cylindrospermopsin ( cyn ) was first obtained from cylindrospermopsis raciborskii isolated from a reservoir in australia where the water had caused human hepatotoxicity ( byth 1980 ; bourke et al . cyn has been found to be genotoxic in in vitro systems and carcinogenic in mice ( falconer and humpage 2006 ) . it is now known to be a metabolite of several other freshwater cyanobacteria belonging to the genera anabaena , umezakia , raphidiopsis , and aphanizomenon ( falconer and humpage 2006 ; spoof et al . the related compounds deoxy - cyn and 7-epi - cyn have also been isolated as co - metabolites ( norris et al . 2007 ) . of particular interest is the formation of cyn by the species aphanizomenon flos - aquae isolated from german lakes ( preuel et al . 2006 ; fastner et al . 2007 ) and which is harvested from natural blooms in klamath lake ( oregon , usa ) to be marketed as a food supplement ( carmichael et al . ( 2006 ) reviewed levels of cyn in cyanobacteria ; 2.36.6 mg g of cyn have been found in lyophilized culture material of a. flos - aquae ( preu et al . more recently , 3.449.33 mg cyn g was determined in freeze - dried a. ovalisporum and cylindrospermopsis raciborskii ( yilmaz et al . procedures for the detection and determination of cyn isolated from water and cyanobacteria include enzyme - linked immunosorbent assay ( elisa ) ( blhov et al . 2009 ) , liquid chromatography ( lc ) with ultraviolet ( uv ) detection ( harada et al . 1994 ; li et al . 2002 ; kubo et al . 2005 ; spoof et al . 2006 ; kokociski et al . 2009 ; wormer et al . 2009 ) , lc - mass spectrometry ( ms ) ( kubo et al . 2005 ) , lc - ms / ms ( eaglesham et al . 1999 ; li et al . 2009 ; kokociski et al . 2009 ) , hydrophilic interaction lc - ms ( dell'aversano et al . 2004 ) , and capillary electrophoresis ( vasas et al . 2004 ) . cyn is not retained by c18 solid - phase extraction ( spe ) adsorbents , but graphite columns do retain it , so they have been used for clean - up of water , usually in series with c18 spe ( norris et al . 2001 ; metcalf et al . 2002 ; metcalf and codd 2005 ; wormer et al . ( 2007 ) , styrene polymer and anion exchange cartridges in series by kubo et al . ( 2005 ) , and c18 and hp-20 polymer resin columns by harada et al . there is no method previously reported for analysis of blue  green algal ( bga ) food supplements for cyn . we have adapted a method that incorporates both a combined  in - series spe system with a c18 column connected to a polygraphitized carbon ( pgc ) column and lc - uv . this method uses lc - uv rather than more expensive lc - ms and should be useful for screening these products for cyn . samples of bga products with different brand names and from five manufacturers were purchased through the internet . they included tablets , powder , capsules , food bars , and one sample of chocolates , which were ground with a coffee grinder and mixed . the bga in most samples was stated to be a. flos - aquae or came from klamath lake , which would be this species ( carmichael et al . 2000 ) . for each sample container , the complete contents were processed , and a representative subsample was taken for extraction . nrc - crm - cyn stock solution ( 12.6 g ml ) was purchased from the institute for marine biosciences ( national research council , halifax , ns , canada ) . cyn working solutions with different concentrations were prepared by dilution of the stock solution with water . spe columns were c18 ( 500 mg/3 ml , supelco lc-18 ; oakville , on , canada ) and polygraphitized carbon ( pgc ) ( hypersep pgc , 100 mg/1 ml ; thermo scientific , waltham , ma , usa ) . bga products ( 0.4 g ) were weighed into 15 ml polyethylene centrifuge tubes , and 6 ml of 5.0% aqueous formic acid added . the mixture was homogenized for 3 min using ultrasonication ( sonic dismembrator model 100 ; fisher scientific , pittsburgh , pa , usa ) . after centrifuging at 11,000 rpm for 10 min ( eppendorf centrifuge 5804r with f-34 - 6 - 38 rotor , mississauga , on , canada ) , the supernatant was collected and transferred to a 15 ml glass graduated test tube . another 3 ml of extraction solvent were added to the residue in the centrifuge tube , vortex mixed and centrifuged . the supernatants were combined and made up to a final volume of 10 ml with water . clean - up of an aliquot of the crude extract was performed by a combined  in - series spe system with the c18 column connected to the pgc column . the spe columns were conditioned with 10 ml methanol containing 0.1% ( v / v ) tfa , followed by 10 ml of water . a total of 2 ml of water were added to 0.25 ml of the crude extract of bga ( 0.01 g of bga powder equivalent ) and the solution loaded onto the combined spe system . the solution was passed through the columns at a flow rate of approximately 1 ml min . after washing the columns with water , the pgc column was removed from the first ( c18 ) column and eluted with 4  4 ml fractions of 0.1% tfa in methanol . on analysing each fraction , no cyn was found in the first two fractions ( 8 ml of eluted solvent ) , but it started to elute in the third fraction and was completely eluted after the fourth fraction . the residue was resuspended in 0.4 ml of water for lc - diode array detection ( dad ) analyses . ( as an alternative , we propose that the total eluate could be evaporated to dryness on a rotary evaporator with a water bath set at 40c . ) lc of cyn was carried out on an agilent 1100 system which included a quaternary pump , autosampler , on - line vacuum degasser , and diode array detector ( agilent technologies canada , mississauga , on , canada ) with a 2.1  250 mm genesis aq column ( grace jones , 120  , 4.0 m ; chromatographic specialties , brockville , on , canada ) and attached guard c18 column . the lc gradient consisted of two mobile phases : a , 5 mm ammonium acetate in water ; and b , 5 mm ammonium acetate in methanol , used under the following conditions : 560% b for 10 min , then held at 60% b for the next 5 min ; returned to 5% b over 0.5 min , then held for another 14.5 min with 5% b before the next injection . the injection volume was 20 l , and the flow rate was 0.2 ml min . cyn standard was added to a cleaned extract of klamath bga then diluted with the cleaned extract to make a series of concentrations . calibration curves in the range 6.063 ng injected were linear ( r  0.9980 ) . the lod was about 2.0 ng cyn injected ( signal - to - noise ratio = 3:1 ) and the loq was 6.5 ng cyn injected ( signal - to - noise ratio = 10:1 ) in bga supplements . method performance was checked by spiking an extract of klamath bga in which no cyn could be detected with different levels of cyn ( i.e. , a certain volume of cyn stock solution spiked in 0.25 ml bga extract and 2 ml of water added , then the solution loaded onto the combined spe system ) . the spiking levels of 25500 g g ( ppm ) were calculated based on the 0.4 g of product which was extracted . spikes were carried out in triplicate at 126 g g ( i.e. , 100 l of cyn standard spiked in 0.25 ml bga extract ) ; other spiked experiments ( 19.8400 l of cyn standard spiked in 0.25 ml bga extract ) were single at 25 , 100 , 252 and 504 g g. recoveries ( % ) were calculated by the equation : 

 where ax is the peak area of the cyn toxin in the bga extract subjected to clean - up and analysed by lc ; as is the peak area of the diluted cyn standard ; cs is the concentration of the diluted cyn standard ( g ml ) , i.e. 0.525 g ml ;  is the equivalent amount of bga ( g ) carried through the clean - up procedure , i.e. 0.01 g of dried bga powder ; and csp is the concentration of the spiked cyn in the crude bga extract , for example , 25 , 100 , 126 , 252 , 504 g g. samples of bga products with different brand names and from five manufacturers were purchased through the internet . they included tablets , powder , capsules , food bars , and one sample of chocolates , which were ground with a coffee grinder and mixed . the bga in most samples was stated to be a. flos - aquae or came from klamath lake , which would be this species ( carmichael et al . 2000 ) . for each sample container , the complete contents were processed , and a representative subsample was taken for extraction . nrc - crm - cyn stock solution ( 12.6 g ml ) was purchased from the institute for marine biosciences ( national research council , halifax , ns , canada ) . cyn working solutions with different concentrations were prepared by dilution of the stock solution with water . spe columns were c18 ( 500 mg/3 ml , supelco lc-18 ; oakville , on , canada ) and polygraphitized carbon ( pgc ) ( hypersep pgc , 100 mg/1 ml ; thermo scientific , waltham , ma , usa ) . bga products ( 0.4 g ) were weighed into 15 ml polyethylene centrifuge tubes , and 6 ml of 5.0% aqueous formic acid added . the mixture was homogenized for 3 min using ultrasonication ( sonic dismembrator model 100 ; fisher scientific , pittsburgh , pa , usa ) . after centrifuging at 11,000 rpm for 10 min ( eppendorf centrifuge 5804r with f-34 - 6 - 38 rotor , mississauga , on , canada ) , the supernatant was collected and transferred to a 15 ml glass graduated test tube . another 3 ml of extraction solvent were added to the residue in the centrifuge tube , vortex mixed and centrifuged . the supernatants were combined and made up to a final volume of 10 ml with water . clean - up of an aliquot of the crude extract was performed by a combined  in - series spe system with the c18 column connected to the pgc column . the spe columns were conditioned with 10 ml methanol containing 0.1% ( v / v ) tfa , followed by 10 ml of water . a total of 2 ml of water were added to 0.25 ml of the crude extract of bga ( 0.01 g of bga powder equivalent ) and the solution loaded onto the combined spe system . the solution was passed through the columns at a flow rate of approximately 1 ml min . after washing the columns with water , the pgc column was removed from the first ( c18 ) column and eluted with 4  4 ml fractions of 0.1% tfa in methanol . on analysing each fraction , no cyn was found in the first two fractions ( 8 ml of eluted solvent ) , but it started to elute in the third fraction and was completely eluted after the fourth fraction . the residue was resuspended in 0.4 ml of water for lc - diode array detection ( dad ) analyses . ( as an alternative , we propose that the total eluate could be evaporated to dryness on a rotary evaporator with a water bath set at 40c . ) lc of cyn was carried out on an agilent 1100 system which included a quaternary pump , autosampler , on - line vacuum degasser , and diode array detector ( agilent technologies canada , mississauga , on , canada ) with a 2.1  250 mm genesis aq column ( grace jones , 120  , 4.0 m ; chromatographic specialties , brockville , on , canada ) and attached guard c18 column . the lc gradient consisted of two mobile phases : a , 5 mm ammonium acetate in water ; and b , 5 mm ammonium acetate in methanol , used under the following conditions : 560% b for 10 min , then held at 60% b for the next 5 min ; returned to 5% b over 0.5 min , then held for another 14.5 min with 5% b before the next injection . the injection volume was 20 l , and the flow rate was 0.2 ml min . cyn standard was added to a cleaned extract of klamath bga then diluted with the cleaned extract to make a series of concentrations . calibration curves in the range 6.063 ng injected were linear ( r  0.9980 ) . the lod was about 2.0 ng cyn injected ( signal - to - noise ratio = 3:1 ) and the loq was 6.5 ng cyn injected ( signal - to - noise ratio = 10:1 ) in bga supplements . method performance was checked by spiking an extract of klamath bga in which no cyn could be detected with different levels of cyn ( i.e. , a certain volume of cyn stock solution spiked in 0.25 ml bga extract and 2 ml of water added , then the solution loaded onto the combined spe system ) . the spiking levels of 25500 g g ( ppm ) were calculated based on the 0.4 g of product which was extracted . spikes were carried out in triplicate at 126 g g ( i.e. , 100 l of cyn standard spiked in 0.25 ml bga extract ) ; other spiked experiments ( 19.8400 l of cyn standard spiked in 0.25 ml bga extract ) were single at 25 , 100 , 252 and 504 g g. recoveries ( % ) were calculated by the equation : 

 where ax is the peak area of the cyn toxin in the bga extract subjected to clean - up and analysed by lc ; as is the peak area of the diluted cyn standard ; cs is the concentration of the diluted cyn standard ( g ml ) , i.e. 0.525 g ml ;  is the equivalent amount of bga ( g ) carried through the clean - up procedure , i.e. 0.01 g of dried bga powder ; and csp is the concentration of the spiked cyn in the crude bga extract , for example , 25 , 100 , 126 , 252 , 504 g g. recoveries for spe clean - up were in the range 7090% for cyn and the standard deviation ( sd ) for the triplicate spike was acceptable ( table 1 ) . the c18 plus pgc double - column clean - up procedure provided an effective , reasonably accurate , and useful method for screening cyn in bga food supplements by lc - uv . for other spe columns mixed ion exchange polymers ( oasis mcx ; waters ) , weak anion exchange ( oasis wax ; waters ) and nh2the breakthrough from these columns either during application of the extract solution or during the subsequent wash was unacceptably high . different types of lc columns with dimension of 4.6  150 mm , such as supelcosil lc-18 , c18 phenomenex ultracarb column , and zorbax c18 , were also tried ; however , even with clean - up by the double columns , co - eluted compounds affected the detection of cyn at 262 nm . therefore , a longer column , genesis aq 120  , 2.1  250 mm , 4.0 m , was selected for the lc - uv method development . typical chromatograms are shown in figure 1(a  d ) . as shown in figure 1(b ) and ( c ) , the chromatograms indicate that quantitative analysis of cyn in crude bga is very difficult due to the interference of co - eluted compounds close to cyn at rt = 10.85 min . in contrast , the chromatogram in figure 1(d ) indicates most of the interferences close to cyn were efficiently removed after clean - up by the double columns . these results showed that this clean - up was effective for the determination of cyn in complex bga matrices . spe clean - up recoveries of cyn from blue - green algal food supplement extract . note : the spiking procedure is described in the text . lc - uv chromatograms of ( a ) cyn analytical standard , 10.5 ng injected ; ( b ) crude bga extract , 0.125 mg matrix - equivalent injected ; ( c ) crude bga extract spiked with cyn at 126 g g , 0.125 mg matrix - equivalent injected ; and ( d ) crude bga extract spiked with cyn at 126 g g , followed by c18 plus pgc clean - up , 0.125 mg matrix - equivalent injected . we analysed ten algal food supplements and one sample of chocolates containing bga , all of which were found to be free of cyn . the lod of the overall method was 16 g g of food supplement , with loq = 52 g g. although we do not know the concentrations of algae in the supplements , 16 g gis a useful lod as cyn can be present at mg gconcentrations in dried cyanobacteria , e.g. up to 6.6 mg g dry weight in a. flos - aquae ( preu et al . the clean - up procedure using lc - uv for detection rather than the more expensive lc - ms should be useful for screening algal food supplements for cyn . the authors are grateful to the office of the chief scientist , health canada , for a fellowship awarded to h.l .
for the analysis of blue  green algal food supplements for cylindrospermopsin ( cyn ) , a c18 solid - phase extraction column and a polygraphitized carbon solid - phase extraction column in series was an effective procedure for the clean - up of extracts . 
 determination of cyn was by liquid chromatography with ultraviolet light detection . at extract spiking levels of cyn equivalent to 25500 g g1 , blue 
 green algal supplement recoveries were in the range 7090% . 
 cyn was not detected in ten samples of food supplements and one chocolate product , all containing blue  green algae . 
 the limit of detection for the method was 16 g g1 , and the limit of quantification was 52 g g1 .