Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high-performance liquid chromatography/electrospray ionization ion trap mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 2447–2462

) DOI: 10.1002/rcm.2607
Published online in Wiley InterScience (www.interscience.wiley.com
Detection of new fumonisin mycotoxins

and fumonisin-like compounds by reversed-phase

high-performance liquid chromatography/electrospray

ionization ion trap mass spectrometry

Tibor Bartok1*, Arpad Szecsi2, Andras Szekeres1, Akos Mesterhazy1 and Mihaly Bartok3

1Cereal Research Non-Profit Company, P.O. Box 391, H-6701 Szeged, Hungary2Department of Plant Pathology, Plant Protection Institute of the Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary3Department of Organic Chemistry, University of Szeged, Dom ter 8, H-6720 Szeged, Hungary

Received 8 April 2006; Revised 2 June 2006; Accepted 14 June 2006

*Correspopany, P.OE-mail: bContract/contract/Contract/ALAP1-0

Fumonisins were produced in a rice culture infected with Fusarium verticillioides. To decrease the

possibility of the formation of artifacts, the fumonisins were analyzed by reversed-phase high-

performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry

(RP-HPLC/ESI-IT-MS2) immediately after the extraction of the culture material without any sample

clean-up. In addition to already known fumonisins, numerous new fumonisin mycotoxins and

fumonisin-like compounds were detected. On the basis of the IT-MS2 data, detailed fragmentation

pathways including new mechanisms were proposed for the different series of fumonisins. The

retention times, the masses of the protonated molecules and of the product ions including the

backbones and the characteristic neutral mass losses from the protonated molecules of the new

compounds suggested their structures (applying the well-known designation): iso-FA1a,b, iso-FB1a–d,

iso-FB2,3a–e, PHFB2a–c, PHFB4a–d, FB5/iso-FB5a–d, FBK1 2TCA, FBK4 2TCA, FC2, iso-FC2,3, PHFC4, FD

and FBX series. The relative quantities of fumonisins and fumonisin-like compounds found in the

sample extract were expressed as percentages of FB1 (0.02–100%). The backbone of the compound

denoted FD contained fewer carbon atoms than the well-known fumonisins with the C19 or C20

backbone and may well be a precursor of the longer compounds. For the compounds denoted FBX (12

compounds), one or two OH groups attached to the fumonisin backbone were esterified by carboxylic

acids other than tricarballylic acid, such as cis-aconitic acid, oxalylsuccinic acid and oxalylfumaric

acid. Copyright # 2006 John Wiley & Sons, Ltd.

The fumonisins1,2 are a group of structurally related

mycotoxins that are mainly produced by Fusarium verticil-

lioides (Sacc.) Nirenberg, formerly known as F. moniliforme

Sheldon.3 This fungus is one of the most common molds

colonizing maize crops throughout the world before

harvesting, during the time between harvesting and drying,

and during storage.4,5 Fumonisins are most frequently found

in maize and maize-based foodstuffs6 and feedstuff, and less

commonly in other grains (i.e. sorghum4, rice7 and wheat8).

The fumonisins cause leukoencephalomalacia9 and pul-

monary edema in swine.10 One of the mechanisms of

fumonisin toxicity may be inhibition of the enzyme

sphinganine N-acetyltransferase, which results in the

accumulation of free sphinganine, and a decrease in the

level of sphingosine, intermediates in the biosynthetic

ndence to: T. Bartok, Cereal Research Non-Profit Com-.Box 391, H-6701 Szeged, [email protected] sponsor: Hungarian State Research grant;grant number: OTKA 46739.grant sponsor: GAK grant; contract/grant number:0073/2004.

pathway for complex sphingolipids leading to disruption

of the membranes.11 The consumption of fumonisin-

contaminated maize has been associated statistically with

the high incidence of esophageal cancer in rural areas of

South Africa,12 China13 and Italy.14 Moreover, FB1 is

considered by the IARC to be a possible carcinogen to

humans (class 2B).15

The fumonisin analogs that have been characterized since

1988 can be classified into four main groups, identified as the

fumonisin series A, B, C, and P.16,17 The fumonisin B (FB)

analogs, comprising toxicologically important FB1, FB2 and

FB3, are the most abundant naturally occurring fumonisins,

with FB1 predominant, and are usually found at the highest

levels.18 FB1 typically accounts for 70–80% of the total

fumonisin produced, while FB2 usually makes up 15–25%

and FB3 from 3–8% when cultured on maize or rice or in

liquid medium.19–21 Apart from the FB series, some of the

other analogs may occur in naturally contaminated maize, at

relatively low levels (<5% of the total fumonisin present).22

Copyright # 2006 John Wiley & Sons, Ltd.

2448 T. Bartok et al.

These lesser known fumonisin analogs are difficult to detect

with most analytical techniques due to the necessary

derivatization processes, but they can be analyzed by liquid

chromatography/mass spectrometry with electrospray ioni-

zation (LC/ESI-MS) and ESI with tandemmass spectrometry

(MS/MS). ESI-MS has gained general acceptance in fumo-

nisin analysis.5,7,23–32 The significance of the method is

especially well demonstrated by pertinent reports published

in 2005.17,23,33,34

Since reversed-phase high-performance liquid chromato-

graphy/electrospray ionization ion trap multistage mass

spectrometry (RP-HPLC/ESI-IT-MSn) is suitable29,35,36 for

the detection, without isolation, of minute amounts of

compounds with unknown structures, we chose this method

for the determination of fumonisins extracted from a 28-day-

old rice culture which had been inoculated with a conidial

suspension of Fusarium verticillioides strain FV16 isolated

from a maize stalk. The results of these studies are presented

here. The experiments furnished the unexpected result that,

in addition to already known mycotoxins of the fumonisin

type, the culture extract also contained numerous other

fumonisin analogs and fumonisin-like compounds.

EXPERIMENTAL

ChemicalsPotato dextrose agar (PDA), HPLC-grade acetonitrile

(MeCN), fumonisin B1, B2 standards and the components

of the modified pentachloronitrobenzene (PCNB) medium

were purchased from Sigma-Aldrich Ltd. (Budapest,

Hungary). Fumonisin B3 and B4 standards were gifts from

Prof. W. C. A. Gelderblom (PROMEK Medical Research

Council, Tygerberg, South Africa) and Prof. R. D. Plattner

(National Center for Agricultural Utilization Research,

USDA, Peoria, IL, USA). HPLC-grade water with a resistivity

of 18MV was produced with a Nanopure II (Barnstead/

Thermolyne Co., Dubuque, IA, USA) cartridge-type water

purification equipment.

Fusarium verticillioides strainThe isolate of F. verticillioides (FV16, Iregszemcse, Hungary)

was obtained from a maize stalk, isolated on a modified

PCNB medium selective for Fusarium species.37 After

growth, the isolate was observed, mass-transferred to freshly

prepared PDA and identified as F. verticillioides (Sacc.)

Nirenberg according to Nirenberg and O’Donnell.38 A

culture was then initiated from a single conidium, and

maintained at 48C on PDA slants. For fermentation studies,

slants of F. verticillioides were washed with sterile distilled

water and filtered through four layers of cheesecloth, which

resulted in a conidial suspension containing approximately

107 conidiamL�1.

Fumonisin productionLong-grain rice (50 g; Uncle Ben’s) and HPLC-grade water

(50mL)were added to Erlenmeyer flasks (500mL). The flasks

were kept at room temperature overnight, and the excess

water was then decanted. The flasks were autoclaved at

1218C for 15min on each of 2 consecutive days, inoculated


with 5mL of the conidial suspensions of F. verticillioides FV16

and incubated at 288C in the dark. The cultures were shaken

once daily for the first 3 days after incubation to distribute the

inoculum and to prevent the grains from adhering. After

4 weeks, the cultures were removed from the incubator,

immediately frozen and freeze-dried, then ground to a fine

meal and stored in a deep freezer (�808C) until analysis.

Extraction of fumonisinsFreeze-dried rice culturematerial (3 g) was homogenized in a

polypropylene centrifuge tube (30mL) with a mixture of

MeCN/H2O (25mL; 75/25, v/v), using a UltraTurrax T25

(IKA, Staufen, Germany) high-speed homogenizer at

13 500 rpm for 1min, and was subsequently extracted on a

vertical shaker at room temperature for 1 h. After extraction,

the sample was centrifuged at 10 000 g for 10min and

membrane-filtered through a 0.45mmPTFEmembrane into a

HPLC autosampler vial. To decrease the possibility of the

formation of artifacts, the fumonisins from the crude extract

were analyzed directly by RP-HPLC/ESI-IT-MS2 without

any sample clean-up.

HPLC separation of fumonisinsRP-HPLC analysis was carried out with an Agilent (Palo

Alto, CA, USA) 1100 Series HPLC system equipped with a

binary pump, a vacuum degasser and a mWell-plate

autosampler. Gradient HPLC separation was performed

on a Supelcosil ABZ Plus analytical column (250� 2.1mm,

5mm; Supelco, Bellefonte, PA, USA). A guard column

(Supelcosil ABZ Plus 20� 3mm, 5mm) was attached to the

analytical column without any capillaries. The end fitting of

the analytical column was attached directly to the nebulizer

through a 40mm PEEK capillary (127mm i.d.). The columns

were thermostatted to 408Cusing amodel 7990 Space column

heater (Jones Chromatography Ltd., Hengoed, UK). The

gradient solvent-delivery system consisted of two solvents.

Solvent A was H2O and solvent B was MeCN, both

supplemented with 0.1% formic acid. The gradient elution

at a flow rate of 300mLmin�1 started with 25% B and was

increased linearly to 40% B at 22min, and then to 100% B at

27min, which value was held at 3min. The injection volume

was 1mL.

Mass spectrometric detection of fumonisinsAuto MS2 measurements of the protonated molecules were

made in full-scan mode with an Agilent 1100 MSD Trap SL

mass spectrometer equipped with an atmospheric-pressure

ESI source and an IT mass analyzer. The instrument

parameters were as follows: Ion source parameters: capillary

high voltage, 3500V; nebulizer gas (N2) pressure, 40 psi;

drying gas (N2) flow and temperature, 9 Lmin�1 and 3508C,respectively; capillary exit voltage, 200V; Detector voltages:

electron multiplier voltage, 1185V; high-energy dynode

(HED) voltage, 7 kV; IT parameters, trap drive: 53.9; max.

accumulation time, 300ms; full-scan, m/z 50–1100; averages,

3 spectra; ion charge control, on; fragmentation amplitude,

1.3V. Analyses were performed in positive ion mode. The

parameters of the mass spectrometer were optimized by the


DOI: 10.1002/rcm

Detection of new fumonisin mycotoxins 2449

continuous infusion (5mLmin�1) of an FB1 standard solution

(10 ngmL�1) directly into the ESI source by a KDS model 101

syringe pump (KD Scientific, Holliston, MA, USA). The

nebulizing gas pressure and the drying gas flow and

temperature were optimized in automated flow injection

analysis (FIA)without a column. During continuous infusion

and optimization by FIA, the MþHeþ ion of FB1 was

monitored at m/z 722.4. Complete system control and data

evaluation were performed with Agilent ChemStation soft-

ware (A09.03) and Bruker ITMS software (v.5.2). The relative

quantities of fumonisins and fumonisin-like compounds

found in the sample extract (based on the detector signal of

the protonated molecule) were expressed as percentages of

FB1.

RESULTS AND DISCUSSION

Results are grouped according to fumonisin types (FB, FA,

FC, and the previously unknown FBX). The present results

on the most intensively studied andmost abundant FB series

are reported for the first time. Since the new compounds have

not yet been isolated, precise determination of their

Scheme 1. (a) Theoretical CID fragmentation pattern of FB1

fragmentation of FB1þHeþ (for abbreviations, see Scheme 5


structures has not been possible. However, the chromato-

graphic retention time on a C18 HPLC column, the masses

of the protonated molecules (obtained via the MS spectra)

and the characteristic product ions including the backbones

and the characteristic neutral mass losses in the automati-

cally recorded MS2 spectra of the MþHeþ ion of the new

compounds allowed identification of the compound type

and, hence, a tentative assignment to be made regarding the

structure.

When the structures of novel compounds were postulated

without their isolation, a common starting point was the

generally accepted consideration that the tricarballylic acid

(TCA) is bound to the same carbon atoms of the fumonisin

backbone in every case. In certain cases, however, in

consequence of the wide range of retention times for the

isomers (eluting peaks) within a given fumonisin type

(e.g. FB1 isomers: 4.7–14.2min), a correction of the commonly

accepted consideration mentioned above may appear

necessary. This correction, however, cannot be carried out

without further studies.

Let us first review the structures of the fumonisins. As

shown in Scheme 1(b), there are several chiral centers in FB1.

It is well known that MS techniques are unable to

þHeþ. (b) Assumed structures of ions formed in the CID

).


DOI: 10.1002/rcm

Figure 1. Product ion spectra (CID) of FB1þHeþ (1), FBK1 2TCAþHeþ (11), PHFB2cþHeþ (16), iso-FB2,3bþHeþ (19),

PHFB4bþHeþ (25) and FB5þHeþ (28) (Tables 1 and 2) (for conditions, see Experimental section).


differentiate between the individual isomers and to deter-

mine the absolute configurations of chiral carbon atoms.

Methods serving this purpose (e.g. NMR, XRD and ORD),

however, have made possible the determination of the

spatial structures of the fumonisins identified so far,

including the absolute configurations of the carbon

atoms.2,39–47 The configurations of the carbon atoms of the

fumonisins are not indicated here, partly for reasons of

simplicity, and partly because the full spatial structures of

the fumonisins described are not known in all cases.

able 1. Fumonisin analogs: B series, known and proposed structures

Side chains to fumonisin backbone

Compound a b c d e f Ref.

FB1 TCA TCA OH OH H OH 48, twiso-FB1 TCA TCA OH H OH OH 27, tw

–6a iso-FB1a–d TCA TCA OH H H OH tw, 8 PHFB1a,b OH TCA OH OH H OH 26, tw, 10 FBK1a,b ¼O ��!TCA OH OH H OH 49, tw1a FBK1 2TCA TCA TCA OH OH H ¼O tw2 FBK4 2TCA TCA TCA H OH H ¼O tw3 FB2 TCA TCA H OH H OH 48, tw4–16a PHFB2a–c TCA ��!OH H H H OH tw7 FB3 TCA TCA OH H H OH 50, tw8–22a iso-FB2,3a–e TCA TCA H H H OH tw3 FB4 TCA TCA H H H OH 50, tw4–27a PHFB4a–d TCA ��!OH H H H H tw8–32b FB5/iso-FB5a–d TCA TCA OH H H OH tw

There is another OH group on the backbone; tw¼ this work.There are two other OH groups on the backbone.

T

1

2

37

9

1

11

1

1

12

2

2

a

b


FB analogsAs mentioned above, the model compound in MS studies on

the fumonisins was FB1 and the characteristic ESI-MS2

spectrum of FB1þHeþ is presented in Fig. 1; the ESI-MS2

spectra of the MþHeþ ions of some new FB analogs are also

shown in Fig. 1.

The fumonisins detected are listed in Table 1 and their

characteristic data (retention time (Rt), amount as percentage

of FB1 andMS2 product ion data) are summarized in Table 2.

The tabulated spectra of the FB1, FB2, FB3, and FB4 standards


DOI: 10.1002/rcm

Table

2.Characteristicionsofproductionspectraoffumonisin

Bseries(forabbreviations,seeScheme5)

Fumonisin

or

fumonisin-like

compounds

Ret.time(min)

Rqð%ofFB1Þ

MþHeþ

MþH-H2Oeþ

MþH-2H2Oeþ

MþH-3H2Oeþ

MþH-4H2Oeþ

MþH-TCAKeþ

MþH-TCAeþ

MþH-TCA-H2Oeþ

MþH-TCA-2H2Oeþ

MþH-TCA-3H2Oeþ

MþH-TCA-TCAKeþ

MþH-2TCAeþ

MþH-2TCA-H2Oeþ

MþH-2TCA-2H2Oeþ

MþH-XTCA-YH2Oeþ

MþH-XTCA-YH2O-NH3eþ

ðbackboneÞ

m/z

values

andrelativeionab

undan

ce(%

)

1FB1a

8.2

100

722

704(100

)68

6(78)

668(26)

650(0)

564(5)

546(81)

528(83)

510(67)

492(6)

388(3)

370(22)

352(47)

334(76)

316(14)

299(3)

2iso-FB1

9.7

1.2

722

704(71)

686(37)

668(3)

650(0)

564(20)

546(100

)52

8(73)

510(23)

492(17)

388(3)

370(67)

352(45)

334(58)

316(23)

299(3)

3iso-FB1a

4.7

0.1

722

704(100

)68

6(41)

668(15)

650(1)

564(2)

546(39)

528(75)

510(94)

492(30)

388(3)

370(8)

352(55)

334(55)

316(0)

299(1)

4iso-FB1b

6.7

1.1

722

704(86)

686(43)

668(9)

650(0.3)

564(8)

546(44)

528(100

)51

0(49)

492(18)

388(0)

370(27)

352(41)

334(16)

316(18)

299(2)

5iso-FB1c

11.2

0.2

722

704(77)

686(61)

668(14)

650(0.4)

564(5)

546(50)

528(100

)51

0(14)

492(17)

388(4)

370(42)

352(85)

334(49)

316(42)

299(3)

6iso-FB1d

14.2

0.5

722

704(15)

686(3)

668(0)

650(0)

564(20)

546(100

)52

8(24)

510(0.2)

492(2)

388(6)

370(45)

352(25)

334(8)

316(0.3)

299(0)

7PHFB1a

4.1

0.03

564

546(100

)52

8(9)

510(6)

492(0)

406(3)

388(40)

370(42)

352(22)

334(21)

——

——

316(4)

299(2)

8PHFB1b

5.3

2.1

564

546(100

)52

8(15)

510(3)

492(0)

406(0.1)

388(4)

370(11)

352(12)

334(5)

——

——

316(0.7)

299(0.4)

9FBK1ab

6.5

0.1

562

544(100

)52

6(27)

508(14)

490(1)

404(3)

386(11)

368(10)

350(11)

——

——

——

297(3)

10

FBK1bc

7.8

4.1

562

544(100

)52

6(48)

508(7)

490(0)

404(3)

386(3)

368(11)

350(7)

——

——

——

297(0.1)

11

FBK12TCA

11.3

0.2

736

718(99)

700(68)

682(2)

664(0)

578(6)

560(26)

542(100

)52

4(29)

506(10)

402(0)

384(19)

366(62)

348(50)

330(5)

313(0.2)

12

FBK42TCA

14.2

0.8

704

686(52)

668(29)

650(0.2)

—54

6(10)

528(34)

510(100

)49

2(16)

—37

0(6)

352(6)

334(43)

316(13)

—29

9(3)

13

FB2

15.6

72.2

706

688(96)

670(28)

652(2)

—54

8(10)

530(32)

512(100

)49

4(23)

476(2)

372(0)

354(46)

336(68)

318(32)

—30

1(0)

14

PHFB2a

9.8

0.8

548

530(100

)51

2(10)

494(0.1)

476(0)

390(7)

372(7)

354(36)

336(9)

318(0.1)

——

——

—30

1(1)

15

PHFB2b

11.9

0.4

548

530(100

)51

2(15)

494(2)

476(0)

390(0)

372(35)

354(36)

336(21)

318(11)

——

——

—30

1(0)

16

PHFB2c

13.7

5.7

548

530(100

)51

2(10)

494(1)

476(0)

390(2)

372(26)

354(29)

336(15)

318(3)

——

——

—30

1(1)

(Continues)

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Comm


un. Mass Spectrom. 2006; 20: 2447–2462

DOI: 10.1002/rcm

Table

2.(C

ontinued)

Fumonisin

or

fumonisin-like

compounds

Ret:timeðminÞ

Rqð%ofFB1Þ

MþHeþ

MþH-H2Oeþ

MþH-2H2Oeþ

MþH-3H2Oeþ

MþH-4H2Oeþ

MþH-TCAKeþ

MþH-TCAeþ

MþH-TCA-H2Oeþ

MþH-TCA-2H2Oeþ

MþH-TCA-3H2Oeþ

MþH-TCA-TCAKeþ

MþH-2TCAeþ

MþH-2TCA-H2Oeþ

MþH-2TCA-2H2Oeþ

MþH-2TCA-3H2Oeþ

MþH-2TCA-4H2Oeþ


ðbackboneÞ

m/z

values

andrelativeionab

undan

ce(%

)

17

FB3

12.9

37.7

706

688(100

)67

0(31)

652(8)

634(0)

548(25)

530(84)

512(53)

494(37)

476(10)

372(2)

354(53)

336(56)

318(58)

——

301(3)

18

iso-FB2,3a

10.8

0.1

706

688(25)

670(16)

652(3)

634(0)

548(2)

530(100

)51

2(8)

494(8)

476(4)

372(11)

354(36)

336(84)

318(20)

——

301(5)

19

iso-FB2,3b

12.1

0.2

706

688(57)

670(11)

652(2)

634(0)

548(22)

530(71)

512(96)

494(53)

476(2)

372(24)

354(65)

336(100

)31

8(32)

——

301(3)

20

iso-FB2,3c

16.5

0.2

706

688(76)

670(22)

652(5)

634(0)

548(10)

530(42)

512(100

)49

4(34)

476(2)

372(0)

354(23)

336(91)

318(38)

——

301(0)

21

iso-FB2,3d

17.0

1.6

706

688(26)

670(12)

652(4)

634(0)

548(14)

530(100

)51

2(14)

494(8)

476(3)

372(24)

354(79)

336(19)

318(19)

——

301(0)

22

iso-FB2,3e

18.8

0.9

706

688(52)

670(31)

652(1)

634(0)

548(0)

530(54)

512(84)

494(7)

476(0.5)

372(50)

354(26)

336(100

)31

8(28)

——

301(3)

23

FB4

20.8

15.3

690

672(44)

654(9)

636(0)

—53

2(10)

514(66)

496(16)

478(28)

460(0)

356(11)

338(72)

320(100

)—

——

303(8)

24

PHFB4a

16.4

0.2

532

514(67)

496(14)

478(3)

—37

4(0.4)

356(81)

338(100

)32

0(18)

——

——

——

—30

3(0.3)

25

PHFB4b

18.7

1.3

532

514(70)

496(5)

478(0.2)

—37

4(2)

356(100

)33

8(74)

320(26)

——

——

——

—30

3(0.1)

26

PHFB4c

22.5

0.7

532

514(100

)49

6(8)

478(0.1)

—37

4(1)

356(50)

338(38)

320(9)

——

——

——

—30

3(1)

27

PHFB4d

25.3

0.2

532

514(42)

496(7)

478(0)

—37

4(1)

356(100

)33

8(39)

320(2)

——

——

——

—30

3(2)

28

FB5

7.1

0.4

738

720(100

)70

2(17)

684(0.1)

666(0.1)

580(12)

562(37)

544(41)

526(11)

508(2)

404(1)

386(39)

368(52)

350(19)

332(4)

314(2)

297(0)

29

iso-FB5a

3.0

0.02

738

720(100

)70

2(45)

684(0)

666(0)

580(0)

562(11)

544(100

)52

6(54)

508(16)

404(0)

386(2)

368(3)

350(23)

332(14)

314(44)

297(2)

30

iso-FB5b

3.9

0.02

738

720(79)

702(36)

684(40)

666(5)

580(0)

562(41)

544(100

)52

6(83)

508(5)

404(0)

386(18)

368(35)

350(14)

332(4)

314(13)

297(0)

31

iso-FB5c

6.0

0.1

738

720(100

)70

2(40)

684(11)

666(0.5)

580(5)

562(9)

544(56)

526(21)

508(6)

404(3)

386(20)

368(30)

350(14)

332(3)

314(6)

297(0)

32

iso-FB5d

11.5

0.06

738

720(100

)70

2(25)

684(15)

666(13)

580(12)

562(26)

544(65)

526(35)

508(0)

404(0)

386(11)

368(53)

350(17)

332(0.5)

314(0)

297(0)

Rq:relativequan

tity

(%ofFB1,FB1¼10

0%).

X:1(7–1

0,1

4–1

6,2

4–2

7)or2(1–6

,1

1–1

3,

17–

23,

28–

32).

Y:1(2

3),2(1

2,1

3,1

7–

22,

24–2

7),3(1

-6,

9–1

1,1

4–1

6)or4(7,

8,2

8–3

2).

aþ

m/z

588(2.9)¼

MþH-FA-H

2Oeþ

;þ

m/z

412(0.8)¼

MþH-TCA-FA-H

2Oeþ

bþ

m/z

333(3)¼

MþH-TCA-2H

2O-N

H3eþ

;þ

m/z

315(0.5)¼

MþH-TCA-3H

2O-N

H3eþ

cþ

m/z

333(5)¼

MþH-TCA-2H

2O-N

H3eþ

;þ

m/z

315(0.2)¼

MþH-TCA-3H

2O-N

H3eþ

Copyright # 2006 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2006; 20: 2447–2462

DOI: 10.1002/rcm


Scheme 2. (a) Theoretical CID fragmentation pattern of FA1þHeþ (aDetailed fragmentation of ion atm/z 704 is shown

in Scheme 1(a). (b) Assumed structures of ions formed in the CID fragmentation of FA1þHeþ (for abbreviations, see

Scheme 5).


(1, 13, 17, 23) are also included in Table 2 (abbreviations

referring to the structures of some compounds are to be

found in Scheme 5). In most cases, the carbon backbone,

which has an important role during the identification of the

fumonisins, was detected. Schemes similar to the low-energy

collision induced dissociation (CID) fragmentation pattern

drawn up for FB1þHeþ (Schemes 1(a) and 1(b)) can be

constructed for the other fumonisin analogs and can serve as

starting points for the assignment of the proposed structures

of the new compounds (Table 1).

It follows from the structures of the fumonisins that, in the

course of fragmentation, groups modifying the carbon

backbone are split off, i.e. C–O and C–N bond scission leads

to the elimination of H2O, TCA andNH3 (Scheme 1(b)). From

the product ions formed as a result of fragmentation, it can be

established that TCA is gradually eliminated, i.e. H2O is

released with the concomitant formation of the correspond-

ing anhydride (TCAD¼ 158Da), followed by the elimination

of TCAD. The splitting-off of TCA in the form of its ketene


(TCAK¼ 158Da) and H2O, and in the form of fumaric acid

(FA¼ 116Da) and AcOH (60Da), was also observed. These

fragmentation steps are included in Scheme 1(b). To make

Scheme 1(b) more simple, the fragmentation path occurring

through TCAK is outlined in Scheme 3(b).

As the studies published to date do not allow the

individual structural isomers (e.g. FB1 and iso-FB1) to be

differentiated via the fragmentation patterns, the differentia-

tion was based on retention times. According to the earlier

reports, the retention time (Rt) of a fumonisin isomer

obtained after RP-HPLC separation on a C18 column is

higher than that of the basic compound (e.g. Rt FB1<Rt

iso-FB127), though we did find FB1, FB2,3 and FB5, isomers with

lower retention times than that of the basic one (see Table 2).

FA analogsSimilarly to the FB series, the corresponding data on the FA

series are presented in Tables 3 and 4 and Schemes 2(a) and

2(b). A spectrum characteristic of the series, the MS2


DOI: 10.1002/rcm

Scheme 3. (a) Theoretical CID fragmentation pattern of FC1þHeþ. (b) Assumed structures of ions formed in the

CID fragmentation of FC1þHeþ. (c) Assumed structures of ions formed in the CID fragmentation of FD

(C18)þHeþ. (d) Assumed structures of ions formed in the CID fragmentation of FD (C17 ketone)þHeþ. Forabbreviations, see Scheme 5.


DOI: 10.1002/rcm


Scheme 4. (a) Theoretical CID fragmentation pattern of FB4 2AAþHeþ.(b) Assumed structures of ions formed in the CID fragmentation of FB4

2AAþHeþ (for abbreviations, see Scheme 5).


spectrum of the MþHeþ ion of the FA1 analog, is depicted in

Fig. 2. Significantly fewer FA analogs than FB analogs were

detected, which is in agreement with data published in this

field. As expected, the fragmentation pattern is unlike that of

the FB series. The main reason for this difference is that,

instead of amino groups, the FA analogs contain the


acetylated derivative of amines, i.e. the FA analogs are

acetamides rather than amines. As shown in Scheme 2(b),

two different fragmentation sequences may develop,

depending on the phase in which the acetyl group is split

off the backbone. This is well illustrated by the m/z values of

the individual product ions.


DOI: 10.1002/rcm

Scheme 5. Structures of compounds formed in the CID fragmentation process.

Table 3. Fumonisin analogs: A series, known and proposed structures



1 FA1 TCA TCA OH OH H OH 51, tw2, 3a iso-FA1a,b TCA TCA H H H OH tw4 FA2 TCA TCA OH H H OH 51, tw5 FA3 TCA TCA H OH H OH 49, tw

a There are two other OH groups on the backbone; tw¼ this work.


FC analogs and FDData similar to those reported for the FB and FA series

are presented in Fig. 3, Tables 5 and 6 and Schemes 3(a)–

3(d).

Again, significantly fewer compounds could be detected

than the number of FB analogs, but somewhat more than the

number of FA analogs. A noteworthy feature is the formation

of the fumonisin analogs designated PHFC4 and FD. Known

acetylated FC analogs16 were not detected.


A simplified CID fragmentation pattern via TCAK is

outlined for FC1, because several steps not indicated in

Scheme 3(b) are already known from the cases of FB1 and

FA1. Unlike in the previously described cases, the last

fragmentation step may also be the formation of methy-

leneimine (MI), with the concomitant splitting-off of NH3.

For the compound designated PHFC4 (Fig. 3), fumonisin

analogs of this type have also been identified in the FB series

(see Table 1).


DOI: 10.1002/rcm

Table


Aseries(forabbreviations,seeScheme5)

Fumoni-

sinor

fumoni-

sin-like

com-

pounds

Ret:timeðminÞ

Rqð%ofFB1Þ

MþHeþ

MþH-H2Oeþ

MþH-2H2Oeþ

MþH-3H2Oeþ

MþH-4H2Oeþ

MþH-AcOHeþ

MþH-AcOH-H2Oeþ

MþH-AcOH-2H2Oeþ

MþH-AcOH-3H2Oeþ

MþH-TCAKeþ

MþH-TCAeþ

MþH-TCA-H2Oeþ

MþH-TCA-2H2Oeþ

MþH-TCAK-AcOHeþ

MþH-TCA-AcOHeþ

MþH-TCA-AcOH-H2Oeþ

MþH-TCA-AcOH-2H2Oeþ

MþH-2TCAeþ

MþH-2TCA-H2Oeþ

MþH-2TCA-AcOHeþ

MþH-2TCA-AcOH-H2Oeþ

MþH-2TCA-AcOH-2H2Oeþ

MþH-2TCA-AcOH-2H2O-NH3eþ

ðbackboneÞ

m/z

values

andrelativeionab

undan

ce(%

)

1FA

112.4

0.7

764

746(27)

728(5)

710(2)

692(0)

704(100)686(44)

668(16)

650(3)606(4)

588(8)

570(10)

552(7)

546(11)

528(48)

510(24)

492(6)412(1)

394(0)

352(34)

334(24)

316(4)299(5)

2iso-FA

1a

21.7

3.3

764

746(100)728(82)

710(34)

692(34)

704(0)

686(93)

668(72)

650(5)606(36)

588(0)

570(54)

552(10)

546(0)

528(23)

510(3)

492(0)412(5)

394(9)

352(0)

334(18)

316(0)299(4)

3iso-FA

1b

22.2

0.3

764

746(55)

728(60)

710(0)

692(0)

704(0)

686(30)

668(0)

650(0)606(0)

588(100)57

0(53)

552(51)

546(0)

528(0)

510(0)

492(0)412(0)

394(38)

352(0)

334(0)

316(0)299(0)

4FA

227.9

8.0

748

730(100)712(0)

694(0)

—688(0)

670(58)

652(0)

—590(10)

572(0)

554(17)

—530(0)

512(11)

494(0)

—396(0)

378(5)

336(12)

318(29)

—301(0)

5FA

319.6

1748

730(18)

712(2)

694(4)

—688(100)670(48)

652(2)

—590(4)

572(2)

554(13)

—530(3)

512(84)

494(28)

—39

6(0.5)378(0.2)336(13)

318(21)

—301(0)

Rq:relativequan

tity

(%ofFB1,FB1¼10

0%).


DOI: 10.1002/rcm


Figure 2. Product ion spectra (CID) of FA1þHeþ (1) (Tables 3and 4) (for conditions, see Experimental section).

Figure 3. Product ion spectra (CID) of FC1þHeþ (1),

PHFC4þHeþ (8) and FDþHeþ (9) (Tables 5 and 6) (for con-

ditions, see Experimental section).


Schemes 3(c) and 3(d) indicate a previously unknown

fumonisin analog or precursor (designated FD). The known

fumonisins contain a C19 or C20 backbone. In the case of FD,

the ion at m/z 678 is either from a compound with a C18

backbone (Scheme 3(c)) or from a ketone with a C17

backbone (Scheme 3(d)). Asmay be seen in both schemes, it is

impossible to differentiate the two compounds by MS2.

Planned MSn (n> 2) measurements will, it is hoped, provide

support for one or other of the possible structures.

FBX analogsTable 7 presents detected compounds in which the OH

groups attached to the fumonisin backbone are esterified by

other carboxylic acids, such as cis-aconitic acid (AA),

oxalylsuccinic acid (OSA) and oxalylfumaric acid (OFA),

rather than TCA. In our opinion, the presence of other

carboxylic acids (e.g. citric acid, isocitric acid, perhaps

ascorbic acid) attached to the fumonisin backbone may also

be expected.

For the compounds in Tables 7 and 8, the MS2 spectra of

MþHeþ ions of FBX analogs (1–12) are shown in Fig. 4.

Scheme 4(a) gives the proposed fragmentation pattern of

compound FB4 2AA. As only an OH group in compound 3 is

esterified by OSA and there is no OH group at position c, d, e

Table 5. Fumonisin analogs: C series and FD, known and proposed structures



1 FC1 TCA TCA OH OH H OH 52, tw2 iso-FC1 TCA TCA OH H OH OH 53, tw3 FC2 TCA TCA H OH H OH tw4 FC3 TCA TCA OH H H OH 54, tw5, 6a iso-FC2,3 TCA TCA H H H OH tw7 FC4 TCA TCA H H H OH 28, tw8 PHFC4 TCA ��!OH H H H OH tw9b FD TCA TCA H H H H tw

aThere is another OH group on the backbone; tw¼ this work.b C17 ketone or C18 compound (there are two OH groups on the backbone).


DOI: 10.1002/rcm

Table


CseriesandFD

(forabbreviations,seeScheme5)

Fumonisin

orfumonisin-

like

compounds

Ret:timeðminÞ

Rqð%ofFB1Þ

MþHeþ

MþH-H2Oeþ

MþH-2H2Oeþ

MþH-3H2Oeþ

MþH-TCAKeþ

MþH-TCAeþ

MþH-TCA-H2Oeþ

MþH-TCA-2H2Oeþ

MþH-TCA-3H2Oeþ

MþH-TCA-TCAKeþ

MþH-2TCAeþ

MþH-2TCA-H2Oeþ

MþH-2TCA-2H2Oeþ

MþH-2TCA-3H2Oeþ


ðbackboneÞ

MþH-XTCA-YH2O-MIeþ

m/z

values

andrelativeionab

undan

ce(%

)

1FC1

7.6

2.3

708

690(100

)67

2(41)

654(16)

550(6)

532(8)

514(62)

496(37)

478(9)

374(1)

356(5)

338(34)

320(25)

302(3)

285(1.5)

273(2)

2iso-FC1

8.9

0.2

708

690(54)

672(21)

654(0)

550(0)

532(69)

514(46)

496(5)

478(9)

374(3)

356(100

)33

8(15)

320(25)

302(0)

285(0)

273(4)

3FC2

15.0

4.2

692

674(54)

656(17)

638(5)

534(7)

516(37)

498(83)

480(12)

462(0)

358(9)

340(45)

322(100

)30

4(26)

—28

7(5)

275(0.4)

4FC3

11.8

2.1

692

674(100

)65

6(39)

638(13)

534(11)

516(45)

498(37)

480(36)

462(28)

358(9)

340(34)

322(57)

304(20)

—28

7(5)

275(5)

5iso-FC2,3

13.9

0.9

692

674(47)

656(31)

638(3)

534(7)

516(68)

498(92)

480(10)

462(0)

358(0)

340(21)

322(100

)30

4(30)

—28

7(8)

275(4)

6iso-FC2,3

16.1

0.9

692

674(9)

656(11)

638(0)

534(30)

516(100

)49

8(14)

480(3)

462(0)

358(60)

340(55)

322(17)

304(33)

—28

7(3)

275(3)

7FC4

19.5

5.6

676

658(37)

640(6)

622(0.3)

518(22)

500(75)

482(13)

464(10)

—34

2(10)

324(100

)30

6(46)

——

289(8)

277(0.7)

8PHFC4

20.5

0.1

518

500(100

)48

2(27)

464(0)

360(0.2)

342(32)

324(62)

306(10)

——

——

——

289(0.5)

277(0)

9FD

10.1

0.2

678

660(64)

642(39)

624(5)

520(7)

502(19)

484(100

)46

6(15)

—34

4(1)

326(9)

308(48)

290(23)

—27

3(3)

—

Rq:relativequan

tity

(%ofFB1,FB1¼10

0%).

X:1(7)or2(1

–6,

8),Y:1(6),2(3

–5,

7,8)

or3(1,2).


DOI: 10.1002/rcm


2

Table 7. Fumonisin analogs: FBX series, proposed structures



1 PHFB4 AA OH ��!AA H H H OH tw2 iso-PHFB4 AA OH ��!AA H H H OH tw3 PHFB0 OSA OH ��!OSA H H H H tw4 PHFB4 OFA OH ��!OFA H H H OH tw5a PHFB2,3 OSA OH ��!OSA H H H OH tw6 FB4 2AA AA AA H H H OH tw7a iso-FB4 2AA AA AA H H H H tw8 FB4 AA, TCA AA ��!TCA H H H OH tw9a iso-FB4 AA, TCA AA ��!TCA H H H H tw

10a FB2,3 2AA AA AA H H H OH tw11, 12a FB2,3 AA, TCA AA ��!TCA H H H OH tw

aThere is another OH group on the backbone; tw ¼ this work.

Figure 4. Product ion spectra (CID) of PHFB4 AAþHeþ (1), iso-PHFB4 AAþHeþ (2), PHFB0 OSAþHeþ (3), PHFB4 OFAþHeþ(4), PHFB2,3 OSAþHeþ (5), FB4 2AAþHeþ (6), iso-FB4 2AAþHeþ (7), FB4 AA, TCAþHeþ (8), iso-FB4 AA, TCAþHeþ (9), FB2,3

2AAþHeþ (10), FB2,3 AA, TCAþHeþ (11) and FB2,3 AA, TCAþHeþ (12) (Tables 7 and 8) (for conditions, see Experimental

section).


or f, in contrast with fumonisin B1–5, we suggest the

designation PHFB0 OSA for this compound (Table 7). Since

fumonisin-like compounds of type FBX contain other

esterifying acids in addition to TCA, these are designated

ACID1 and ACID2 (corresponding ketene: ACK1 and ACK2)

in Table 8. Elimination of ACID1 and ACID2 was usually

observed to occur simultaneously rather than sequentially.

Unlike the fumonisin analogs of types FA, FB and FC, the

fragmentation of compound FB4 2AA may be characterized

by the fragmentation pattern shown in the upper part of

Scheme 4(b), but another pattern associated with CO2


elimination may also be drawn up, based on the product

ions with identified m/z values (detailed lower part of

Scheme 4(b)).

CONCLUSIONS

The experimental data reported in this manuscript repeat-

edly reveal that RP-HPLC/ESI-IT-MS2 is suitable for the

identification of unknown compounds present at low

concentrations and for suggesting their structures. The

detection of new fumonisin-type mycotoxins and their


DOI: 10.1002/rcm

Table


FBX

series(forabbreviations,seeScheme5)

Fumonisin

or

fumonisin-like

compounds

Ret:timeðminÞ

Rqð%ofFB1Þ

MþHeþ

MþH-H2Oeþ

MþH-2H2Oeþ

MþH-3H2Oeþ

MþH-H2O-CO2eþ

MþH-ACK1eþ

MþH-ACID1eþ

MþH-ACID1-H2Oeþ

MþH-ACID1-2H2Oeþ

MþH-ACID1-3H2Oeþ

MþH-ACID1-ACK

2eþ

MþH-ACID1-ACID

2eþ

MþH-ACID1-ACID

2-H2Oeþ

MþH-ACID1-ACID

2-2H2Oeþ

MþH-ACID1-ACID

2-2H2O-NH3eþ

ðbackboneÞ

m/z

values

andrelativeionab

undan

ce(%

)

1PHFB4AA

19.0

0.3

530

512(31)

494(0)

476(0)

468(5)

374(1)

356(100

)33

8(44)

320(22)

——

——

—30

3(6)

2iso-PHFB4AA

25.9

0.2

530

512(18)

494(36)

476(0)

468(0)

374(0)

356(100

)33

8(61)

320(6)

——

——

—30

3(1)

3PHFB0OSA

27.5

4.1

530

512(71)

494(3)

—46

8(0)

358(2)

340(100

)32

2(64)

——

——

——

305(4)

4PHFB4OFA

23.4

0.1

544

526(100

)50

8(36)

490(0.5)

482(0)

—35

6(100

)33

8(30)

320(7)

——

——

—30

3(0.3)

5PHFB2,3OSA

17.4

0.3

562

544(100

)52

6(19)

508(5)

500(0)

390(0.2)

372(24)

354(45)

336(9)

318(5)

——

——

301(0.1)

6FB42A

Aa

24.6

0.1

686

668(18)

650(2)

632(1)

624(1)

530(1)

512(18)

494(23)

476(8)

—35

6(2)

338(94)

320(41)

—30

3(24)

7iso-FB42A

A28

.51.2

686

668(43)

650(18)

632(3)

624(0)

530(13)

512(10)

494(0)

476(60)

—35

6(4)

338(74)

320(100

)—

303(7)

8FB4AA,TCA

c23

.00.1

688

670(100

)65

2(15)

634(0)

626(2)

532b

(30)

514(61)

496(45)

478(16)

—35

6(7)

338(61)

320(65)

—30

3(11)

9iso-FB4AA,TCA

27.4

0.8

688

670(64)

652(20)

634(0)

626(0)

532b

(3)

514(74)

496(46)

478(11)

—35

6(2)

338(66)

320(100

)—

303(8)

10

FB2,32A

Ad

24.8

0.2

702

684(100

)66

6(42)

648(10)

640(1)

546(7)

528(7)

510(15)

492(25)

474(16)

372(3)

354(43)

336(76)

318(32)

301(12)

11

FB2,3AA,TCA

e22

.80.2

704

686(100

)66

8(69)

650(9)

642(0)

548(14)

530(21)

512(61)

494(16)

476(0)

372(0)

354(29)

336(64)

318(6)

301(0.6)

12

FB2,3AA,TCA

e25

.10.2

704

686(62)

668(42)

650(14)

642(0)

548(7)

530(70)

512(100

)49

4(18)

476(22)

372(5)

354(29)

336(61)

318(77)

301(2)

Rq:relativequan

tity

(%ofFB1,FB1¼10

0%).

ACID

1:AA

(1,

2,6

–1

2),OSA

(3,

5),OFA

(4);ACID

2:TCA

(8,

9,1

1,1

2),AA

(6,

7,1

0).

ACK1:A

AK

(1,

2,6

–1

2),OSAK

(3,

5);ACK2:AAK

(6,

7,1

0),TCAK:(8

,9

,1

1,

12).

aþ

m/z

468(100

),45

0,43

2,42

4,39

0(see

6in

Fig.4b

,Sch

eme4a

andSch

eme4b

).bþ

m/z

530.

cþ

m/z

468,

450.

dþ

m/z

512,

448,

430.

eþ

m/z

546,

510,

370,

338,

334,

320.


DOI: 10.1002/rcm



precursors may yield further data for a more detailed

knowledge of their biosynthesis. The experimental data

described here draw attention to the need to study the

biological effects of toxins present at low concentrations.

After the detection of numerous novel fumonisin analogs, it

appears important to conduct a more detailed mass

spectrometric study of their structures (utilizing MSn), to

isolate analogs present at relatively high concentrations

and to determine their structures by appropriate methods

(e.g. NMR, XRD and ORD).

AcknowledgementsThe authors would like to thank Prof. W. C. A. Gelderblom

(PROMEK Medical Research Council, Tygerberg, South

Africa) and Prof. R. D. Plattner (National Center for Agri-

cultural Utilization Research, USDA, Peoria, IL, USA) for the

kind gifts of the FB3 and FB4 standards. This research was

supported by a Hungarian State Research grant (OTKA

46739) and a GAK grant (ALAP1-00073/2004).

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DOI: 10.1002/rcm

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Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high-performance liquid chromatography/electrospray ionization ion trap mass spectrometry