Multidimensional Biochemical Detection of Microcystins in Liquid

and protein phosphatase 1 (PP1) were used as off-line detectors for the HPLC separation of microcystins and nodularin in comparison to UV detection...
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Anal. Chem. 2001, 73, 5509-5517

Multidimensional Biochemical Detection of Microcystins in Liquid Chromatography Anne Zeck, Michael G. Weller,* and Reinhard Niessner

Institute of Hydrochemistry, Technical University of Munich, Marchioninistrasse 17, D-81377 Mu¨nchen, Germany

The coupling of antibody-, receptor-, or enzyme-based inhibition assays postcolumn to chromatographic systems provides biological detectors with extraordinary high sensitivity and specificity. Three monoclonal antibodies (MC10E7, AD4G2, M8H5) directed against microcystins and protein phosphatase 1 (PP1) were used as off-line detectors for the HPLC separation of microcystins and nodularin in comparison to UV detection. For HPLC/ ELISA coupling using antibody MC10E7, a detection limit of 0.04 ng microcystin-LR was achieved. The provisional guideline value for microcystin-LR (1 µg/L, WHO) could be monitored without prior sample concentration, in contrast to UV detection. Quantification of microcystinLR and two cross-reactants was demonstrated. Furthermore, cross-reactivity or enzyme inhibition of new microcystins, only available in small amounts, can be determined by this method. Using a cyanobacterial extract, HPLC/ ELISA coupling was compared to HPLC/UV and electrospray ionization mass spectrometry (ESI-TOFMS). The analysis of microcystins, extremely hepatotoxic cyclic heptapeptides, is of growing interest for water works and surveillance authorities, because microcystins can contaminate drinking water reservoirs or lakes belonging to recreational areas. Microcystins are produced by cyanobacteria (“blue-green algae”) and are released into the surrounding water when cyanobacteria die naturally or as a result of algicide treatment. To assess the hazard potential of surface water, the concentration of microcystins released in water as well as the cellular microcystin concentration have to be determined. Thus, for analysis of cellular microcystins, sample extraction and separation (cleanup) from complex biological matrixes has to be performed prior to their detection and quantification. A widely used cleanup procedure is solid-phase extraction (SPE) with reversed-phase cartridges, as described by Lawton et al.1 As in many cases, a one-step cleanup is not effective in completely removing biological matrixes; the solid-phase extraction is often combined with additional cleanup steps. Tsuji et al.2 combined C18 SPE with a cleanup step on silica gel, but more often, reversed-phase chromatography is used in combination with size exclusion chromatography on Sephadex G-25 or LH-20, Superdex Peptide, or Toyopearl HW-40F.3-7 Sometimes * E-mail: [email protected]. (1) Lawton, L. A.; Edwards, C.; Codd, G. A. Analyst 1994, 119, 1525-1530. (2) Tsuji, K.; Naito, S.; Kondo, F.; Watanabe, M. F.; Suzuki, S.; Nakazawa, H.; Suzuki, M.; Shimada, T.; Harada, K. I. Toxicon 1994, 32, 1251-1259. (3) Gregson, R. P.; Lohr, R. R. Comp. Biochem. Physiol. 1983, 74C, 413-417. 10.1021/ac015511y CCC: $20.00 Published on Web 10/06/2001

© 2001 American Chemical Society

up to five cleanup steps have been performed prior to analysis of microcystins. In view of these procedures including steps with low efficiency, more selective cleanup methods seem to be desirable. Antibodies that recognize microcystins can serve this purpose. To date, several attempts of microcystin cleanup and enrichment with immunoaffinity cartridges have been published.8-10 A limiting point in using immunoaffinity extraction is the availability of a sufficient amount of antibody with a broad recognition pattern for the analytes of interest. However, another approach using immunological methods and conventional HPLC could be helpful. Antibodies can be used for immunodetection coupled postcolumn to chromatography. Performing an immunoassay after chromatography has the advantage that sensitivity can be 501000-fold better in comparison to UV detection. Since the matrix effects can be minimized by dilution, lavish sample enrichment and cleanup are no longer necessary. The procedure can be performed in all laboratories possessing HPLC and ELISA equipment. It can even be performed with usual ELISA test kits. If more than one antibody is available, the sample can be analyzed in parallel by splitting the HPLC eluate or the collected HPLC fractions. Immunological quantification of microcystins can be achieved if the cross-reactivity pattern of the employed antibody is known. In the case of microcystin analysis, an enzyme inhibition assay using protein phosphatases can be coupled to the chromatography, as well. This provides a direct monitoring of a toxicityrelated parameter. Identification and quantification of biomolecules in complex mixtures are a challenge not only in environmental analysis but also in medical research or natural-products-based drug discovery. Several attempts have been made to combine separation techniques with biosensor detection. Early works using steroids, such as digoxin,11,12 19-nortestosterone,13,14 or eicosanoids15 as analytes (4) Krishnamurthy, T.; Carmichael, W. W.; Sarver, E. W. Toxicon 1986, 24, 865-873. (5) Brooks, W. P.; Codd, G. A. Lett. Appl. Microbiol. 1986, 2, 1-3. (6) Namikoshi, M.; Rinehart, K. L.; Sakai, R.; Stotts, A. M.; Dahlem, V. R.; Beasley, V. R.; Sivonen, K.; Carmichael, W. W.; Evans, W. R. J. Org. Chem. 1992, 57, 866-872. (7) Hummert, C.; Reichelt, M.; Legrand, C.; Graneli, E.; Luckas, B. Chromatographia 1999, 50, 173-180. (8) Rivasseau, C.; Hennion, M. C. Anal. Chim. Acta 1999, 399, 75-87. (9) Kondo, F.; Matsumoto, H.; Yamada, S.; Tsuji, K.; Ueno, Y.; Harada, K. Toxicon 2000, 38, 813-823. (10) Tsutsumi, T.; Nagata, S.; Hasegawa, A.; Ueno, Y. Food Chem. Toxicol. 2000, 38, 593-597. (11) Nelson, H. A.; Lucas, S. V.; Gibson, T. P. J. Chromatogr. 1979, 163, 169177. (12) Stone, J. A.; Soldin, S. J. Clin. Chem. 1988, 34, 2547-2551. (13) Meyer, H. H. D.; Hartmann, F. X.; Rapp, M. J. Chromatogr. 1989, 489, 173-180.

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were published in the biomedical field as well as in food chemistry performing mycotoxin analysis.16,17 In most cases, coupling of chromatography with immunoassay was achieved by manual fraction collection, evaporation of solvent, and subsequent dilution of fractions with a suitable buffer. For environmental analysis, HPLC/ELISA coupling was first published by Kraemer et al.18 They used nitrophenols as analytes and performed isocratic HPLC with off-line coupling. The acidic HPLC fractions were neutralized and measured without dilution. For quantification, standard solutions with an equal solvent content were used. This method was, therefore, limited to isocratic elution. Application of HPLC coupled postcolumn with protein phosphatase inhibition assay was first performed for okadaic acid and related diarrhetic shellfish toxins by Holmes19 and for microcystin analysis by Sherlock et al.20 The latter work shows clearly the superiority in selectivity and specificity of biological detection over UV detection. The authors compared the UV and the enzyme inhibition assay chromatograms obtained from a bloom extract purified by size exclusion chromatography. The enzyme inhibition trace was obtained after 1000-fold dilution of the HPLC fractions and shows certain sharp peaks, whereas the UV chromatogram shows many peaks. Similar results were published by Nagata et al.21 with HPLC/ELISA coupling. The authors used the monoclonal anti-microcystin-LR antibody M8H5 for qualitative immunological detection of microcystins coupled postcolumn to HPLC. Nowadays, the efforts in this working field are mainly concentrated on the postcolumn on-line coupling of enzyme-, antibody-, or receptor-based assays to HPLC.22-26 In most bioassays that are coupled on-line to HPLC, free and bound label used for the detection have to be separated. This can be achieved with an additional chromatographic step or by using hollow fibers. Another approach published recently27 gets by without a separation step but takes advantage of the phenomenon of fluorescence energy transfer for the separate detection of bound and free label. Reviews dealing with these new analytical tools have been published by Fishman et al.,28 Shahdeo et al.,29 Hage,30 and Weller.31 (14) Rapp, M.; Meyer, H. H. D. J. Chromatogr. 1989, 489, 181-189. (15) Gelpi, E.; Ramis, I.; Hotter, G.; Bioque, G.; Bulbena, O.; Rosello, J. J. Chromatogr. 1989, 492, 223-250. (16) Park, J. J.; Chu, F. S. J. AOAC Int. 1996, 79, 465-471. (17) Yu, J.; Chu, F. S. J. Assoc. Off. Anal. Chem. 1991, 74, 655-660. (18) Kraemer, P. M.; Li, Q. X.; Hammock, B. D. J. AOAC Int. 1994, 77, 12751287. (19) Holmes, C. F. B. Toxicon 1991, 29, 469-478. (20) Sherlock, I. R.; James, K. J.; Caudwell, F. B.; Mackintosh, C. Nat. Toxins 1997, 5, 247-254. (21) Nagata, S.; Tsutsumi, T.; Hasegawa, A.; Yoshida, F.; Ueno, Y.; Watanabe, M. F. J. AOAC Int. 1997, 80, 408-417. (22) Irth, H.; Oosterkamp, A. J.; van der Welle, W.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 633, 65-72. (23) Irth, H.; Oosterkamp, A. J.; Tjaden, U. R.; van der Greef, J. Trends Anal. Chem. 1995, 14, 355-361. (24) Lutz, E. S.; Irth, H.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. A 1996, 755, 179-187. (25) Oosterkamp, A. J.; Herraiz, M. T. V.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 1201-1206. (26) Oosterkamp, A. J.; Irth, H.; Heintz, L.; Marko-Varga, G.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 4101-4106. (27) Graefe, K. A.; Tang, Z.; Karnes, H. T. J. Chromatogr. B 2000, 745, 305314. (28) Fishman, H. A.; Greenwald, D. R.; Zare, R. N. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 165-198. (29) Shahdeo, K.; Karnes, H. T. Mikrochim. Acta 1998, 129, 19-27. (30) Hage, D. S. J. Chromatogr. B 1998, 715, 3-28.

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Figure 1. General structure of microcystins. R1, R2, R3 ) CH3 or H. X, Y ) L-amino acids.

This work describes results obtained with off-line coupling of postcolumn immuno- and enzyme assays to chromatography of microcystin-containing samples. The method described here is robust and easy to perform, rather than technically sophisticated. Nevertheless, it has a certain degree of automation, because fractions are collected with a fraction collector into the wells of up to three 96-well microtiter plates. The fractions can easily be transferred to antibody-coated microtiter plates using multichannel pipets. It could be shown that HPLC/ELISA is a valuable tool for quantification and structural characterization of low concentrations of microcystins in a mixture. Furthermore, the HPLC/ELISA can be used for a rapid estimation of cross-reactivities. EXPERIMENTAL SECTION Abbreviations. Adda, (2S,3S,8S,9S)-3-Amino-9-methoxy-2,6,8trimethyl-10-phenyldeca-(4E,6E)-dienoic acid; BSA, bovine serum albumin; CR, cross-reactivity; DTT, DL-dithiothreitol; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; IC50, 50% inhibition, midpoint of the sigmoidal calibration curve; MC, microcystin; PP, protein phosphatase; SPE, solid-phase extraction; TFA, trifluoroacetic acid; TOF, time-of-flight. Microcystins. Microcystins have the general structure (Figure 1) cyclo(-D-Ala-L-X-D-erythro-β-methylisoAsp-L-Y-Adda-D-iso-Glu-NmethyldehydroAla), where X and Y are the one-letter codes of the variable amino acids (microcystin-XY). MC-LR (purity g98%), MC-RR (purity g97%), MC-LF (purity g95%), and MC-LW (purity g95%) isolated from Microcystis aeruginosa were supplied by Alexis (La¨ufelfingen, Switzerland, purity tested by HPLC, 238 nm). MCRR, MC-YR, MC-LA isolated from M. aeruginosa and nodularin (cyclo(-D-erythro-β-methylisoAsp-L-Arg-Adda-D-iso-Glu-N-methyldehydrobutyrine)) isolated from Nodularia spumigena (all g95% by HPLC) were supplied by Calbiochem (La Jolla, CA). Adda32,33 was obtained from Dr. D. Cundy and Dr. T. McCarthy (CSIRO Molecular Science, Clayton South, Victoria, Australia). Reagents. Tween 20, 3,3’,5,5’-tetramethylbenzidine (TMB), and hydrogen peroxide (30%) were purchased from Merck (Darmstadt, Germany); goat anti-mouse IgG (Fc fragment), from ICN Pharmaceuticals (Eschwege, Germany); and horseradish (31) Weller, M. G. Fresenius’ J. Anal. Chem. 2000, 366, 635-645. (32) Cundy, D. J.; Donohue, A. C.; McCarthy, T. D. Tetrahedron Lett. 1998, 39, 5125-5128. (33) Cundy, D. J.; Donohue, A. C.; McCarthy, T. D. J. Chem. Soc., Perkin Trans. 1 1999, 559-567.

peroxidase (HRP), from Roche Diagnostics (Mannheim, Germany). Chemicals for the preparation of the buffers as well as trifluoroacetic acid (puriss. p.a. for HPLC; g99.0%) were supplied in the highest purity available from Fluka (Neu-Ulm, Germany). Acetonitrile (Chromasolv for HPLC, gradient grade) was purchased from Riedel-de Hae¨n (Seelze, Germany); protein phosphatase 1 (catalytic subunit, R-isoform) and p-nitrophenyl phosphate (di(tris) salt) and DL-dithiothreitol (>99%) were purchased from Sigma (Steinheim, Germany). The peroxidase tracer (MCLR-peroxidase conjugate) was synthesized as previously reported.34 Antibodies. The monoclonal anti-microcystin-LR antibody MC10E7 was described in ref 34 and is available from Alexis (La¨ufelfingen, Switzerland). Cell culture supernatant from hybridoma cells containing 250 mg/L MC10E7 was stored after an addition of 0.1% (w/v) NaN3 at 4 °C. Monoclonal anti-microcystinLR antibody M8H5 (9.87 g/L, affinity purified) was kindly provided by Dr. S. Nagata, was characterized in refs 35, 36, and is commercially available as Microcystin ELISA Kit37 from Wako (Richmond, VA). The monoclonal anti-Adda antibody AD4G2 was described in ref 38. The cell culture supernatant containing 4.7 mg/L AD4G2 was stored at 4 °C after addition of 0.1% (w/v) NaN3. Apparatus. Flat-bottomed polystyrene ELISA plates (96-well) with high binding capacity were purchased from Greiner (Nu¨rtingen, Germany). A Columbus washer for microtiter plates, an Easyshaker EAS 2/4, and a Reader 340 ATTC for microtiter plates controlled by a personal computer containing the standard software package EasySoftware from SLT (Gro¨ding/Salzburg, Austria) were used. All data processing was done using Origin 6.0 (Microcal Software Inc., Northampton, MA). HPLC measurement was performed using an on-line degasser from ERC (Alteglofsheim, Germany), an L-6200A Intelligent Pump and a UV-vis detector L-4250 from Merck (Darmstadt, Germany), using ChromStar software from SCPA (Stuhr, Germany). HPLC fractions were collected in microtiter plates using a fraction collector (model 2128) from Bio-Rad Laboratories (Hercules, CA). The column used for chromatography was a Discovery RP Amide C16, 5 µm, 25 cm × 4.6 mm from Supelco (Bellefonte, PA). Mass spectra were obtained using an electrospray time-of-flight mass spectrometer LCT, Micromass (Manchester, U.K.). Liquid flow (18 µL/min) was achieved with a syringe pump, model 11, from Harvard Apparatus (Holliston, MA). Parameters for ESI-TOF: polarity, ES-; capillary voltage, 3100 V; sample cone voltage, 60 V; RF lens, 200 V; extraction cone voltage, 2 V; source temp., 100 °C; desolvation temp., 350 °C; nebulizer gas flow, 96 L/h; and desolvation gas flow, 412 L/h. HPLC Method for Microcystins and Collection of Fractions. Water containing 0.1% TFA (A) and acetonitrile containing 0.04% TFA (B) were used as solvents. Starting condition (minute 0 to 1) was 90% A and 10% B, and thereafter, the concentration of A decreased linearly to 10% A at minute 60. UV absorbance was (34) Zeck, A.; Eikenberg, A.; Weller, M. G.; Niessner, R. Anal. Chim. Acta 2001, 441, 1-13. (35) Nagata, S.; Soutome, H.; Tsutsumi, T.; Hasegawa, A.; Sekijima, M.; Sugamata, M.; Harada, K. I.; Suganuma, M.; Ueno, Y. Nat. Toxins 1995, 3, 78-86. (36) Weller, M. G.; Zeck, A.; Eikenberg, A.; Nagata, S.; Ueno, Y.; Niessner, R. Anal. Sci. 2001, in press. (37) Wako BioProducts. Product Information; Wako Chemicals: Richmond, VA, 1998. (38) Zeck, A.; Weller, M. G.; Bursill, D.; Niessner, R. Analyst 2001, in press.

recorded at 238 nm. The fraction collector was connected to the outlet of the UV detector and was programmed to collect 5 (or 2) drops (∼130 or 50 µL) per well of the microtiter plates in the full retention time window of microcystins. In this case, 2 microtiter plates are needed for collection. It has to be stressed that the UV signal is not necessary for fractionation. Prior to the collection of fractions, 120 µL (or 100 µL) of a neutral buffer (phosphatebuffered saline, pH 7.6, or TRIS buffer, pH 7.4) was added to the wells of the microtiter plate to neutralize the acidic HPLC eluate immediately. After fraction collection, the microtiter plates were sealed and stored at 3 °C until the antibody or enzyme inhibition assay was carried out. HPLC/ELISA and ESI-TOF of an Environmental Sample. To 7.2 mg of freeze-dried cyanobacteria (Microcystis sp., bloom 1993, Australia), 1 mL of a methanol/water mixture (3:1) was added and sonicated for 8 min. After centrifugation, the supernatant was diluted 1:1 with water, and 20 µL was injected into the HPLC. Five drops/well (∼120 µL) of HPLC eluate was added to 120 µL of PBS present in the wells. Subsequently, the fractions were further diluted 1:40 with PBS for ELISA. For ESI-TOF spectra, the sample was diluted 1:100 in acetonitrile/water (1:1) and infused via syringe pump using a flow rate of 18 µL/min and a fused-silica transfer capillary. As an internal standard, the peptide H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH (Calbiochem-Novabiochem AG, La¨ufelfingen, Switzerland, mass 952.45) in the negative ion mode was used. Scans were accumulated over 35 s. Protocol for Direct, Competitive ELISA with MC10E7, M8H5, and AD4G2. Microtiter plates were coated with 250 µL/ well of goat anti-mouse IgG diluted 1:3000 in coating buffer (40 mmol/L of sodium carbonate, pH 9.6) by overnight incubation at room temperature. The plates were washed three times with washing solution (7 mmol/L of phosphate-buffered saline (PBS), pH 7.6, containing 15 mmol/L of sodium chloride and 0.05% v/v of Tween 20). Then the plates were incubated for 1-3 h with 200 µL/well of monoclonal antibody (MC10E7 cell culture supernatant diluted 1:15 000; affinity purified M8H5, diluted 1:500 000; and AD4G2 cell culture supernatant, diluted 1:800, respectively) in PBS (80 mmol/L of sodium phosphate, pH 7.6) containing 8.5 g/L of sodium chloride. After washing the plates three times with washing solution, 200 µL of MC-LR standard solutions and diluted, neutralized HPLC fractions were pipetted into the wells and preincubated for 2 h. The peroxidase tracer (MC-LR-peroxidase conjugate) was diluted 1:2000 in PBS (pH 7.6), and 50 µL was added to each well. After a further incubation of 15 min., the plates were washed as described above. A 200-µL portion of a freshly prepared substrate solution (hydrogen peroxide/TMB in citrate buffer, 0.2 mol/L citrate, 0.01% sorbic acid potassium salt, pH 3.8) was added. The enzyme reaction was stopped by adding 100 µL of 5% v/v H2SO4 per well, and the absorbance at 450 nm was measured on a microtiter plate reader. Standards were prepared in purified water (Milli-Q Plus 185 from Millipore). Their concentration levels ranged from 1000 to 0.001 or 100 to 0.0001 µg/L. Protein Phosphatase Inhibition Assay. The assay was performed according to the procedure published by Wirsing et al.39 The enzyme stock solution was prepared by dilution of 25 (39) Wirsing, B.; Flury, T.; Wiedner, C.; Neumann, U.; Weckesser, J. Environ. Toxicol. 1999, 14, 23-29.

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µg of enzyme in 100 µL of TRIS-HCl buffer (50 mM, pH 7.0, 200 µM MnCl2, 5 mM DTT, 200 µM EDTA, and 200 mg/L of BSA). BSA was added just before use. A 144-µL portion of 33 mM p-nitrophenyl phosphate in TRIS-HCl buffer (a) (50 mM, pH 8.1, 1 mM DTT, 20 mM MgCl2, 200 µM MnCl2, and 0.5 g/L of BSA) was added to the wells and incubated for 12 min at 30 °C with 50 µL of the HPLC fractions diluted 2-fold in TRIS-HCl buffer (0.1 M, pH 7.4). Then 20 µL of the enzyme stock solution, diluted 1000fold in TRIS-HCl buffer (a), was added to the wells and incubated at 30 °C. Absorbance (405 nm) was measured after 2 h. Evaluation of Standard Curves. Each calibration point of standard curves and each sample value was determined by calculation of the median of the data (n ) 3 or 4). Standard curves of different microcystin variants were obtained by fitting calculated medians to the four-parameter function.

Y)

(A - D) +D X B 1+ C

[ ( )]

(1)

X, concentration of analyte [µg/L]; Y, absorbance at 450 nm with reference wavelength 620 nm; A, maximum absorbance (upper asymptote); D, minimum absorbance (lower asymptote); C, midpoint (IC50 value) [µg/L]; and B, slope parameter. Data Processing for Quantification of Microcystins from Immunological HPLC Detection. For quantification of microcystin-containing fractions in the HPLC, eluate and standards consisting of eight different concentrations of MC-LR measured in duplicate or triplicate were included on each plate. Standard curves obtained with different antibodies and the enzyme are shown in Figure 2. The minimal absorbance (D in eq 1) was subtracted from all absorbance values, and normalization to a maximal absorbance of 1 was performed. Thereafter, each absorbance value was converted to the corresponding concentration value by applying the reversed four-parameter fit function (eq 1). This procedure inverts the original inhibition (“negative”) peaks into “positive” peaks. The amount of microcystin was calculated according to eq 2. n

Vd m)

∑c 1

10CR

(2)

m, amount of microcystin present in the sample (ng); V, volume of one HPLC fraction (µL); d, dilution ratio of HPLC fractions for ELISA n, number of positive fractions; c, concentration of fractions [µg/L] determined by ELISA using eq 1; and CR, mass-related cross-reactivity (%). The calculated amount was compared to the amount obtained from peak integration of the UV spectra. Determination of Cross-Reactivities with HPLC/UV/ ELISA. If unknown microcystins occur in environmental samples or in the case that an antibody with unknown cross-reactivity 5512

Figure 2. Normalized calibration curves obtained for different monoclonal antibodies and the enzyme protein phosphatase 1 (PP1). Fitting parameters obtained from the curves were used for calculation of MC-LR equivalents to create the enzymo- and immunograms.

Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

pattern is used, cross-reactivities can be calculated from the HPLC/UV/ELISA chromatogram. On the condition that all microcystins have similar extinction coefficients at 238 nm and MCLR is present in the sample, the following equation can be applied. n

∑c CR(%) )

MC-XY

1

‚

AMC-XY

m

∑c

AMC-LR

(3)

MC-LR

1

∑nl cMC-XY: concentration of cross-reacting substance, determined by addition of the concentrations of all positive fractions belonging to the peak of MC-XY by immunodetection as MC-LR equivalents according to eq 2; ∑m l cMC-LR: concentration of MC-LR, determined by the addition of the concentrations of all positive fractions belonging to the peak of MC-LR; and A, peak area determined by UV detection at 238 nm. RESULTS AND DISCUSSION Measurement of a Microcystin Standard Mixture with Three Monoclonal Antibodies and a Protein Phosphatase Postcolumn to HPLC. There are some critical points when coupling immuno- or enzyme assays to HPLC. First, one has to consider that the measuring range of the sigmoidal calibration curve of the ELISA covers only about 2 orders of magnitude and is, therefore, smaller than the linear range of the UV absorbance. Moreover, the measuring range of microcystins depends on their cross-reactivity and the antibody used (compare Figure 2). As a result, HPLC fractions may have to be measured in different dilutions to determine their concentrations. If the concentration can be measured by UV, the dilution needed for ELISA can be estimated. Another point is the organic solvent content present in the HPLC fractions, as well as the acidic pH used for chromatography of microcystins. Both can denature the antibody and the enzyme label or change the affinity of the antibody-analyte bond; therefore, it is crucial to neutralize the eluate with a suitable buffer and to guarantee that the dilution with buffer is sufficient

Figure 3. Chromatographic separation of a microcystin standard mixture (1 mg/L; 20 µL) on reversed-phase (C16-Amide) and detection with UV (238 nm); three monoclonal anti-microcystin antibodies (M8H5, MC10E7, and AD4G2) with different cross-reactivity pattern and an enzyme (PP1).

to avoid denaturation effects caused by the organic solvent. The third point to be discussed here is the fraction size. A compromise between resolution and number of fractions to be handled in ELISA has to be found. A comparison between immunodetection of two close peaks of MC-YR and MC-LR was performed. Narrow (50 µL/well, resulting in 12 fractions per peak) and broader fractionation (120 µL/well, resulting in 5 fractions per peak) were compared. Even with only 5 fractions per peak, sufficient resolu-

tion was achieved to separate the peaks (data not shown). The fact that quantification does not depend on the fraction size will be discussed later (Table 2). Because the fraction collector rack had a capacity of three microtiter plates, it was possible to collect automatically fractions over a period of 35 min (flow rate, 1 mL/ min; fraction size, 120 µL). This time window is more than sufficient to cover the full elution window of microcystins under the chosen chromatographic conditions. Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Table 1. Comparison between Microcystins Determined by HPLC/UV and HPLC/ELISAa m, ng

microcystin (mass-related CR, %)

by UV

by ELISA

MC-LR (100) MC-YR (68 ( 7) MC-RR (96 ( 22) nodularin (7.3 ( 1.3)

23.8 ( 0.9 21.5 ( 0.7 13.0 ( 0.5 30.5 ( 1.2

26.3 ( 1.2 23.1 ( 1.0 11.6 (1.6 27.5 ( 1.4

a All values have been determined at least in triplicate. For ELISA values, fractionation resulted in 5-7 fractions/peak

Table 2. Comparison of MC-LR Amount Found for Different Fraction Sizes MC-LR fractions/peak

m (ng; n)6)

15 6 1 (pooled)

22.4 ( 2.4 21.1 ( 1.2 22.9 ( 2.7

To show that coupling of HPLC with ELISA can be performed in a multidimensional way, HPLC fractions of one single run were measured with all available antibodies and an enzyme (protein phosphatase 1). A microcystin standard mixture of 1 mg/L (20 µL) containing MC-RR, nodularin, MC-YR, MC-LR, MC-LA, MCLW, and MC-LF was injected onto the column. The traces of all “detectors” are shown in Figure 3. UV Detection. The UV spectra of the solvent additive TFA in water and in acetonitrile differ as a result of differences in dielectric constants that modify π-π* electron transitions.40,41 As a result, elution gradients with a constant concentration of TFA in water and acetonitrile show a considerable drift in the baseline, especially when monitored at 238 nm, the wavelength of the maximum absorbance of microcystins. Using 0.1% TFA in the water phase and 0.04% TFA in the acetonitrile phase results in a flat baseline until approximately 55% organic content. As a consequence, the UV chromatogram shows a flat baseline at the retention times of hydrophilic microcystins and an increasing baseline at the retention times of the hydrophobic microcystins, as shown in Figure 3. Immuno- and Enzyme Detection. If a biochemical inhibition assay is used, the peaks are inverted relative to the peaks obtained by UV detection. Low absorbance values obtained in the competitive assay indicate high analyte concentrations. Furthermore, the peak area or height is not simply inversely proportional to the concentration. If a concentration-related chromatogram with positive peaks is desired, a calibration curve has to be included on the microtiter plate used to monitor the HPLC fractions. The absorbance values obtained in the biochemical assay have to be processed as described in the Experimental Section. Figure 3 represents a visual overview over the cross-reactivity pattern of three monoclonal antibodies and the enzyme protein phosphatase 1. Quantification of Microcystins by HPLC/ELISA. Three questions regarding the quantification of HPLC fractions with (40) Winkler, G. LC-GC 1987, 5, 1044-1045. (41) The Handbook of Analysis and Purification of Peptides and Proteins by ReversedPhase HPLC, 2nd ed; Vydac: Hesperia, CA, 1995.

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Figure 4. Sensitivity of UV (lower line) in comparison to ELISA detection (upper line) for MC-LR. A 20-µL portion of a solution of 10 µg/L MC-LR (200 pg) was injected, monitored by UV, fractionated, diluted 1:10 with buffer, and measured with ELISA using antibody MC10E7. Table 3. Comparison of Mass-Related Cross-Reactivity Data Obtained by the Usual Determination Method Using the IC50 Values of Standard Curves and by HPLC/ELISA CR, % toxin

using IC50 values

by HPLC/ELISA

MC-LR MC-YR MC-RR nodularin

100 68 ( 7 96 ( 22 7.3 ( 1.3

100 66 ( 6 76 ( 14 5.5 ( 0.5

bioassays were investigated. First, the possible influence of the fraction size on the quantification was examined. Then quantification of different microcystins using their cross-reactivity values calculated from competitive ELISA34 was tried. Finally, the sensitivity of immunodetection was compared with UV detection. For these experiments, the antibody MC10E7 was used. It is likely that results obtained using other antibodies are similar. The monoclonal antibody MC10E7 recognizes MC-YR and MC-RR, whereas nodularin is partially discriminated, showing a crossreactivity of only 7%. Table 1 shows that quantification of analytes is possible over a wide range of cross-reactivities, because good agreement between UV and ELISA quantification has been found. Table 2 shows the quantification results obtained with different fraction sizes of HPLC eluent. Because the microcystin present in one peak is completely collected in the wells of the microtiter plate, the quantification should be independent of the fraction size, which could be proven by this experiment. A comparison between the sensitivity of UV and ELISA detection is shown in Figure 4. A 20-µL portion of a solution of 10 µg/L of MC-LR were injected into the HPLC, monitored by UV (238 nm), fractionated, and tested by ELISA. For ELISA measurement, the HPLC fractions were diluted 1:10 with PBS buffer. Although the UV detection shows a flat baseline, the ELISA detection shows a peak significantly above the noise. The limit of detection for the HPLC/ELISA method using the antibody MC10E7 was calculated to be 2 µg/L MC-LR (signal-to-noise ratio,

Figure 5. Chromatographic separation of an extract of freeze-dried cyanobacteria on reversed-phase (C16-Amide) and parallel detection with UV (238 nm) and two monoclonal anti-microcystin antibodies (M8H5, MC10E7).

3) under the conditions given above. If the guideline value for MC-LR proposed by the WHO (1 µg/L) needs to be reached, one might inject 200 µL of the water sample and dilute HPLC fractions 1:10 with buffer. Although the complete optimization of a HPLC with a multidimensional biochemical detection seems to be a complex task, in practice, a straightforward “recipe” can be followed. At first, some general parameters have to be fixed, such as the chromatographic conditions (column, solvents, gradient, etc.), the fraction size, and the minimal dilution factor. The latter, as well as crossreactivity data, can be taken from the literature if the antibody has already been characterized. Furthermore, the buffer concentration may have to be adjusted to avoid a pH drift. Finally, the retention time window has to be determined or estimated either using analytical standard compounds or on the basis of literature data. All of these parameters are optimized only once during the setup of the system. The measurement itself is quite simple to perform. The filtered sample is injected into the HPLC and fractionated on 1-3 microtiter plates (depending on the chosen retention time window). Subsequently, the fractions are diluted (e.g., 1:10 ) minimum dilution factor) in a neutral buffer and examined by

immunoassay or enzyme inhibition assay or by both. Further dilution of some fractions should be considered only if very high concentrations of analyte had been detected, which lead to a full inhibition of some of the tests. The complete procedure of HPLC, fractionation, and off-line biochemical detection can be completed in 3-4 h. This compares favorably with other techniques requiring complex cleanup and enrichment steps. Determination of Cross-Reactivity by HPLC/ELISA. As mentioned in the Experimental Section, coupling of HPLC with ELISA can serve not only for the quantification of a microcystin mixture at low concentration levels but also for the rapid measurement of unknown cross-reactivities, either for unknown toxins or for novel antibodies. We assessed this method by calculating the cross-reactivities of MC-YR, MC-RR, and nodularin with antibody MC10E7 according to eq 3 in the Experimental Section. Requirements for the measurement of cross-reactivities with this method are the presence of MC-LR in the sample (spiked or naturally) and that both UV and immunodetection are performed. Table 3 shows a comparison between cross-reactivities calculated with the new method and cross-reactivities obtained by the conventional method using the midpoint of calibration curves.34 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 6. Negative ion electrospray mass spectrum of a cyanobacterial extract.

Measurement of Real Samples by HPLC/ELISA and ESITOFMS. The HPLC/ELISA analysis of an extract of freeze-dried cyanobacteria is shown in Figure 5. The superiority of immunological over UV detection is obvious. Whereas the UV detector shows no signal at the retention time 25.61, both antibodies M8H5 and MC10E7 detect a microcystin. Antibody MC10E7 shows a higher signal than M8H5 as a result of higher sensitivity. On the other hand, the peak at 27.85 min is not recognized by both antibodies and is, therefore, very probably not a microcystin. The peak of microcystin-LR at 29.03 min is detected by all three detectors. At longer retention times, two peaks at 33.84 and 34.95 min are detected by UV and the antibody M8H5. Because MC10E7 does not show peaks at this retention time, these microcystins do not possess an arginine at position 4 of the cyclic peptide. These findings suggest that using UV absorbance as the sole detection method for microcystins can lead both to underestimation because of lack of sensitivity and to overestimation because of other substances that show absorbance at 238 nm. The negative ion electrospray mass spectrum of the cyanobacteria extract is shown in Figure 6. Because microcystins are singly charged in the negative ion mode, the signals of microcystins are limited to a range between 900 and 1200 m/z. The negative ion mode was chosen to minimize the formation of sodium adducts, which can occur if acids are used as solvent additives in the positive ion mode. The mass spectrum shows numerous peaks in the mass range of microcystins; however, only few of them could be assigned to known microcystins (Table 4). The mass spectrum and the assignment of the detected peaks to known microcystins demonstrate that there are still unidentified 5516 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

Table 4. Tentative Assignment of Peaks in the Mass Spectruma to Known Microcystins m/z [M - H](26)b

908.5009 922.5088 (17)

924.4459 931.3954 936.5220 (15) 938.4739 950.5381 (15) 952.4529 (0) 974.4281 (7) 990.4143 993.5849(44) 1004.4023 1007.5962 (75) 1018.4342 (-25) 1026.3726 1040.3912 (-48) a

microcystin microcystin-LA microcystin-LA methyl ester or microcystin-LAba unknown unknown microcystin-LA dimethyl ester unknown microcystin-LL peptide (internal standard) peptide, sodium adduct (internal standard) unknown microcystin-LR unknown [D-Asp3, ADMAdda]microcystin-LR microcystin-YM sodium adduct of 1004.4 microcystin-YM, sodium adduct

Figure 6. b ∆m from calculated mass (ppm).

peaks that might be microcystins. To identify the microcystin peaks, the microcystins should be separated by chromatography prior to mass detection, fragmented, and examined for typical fragments, such as m/z 135, in the positive ion mode. However, this confirmation by fragmentation is not possible at very low concentrations. With the prior knowledge resulting from the HPLC/ELISA coupling, the maximum number of microcystins present in the sample and the number of microcystins with and without arginine at position 4 of the cyclopeptide are known. In the case of the investigated sample, microcystin-LR and micro-

cystin-LA could be confirmed. The other two peaks found in the HPLC/ELISA data might be the mono- and the dimethyl esters of microcystin-LA, which could be an artifact of the methanolic extraction method. The first peak is a microcystin containing arginine at position 4. Such a microcystin could not be assigned in the mass spectrum and, therefore, remains unknown. These results indicate that HPLC/ELISA coupling gives useful additional information, even if a mass spectrometer is available. The chromatographic eluate is often split postcolumn to obtain a lower flow rate for mass spectrometric detection. The other part of the eluate could be used for fractionation and immunological and enzymatic detection, as shown in this paper. CONCLUSION This work demonstrates that coupling of biochemical assays, such as immunological or enzyme-inhibition assays, postcolumn to chromatography provides a highly sensitive and selective method for the analysis of a class of substances, for example, microcystins. The coupling is particularly advantageous if the number of available standards is limited in comparison to the maximal number of potential analytes in the sample, if a highly sensitive analysis is required, or in cases when biologically active components need to be analyzed. The off-line coupling is easy to perform and enables detection using different biomolecules or later analysis of the fractions. In addition, quantification can easily be achieved, because standards needed for calibration can be included on each microtiter plate. Quantification was demonstrated for microcystin-LR and two cross-reactants having high and low

cross-reactivity. Data processing is relatively complex, because the immunoassay calibration curve is sigmoidal and the raw peaks are negative as a result of the inhibition mechanism. However, the data evaluation can be fully automated. It could also be proven that quantification is not dependent on the fraction size as long as the peaks are sufficiently separated. The provisional guideline value of 1 µg/L for microcystin-LR proposed by the WHO could be achieved without prior sample enrichment. This concentration could not be detected using UV absorbance. Simple immunological methods, such as ELISA, only provide microcystin equivalents. Finally, a peak-assigned toxicity-related parameter (protein phosphatase inhibition) could also be obtained, which is information not available by any conventional technique. ACKNOWLEDGMENT The financial support by the Federal Ministry of Education, Science, Research, and Technology of Germany (BMBF) is gratefully acknowledged. We thank our project partners, Prof. Dr. D. Bursill, Dr. J. Morrall, Dr. B. Nicholson, and Dr. D. Steffensen from the Australian Water Quality Centre, AWQC, Adelaide, Australia. We also thank Dr. S. Nagata from National Cancer Institute, National Institute of Health, Bethesda, MD for kindly providing the monoclonal antibody M8H5.

Received for review April 19, 2001. Accepted September 5, 2001. AC015511Y

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