Mass Spectrometry of the Copper Salt of Tenuazonic Acid

Avinash Joshi,? Zhao Min,S William C. Brumley, Peter A. Dreifuss, George C. Yang and James A. Sphon Division of Chemistry and Physics, Food and Drug Administration, Washington, DC 20204, USA

The copper salt of tenuazonic acid was studied by both positive and negative ion techniques. Fragmentation was elucidated with the aid of collision-induced dissociation/mass-analysed ion kinetic energy spectroscopy. During the course of the investigations, a homolog of tenuazonic acid was tentatively identified by using a combination of mass spectral techniques.

INTRODUCTION

Tenuazonic acid is a toxic metabolite of Alternaria tenuis.’ It is roduced by other Alternaria species and other molds commonly found in tomato products, including species of Aspergillus, Botrytis, Cladosporium, Colletotrichum, Fusarium, Geotrichum, Mucor, Penicil- lium, Phytophthora, Rhizopus and Stemphyl i~m.~ Tenuazonic acid has been shown to possess physiological activity in plants: and antitumor, cytotoxic and antibacterial a~t ivi ty .~

A number of researchers have described methods for the detection of tenuazonic acid. Harwig et d3 used a thin-layer chromatography (TLC) method for initial- screening and gas chromatography/mass spectrometry (GC/MS) for the determination of the trimethylsilyl (TMS) ether derivative. The TMS derivatives of both tenuazonic acid and its copper salt, Cu(TA)’ where TA represents the tenuazonic acid ligand, were used by Chu6 for gas chromatographic/mass spectrometric confirma- tion. Recently a high-performance liquid chromatogra- phy (HPLC) method was reported for the determination of tenuazonic acid,’ which is known to lose its biological activity’ and possibly degrade on long standing;’ thus, it is commonly stored as the relatively stable copper salt.

A general reference to the behavior of inorganic com- plexes under electron ionization (EI) conditions is the book by Litzow and Spalding.’ The EI fragmentation of metal chelates in particular was recently reviewed.’ Positive ion chemical ionization (CI) mass spectrometry has been ap lied to the determination of volatile chelate complexes, ‘J’ and such complexes have also been determined by gas chromatography with electron cap- ture (EC) detection,” a technique which suggests the possibility of mass s ectral detection by negative ion CI mass spectrometry.

In this work we report the mass spectral behavior of the underivatized copper salt studied under positive and negative ion conditions and with the use of collision- induced dissociation/mass-analysed ion kinetic energy

P

P,

t Author to whom correspondence should be addressed. Present address: Muskegon County Wastewater Management System, 8301 White Road, Muskegon, Michigan 49442, USA.

$ Visiting Scientist from the National Institute of Metrology, Beijing, China.

spectroscopy (CID/MIKES). In the course of our investigations of Cu(TA)’, we tentatively identified the copper salt of a homolog of tenuazonic acid which to our knowledge has not been previously reported. The mass spectral data relating to this compound are also discussed and a likely structure is proposed.

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EXPERIMENTAL

Low resolution EI spectra were obtained with a Finnigan 3300F mass spectrometer using an electron energy of 70 eV and an emission current of 0.50 mA. Positive and negative ion CI mass spectra were obtained with a modified14 Finnigan 3300F instrument operated in the positive or negative ion mode. Eiectron energy was 140 eV and emission current was 0.50 mA from a heated rhenium filament. Source pressure was approximately 0.8 Torr CH4 (99.97% purity, Matheson Gas Products, Inc., East Rutherfod, New Jersey), and chloride ion was generated by introduction of CF2C12 (99.0% purity, Matheson). Source temperature was between 110 and 130 “C. Samples were introduced by direct probe heated independently of the source.

Low and high resolution mass measurements were carried out on a VG Analytical ZAB-2F instrument operated in the positive or negative ion mode.” Operat- ing parameters for positive and negative ion work were 100 eV electron energy, 1.5-2.0 X loe5 mbar source region pressure and 180-200 “C source temperature. High resolution measurements were made at 10 000 resolution with 5 YO valley. Peak-matching calibration was obtained with m / z 185 and 178 of polyper- fluoropropylene oxide. l6 CID/MIKE spectra were obtained with a second field-free collision cell region pressure of 3.5 x lop7 mbar He by scanning the electric sector voltage using the digital MIKES unit. 1s~17

Tenuazonic acid was isolated as follows from Alter- naria tenuis mold grown on polished rice by Dr F. Chu, University of Wisconsin-Madison.6 The methanolic mold extract was filtered, acidified to pH2, and ex- tracted with methylene chloride. The solution was readjusted to pH2 and further extracted into 5% sodium bicarbonate solution. The aqueous portion was then acidified by adding a 1/3 volume of HCl: H 2 0 ( 1: 1)

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and extracted twice with equal volumes of hexane. The hexane portion was evaporated to dryness and redissol- ved in methanol, followed by adjustment of its pH to 7.0 with 0.1 N NaOH. A 10-fold volume of 0.1 M Cu(Ac), was added and extracted with equal volumes of methylene chloride. Cu(TA), crystals were formed upon standing at room temperature. The purity of the Cu(TA), so obtained was estimated to be greater than 95% from TLC results.

RESULTS AND DISCUSSION

Positive ion spectra

The E l mass spectrum of Cu(TA), given in Fig. 1 is dominated by fragmentation associated with the TA ligand or with the uncomplexed tenuazonic acid, most of which is produced by decomposition (see below). Thus the base peak is m / z 141, representing the loss of C4Hs from the tenuazonic acid (1) moiety (197 u). Ions

at m / z 123,112 and 98 represent additional fragmenta- tion corresponding to losses of 18,29 and 43 u, respec- tively, from m / z 141 (see below). The highest mass ion observed of significant relative abundance was m / z 260

[M- 195]+', which corresponds to the loss of [TA-HI from the complex. (In subse uent discussions, ions pos- tulated to contain copper ( CU) also have an isotopic contribution due to the presence of 6sCu although this will not be explicitly mentioned.) An ion of very low relative abundance was also observed at m / z 399 [M-56]".

The positive ion CI (methane) mass spectrum (Fig. 2) does provide molecular weight information with the weak [M+H]+ionat m / z 456 and the[M+29]+adduct at m / z 484. The base peak at m l z 198 is due to proto- nated tenuazonic acid. The presence of the adduct ions at m / z 226 [197+29]+ and 238 [197+41]+ suggests that uncomplexed tenuazonic acid is produced by ther- mal decomposition followed by ionization rather than by ionization of Cu(TA),, and this process accounts for most of the ion current representing the TA ligand. It is possible that a small amount of uncomplexed tenuazonic acid is also present before heating. A less abundant ion at m / z 196, [197-H]+ or [TA]+, is also present. The ion at m / z 141, the base peak under EI, is less abundant under CI.

The positive ion CI mass spectrum also contains an ion at m / z 184. We propose that this ion represents a protonated homolog of tenuazonic acid that is 14 u less in mass than the m / z 198 ion. It is unlikely that m l z 184 is a fragment arising from m / z 198, since a loss of 14 u is not commonly observed. In addition, an ion at m / z 212 corresponds to the adduct [183+29]+ of the homolog. The ion at m / z 182 is analogous to m / z 196 of tenuazonic acid and therefore represents [183 - H]+ or [TA']+. A very weak ion at m l z 442 represents the [M + HI+ ion of the complex Cu(TA, TA'), incorporating

69

102 BIOMEDICAL MASS SPECTROMETRY, VOL. 11, NO. 3, 1984

COPPER SALT OF TENUAZONIC ACID

[M*H]* 456

Figure 2. Positive ion CI (methane) mass spectrum of Cu(TA), (Enlarged 3OX above m/z350.)

one ligand of the homolog. The use of derivatives and EI mass spectrometry in previous work may have pre- cluded observation of this homolog.

The observation of distinct ions characteristic of tenuazonic acid and its homolog at m / z 198 and 184 makes possible a parallel study of their fragmentations by CID/MIKES without further chemical separation. A possible structure of the homolog (2) is shown. The

CID/MIKE spectrum of m / z 198 revealed many decompositions including those corresponding to m/z 181 [198-17]', 168 [198-30]+, 153 [198-45]+, 141 [198-57]+, 125,123,110,98, 84,69 and 68. The most abundant daughter ion, m / z 141, corresponded to [198 -C4H9]+ and was rationalized as 3. Similarly,

3

the CID/MIKE spectrum of m/z 184 also revealed many decompositions including those corresponding to m/z 167 [184-17]+, 154 [184-30]+, 141 [184-43]+,

125, 123, 110,98, 84, 69 and 68. The most abundant daughter ion, m / z 141; corresponded to [184-C3H,]+. These data are consistent with similar ion structures for m/z 198 and 184 and with the proposed structure of the homolog.

For further study of the fragmentation, the CID/MIKE spectrum of m / z 141, which revealed decompositions corresponding to m / z 126, 123, 112, 98, 85 and 69, was also obtained. These data suggest a common origin for the lower mass ions observed in the EI spectrum ( m / z 123,112,98, 85 and 69) and in the CID/MIKE spectra of both m / z 198 and 184. In sum- mary, the positive ion data are consistent with a structure of TA' that has a propyl or isopropyl side chain and differs from TA by the elements of one methylene group.

Negative ion CI mass spectra

The negative ion CI mass spectrum of CU(TA)~ (Fig. 3) reflects information complementary to that found in the positive ion spectrum. The base peak at m / z 455 [MI-' represents the intact complex ionized by resonance EC13. The ions at m / z 196 and 195 arise from the TA ligand corresponding to [TAI- and [TA - H I-., respec- tively. Again, the ligand negative ions are presumably formed as a result of thermal decomposition of the complex followed by resonance or dissociative EC. l4

The ion at m / z 441 clearly indicates the presence of the homolog TA' in a complex represented as Cu(TA,TA'). The relative abundance of m/z441 is 14%, compared to that of m / z 455. If the homolog TA' is randomly distributed, we calculate that its relative abundance is 6.5% and that of TA is 93.4%, and we

BIOMEDICAL MASS SPECTROMETRY, VOL. 11, NO. 3, 1984 103

A. JOSHI, Z. MIN, W. C. BRUMLEY, P. A. DREIFUSS, G. C. YANG AND J. A. SPHON

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Figure 3. Negative ion CI (methane) mass spectrum of Cu(TA),

predict that the compound CU(TA’)~ at m / z 427 should have a relative abundance of 0.50%. Experimentally, m / z 427 was found to have 0.55% relative abundance. Although the agreement may be fortuitous, it is con- sistent with the random distribution of TA’ in the com- plex, assuming all other factors are constant (e.g., ioniz- ation efficiency, association constants, etc.). The TA’ ligand is observed predominantly as [TA’I- and [TA’ - H]-’ at m / z 182 and 181, respectively. Again, this occurs primarily by thermal decomposition of the com- plex followed by ionization.

The CID/MIKE spectra of the negative ions at m / z 196 and 182 were obtained and compared to posi- tive ion results. Major decompositions of m / z 196 cor- responded to m / z 195 [TA-H’I-’, 181 [TA-CHJ’ and 139 [TA- C4H9]-’. Similarly, decompositions of m / z 182 corresponded to m / z 181 [TA’-H’I-’, 167 [TA’ - CH3]-’ and 139 [TA’ - C3H7]-‘. These data are also consistent with 2, the compound postulated to account for the positive ion data, and with 4, the struc- ture proposed to explain the ion observed at m / z 139.

H WCH3 H 0.

4

In addition, high resolution mass measurements revealed an elemental composition of C9HI2O3N1 (182.0817 calc., 182.0814 found) at m / r 182 that is consistent with a difference of CH2 between the corres- ponding ions of 196 and 182 u.

The C1- attachment spectrum18 confirms the presence of the distinct species Cu(TA, TA’) and CU(TA)~ by the observation of ions at m / z 476 and 490, corresponding to [Cu(TA, TA’) + C1]- and [Cu(TA), + C1]-. In addi- tion, ions representing the intact complex are observed at m / z 441 and 455 together with ions representing the ligands at m/z 182 [TA’I- and 196 [TAI-. That thermal decomposition occurs prior to ionization is substantiated by the observation of C1- adducts of tenuazonic acid ,andthehomolog at m / z 232[197+Cl]-and218[183+

In any event, the possible presence of the homolog should be checked in confirmative or determinative assays. The mode of TA’ incorporation in the copper complex is not entirely clear; however, biological origin seems most likely. An isopropyl side chain, which is consistent with the proposed structure of TA’, has been observed in the biosynthesized pigment called erythroskyrine. 1920 The application of negative ion CI mass spectrometry appears to be useful for examining standards of Cu(TA), for the presence of this homolog and for verifying structure without derivatization or isolation of the free acid.

c11-.

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COPPER SALT OF TENUAZONIC ACID

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Received 10 February 1983; accepted 20 May 1983

BIOMEDICAL MASS SPECTROMETRY, VOL. 11, NO. 3, 1984 105