Effects of Organic Solvents on the High Performance Liquid

FEMS Microbiology Letters 216 (2002) 159^164 www.fems-microbiology.org

E¡ects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic

waters by solid phase extraction Downloaded from https://academic.oup.com/femsle/article/216/2/159/483493 by guest on 24 September 2021

J.S. Metcalf a;Ã, K.A. Beattie a, M.L. Saker b, G.A. Codd a

a Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, UK b Departamento de Zoologia e Antropologia, Faculdade de Cie“ncias, Prac°a Gomes Teixeira, 4050 Porto, Portugal

Received 24 June 2002; accepted 6 September 2002

First published online 3 October 2002

Abstract

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 Wgl31. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Cylindrospermopsin; Cyanobacterial toxin; Analysis; Solid phase extraction

1. Introduction and 7-epicylindrospermopsin appears to have a similar toxicity to cylindrospermopsin [9]. Studies such as these Cylindrospermopsin is a cyanobacterial guanidine alka- have indicated that the uracil moiety of cylindrospermop- loid hepatotoxin [1] originally isolated from cultures of sin may be required to express toxicity [10]. Cylindrospermopsis raciborskii [2]. The toxin has since Several methods are used to detect and analyse cylin- been isolated from cultures of Aphanizomenon ovalisporum drospermopsin, including mouse bioassay [11], and high [3,4], Umezakia natans [5] and Raphidiopsis curvata [6].In performance liquid chromatography (HPLC) employing comparison to the more widely known hepatotoxic micro- either photodiode array detection (HPLC-PDA) [5] or cystins of cyanobacteria, which include over 70 variants mass spectrometry (HPLC-MS/MS) [12]. In£uences of or- [7], only two further naturally occurring analogues of cy- ganic solvent concentration on the quanti¢cation of cylin- lindrospermopsin have been identi¢ed, namely deoxycylindrospermopsin by HPLC-PDAare identi¢ed in the drospermopsin [8] and 7-epicylindrospermopsin [9].Of present communication. these, deoxycylindrospermopsin has been shown to be ap- The recovery of cylindrospermopsin from solution has proximately one tenth as toxic as cylindrospermopsin [8] been performed using resins [5] and graphitised carbon solid phase extraction (SPE) cartridges have recently been applied to recover the toxin from the cells and

* Corresponding author. Tel.: +44 (1382) 34 48 66; growth medium of cultures of C. raciborskii [13]. In this Fax: +44 (1382) 34 42 75. study, we further optimised the extraction of cylindrosper- E-mail address: [email protected] (J.S. Metcalf). mopsin from spent C. raciborskii medium by combining

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C18 and graphitised carbon solid phases. This procedure analysed. Finally, 10-Wg aliquots were dissolved to a con- was also shown to be e¡ective to extract the toxin from centration of 10 Wgml31 in either 100% (v/v) MilliQ water, spiked environmental waters. These procedures are recom- 50% (v/v) aqueous methanol or 100% (v/v) methanol in mended to extract and purify cylindrospermopsin for an- glass vials. The contents of the vials were then removed alytical and research purposes, since the toxin is not avail- and placed in fresh glass vials and 100 Wl of water were able commercially. added to the original vials. After vortexing, the contents were removed and all solutions were analysed by HPLC- PDA( n = 3). 2. Materials and methods 2.3. E¡ect of tap and eutrophic environmental waters on the 2.1. Puri¢cation and HPLC-PDA analysis of HPLC analysis of cylindrospermopsin cylindrospermopsin

Dundee municipal tap water and environmental water Downloaded from https://academic.oup.com/femsle/article/216/2/159/483493 by guest on 24 September 2021 C. raciborskii strain CR3 [14] was grown in 10-l batch from eutrophic lakes (Lochs Rescobie and Balgavies, and cultures in BG11 medium minus nitrate [15]. The cells Monikie Island Pond, Angus, UK) were ¢ltered through were harvested during late growth phase and removed GF/C ¢lters (Whatman, Maidstone, UK) and spiked with from the culture medium by tangential £ow ¢ltration using puri¢ed cylindrospermopsin at concentrations of 10, 5, 2, a Millipore Pellicon 2 tangential ¢lter (Millipore, Watford, 1 and 0.5 Wgml31. All solutions were analysed by HPLC- UK). The ¢ltrate was retained and stored for SPE and PDA( n = 3). toxin puri¢cation. Cylindrospermopsin was puri¢ed using Waters equipment including a 510 pumping system, 471 2.4. SPE procedure for the recovery of cylindrospermopsin WISP, 484 UV detector and a Waters fraction collector from culture medium and environmental waters employing a Phenomenex Luna semiprep column (C18, 5 Wm, 150U10 mm i.d.). Analytical detection of cylindro- For the recovery of cylindrospermopsin from spent cul- spermopsin was performed using a Waters 2690 separa- ture medium and spiked water, stacked SPE cartridges tions module and a 996 photodiode array detector. Sepa- were used with C18-packed material (Jones Chromato- ration was achieved using a method modi¢ed from that of graphy, Mid Glamorgan, UK) placed in series before Hy- Harada et al. [5] employing a Cosmosil C18 column (Phe- persep Hypercarb SPE cartridges (Thermo Hypersil- nomenex; 5 Wm, 150U4.6 mm i.d.) with a linear gradient Keystone, Runcorn, Cheshire, UK). For all samples, the of 1^12% (v/v) methanol/water over 24 min at 40‡C. Chro- cartridges were primed with 100% methanol and washed matograms were monitored at 262 nm with spectra from with 100% water at £ow rates speci¢ed by the manufac- 200 to 300 nm. turer. The samples were applied after priming at a £ow rate of 1 ml min31 and after application, were eluted from 2.2. E¡ect of solvents on the UV absorbance and the solid phases. For cylindrospermopsin isolated from HPLC-PDA analysis of cylindrospermopsin cell-free spent culture medium, the toxin was extracted by the addition of 10 ml of 100% (v/v) methanol or 10% Aliquots of puri¢ed cylindrospermopsin (10 Wg) were incremental steps of methanol in water from 0 to 100% dispensed into glass vials and dried down under N2 gas (v/v). For the elution of extracted cylindrospermopsin at 50‡C. These were then resuspended to 10 Wgml31 with from spiked environmental waters, 100% (v/v) methanol solutions of increasing methanol concentration in 10% in- plus 0.1% (v/v) tri£uoroacetic acid was used. Once ex- crements, from 100% (v/v) MilliQ water up to 100% (v/v) tracted, the solvents were dried down and resuspended methanol (HPLC grade; Rathburn, Walkerburn, UK). in MilliQ water before HPLC-PDAanalysis. The absorbance of these solutions was measured in quartz cuvettes by a UV spectrophotometer at 262 nm (Pharma- cia LKB Ultrospec II, Uppsala, Sweden), blanked against 3. Results the appropriate concentration of methanol. These same solutions were then analysed by HPLC-PDAas described The absorbance at 262 nm of cylindrospermopsin pre- above. After analysis, the samples were dried down and pared in methanol increased to a maximum, with increas- resuspended in methanol/water to the same concentration ing methanol concentration up to 50% v/v (Fig. 1A), in the reverse solvent direction and analysed again by above which absorbance remained approximately con- HPLC-PDA. Further 10-Wg aliquots were prepared and stant. Polynomial regression analysis (second and fourth dried down. These were resuspended as for the methanol order) showed good correlation with R2 values of at least solutions, except that methanol was replaced by HPLC 0.94. HPLC-PDAanalysis of these same solutions showed grade acetonitrile (Rathburn). These solutions were ana- the opposite trend with a decrease in peak height, with the lysed by HPLC-PDA, dried down, resuspended in the re- subsequent indicated cylindrospermopsin concentration verse solvent order to the same concentration and again reaching a minimum at 50% (v/v) methanol which contin-

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A C

0.060 ) -1 12 0.055 10 µ 0.050 8

0.045 6

4 0.040 2 0.035

Absorbance (262nm) 0 Downloaded from https://academic.oup.com/femsle/article/216/2/159/483493 by guest on 24 September 2021 0.030 0 20 40 60 80 100 0 20406080100 Cylindrospermopsin ( g ml % methanol (v/v) % Acetonitrile (v/v) ) ) D -1 B -1

8 10 µ µ

6 8

6 4 4

2 2

0 0 20 40 60 80 100 0 Cylindrospermopsin ( g ml 0 20 40 60 80 100 Cylindrospermopsin ( g ml % methanol (v/v) % methanol (v/v) Fig. 1. E¡ects of solvents on the analysis of cylindrospermopsin. Cylindrospermopsin resuspended in methanol from 0 to 100% (v/v) methanol was analysed by a spectrophotometer (A) at 262 nm with second order (8) and fourth order (ööö) polynomial regression. The cylindrospermopsin solutions were also analysed by HPLC-PDA, resuspended in methanolic (B), and acetonitrile-containing (C) solutions, resuspended from 0 to 100% (v/v) solvent (F), or dried and again resuspended from 100% to 0% (v/v) organic solvent (b). D: To rule out the possibility of precipitation in organic solvent, cylindrospermopsin was redissolved in 0, 50 and 100% (v/v) methanol (F), supernatant removed and the MilliQ water added to the vials (b). All samples were analysed by HPLC-PDA(n = 3). Vertical error bars represent standard deviation. ued to 100% (v/v) methanol (Fig. 1B). However, after with organic solvents were due to precipitation of cylin- these solutions were dried down and resuspended in the drospermopsin within the glass vial, new vials containing reverse solvent order, the opposite trend was apparent, dried cylindrospermopsin were resuspended in water, 50% indicating that the higher the methanol concentration, (v/v) methanol and 100% (v/v) methanol (Fig. 1D). Once the lower the HPLC response (Fig. 1B). The e¡ect of resuspended, the solutions were removed and water was methanol on analysis by HPLC was compared to the e¡ect added to all vials to dissolve potentially precipitated cylin- of acetonitrile (Fig. 1C). Again, the same response oc- drospermopsin. With the initial re-dissolving, a decrease in curred although concentrations of 30% (v/v) acetonitrile the absorbance by HPLC was observed, although when were required to obtain the same response as with 50% the vials were rinsed with water and absorbances deter- methanol. Finally, to ascertain whether the observations mined, the observed toxin concentrations for all three re-

Table 1 Mean recoveries of cylindrospermopsin from tap and environmental eutrophic waters spiked with toxin at concentrations between 0.5 and 10 Wgml31 as analysed by HPLC (n =3) Spike concentration (Wgml31) Tap water Monikie Island Pond Loch Rescobie 10 10.14 þ 0.07 8.98 þ 0.08 10.26 þ 0.04 5 5.02 þ 0.04 4.68 þ 0.05 5.21 þ 0.00 2 1.95 þ 0.00 1.80 þ 0.01 2.07 þ 0.00 1 0.96 þ 0.02 0.87 þ 0.01 1.05 þ 0.02 0.5 0.47 þ 0.02 0.43 þ 0.00 0.55 þ 0.00 Values are means þ S.D.

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the puri¢ed toxin to ¢nal concentrations of between 0.5 and 10 Wgml31 (Table 1). The HPLC response for tap water and untreated Loch Rescobie water was good, with recovered cylindrospermopsin concentrations similar to the spike concentration. The recovered toxin concentrations for spiked waters from Monikie were lower than the spike concentrations, although the recovery was at least 86%, indicating that environmental waters did not interfere greatly with the HPLC analysis of cylindrospermopsin. HPLC analysis of actively growing cultures of C. raciborskii strain CR3 has indicated that at least 60% of the total detectable cylindrospermopsin is present in the extra-

cellular growth medium (data not shown). Methods were Downloaded from https://academic.oup.com/femsle/article/216/2/159/483493 by guest on 24 September 2021 developed to recover this abundant source of toxin. The combination of SPE using C18, followed by Hypercarb cartridges, facilitated the isolation of cylindrospermopsin, and HPLC-PDAanalysis indicated that a large proportion of the impurities were removed by the C18 packing. Ap- plication of 4 mg of cylindrospermopsin to this combined setup resulted in association of cylindrospermopsin with both C18 and Hypercarb (Fig. 2A) with cylindrospermopsin quantitative ratios of 1:80 respectively, according to HPLC-PDAanalysis. Elution of the Hypercarb cartridges with volumes of methanol increasing by 10% increments resulted in the release of cylindrospermopsin between 0 and 50% (v/v) with a maximal concentration eluted at 30% (v/v) methanol. Due to the high concentrations of cylindrospermopsin present in the spent medium, the use of modi¢ers for solid phase elution was not necessary, as other compounds eluted from the cartridges did not inter- Fig. 2. SPE of cylindrospermopsin from spent extracellular growth me- fere with the HPLC analysis of cylindrospermopsin. How- dium after batch culture of C. raciborskii to late growth phase (A) and ever, the use of this system for the recovery of cylindro- spiked environmental eutrophic waters (B) using combined C18, fol- spermopsin from environmental eutrophic waters required lowed in series by polygraphite carbon cartridges. A: Extracellular growth medium containing cylindrospermopsin (grey bar) was passed method development to permit the recovery of low con- through the SPE system and eluted from C18 (white bar) and poly- centrations of the toxin. In order to elute these low con- graphite carbon (hatched bars) before analysis by HPLC-PDA. B: Envi- centrations, and allow sample concentration, 100% meth- ronmental water samples from Lochs Balgavies (white bar) and Resco- anol supplemented with 0.1% (v/v) TFAwas found to be bie (light grey bar) and Monikie Island Pond (dark grey bar) spiked the most suitable. When cylindrospermopsin isolated from with cylindrospermopsin at 1 Wgl31 were eluted with 1 ml incremental volumes of 100% (v/v) methanol+0.1% (v/v) TFAand analysed by environmental water was eluted from the Hypercarb car- HPLC-PDA. Asterisks represent undetected cylindrospermopsin due to tridge, 8 ml of elution solvent were required (Fig. 2B). In the presence of interfering compounds. some fractions, interfering compounds prevented the detection of cylindrospermopsin by HPLC-PDA, although dissolution treatments were the same, indicating that pre- this was only a problem for the second millilitre of solvent cipitation of cylindrospermopsin from solution had not applied (Fig. 2B). When the cylindrospermopsin was taken place. eluted as one pool using 100% methanol/0.1% TFA, For subsequent investigations into the analysis of cylin- good recoveries were found (Table 2). In all cases, full drospermopsin in eutrophic waterbodies, ¢ltered lake recovery was achieved for environmental waterbody sam- water, with tap water for comparison, was spiked with ples spiked to 1.14 Wgl31.

Table 2 Recovery of cylindrospermopsin from three raw waters obtained by SPE (n = 2) and analysis by HPLC-PDA Source Spike concentration (Wgl31) Recovered concentration (Wgl31) Spike recovery (%) Loch Balgavies 1.14 1.17 103 Loch Rescobie 1.14 1.22 107 Monikie Island Pond 1.14 1.28 112

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4. Discussion mopsin in environmental waters, a variety of solvents may be employed in chromatographic methods such as HPLC- In comparison to the microcystins, methods for the re- PDA. When cylindrospermopsin was redissolved in a covery and analysis of cylindrospermopsins of cyanobac- range of solvent concentrations, a loss of the HPLC- teria have received little attention. The early method of PDAabsorbance signal occurred at concentrations greater Harada et al. [5] was a rapid screening procedure involving than 50% methanol and 30% acetonitrile. Organic solvents four steps: extraction, cleanup, separation and determina- have been found to in£uence the detection and quanti¢cation. The determination was performed by HPLC-PDA tion of microcystin-LR [16]. Methanol is widely used to and cleanup involved the use of HP resin, after which extract microcystins, 70^75% methanol being optimal the cylindrospermopsin was eluted and applied to a C18 [17,18]. Moreover, the UV absorbance of microcystin-LR cartridge to remove lipophilic compounds. However, the is maximal in 70% (v/v) methanol when determined by use of resins and C18 separately is more time-consuming HPLC-PDAand UV spectrophotometry. For cylindro- and more complicated than in the present study and the spermopsin, absorbance in the UV spectrophotometer at Downloaded from https://academic.oup.com/femsle/article/216/2/159/483493 by guest on 24 September 2021 unit costs are more expensive. Since the Harada et al. high methanol concentrations has higher absorbance at study [5], other methods of SPE have been used to isolate 262 nm, unlike the response by HPLC detection. As pre- and concentrate this toxin. In particular, Norris et al. [13] cipitation of cylindrospermopsin was not observed with investigated a wide range of commercially available SPE high solvent concentrations, the loss of the HPLC signal sorbents over a wide range of pH values. Of these, graphi- indicates that the HPLC procedure is involved in the re- tised carbon was found to recover cylindrospermopsin duced response visualised with cylindrospermopsin. The from spent growth medium when analysed by HPLC- use of C18 analytical columns with cylindrospermopsin MS/MS. However, as the graphitised carbon cartridge dissolved in organic solvents at high concentrations may was used on its own, there was a greater potential for result in dissociation or association of cylindrospermopsin breakthrough with small applied volumes, compared to a molecules due to their planarity resulting in the reduced combined system having a C18 cartridge to remove lipo- responses visualised. Furthermore, retention time di¡eren- philic compounds [5] and other components which may ces which could be attributed to poor retention with in- interfere with the HPLC-PDAanalysis of cylindrosper- creasing organic solvent concentrations were not observed. mopsin. The concentration of cylindrospermopsin used for envi- The ability to recover and concentrate cylindrospermop- ronmental raw water spiking and achieved by analysis in sin from environmental waters can help to accurately this study (V1 Wgl31) is in the range advocated as a monitor toxin concentrations when sensitive analytical potential guideline value for the protection of human methods such as HPLC-MS/MS, which do not require health [19] and well below the limits of detection for sample concentration, are not available. The use of C18 HPLC-PDA(400 Wgl31 ; 25-Wl injection, no sample pre- followed by Hypercarb cartridges in series permits other concentration). Further assessment of this SPE system to- organic substances to be removed in one step, allowing the gether with HPLC-PDAis necessary to assess the lower HPLC detection of cylindrospermopsin after the removal limits of detection and linearity for cylindrospermopsin of a large number of other organic compounds that may analysis in diverse raw and treated waters that may con- be present in environmental waters. As reported by Norris tain a wide range of total organic carbon concentrations, et al. [13], breakthrough of cylindrospermopsin from which could interfere with this system for environmental graphite cartridges was found to occur after 4^5 l of me- use. dium had been passed through the cartridges. However, by preceding the graphite cartridges with C18 cartridges, we found that the volume of spent growth medium through Acknowledgements the cartridges could be greatly increased. At least 10 l of spent medium could be passed through without break- We thank Dr. G.K. Eaglesham for a puri¢ed cylindro- through (data not shown), although the maximum volume spermopsin standard and Prof. R.A. Herbert for the kind would be expected to depend on the concentration of total loan of the tangential £ow equipment. We also thank the organic compounds within the sample for extraction. Or- European Union for ¢nancial assistance (CYANOTOX ganic products in the spent medium might have the ca- project ENV4-CT-98). pacity to displace cylindrospermopsin from the cartridge. If so, a two-stage system might have the advantage of preventing these compounds from interfering with cylindrospermopsin for potential binding sites within the car- References bon cartridge, therefore permitting a larger volume of me- [1] Ohtani, I., Moore, R.E. and Runnegar, M.T.C. (1992) Cylindrosper- dium to be passed through the system and a greater mopsin ^ Apotent hepatotoxin from the blue-green-alga Cylindro- amount of cylindrospermopsin to be retained. spermopsis raciborskii. J. Am. Chem. Soc. 114, 7942^7944. In order to determine the concentration of cylindrosper- [2] Hawkins, P.R., Runnegar, M.T.C., Jackson, A.R.B. and Falconer,

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