562 Anal. Chem. 1986, 58, 562-565

Fast Atom Bombardment Mass Spectrometry of 12 Marine Toxins Isolated from Profogonyaulax

Kevin D. White* and James A. Sphon

Food and Drug Administration, Division of Chemical Technology, Washington, D.C. 20204

Sherwood Hall Food and Drug Administration, Division of Chemistry a n d Physics, Washington, D. C. 20204

Twelve of the neurotoxins, including saxitoxin, that comprise paralytic shellfish poison have been characterized using positive ion fast atom bombardment (FAB) mass spectrome- try. The positive ion FAB spectra of these toxins afforded [M + HI+ ions and adduct Ions. Ions corresponding to the loss of SO, and water were observed. A series of fragment ions, probably produced by analogous processes, were also ob- served in the spectra of all toxins analyzed.

Paralytic shellfish poisons (PSP) consist of a group of neurotoxins produced by marine microorganisms and deriv- atives resulting from metabolism by accumulating organisms (1-3). The principal source of these compounds in shellfish are dinoflagellates of the genus Protogonyaulax (4, 51, the toxins of which (Table I) include saxitoxin (STX) and 11 derivatives of STX formed by the addition of N-1-hydroxyl, 11-hydroxysulfate, and 21-sulfo groups. Interest in the toxins centers around their biosynthesis and entry into the food chain. When toxic dinoflagellates are consumed by shellfish, such as mussels and clams, the toxins are bioaccumulated with little apparent effect on the shellfish (5-7). However, these toxins have a severe neurologic effect on humans when they consume the shellfish. Death can occur in severe cases due to paralysis of the respiratory system (8, 9).

The purpose of this study was to find a rapid means of confirming the presence of PSP as well as to provide mass spectral information not previously available. Past efforts to obtain mass spectra of the saxitoxins were fruitless due to the highly polar, thermolabile nature of the compounds (10). This difficulty has now been reduced with the advent of fast atom bombardment (FAB) ionization (12,12) . While this manu- script was in preparation, two reports have appeared from other laboratories describing FAB mass spectrometry (MS) of compounds STX, GTX2, C1, and GTXl (13,14).

EXPERIMENTAL SECTION Sample Preparation. The toxins used in this study were

isolated from cultured dinoflagellates of the genus Protogonyaulax ( I , 15) and purified either by extensive chromatography (com- pounds STX, B1, NEO, and B2) or by chromatography followed by crystallization (compounds C1, C2, C3, and C4). Purity was variously assessed by nuclear magnetic resonance spectrometry, combustion analysis, and bioassay. With the exception of GTX1, GTX2, GTX3, and GTX4, purities were >95%. In the cases of these four compounds, in which epimerization at carbon 11 occurs readily, gross purity of the epimeric pairs was >95% but epimeric purity a t the time of analysis may have been slightly less, par- ticularly for the l l p epimers, GTXS and GTX4. Compounds GTX1, GTX2, GTX3, and GTX4 were most conveniently ob- tained by hydrolysis of their sulfamate counterparts, C1, C 2 , C3, and C4.

Mass Spectrometry. An Ion Tech (San Jose, CA) saddle field source attached to a Varian-MAT CH5 double focusing mass spectrometer operated in the positive ion mode was used. The Glassman (Whitehouse Station, NJ) voltage-regulated high-voltage supply was operated a t I kV. Xenon gas was introduced until

0003-2700/86/0358-0562$01.50/0

Table I. Constituents of Paralytic Shellfish Poisons

compound compound abbreviation name

STX saxitoxin B1 GTX2 gonyautoxin 2 c1 GTXS gonyautoxin 3 c2

N E 0 neosaxitoxin B2 GTXl gonyautoxin 1 e 3

GTX4 gonyautoxin 4 c 4

semisystematic name

saxitoxin 21-sulfosaxitoxin lla-hydroxysaxitoxin sulfate 21-sulfo- 1 la-hydroxysaxitoxin

llp-hydroxysaxitoxin sulfate 21-sulfo-ll~-hydroxysaxitoxin

N - 1-hydroxysaxitoxin 21-sulfoneosaxitoxin lla-hydroxyneosaxitoxin sulfate 21-sulfo-1 la-hydroxyneosaxitoxin

110-hydroxyneosaxitoxin sulfate 21-sulfo-1 I@-hydroxyneosaxitoxin

sulfate

sulfate

sulfate

sulfate

a source pressure reading of 1 X lom5 torr was obtained. Current emission under these conditions was approximately 1 mA. Xenon gas was research grade 99.995% minimum purity obtained from Matheson (Dorsey, MD). Source lenses were initially tuned on the sodium iodide cluster ion at m/z 323 corresponding to Na31,+. An equimolar mixture of sodium fluoride and sodium iodide in water was used for calibrating the magnet. Glycerol (Baker Analyzed 99.5% minimum purity) was then introduced and the source lenses were retuned on m / z 185 corresponding to [G, + HI+, where G is glycerol. Calibration was verified after tuning. Glycerol blanks were run before each sample. Approximately 0.1 fig of sample was mixed with about 2 pL of glycerol. Acquisition was started immediately upon introduction of the sample. In- strument scan was computer-controlled from 15 to 800 daltons. Data were acquired on a Finnigan Incos 2300 data system with revision 3.1 software.

Data Processing. The spectra obtained by FAB MS vary depending on sample characteristics. These variations have been discussed at a recent symposium on fast atom and ion induced MS of nonvolatile organic solids (16). With certain compounds, a constant yield of molecular ions is observed throughout the lifetime of the glycerol, after which ion production ceases. With other compounds, molecular ions do not begin to appear until the glycerol has partially evaporated. These molecular ions then continue to increase in abundance until they reach a maximum before complete evaporation of both the glycerol and the com- pound. Still other compounds desorb as follows: Molecular ion yields are most intense in the first few scans and then trail off until only glycerol ions are observed. The shellfish toxins behave in the latter manner.

The formation of fragment ions is infrequently discussed in FAB literature; however, such ions are probably as varied in production as the [M + HI+ ions. For example, [M + H - H201' and [M + H - H20 + G]+ ions of PSP increase proportionately with the decrease of [M + HI+ ions. On the other hand, [M + H - SO3]+ ions decrease along with [M + HI+ ions. Therefore, to obtain a representative spectrum, acquisition was started im- mediately upon insertion of the target. Then, ten spectra were typically summed. Next, the glycerol ions were subtracted, using spectra from the glycerol blanks run immediately preceding each

0 1986 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986 563

R l R 2 R 3 R4 STX H H H H E1 H H H SO, GTX2 H H OSO, H c 1 H H OSO, SO, GTXJ H OSO, H H c 2 H OSO, H SO, N E 0 OH H H H 82 OH H H SO, G T X l OH H OSO, H

G T X 4 OH OSO, H H

c 4 OH OSO, H SO,

c 3 on n oso, so,

Figure 1. Basic structure, compound abbreviation (see Table I), and R substitutlon.

L J

Figure 2. Depiction of molecular ion of C4, 2l-sulfo-l l@-hydroxy- neosaxitoxin sulfate.

Table 11. Net Charge of N-1-Hydroxylated Toxins at Various pH Values

approximate net charge compound below pH 6 pH 7-8

N E 0 +2 +1 B2 +1 0 GTXl +1 0 c3 0 -1 GTX4 +1 0 c4 0 -1

sample. To ensure proper subtraction, both spectra were nor- malized to the m/z 93 ion, i.e., base peak of glycerol spectra.

RESULTS AND DISCUSSION PSP toxins exist in nature as ions, with their net charge

dependent on the number of ionic groups present, e.g., SO3-, OSOB-, NH2+, 0- (see Figure 1). pH is also a factor on the net charge of toxins containing N-1-hydroxyl since this group loses its proton in the p H 6-7 range. For example, S T X is a doubly charged cation with the charges retained on the two guanido groups. Addition of one negatively charged 21-sulfo or 11-hydroxysulfate group to the STX structure, as with B1, GTX2, and GTX3, results in a net charge of +1. Addition of both an SO3- and OSOB-, as with C1 and C2, results in a net charge of zero. The net charge of toxins containing the N-1-hydroxyl will vary depending on the pH. Table I1 sum- marizes these charges. Although there are differences in the charges on these compounds, all were observed by FAB as singly charged ions, which is a common observation with FAB (12,17). For simplicity, these ions are labeled [M + H]+ and their structures are illustrated as C4 is in Figure 2.

The positive ion FAB spectra of these toxins (Table 111) all afforded [M + H]+ ions and ions corresponding to the loss of H 2 0 from [M + H]+ or from a fragment ion of [M + HI+. Compounds containing either sulfamate or 0-sulfate groups show a loss of 80 daltons, corresponding to SO,. Compounds containing both groups show the successive loss of both.

Ion abundances relative to the glycerol base peak, m/z 93, varied from 1 to 50%. Most of the glycerol ions could be removed from the spectra by background subtraction as ex- plained previously; however, [G, + Na]+ ions, where n = 1-4,

2 E N \

E

564 ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986

Table IV. Fragment Ions of Saxitoxin That Result from Cleavage of the Upper Side Chain

cleavage calcd obsd site mlz m lz

proposed species

spectrum type (where observed)

a 256 255 (M + H - NH&O)+ normal only b 240 240 (M + H - NHZCO,)+ MIKES only

226 226 (M + H - NH&OZCH2)+ normal and MIKES 225 (M + H - NH,COZCHz)+ normal and MIKES

a’ 238 238 (M + H - H20 - NH,CO)+ normal and MIKES b’ 222 223 (M + H - HzO - NHC02)+ normal and MIKES C’ 208 208 (M + H - HZO - NHZCOzCHz)+ MIKES only

207 (M + H - HzO - NH3CO,CHZ)+ normal only d’ 266 265 (M + H - HzO - NHJ+ MIKES only

C

1

I

I3ag. 430 j I 50 2CIE 250 300 350 400 450

M/Z

Flgure 3. Positive ion FAB spectrum of saxitoxin. Asterisks indicate ion series common to all toxins analyzed.

a b - H /N\A I

0 H+

H LB H L A

- H+

Figure 4. Fragmentation of upper side chain shown by M I K E S of (A) mlz 300 and (B) mlz 282.

3 / 6 100 0 1

could not. These results are similar to those obtained on other compounds by FAB MS in that usually [M + HI+ ions are observed (11, 18), and the spectra are plagued with glycerol peaks (19).

Adduct ions formed between the sample and the matrix are also observed with PSP toxins. Glycerol adducts are com- monly observed by FAB but are usually observed 92 daltons higher than the protonated molecular ion (20). Glycerol ad- ducts with PSP are observed 92 daltons above fragments as well, particularly fragments that have lost HzO, e.g., [M + H

These adduct ions are quite substantial, in some instances reaching base peak abundances. It is not clear from the data whether H 2 0 is lost before or after adduct formation.

Figure 3 shows the positive FAB spectrum of STX, in which the protonated molecular ion [M + H]+ a t mlz 300, the loss of the H 2 0 fragment a t mlz 282, and the glycerol adduct formed with that’fragment a t mlz 374 are observed. Elim- ination of H20 from the gem diol a t carbon 12 to form the corresponding ketone accounts for the loss of H 2 0 observed with STX. This observed fragmentation was substantiated by the use of lSO. Incorporation of lSO a t both hydroxyls resulted in a shift of the [M + HI’ to mlz 304 and m / z 282 to mlz 284, demonstrating that one of the lSO atoms was lost from the carbon 12 position. This elimination is also con- sistent with the known solution chemistry of STX (10, 21,22).

Finally, the FAB spectrum of STX reveals a series of characteristic fragment ions in the 200-270 region. These characteristic fragment ions result from the fragmentation of the upper side chain (Figure 4). Mass analyzed ion kinetic energy scans (MIKES) on the molecular ion [M + H]+ of STX, for example, showed peaks corresponding to cleavages at sites a, b, and c (Figure 4A). Likewise, MIKES on the fragment ion [M + H - HzO]+ on STX again showed peaks corre- sponding to cleavages a t sites a’, b’, and c’ and , in addition, cleavage a t d’ (Figure 4B). The mlz 240, corresponding to

- HzO]+, [M + H - SOB - H,O]+, and [M + H - 2S03 - H,O]+.

S E hjd li cM+HJ+ t 4w ’ 284

328 353 378

200 250 300 350 400 450 500 M/Z

Figure 5. Positive ion FAB spectrum of C1, 21-sulfo-1 la-hydroxy- saxitoxin sulfate. Asterisks indicate ion series common to all toxins analyzed.

cleavage at site b, although not present in the normal spectrum of STX, was very abundant in the MIKE spectrum of mlz 300. Similarly, mlz 208, corresponding to cleavage a t site d’, was abundant in the MIKE spectrum but weak in the normal spectrum of STX. Table IV lists the ions and proposed losses for the ion series of STX in the 200-270 region.

Similar patterns were observed in the normal spectra of each toxin analyzed. Table I11 lists these ions under other sig- nificant ions. The fragmentation patterns for C1, C2, C3, and C4 differ from those observed for the other eight toxins in that there is no significant loss of H 2 0 from the [M + HI+ ion. In all other respects, the normal spectrum of C1, C2, C3, and C4 typifies PSP with two consecutive losses of the elements of SO3, the loss of H20 from these two fragments, and the series of fragment ions in the 200-270 region. This is illustrated for the toxin C1 in Figure 5.

Registry No. B1,64296-25-9; B2, 82810-44-4; C1,80173-30-4; C2,80226-62-6; C3,89614-45-9; C4, 89674-98-6; GTX1,60748-39-2; GTX2, 60508-89-6; GTX3,60537-65-7; GTX4,64296-26-0; NEO, 64296-20-4; STX, 35523-89-8.

LITERATURE CITED ( 1 ) Hall, S. Ph.D. Dissertation, University of Alaska, Fairbanks, 1982. (2) Shimizu, Y.; Yoshioka, M. Science 1981, 212, 547-549.

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(4) Sommer, H.; Whedon, W. F.; Kofoid, C. A,; Stohler, R. Arch. Pathol.

(5) Taylor, D. L.; Selinger, H. H. "Toxic Dinoflagellate Blooms. Develop- ments In Marine Biology 1"; Elsevier/North Holland: New York, 1979; p 505.

(6) Prakash, A.; Medcof, J. C.; Tennant, A. D. Bull.-Fish. Res. Board Can. 1971, No. 177, 87 pp.

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(12) Williams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.; Taylor, L. C. E.

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(18) Ligon, W. V., Jr. Int. J. Mass Specfrom. Ion Phys. 1983, 52,

(19) Rinehart, K. L., Jr. Science 1982, 218, 254-260. (20) Puzo, G.; Prome, J. C. Org. Mass Spectrom. 1984, f9 , 448-451. (21) Shimlzu. Y.; Hsu, C. P.; Genanah, A. J. Am. Chem. SOC. 1981, 103,

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RECEIVED for review June 4,1985. Accepted October 10,1985.

Determination of Tobacco-Specific N-Nitrosamines by Capillary Gas Chromatography/Selected Ion Monitoring Mass Spectrometry

R. F. Arrendale,* W. J. Chamberlain, 0. T. Chortyk, and J. L. Baker

Tobacco Safety Research Unit, Agricultural Research Service, United States Department of Agriculture, P.O. Box 5677, Athens, Georgia 30613

M. G. Stephenson

Crops Research, Agricultural Research Service, United States Department of Agriculture, Coastal Plains Experiment Station, Tifton, Georgia 31 793

Methodology was developed to determine the concentrations of the tobacco-speclflc N-nltrosamlnes, N-nitrosonornlcotlne (NNN) and 4-(Nmethyl-N -n~rosamlno)-I-(3-pyrldyl)-l-buta- none (NNK), In tobacco and tobacco smoke, based upon gas chromatography/mas spectrometry (GC/MS) analyses In the selected Ion monltorlng (SIM) mode. The N-nltrosamlnes were separated by caplllary GC on a fused silica Superox-4 caplllary column. Both splitless and cold on-column Injection modes were evaluated, with the latter produclng the better resutts. Our recent advances In analytical methodology, such as a laboratory-constructed cold on-column capillary GC inlet and an open-spllt interface for caplllary GWMS application, were Instrumental In the successful analyses of these tobac- co-speciflc N-nltrosamlnes. The development of this GC/ SIM-MS method and its appllcatlon to analyses of N-nltros- amines from tobacco and tobacco smoke are presented and discussed.

The determination of concentrations of the two major to- bacco-specific N-nitrosamines, N-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-l-(3-pyridyl)-l-butanone (NNK), is very important, as both compounds are carcinogens and are found in tobacco and tobacco smoke (1-4). A variety of methods for the determination of these important com- pounds have been developed (5-14). Separation of N- nitrosamines by GC or high-pressure liquid chromatography (HPLC) and detection with a thermal energy analyzer (TEA) have long been the methods of choice (11,13,15). However, many laboratories do not possess a TEA, mainly due to its expense and limited application to other areas of research. But, most laboratories have access to a GC/MS system. Our

laboratory falls into this latter category. Consequently, we have developed a nitrosamine analysis method, using our GC/MS system in the selected ion monitoring (SIM) mode. In this technique, the mass spectrometer is adjusted to selected masses for defined time periods, during which time the signal response is recorded (26-19). This technique has two major advantages; (1) it provides detection limits that are 2 or 3 orders of magnitude lower than are possible in the normal scannling mode, and (2) by choosing ions that are charac- teristic of the compound of interest, the mass spectrometer in the SIM mode functions as a selective detector. Integration of the recorded signal response can then be used for the de- termination of component concentrations.

We have also utilized the many advantages of capillary GC in the development of this method, especially the use of cold on-column injection. This technique has several advantages over the now conventional split/ splitless techniques (20-28). Some of these advantages are (1) elimination of discrimination between low and high boiling components; (2) reproducibility and linear response; (3) excellent determination of concen- tration of individual sample components; (4) elimination of thermal or catalytic decomposition of sample components due to rapid heating; and (5) analysis of sample components a t ng/pL concentrations using a flame ionization detector. We also compared our recently developed cold on-column inlet and a commercial inlet, operating in the splitless mode, for the determination of concentrations of NNN (29).

An analytical capillary GC/MS technique must also include a capillary GC/MS interface, that provides reproducible, linear, and quantitative transfer of the components eluting from the capillary GC column to the MS source for detection. We have designed and constructed an open split interface (OSI), which satisfied these requirements (30). These im-

This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society