of the bromate or the peroxide introduces complications into the extraction procedure. The bromate slowly decomposes producing bromine gas and the silver peroxide produces a precipitate during the extraction process. Tests indicate that a fuming time of 80 min and an oxidation time of 10 min were optimal for the sample sizes used in this study. Samples of equilibrium 103R~-103mRh were evaporated to dryness, reconstituted with hot sulfuric acid or with hot hydrochloric acid, and then carried through the extraction procedure. Fuming a sample to dryness was very detrimental to the subsequent extraction of l03Ru, but it had no effect on 103mRhyields. Approximately 40% of reconstituted lo3Ru was not extracted by the organic phase. I t should be mentioned that, even with repeated evaporation to dryness, there were no isotopic losses by volatilization, nor was secular equilibrium disturbed. The extraction is fast and simple. Rhodium-103m yields are good and the lo3Ru can be completely recovered and used again. The fact that the loBrnRhsulfato complex is highly stable gives reason to believe that logmRhcan be successfully isolated in a pure form suitable for biological usage. It is believed that the procedure is sound, and that, with some additional work, the extraction of loSmRhfrom lo3Ru can be

made quantitative. Furthermore, the extraction system used in this study might be applicable to the large scale recovery of rhodium.

LITERATURE CITED T. Lengyel, "Preparation and Control of Rhodium-103m Radiopharmaceuticals", IAEA. STIIPUB/294, 137, 1971. C. Rohrmann, /sot. Radiat. Techno/.,6, 352 (1969). A. LeRov. Ed., "A ComDrehensive EibliwraDhy of Element 44, Ruthenium", BRHKFS 70-1, 1969: J. Armstrong and G. Choppin, "Radiochemistry of Rhodium", NASNS 3008, IF165

K:&ura, N. Ikeda.and K. Yoshihara, Bull. Chem. Soc. Jpn, 29,395 (1956). T. Autokratova, "Analytical Chemistry of Ruthenium", Ann Arbor-Humphrey, Ann Arbor, Mich., 1969, p 144. F. Eeamish, Talanfa, 14,991 (1967). J. Korkisch. "Modern Methods for the Separation of Rarer Metal Ions", Pergamon, New York, 1969, p 524. E. A. Hausman, US. Patent 3,166,404, Jan. 19. 1965. C. Surasiti and E. Sandell, Anal. Chim. Acta, 22, 261 (1960). M. Khan and D. Morris, J. Less-Common Met., 13, 53 (1961). M. Khan and D. Morris, Sep. Scl., 2, 635 (1967). I. Kolthoff and E. Sandeil, "Textbook of Quantitative Inorganic Analysis", 3rd ed., Macmillan, New York. 1952, p 582.

RECEIVEDfor review January 8, 1976. Accepted March 4, 1976. Work supported in part by the United States Public Health Services under Grant No. 3-T01-FDO-1008.

Field Desorption Mass Spectrometry of Biogenic Amines and lsoquinoline Alkaloids: Some Comparisons with Chemical Ionization Results Gordon W. Wood* and Ning Mak Department of Chemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4

Alan M. Hogg Department of Chemistry, The University of Alberta, Edmonton, Alberta, Canada

Field desorption mass spectrometry has been shown to give molecular ions for several structural types of 2-arylethylamines, both as free bases and as salts. Synthetic samples of isoquinoline alkaloid derivatives, which may arise from in vivo condensation with aldehydes, were also studied. Comparative results obtained using other ionization techniques are included.

Field desorption mass spectrometry (FDMS) is a relatively new technique with potential for analysis of a number of important classes of biological compounds. Biogenic amines constitute one such class, the chemical ionization mass spectral (CIMS) properties of which have been published recently ( 11. Since a t least part of the advantage of CIMS arises from stability of the protonated molecular ion, we undertook a comparative FD study where additional advantage may be expected from the fact that gentle heating is sufficient to cause desorption. One context in which analysis of biogenic amines is of interest involves the alleged formation of isoquinoline alkaloids from condensation with acetaldehyde derived from ethanol ingestion (2, 3 ) . We therefore include a number of synthetic isoquinoline alkaloids in this study. In addition, several cross comparisons between various methods of ionization-electron impact (EI), CI, and field ionization (F1)-are included.

EXPERIMENTAL Field desorption and electron impact mass spectra were obtained on a Varian Model CH 5 DF Spectrometer with FD/CI/EI source. For field desorption measurements, samples were applied by the dipping technique ( 4 )to a 10-k tungsten wire which was conditioned (PhCN) at 40 mA (Varian procedure). The source was unheated (-80 "C) except for a current applied directly to the tungsten wire anode. Anode potential was +3 kV and the cathode was generally at -7 kV. Spectra were recorded electrically on light-sensitive paper calibrated by a Hall probe mass marker. The mass scale was established by comparison with the spectrum of perfluorokerosene run under identical scanning conditions in the E1 mode. Scan rate was generally 25 amu/sec. Spectra were qualitatively reproducible but, since fragmentation is a sensitive function of anode heating current, some quantitative differences were observed in spectra run with different anodes at the same current. This effect no doubt arises from temperature differences related to varying anode resistance. Chemical ionization spectra were obtained on a modified AEI MS12 mass spectrometer ( 5 ) combined with an AEI DS50 on-line data system. In most cases, the source was held at 150 "C and the sample heated independently to a temperature giving adequate vapor pressure. Where temperatures in excess of 150 "C were required, both source and sample were heated to about the same temperature. Ammonia at a pressure of about 0.6 Torr was used as a reagent gas throughout because experience in this laboratory has shown that it gives the greatest ion current due to the protonated molecular ion with amines, and its relatively high boiling point is well suited to our liquid nitrogen trapped vacuum system. Methane and isobutane were also evaluated but, as was to be expected, a greater degree of fragmentation ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

981

was encountered which was considered undesirable in a direct comparison with FDMS. Biogenic amine samples were obtained from Sigma Chemical Corporation with the exception of the following: dopamine was obtained as the free base by a published procedure (61,and tyramine salts were prepared from 40 mg of free base dissolved in absolute ethanol (8ml) by addition of the appropriateacid in absolute ethanol until the pH reached 2. Salts were isolated by concentration of the solution and addition of ethyl ether (ethyl ether was replaced by benzene for the iodide and sulfate).The crude salts were recrystallized (mixture of ethanol and precipitating solvent) and their identity was checked by infrared spectrometry. Isoquinolines were prepared by the condensation of an aldehyde with the appropriate 2-phenylethylamine (Pictet-Spengler reaction) in the laboratory of Maurice Hirst, Department of Pharmacology, University of Western Ontario.

Table I. Fragmentation of Tyramine by Various Methods of Ionization Ionization M+1 M M - 30 30 Ref. E1 CI FD

15

25

I

100

100

1

This work " Result obtained on free amine at 16-mA anode current. This value is variable, but it is clearly in excess of the 13C isotope contribution. 35"

100

16

&cleavage somewhat under FD conditions (compare A = C1, Br, I). Several common biogenic amines were studied as free bases RESULTS AND DISCUSSION and hydrochlorides to establish the influence of various The following amines were studied in the present work: structural parameters on FDMS. The results are reported in tyramine, 1; dopamine, 2; 3-methoxytyramine, 3; epinephrine, Table 111, along the data for serotonin, 8, in its common cre4; normetanephrine, 5; tryptamine, 6; 5-methoxytryptamine, atinine sulfate form. 7; serotonin, 8. Of the several conclusions to be drawn from these data, perhaps most important is the fact that, with controlled anode heating, every one of the amines and salts tested gave the molecular ion of the free amine as base peak. Considering the range of structures presented, and especially the aliphatic hydroxyl groups in 4 and 5, this represents a significant finding. Second, the operating parameter to which fragmen6 2-H 2 R-R'=R'hX'H tation is most sensitive appears to be anode heating. I t is 3 R'R'WH, RtCH3 7 t-oCH3 perhaps worth noting in this connection that tyramine, 1, and 4 R-R'", R ' C C H 3 . X-OH 8 zton tryptamine hydrochloride, 6, are much less sensitive than dopamine, 2. In the case of the latter, careful control of anode 5 R=R"=H. R"CHJ X'OH heating is essential if the molecular ion is to be recorded as Synthetic isoquinolines had the structures 9-15 as shown base peak. below: Serotonin, 8, is of considerable interest because it is handled as a mixed sulfate with creatinine being the other base. FDMS of this complex salt as received gave a remarkably clear-cut spectrum with two base peaks corresponding to the two amines, albeit a t a somewhat higher than usual anode tem14 perature. We associate the relatively intense P-cleavage peak with this latter circumstance. Direct comparison of the four methods of ionization (CI, EI, FI, FD) was made on 5-methoxytryptamine 7, with the 13 results shown in Table IV. Each of the three newer methods n no o w n c o n of ionization yields a baseo peak related to the molecular ion, whereas the more conventional electron impact technique has I5 one of the 6-cleavage fragments (M - 30) as the base peak. In both CI and FD this cleavage is quite effectively suppressed, The major fragmentation pathway of 2-phenylethylamines while FI gives an intermediate result. The various ratios of M involves 6-cleavage with the formation of ions M - 30 or m/e 30 to 30 may be rationalized by assuming a strong prefer30 or both. ence for placing the charge on M - 30 under gas-phase (unimolecular) conditions (EI),a preference which no longer exists in condensed phase (FD). Field ionization spectra determined in the usual way are composites of FI (gas phase) and FD (condensed phase). Further study is required t o determine Y M- 30 m h 30 whether this factor or thermal effects arising from the different operating temperatures for FI and FD are primarily The relative importance of this process in underivatized tyramine after various methods of ionization is shown in Table responsible for the observed differences. I. For this example, only CI prevents P-cleavage although FD Mass spectra obtained in FD mode of the seven isoquinolshows a substantial reduction in this mode of fragmentation ines are presented in Table V. These spectra would be of some by comparison with EI. This same amine was used to detervalue analytically, since the base peak is M or M 1in every mine the effect on the FD spectrum of protonation by various case. The relative intensity of these ions is not simply related acids. These results are shown in Table 11. to structure. The presence of OH, COOH, or additional aroThe amine FD spectrum is significantly influenced by the matic rings appears to lead to varying amounts of M H as properties of the acid used for salt formation. The general well as more complex fragmentation by comparison with 11 trend can most readily be accounted for on the basis of acid and 12. volatility. Thus, the hydrochloride does not differ from the One goal of this study was to compare the nature and degree free amine, whereas the sulfate and sulfonate spectra are of fragmentation of CI and FD for these compounds. For this dominated by the protonated amine ion. Increasing the acid purpose, the results of Milne et al. ( I ) are supplemented by results which are reported in Table VI. CI ("3) strength also may favor the protonated species and reduce I

+

+

962

ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

Table 11. FD Mass Spectra of Tyramine Salts (BH+.A-)"

A

Anode, mAb

BH

B

B-30

30

Others (rnle)

...

10 15 100 33 22 13 25 100 14 139 100 32 . 43 80,' 82c Br 18.5 27 I 15 51 100 43 7 12P 100 25 87 63,c 64c NO3 14 47 HS04 10 100 5 218, 235,d 372e p -CH3CsH4S03 8 100 4 309,d 449,e 757e " Peaks above mle 28 with relative intensity 5% or greater are reported. The free amine is symbolized by B, its conjugate acid,by BH+, and the accompanying anion by A-. Heating current applied to field anode. Peaks derived from HA. Peaks derived from BH-A. ePeaks derived from BH-AB, BH-2AB.

c1

Table 111. FD Mass Spectra of Selected Biogenic Amines" Compound (mol wt) Anode, mAb M+1 (137)

M - 30

M

Others

100 22 100 33 rnle 30 = 22 2 (153) 100 10 7 100 2c 100 M + 1 - 17 = 1 3c (167) 100 11 M + 1- 1 7 = 3 4 (183) 100 M-44=11 100 11 M + 1 - 17 = 2; m/e 30 = 7 5c (183) 6c (160) 100 18 17 100 8 7 (190) 12 22 100 6 mle 30 = 7 8d (176) 20 36 100 60 m/e 113 = 100; 114 = 68 a All intensities are expressed as a percentage of the base peak (100). Peaks above mle 28 with relative intensity 5% or greater are reported, along with certain others for comparison with CI. Heating current applied to field anode. Run as hydrochloride. Run as serotonin creatinine (mol w t 113) sulfate. 1

0 10 16 17 15 17 16 17 0

15 33 18 30 9 54 14

Table IV. Mass Spectra of 5-Methoxytryptamine by Various Ionization Methods Ionization M+2 M+1 M M-29 M-30 M-31 FD; 1 2 mA FI; probe 200 "C OC" CI; "3,150 EI; probe 200 OC " Peaks at rnle 208 (M

22

22

100 100

+ 1 + "3)

M-45

M-73

30 7

25

5

11

9

100

8

6 33

M-43

31 = 24%, 78 = 48,78

22

100

+ NH3 = 11also observed.

11

8

Table V. FD Mass Spectra of Isoquinoline Alkaloids" Compound

(mol wt)

9

(165)

Anode, mA

M-1

M

M+1

M+2

M+3

2M

2M+1 2M+22M+3

18 19 100 90 9 5 4 (179) 16 4 13 100 48 13 4 7 11 (193) 17 100 28 12 (207) 14 100 17 13 (339) 12 100 38 4 8 25 13 4 14 (177) 15 4 7 100 57 4 15c (223) 24 44 89 100 25 a Compounds run as hydrochlorides.All ions 3% relative intensity and common to at least two compounds are reported. Ions peculiar to one compound are listed in footnotes. Hydrobromide gives M (loo),M + 1 (84),M 2 (8),2MH+.Br- (18,22). This compound gives a total of 14 other ions, including 4 which are doubly charged, representing weights from M - 2 to M - 48. The most intense are M - 45 (31%),M - 44 (31), M - 44'+ (33). lob

+

The ammonia chemical ionization spectra in Table VI show, in the main, intense peaks corresponding to the protonated molecular ion with the exception of serotonin creatinine sulfate which shows only the MH+ for creatinine below 200 OC with the serotonin MH+ appearing to a lesser extent a t higher temperatures. This may well be a manifestation of the problem frequently encountered in CIMS where one component of a mixture (even a very minor one) may suppress the ionization of other compounds of lower proton affinity. Similarly

a number of low mass ions, mainly 78+ (together with its ammoniated 95+), 49+ (66+) and.46+ (63+),are observed with vaying abundances in the spectra. While these are not present in the spectrum of ammonia they are probably due to impurities in the system and are unrelated to the compounds under study. I t would no doubt be possible by careful sample purification and adjustment of the CI operating conditions to produce spectra which show virtually only the MH+ ion together with ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

983

Table VI. CI(NH3) Mass Spectra of Selected Amines Major peaksa ComTemp, pound (mol wt) O C M+1 Others: m/e (96) 4 7

(183) (190) (176)

66(12),49(100) 161(9),95(11), 78(48) 8* 160 0 131(58),114(100), 69(13), 63(11),49(11) 220 17 227(14), 131(56), 114(100),66(8), 49(154) 10 (179) 160 100 78(9),61(55) 12 (207) 125 100 95(19),78(29) 14c (177) 150 100 176(4),63(3), 46(4) a (M 1)+-NH3peaks are generally present with relative intensities 5 2 5 % as are (2M 1)+ peaks particularly at higher sample pressures. These peaks are omitted together with all those arising from the spectrum of NH3 reagent gas. Run as serotonin creatinine (mol wt 113) sulfate. This spectrum was obtained only after allowing the sample to evaporate in the direct probe for 9 min. A t shorter times, the spectrum was dominated by a peak at m / e 192, apparently resulting from a more volatile aryl methyl ether impurity present in the sample (E1and FD suggests that about 2% of this impurity is present).

+

160

75 100

150

+

the reagent gas ions. However, in this case, the samples were handled in a completely routine manner to better approximate the results which would be obtained in the case of an unknown of biological origin. Thus the characteristics of CIMS when used in these circumstances can be summarized as simple and reliable operation and high sensitivity. Ion currents due to sample are a t least as high for the same sample flow as in EIMS, and protonated molecular ion currents greater than A a t the collector are obtainable. CONCLUSIONS The present work demonstrates the potential of FDMS for analysis and structure determination of biogenic amines and

related compounds. The comparisons with spectra obtained by other techniques allow for some assessment of their relative strengths. The caveat contained in the last paragraph of the paper by Milne et al. (1)emphasizes the difficulties inherent in the application of CIMS to biological samples and it is likely that formidable problems would be associated with FDMS also. In introducing this latter alternative, one escapes the problems related to vaporization of an unstable, polar sample and thereby gains an element of flexibility in that nonvolatile salts which have improved spectral characteristics may be employed. To counter this, one may expect a loss in sensitivity and, perhaps, a new set of sample preparation difficulties. We have not explored this latter area, except to show that the presence of alkali metal salts (NaCl, CsC1) does not interfere with smooth field desorption of isoquinoline 13, even when massive amounts are added to the sample. It may also be noted that no evidence of sodium containing organic ions was found in any of the commercial samples studied, a result which our experience suggests is more likely to arise from favorable field desorption characteristics of the compounds than from scrupulous absence of sodium salts. ACKNOWLEDGMENT The authors acknowledge Maurice Hirst for generous samples of isoquinolines. We thank Robert Charlton, Pui-Yan Lau and John Olekszyk for technical assistance. LITERATURE C I T E D (1) G. W. A. Milne, H. M. Fales,andR. W. Colburn, Anal. Chem., 45, 1952(1973). (2)G.Cohen and M. Collins, Science, 167, 1749 (1970). (3)V. E. Davis, M. J. Walsh, and Y-L. Yamanaka, J. Pharmcol. Exp. mer., 174, 401 (1970). (4) H. D. Beckey, Int. J. Mass Spectrom. /on Phys., 2, 500 (1969). (5) A. M. Hogg, Anal. Chem., 44, 227 (1972). (6) E. Waser and H. Sommer, HeIv. Chim. Acta, 6, 61 (1923). (7) J. Reisch, R. Pagnucco, H. Alfes, N. Jantos, and H. Mollmann, J. Pharm. Pharmacol., 20, 81 (1968).

RECEIVEDfor review December 1,1975.Accepted March 8, 1976. We are grateful to the National Research Council of Canada for generous support.

Determination of Chlorinated Dibenzo-p-dioxins in Pentachlorophenol by Gas Chromatography-Mass Spectrometry W. W. Blaser,” R. A. Bredeweg, L. A. Shadoff, and R. H. Stehl Analytical Laboratories, Dow Chemical U.S.A., Midland, Mich. 48640

A gas chromatographic-mass spectrometric procedure has been developed for the determination of chlorinated dibenzo-pdioxins in pentachlorophenol. The chlorinated dibenzo-pdioxins are isolated from the sample matrix using ion exchange chromatography. Since the detection system is highly specific, no further sample preparation is necessary.

Several papers (1-5) have drawn attention to the chlorinated dibenzo-p-dioxin (CDD) content in technical grades of domestically manufactured pentachlorophenol. In these, the analytical method employed to measure CDD has been electron capture gas chromatography. This detection system is relatively nonspecific to the large number of structu984

ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976

rally similar components found in these samples (6);thus, a multistep clean-up procedure was employed to prepare the samples prior to quantitation. The purpose of this paper is to describe a simple analytical technique for the accurate determination of CDD employing ion exchange chromatography followed by gas chromatography-mass spectrometry (GUMS) determination. Since the mass spectrometer can monitor the molecular ions from the particular component of interest to the exclusion of other ions, the specificity attained allows minimal sample clean-up prior to quantitation with high sensitivity. EXPERIMENTAL Solvents. All solvents used in the procedure were “distilled in glass” quality obtained from Burdick and Jackson Company,