The combination of liquid chromatography and mass ... calibration standards and certified reference materials. (CRMs) ... chromatography-mass spectrometry.
IN: HARMFUL ALGAE MANAGEMENT AND MITIGATION. S. HALL, S. ETHERIDGE, D. ANDERSON, J. KLEINDINST, M. ZHU, Y. ZOU (EDS.), ASIA-PACIFIC ECONOMIC COOPERATION (SINGAPORE): APEC PUBLICATION #204-MR-04-2, PP. 171-173 (2004). LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS): A UNIVERSAL METHOD FOR THE ANALYSIS OF TOXINS?
Michael A. Quilliam Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford St., Halifax, Nova Scotia, B3H 3Z1 ABSTRACT The utility of the liquid chromatography-mass spectrometry (LC-MS) is first reviewed in terms of its advantages and disadvantages. Then a method is presented for the simultaneous analysis of a wide range of shellfish toxins in a single analysis. INTRODUCTION The combination of liquid chromatography and mass spectrometry (LC-MS) has proven to be a powerful tool for the detection and quantitation of toxins in plankton and shellfish at part-per-billion levels, the identification of new toxins, and the investigation of toxin metabolism in shellfish [1-3]. LC-MS methods have been developed for the following toxins: domoic acid and other ASP toxins; okadaic acid and related DSP toxins, including DTX1-5 toxins; pectenotoxins; azaspiracids; yessotoxin; saxitoxin and other PSP toxins; brevetoxins; gymnodimine and spirolides; and ciguatoxins. LC-MS is in fact the only analytical method that has been shown to be suitable for the analysis of all toxins. LC-MS meets all the needs of laboratories involved in both monitoring and toxin research: • universal detection capability • high sensitivity • high selectivity and specificity • minimal sample preparation • ability to deal with the structural diversity and labile nature of toxins • separation of complex mixtures of toxins • precise and accurate quantitation • wide linear range • automation • high throughput • rapid method development • legal acceptability for confirmation • structural information for identification of new toxins, analogs and metabolites The high capital cost of LC-MS systems does present difficulties for many laboratories but the recent introduction of less expensive, easy-to-use “bench-top” instruments should help to reduce these problems. In addition, the actual cost per sample must be considered carefully when comparing different methods. Analyses by LC-MS can be very rapid (as low as 2-3 min in some cases) and can be totally automated, resulting in a very
low cost per sample. The cost of LC-MS analyses is also low because minimal sample preparation is required, compared to other analysis methods based on complicated cleanup and derivatization schemes. This feature is also important for sample throughput, as sample preparation is usually the major bottleneck in most laboratories. An additional item of importance to some laboratories is that an LC-MS system can be used for both research and monitoring work, and for a variety of analytes, not just toxins. An LC-MS instrument certainly does need a steady flow of samples to justify its purchase, unless it is also used for research. Obviously the LC-MS option is best suited to a central laboratory that would have the technical expertise and the workload to justify such an instrument. One of the most appealing features of LCMS to many laboratories is the possibility that a wide range of methods could be replaced by just one instrument. Caution must be exercised here, as complete reliance on one instrument could present difficulties in that any breakdown could result in delays during a crucial monitoring program. However, our experience is that the relatively inexpensive modern LC-MS systems are very reliable and give a 90%+ up-time; service contracts on such instruments usually allow a 24-48 hr repair. The bottleneck of using just one instrument can also be a problem, but with automation and high-speed analyses, an LC-MS system can deal with many emergencies. A laboratory basing its entire operation on LC-MS, may wish to have 2 instruments in any case, one for routine monitoring and another for research and methods development as well as being a backup to the routine system. Of course this is a difficult proposition for many institutions that cannot even afford an LC system! The combination of rapid assay methods for screening and an LC-MS system in a central laboratory for research and confirmation may be the best route to follow for many economies. Another cautionary note is that a fair amount of technical expertise is involved in operating an LC-MS system, although the newer bench-top LC-MS systems are becoming very easy to use and maintain. In our laboratory, we have many instrument users, including temporary summer student assistants. Training usually takes no more than 1-2 days for routine use. On the other hand, research analytical chemists generally carry out the method development work. One final concern, which applies to all instrumental methods and many assay methods, is the shortage of calibration standards for marine toxins. The development of readily availalable
calibration standards and certified reference materials (CRMs) will be essential for the full implementation of LC-MS methods in regulatory laboratories. Whatever the opinions about investing in LC-MS systems as a tool for routine toxin analysis, most people will agree that this technique will continue to play a significant role in the future of toxin research. This paper will present an LC-MS method for the simultaneous analysis of many shellfish toxins, a procedure that could replace several individual methods. EXPERIMENTAL Plankton and shellfish samples were extracted with three portions of methanol/water (8:2) with a final volume/sample ratio of 10 (e.g., 1 g extracted into 10 ml solvent). After filtration, the extract was injected directly into the LC-MS system. The LC column was a Keystone Scientific 50 x 2 mm Quicksilver cartridge packed with 3 µm Hypersil-BDS-C8, equipped with a guard column. The mobile phases were as follows: A = water and B= acetonitrile:water (95:5), both containing 2 mM ammonium formate and 50 mM formic acid. Gradient elution from 5% to 100% B was performed over 10 min and then held at 100% B for 10 min, before returning to initial conditions and equilibrating for 7 minutes. The flow rate was 0.2 ml min-1 and the injection volume was 5 µl. The LC system was a HP 1100 with binary pump, variable volume injector and autosampler. The mass spectrometer was a Perkin-Elmer Sciex API165 single quadrupole bench-top system equipped with ion-spray source. Eluent from the LC was split 10:1 with 20-30 µl min-1 going to the MS. Selected ion monitoring was used for data acquisition. RESULTS AND DISCUSSION My current research has been directed towards finding a single column and mobile phase that would be suitable for a wide range of toxin families, in both plankton and shellfish samples. Using short columns and rapid gradients, it is possible to analyze several toxins in one extract by one LC-MS analysis. Of course, one limitation of this multi-toxin approach lies not in the LC-MS system, but in sample preparation – i.e., finding a universal extraction solvent and cleanup scheme that will give good recovery for all
toxins. In reality, however, it is unlikely that all toxins would have to be monitored at once – most locations only suffer from one to three different types of toxins. We have now developed preliminary extraction and LCMS methods for domoic acid, gymnodimine, spirolides, the okadaic acid family of toxins, pectenotoxins, azaspiracids, and yessotoxin in one group and the very polar PSP toxins and tetrodotoxin in a second group. Figure 1 illustrates application of the technique to a mussel tissue extract spiked with assorted toxins. The method has proven to be very sensitive and provides a high degree of certainty in toxin detection. Further confirmation of toxin identity or the identification of new toxin analogues can be assisted with the use of tandem mass spectrometry (MS/MS). Accurate quantitation is reliant on the availability of accurate calibration standards. However, estimation of concentrations of toxin analogues can be achieved through the use of estimated relative molar response factors. Further method validation in a regulatory setting is still required of course. Additional research at IMB, under the auspices of the Certified Reference Materials Program, is being directed towards the development of a series of calibration solution CRMs for world-wide distribution. REFERENCES 1. Quilliam, M. A. 1996. Liquid chromatography-mass spectrometry of seafood toxins. In: Barcelo, D. (ed.), Applications of LC-MS in Environmental Chemistry, pp. 415-44. Elsevier Science Publ. BV, Amsterdam. 2. Quilliam, M. A. 1998, Liquid chromatography-mass spectrometry: a universal method for the analysis of toxins?, In: Reguera, B.; Blanco, J.; Fernandez, M. L., Wyatt, T. (eds.), Harmful Algae (Proc. Eighth International Conference on Harmful Algae, Vigo, Spain, June 25-29, 1997), pp. 509-14. Xunta de Galicia and IOC/UNESCO. Vigo. 3. Quilliam MA, Hess P, Dell’Aversano C. 2001, Recent developments in the analysis of phycotoxins by liquid chromatography-mass spectrometry. In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Century, deKoe WJ, Samson RA, van Egmond HP, Gilbert J, Sabino M (Eds.). deKoe WJ, Wageningen, The Netherlands., pp. 383-391.
C16:0-OA AZA1
DTX1
PTX2 C18:1
OA DTX2 SpiroD SpiroB DA
C16:0-DTX2
"DTX3"
PTX2sa
C16:1
AZA2
1086.7 1060.7 1158.7
AZA3
856.5
842.5 828.5
m/z 876.5 894.5 836.5 822.5 708.5 694.5 312.1
0
5
10 Time (min)
15
20
Figure 1. A gradient elution LC-MS analysis of an extract of mussel tissue spiked with assorted toxins: domoic acid (DA), spirolides (B and D), okadaic acid (OA), dinophysistoxin-1 and –2 (DTX1 and DTX3), pectentoxin-2 (PTX2) and its seco acid (PTX2sa) and azaspiracids (AZA).
