Anal. Chem. 1995, 67, 1815-1823
On-Line High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry for the Determination of Brevetoxins in “Red Tide” Algae Yousheng Hua,t Wenzhe Lu,~ Michael S. Henry,* Richard H. Pierce,* and Richard B. Cole*it
Department of Chemistry, Universify of New Orleans, Lakefront, New Orleans, Louisiana 70148, and Mote Marine Laboratory, 1600 Thompson Parkway, Sarasota, Norida 34236
On-line high-performance liquid chromatography-electrosprayionization mass spectrometry (HPLC-ESMS) has been successfully applied to the separation and identification of brevetoxins associated with “red tide“ algae. Brevetoxins are toxic polyethers produced by the marine dinoflagellate Gymnodinium breve. They are responsible for fish kills, and they pose certain health risks to humans, The LC-MS method employs reversed-phase microbore HPLC on a cl8 column with a mobile phase consisting of 8 5 1 5 methanol/water, a flow rate of 8 pW min, and a postcolumn split ratio of 3:l (w absorbance detector/mass spectrometer). A brevetoxin culture sample was found to contain at least six components, including two well-separated peaks correspondingto the brevetoxins Pb’Ik-2 and PbTk-1, as well as several unknown compounds, including one with a molecular mass of 899 Da (possibly an isomer of PbTx-9). The brevetoxin molecules exhibited a high tendency to bind to alkali cations in positive ion ESMS. For standard PbTx-9, Pb’Ik-2, and Pb’Ik-1 brevetoxins analyzed on our LC-MS system, the detection limits (employing mass spectrometer scans of 100 m / z units) were determined to be less than 600 fmol, 1 pmol, and 50 h o l , respectively (S/N = 3); the total analysis time was about 35 min. Brevetoxins are naturally occurring cyclic polyether toxins produced by the marine dinoflagellate, Gymnodinium breve,’V2 formerly named Ptychodiscus brevis? Blooms of this organism in seawater, referred to as “red tides”, are most prevalent along the Gulf coast of Florida, although they have been observed in other parts of the Gulf of Mexico, the Atlantic coast of Florida, and the coasts of North and South Carolina.*J The brevetoxins produced during these red tides cause massive fish kills and contamination of shellfish.6 In addition, the toxins become airborne in the form of aerosolized particles, causing severe respiratory problems and +
University of New Orleans, Lakefront.
* Mote Marine Laboratory.
(1) Davis, C. C. Bot. Gaz. (Chicugo) 1948, 109, 358-360. (2) Poli, M. A; Mende, T. J.; Baden, D. G. Mol. Phanacol. 1986, 30, 129135. (3) Steidinger, K. A In Toric Marine Phytoplankton; Graneli, E., Sundstrom, B., Edler, L., Anderson, D., Eds.; Elsevier Publishing Co.: New York, 1990; pp 11-16. (4) Steidinger, K A; Baden, D. G. In DinofEagellates; Spector, D. L., Ed.; Academic Press: New York, 1984; pp 201-261. (5) Tester, P. A; Stumpf, R P.; Vukovick, F. M.; Fowler, P. K; Turner, J. T. Limnol. Oceanogr. 1 9 9 1 , 3 6 , 1053-1061. 0003-2700/95/0367-1815$9.00/0 0 1995 American Chemical Society
eye irritation to humans and other mammals along the b e a ~ h . ~ - ~ Skin irritation has also been reported as a result of exposure to brevetoxin-containing seawater.l0 So far, nine brevetoxins have been identitled as being produced by the red tide dinoflagellate G. breve.3~9 The structures of four brevetoxin varieties3J1J2relevant to this study are shown in Figure 1. In addition to the brevetoxins, other cyclic polyether toxins are produced by different species of harmful marine algae. Some of the more prevalent include ciguatoxin, produced by the benthic dinoflagellate Gamberdiscus toxicus, and okadaic acid, produced by several planktonic dinoflagellates of the genus Din~$hysis.~~J~ Development of reliable techniques for detection of these toxins in the marine environment is of special concern for public health and for fisheries management. Current methods of brevetoxin analysis can be divided primarily into three types. One classification is based on response of a living organism to the toxic effects elicited by these compounds. This category includes the “mouse bioassay”15 and the “cell bioassay”.16 These methods are intended to serve as screening procedures to verify the integrity of seafood products destined for human consumption. The mouse bioassay uses intraperitoneal (stomach) injections of aqueous extracts to stimulate toxic reactions. However, the mice may be insensitive to small quantities of brevetoxins, and there is also a danger of false positives and failure to notice adverse responses of the test a n i m a l ~ . ~The ~ J cell ~ bioassay, relying upon the different activities of various toxins in perturbing normal membrane properties of mouse neuroblastoma cells,16 can distinguish brevetoxins from (6) Shimizu, Y. In Marine Natural Products; Scheuer, J. P., Ed.; Academic Press: New York, 1978 Vol. I, Chapter 1. (7) Steidinger, K. A Prog. Phycol. Res. 1983, 2, 147-188. (8) Pierce, R H. Toxicon 1986, 24, 955-965. (9) Pierce, R H.; Henry, M. S.; Proffitt, L. S.; Hasbrouck, P. A In Toric Marine Phytoplankton; Granbli, E., Sundstrom, B., Edler, L., Aiderson, D., Eds.; Elsevier Publishing Co.: New York, 1990; pp 397-402. (10) Kemppainen, B. W.; Mehta, M.; Stafford, R; Riley, R T. Toricon 1992,30, 931-935. (11) Lin, Y. Y.; Risk, M.; Ray, S. M.; Van Engen, D.; Clardy, J.; Golik, J.; James, J. C.; Nakanishi, IC]. Am. Chem. SOC. 1 9 8 1 , 103, 6773-6775. (12) Shimizu, Y.; Chou, H.; Bando, H.; Van Duyne, G.; Clardy, J. C. J. Am. Chem. SOC.1986, 108, 514-515. (13) Yasumoto, T. In Toric Dinoflagellates;Anderson, D. M., White, A W., Baden, D. G., Eds.; Wiley & Sons: New York, 1985, pp 259-270. (14) Juranovic, L. R; Park, D. L. Reo. Enuiron. Contam. Toricol. 1991,117,l-44. (15) Oficial Methods of Analysis, 14th ed., Association of Official Analytical Chemists: Arlington, VA, 1984; Section 18.086-18.092. (16) Manger, R L.; Leja, L. S.; Lee, S. Y.; Hungerford, J. M.; Wekell, M. M. Anal. Biochem. 1 9 9 3 , 214, 190-194.
Analytical Chemisity, Vol. 67, No. 11, June 1 , 1995 1815
PbTx-2 ( M I YO..
' 0 ~
Figure 1. Structures of selected brevetoxins.
saxitoxins while offering high sensitivity with detection limits of 2 ng for the brevetoxin PbTx-3. The cell bioassay method, however, cannot distinguish between individual brevetoxins (e.g., those appearing in Figure l),and the analytical procedures take 4-6 h to complete. Methods in the second category of approaches to brevetoxin analysis rely on the unique biochemical properties of the compounds; they can be further divided into two subgroups. The first subgroup employs biochemical immunoassay analysis, such as radioimmunoassay (RIA) ,I9 which counts the y-ray emission of radioactive products produced by specific antibody responses toward toxins of G. breve. Enzyme-linked immunosorbentassays ELI IS AS)^^^^^ measure light absorbance changes (visible region) resulting from the formation of complexes due to brevetoxin antibody reactions with peroxidase-linked coenzyme. These biochemical methods grant high sensitivity and high selectivity but usually can only detect one compound or one class of compounds in each determination employing a specially prepared antibody. The second subgroup relies upon pharmacologic activity of the toxins, focusing on spec& receptor binding assays for ciguatoxinslbrevetoxinsand domoic acid.22 (17) Halstead, B. W. Poisonous and Venomous Marine Animals of the World; Darwin Press: Princeton, NJ, 1988; Chapter 10. (18) Rakotoniaina. C. A; Miller D. M. In Ciguatera Seafood Toxins;Miller D. M., Ed.; CRC Press: Boca Raton, FL, 1991; Chapter 6. (19) Baden, D. G.; Mende, T. J.; Walling, J.; Schultz, D. R Toxicon 1984, 22, 783-789. (20) Trainer, V. L.; Baden, D. G. Toxicon 1991, 29, 1387-1394. (21) Baden, D. G.; Mende, T. J.; Szmant, A M.; Tranier, V. L.; Edwards, R A; Rozell, L. E. Toxicon 1988, 26, 97-103. (22) Van Dolah, F. M.; Finley, E. L.; Zevotek, N. L.; Doucette, G. J.; Moeller, P. D. E.: Ramsdell, J. S. In Proceedings ofthe 5th International Conference on Toxic Phytoplankton, Nice, France, in press.
1816 Analytical Chemistry, Vol. 67,No. 11, June 1, 1995
The third category of analytical methodology for brevetoxin determinations is chromatographic analysis, such as thin-layer chromatography (TLC),23 and high-performance liquid chromatography (HPLC) ?*sz5 usually with UV detection. Identification is based upon matching component mobility (retention) with that of a standard. These methods offer ng/pL sensitivity and can separate and detect a series of toxins in a single run. However, they are subject to interferences from coeluting species, and limited structural information is provided for unknown compounds. Although the above methods for brevetoxin analysis have considerable utility, there is still a need for the further develop ment of analytical methods which offer short analysis times, high sensitivities, and specifk detection of individual components contained in brevetoxin mixtures. In real samples, several toxic compounds often coexist, and it may be necessary to distinguish between species if the elicited toxic effects are significantly different for the individual toxins. For example, it was reported that PbTx-1 was the most powerful ichthytoxin among the brevetoxins, with a lethality of 3 ppb against z e b r a f i ~ h . 2PbTx-3 ~~~~ exhibited toxic effects at 1/10 the dosage (orally administered to mice) of PbTx-2.2* One of the promising methods capable of fulslliig the above requirements of detection specificity is on-line HPLC-mass spectrometry (MS). Recently, HPLC-pneumatically assisted electrospray MS was used in the analysis of certain marine toxins including domoic acid, saxitoxins, tetrodotoxin,29okadaic acid and, diophysistoxin-1,30with detection limits of 2 ng for okadaic acid. In this paper, we report on the use of electrospray ionization mass spectrometry (ESMS) for the analysis of brevetoxins, as well as on-line HPLC-ESMS for the analysis of red tide algae cultures containing brevet ox in^.^^ EXPERIMENTALSECTION HPLC-UV. Microbore HPLC separation was conducted using a reversed-phase CIS3-pm Spherisorb microbore column (Isco, Lincoln, NE), lO@mm x 1-mm4.d. The mobile phase was delivered by two precision syringe pumps (Isco, Model 100D) at room temperature. Samples were injected using a high-pressure, low-dispersion injection valve (Rheodyne 7520, Cotati, CA) that was fitted with a l.O-pL sample loop. UV detection was achieved using a CV4 capillary electrophoresis absorbance detector (Isco) operating at 215 nm. Eledrospray MS. A quadrupole electrospray ionization mass spectrometer (Vestec 201, PerSeptive Biosystems, Houston, TX) was employed in all mass spectrometry experiments. The mass spectrometer data system was a Vector Two system r e h i v e n t (23) Baden, D. G.; Mende, T. J.; Bikhazi, G.; Leuny, I. Toxicon 1982,20, 929932. (24) Alam, M.; Trieff, N. M.; Hudson, J. E. /. Pharm. Sci. 1975, 64, 865. (25) Pierce, R H.: Brown, R C.; Kucklick, J. R In Toxic Dinbflagellates;Anderson, D. M., White, A W., Baden, D. G., Eds.; Wiley and Sons: New York, 1985; p 309. (26) Shimizu, Y.; Gupta, S.; Norte, A; Genenah, A; Kobayashi, M. Proc. Int. Conf Toxic Dinoflagellates 1985, 3, 271. (27) Nakanishi, K. Toxicon 1985,23, 473-479. (28) Baden, D. G.; Mende, T. J. Toxicon 1982, 20, 457-461. (29) Quilliam, M. A; Thompson, B. A; Scott, G. J.; Siu, IC W. M. Rapid Commun. Mass Spectrom. 1989, 3, 145-150. (30) Pleasance, S.; Quilliam, M. A; de Freitas, A S. W.; Marr, J. C.: Cembella, A D. Rapid Commun. Mass Spectrom. 1990, 4, 206-213. (31) Presented in part: Hua, Y.; Lu,W.; Henry, M. S.; Pierce, R H.; Cole, R B. 13th International Mass Spectrometry Conference, Budapest, Hungary, August 30, 1994.
Corp., Maryland Heights, MO). In the direct infusion mode, sample solutions were delivered into the electrospray ion source by a 6km-long, lWpm-i.d. deactivated fused silica tube via a syringe pump (Sage Instruments, Cambridge, MA). Electrospray ionization source conditions were similar to those described below for on-line HPLC-ESMS. For all experiments, the quadrupole mass analyzer was calibrated to indicate average mass values at low resolution. On-Line HPLC-ESMS. The HPLC-ESMS interface for online analysis of brevetoxins was constructed in-house at the University of New Orleans. The HPLC was simultaneously interfaced to ESMS and W detectors via a tee valve (0.1pL dead volume, Valco, Houston, TX). One outlet of the tee valve was connected via a 2O-cm-long, 100-pm4.d. deactivated fused silica capillary to the UV detector. The other outlet was connected to the electrospray mass spectrometervia the same type of capillary, 32 cm in length. The LC effluent was controlled in an a p proximately 3:1 ratio W detector/ESMS detector. During HPLC-ESMS operation, HPLC effluent was introduced into the mass spectrometer ion source through a stainless steel capillary (lWpm-i.d., 2cm-long "needle") held at a high voltage (+2.6 i 0.1 kv). The distance between the needle and the counter electrode (flat plate) was held constant. The voltage at the flat plate and the skimmer-collimator voltage difference were held at 297 and 20 V, respectively. The temperature in the source block was 247 k 1 "C (measured by thermocouple), and the temperature in the vicinity of the needle (electrospraychamber) was controlled at 50 iz 1 "C. Gymnodinium breve Culture. An extract containing brevetoxins was obtained by solid phase extraction32from a laboratory G. breve culture maintained at Mote Marine Laboratory. The original inoculum was obtained in 1984 from the Florida Department of Natural Resources, St. Petersburg, FL, from the W. B. Wilson 1953 isolate. Cultures were maintained in a temperaturecontrolled room at 25 f 2 "C under fluorescent lights with a 12-h light/dark cycle under static conditions. Cultures were reinoculated into fresh media on a monthly basis to maintain log growth conditions. The G. breve cultures were maintained in &L volumes of NH-15 medium in 12-Lglass carboys. The NH-15 is a distilled water, artifkid seawater medium that maintains slow gr0wth.3~Toxins produced by the G. breve isolate in laboratory culture have been shown to be the same as those produced during natural blooms.32 After the extraction, the extract sample solution was kept refrigerated at about -5 "C for 7 months. Chemicals. Solutions of the brevetoxin standards PbTx-2, PbTX-3, and PbTx-9 were prepared for direct infusion by individually dissolving 50 pg of the commercially purchased chemical (Chiral Corp., Miami, FL) in 1mL of methanol. PbTX-1, PbTX-2, and PbTx-9 standard solutions for LC-MS were prepared individually by dissolving 100 pg in 0.2 mL of acetone. Sample concentrationswere adjusted by diluting the above solutions and standard brevetoxin mixtures with mobile phase, 8515 methanol/ water. Acetone, methanol, and water (HPLC grade) were purchased from EM Science (Gibbstown, NJ). RESULTS AND DISCUSSION
Development of Chromatographic Conditions for Analysis of Brevetoxin Standards. As a fist step toward developing an (32) Pierce, R H.; Henry, M. S.; Proffitt, L. S.; deRosset, A. J. Bull. Enuiron. Contam. Toxicol. 1992,49, 479-484. (33) Aldrich. D. V.; Wilson, W. B. Biol. Bull. 1960,119,57-64.
0.006
,
0.0055
-5
e
0.005 0.0045 0.004
e
0.0035
e
0.0025
0
5
10 15 20 25 Flow Rate (ul/min)
30
35
Figure 2. Van Deemter plot of plate height ( H ) vs mobile phase flow rate for PbTx-9 in a PbTx-9 solution containing some decomposed products. Conditions: 100-mm x 1-mm4.d. CIS column; methanol/water (8515 v/v) mobile phase; injection volume, 1 pL.
on-line HPLC-ESMS method for brevetoxin analysis, off-line microbore HPLC-W was used to find suitable separation conditions. Literature reports of chromatographicseparations of brevetoxins describe the use of normal-phase silica g e P and reverse-phase c18 ~ o l u m n s . 9 JThe ~ ~ ~silica ~ gel column was used mainly for isolation and purification purposes, while CIScolumns were often used for quantitative purposes. In the current study, a CIS column was employed to separate brevetoxins. The composition of the methanol/water mobile phase was optimized for standard brevetoxin analysis by testing three solutions with methanol/water ratios of 90:10, 85:15, and 75:25 at a flow rate of 20 pL/min. The capacity factors (k') measured for PbTx-9, PbTx2, and PbTx-l were 0.9, 1.0, and 1.4 for the 9010 mixture; 1.2, 1.4, and 2.2 for the 8515 ratio; and 6.2, 7.3, and 10.2 for the 75:25 mixture, respectively. The k' values for the 85:15 mixture were deemed most suitable for chromatographic analysis, as the k' values for the 9O:lO mixture were too low for adequate separation, while those of the 7525 mixture resulted in excessively long analysis times. To determine the optimum flow rate for the 85: 15 methanol/water mixture, a PbTX-9 solution containing some decomposed products was analyzed at different flow rates from 2 to 30 pL/min. Van Deemter plots were obtained for PbTx-9 (Figure 2) and two other components. The optimum flow rate was approximately 4 pL/min for all three peaks (i.e., minimum plate height, H = 0.0024 cm for PbTx-9; H = 0.0021 and 0.0014 cm for the two earlier-eluting contaminants). For LC-MS work, a higher flow rate of 8 pL/min was employed to shorten analysis time and to reduce band broadening in the additional dead volume. The experimentally determined optimum flow rate was rather low compared to typical specifications of other c18 microbore columns.34
Modification of Electrospray Ionization Source for LCMS. A modfication of the commercially available electrospray ion source35was performed wherein the nozzle normally used as the counter electrode for the electrospray needle was replaced by a flat stainless steel plate with an aperture of 0.4 mm. To maintain adequate pumping in the first stage of the ion source and to enable an appropriate needle-to-counter electrode distance for electrospray operation, the spacer which guides the movement of the counter electrode on the interior of the source was modified (34) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987,59, 26422646.
(35) Allen, M. A; Vestal, M. L. 1.Am. SOC.Mass Spectrom. 1992,3, 18-26.
Analytical Chemistry, Vol. 67, No. 11, June I , 7995
1817
in-house according to manufacturer specificationsto accommodate the stainless steel plate. Another modification was the replacement of the standard 200 Wmin mechanical pump used to evacuate the first stage of the ion source with a 500 Wmin pump. Installation of the flat plate and increasing of the pumping capacity in the first stage of the ion source were performed to allow greater sensitivity and greater electrospray stability at higher flow rates. With these modifications, the detection limit was lowered by a factor of 4 compared to the regular nozzle design under an optimized direct infusion rate of 2 pWmin for a 1.0 x 10-6 M standard PbTx-l solution. Alternative approaches to accommodating higher flow rates in ESMS include the use of pneumatic nebulization, counter flow curtain and ultrasound neb~lization.~~-~~ The electrospray probe was also modified by removing the 2km-long stainless steel connection tube and directly connecting the fused silica capillary to the ES needle (where high voltage was directly applied). This modAcation minimized the HPLCESMS connection tube length (32 cm), corresponding to a dead volume of 2.5 p L for a 1Wpmi.d. connecting capillary. Direct Infusion of Brevetoxin Standards. Three brevetoxins, PbTx-2, PbTx-3, and PbTx-9, were run in the direct infusion mode to demonstrate the utility of ESMS for brevetoxin analysis. For all three high-concentration standards, ES mass spectra (Figure 3) exhibit singly charged peaks corresponding to sodium adducts of the brevetoxin monomer. In addition, it is interesting to note observation of singly charged noncovalently bound dimers and doubly charged noncovalently bound trimers (two and three brevetoxin molecules solvating one and two sodium ions, respectively) for each standard. Ions of this form were not observed in ES mass spectra of other ionophoric substances such as okadaic acid and dinophysistoxin-1.30No attempt was made to remove sodium (ubiquitous in the environment) prior to acquiring the mass spectra shown in Figure 3. There are some additionalpeaks in the mass spectrum of PbTx-2 which originated from potassium ion contamination; assignment of ions is given in Table 1. Accompanying the high level of sodium attachment, it is also noted that the abundances of protonated brevetoxin molecules (appearing at 22 m/z units below the corresponding base peaks) are very low (less than 1/10 the abundance of the sodium adduct) for all three brevetoxins. The protonated PbTx-2 molecule was previously observed" by desorption/chemical ionization mass spectrometry (D/CI/MS). If no sodium was present, protonated PbTx-2 molecules would be expected to exhibit a much higher relative abundance. Figure 3 demonstrates that the polyether structure of the brevetoxins is quite adept at binding to Na+ ions. This behavior is likely related to the mode of action underlying the toxic effects associated with the compounds because brevetoxins are known to disrupt normal membrane properties of excitable cells by activating sodium channels at inappropriate times.2m4 Strong binding of alkali cations to other cyclic polyethers (36) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1990,62, 957967. (37) Hiraoka, K; Kudaka, I. Rapid Commun. Mass Spectrom. 1990,4, 519526. (38) Banks, J. F., Jr.; Shen, S.; Whitehouse, C. M.; Fenn, J. B. Anal. Chem. 1994, 66, 406-414. (39) Straub, R;Linder, M.; Voyksner, R D. Anal. Chem. 1994,66, 3651-3658. (40) Banks, J. F., Jr.; Quinn, J. P.; Whitehouse, C. M. Anal. Chem. 1994,66, 3688-3695. (41) David, L.; Della Negra, S.; Fraisse, D.; Jeminet, G.; Lorthiois, I.; Le Beyec, Y.; Tabet, J. C. Int. J. M a s Spectrom. Ion Phys. 1983,46, 391-394.
1818 Analytical Chemistry, Vol. 67,No. 1 1, June 1, 1995
[ PbTx-2+Nal'
918
I
I
10000~
4
f
[?PbTx-Z+NaI+
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al U
500
1000
1500
2000
4000-
il a
1
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[ZPbTx-S+Nal' 1817
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500
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1000
2000
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3000
I
I
[ 3PbTx-S+ZNal"
1000
[ZPbTx-S+Nal+
200
200
500
1500
1000
2000
m/ z
Figure 3. Direct infusion electrospray ionization mass spectra obtained as averages of 10 scans for standard brevetoxin solutions: (a) PbTx-2, (b) PbTx-3, and (c) PbTx-9. Table 1. Assignments for Brevetoxin-RelatedIons
PbTx-2
PbTx-1
m/z
assignment
m/z
assignment
896 918 926 934 1366 1374 1382 1813
[PbTx-2 H]+ [PbTx-2 + Nal+ [PbTx-2 Na + KI2+ [ PbTx-2 K] [3PbTx-2 2NaI2+ [3PbTx-2 Na KI2+ [3PbTx-2 2KI2+ [2PbTx-2+ Nal+
+ + + ' + + + +
868 890 898 906 1324 1332 1340
[PbTX-l+ HI+ [PbTx-1+ Na]+ [PbTx-1+Na KI2+ [PbTX-l+ K1+ [SPbTx-1 2NaI2+ [SPbTx-lSNa + K12+ [3Pb?k-l+ 2KI2+
+
+
has been observed to occur readily in D/CI, FAB, and z52Cfmass spectrometries.41 LC-W and LC-ESMS Analysis of Brevetoxin Standards. Three known brevetoxins, PbTx-1, PbTx-2, and PbTx-9, were tested as standards (separately and as a mixture) to examine the capability of this on-line LC-ESMS system for brevetoxin analysis. The standard solutions were simultaneously analyzed by HPLCUV and HPLC-ESMS employing the postcolumn split arrangement described previously. To reduce sodium and especially
respectively; these assignments were confirmed by injections of the individual brevetoxins. An unanticipated peak (7'') (unknown compound assigned as UJ appeared with a retention time of 27.8 min. Individual compound injections revealed this unknown to be a contaminant of the PbTx-l standard. The mass spectra taken from the four regions of peak maxima exhibit characteristics similar to those of spectra obtained via direct infusion (Figure 3). All mass spectra show four related ions with overlapping elution profiles, corresponding to MH+ (low abundance), [M + Nal+ (base peak), [3M 2Na12+,and [2M Nal+. The unknown Ua and PbTx-9 were well-separated on the column, but their mass spectra exhibited exactly the same mass values corresponding to the above four ions, suggesting that U, is an isomer of PbTx-9, with an average molecular weight of 899. Signi6cant differences existed in the intensity ratios of the listed peaks, e.g., the intensity of m/z 1371 ([3Ua 2Na12+)is much lower than that for the corresponding peak of PbTx-9. The unknown Ua was also found in red tide extract samples. The UV trace of the same run of this mixture is shown in Figure 4a. Peak retention times for UV detection were about 1 min shorter than for the corresponding ESMS peaks because of the faster flow rate and shorter capillary distance to the UV detector. According to the retention times, peaks 4, 5, 7, and 8 correspond to PbTx-9, PbTx-2, U,, and PbTx-1. Additional UV peaks labeled 1,2,3,and 6 indicate that the brevetoxin standards may have decomposed and/or contain other impurities that were absent from a solvent blank. The instability of brevetoxin molecules has been reported (e.g., PbTx-1), and the half-lives for active material range from a few minutes to several hours in protic solvents.42 These extra components giving UV peaks did not exhibit strong responses in the corresponding ES total ion chromatogram (Figure 4b) under the employed operating conditions, possibly due to low aftinities for Na+ and H+. The UV peaks of brevetoxins (Figure 4a) are about 20% narrower than the corresponding ESMS peaks (Figure 4b). The degree of extracolumn band broadening in the UV connecting capillary of this on-line system is not severe because the peak width is similar to that of off-line LC-UV. The peak band broadening is more severe in the extracolumn ESMS connecting capillary because the flow rate (2 pL/min) is lower than in the UV connecting capillary (6 pL/min), resulting in an extra minute of residence and additional longitudinal diffusion in the ES capillary dead volume. Detection limits and linear dynamic ranges for PbTx-9, PbTx2, and PbTx-1 were investigated with this system by injecting brevetoxin mixtures in concentrations from 60 pg/pL to 20 ng/ p L with the mass spectrometer scanning from m/z 850-950 to detect [M Nal+ forms of the various brevetoxins. Intensities of other related ions (mainly [3M 2NaI2+)were small in the investigated concentration ranges. The relative abundance of [3M 2Na12+is 20%of the [M Nal+ base peak for 20 ng/pL PbTx-2 solution, and 10% of that for 10 ng/pL PbTx-2 solution. The trimeric and dimeric forms decreased in abundance with dilution more rapidly than [M + Nal+ for all tested brevetoxins. Response (peak area of [M + Nal+) vs concentration curves are shown in Figure 5. The detection limits for PbTx-9, PbTx-2, and PbTx-1 were less than 600 fmol, 1 pmol, and 50 fmol, respectively (S/N = 3), employing mass spectrometer scans of 100 m/z units.
+
I I
"
"
l
"
"
1
'
~
20 Retention Time (min)
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+
+
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4x1O4 A
.-3 C
a
p
2. 2
; .- 2 x 1 0 ~ c
ul C
3 C
-- 0
x 10
1
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Figure 4. Simultaneous HPLC-UV and on-line HPLC-ESMS analysis of a mixture of 17 ng/pL PbTx-9, 20 ng/pL PbTx-2, and 20 ng/pL PbTx-1 brevetoxin standards: (a) UV chromatogram; (b) total ion chromatogram, m/z 300-1900; and (c) selected ion chromatogram for three ions, m/z922,918, and 890. The three chromatograms were obtained from a single run. Conditions: 100-mm x 1-mm4.d. CIS column; methanoi/water (8515 v/v) mobile phase at 8 pUmin; postcolumn split ratio, 3:l (UV absorbance detector/mass spectrometer); injection volume, 1 pL.
potassium ion contaminations, the ES ion source, probe, and introduction lines were rigorously cleaned prior to analysis. The on-line analysis of a mixture of 17 ng/pL Pb?k-9,20 ng/ pL PbTx-2, and 20 ng/pL PbTx-1 appears in Figure 4. The total ion chromatogram from m/z 300-1900 is shown in Figure 4b with four major peaks. These peaks correspond chiefly to sodium adduct ions of the respective brevetoxins, appearing at m/z 922, 918, and 890. The selected ion chromatogram corresponding to these three ions is shown in Figure 4c. Peaks at 20.7 (4'7, 22.7 (Y), and 29.7 min (8") were from PbTx-9, PbTx-2, and PbTx-1,
+
+
+
+
(42) Trainer, V. L.; Edwards, R A; Szmant, A M.; Stuart, A M.; Mende. T. J.; Baden, D. G. ACS Symp. Ser. 1990,418, 166.
Analytical Chemistry, Vol. 67, No. 7 7 , June 1, 7995
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300 0.5
10 Concentration (ngiul)
1
f
m
2
5
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1000
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,
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laboratory red tide culture maintained at Mote Marine Laboratory was simultaneously analyzed by HPLC-W and HPLC-ESMS with the mass spectrometer scanning m/z 300-2000. The analysis of extract A was conducted before the aforementioned modifkation to the electrospray probe. Additional dead volume thus existed in the connection capillary (5C-cm-long, 100-pm4.d. deactivated fused silica) after the tee valve and in the electrospray connecting tube. The postcolumn split ratio was approximately 101 (w absorbance detector/mass spectrometer). Other conditions are described in the Experimental Section. By carefully searching the obtained mass spectra over the entire chromatographic time range, six distinct peaks with relatively high intensities and different retention times were uncovered. Figure 6 shows reconstructed ion chromatograms for each of six separated components (apparently corresponding to sodium adducts of intact molecules), while Figure 7 shows the corresponding ES mass spectra representing the averages of at least five consecutive scans taken from the regions of maximum ion current of the reconstructed ion chromatograms displayed in Figure 6. Based on the known molecular weights (and structures) and by comparing the retention times of certain individual brevetoxin standards, several peaks appearing in the ES ion chromatograms could be identified. The retention times in ES chromatogramsare somewhat longer than those of the standards because of the lower flow rate (about 0.7 pL/min), but the differences in retention times are rather uniform. The distinct peak exhibiting a retention time (tR) of 33.7 min Corresponding to m/z 918 in Figure 6c was assigned to the sodium adduct of PbTx-2, i.e., [PbTx-2 + Nal’; PbTx-2 has an average mass value of 895.1 Da. The ions appearing in the corresponding mass spectrum (Figure 7c) have been assigned in Table 1. It was found that reconstructed ion chromatogramsof m/z 896,918, 926, 934, 1366, 1374, 1382, and 1813 all exhibited overlapping elution profiles, suggesting that they originated from the same compound. Comparison of the mass spectral peaks shown in Figure 7c with those of the direct infusion ES mass spectrum of the PbTx-2 standard (Figure 3a) confirms the identity, as the main peaks are the same. Subtle differences in the relative intensities of minor peaks observed on comparing Figure 7c and Figure 3a can be explained on the basis of different levels of contaminating salts. Notably, several intense peaks representing potassium adducts (presumably from contamination of the ESMS system) are observed in both mass spectra. The peaks at m/z 680 and 413 in Figure 7c have been assigned to coeluting components and/or “preanalyzer” collision-induced dissociation (CID) fragment ions. The last peak shown in the ES ion chromatograms appeared at 42.2 min (m/z 890 ion chromatogram, Figure Sf). The m/z 890 peak corresponds to the sodium adduct of Pb?k-1, i.e., [PbTx1 + Nal+, as subsequently verified by LC-MS of the individual standard; the average molecular mass of PbTx-1is 867.1 Da. Peaks in the corresponding mass spectrum (shown in Figure 7f) are assigned in Table 1 in an analogous manner to the previous assignments for PbTx-2. The other four ions whose reconstructed ion chromatograms appear in Figure 6 do not correspond to sodium adducts of known brevetoxin molecule^.^^^^^^ However, they may represent other intact compounds, reaction products, or decomposition products from brevetoxins. The distinct peak appearing at a retention time
m lOOo0j p
3
J
i 1000Y
i
Y
I
P
0.1 1 10 100 Concentration (nglul) Figure 5. Signal response (peak area [M+Na]+) vs concentration for brevetoxin mixtures during on-line HPLC-ESMS. Each data point represents the average of three replicate 1-pL injections; error bars show fa.(a) PbTx-9, (b) PbTx-2, (c) PbTx-1. Insets show responses obtained from injections of (a) 1.1 pmol of PbTx-9, (b) 1.1 pmol of PbTx-2, and (c) 70 fmol of PbTx-1. 0.01
Because the standards contained some impurities and decomposed compounds, actual detection limitsare somewhat lower than these values. Correlation coefficients of the signal response vs concentration curves showed good linearity with r = 0.989 (n = 4), 0.988 (n = 4), and 0.995 (n = 6) for PbTx-9, PbTx-2, and PbTx-1 (Figure 5a-c, respectively). Linearity was maintained over the entire displayed range, i.e., until the concentrationsexceeded 20 pg/mL, where the column became overloaded. A mixture of 50 pg/mL standards exhibited wide unsymmetric chromatographic peak shapes with shorter centroid retention times and peak splitting characteristic of column overload.40 E-ESMS Analysis of Red Tide Algae -acts. The unknown sample consisting of a brevetoxin extract A from a 1820 Analytical Chemistry, Vol. 67, No. 1 1 , June 1 , 1995
(43) Yasumoto, T.; Murata, M. Chem. Rev. 1993,93, 1897-1909.
15000
7
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986
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10
20
30
40
50
300
I000
RETENTION TIME (YIN) Figure 6. Reconstructed ion chromatograms for on-line HPLCESMS analysis of red tide extract A containing brevetoxins. Conditions: 100-mm x 1-mm-i.d.Cla column; methanol/water (85:15v/v) mobile phase at 8 pUmin; postcolumn split to about 0.7 pUmin into the mass spectrometer; injection volume, 1 pL.
of 39.5 min (m/z 922 reconstructed ion chromatogram (Figure 6e)) was assigned to the sodium adduct of unknown U,, i.e., [U, Nal +;U, has an average mass value of 899 Da. This unknown U, is the same as that which appeared as a contaminant in the P b n - 1 standard (Figure 4). The relative retention time of U, (about 5 min longer than that of PbTk-2 and 2 min shorter than that of PbTx-1) was maintained in the standard mixture, extract A, and extract B (see Figure 8). Its corresponding mass spectrum (Figure 7e) has already been interpreted in the preceding description of the analysis of brevetoxin standards. Evidence that U, belongs to the brevetoxin class of compounds is provided by similarities in mass spectral features. An unknown compound ub, exhibiting a clear peak with retention time of 31.7 min, is shown as the reconstructed ion chromatogram of m/z 982 (Figure 6b). Ions at m/z 413,680,696, 982, 998, and 1462 were observed in the corresponding mass spectrum (Figure 7b). Except for m/z 413, 680, and 696, presumably coeluting impurities, all reconstructed ion chromato-
+
m/ z
1500
2000
Figure 7. Electrospray ionization mass spectra obtained as averages of five scans at peak maximum from corresponding selected ion chromatograms (Figure 6).
grams of these ions show peaks with identical retention times. By analogy to the identified known brevetoxins, m/z 982 is proposed to represent the sodium adduct of an unknown molecule of average mass 959 Da. If this proposition is correct, then ions which are present at m/z 998 and 1462 can be assigned to [ u b Kl+ and [3ub 2NaI2+,respectively. Note that analogous ions appeared for all three brevetoxin standards. The peak corresponding to [ub + Nal+ (Le., reconstructed ion chromatogram of m/z 982, Figure 6b) is overlapped by the large peak at m/z 680 (Figure 6a). Therefore, at least a portion of the signal at m/z 680 in the ES mass spectrum of u b (Figure 7b) originates from the "tailing" previous component. Some of the m/z 680 signal in Figure 7b, however, may originate from fragmentation of u b by preanalyzer CID. Another unknown peak appearing at a retention time of 36.2 min is shown in Figure 6d (the reconstructed ion chromatogram of m/z 986). The correspondingmass spectrum of this unknown, U,, appears in Figure 7d. This mass spectrum shows ions at m/z 680,696,986,1002,and 1468. For these ions, each reconstructed ion chromatogram gives peaks having the same retention time of
+
Analytical Chemistry, Vol. 67, No. 7 1, June 1, 1995
+
1821
20000
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15 2 0 2 5 30 3 5 4 0 RETENTION TIME (WIN) Figure 8. Reconstructed ion chromatograms for on-line HPLCESMS of red tide extract B containing brevetoxins. Conditions: 100mm x 1-mm4.d.CIS column; methanowwater (8535 v/v) mobile phase at 8pUmin; postcolumn split to 2 yUmin into the mass spectrometer; injection volume, 1 yL.
36.2 min. Again, by analogy to the previous results concerning the ident3ed compounds, we propose that the peak at m/z 986 represents the sodium adduct of U,, an unknown having an average molecular mass value of 963 Da, which does not coincide with the molecular mass of any known brevetoxin. On the basis of this proposition, ions which appear at m/z 1002 and 1468 can be assigned to [U, + Kl+ and [3U, 2Na12+,respectively, analogous to assignments for the known brevetoxins. A rather broad peak appeared around 30 min, shown as the reconstructed ion chromatogram of m/z 680 in Figure 6a. The trailing portion of this peak may be composed of several small overlapping peaks appearing between 35 and 43 min. The corresponding ES mass spectrum taken from the apex of the main peak is shown in Figure 7a. Peaks were observed at m/z 658, 680,696, 712, and 1337; each of them shows rather superimposable reconstructed ion chromatograms. The dominating m/z 680
+
1822 Analyfical Chemistry, Vol. 67, No. 7 7 , June 7, 7995
300
1500
1000
1900
m/z Figure 9. Electrospray ionization mass spectra obtained as averages of four scans at peak maximum from corresponding selected ion chromatograms (Figure 8).
peak may be assigned as the sodium adduct of a fourth unknown, u d , i.e., [ud Nal+, where U d has an average molecular mass of 657 Da. Based on this assignment, m/z 658 may be assigned as [ud HI+,m/z 696 may be assigned as [ u d Kl+, and 1337 as [3ud 2Na12+. The unknown u d is postulated to be a product of brevetoxin decomposition whose formation occurred in solution during the sample storage period. Small peaks at m/z 680 associated with the elution of Ua,Ub, PbTx-9, PbTx-2, and PbTx-1 may be produced by preanalyzer CID. Further study of the structure and properties of these unknowns is currently in progress. The same sample extract was run again after the electrospray probe was modfied as previously described to cut down on postcolumn dead volume. Furthermore, to reduce the contribution of potassium adduct ions to acquired mass spectra, the ES source, probe, and introduction line were rigorously cleaned. The laboratory red tide culture extract sample was also diluted by a
+
+ +
+
factor of 3 with mobile phase (85:15 methanol/water). This diluted sample, extract B, was run just after data were acquired on brevetoxin standards (Figure 5) under the same conditions. Figure 8 shows reconstructed ion chromatograms for each of six selected components (the same ones shown in extract A), while Figure 9 shows the Corresponding ES mass spectra representing the average of four consecutive scans taken from the regions of maximum ion current. Comparing Figures 8 and 9 with Figures 6 and 7, the base peaks for the six components are the same, and the shifts in retention times are quite uniform. There are no potassium adduct ions in Figure 9, as the potassium ions were removed from the ESMS system prior to extract B analysis. Some mass spectral peaks corresponding to noncovalent trimeric and dimeric forms which appeared in Figure 7 were not present in Figure 9 because extract B is diluted 3-fold relative to extract A, and the intensities of trimeric and dimeric species decrease more rapidly upon dilution than those of the [M Nal+ base peaks. The retention times of PbTx-2, U,, and PbTx-1 in extract B correspond to those of the brevetoxin standards (Figure 4), but the peaks are wider in Figure 8, possibly due to chromatographic interferences of other unknown components in this complicated extract sample. The concentrations of PbTx-2 and PbTx-1 in extract B were determined to be 20 and 1.0 ng/pL, respectively, according to calibration curves shown in Figure 5. To confirm the PbTx-1 identity assignment and quantitation from extract B, 10.0pL of a 2Wng PbTx-1 standard was spiked into 50 pL of extract B. The spiked sample was analyzed three times using the same LC-MS conditions. From the increase in the area of the m/z 890 peak after standard addition, the concentration of PbTx-1 in extract B
+
was calculated to be 0.98 f 0.07 ng/pL, which correlates well with the calibration curve value. CONCLUSION This work establishes HPLC-ESMS as a powerful tool for the analysis of brevetoxins in red tide algae extracts. The low detection limits (subpicomole) and high detection speczcity offered by HPLC-ESMS for brevetoxin analysis surpass those of currently used methods. Another clear advantage relative to HPLC-W is that coeluting compounds can be much more readily noticed and possibly identified via mass spectral information. Although the required HPLC-ESMS equipment is relatively expensive, consumed chemicals and reagents are not, and the analysis time is short. Continuing work is centered on increasing the compatibility of HPLC and ESMS with the goal of improving separations and further characterizing structures of brevetoxins and other real-world marine toxins and their decomposition products. ACKNOWLEDGMENT Financial support for this project was provided by the Louisiana Education Quality Support Fund through Grant No. LEQSF (1991-1993)-RD-B15. Received for review August 23, 1994. Revised manuscript received February 17, 1995. Accepted March 16, 1995.@ AC9408383 @Abstractpublished in Advance ACS Abstracts, April 15, 1995.
Analytical Chemistry, Vol. 67,No. 11, June 1, 15’95
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