in calculating this figure.
A molecular ion whose relative intensity is under 570, while entirely satisfactory when dealing with pure substances, may be ambiguous when the mixtures characteristic of biological samples are encountered. Because of this, specific ion detection, rather than the recording of the complete mass spectrum, has been used extensively for such samples (9). The low relative intensity of the molecular ion has, in general, required that fragment ions, notably the one referred to above, be used for this purpose and, when several intense ions can be found, the method is very reliable since their relative intensities can be monitored. This technique is known as multiple specific ion detection. In the case of histamine, it is clear that the ion of m/e 82 is quite satisfactory since it contains most of the molecular structure of the compound. The ion a t m/e 30 in the E1 mass spectrum of dopamine is found, however, in the E1 mass spectra of all unsubstituted pphenethylamines and an analytical method based on this ion would be lacking in specificity.
EXPERIMENTAL All CIMS reported here were measured on a n MS-9 modified for operation in the CI mode (10). Samples were admitted via a direct insertion probe into the ionization chamber which was heated to between 150 and 200 "C to vaporize the sample. The actual source temperature is given in Table I. The reagent gas in all cases was isobutane of specified purity 99.5%. The CIMS reported here are substantially unchanged when measured on a Finnigan 1015 CI quadrupole mass spectrometer.
RESULTS AND DISCUSSION In CI mass spectrometry, there is formed a protonated molecular ion which, if produced using a reagent ion of suitably low energy content ( e . g . , the tert-butyl ion), has little tendency to fragment (11, 12). For example, ephedrine (IV), when examined using the moderately high energy ions CHs+ and C2H5+ derived from methane, gives a protonated molecular ion of relative abundance of only 40% (13), the base peak being due to the ion formed by loss of water from the protonated molecular ion. When examined with isobutane as the reagent gas a t the same temperature, however, ephedrine gives a mass spectrum in which the base peak is the protonated molecular ion. (9)
Boulton and J. R . Majer, Can. J . Biochem.. 49, 993 (1971). (10) F . H . Field, J. Amer. Chem. SOC.,91, 2827 (1969). (11) F. H. Field, J. Amer. Chem. SOC..91, 6334 (1969). (12) H . M . Fales, G . W. A. Milne, and M . L. Vestal, J . Amer. Chem. SOC..91. 3682 (1969) (13) D. Beggs. M . L. Vestal. H . M . Fales, and G . W. A . Milne, Rev. Sci. Instrum.. 42, 1578 ( 1 9 7 1 ) .
OH
CH,
I
I
CH-CH-NHCH3
Iv The isobutane CIMS of fifteen biologically important amines are given in Table I. In the compounds which do not possess aliphatic hydroxyl groups, the isobutane CIMS are extremely simple, consisting mainly of a protonated molecular ion at m l e ( M 1)+and the corresponding isotope satellite ions, mainly a t m l e ( M 2)+. The extensive fragmentation in the EIMS of histidine, especially the loss of the -CHzNHz side chain with formation of the ion at m/e 82, is completely absent from the CIMS. In the CIMS of dopamine, the only fragment ion is a t m l e 137 and it has a relative abundance of 8%. This ion is formed by the protonation of the primary amino group and the subsequent loss of ammonia from the protonated molecular ion probably facilitated by the aromatic ring as noted earlier in studies of the CI mass spectra of amino acids (14).This same fragmentation is seen in the CIMS of tyramine, tryptamine, and 3-methoxy-4-hydroxyphenethylamine and in each of these compounds gives rise to the only fragment ion in the CIMS. In compounds which have a side chain hydroxyl group, the loss of ammonia from the side chain is usually supplanted by the far more facile loss of water from the protonated aliphatic hydroxyl group. The resulting fragment ion gives rise to the base peak in the case of octopamine (V) and normetanephrine (VI). In the case of norepinephrine (W) and metanephrine (VIII), these ions have relative abundances of 47 and 8270,respectively.
+
+
OH
I
V , R = R ' = H , VI, R = CH,O; R' = H VII, R = HO; R' = H VIII, R = CH,O; R ' = CH,
A. A.
(14) G. W. A . Milne, T. Axenrod, and H . M . Fales. J. Amer. Chem. SOC.. 92. 5170 (1970).
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VOL. 45, NO. 11, SEPTEMBER 1973
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Three compounds whose CIMS are given in Table I are amino acids. The fragmentation seen in these spectra is very similar to that observed previously in the methane CIMS of amino acids (14) although considerably less fragmentation is noted here using isobutane. Protonation of the hydroxyl oxygen of the carboxyl group and subsequent loss of water is not an important process, occurring only in the case of tryptophan where an ion of relative intensity 4% results. In all three compounds, protonation of the carbonyl oxygen and loss of the elements of formic acid appears to be a more facile process as is also observed in the methane CIMS of amino acids (14). The corresponding process, loss of the elements of methyl formate, is the only fragmentation observed in the isobutane CIMS of p chlorophenylalanine methyl ester. It must be pointed out that major problems remain in
applying this or any other gas-phase method to biological samples. The compounds in question here are not highly stable and are very polar. They do not therefore evaporate smoothly from the usual biological sample matrix. Derivatization is often resorted to, but this invariably introduces new complications. This communication shows only the inherent advantages of CIMS for such analysis; its ultimate use is contingent upon the development of proper procedures for handling biological samples. ACKNOWLEDGMENT We are grateful to T. Sun for his assistance in measuring several of these spectra. Received for review December 21, 1972. Accepted March 26, 1973.
Polarographic Study of Calomel Electrode in Anhydrous Formic Acid Michel Arnac and Gilles Verboom
Universife du Quebec a Rimouski, Departement des Sciences Pures, 300 Avenue des Ursulines, Rimouski, P.Q., Canada omel electrode, for the equivalent reference is commonly Anhydrous formic acid is a solvent which is interesting used in glacial acetic acid. from an electrochemical point of view. It is a solvent for many organic and inorganic compounds ( I , 2). This solEXPERIMENTAL vent has high ionizing properties and a dielectric constant of 56.1 a t 25 "C (3). In these respects, formic acid is much Direct current polarograms of the mercury system were obtained for anhydrous formic acid solutions containing variable more like water than such a solvent as acetic acid. Its amounts of chloride. The quinhydrone-formic acid electrode was high dielectric constant eliminates the formation of ion used as a reference (7). pairs in dilute solutions. Most alkali salts are completely Apparatus. Dc voltamperograms were recorded using a classidissociated in formic acid solution ( 4 ) . Nonaqueous solcal three-electrode technique. The apparatus included a Tacussel vents change solvation and complexation states of cations. PRT-2XZ potentiostat, a Tacussel Servovit pilot scanner for the In contrast, the solvent offers considerable experiment difpotentials, an electronic millivoltmeter Tacussel S6R, and a Sefram Graphirac BGVAM galvanometric recorder. Controlled curficulties such as its tendency to undergo slow spontaneous rent coulometries were produced with a Tacussel chronoamperodecomposition, its hygroscopicity (5), its volatility and a CEAMD 5. All the measurements were performed a t constant relatively large self ionization constant (about 10- 9.stat temperature in a Tacussel water jacketed cell, Model RM06, with Another major disadvantage is the impossibility to obtain a five-ground joints cover. anhydrous liquid by the addition of an anhydride. The reference electrode was prepared by dipping a bright platinum wire into a solution of 0.05M quinhydrone and 0.25M sodium While abundant literature exists on the use of glacial formate in formic acid (7). This solution was in a compartment, acetic acid as a nonaqueous solvent, the solvent properties separated from the measuring cell by a sintered glass disk of of formic acid remain largely unexplored. Polarographic 10-mm diameter with a pore size of F. The auxiliary electrode studies were published only by Pinfold and Sebba (6). was a platinum foil of large area. The capillary tube of the dropThey have investigated the quinhydrone electrode and deping mercury electrode was equipped with a ground joint which termined half-wave potentials of some transition elements fits into the cap of the cell. A 15-mm diameter glass bulb, filled with mercury was the coulometric electrode. in anhydrous formic acid. They found the quinhydroneReagents. All the chemical reagents were the purest ones comformic acid electrode to fulfill the requirements of a suitmonly found. They were used as such, without purification, exable reference system (7). Pleskov measured the potential cept for formic acid. Anhydrous formic acid, 99-100'70 "purissiof many reversible electrodes and finally chose a silvermum grade," was obtained from Fluka. This acid was dried over silver picrate system as a reference (8), while Mukherjee boric anhydride. It was then fractionally distilled a t room temrecommends the silver chloride electrode (9). perature under a pressure of about 10 mm of mercury according to Popov and Marshall ( 5 ) . Average specific conductance of the We felt it was necessary to investigate by a polaropurified formic acid was 7.0 X 10-5 ohm-' X cm-1. It froze graphic study the formic acid analog of the saturated cal(1) 0. Aschan,Chem. Ztg.. Chem. App., 37, 117 (1913). (2) T. Pavlopoulos and H. Strehlow. Z. Phys. Chem., 202, 474 (1954). (3) J. F. Johnson and R. H. Cole, J. Amer. Chem. SOC.. 73, 4536 (1951 ) . (4) T. C. Wehman and A. I . Popov, J, Phys. Chem., 72, 4031 (19681, (5) A. I . Popov and J . C. Marshall, J . Inorg. Nuci. Chem.. 19, 340 (1961). (6) T. A. Pinfold and F. Sebba, J. Amer. Chem. SOC.,78, 5193 (1956). 78, 2095 (1956). (7) T. A. Pinfold and F. Sebba, J. Amer. Chem. SOC., (8) V. Pleskov, Acta Physicochimica U.R.S.S.. 21, 41 (1946). (9) L. M . Mukherjee.J. Amer. Chem. SOC.. 79, 4040 (1957).
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sharply a t 8.3 i 0.1 "C. These results are in essential agreement with the best values found in the literature ( 4 ) . It was stored in colored sealed glass stoppered bottles and kept under refrigeration to minimize the decomposition. Procedure. The working cell was equipped with a condenser unit, cooled by circulating ice water. Controlled current oxidation (10 mA) of a mercury coulometric electrode of large area introduced mercurous ions in the solution. The drop time of the capillary electrode was in the range of 4-5 seconds. Voltage scanning was 60 mV per minute. Solutions were all initially deaerated with oxygen-free nitrogen for 10 mn.
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 11, SEPTEMBER 1973
