RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6 , 121-127 (1992)
Ionspray Mass Spectrometry of Marine Toxins. IV. Determination of Diarrhetic Shellfish Poisoning Toxins in Mussel Tissue by Liquid Chromatography/Mass Spectrometryt Stephen Pleasance* and Michael A. Quilliam Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 321, Canada
Julie C. Marr Fenwick Laboratories, 5595 Fenwick Street, Suite 200, Halifax, Nova Scotia, B3H 4M2, Canada SPONSOR REFEREE: D. E. Games, Department of Chemistry, The Universitv of Swansea. Swansea. UK
An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.
The development of a combined liquid chromatographidmass spectrometric (LC/MS) method utilizing ion-spray (ISP)’ atmospheric-pressure ionization for the analysis of okadaic acid (OA)’ and dinophysistoxin1 (DTX-1) was recently r e p ~ r t e dThese .~ hydroxylated polyether compounds, the structures of which are shown below, are the principal agents responsible for
~~
Okadaic acid (OA)
H
H
CH3
Dinophysistoxin- 1 @TX-1) H
CH3
CH3
-
CH3
H
Dinophysistoxin 2 @TX-2) H
cases of diarrhetic shellfish poisoning (DSP), a severe gastrointestinal illness caused by the consumption of shellfish contaminated by feeding on toxic strains of certain dinoflagellate^.^-' Using isocratic conditions, the LC/MS method was used to confirm the presence of OA in a culture of the benthic dinoflagellate species Prorocentrum concauum and, for the first time, in natural populations of Dinophysis spp. from Eastern Canadian water^.^ With slight modifications the LC/MS NRCC 33003. Author to whom correspondence should be addressed (under contract from Sciex, 55 Glen Cameron Road, Thornhill, ON, Canada). 0951-4198/92/020121-07 $05.00 01992 by John Wiley & Sons, Ltd.
method was also used to analyse the anthrylmethyl ester of okadaic acid, consequently validating the highperformance liquid chromatographic method with fluorescence detection (HPLC-FLD) which is currently the standard instrumental technique for DSP analysis.’ The presence of DSP toxin producing strains of dinoflagellates in Canadian together with increasing aquaculture operations, provided the impetus to establish a collaborative research program between the National Research Council of Canada (NRC) and Fenwick Laboratories Ltd to investigate the chemistry of DSP toxins and to develop improved analytical procedures for their determination in marine extracts. The emphasis on instrumental techniques has become more urgent with the recent banning of animal testing in several member countries within the European community. Since our earlier report , 3 significant improvements have been made in the LC/MS methodology, resulting in lower detection limits for the underivatized DSP toxins. The technique has since been applied to more complex sample matrices, and in the present work its application to the analysis of DSP-contaminated cultivated mussels (Mytilus edulis) from European and Canadian waters is described. EXPERIMENTAL Materials The toxins OA and DTX-1 were isolated from a laboratory culture of the dinoflagellate species Prorocentrum Lima grown at the NRC Institute for Marine Biosciences. The culture conditions and details of the harvesting of this organism are similar to those previously described for P. c ~ n c a u u mO. ~A is also availReceived 26 November I991 Accepted 29 November 1991
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
122
able through Diagnostic Chemicals Ltd (Charlottetown, PEI, Canada). HPLC-grade acetonitrile was purchased from Anachemia (Lachine, PQ, Canada). A Milli-Q water purification system (Millipore Corp., Bedford, MA, USA), equipped with ion-exchange and carbon filters, was used to further purify glass-distilled water. Cultivated samples of the blue mussel, Mytilus edulis, were used exclusively in this study. Both fresh and frozen mussel tissue were examined, together with cooked mussels from ,a restaurant. Mussels harvested at affected lease sites were randomly picked from socks at varying depths. Except where indicated in the text, fresh mussels were shucked and the digestive glands excised. Frozen mussel meat was thawed at room temperature and the digestive glands similarly removed. Sample extraction The extraction procedure used in the present work was modified from that described by Lee el al.' Approximately 20 g of either whole mussel tissue or digestive glands were drained, accurately weighed and then homogenized (PolytronTM,Brinkman Instruments Ltd, Rexdale, ON, Canada) with methanol (80 mL) for 2 min at room temperature. The homogenate was transferred to a 250 mL centrifuge bottle and centrifuged for 15 min at 3000 rpm. A 50 mL portion of the supernatant liquid was combined with water (10 mL) and extracted twice with hexane (50 mL), discarding the upper layers. The aqueous methanol extract was mixed with water (20mL), and extracted twice with chloroform (80 mL). The combined chloroform extracts were taken to dryness on a rotary evaporator. The residue was transferred in methanol to a 5 mL volumetric flask and made up to the mark to give a solution of 2 g equivalent tissue per mL. For LC/MS analysis of high-level samples this methanolic extract was analysed directly; for trace-level samples a 500 pL portion of this extract was taken to dryness under a stream of dry nitrogen, and redissolved in 1OOpL of 50% aqueous methanol (10 g equivalent tissue per mL). For HPLC-FLD analysis a 50pL portion of the same extract (0.1 g e uivalent tissue) was taken to dryness in a Reacti-vial (Pierce, Rockford, IL, USA) ready for derivatization (see below). Previous concerns over the adsorption of DSP toxins on to glass surfaces' also led us to perform several duplicate extractions of standard solutions of O A (50100 pg/mL) using both glass and plastic sample vials. Results obtained by LC/MS indicated that, at the levels used in these experiments, there was no significant difference between the two materials, and glassware was used throughout this work.
9,
Derivatization and HPLC ADAM deriuatization. The derivatization reagent 9anthryldiazomethane (ADAM), was prepared as described previously.3 Briefly, the tissue extract residue (as prepared above) was dissolved in 10 yL dimethylformamide and mixed with 90 yL of ADAM reagent. After heating at 35 "C for 1 h, the solvent was removed under a stream of nitrogen. This residue was then dissolved in 1 mL hexane + chloroform ( l : l ) , transferred to a silica
solid-phase extraction cartridge (Supelco, Oakville, ON, Canada), and eluted using the procedure described by Lee and c o - ~ o r k e r sThe . ~ final fraction was evaporated and re-dissolved in 100 pL methanol (1 g equivalent tissue per mL) for analysis by HPLC-FLD. NABA deriuatization. Samples were derivatized with the N-(9-acridinyl)-bromoacetamide (NABA) reagent using the method described by Allenmark et a1." Briefly, the residue (as prepared above) was dissolved in 100 yL acetonitrile and mixed with 400 pL of 2 mM NABA in chloroform, 500 pL of 20 mM phosphate buffer (pH 7.0) and 50 pL of 10 mM tetrabutylammonium sulfate. The reaction mixture was heated to 90 "C, with stirring, for 1h. The chloroform phase was removed, taken to dryness under a stream of dry nitrogen and reconstituted in 100 yL acetonitrile prior to LC/MS. Fluorescence HPLC. Analyses were performed using a model HP1090L liquid chromatograph (HewlettPackard, Palo Alto, CA, USA) equipped with a binary DR5 solvent delivery system, a HP1046A fluorescence detector, and a HP79994A data system. Settings for the fluorescence detector were: 249 nm excitation, and 407 nm emission protected by a 280 nm cut-off filter. Separations at ambient temperature were achieved on a Merck LiChrospher 100 RP-18 250 x 4.6 mm I D column (BDH Inc., Toronto, ON, Canada) using isocratic elution with aqueous acetonitrile (10 :90) at a flow rate of 1mL/min. Liquid chromatography/mass spectrometry All LC/MC experiments were performed on a Hewlett-Packard Model 1090 Series I1 liquid chromatograph coupled to a Sciex API I11 triple quadrupole mass spectrometer (Thornhill, ON, Canada) via an Ionspray@ (ISP) interface, as previously d e ~ c r i b e d . ~ Separations of underivatized and NABA derivatized DSP toxins were achieved on a 1mm ID X 25 cm microbore column packed with 5 yrn Vydac 201TP stationary phase (Keystone Scientific, Bellafonte, PA, USA). Aqueous acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used as the mobile phase at a flow rate of 50 pL/min. A linear gradient of 40% to 100% acetonitrile in 20 mins was used, followed by a hold of 5 min. RESULTS AND DISCUSSION Gradient elution LC/MS of DSP toxins In our earlier report on the LC/ISP-MS analysis of DSP toxin^,^ the isocratic reversed-phase separation of OA and DTX-1 with 60% acetonitrile in water containing O.l%trifluoroacetic acid was described. Isocratic conditons and 2.1 mm I D columns were employed for the screening of large numbers of relatively clean plankton extracts. Since then we have focused our efforts on the use of 1mm I D columns and gradient elution to achieve improved sensitivity and compatibility with more complex samples. In addition, a fully-articulated version of the ISP interface has also been developed, allowing more precise control in the positioning of the ISP needle in relation to the sampling orifice, and thus enabling a reduction in background chemical noise. Figure 1presents the improved gradient elution separation of a mixture of OA and DTX-1 using a l mm I D reversed-phase column and selected-ion monitoring
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
75
-E 5
50
a l
i
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
lime (min)
Figure 1. Gradient elution LC/ISP-MS separation of (a) O A (20 ng) and (b) DTX-1 (25 ng) using selected-ion monitoring of their respective protonated molecules. Insert in (a) shows response from injection of 400pg of O A . Conditions: 2 5 0 1~ mm ID column packed with Vydac 201TP stationary phase; aqueous acetonitrile mobile phase containing 0.1% trifluoroacetic acid, linear gradient of 40-100% acetonitrile in 20min, at a flow rate of SOyL/min; 1 pL injection volume; dwell time, 200 ms per ion.
(SIM) of their respective protonated molecules (mlz 805 and 819). While the retention times of the toxins (11.3 and 14.4 min, respectively) are significantly longer than those obtained under the isocratic conditons, the narrower peaks obtained under the elution profile, coupled with the lower flow rate and improved optimization of the ISP interface, result in a 10-fold improvement in detection limit over that previously reported.3 The response obtained for an injection of 400pg of O A is presented in the insert in Fig. 1, and with the observed signal-to-noise ratio of 2 : l this probably represents the detection limit of the LC/MS method, i.e., approximately 0.4 pg/mL (1pL injection). As shown by the calibration curve for OA in Fig. 2, a linear response was obtained between 1 and 75 ng ( r 2= 0.9998). The reproducibility of peak areas for replicate injections of a 5 pg/mL solution of O A over the course of a day was of the order of 2.4% relative standard deviation ( n = 5 ) . The relative molar response of OA and DTX-1 was found to be very close to unity. Mussel tissue extraction The procedure used in the present work for the extraction of DSP toxins from shellfish tissue is similar to that described by Lee and co-workers for the ADAM HPLC-FLD method.' The original procedure consists of an initial dispersion of mussel tissue into aqueous
123
methanol (80%), followed by extraction with hexane, and finally a partition into chloroform. We have modified the procedure slightly, and use 100% methanol in the initial dispersion since we have found that this provides higher and more reproducible recovery of OA. At a level of 1 pg per g of tissue, recoveries of O A spiked into control mussel tissue were typically in excess of 90%. The current legal tolerance level for DSP in Europe is 0.2 pg per g of edible tissue." However, it is common practice to use only the digestive glands of the mussel in the extraction procedure (see Experimental). This stems from the original observation of Yasumoto et aZ.,4 that DSP toxicity appears to be localized to the hepatopancreas, and the fact that the HPLC-FLD method is often not sensitive enough to analyse the entire edible tissue reliably at, or below, the tolerance level. Knowledge of the weight of digestive glands in proportion to that of the whole mussel tissue then allows the concentration of toxin to be calculated in terms of the whole tissue. The HPLC-FLD method is limited by the permissible degree of pre-concentration of the extract prior to analysis due to the derivatization step. With the present clean-up procedure, the maximum amount of sample that can be derivatized reliably is 0.1 g equivalent tissue in 100 pL reagent (1 g/mL). In our hands, the detection limit available with this approach using all edible tissue has been 120 ng per g tissue. If only digestive glands are used in the extraction, and if they represent 25% of the edible tissue, the detection limit in the whole tissue is then in the order of 30 ng/g. However at this level there are numerous peaks in the chromatogram, due to derivatives of endogenous compounds and/or reagent impurities, that are of comparable size to those of the toxins and that make detection and quantitation difficult. The LC/MS method, on the other hand, with its much greater selectivity of detection and ability to analyse the toxins directly without derivatization, can accommodate much higher pre-concentration factors. Thus, in conjunction with gradient elution, it is possible
Peak Area
x 106
0
20
80
100
Amount injected (ng)
Figure2. Calibration curve for the LC/MS analysis of O A using selected-ion monitoring of the protonatcd molecule (mlz 805). Conditions as in Fig. 1.
124
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
to concentrate the final extract to 10 g equivalent tissue per mL. With a detection limit of 0.4yglmL in the extract it is possible to detect 40ng/g in the tissue. If only digestive glands are used in the assay, the detection limit expressed in terms of whole tissue equivalent is approximately 10 ng/g. The LC/MS method therefore provides a better detection limit than HPLC-FLD, allowing the analysis of whole mussel tissue samples, and does not require time-consuming and often troublesome derivatization steps. Application to DSP-toxin-contaminated shellfish Initial experiments with contaminated shellfish extracts were conducted on batches of cooked deep-frozen mussel meats from Sweden, which had been impounded by Dutch authorities after being implicated in the development of DSP symptoms by consumers and subsequently shown to produce a positive (++) response with the DSP rat bioassay.12 As part of the NRC Marine Analytical Chemistry Standards Program (MACSP), we are currently evaluating this material as a potential component of a blended mussel tissue reference material for DSP, to complement our previously released reference material for domoic acid (MUS-1I3), the toxin responsible for amnesic shellfish p ~ i s o n i n g . ' ~ This program relies on reliable analytical methods to provide accurate levels of the toxin(s) in the different matrices, at all stages of production, e.g. blending, homogeneity, stability studies and certification. In order to confirm the localization of the DSP toxins within the digestive gland of the mollusc, individual mussels were dissected and the tissue divided into three main portions, viz. digestive glands, meats, and other tissue including the juices, The three portions were individually weighed, extracted and then analysed by LClMS without preconcentration. The digestive glands of the particular mussels used for this experiment were found to constitute approximately 24% of the weight of the entire edible tissue. This proportion will of course vary from species to species, and with the time and location at which the mussels are harvested. The LC/MS selected-ion monitoring (SIM) chromatogram of rnlz 805 from the digestive gland extract, presented in Fig. 3(a), shows a strong peak at the correct retention time for O A (Fig. l(a)), which was not observed in the corresponding trace obtained from non-toxic, control mussel-tissue extracts. By external calibration it was determined that the concentration of OA was 0.96 pg/g of digestive gland or approximately 0.23 yg/g in the whole tissue. This is slightly higher than the level generally allowed in Europe (0.2 pg/g)." No DTX-1 was detected, as evidenced by the absence of a response in the corresponding mlz 819 trace (Fig. 3(b)). No toxins were detected in the 'meats', and only negligible amounts of O A were found in the 'other tissue' portion. These results clearly support the original findings of Yasumoto et aL4 The sensitivity and selectivity of the LClMS method is illustrated in Fig. 3(c), which shows the corresponding mlz 805 chromatogram obtained from the analysis of a whole tissue extract from the same mussels, concentrated to only 2 g equivalent of tissue per mL. Even without the additional preconcentration a signal is clearly observed at the correct retention time for OA. An opportunity to test the effectiveness of the
z3
30
DTX-1
20
1 1°1 0:o
z2
4:O
8.0
.
12:o
16.0
7s 50
1 1 I 0.0
4.0
8.0
12.0
16.0
Time (min)
Figure 3. Selected-ion chromatograms obtained from the LCIISP-MS analysis of (a) and (b) digestive gland and (c) wholetissue extracts from DSP-toxin-contaminated Swedish mussels. The final extract was concentrated to only 2 g equivalent tissue per mL. Conditions as in Fig. 1.
LC/MS method under more pressing conditions arose in the summer of 1990 when several people developed severe DSP symptoms after eating cultivated mussels 'harvested in Nova Scotia. Mussel samples from an affected restaurant (cooked), a domestic residence, and also the lease site from which the mussels were subsequently traced, were shown by Fisheries and Oceans Canada to be toxic to mice (intra-peritoneal i n j e ~ t i o n ) .Bacterial '~ gastroenteritis was ruled out, and the NRC was asked for analytical support. The results of the LC/MS analysis of a digestive gland extract from mussels harvested at the (quickly closed) lease site are presented in Fig. 4. While no OA was detected ( m / 2805; Fig. 4(a)), the presence of DTX-1 was clearly indicated by the strong signal in the mlz 819 trace (Fig. 4(b)) at the correct retention time for the authentic toxin. DTX-1 was similarly detected in the cooked mussels from the restaurant. Full-scan LC/MS analysis of a concentrated extract provided the mass spectrum shown in Fig. 4(c), in which the protonated molecule (MH') is clearly observed. The ions at mlz 801 and 783, due to successive losses of water, are characteristic of the tetrahydroxylated DSP toxins.3,16, l7 The ion at mlz 837 is assigned to a mobile-phase cluster ion i.e., [MH f H20]'. The toxin was subsequently isolated and its structure confirmed by additional spectroscopic techniques, including nuclear magnetic resonance. Details of this incident, which to our knowledge is the
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
first confirmed incident of DSP in North America, have been reported recently." More recently, we have received samples of mussel digestive glands from Ireland which had already been shown to be contaminated with O A by the ADAM HPLC-FLD method. Interestingly, an additional peak was detected in the fluorescence chromatograms which did not correspond to any of the known DSP toxins. The fluorescence trace subsequently obtained in our laboratory is shown in Fig. 5(a), in which a second peak is clearly observed eluting after that corresponding to the ADAM-OA derivative. The appearance of the peak suggests that this component _also has a free carboxyl functionality which is necessary for the ADAM derivatization. The corresponding LC/MS-SIM chromatograms for this extract are shown in Fig. 5(b) and (c), respectively, and it can be seen that two peaks are observed in the mlz 805 chromatogram of similar relative intensities to those observed in the fluorescence trace. The first peak again appears at the correct retention time for authentic O A , supporting the assignment. The presence of DTX-1 is also indicated by the appearance of a weak signal at the correct retention time in the mlz 819 chromatogram; however, it was not observed in the corresponding fluorescence trace due to its relatively low concentration in the extract. Full-scan analyses were performed on this sample, and the
125
fa)
R"' 0.0
4.0
8.0
12.0
16.0
Figure 5. Analysis of digestive-gland extract of DSP-toxin-contaminated Irish mussels by (a) ADAM derivatization and HPLC-FLD and by (b) and (c) LC/ISP-MS using selected-ion monitoring. The final extract for LC/MS analysis was concentrated to l o g equivalent tissue per mL. HPLC-FLD conditions as in Experimental. LC/MS conditions as in Fig. 1 .
4:O
8:O
16.0
12.0
T i (min) 819
[M+HI+
m/z
Figure4. (a) and (b) Analysis of digestive-gland extract from DSP-toxin-contaminated Canadian mussels by LC/ISP-MS using selected-ion monitoring. The final extract was concentrated to 2 g equivalent per mL. (c) The mass spectrum of the DTX-1 peak obtained from a full-scan analysis. LC conditions as for Fig. 1. Dwell times (a) and (b) 200 ms per ion, (c) 8 ms/Da over a range of 500900 m / z .
second peak in the mlz 805 chromatogram provided a mass spectrum identical to that for O A , suggesting its interpretation as a possible isomer of the toxin as opposed to a cluster ion of a lower or a fragment ion of a higher molecular weight analyte, respectively. This compound has been successfully isolated and purified, and its structure determined to be a previously unreported positional isomer of OA which has been termed dinophysistoxin-2 (DTX-2; see structures).l8 During this investigation, quantitative results on the various mussel samples obtained by LC/MS were compared with those obtained by HPLC-FLD using the ADAM derivatization method. The results obtained by both techniques on the various mussel extracts are presented in Table 1. In general there is good agreement between these two independent techniques. The only serious discrepancy was between values for DTX-1 in the Canadian mussels; this may have been due to the fact that the analyses were performed several days apart. The better sensitivity of the LC/MS method allowed the detection of minor components, such as DTX-1 in the Irish mussel sample, that could not be reliably detected using the HPLC-FLD method. As discussed above, the main problem encountered with the latter method is the presence of numerous small peaks throughout the chromatogram due to derivatives of endogenous compounds and/or reagent impurities, which can complicate analysis. The higher selectivity of
IONSPRAY MASS SPECTROMETRY O F MARINE TOXINS. IV
126
the LC/MS method, which utilizes selected-ion monitoring of the high-mass protonated molecules, effectively eliminates most chemical interference.
7i 5
LC/MS of fluorescent derivatives As part of our research program we are also investigating reagents for the fluorescent labelling of DSP toxins that may be used as alternatives to the ADAM reagent currently used in the HPLC-FLD method described by Lee et al.' The ideal choice would be a universal derivative that can be used for HPLC with either fluorescence, ultraviolet and/or ISP-mass spectrometric detection. One reagent currently being evaluated is N-(9-acridinyl)-bromoacetamide (NABA), previously described by Allenmark and co-workers for the phasetransfer-catalysed fluorescence labelling of carboxylic acids for liquid chromatography." The authors' choice of reagent was based on the fact that the starting material, 9-aminoacridine, is one of the most fluorescent componds known and that the NABA-derivatized analytes, when protonated, have good retention characteristics on reversed-phase columns. It is the protonated nitrogen which is of interest with respect to additional sensitivity under (positiveion) ISP ionization conditions, since the ion-evaporation process19 favours pre-formed ions in solution. Previous experiments with the ADAM-OA derivative were disappointing, in that the LC/MS sensitivity for the derivative was very similar to that of the underivatized toxin. A mixture of A 0 and DTX-1 was derivatized with NABA and analysed by LC/MS, monitoring the protonated molecules of the individual derivatives (mlz 1039 and 1053, respectively). The results are shown in Fig. 6(a) and (b). Both toxins were detected with characteristic shifts in mass and retention time. It can also be seen that the derivatives are well resolved under this elution profile, and have good peak shapes. Comparison of the LC/MS peak areas with those of underivatized standards indicated that a 5-fold increase in sensitivity was obtained using the NABA derivative. Contaminated mussel extracts were also derivatized with the NABA reagent, and Fig. 6 also shows the results of the LC/MS analysis of the Nova Scotian mussels previously found (Fig. 4) to contain DTX-1.
Table 1. Comparison of quantitative results obtained by HPLC-FLD and LC/MS methods for the analysis of OA and its analogues in DSP-toxin-contaminated cultivated blue mussels Sample
Swedish' Canadian
Conccntration (vg/g)'l. DTX- 1
Mcthod
OA
HPLC-FLD LC/MS
1.52 k 0.05 1.40k0.09
HPLC-FLD LC/MS
NDd ND
DTX-2
ND~ ND
ND ND
0.67+0.07 1.08+0.04
ND ND
HPLC-FLD 12.7 k 1.2 ND 8.5 2 0.8 LC/MS 12.420.4 0.3720.01 8.420.4 Concentration is reported for digestive glands on a wet weight basis. Mean f standard deviation reported for triplicate analyses of a single extract. Preliminary reference material having undergone autoclaving heat treatment. N D = not detected. Irish
36
II NABA-DTX-1
iwz 1053
I
21 18
9 0.0
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12.0
16.0
DTX-I 20
IS 10
NABA-DTX-1
; 2
2s
0.0
4.0
8.0
12.0
16.0
Time (min)
Figure6. (a) and (b) Analysis of NABA derivatized O A and DTX-1, and (c) and (d) NABA derivatized digestive-gland extract from DSP-toxin-contaminated Canadian mussels by LC/ISP-MS. Conditions as in Fig. 1.
Comparison of the m/z 1053 traces for the mussel extract (Fig. 6(d)) and the derivatized standard (Fig. 6(b)) clearly indicates the presence of the NABA-DTX-1 derivative. These experiments provide additional confirmatory evidence for the toxin's identity. The protonated molecule of the underivatized toxin (rnlz 819) was also monitored during these experiments, and it can be seen from the corresponding trace for the mussel extract (Fig. 6(c)) that none of the underivatized DTX-1 was detected, indicating that derivatization was complete. This ability to monitor both reactant and product in a derivatization reaction is a useful aid to the chemist in developing the optimal reaction conditions. These preliminary results with the NABA derivative appear encouraging, although further experiments are required to confirm that any LC/MS sensitivity gained with the use of the derivative is not compromised by losses and dilution factors resulting from the additional derivatization step in the methodology, Further details on the fluorescent labelling of DSP toxins, including the use of other derivatizing agents, will be published in due course. CONCLUSION It has been shown that gradient elution in conjunction with the lower flow rates utilized with 1mm ID microbore columns, together with the improved optimization
IONSPRAY MASS SPECTROMETRY O F MARINE TOXINS. IV
of the ionspray interface, provide significantly improved LC/MS detection limits for underivatized DSP toxins over those obtained previously using isocratic elution, and better than those available with the HPLC-FLD method using ADAM derivatization. The improved detection limits allow the confirmatory analysis of whole mussel tissue. While there is no need to derivatize mussel extracts using the LC/MS method, derivatization with NABA can be useful for additional confirmatory evidence and may also provide greater sensitivity under ionspray ionization. The use of gradient elution is particularly valuable for screening complex samples for a wide range of toxins, and one of the real benefits of the combined LC/MS approach in our research program lies in the detection of related compounds at low levels in shellfish extracts. This is illustrated by the detection of the previously unreported DSP toxin, DTX-2, which as a consequence also validates the HPLC-FLD procedure for the analysis of this toxin in shellfish tissue. Numerous other related compounds have been similarly identified and will be reported separately. In many cases the isolation of these compounds has been achieved by simply scaling up the chromatography to preparative columns. The results presented here indicate significant variation in the toxin profiles of DSP-contaminated shellfish from different locations. The improved sensitivity of the LC/MS technique will hopefully allow us to examine more closely the causes of the observed variation in toxin profile, and perhaps lead to a greater understanding of the role of these compounds in the source dinoflagellates.
Acknowledgements The authors are grateful to Ms L. McDowell (Fenwick), Mrs P. Blay (Sciex) and Mr W. Hardstaff (NRC) for technical assistance, and to Dr A. Jackson (NRC) for help with dissections. We thank Drs T. Hu (Fenwick) and J. L. C. Wright (NRC) for the purified DSP toxins, and Dr M. W. Gilgan (Fisheries and Oceans Canada) for help with the development of extraction procedures. The contaminated Irish mussels were kindly provided by Drs J. Doyle and E. Nixon of the Department of the Marine Fisheries Research Centre, Abbotstown,
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Dublin, Ireland. Impounded Swedish mussel tissue was provided by Dr P. Hagel of the Netherlands Institute for Fisheries Research (RIVO), Ijmuiden, the Netherlands.
REFERENCES 1. A. P. Bruins, T. R. Covey and J. D. Henion, Anal. Chem. 59, 2642 (1987). 2. K. Tachibana, P. J. Scheuer, Y. Tsukitani, H. Kikuchi, D. Van Engen, J . Clardy, Y. Gopichand and F. J. Schmietz, J . A m . Chem. Soc. 103, 2469 (1981). 3. S. Pleasance, M. A . Quilliam, A. S. W. de Freitas, J. C. Marr and A. D. Cembella, Rapid Commun. Mass Spectrom. 4, 206 (1990). 4. T. Yasumoto, Y. Oshima and M. Yamaguchi, Bull. Jpn SOC. Sci. Fish. 44,1249 (1978). 5. T. Yasumoto, M. Murata, Y. Oshima, G . L. Matsumoto and J. Clardy, in Seafood Toxins, ed. by E. Ragelis, ACS Symposium Series 262 pp. 207, American Chemical Society, Washington (1984). 6. T. Yasumoto, M. Murata, Y. Oshima, M. Sano, G . K. Matsumoto and J. Clardy, Tetrahedron 41, 1019, (1985). 7. J. S. Lee, T. Yanagi, R. Kenma and T. Yasumoto, Agric. Biol. Chem. 51, 877 (1987). 8. A. D. Cembella, J . App. Phycology 1, 307 (1989). 9. T. Yasumoto (personal communication). 10. S. Allenmark, M. Chelminska-Bertilsson and R. A. Thompson, Anal. Biochem. 185, 279 (1990). 11. J. Haamer, P. 0. Andersson, S. Lange, X. P. Li and L. Edebo, J . Shell. Res. 9, 109 (1990). 12. M. Kat, Sarsia 68, 81 (1983). 13. W. R. Hardstaff, W. D. Jamieson, J. E. Milley, M. A. Quilliam and P. G . Sim, Fres. J . Anal. Chem. 338,520 (1990). 14. M. A. Quilliam and J. L. C . Wright, Anal. Chem. 61, 1053A (1989). 15. S. Pleasance, D. Douglas, A. S. W. deFreitas, L. Fritz, M. W. Gilgan, T. Hu, J. C. Marr, M. A. Quilliam, C. Smyth, J. Walter and J. L. C. Wright, Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics, Nashville, TN, ASMS, East Lansing (1991). 16. R. W. Dickey, S. C. Bobzin, D. J. Faulkner, F. A. Bencsath and D . Andrzejewski, Toxicon 28, 371 (1990). 17. F. A . Bencsath and R. W. Dickey, Rapid Comrnun. Mass Spectrom. 5 , 283 (1991). 18. T. Hu, J. Doyle, D. Jackson, J. C. Marr, E. Nixon, S. Pleasance, M. A. Quilliam, J. Walter and J. L. C. Wright, Chem. Commun. (in press). 19. B. A. Thomson and J . V. Iribarne, J . Chem. Phys. 71, 4451 (1979).
Ionspray Mass Spectrometry of Marine Toxins. IV. Determination of Diarrhetic Shellfish Poisoning Toxins in Mussel Tissue by Liquid Chromatography/Mass Spectrometryt Stephen Pleasance* and Michael A. Quilliam Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 321, Canada
Julie C. Marr Fenwick Laboratories, 5595 Fenwick Street, Suite 200, Halifax, Nova Scotia, B3H 4M2, Canada SPONSOR REFEREE: D. E. Games, Department of Chemistry, The Universitv of Swansea. Swansea. UK
An improved liquid chromatographic/mass spectrometric (LC/MS) method utilizing gradient elution and ion-spray ionization is described for the sensitive determination of okadaic acid and dinophysistoxin-1, the principal toxins implicated in cases of diarrhetic shellfish poisoning. The method was used to confirm the presence of both toxins, together with a recently identified isomer of okadaic acid, dinophysistoxin-2, in various samples of cultivated blue mussels (Mytilus edulis) from Canadian and European waters. The method provided a mass detection limit of 0.4 ng for each toxin, thus allowing detection of 40 ng per g of whole mussel tissue (or approximately 10ng/g if only the digestive glands were used in the assay). Quantitative results obtained by LC/MS were in good agreement with those obtained by derivatization and high-performance liquid chromatography with fluorescence detection.
The development of a combined liquid chromatographidmass spectrometric (LC/MS) method utilizing ion-spray (ISP)’ atmospheric-pressure ionization for the analysis of okadaic acid (OA)’ and dinophysistoxin1 (DTX-1) was recently r e p ~ r t e dThese .~ hydroxylated polyether compounds, the structures of which are shown below, are the principal agents responsible for
~~
Okadaic acid (OA)
H
H
CH3
Dinophysistoxin- 1 @TX-1) H
CH3
CH3
-
CH3
H
Dinophysistoxin 2 @TX-2) H
cases of diarrhetic shellfish poisoning (DSP), a severe gastrointestinal illness caused by the consumption of shellfish contaminated by feeding on toxic strains of certain dinoflagellate^.^-' Using isocratic conditions, the LC/MS method was used to confirm the presence of OA in a culture of the benthic dinoflagellate species Prorocentrum concauum and, for the first time, in natural populations of Dinophysis spp. from Eastern Canadian water^.^ With slight modifications the LC/MS NRCC 33003. Author to whom correspondence should be addressed (under contract from Sciex, 55 Glen Cameron Road, Thornhill, ON, Canada). 0951-4198/92/020121-07 $05.00 01992 by John Wiley & Sons, Ltd.
method was also used to analyse the anthrylmethyl ester of okadaic acid, consequently validating the highperformance liquid chromatographic method with fluorescence detection (HPLC-FLD) which is currently the standard instrumental technique for DSP analysis.’ The presence of DSP toxin producing strains of dinoflagellates in Canadian together with increasing aquaculture operations, provided the impetus to establish a collaborative research program between the National Research Council of Canada (NRC) and Fenwick Laboratories Ltd to investigate the chemistry of DSP toxins and to develop improved analytical procedures for their determination in marine extracts. The emphasis on instrumental techniques has become more urgent with the recent banning of animal testing in several member countries within the European community. Since our earlier report , 3 significant improvements have been made in the LC/MS methodology, resulting in lower detection limits for the underivatized DSP toxins. The technique has since been applied to more complex sample matrices, and in the present work its application to the analysis of DSP-contaminated cultivated mussels (Mytilus edulis) from European and Canadian waters is described. EXPERIMENTAL Materials The toxins OA and DTX-1 were isolated from a laboratory culture of the dinoflagellate species Prorocentrum Lima grown at the NRC Institute for Marine Biosciences. The culture conditions and details of the harvesting of this organism are similar to those previously described for P. c ~ n c a u u mO. ~A is also availReceived 26 November I991 Accepted 29 November 1991
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
122
able through Diagnostic Chemicals Ltd (Charlottetown, PEI, Canada). HPLC-grade acetonitrile was purchased from Anachemia (Lachine, PQ, Canada). A Milli-Q water purification system (Millipore Corp., Bedford, MA, USA), equipped with ion-exchange and carbon filters, was used to further purify glass-distilled water. Cultivated samples of the blue mussel, Mytilus edulis, were used exclusively in this study. Both fresh and frozen mussel tissue were examined, together with cooked mussels from ,a restaurant. Mussels harvested at affected lease sites were randomly picked from socks at varying depths. Except where indicated in the text, fresh mussels were shucked and the digestive glands excised. Frozen mussel meat was thawed at room temperature and the digestive glands similarly removed. Sample extraction The extraction procedure used in the present work was modified from that described by Lee el al.' Approximately 20 g of either whole mussel tissue or digestive glands were drained, accurately weighed and then homogenized (PolytronTM,Brinkman Instruments Ltd, Rexdale, ON, Canada) with methanol (80 mL) for 2 min at room temperature. The homogenate was transferred to a 250 mL centrifuge bottle and centrifuged for 15 min at 3000 rpm. A 50 mL portion of the supernatant liquid was combined with water (10 mL) and extracted twice with hexane (50 mL), discarding the upper layers. The aqueous methanol extract was mixed with water (20mL), and extracted twice with chloroform (80 mL). The combined chloroform extracts were taken to dryness on a rotary evaporator. The residue was transferred in methanol to a 5 mL volumetric flask and made up to the mark to give a solution of 2 g equivalent tissue per mL. For LC/MS analysis of high-level samples this methanolic extract was analysed directly; for trace-level samples a 500 pL portion of this extract was taken to dryness under a stream of dry nitrogen, and redissolved in 1OOpL of 50% aqueous methanol (10 g equivalent tissue per mL). For HPLC-FLD analysis a 50pL portion of the same extract (0.1 g e uivalent tissue) was taken to dryness in a Reacti-vial (Pierce, Rockford, IL, USA) ready for derivatization (see below). Previous concerns over the adsorption of DSP toxins on to glass surfaces' also led us to perform several duplicate extractions of standard solutions of O A (50100 pg/mL) using both glass and plastic sample vials. Results obtained by LC/MS indicated that, at the levels used in these experiments, there was no significant difference between the two materials, and glassware was used throughout this work.
9,
Derivatization and HPLC ADAM deriuatization. The derivatization reagent 9anthryldiazomethane (ADAM), was prepared as described previously.3 Briefly, the tissue extract residue (as prepared above) was dissolved in 10 yL dimethylformamide and mixed with 90 yL of ADAM reagent. After heating at 35 "C for 1 h, the solvent was removed under a stream of nitrogen. This residue was then dissolved in 1 mL hexane + chloroform ( l : l ) , transferred to a silica
solid-phase extraction cartridge (Supelco, Oakville, ON, Canada), and eluted using the procedure described by Lee and c o - ~ o r k e r sThe . ~ final fraction was evaporated and re-dissolved in 100 pL methanol (1 g equivalent tissue per mL) for analysis by HPLC-FLD. NABA deriuatization. Samples were derivatized with the N-(9-acridinyl)-bromoacetamide (NABA) reagent using the method described by Allenmark et a1." Briefly, the residue (as prepared above) was dissolved in 100 yL acetonitrile and mixed with 400 pL of 2 mM NABA in chloroform, 500 pL of 20 mM phosphate buffer (pH 7.0) and 50 pL of 10 mM tetrabutylammonium sulfate. The reaction mixture was heated to 90 "C, with stirring, for 1h. The chloroform phase was removed, taken to dryness under a stream of dry nitrogen and reconstituted in 100 yL acetonitrile prior to LC/MS. Fluorescence HPLC. Analyses were performed using a model HP1090L liquid chromatograph (HewlettPackard, Palo Alto, CA, USA) equipped with a binary DR5 solvent delivery system, a HP1046A fluorescence detector, and a HP79994A data system. Settings for the fluorescence detector were: 249 nm excitation, and 407 nm emission protected by a 280 nm cut-off filter. Separations at ambient temperature were achieved on a Merck LiChrospher 100 RP-18 250 x 4.6 mm I D column (BDH Inc., Toronto, ON, Canada) using isocratic elution with aqueous acetonitrile (10 :90) at a flow rate of 1mL/min. Liquid chromatography/mass spectrometry All LC/MC experiments were performed on a Hewlett-Packard Model 1090 Series I1 liquid chromatograph coupled to a Sciex API I11 triple quadrupole mass spectrometer (Thornhill, ON, Canada) via an Ionspray@ (ISP) interface, as previously d e ~ c r i b e d . ~ Separations of underivatized and NABA derivatized DSP toxins were achieved on a 1mm ID X 25 cm microbore column packed with 5 yrn Vydac 201TP stationary phase (Keystone Scientific, Bellafonte, PA, USA). Aqueous acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used as the mobile phase at a flow rate of 50 pL/min. A linear gradient of 40% to 100% acetonitrile in 20 mins was used, followed by a hold of 5 min. RESULTS AND DISCUSSION Gradient elution LC/MS of DSP toxins In our earlier report on the LC/ISP-MS analysis of DSP toxin^,^ the isocratic reversed-phase separation of OA and DTX-1 with 60% acetonitrile in water containing O.l%trifluoroacetic acid was described. Isocratic conditons and 2.1 mm I D columns were employed for the screening of large numbers of relatively clean plankton extracts. Since then we have focused our efforts on the use of 1mm I D columns and gradient elution to achieve improved sensitivity and compatibility with more complex samples. In addition, a fully-articulated version of the ISP interface has also been developed, allowing more precise control in the positioning of the ISP needle in relation to the sampling orifice, and thus enabling a reduction in background chemical noise. Figure 1presents the improved gradient elution separation of a mixture of OA and DTX-1 using a l mm I D reversed-phase column and selected-ion monitoring
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
75
-E 5
50
a l
i
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
lime (min)
Figure 1. Gradient elution LC/ISP-MS separation of (a) O A (20 ng) and (b) DTX-1 (25 ng) using selected-ion monitoring of their respective protonated molecules. Insert in (a) shows response from injection of 400pg of O A . Conditions: 2 5 0 1~ mm ID column packed with Vydac 201TP stationary phase; aqueous acetonitrile mobile phase containing 0.1% trifluoroacetic acid, linear gradient of 40-100% acetonitrile in 20min, at a flow rate of SOyL/min; 1 pL injection volume; dwell time, 200 ms per ion.
(SIM) of their respective protonated molecules (mlz 805 and 819). While the retention times of the toxins (11.3 and 14.4 min, respectively) are significantly longer than those obtained under the isocratic conditons, the narrower peaks obtained under the elution profile, coupled with the lower flow rate and improved optimization of the ISP interface, result in a 10-fold improvement in detection limit over that previously reported.3 The response obtained for an injection of 400pg of O A is presented in the insert in Fig. 1, and with the observed signal-to-noise ratio of 2 : l this probably represents the detection limit of the LC/MS method, i.e., approximately 0.4 pg/mL (1pL injection). As shown by the calibration curve for OA in Fig. 2, a linear response was obtained between 1 and 75 ng ( r 2= 0.9998). The reproducibility of peak areas for replicate injections of a 5 pg/mL solution of O A over the course of a day was of the order of 2.4% relative standard deviation ( n = 5 ) . The relative molar response of OA and DTX-1 was found to be very close to unity. Mussel tissue extraction The procedure used in the present work for the extraction of DSP toxins from shellfish tissue is similar to that described by Lee and co-workers for the ADAM HPLC-FLD method.' The original procedure consists of an initial dispersion of mussel tissue into aqueous
123
methanol (80%), followed by extraction with hexane, and finally a partition into chloroform. We have modified the procedure slightly, and use 100% methanol in the initial dispersion since we have found that this provides higher and more reproducible recovery of OA. At a level of 1 pg per g of tissue, recoveries of O A spiked into control mussel tissue were typically in excess of 90%. The current legal tolerance level for DSP in Europe is 0.2 pg per g of edible tissue." However, it is common practice to use only the digestive glands of the mussel in the extraction procedure (see Experimental). This stems from the original observation of Yasumoto et aZ.,4 that DSP toxicity appears to be localized to the hepatopancreas, and the fact that the HPLC-FLD method is often not sensitive enough to analyse the entire edible tissue reliably at, or below, the tolerance level. Knowledge of the weight of digestive glands in proportion to that of the whole mussel tissue then allows the concentration of toxin to be calculated in terms of the whole tissue. The HPLC-FLD method is limited by the permissible degree of pre-concentration of the extract prior to analysis due to the derivatization step. With the present clean-up procedure, the maximum amount of sample that can be derivatized reliably is 0.1 g equivalent tissue in 100 pL reagent (1 g/mL). In our hands, the detection limit available with this approach using all edible tissue has been 120 ng per g tissue. If only digestive glands are used in the extraction, and if they represent 25% of the edible tissue, the detection limit in the whole tissue is then in the order of 30 ng/g. However at this level there are numerous peaks in the chromatogram, due to derivatives of endogenous compounds and/or reagent impurities, that are of comparable size to those of the toxins and that make detection and quantitation difficult. The LC/MS method, on the other hand, with its much greater selectivity of detection and ability to analyse the toxins directly without derivatization, can accommodate much higher pre-concentration factors. Thus, in conjunction with gradient elution, it is possible
Peak Area
x 106
0
20
80
100
Amount injected (ng)
Figure2. Calibration curve for the LC/MS analysis of O A using selected-ion monitoring of the protonatcd molecule (mlz 805). Conditions as in Fig. 1.
124
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
to concentrate the final extract to 10 g equivalent tissue per mL. With a detection limit of 0.4yglmL in the extract it is possible to detect 40ng/g in the tissue. If only digestive glands are used in the assay, the detection limit expressed in terms of whole tissue equivalent is approximately 10 ng/g. The LC/MS method therefore provides a better detection limit than HPLC-FLD, allowing the analysis of whole mussel tissue samples, and does not require time-consuming and often troublesome derivatization steps. Application to DSP-toxin-contaminated shellfish Initial experiments with contaminated shellfish extracts were conducted on batches of cooked deep-frozen mussel meats from Sweden, which had been impounded by Dutch authorities after being implicated in the development of DSP symptoms by consumers and subsequently shown to produce a positive (++) response with the DSP rat bioassay.12 As part of the NRC Marine Analytical Chemistry Standards Program (MACSP), we are currently evaluating this material as a potential component of a blended mussel tissue reference material for DSP, to complement our previously released reference material for domoic acid (MUS-1I3), the toxin responsible for amnesic shellfish p ~ i s o n i n g . ' ~ This program relies on reliable analytical methods to provide accurate levels of the toxin(s) in the different matrices, at all stages of production, e.g. blending, homogeneity, stability studies and certification. In order to confirm the localization of the DSP toxins within the digestive gland of the mollusc, individual mussels were dissected and the tissue divided into three main portions, viz. digestive glands, meats, and other tissue including the juices, The three portions were individually weighed, extracted and then analysed by LClMS without preconcentration. The digestive glands of the particular mussels used for this experiment were found to constitute approximately 24% of the weight of the entire edible tissue. This proportion will of course vary from species to species, and with the time and location at which the mussels are harvested. The LC/MS selected-ion monitoring (SIM) chromatogram of rnlz 805 from the digestive gland extract, presented in Fig. 3(a), shows a strong peak at the correct retention time for O A (Fig. l(a)), which was not observed in the corresponding trace obtained from non-toxic, control mussel-tissue extracts. By external calibration it was determined that the concentration of OA was 0.96 pg/g of digestive gland or approximately 0.23 yg/g in the whole tissue. This is slightly higher than the level generally allowed in Europe (0.2 pg/g)." No DTX-1 was detected, as evidenced by the absence of a response in the corresponding mlz 819 trace (Fig. 3(b)). No toxins were detected in the 'meats', and only negligible amounts of O A were found in the 'other tissue' portion. These results clearly support the original findings of Yasumoto et aL4 The sensitivity and selectivity of the LClMS method is illustrated in Fig. 3(c), which shows the corresponding mlz 805 chromatogram obtained from the analysis of a whole tissue extract from the same mussels, concentrated to only 2 g equivalent of tissue per mL. Even without the additional preconcentration a signal is clearly observed at the correct retention time for OA. An opportunity to test the effectiveness of the
z3
30
DTX-1
20
1 1°1 0:o
z2
4:O
8.0
.
12:o
16.0
7s 50
1 1 I 0.0
4.0
8.0
12.0
16.0
Time (min)
Figure 3. Selected-ion chromatograms obtained from the LCIISP-MS analysis of (a) and (b) digestive gland and (c) wholetissue extracts from DSP-toxin-contaminated Swedish mussels. The final extract was concentrated to only 2 g equivalent tissue per mL. Conditions as in Fig. 1.
LC/MS method under more pressing conditions arose in the summer of 1990 when several people developed severe DSP symptoms after eating cultivated mussels 'harvested in Nova Scotia. Mussel samples from an affected restaurant (cooked), a domestic residence, and also the lease site from which the mussels were subsequently traced, were shown by Fisheries and Oceans Canada to be toxic to mice (intra-peritoneal i n j e ~ t i o n ) .Bacterial '~ gastroenteritis was ruled out, and the NRC was asked for analytical support. The results of the LC/MS analysis of a digestive gland extract from mussels harvested at the (quickly closed) lease site are presented in Fig. 4. While no OA was detected ( m / 2805; Fig. 4(a)), the presence of DTX-1 was clearly indicated by the strong signal in the mlz 819 trace (Fig. 4(b)) at the correct retention time for the authentic toxin. DTX-1 was similarly detected in the cooked mussels from the restaurant. Full-scan LC/MS analysis of a concentrated extract provided the mass spectrum shown in Fig. 4(c), in which the protonated molecule (MH') is clearly observed. The ions at mlz 801 and 783, due to successive losses of water, are characteristic of the tetrahydroxylated DSP toxins.3,16, l7 The ion at mlz 837 is assigned to a mobile-phase cluster ion i.e., [MH f H20]'. The toxin was subsequently isolated and its structure confirmed by additional spectroscopic techniques, including nuclear magnetic resonance. Details of this incident, which to our knowledge is the
IONSPRAY MASS SPECTROMETRY OF MARINE TOXINS. IV
first confirmed incident of DSP in North America, have been reported recently." More recently, we have received samples of mussel digestive glands from Ireland which had already been shown to be contaminated with O A by the ADAM HPLC-FLD method. Interestingly, an additional peak was detected in the fluorescence chromatograms which did not correspond to any of the known DSP toxins. The fluorescence trace subsequently obtained in our laboratory is shown in Fig. 5(a), in which a second peak is clearly observed eluting after that corresponding to the ADAM-OA derivative. The appearance of the peak suggests that this component _also has a free carboxyl functionality which is necessary for the ADAM derivatization. The corresponding LC/MS-SIM chromatograms for this extract are shown in Fig. 5(b) and (c), respectively, and it can be seen that two peaks are observed in the mlz 805 chromatogram of similar relative intensities to those observed in the fluorescence trace. The first peak again appears at the correct retention time for authentic O A , supporting the assignment. The presence of DTX-1 is also indicated by the appearance of a weak signal at the correct retention time in the mlz 819 chromatogram; however, it was not observed in the corresponding fluorescence trace due to its relatively low concentration in the extract. Full-scan analyses were performed on this sample, and the
125
fa)
R"' 0.0
4.0
8.0
12.0
16.0
Figure 5. Analysis of digestive-gland extract of DSP-toxin-contaminated Irish mussels by (a) ADAM derivatization and HPLC-FLD and by (b) and (c) LC/ISP-MS using selected-ion monitoring. The final extract for LC/MS analysis was concentrated to l o g equivalent tissue per mL. HPLC-FLD conditions as in Experimental. LC/MS conditions as in Fig. 1 .
4:O
8:O
16.0
12.0
T i (min) 819
[M+HI+
m/z
Figure4. (a) and (b) Analysis of digestive-gland extract from DSP-toxin-contaminated Canadian mussels by LC/ISP-MS using selected-ion monitoring. The final extract was concentrated to 2 g equivalent per mL. (c) The mass spectrum of the DTX-1 peak obtained from a full-scan analysis. LC conditions as for Fig. 1. Dwell times (a) and (b) 200 ms per ion, (c) 8 ms/Da over a range of 500900 m / z .
second peak in the mlz 805 chromatogram provided a mass spectrum identical to that for O A , suggesting its interpretation as a possible isomer of the toxin as opposed to a cluster ion of a lower or a fragment ion of a higher molecular weight analyte, respectively. This compound has been successfully isolated and purified, and its structure determined to be a previously unreported positional isomer of OA which has been termed dinophysistoxin-2 (DTX-2; see structures).l8 During this investigation, quantitative results on the various mussel samples obtained by LC/MS were compared with those obtained by HPLC-FLD using the ADAM derivatization method. The results obtained by both techniques on the various mussel extracts are presented in Table 1. In general there is good agreement between these two independent techniques. The only serious discrepancy was between values for DTX-1 in the Canadian mussels; this may have been due to the fact that the analyses were performed several days apart. The better sensitivity of the LC/MS method allowed the detection of minor components, such as DTX-1 in the Irish mussel sample, that could not be reliably detected using the HPLC-FLD method. As discussed above, the main problem encountered with the latter method is the presence of numerous small peaks throughout the chromatogram due to derivatives of endogenous compounds and/or reagent impurities, which can complicate analysis. The higher selectivity of
IONSPRAY MASS SPECTROMETRY O F MARINE TOXINS. IV
126
the LC/MS method, which utilizes selected-ion monitoring of the high-mass protonated molecules, effectively eliminates most chemical interference.
7i 5
LC/MS of fluorescent derivatives As part of our research program we are also investigating reagents for the fluorescent labelling of DSP toxins that may be used as alternatives to the ADAM reagent currently used in the HPLC-FLD method described by Lee et al.' The ideal choice would be a universal derivative that can be used for HPLC with either fluorescence, ultraviolet and/or ISP-mass spectrometric detection. One reagent currently being evaluated is N-(9-acridinyl)-bromoacetamide (NABA), previously described by Allenmark and co-workers for the phasetransfer-catalysed fluorescence labelling of carboxylic acids for liquid chromatography." The authors' choice of reagent was based on the fact that the starting material, 9-aminoacridine, is one of the most fluorescent componds known and that the NABA-derivatized analytes, when protonated, have good retention characteristics on reversed-phase columns. It is the protonated nitrogen which is of interest with respect to additional sensitivity under (positiveion) ISP ionization conditions, since the ion-evaporation process19 favours pre-formed ions in solution. Previous experiments with the ADAM-OA derivative were disappointing, in that the LC/MS sensitivity for the derivative was very similar to that of the underivatized toxin. A mixture of A 0 and DTX-1 was derivatized with NABA and analysed by LC/MS, monitoring the protonated molecules of the individual derivatives (mlz 1039 and 1053, respectively). The results are shown in Fig. 6(a) and (b). Both toxins were detected with characteristic shifts in mass and retention time. It can also be seen that the derivatives are well resolved under this elution profile, and have good peak shapes. Comparison of the LC/MS peak areas with those of underivatized standards indicated that a 5-fold increase in sensitivity was obtained using the NABA derivative. Contaminated mussel extracts were also derivatized with the NABA reagent, and Fig. 6 also shows the results of the LC/MS analysis of the Nova Scotian mussels previously found (Fig. 4) to contain DTX-1.
Table 1. Comparison of quantitative results obtained by HPLC-FLD and LC/MS methods for the analysis of OA and its analogues in DSP-toxin-contaminated cultivated blue mussels Sample
Swedish' Canadian
Conccntration (vg/g)'l. DTX- 1
Mcthod
OA
HPLC-FLD LC/MS
1.52 k 0.05 1.40k0.09
HPLC-FLD LC/MS
NDd ND
DTX-2
ND~ ND
ND ND
0.67+0.07 1.08+0.04
ND ND
HPLC-FLD 12.7 k 1.2 ND 8.5 2 0.8 LC/MS 12.420.4 0.3720.01 8.420.4 Concentration is reported for digestive glands on a wet weight basis. Mean f standard deviation reported for triplicate analyses of a single extract. Preliminary reference material having undergone autoclaving heat treatment. N D = not detected. Irish
36
II NABA-DTX-1
iwz 1053
I
21 18
9 0.0
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DTX-I 20
IS 10
NABA-DTX-1
; 2
2s
0.0
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Time (min)
Figure6. (a) and (b) Analysis of NABA derivatized O A and DTX-1, and (c) and (d) NABA derivatized digestive-gland extract from DSP-toxin-contaminated Canadian mussels by LC/ISP-MS. Conditions as in Fig. 1.
Comparison of the m/z 1053 traces for the mussel extract (Fig. 6(d)) and the derivatized standard (Fig. 6(b)) clearly indicates the presence of the NABA-DTX-1 derivative. These experiments provide additional confirmatory evidence for the toxin's identity. The protonated molecule of the underivatized toxin (rnlz 819) was also monitored during these experiments, and it can be seen from the corresponding trace for the mussel extract (Fig. 6(c)) that none of the underivatized DTX-1 was detected, indicating that derivatization was complete. This ability to monitor both reactant and product in a derivatization reaction is a useful aid to the chemist in developing the optimal reaction conditions. These preliminary results with the NABA derivative appear encouraging, although further experiments are required to confirm that any LC/MS sensitivity gained with the use of the derivative is not compromised by losses and dilution factors resulting from the additional derivatization step in the methodology, Further details on the fluorescent labelling of DSP toxins, including the use of other derivatizing agents, will be published in due course. CONCLUSION It has been shown that gradient elution in conjunction with the lower flow rates utilized with 1mm ID microbore columns, together with the improved optimization
IONSPRAY MASS SPECTROMETRY O F MARINE TOXINS. IV
of the ionspray interface, provide significantly improved LC/MS detection limits for underivatized DSP toxins over those obtained previously using isocratic elution, and better than those available with the HPLC-FLD method using ADAM derivatization. The improved detection limits allow the confirmatory analysis of whole mussel tissue. While there is no need to derivatize mussel extracts using the LC/MS method, derivatization with NABA can be useful for additional confirmatory evidence and may also provide greater sensitivity under ionspray ionization. The use of gradient elution is particularly valuable for screening complex samples for a wide range of toxins, and one of the real benefits of the combined LC/MS approach in our research program lies in the detection of related compounds at low levels in shellfish extracts. This is illustrated by the detection of the previously unreported DSP toxin, DTX-2, which as a consequence also validates the HPLC-FLD procedure for the analysis of this toxin in shellfish tissue. Numerous other related compounds have been similarly identified and will be reported separately. In many cases the isolation of these compounds has been achieved by simply scaling up the chromatography to preparative columns. The results presented here indicate significant variation in the toxin profiles of DSP-contaminated shellfish from different locations. The improved sensitivity of the LC/MS technique will hopefully allow us to examine more closely the causes of the observed variation in toxin profile, and perhaps lead to a greater understanding of the role of these compounds in the source dinoflagellates.
Acknowledgements The authors are grateful to Ms L. McDowell (Fenwick), Mrs P. Blay (Sciex) and Mr W. Hardstaff (NRC) for technical assistance, and to Dr A. Jackson (NRC) for help with dissections. We thank Drs T. Hu (Fenwick) and J. L. C. Wright (NRC) for the purified DSP toxins, and Dr M. W. Gilgan (Fisheries and Oceans Canada) for help with the development of extraction procedures. The contaminated Irish mussels were kindly provided by Drs J. Doyle and E. Nixon of the Department of the Marine Fisheries Research Centre, Abbotstown,
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Dublin, Ireland. Impounded Swedish mussel tissue was provided by Dr P. Hagel of the Netherlands Institute for Fisheries Research (RIVO), Ijmuiden, the Netherlands.
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