Structure and natural occurrence of stereoisomers of the fumonisin B series mycotoxins

Article abstract of DOI:10.1021/jf070061h

¹H and ¹³C NMR spectroscopy of both fumonisin B ₃ and B₄, as well as high-performance liquid chromatography (HPLC) analysis of samples of fumonisin B₃ used as standards, showed in each case the presence of two stereoisomers, which could not be separated by preparative chromatography. The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B₃ and B₄ was deduced from the NMR data, and their 2S,3R absolute configurations were established by application of Mosher's method using the fumonisin B₃ sample. Samples of fumonisin B₃ and B ₄ can contain between 10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB₃, determined by HPLC with fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much lower than FB₃ (<20%). The identification of members of the 3-epi-fumonisin B series provides insight into the order and selectivity of steps in fumonisin biosynthesis.

Full text of DOI:10.1021/jf070061h

4388 J. Agric. Food Chem. 2007, 55, 4388−4394
Structure and Natural Occurrence of Stereoisomers of the
Fumonisin B Series Mycotoxins
WENTZEL C. A. GELDERBLOM,†,‡ VIKASH SEWRAM,† GORDON S. SHEPHARD,†
PETRA W. SNIJMAN,† KENNY TENZA,§ LIANA VAN DER WESTHUIZEN,† AND
ROBERT VLEGGAAR*,§
Programme on Mycotoxins and Experimental Carcinogenesis, Medical Research Council, P.O. Box
19070, Tygerberg 7505, Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600,
and Department of Chemistry, University of Pretoria, Pretoria 0002, South Africa
1H and 13C NMR spectroscopy of both fumonisin B3 and B4, as well as high-performance liquid
chromatography (HPLC) analysis of samples of fumonisin B3 used as standards, showed in each
case the presence of two stereoisomers, which could not be separated by preparative chromatography.
The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B3 and B4 was deduced
from the NMR data, and their 2S,3R absolute configurations were established by application of
Mosher’s method using the fumonisin B3 sample. Samples of fumonisin B3 and B4 can contain between
10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB3, determined by HPLC with
fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-
tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much
lower than FB3 (<20%). The identification of members of the 3-epi-fumonisin B series provides insight
into the order and selectivity of steps in fumonisin biosynthesis.
KEYWORDS: Fumonisins; corn; NMR; LC-MS; biosynthesis
INTRODUCTION
The fumonisins of the B series, for example, B1-4 (1-4) (see
Figure 1), are a family of structurally related mycotoxins first
isolated from cultures of Fusarium Verticillioides (strain MRC
826) (1, 2). These secondary metabolites are common contami-
nants of corn throughout the world (3) and the causative agents
of equine leucoencephalomalacia (4) and porcine pulmonary
edema (5). The carcinogenic nature of these mycotoxins in
rodent studies (6, 7), their association with human esophageal
cancer (8), and the recent demonstration of their possible role
in neural tube defects (9, 10) have heightened international
concern over their natural occurrence. The International Agency
for Research on Cancer has recently declared FB1, the most
abundant of the fumonisins, to be a group 2B carcinogen (i.e.,
possibly carcinogenic to humans) (11). Since the initial discov-
ery of FB1 (1) and FB2 (2) in 1988, the number of known
fumonisin analogues has greatly increased and a recent review
of this family of compounds listed 28 members (12). The
fumonisins are generally divided into the A, B, C, and P series.
The significance of many of these analogues as natural contam-
inants of food is uncertain, and reports of fumonisin analysis
are frequently restricted to FB1 (1) and FB2 (2), although some
* To whom correspondence should be addressed. Tel: +27 12 420-
3095. Fax: +27 12 362-5297. E-mail: robert.vleggaar@up.ac.za.
† Medical Research Council.
Figure 1. Chemical structures of the fumonisins.
reports include FB3 (3). The necessity for including FB3 in total
fumonisin analysis has recently been highlighted by the inclusion
‡ University of Stellenbosch.
§ University of Pretoria.
10.1021/jf070061h CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/01/2007

Fumonisin B Series Mycotoxins
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4389
Figure 2. Chemical structures of FB3 and 3-epi-FB3 tetramethyl esters and their MTPA derivatives.
Figure 3. Chemical structures of natural products with the terminal 2-amino-3-hydroxy motif.
MATERIALS AND METHODS
of all three analogues in the setting of Food and Drug
Administration guidelines for industry in the United States (13)
as well as in the provisional maximum tolerable daily intake
determined in the recent risk assessment of fumonisins con-
ducted by the Joint FAO/WHO Expert Committee on Food
Additives (14).
Analytical standards of FB1 (1), FB2 (2), and FB3 (3)
have been prepared by scientists at the MRC, Tygerberg,
South Africa, on a commercial basis since the early 1990s.
Analysis of fumonisin B3 samples obtained from different
batches of F. Verticillioides (MRC 826) has recently shown the
presence of 10-40% of a stereoisomer of FB3 that elutes
immediately prior to FB3 in the analytical reversed-phase high-
performance liquid chromatography (HPLC) chromatogram of
the o-phthaldialdehyde (OPA)-derivatized standards. In this
paper, we report on the identification and absolute configuration
of the stereoisomers 3-epi-FB3 (5) and 3-epi-FB4 (6) (Figure
1) present in samples of FB3 and FB4, respectively, and the
natural occurrence of 3-epi-FB3 (5).
HPLC-Tandem Mass Spectrometry (MS-MS) Analyses of Fu-
monisin Standards. The fumonisin standards (FB1, FB2, FB3, and FB4)
were isolated from corn cultures of F. Verticillioides as described by
the method of Cawood et al. (2). The isolated FB3 standard and the
dried residues of corn sample extracts, prepared as described below
for determination by fluorescence detection, were dissolved in aceto-
nitrile-water-formic acid (10:90:0.1). The two stereoisomers of FB3
were separated by binary gradient reversed-phase HPLC on a Luna
C18 column (Phenomenex, Torrance, CA). The individual elution
solvents were mixtures of water-acetonitrile-formic acid in the ratios
(90:10:0.1; solvent A) and (10:90:0.1; solvent B), respectively. The
mobile phase was pumped at a flow rate of 0.7 mL/min. The initial
composition of 80% solvent A was adjusted linearly to 75% solvent A
over 35 min. The fumonisins were detected by mass spectrometry using
a Finnigan MAT (San Jose, CA) LCQ ion trap instrument with positive
ion electrospray (ES) ionization. The HPLC eluate was passed directly
into the MS at a source voltage of 4.5 kV and a capillary voltage of 40
V. The heated capillary temperature was maintained at 220 °C, and
the sheath to auxiliary gas ratio was set at 4:1. Fumonisins were

4390 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Gelderblom et al.
Table 1. NMR Data for the Normal and 3-epi Series of Fumonisin B3
and B4
Table 3. Fumonisin Levels in Samples of Corn and Commercial Corn
Meal
a
FB3 (3)
3-epi-FB3 (5)
fumonisin levels
FB2 FB3
100 30
µ
g/kg)a
atom
δC
δH
δC
δH
sample
FB1
380
625
240
epi-FB3
total
512
905
327
% epi/FB3
1
2
3
4
15.61 Q
51.36 D
71.08 D
32.90 T
1.113 d (J 6.7)
2.956 qd (J 6.7, 6.8)
3.336 m
12.01 Q
50.63 D
69.65 D
32.46 T
1.054 d (J 6.7)
3.125 qd (J 6.8, 3.1)
3.568 m
commercial corn meal
2
5
2
6.7
8.3
13
16
12
14
19
19
18
17
21
8.1
15
16
15
6.7
215
70
60
15
265
80
25
4
374
Brazilian corn
1960
4515
3635
2405
2350
1370
2980
720
255
900
1985
540
465
780
370
665
320
400
200
120
215
45
95
60
75
35
20
45
200
25
285
75
30
3095
5530
4915
4865
3125
1975
4020
39000
3685
21365
7655
7865
FB4 (4)
3-epi-FB4 (6)
atom
δC
δH
δC
δH
Transkei home-grown,
good corn
1
2
3
4
15.42 Q
51.23 D
70.96 D
32.75 T
1.120 d (J 6.6)
2.957 qd (J 6.7, 6.7)
3.33 m
11.93 Q
50.56 D
69.57 D
32.37 T
1.060 d (J 6.7)
3.120 qd (J 6.8, 3.1)
3.58 m
Transkei home-grown, 26240 10070 2490
moldy corn
2665
12480
5035
830
6860 1740
2050
2025
165
a Solvent DMSO-d6. NMR analyses were performed on mixtures of stereoisomers
of FB3 and FB4.
495
445
5365
Table 2. 1H NMR Data for the MTPA Amides (
δ
, CDCL3)
a FB4 was not detected.
compound
atom
(S)-MTPA (b)
(R)-MTPA (a)
∆δ
(
δS − δR)
A solution of the tetramethyl esters 7 and 8 (265 mg, 0.35 mmol),
Et3N (352 mg, 3.48 mmol), and DMAP (5 mg) in CH2Cl2 (5 mL) was
added to a solution of the (S)-MTPA chloride in CH2Cl2 (2 mL)
solution. The mixture was stirred for 30 min and then quenched by
addition of water (2 mL). The organic solution was washed with 0.5
M HCl, followed by saturated NaHCO3 solution and water. The CH2-
Cl2 solution was dried (Na2SO4), filtered, and evaporated. The product
was purified by column chromatography on silica gel with EtOAc-
hexane (3:2) as the eluent to give the (R)-Mosher amide derivative
(180 mg, 59%), an oil, as a mixture of two diastereomers (9a and 10a)
(d.r. 82:18); Rf 0.34.
9
H(1)
H(3)a
H(1)
H(3)a
1.110
3.49
1.025
3.60
1.179
3.47
1.095
3.54
−
+
−
+
0.069
0.02
0.070
0.06
10
a Chemical shift values obtained from COSY and HETCOR data.
monitored by full-scan MS-MS between m/z 330 and m/z 730. A
collision energy of 32% was used to fragment the molecular ions, and
the resultant product ions were monitored as diagnostic indicators for
the presence of the toxins.
The same protocol was followed but using (S)-(+)-MTPA (e.e.
g99%), to convert a sample of the tetramethyl esters 7 and 8 (265 mg,
0.34 mmol) to a mixture of the two diastereomeric (S)-Mosher amide
derivatives (9b and 10b) (214 mg, 63%) (d.r. 82:18) (Figure 2).
NMR Analysis. NMR spectra were recorded on a Bruker Avance-
500DRX spectrometer operating at 500 MHz for 1H and 125 MHz for
13C nuclei using standard Bruker pulse sequences. Spectra acquired
for DMSO-d6 solutions were referenced to the signals at δH 2.49 and
δC 39.50 and for spectra in CDCl3 to the signals at δH 7.24 and δC
77.00.
RESULTS AND DISCUSSION
The terminal 2-amino-3-hydroxy motif of the fumonisin C20
backbone is also present in a number of marine natural products
(see Figure 3). In 1989, Gulavita and Scheuer (16) isolated two
epimeric aliphatic amino alcohols from a Papua New Guinea
sponge, Xestospongia sp., and proposed their structures as
(2S,3S,5E,7E)- and (2S,3R,5E,7E)-2-aminotetradeca-5,7-dien-
3-ol, ent-(11) and ent-(12), respectively. The relative stereo-
chemistry followed from nuclear Overhauser effect studies on
the oxazolidinone derivative and the absolute configuration at
C(2) by degradation of the diacetyl derivatives to alanine and
HPLC analysis of the derivative formed with 1-fluoro-2,4-
dinitrophen-5-yl-(2S)-alanine amide. Mori and Matsuda (17)
reported the total synthesis of both enantiomers of 11 and 12
and assigned the enantiomeric stereochemistry to the natural
products isolated by Gulavita and Scheuer (16), since the 2R
stereoisomers showed the same sign of optical rotation as the
natural products. The stereochemistry of the xestoaminols A
(13), B (14), and C (15) isolated by Jime´nez and Crews (18)
from two different sponges of the genus Xestospongia was
determined by Garrido et al. (19) in their study on the
obscuraminols, for example, obscuraminol C (16) obtained from
the tunicate Pseudodistoma obscurum. Sata and Fusetani (20)
isolated two new cytotoxic stereoisomeric 2,3-amino alcohols,
amaminol A (17) and B (18), from an unidentified tunicate of
the family Polyclinidae. The stereochemical relationship between
these two compounds, as in the case of the amino alcohols 11
and 12 isolated by Gulavita and Scheuer (16), is of particular
Determination of Fumonisin Analoques by HPLC with Fluores-
cence Detection. Determination of fumonisin analogues in corn was
conducted by the method of Sydenham et al. (15). Briefly, corn samples
were homogenized in methanol-water (3:1), centrifuged, and filtered.
An aliquot of the clear extract was applied to a strong anion exchange
solid-phase extraction cartridge (Varian, Harbor City, CA) and washed
with methanol, and the fumonisins were eluted with 1% acetic acid in
methanol. The purified extracts were dried down under nitrogen at
60 °C. Immediately prior to analysis, the residues were redissolved in
methanol and an aliquot was derivatized with OPA. The fumonisin
analogues were separated and detected by reversed-phase HPLC
(Ultracarb 5 ODS column; Phenomenex) with fluorescence detection
(Figure 4). The mobile phase was pumped at 1 mL/min and consisted
of methanol-sodium dihydrogen phosphate (0.1 M, pH 3.3) (77:23)
as generally used for fumonisin determination.
Preparation of the FB3 Derivatives. Fumonisin B3 Tetramethyl
Ester. A solution of FB3 standard (549 mg) containing a mixture of
FB3 (3) and 3-epi-FB3 (5) was treated with an excess of a diethyl ether
solution of diazomethane (prepared from 4.28 g of Diazald). After 10
min, the excess diazomethane was evaporated in a stream of nitrogen
and the residue was dried in vacuo to give the tetramethyl esters 7 and
8 (590 mg, 99%) (Figure 2).
Preparation of the Mosher Amide DeriVatiVes. Oxalyl chloride (220
mg. 1.74 mmol) was added to a solution of (R)-(-)-MTPA (e.e. g99%,
82 mg, 0.35 mmol) and dimethyl formamide (DMF) (25.4 mg, 0.35
mmol) in hexane (5 mL) at room temperature. A white precipitate
formed immediately. After 60 min, the mixture was passed through a
small cotton plug to filter off the formed DMF-Cl. The filtrate was
concentrated under reduced pressure to yield the (S)-MTPA chloride.

Fumonisin B Series Mycotoxins
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4391
Figure 4. HPLC chromatogram with fluorescence detection of the OPA derivatives of the isolated FB3 standard showing the relative areas of the two
stereoisomers.
significance to the structure elucidation of the minor metabolites
1
of the fumonisins. A detailed analysis of the H NMR spectra
of 17 and 18 disclosed that the relative stereochemistry of the
C(2) and C(3) stereogenic centers differed as indicated by the
coupling constant between H(2) and H(3) [J2,3 3.1 Hz for (17)
vs 7.3 Hz for (18)]. The Mosher ester analysis (21-23) indicated
that the two compounds had the same 3S absolute configuration
and were epimeric at C(2). This is in direct contrast to the results
obtained by Gulavita and Scheuer (16) on the amino alcohols
11 and 12, which are C(3) epimers. The absolute configuration
of the crucigasterins 19-21, isolated by Jarez et al. (24) from
a Mediterranean tunicate Pseudodistoma crucigaster, was as-
signed by chemical degradation to (3S,4R)-3-hydroxy-4-ami-
nopentanoic acid. The anti stereochemistry of the 2-amino-3-
hydroxy group of the crucigasterins is reflected by the 3.0 Hz
coupling constant for the C(2) and C(3) protons. Analysis of
the 13C NMR data reported by Gulavita and Scheuer (16) and
Mori and Matsuda (17) in the work on the amino alcohols 11
Figure 5. Biosynthetic formation of the fumonisins and the 3-epi
and 12 and by Sata and Fusitani (20) on the amaminols 17 and
stereoisomers.
18 showed characteristic chemical shift values for the methyl
group of the syn- and anti-2,3-amino alcohols. Thus, the methyl
15.61Q) and the minor component 3-epi-FB3 (5) (δC 12.01Q)
group, C(1), in the diacetyl derivative of 11 appeared at δC 18.49
(syn) whereas in the diacetyl derivative of 12 it appeared at δC
14.93 (anti). The same trend is observed for the amaminols:
δC 16.0 for the syn compound (11) and δC 12.1 for the anti
compound (12). The anti-amino alcohol (22) (25) showed the
methyl groups at δC 12.1 and 11.9, whereas in the syn-amino
alcohols such as fumonisin B3 (3) and B4 (4) the methyl group
appears at δC 15.61 and 15.42. It is thus evident that the 13C
chemical shift values for the methyl group, C(1), define the
relative stereochemistry of the 2,3-amino alcohol moiety in these
compounds.
confirmed the anti-2,3 stereochemistry of the latter.
1
The H and 13C NMR spectra of samples of fumonisin B4
(4) isolated over the years sometimes also showed the presence
of up to 40% of a minor metabolite 3-epi-FB4 (6) with the 2,3-
anti stereochemistry (Table 1). The anti stereochemistry of
3-epi-FB4 (6) followed once again from the coupling constant
of 3.1 Hz for the C(2) and C(3) protons and the 13C chemical
shift value of 11.93 for the methyl group, C(1) (see Table 1).
In contrast, the 1H and 13C NMR spectra of samples of FB1 (1)
and FB2 (2) prepared by us have always lacked the signals for
the corresponding 3-epi stereoisomers with the anti-2,3 stereo-
chemistry.
The absolute configuration of the C(2) and C(3) stereogenic
centers of FB1-4 has been established as (2S,3S) (26-32), and
consequently, the minor metabolites 3-epi-FB3 (5) and 3-epi-
FB4 (6) must either have the (2S,3R) or (2R,3S) configuration.
The C(2) absolute configuration for the FB3 minor metabolite,
3-epi-FB3 (5), was determined using Mosher methodology (21-
23). A sample of FB3 (3) [containing 18% of the minor
metabolite 3-epi-FB3 (5)] was converted to the tetramethyl ester
derivatives 7 and 8 (minor) by treatment with an excess of
diazomethane. The amino group was then converted to the (R)-
and (S)-MTPA amides (7a,b and 8a,b) (minor), respectively,
by reaction with the (S)- and (R)-MTPA chlorides [prepared
from the (R)- and (S)-MTPA acid, respectively, following the
protocol developed by Ward and Rhee (33)]. The ∆δ (δS -
δR) values observed for H(1) (-0.069 ppm) and H(3) (+0.02
Fast atom bombardment and ES mass spectrometry of FB3
samples, which exhibited two peaks in the HPLC analysis,
showed the [M + H]+ ion at 706 and established the molecular
1
formula C34H59NO14 for each of the two compounds. The H
and 13C NMR spectra showed in all cases the presence of a
minor component 3-epi-FB3 (5). The use of two-dimensional
correlation spectroscopy (COSY) and heteronuclear correlation
(HETCOR) experiments established that the discernible signals
of the minor component in the 1H and 13C spectra represent the
C(1)-C(4) unit of the backbone. The coupling constant of 6.8
Hz between the C(2) and the C(3) protons of FB3 is character-
istic of the syn-2,3-amino alcohol in these metabolites. The anti
stereochemistry for the 2,3-amino alcohol unit of the minor
metabolite 3-epi-FB3 (5) followed from the 3.1 Hz coupling
observed for the C(2) and C(3) protons (Table 1). The 13C
chemical shifts for the methyl group in fumonisin B3 (δC

4392 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Gelderblom et al.
Figure 6. Total ion chromatogram of a Transkeian moldy corn sample and mass spectra of the protonated FB3 and 3-epi-FB3 analogues.
ppm) of the Mosher amide derivatives (9) (see Table 2) are in
agreement with the 2S absolute configuration of the fumonisins.
The fact that similar values are observed for the corresponding
protons of the Mosher amide derivatives (10) implies that the
minor metabolite 3-epi-FB3 (5) present in samples of FB3 (3)
also has the 2S absolute configuration and therefore on the basis
of its 2,3-anti relative stereochemistry, the 3R absolute config-
uration. As a consequence of this result, the assignment of the
2S,3R absolute configuration to the minor metabolite 3-epi-FB4
(6) present in samples of fumonisin B4 (4) is reasonable.
Biosynthetic studies on the fumonisins using [1-13C]-, [2-13C]-
, and [1,2-13C2]acetate, [methyl-13C]-(2S)-methionine, and [3-13C]-
(2S)-alanine precursors established that C(3)-C(20) are derived
from acetate and C(1), C(2), and the 2-NH2 group are derived
from alanine. The C(12) and C(16) methyl groups are derived
from methionine (34-38). An acetate-derived carbonyl group
is the source of the C(3) hydroxyl group, whereas the C(5),
C(10), C(14), and C(15) hydroxyl groups are likely derived from
molecular oxygen (39). The citric acid cycle is believed to be
the source of the tricarballylic acid moiety (37). Recently, a
15-gene cluster (FUM1-FUM3, FUM6-FUM8, FUM10,
FUM11, and FUM13-FUM19) required for the biosynthesis
of the fumonisins in F. Verticilloides was cloned and character-
ized (40-42). The data obtained from direct studies of these
biosynthetic genes by genetic and biochemical approaches (40-
48) provided the basis for a biosynthetic pathway (46-48). The
biosynthesis starts with the formation of a dimethylated,
saturated C18 polyketide chain catalyzed by the Fum1p polyketide
synthase (see Figure 5) The carbon-carbon bond formation
between the (2S)-alanine and the enzyme-bound C18 polyketide
chain by the pyridoxal-phosphate-dependent aminoacyl trans-
ferase enzyme Fum8p, encoded by the FUM8 gene (42, 43),
involves the loss of CO2 to form the 3-keto intermediate (23)
with overall retention of the C(2) configuration. Stereospecific
reduction of the 3-keto intermediate at the ReS face of the
carbonyl group by ketoreductase Fum13p results in the forma-
tion of the 3S hydroxyl group and the syn-2,3 amino alcohol
motif (24) (43, 45, 47). This reduction is analogous to that of
3-ketosphinganine to sphinganine (43) except that the reduction
of the carbonyl group with 3-ketosphinganine reductase must
occur at the Si face to generate the 3R hydroxyl group and the
anti-2,3 amino alcohol of sphinganine.
The formation of 3-epi-FB3 (5) with its 3R hydroxyl group
and thus the 2,3-anti stereochemistry is postulated to occur by
reduction at the SiS face of the carbonyl group of the 3-keto
intermediate (23) to give the intermediate (25) in the fumonisin
biosynthesis by a 3-ketosphinganine type reductase. The pres-
ence of such a 3-ketosphinganine type reductase has been
proposed to account for the low levels of FB1-4 production in
FUM13 deletion mutants (43).
Evidence has been presented that the reduction of the 3-keto
group is an early step in the biosynthetic pathway, whereas the
introduction of the C(5) hydroxyl group by a 2-ketoglutarate-
dependent dioxygenase Fum3p, encoded by the FUM3 gene, is
the last step (43, 47, 48). On the basis of this proposed
biosynthetic pathway, the presence of 3-epi-FB3 (5) and 3-epi-
FB4 (6) should lead to the formation of the 3-epi stereoisomers
of FB1 and FB2 by the Fum3p enzyme. This is not the case as
these 3-epi stereoisomers of FB1 and FB2 have never been
detected by us. This failure of Fum3p to convert 3-epi-FB3 (5)
and 3-epi-FB4 (6) to 3-epi-FB1 and 3-epi-FB2, respectively,
suggests that the metabolites with the 3R hydroxyl group, that
is, the 2,3-anti stereochemistry, are not recognized by Fum3p
and thus do not serve as substrates.
The newly described 3-epi-FB3 (5) was identified from the
FB3 standard isolated in the PROMEC Unit from various batches
of corn patty culture material prepared from F. Verticillioides
MRC 826. In an attempt to avoid the presence of this analogue
in the analytical standard, a range of other F. Verticillioides
strains, including MRC 8041 that only produces FB3, have been
cultured. The 3-epi-FB3 (5) has been found to occur in all of
these at levels ranging from 21 to 42% of the level of the normal
FB3. In a limited comparison, two strains grown in liquid
medium showed lower levels (around 7-8%) of the epi
stereoisomer. Of greater concern is the potential natural occur-
rence of this new fumonisin analogue. A range of corn samples
from various sources have recently been analyzed by HPLC
with fluorescence detection. 3-epi-FB3 (5) was identified by its

Fumonisin B Series Mycotoxins
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4393
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retention time as a small peak eluting from the HPLC column
immediately prior to normal FB3. Table 3 shows the results of
the analytical determination of FB3 (3) and 3-epi-FB3 (5) for
16 samples of corn and commercial corn meal. The presence
of the stereoisomer was confirmed by LC-MS-MS analysis and
the presence of the characteristic fragmentation pattern for
fumonisins. Figure 6 shows the total ion chromatogram for a
sample of Transkeian corn, as well as a comparison of the mass
spectra for FB3 (3) and for the peak corresponding to 3-epi-
FB3 (5). The similarity of the spectra and the presence of the
fragment ions corresponding to [M + H - H2O]+ (m/z 688),
[M + H - 2H2O]+ (m/z 670), [M + H - TCA]+ (m/z 530),
[M + H - H2O - TCA]+ (m/z 512), [M + H - 2TCA]+ (m/z
354), and [M + H - H2O - 2TCA]+ (m/z 336) provides
unequivocal identification of the compounds. The level of 3-epi-
FB3 (5) in these samples ranged from 6.7 to 21% of the level
of FB3 and contributed only marginally to the total level of
fumonisins. On the basis of these data and the fact that it also
only occurs at lower levels than FB3 in culture, it can be
concluded that, in general, the influence of 3-epi-FB3 on
fumonisin exposure will be small.
(13) Food and Drug Administration, 2000; http://vm.cfsan.fda.gov/
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Received for review January 9, 2007. Revised manuscript received
March 22, 2007. Accepted March 22, 2007. Financial support from the
University of Pretoria and the National Research Foundation, Pretoria,
as well as the Wellcome Trust toward the purchase of the LC-MS
system is gratefully acknowledged.
JF070061H