Anal. Chem. 1987, 59,614-617
614
Determination of Sites of Incorporation of Oxygen- 18 Atoms in Maduramicin a by Fast Atom Bombardment Mass Spectrometry Ted T. Chang* Chemical Research Diuision, American Cyanamid Company, Stamford, Connecticut 06904
Hwei-Ru Tsou and Marshall M. Siege1 Medical Research Division, American Cyanamid Company, Pearl Riuer, New York 10965
Eiucldation of the bioorigln of the molecular structure is Important for the understanding d biosynthetic mechanisms. To investigate the function of molecular oxygen, an 180-iabeled maduramlcin a was blosyntheslzed under '*O gas and analyzed by fast atom bombardment mass spectrometry. Five "0 atoms were found to be Incorporated in the biosynthesized maduramicin a structure. The exact iocatlons of these five "0 atoms were determlned from fragmentation patterns as 0 ( 5 ) , O( IO), O( l l ) , O( 12), and O(13) positions.
Maduramicin a is a potent coccidiostat, poultry antibiotic, effective a t a level of 5 ppm. It is a polyether ionophore antibiotic possessing a polyoxygenated carbon backbone and a 2,6-dideoxysugar ( I , 2 ) with the structure shown in Figure 1.
Elucidation of the bioorigin of carbon atoms and oxygen atoms of maduramicin a (simplified as maduramicin in the following text) has been a major research subject of our laboratories. It is important not only for the understanding of the biosynthetic mechanism but also for the optimization of the fermentation processes. With 13C labeled precursors fed to cultures of Actinomadura yumaensis and a subsequent assignment of 13C NMR spectrum, it was recently established that the basic aglycone carbon skeleton of maduramicin (excluding the G ring) is derived from eight acetate and seven propionate units and that the methoxy carbons are derived from methionine (3, 4 ) . The investigations on the bioorigin of some oxygen atoms were also carried out with isotope labeled maduramicin. They were studied by incorporating [ l-13C,180]acetate and [ 113C,180]propionatein the fermentation cultures. From the 13C NMR spectra of carbon atoms adjacent to the oxygen atoms, it was concluded that five oxygen atoms, O(l), 0 ( 6 ) , 0 ( 8 ) ,0(9), and 0(14), are derived from acetate while three oxygen atoms, 0(3), 0(4), and 0 ( 7 ) ,originated from propionate (3).
Our current objective is to understand the contribution of molecular oxygen in the biosynthesis of maduramicin. In the past, biosynthesis of antibiotics in l80gas has usually resulted in poor yields. We have, therefore, designed a special closed fermentation system which is capable of producing highly enriched 180-labeled maduramicin. This fermentation system and the purification procedures will be reported elesewhere (5).
Mass spectrometry has been a major analytical tool for the structural studies of polyether ionophores (6, 7). Unfortunately, in many cases, its application was limited by the volatility and thermal stability of ionophores. This limitation was considerably minimized after the introduction of fast atom bombardment (FAB) (8). FAB was found to be very suitable
for the analysis of ionophores because ionophores produce intense and steady FAB spectra (9,IO). For this report, we have applied FAB to determine the number of molecular oxygen atoms incorporated in the structure of maduramicin. Furthermore, we successfully located the exact positions of these five oxygen atoms from the fragmentation patterns of maduramicin.
EXPERIMENTAL SECTION The mass spectrometer used for this study was a Kratos MS-50 high-resolution mass spectrometer, Kratos Analytical, Manchester, England. The instrument was equipped with a FAB source and gun supplied by M-Scan, Ltd., Ascot, Berkshire, England. Xenon gas was used as the gas of the FAB gun and thioglycerol as the FAB matrix liquid. The primary beam energy was 8 keV. The instrumental resolution was set at 3000 for the low-resolution studies and 10000 for the high-resolution studies. Exact masses were obtained by the peak matching technique. The molecular ion of maduramicin was peak matched against the molecular ion of septamycin, a known ionophore, where MNa' ion is 937.5500, C,Hs201sNa. The fragment ions were either peak matched against two other known ionophores, monensin, MNa' = 693.4190 (C36H620,,Na),and lasalosid, MNa' = 613.3716 (C3H,08Na), or against fragment ions whose exact masses have been determined. Correction for instrumental deviation was made for each measurement by using a correction factor established from known compounds in the mass range similar to that of the unknown mass. Ethoxylated alkylphenols
were found to be convenient for establishing the correction factor, because they have a repeating unit of 44.0262 (C,H,O), cover the mass range of 400-1000, and produce steady signals.
RESULTS AND DISCUSSIONS Maduramicin produces intense and steady FAB mass spectra especially in the presence of alkali salts. In the presence of sodium ion, the FAB mass spectrum consists of MNa+, (MNa' - CO,), and the base peak (MNa' - H20- C 0 2 ) ions as shown in Figure 2. It also consists of many other fragment ions of lesser abundance as listed in Table 11, which will be discussed later. Analysis of the biosynthesized 180-labeled maduramicin by FAB showed that the cationized molecular ion (MNa') of the unlabeled maduramicin was shifted from m l z 939 to m l z 949 as illustrated in Figure 2, indicating that the major enriched product molecule contained five l80atoms in the structure. By use of the high-resolution peak-matching technique, the MNa+ ion of the 180-labeledmaduramicin sodium adduct ion, mlz 949, was determined as 949.5467, which again confirmed the incorporation of five l80atoms in the structure. (Calculated value of C47H800121805Na = 949.5502; deviation of measured value was 3.7 ppm.) Two major fragment ions of
0 1987 American Chemical Society 0003-2700/87/0359-0614$01.50/0
ANALYTICAL CHEMISTRY, VOL. 59, NO. 4, FEBRUARY 15, 1987 I 6
8
2
M
Flgure 1.
Table I. Relative Contribution of Peak Intensities from Various ls0-Labeled Maduramicins
17 OCH,
OH
1
16
916.5395
C,,H,,O,,
Structure of maduramicin a as free acid.
615
peak intens contributed fromb
m/zn
total re1 peak intens
883 885 887 889 891
10.7 40.0 100.0 37.0 10.5
1804
1805
'SO6
4.2 17.5 5.1 1.0
4.3 22.1 92.1 26.1 5.0
0.4 2.2 9.4 2.7
The most intense high-mass fragment ion (MH+ - COz - HzO) and I8O6 maduramicins are m / z 885, 887, and 889, respectively. bThe fragment pattern around the (MNa+- COP HzO) ions are estimated according to the fragment pattern around the m / z 887 of the unlabeled maduramicin P O 5 ) . In the unlabeled maduramicin, the relative peak intensities of m / z 873, 875, 877, 879, and 881 are 4.7, 24, 100, 29, and 5.4, respectively. of
1804,
Table 11. Major Fragment Ions in Maduramicin and [ lsO]Maduramicin 180-
Flgure 2. FAB
mass spectra of unlabeled and '80-labeled maduram-
icins. unlabeled maduramicin were m/z 895 (MNa+ - COP)and m / z 877 (MNa+ - COS- HzO),as shown in Figure 2. The m / z 877 was formed by simultaneous losses of C02 [including O(1) and 0(2)] and HzO [including 0(3)] via a McLafferty-type rearrangement. This hypothesis was substantiated by MS/MS investigations (10, 11) and the observation of an intense metastable ion at m/z 819.1. In the FAB spectra of la0-labeled maduramicin, m / z 895 and m/z 877 also shifted 10 Da to m/z 905 and m / z 887, showing O(l), 0(2), and O(3) are not l80 labeled. The biosynthesized 180-labeled maduramicin also contains some four and six l80compounds in addition to the predominant five l80compounds. The relative concentration of these 180-labeled compounds was established by careful comparison of the fragment pattern around the (MNa+ - COz - HzO) ion, i.e., m / z 877 for the control and m / z 887 for the labeled sample. The (MNa+ - COz- HzO) ions were selected over the molecular ion species for this study because of their intensities and better signal to noise ratios. Twenty FAB spectra were recorded and averaged under the same operational conditions for both the control and labeled samples. By comparing their ion intensity patterns, we have estimated that among the 180-labeled molecules, about 77% of the molecules contain five l80atoms, while almost 15% and 8% of the molecules contain four and six atoms, respectively (see Table I). The exact locations of the five l80atoms were determined by studying the fragment ions of both control and 180-labeled maduramicins. Table I1 lists the major fragment ions of unlabeled maduramicin in the presence of sodium and potassium ions, the major fragment ions of 180-labeled maduramicin in the presence of sodium ions, the mass difference between the labeled and unlabeled fragment ions, the number of l80atoms incorporated in the fragment ions, and the elemental composition of the unlabeled fragment ions. These
labeled madura. micin
elemental composition
maduramicin with NaCl
maduramicin with
with
'80
of Na
KC1
NaCl
atoms
maduramicin'
939 895 877 763 735 701 593 59 1 559 537 535 519 493 489 453 451 433 159
955 911 893 779 751 717 609 607 575 553 551 535 509 505 469 467 449 159
949 905 887 773 743 709 601 599 565 545 543 525 501 493 461 457 435 159
no. of
C47H80017Na
C46H80016Na C46H78014Na C40H68012Na C38H64012Na
C38H62010Na C30H50010Na C31H6209Na
3
C30Ha08Na C27H4609Na C28H4808Na C27H4408Na
C25H4208Na C26H4207Na
C22H3808Na
C23H4007Na 1
C23H3806Na C8H1503
These elemental compositions were confirmed by exact mass measurement at 10K resolution. elemental compositions were confirmed by high-resolution peak matching. In the following discussions, the fragment ions are identified based on the observed masses from the unlabled maduramicin in NaCl matrix, the first column of the Table 11. When the maduramicin was examined in the presence of KC1, all ions except the m / z 159 shifted 16 Da higher (replacing the Na atom with a K atom), indicating the presence of a sodium atom in all ions except m / z 159. It is also interesting to note that m/z 159 originates from the G ring which does not belong to the basic aglycone skeleton. Since the l8O atoms are dispersed throughout the maduramicin structure, it is possible to locate the labeled oxygen atoms from the favorable fragmentation. Most fragment ions of maduramicin can be categorized into two series, namely, A series and F series. The A series are those fragment ions retaining the A ring and the F series are fragment ions retaining the F ring. The fragmentation schemes of these fragment ions listed in Table I1 are shown in Figure 3. The A series fragment ions originated from the (MNa+ - COz H20) ion, m / z 877 for the control and m / z 887 for the labeled sample. The F series fragment ions probably also originated from the (MNa+ - COz - HzO) ion but could also be derived
616
ANALYTICAL CHEMISTRY, VOL. 59, NO. 4, FEBRUARY 15, 1987 F SERIES IONS
0413)
1 '
39"64O1SNa 763.4244
fLdi
A SERIES IONS 6
ocn,
E OH
17 OCH.
-H = 489
519
55; 763'
'
'-
NORMAL PEAK
Figure 3. Fragmentation schemes of maduramicin.
c directly from the MNa+ ion. No attempt was made to investigate the contribution from these two pathways. It was found that most oxygen atoms in the aglycone skeleton can be singled out by the combination of two or three fragment ions listed in Table 11. The exact locations of the atoms, therefore, were determined from the change of l8O atoms in neighboring fragment ions. Accordingly,the locations of five '80 atoms were determined as 0(5), 0(10), O(ll), 0(12), and O(13) positions. The confirmation of the O(5)position was based on two F series ions, mlz 763 and mlz 735. The mlz 763 has five lSO, indicating O(4) and O(6) to be leO atoms as are O W , 0 ( 2 ) , and O(3). However, the mlz 735, which excluded the 0(5), had only four l80atoms, thus proving the O(5)position to be l80labeled. By the same argument, the 180-labeledatom at the O(l0) position was established based on the observation of two F series ions, mlz 451 and 453; the O(l1) position was established based on the observation of two A series ions, mlz 519 and mlz 489. The O(12) position was confirmed by two ions, mlz 701 of the A series and mlz 159 (ring G). Since the mlz 701 contained four '%) atoms, it suggested that there was one '%) atom in the excluded part, the G ring or the O(12) position. No isotope shifts were observed for the mlz 159 ion, indicating no l80atom in the G ring. Therefore, the l80atom had to be located at the O(12) position. Although high-resolution results were used to confirm the proposed fragmentation schemes, the effective utilization of the high-resolution experiment was best demonstrated by the simultaneous confirmation of I%) atoms at the O(5) and O(13) positions. The mlz 763 could have originated by either the cleavage of the A ring to form an F series ion or by the cleavage of the F ring to form an A series ion as exhibited in Figure 3. The exact origin of this fragment ion is difficult to establish based solely on the low-resolution results. A speculative assignment could only establish the location of an lSO atom in one ring, for instance, the A ring, and leave the assignment of the l80atom in the other ring ambiguous, for instance, the F ring. The high-resolution results eliminated this ambiguity and clearly established the presence of l80atoms at both the O(5) and O(13) positions. Figure 4 exhibits the peak shapes of m / z 763 of unlabeled maduramicin, mlz 773 of l80-labeled maduramicin, and a normal peak shape acquired at the instrumental resolution of 10000. It shows that the mlz 735 is an unresolved doublet of mlz 763.4244, C3BHe4013Na, and mlz 763.4608, C40H6sOlzNa.Similarly, the mlz 773 is the unresolved doublet of mlz 773.4457, CSI&0J8O5Na, and mlz 773.4820, C40H,8071805Na.The first peak is an A series ion which confirms the O(13) position and the latter peak is an
.I'l
Jr..h..
I.
'!
f i w w
Flgure 4. Peak shapes at 10K resolution: (a) m / z 763 of maduramicin; (b) m / r 773 of '80-labeledmaduramicin; (c)a normal peak shape.
Table 111. Summary of the Oxygen Isotope in ['*O]Maduramicin
position of 0 atom 1 2 3 4 5 6 7 8 9 10 10
11 12 12 13
type of
significant atom fragment ion 'W l80 (1800-1806) X X X X X
X X X X X X X X X X
series
895-995 895-995 877-887 763-773 735-743 763-773 591-599 593-601 535-543 451-457 489-492 519-525 159-159 701-709 763-773
F F F F F F F F F F A A
A A
no. of 180 atoms in significant ions 5 5 5 5 4 5 4
4 4 3 2 3 0
4 5
F series ion which confirms the O(5) position. Table I11 summarizes the above findings. It tabulates the type of oxygen isotopes for all oxygen atoms in the aglycone structure from O(1) to O(13) positions, the most significant fragment ion used for the assignment in the unlabeled and labeled maduramicin, the series that this fragment belongs to, and the number of '80 atoms incorporated in this particular fragment ion. In conclusion, FAB-MS has proven to be a powerful tool for the structural elucidation of maduramicin. This study further demonstrates that, with a highly enriched isotopelabeled sample, it is possible not only to determine the number of isotope atoms but also to establish their exact locations. Registry NO. Oz,7782-44-7; maduramicin a,79356-08-4.
LITERATURE CITED (1) Liu, C. M.; Hermann, T. E.; Downey, A.; Prosser, B. La T.; Schildknecht, E.; Palleroni, N. J.; Westley, J. W.; Miller, P. A. J . Antibiot. 1983, 3 6 , 343. (2) Labeda, D. P.; Martln, J. H.; Goodman, J. J. U.S. Patent, 4407946, 1983. (3) Tsou. H.-R.; Rajan, S.;Fiala, R.; Mowery, P. C.; Bullock, M. W.; Borders, D. B.; James, J. C.; Martln, J. H.;Morton, G. 0.J . Antibiot. 1984, 3 7 , 1000. (4) Rajan, S.; Tsou. H.-R.; Mowery, P. C.; Bullock, M. W.; Stockton, G. W. J . Antiblot. 1984, 3 7 , 1495. (5) Tsou, H.-R.; Chang, T. T., submitted for publication in J . Antibiot
Anal. Chem. 1987, 59, 617-623 (6) Occobwltz. J. L.; Hamill, R. L. fdyettmf Antibiotics; Wlley: New York, 1982. (7) Tabet, J. C.; Fraisse, D.; David, L. Int. J . Mess Spechom. Ion fromsses 1085, 63, 29. (8) Barber, M.; Bordoll, R. S.; Elliot, 0. J.; Sedgwlch, R. D.; Tyler, A. N. .Anal. .. . Chem. -. .- ... . W82. . - -, 54. .. , 645A. . . (9) Chang, T. T.; Lay, J. O., Jr.; Francel, R. J. Anal. Chem. 1984, 56, 109.
617
(10) Slegel, M. M.; McGahren, W. J.; Tomer, K. B.; Chang, T. T. The 33rd Annual Conference on Mass Spectrometry, San Dlego, CA, 1985. (11) Slegel, M. M.; Tomer, K. B.; Chang, T. T. J . Biomed. Environ. Mess Specfrom ., In press.
RECEIVED for review July 23,1986. Accepted October 15,1986.
Comprehensive Trace Level Determination of Organotin Compounds in Environmental Samples Using High-Resolution Gas Chromatography with Flame Photometric Detection Markus D. Muller Swiss Federal Research Station, CH-8820 Wadenswil, Switzerland
A comprehensive method for trace analysis of mono-, dC, trl-, and some tetrasubstltuted organotln compounds is presented. The Ionic compounds are extracted from diluted aqueous 80lutlons as chlorides by using a Tropoion-C,, silica cartrldge and from sediment and sewage sludge by using an ethereal tropoion solution. The extracted organotln compounds are ethyiated by a Grlgnard reagent and analyzed by using hlghresolutlon gas chromatography with flame photometric detectlon (HRGWFPD). Gas chromatography/mass spectrometry (GC/MS) was used for conflrmatlon. The extraction behavior, gas chromatographk retention, and photometric response of a series of organotln compounds are descrlbed, and the ldentlfication via electron Impact (EI) and chemlcal Ionization (CI) mass spectrometry Is discussed. The main organotln compounds detected In varlous samples are butyltlns; cyciohexyi- and phenyltins were ldentlfled In some of the sediment and sewage sludge samples. Methylbutyltlns and tetrabutyltln were not detected. Concentratlons were found to range from low ng/L (parts per trillion) In surface water to low mg/kg (parts per million) In sewage sludge.
Organotin compounds have found applications in many fields, such as stabilizers for PVC, fungicides and miticides in agriculture, and biocides (1-4), because their properties can be tailored by the variation of the type and the number of substituents to meet widely different requirements. Annual world production was estimated to be 33000 tons in 1983, most of it dioctyltin maleate (2,5). The toxicity and degradation in the environment depend strongly on the number and nature of the substituents ( 1 , 5 ) . Organotin compounds with short alkyl chains or phenyl substituents generally exhibit considerable toxicity toward both aquatic organisms and mammals. Alkyltins with small alkyl chains degrade slowly in the environment (6, 7);phenyltins are less stable and may, under certain conditions, rapidly loose the phenyl substituents (3). Organotin compounds may accumulate in sediments and aquatic organisms (6). Trace.determination of organotin compounds in environmental samples is complicated by the fact that organotins with one to three substituents are polar, involatile substances due to their ionic character. In the last few years, a series of publications dealing with different approaches for trace 0003-2700/87/0359-0617$01.50/0
analysis of organotin compounds appeared, marking a growing concern over the fate of these persistent and toxic compounds in the environment and their impact especially on aquatic organisms. Trace level determination of these compounds can be carried out either by nonchromatographic methods (e.g., electrochemical or fluorometric assays ( 8 , 9 ) )or by chromatography with an appropriate detection method. High-performance liquid chromatography (HPLC) coupled with fluorescence detection ( 1 0 , I I ) or ion-exchange HPLC with detection by graphite furnace atomic absorption spectroscopy (GF/AAS) (12)proved to be sensitive methods, but may lack from limitations in separation power and ease of identification of unknown products. The preparation of volatile derivatives makes the ionic organotin compounds amenable to evaporative separation techniques (purge and trap or gas chromatography (GC)). Hydride formation in dilute aqueous solutions is becoming a routine method for determination of methyltins (13-16), methyl- and butyltins (17-19),and phenyl- and various other organotin compounds (20-22) to form the volatile hydrides (stannanes), which are analyzed either by purging and AAS or flame photometric detection (FPD) or by liquid-liquid extraction with subsequent GC analysis. Unfortunately, stannanes are rather labile thus preventing further cleanup steps (2). Therefore, alkylation is often preferred over hydride formation, as the resulting tetrasubstituted organotin compounds can easily be purified and concentrated, which is necessary for low-level samples and complex matrices such as animal tissue or sewage sludge. A Grignard reagent or an alkyllithium compound is used to convert the ionic mono-, di-, or triorganotin compounds into the corresponding nonpolar tetrasubstituted compound. The reaction has to be carried out in aprotic solvents and thus requires extraction of aqueous samples prior to derivatization. Procedures have been described for the analysis of methyltins (23), butyltins (24,25),mixed methylbutyltins (26), various alkyltins (27),cyclohexyltins (28),and phenyltins (29). Alkylation also offers the possibility for selection of the volatility range of the derivatives, which are in most cases analyzed by GC. However, there are few methods for the sensitive determination of a broad range of organotin compounds in environmental samples. Recently, the sensitive determination of butyltin residues in sediment and surface water was described on the basis of extraction/methylation and high@ 1987 American Chemical Society
