Anal. Chem. 1983, 55, 1033-1036 (3) Winterstein, A.; Schon, K. Hoppe-Seyler's Z. Physlol. Chem. 1934, 230. 146-1!58. (4) Herlan, A. Zbl. Eakt. Hyg., I Abt. Orig. 6.1977, 165, 174-191. (5) Herlan, A. €dol Kohls 1974, 27, 138-145. (6) Glger, W.; Bllumer, M. Anal. Chem. 1974, 46, 1683-1871. (7) Matsushita, H.; Esunii, Y.; Yamada, K. Japan Analyst 1970, 19, 951-966. (8) McKay, J. F.; Latham, D. R. Anal. Chem. 1973, 45, 1050-1055. (9) Thorns, R.; Zander, M. Fresenius' Z. Anal. Chem. 1978, 262, %. 443-445. (IO) Blumer, G.-F'.; Zander, M. Fresenlus' Z. Anal. Chem. 1977, 288, 277-280. (1 1) Sauerland, A. D.; Stedelhofer, J.; Thorns, 13.; Zander, M. Erdol Kohle 1977, 30, 215-216. (12) Blumer, G.-P.; Zander, M. Proceedings, 26th DGMK-Haupttagung, Berlin, 1978, pp 1472-1403. (13) Peaden, P. 11.;Lee, M. L.: Hlrata, Y.; Novotny, M. Anal. Chem. 1980, 52, 2268-2271. (14) Bj~rseth,A. VDI-Eerichte 1980, 358, 81-93. (15) Hlrata, Y.; Novotny, M. J. Chromatogr. 1979, 166, 521-528. (16) Hlrata, Y.; Novotny, M.; Peaden, P. A,; Lee, M. L. Anal. Chlm. Acta 1981, 727, 55-61.
1033
(17) Peaden, P. A.; Wright, B. W.; Lee, M. L. Chromatographla 1982, 75, 335-340. (18) Snook, M. E.; Severson, R. F.; Arrendaie, R. F.; Higman, H. C.; Chortyk, 0. T. Beltr. Tabakforsch. 1979, 9 ,79-101. (19) Lee, M. L.; Hltes, A. Anal. Chem. 1978, 48, 1890-1893. (20) Stenberg, IJ.; Alsberg, T.; Blomberg, L.; Wannman, T. "Polynuclear Aromatic Hydrocarbons"; Ann Arbor Science: Ann Arbor, MI, 1979; pp 313-328. (21) Romanowskl, T.; Funcke, W.; Konig, J.; Balfanz, E. HRC CC J. High Resolut. Cliromatogr. Chromatogr, Commun. 1981, 4 , 209-214. (22) Baifanz, E.: Konlg, J.; Funcke, W.; Romanowski, T. Fresenlus' Z. Anal. Chem. 1981, 306,340-346. (23) Schomburg, G.; Husmann, H.; Weeke, F. J. Chromatogr. 1974, 99, 63-79. (24) Vangaever, F.; Sandra, P.; Verzeie, M. Chromatographia 1979, 12, 153-154. (25) Blomberg, I..; Wannman, T. J. Chromatogr. 1979, 786,159-166.
RECEIVED for review November 1, 1982. Accepted March 1, 1983.
Fast Atom Bombardment and Mass Spectrometry/Mass Spectrometry for Analysis of a Mixture of Ornithine-Containing Lipids from Thiobacillus thiooxidans K. B. Tomer, F. W. Crow, H. W. Kmoche,' and M. L. Gross* Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588
Two thermally lablle, zwitterionic ornlthlne-containing lipids Isolated as a mlxture from Thlobac///ustMooxidans have been analyzed by using fast atom bombardment (FAB) for vaporlzatlon and maim spectrometry/mass spectrometry (MS/MS) for analysls. E,achllpld component ylelded a gas-phase protonated molecule whose composltlons were determined to be C,,H,,N,O, andl C30H7SN20B' Colllslon Induced decomposltlon (CID) spectra of the lower molecular weight homologue obtained In an MSi/MS mode revealed that part of the molecule which has one less methylene group. The molecular mass assignments of the constltuents were verlfled by a study of the (M H) negative lons. C I D spectra of these negatlve lons gave new structural lnformatlon whlch was complementary to that from the C I D spectra of the posltlve Ions.
-
The recently introduced technique o:f fast atom bombardment (FAB) (1,2) mass spectrometry has rapidly developed into an extremely useful analytical technique. It is especially useful for proviiding molecular weight information for polar, zwitterionic, and/or labile compounds whose mass spectra have heretofore been determined only with extreme difficulty. In this technique the analyte is typically dissolved in glycerol, and the solution is subjected to bombardment by fast atoms (6-8 keV). By a sputtering process, the analyte molecules are ejected from the surface often as (M + H)+or (M - H)-ions. Mass spectrometry/mass spectrometry (MS/MS) (3) is also an important instrumentation advance and has developed in parallel with FAB. It offers some distinct advantages which, Department of Agricultural Biochemistry, University of Nebraska-Lincnln, Lincoln, NE.
if used in combination with FAB, produce a combination suitable for analysis of many polar molecules of biological importance. In fact, many of the major drawbacks of FAB are overcome by the combination. For example, FAB mass spectra show considerable chemical noise from the glycerol matrix which can be removed by MS/MS analysis. Furthermore, FAB spectra are often very simple and contain little structural information. With MS/MS, the molecular ion or protonated molecular ion can be selected and collisionally activated and a "mass spectrum" produced. Finally, isolates of biological origin are often mixtures. In that case, the MS-I of the MS/MS can serve as a separation stage for each component and MS-I1 used for structural analysis or identification provided all components are amenable to FAB desorption. The feasibility of coupling MS/MS with desorption methods of ionization such as FAB for obtaining collision induced decomposition (CID) spectra has been demonstrated by other workers (4-9). CID spectra have been acquired for various small peptides ionized by field desorption (4-6) and by FAB (7). The FD MS/MS experiments were carried out on a forward geometry double focusing mass spectrometer by using linked B / E scans, and the FAB experiments were done by using tandem quadrupole analyzers as the MS/MS. FD ionization of benzo[a]pyrene/DNA adducts has been shown to produce sufficient ion beam for MS/MS analysis by BIE linked scans (8). A method of analysis of mixtures of surfactants has been developed recently which makes use of FD ionization and MS/MS analysis with a tandem magnetic sector instrument equipped with a channeltron electron multiplier array (CEMA) detector (9). The instrument permits simultaneous detection of small portions of the CID spectrum which is a real advantage for study of the weak, short-lived, and often fluctuating ion currents produced by FD.
0003-2700/83/0355-1033$01.50/00 1983 American Chemical Society
1034
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, O=C-CH-ICH2jgI
F Y;
C=O
t
CH-CH-( \ /
JUNE 1983 CH2)5-
CY3
I 0
100 90 -
-
60 70 60
,681
-
50
-
513
5 IC
G6H+
0
Flgure 1. Structure of ornithine-containing lipid.
MS/MS has also been combined with laser desorption (10) and with liquid ion evaporation (11) to produce a method which promises to be suitable for study of various thermally labile molecules of biochemical interest. In this paper, we report the analysis of a binary mixture of amino acid containing lipids isolated from Thiobacillus thiooxidans. CID spectra were obtained with a triple analyzer MS/MS instrument (12)equipped with FAB ionization. The lipids did not give molecular ions when ionized by electron ionization (13)or chemical ionization (14) but instead thermally decomposed when heated on the sample introduction probe losing both water and one of the constituent fatty acids. As a result, only fragments of the themolysis were observed. This study provides the first direct evidence for the molecular weights of the compounds. Moreover, using the exact mass measurement capabilities of the MS-I of the triple analyzer MS/MS (12),the exact masses of the molecular species were determined to verify the chemical formulas of the two lipid components. Finally, the steady ion beams produced by FAB permitted CID spectra to be obtained of both the (M + H)' and (M - H)- of each component. These CID spectra are complementary in information content and have been interpreted in terms of structures to show that the minor constituent is a homologue of the major component of the isolate.
EXPERIMENTAL SECTION The ornithine-containing lipid, which is the subject of this investigation, was isolated and purified as described previously (15-1 7).
Mass spectra were obtained with a Kratos MS-50 triple analyzer mass spectrometer which has been recently described (12). Briefly, the instrument consists of a high-resolution MS-I of Nier-Johnson geometry followed by an electrostatic analyzer used as MS-11. MS-I is a standard Kratos MS-50 with an upper limit of mass resolving power of greater than 100000. The FAB ion source was of the standard Kratos design and was used with an Ion Tech atom gun. The sample (approximately 20 wg) was dissolved in glycerol, and a small drop of the solution was placed on the copper target end of a direct-insertion probe. The bombardment was with 7-keV argon atoms. The FAB produced ions were accelerated through 8 keV. CID spectra were taken by scanning MS-I1 following activation in the third field-free region. CID spectra were acquired and signal averaged with a standard Kratos DS-55 computer system using software written in this laboratory. In the case of the negative ion CID spectra, corroboration of the mass assignment is found from the observation of the same ions in the full scan spectrum. A spectrum of glycerol was obtained in the absence of the lipid, and peaks arising from the glycerol are so indicated in the spectra. The elemental compositions of the two lipid components were confirmed from their exact masses which were measured by peak-matching. The m / z 681.5781 was measured with respect to m / z 645.3392 from glycerol [ (glycer~l)~H+]. The m / z 645.3392 is not seen in Figure 2 because the sample concentration in glycerol was greater for this experiment than for peak matching. A lower concentration of sample led to more abundant glycerol cluster ions.
RESULTS AND DISCUSSION The FAB spectrum, obtained of the lipid (see Figure 2), showed a protonated molecular ion a t m / z 681 which is consistent with the previously assigned structure (13). In addition
:sa
I
667
:20 450
4
500
,
600
550
130
650
750
.IO
I85 G"i+
i :: j 60
30
Flgure 2. Positive ion FAB mass spectrum of the ornithine-containing lipid. Intensities of weaker ions (such as 13C isotope peaks) are susceptible to variations due to ion statistics. Only noise peaks were observed above m l z 750. .2L
T' 297
1
m ,,
Figure 3. Negative ion FAB mass spectrum of the ornithine-containing lipid. Spectrum shown is a single scan. Intensities of weaker ions (such as 13C isotope peaks) are susceptible to variations due to ion statistics. Only noise peaks were observed above m l z 700.
to the molecular ion envelope, an envelope of ions 14 amu lower (mlz 667) was observed which may be indicative of the presence of a homologous compound. Some evidence for the presence of this homologue was also seen after careful interpretation of the fragment ions in the electron impact spectrum (13). The negative ion FAB spectrum (Figure 3) shows (M - H) anions of the two homologous compounds a t m / z 679 and 665. Thus, the negative ion spectrum complements the spectrum obtained in the positive ion mode and serves as confirmation that m / z 667 and 681 are (M H)' ions and not other species such as (M Na)' or (M K)'. T o confirm the proposed elemental compositions, peak matching experiments in the positive ion mode were performed. The elemental composition of the m / z 681 was found to be C40H77N206 (681.5781) with an error in the mass measurement of 8 ppm. Peak matching the m / z 681 and the homologous ion, m / z 667, showed the difference to be a CH2 group with a mass measurement error of 1.6 ppm. MS/MS experiments were performed on both the m / z 667 and m/z 681 ions in the positive ion mode. The resulting CID spectra are shown in Figure 4. The major fragment ions are identical in each case. There is a peak due to loss of water in both spectra. The first major fragment ion is m/z 387. This ion is due to loss of the fatty acid moiety possibly as a ketene to give ion A (Scheme I). Successive losses of two water molecules give m / z 351 (ion B). This ion is the highest mass ion of significant intensity observed in the electron impact
+
+ +
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Figure 4. (a) M S M S spectrum of m l z 681 in the positive ion mode (average of 20 $;cans). The major fragment ions are m l z 35 1, m l z 369, and rnlz 387. (b) MSIMS spectrum off rnlz 667 in the positive ion mode (average of 20 scans). The major fragment ions are m l z 115, m l z 351, m / z 369, and r n l z 387.
1035
Figure 5. (a) MSIMS spectrum of m l r 679 in the negative ion mode (average of 20 scans). The major fragment ions are rnlz 31 1 and m l z 367. (b) MSlMS spectrum of m l z 665 in the negative ion mode (average of 20 scans). The major fragment ions are rnlz 297 and m l z 367.
Scheme I1
1
0-c-
/"*
Scheme I
I
O-
Yo
i"'
1 -cn-cn-icH2ig-cn3
H ICH
Fbn-
2a
0
/I
CH2 m/r 679
I
I
I
CH-NH-C-CH2-CH-(CH2112-Ch3
+
7-
r r /"3
/
CH2
1
-KETENE
mi2
:: Y
367
'O-C-CH-C16H31
F
H,p
CH-NH-C-CH2 1 I 0
ul
0
I -C H - ( C H z ) 1 2 -
A CH3
m T 3 ~ 7
-2Hz0
B
a
m/z 351
NH-C- CH=CH-(CH2)12-CH3
+I H
0.HF
s,
m/z 115
spectrum (1). The ion of mass 115 arises from the ornithine moiety and is proposed to have the structure shown as ion C. Since the positive ion CID spectra of the two homologous lipids exhibit identical major ions resulting from the loss of the ketene moiety, and consecutive losses of HzO, we may conclude that the methylene unit difference between them is located on t,he fatty acid chain containing the cyclopropyl ring. Confirmation of this proposal was found in the CID spectra obtained of the negative (M - H) ions, of mlz 679 and mlz 665 (Figure 5). Both spectra exhibit an mlz 367 ion (proposed as structure D) as the base peak (Scheme 11). This presumably arises from loss of a carboxylic acid molecule from the molecular anions. A second intense peak a t m / z 311 is observed in the spectrum of the higher molecular mass com-
m h 297
ponent, and this peak shifts to mlz 297 in the spectrum of the homologue (see Figure 5). The mlz 311 and 297 ions correspond to ions E and F and arise from the carboxylate moiety thus confirming that the difference in the homologue structure occurs in this moiety. We also note that mlz 311 and 297 are found also in the FAB negative ion spectrum, whereas no detectable fragmentation is found in the positive ion spectrum. Of course the origins of these peaks can only be assigned after obtaining the MS/MS spectra. In conclusion, a mixture of ornithine-containing lipids from Thiobacillus thiooxidans have proven amenable to analysis by FAB and FAB MSJMS. The data obtained serve to support the previously postulated structure as well as to demonstrate the existence of a homologous compound which differs by a methylene group on one of the fatty acid moieties. Furthermore, the results are a demonstration of the suitability of FAB MS/MS for studies of a simple mixture of amino acid containing lipids. Registry No. Ornithine-containing lipid (C40H76N206), 61574-56-9; ornithine, 70-26-8.
LITERATURE CITED (1) Devienne, F. M.; Roustan, J . 4 . C . R . Acad. Sci. Paris, Ser. B 1976, 283. 397-399. (2) Barber, M.; Bordeli, R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. Soc., Chem. Commun. 1981, 325. (3) Cooks, R. G.; Glish, G. L. Chem. Eng. News 1981, 54 (48), 40-52. (4) Weber, R.; Levsen, K. Biomed. Mass Spectrom. 1980, 7 , 314. 15) Matsuo. T.: Matsuda. H.: Katakuse. I.: Shimonishi. Y,: Maruvama. Y.: Higuchi; T.f Kubota, E. Anal. Chem. 1981, 5 3 , 4i6-421. ' ~' ' (6) Desiderio, D. M.; Sabbatini, N. Z . Blomed. Mass Spectrom. 1981, 8 , 565-568. (7) Hunt, D. F.; Bone, W. M.; Shabanowitz, J.; Rhodes, J.; Ballard, J. M. Anal. Chem. 1981, 5 3 , 1704-1706. I
,
1036
Anal. Chem. 1983, 55, 1036-1040
(8) Straub, K. M.; Burlingame, A. L. Biomed. Mass Spectrom. 1981, 8 , (9) (10) (11)
(12)
(13)
431-435. Weber, R.; Levsen, K.; Louter, G. J.; Boerboom, A. J. H.; Haverkamp, J. Anal. Chem. 1982, 54, 1456-1466. Zakett, D.;Schoen, A. E.; Cooks, R. G.; Hemberger, P. H. J. Am. Chem. SOC.1981. 103. 1295-1297. Thomson, B. A,; Iiibarne, J. V.; Dzledzlc, P. J. Anal. Chem. 1982, 54. 2219-2225. Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge, H. Int. J . Mass Spectrom. Ion Phys. 1982, 42, 243-254. Hilker, D. R.; Gross, M. L.; Knoche, H. w.; Shlvely, J. M. Bjomed, Mass Spectrom. 1978, 5 , 64-71.
(14) Hilker, D. R.; Knoche, H. W.; Gross, M. L. Biomed. Mass Spectrom. 1979, 6 , 356-358. (16) Shively, J. M.; Knoche, H. W. J. Bacteriol. 1969, 96, 829-830. (16) Knoche, H. W.; Shively, J. M. J. BlOl. Chem. 1969, 224, 4773-4778. (17) Knoche, H. W.; Shively, J. M. J. Bioi. Chem. 1972, 247, 170-178.
RECEIVED for review December 6, 1982. Accepted March 1, 1983. This work was supported by the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CHE 78-18572).
Negative Ion Chemical Ionization Mass Spectrometry of Pyrrolizidine Alkaloids with Hydroxide Reactant Ion Peter A. Drelfuss," Wllllam C. Brumley, and James A. Sphon Division of Chemistry and Physics, Food and Drug Administration, Washington, D.C. 20204
Edward A. Caress Department of Chemistry, George Washington University, Washington, D.C. 20052
Macrocyclic dlester and noncycllc monoester pyrrollzldlne alkaloids were examlned by hydroxlde reactant ion negatlve Ion chemlcal lonlzation mass spectrometry. I n addltlon to abundant (M - H)- ions, the macrocyclic diester alkaloids produced (M OH)- adducts and extenslve fragmentatlon. The fragmentatlon often provlded the masses of the Intact necic acld side chaln and pyrrollzidlnenucleus (neclne). The spectra of the monoester pyrrohldlne alkalolds were slmllar to those of the macrocycllc dlesters, but no (M OH)- Ions were observed. The study was aided by hlgh-resolutlon exact mass measurements, colllslonally Induced dlssoclatlon/mass analyzed ion klnetlc energy spectrometry, and B2/E linked scans.
+
+
Pyrrolizidine alkaloids (PAS) are naturally occurring toxins present in about 3% of the world's flowering plant species (1). These plants are mainly from the families Boraginaceae (all genera), Compositae (tribes Senecioneae and Eupatorieae), and Leguminosae (genus Crotalaria) (2). Major outbreaks of liver disease in South Africa, the Soviet Union, Jamaica, Afghanistan, and India have been correlated with the direct or indirect consumption of these plants ( I ) . PAS have been found in some herbal teas, milk, and honey a t levels equal to, or greater than, those which have induced chronic liver damage and cancer in laboratory animals (2). Chronic effects include carcinogenicity ( 3 ) ,mutagenicity (4),and teratogenicity (5). Perhaps the greatest threats posed by PAS to inhabitants of industrialized countries are the chronic effects that can be produced by continuous low level consumption of PAS. Huxtable et al. (6) have suggested that low level exposure to PAS may be a cause of lung disease. To assess the extent of any public health hazard, methods with high sensitivity and specificity are necessary to detect and identify low levels of PAS (1 ppm and less) present in foods. Mass spectrometry (MS) has played a significant role in the detection and identification of PAS because of the structural information and sensitivity which can be obtained.
Electron ionization (EI) MS (7-10) mainly has been used, but positive ion (PI) chemical ionization (CI) MS employing B2/E linked scans has also been used (11). The ion current produced by E1 and PI CI is mostly restricted to ions characteristic of the pyrrolizidine ring moiety. Therefore, structural elucidation of the side chain(s) is often difficult. To simplify the structure elucidation, PAS are sometimes hydrolyzed or hydrogenolyzed to produce the resulting mono- or dibasic carboxylic acids, known as necic acids, and the pyrrolizidine nucleus amino alcohols, known as necines ( 4 ) . Hydroxide reactant ion (OH-) was first described and applied as a reactant ion for negative ion (NI) CI MS by Smit and Field (12). OH-, a strong Bronsted base, has often been used because of its proton abstraction abilities, but substitution and elimination reactions have also been observed (13). OH- is known to produce a gas phase analogue of base hydrolysis of esters and therefore has significant potential in the structural elucidation of complex esters (12, 14). Here, we present the analysis of PAS by OH- NI CI MS, a single step instrumental alternative to base hydrolysis followed by P I MS techniques. The structural significance of the fragmentation is explained, aided by collisionally induced dissociation/mass analyzed ion kinetic energy (CID/MIKE) spectrometry (15, 16), high-resolution mass measurements, and B 2 / E linked scans (17). EXPERIMENTAL S E C T I O N Standards. All of the PAS were gifts and were used without further purification. Purity was judged to be 99% or higher on the basis of their E1 and OH- NI CI mass spectra. Senecionine was supplied by Russell J. Molyneux, U.S. Department of Agriculture, Western Regional Research Center, Berkeley, CA. Jaconine, jacoline, anacrotine, and lycopsamine were obtained from C. C. J. Culvenor, CSIRO, Parkville, Victoria, Australia. Fulvine, heliotrine, crocandine, and ehretinine were supplied by C. K. Atal, Regional Research Laboratory, Jammu Tawi, India. Heliotrine, rosmarinine, and senecionine were supplied by Abdel-Fattah M. Rizk, National Research Center, Cairo, Egypt. Peak Matching Reference Materials. A low boiling fraction of Fomblin (18) was supplied by Gary A. McCluskey and Mary Shorter of the Frederick Cancer Research Center, Frederick, MD.
0003-2700/83/0355-1036$01.50/00 1983 American Chemical Society
