Abstract:
Kits and methods for the detection of toxins produced by cyanobacteria are disclosed. The methods include preparing a sample that potentially includes cyanobacterial toxins on a solid phase extraction device. In some embodiments, the sample extract can be formed using a weak cationic exchange process and a weak anionic exchange process.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the National Stage of International Application No. PCT/US2009/062299, filed Oct. 28, 2009, filed on and designating the United States, which claims benefit of a priority to U.S. Provisional Patent Application No. 61/110,021, filed Oct. 31, 2008. The contents of these applications are expressly incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The inventions of the present application were not made with Federal or state funds or grants. 
     THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
     The inventions of the present application were not made under a joint research agreement. 
     REFERENCE TO SEQUENCE LISTING 
     The present application does not have any nucleic acid, peptide or protein sequence. 
     BACKGROUND OF THE INVENTION 
     Embodiments of the present invention are directed to kits and methods for the detection of toxins produced by cyanobacteria. Cyanobacteria are commonly found in surface fresh water. Toxic cyanobacteria blooms are problems where such blooms may cause toxins to be released in water supplies. The major cyanobacterial toxins comprise cyclic peptides, alkaloids and lipopolysaccharides. 
     By way of example, without limitation, the major cyclic peptides comprising cyanobacterial toxins are nodulin, microcystin-LR, microcystin RR, and microcystin YR. The formula for nodulin is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin LR is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin RR is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin YR is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin LA is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin LY is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin LW is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for microcystin LF is set forth below: 
     
       
                 
         
             
             
         
      
     
     The alkaloid cyanobacterial toxins comprise, by way of example, without limitation, anaroxins and saxitoxins. Anaroxins comprise, by way of example, without limitation, anatoxin a, anatoxin a(S), homoanatoxin-a, cylindrospermopsin. 
     The formula for anatoxin a is set forth below: 
     
       
                 
         
             
             
         
      
     
     The formula for cylindrospermopsin is set forth below: 
     
       
                 
         
             
             
         
      
     
     The cyanobacterial lipopolysaccharides toxins are not as well characterized and appear to be less toxic than the cyclic peptides, alkaloids. 
     This paper will use the term “analyte” to denote a compound which one desires to determine the presence of absence of. 
     This paper will use the term “sample” to mean a material which one desires to test for the presence or absence of ergot alkaloids. The sample may be obtained as tissues or fluids from animals or plants. For example, without limitation, the sample may comprise leaves, seeds or other plant tissues or blood, urine, saliva or tissues obtained from animal sources. 
     An “extract” is a solution obtained by subjecting a sample to a solvent such that one or more compounds held in the sample are dissolved in the solution. 
     An “aliquot” is used to denote a subpart or fraction of a sample. 
     Chromatography is a method of separating compounds in a solution. Chromatography can be performed in different devices. This paper will use the term “cartridge” to refer to low pressure devices comprising a column and/or funnel in which a solid phase is placed. The sample is applied to the solid phase and passes through under low pressure or gravity. These devices are typically used to prepare a sample by removing particulates and concentrating desired compounds. 
     For the purpose of this paper, the term “column” will be used in the sense of a high pressure device in which solutions are forced through a solid phase matrix under pressure. The solid phase can be particulate or a porous monolith. 
     Mass spectrometry is used to determine the mass to charge ratio of ions formed by compounds. Mass spectrometers are used to form fragments of larger molecules and such fragments and complete ions are used to identify such compounds. 
     Standards are solutions with known amounts of compounds which solutions are used to compare data to data derived from non-standard samples. Standards can use compounds with labels comprising heavy isotopes which allow the operator of the mass spectrometer to differentiate between the standard and the analyte. 
     Interest in reliable and fast analysis of cyanobacterial toxins is needed to monitor water supplies, protect public health and fisheries. Prior to the present invention, the methods used to detect cyanobacterial toxins were not specific or sensitive to analyze for these compounds in the environment. Prior to the present invention, the methods were time consuming and labor intensive. 
     BRIEF SUMMARY OF THE INVENTION 
     Embodiments of the present invention are directed to reliable and fast analysis of cyanobacterial toxins. Embodiments of the present invention have utility for monitoring water supplies, protecting public health and fisheries. Embodiments of the present invention are directed to methods and kits for the detection of cyanobacterial toxins. 
     One embodiment of the present invention, directed to a method for detecting the presence or absence of cyanobacterial toxins in a sample comprises the steps of preparing a water sample potentially comprising cyanobacterial toxins to form a retained or concentrated toxin sample on a solid phase extraction device. Next, the retained or concentrated toxin sample is eluted in a mobile phase to form a sample extract. The sample extract is placed on the head of a chromatographic column packed with particles having a mean particle size of 1 to 3 microns under pressure of 6,000 to 15,000 psi to form a retained sample potentially comprising cyanobacterial toxin, in the event said sample extract contained such cyanobacterial toxins. Compounds from said retained sample, potentially comprising cyanobacterial toxins, are eluted from said chromatographic column under a gradient of organic solvent to form at least one eluted compound. This eluted compound comprises a cyanobacterial toxin, in the event said sample extract contained such cyanobacterial toxin. And, the eluted compound, potentially comprising a cyanobacterial toxin, is placed in a mass spectrometer to form a mass spectra. The mass spectra are used to determine the presence or absence of the cyanobacterial toxin. 
     Embodiments of the present method can be performed from said steps of placing the sample extract on the head of a chromatographic column to eluting and placing said cyanobacterial toxin in a mass spectrometer in a time period of less than fifteen minute and, preferably, ten minutes. 
     Preferably, the mass spectrometer forms one or more fragments of the cyanobacterial toxin and the spectra of the fragments are used to identify and determine the presence or absence of the cyanobacterial toxin. Preferably, the method comprises comparing the spectra from the parent ions and fragments to those obtained with standards. 
     Preferably, the column has a particle is selected from the group consisting of a bridged ethyl hybrid and high strength silica. Preferred particles have a mean average diameter of less than three microns. 
     Preferably, the sample extract is formed by extracting alkaloid cyanobacterial toxins on weak anion exchange resin. Preferably, the sample extract is formed by extracting cyclic peptide cyanobacterial toxins on weak cation exchange resin. The sample extract is preferably formed with an extraction cartridge or extraction device having a weak anion exchange resin or a weak cation exchange resin. Most preferably, the method comprises the step of forming at least one sample extract with both a weak cation exchange resin and a weak anion exchange resin. A preferred resin capable of being functionalized with weak anionic and weak cationic functional groups comprising a polymer, poly(divinylbenzene-co-N-vinylpyrrolidone). 
     A further embodiment of the present invention is directed to a kit for performing an analysis of a sample for the presence or absence of cyanobacterial toxins. As used herein, the term “kit” refers to an article of manufacture, an assembly of parts, reagents, and components for performing a method. Kits are typically packaged in suitable packaging such a wrap, box, clam shell or bag with instructions for use. Embodiments of the present invention comprise one or more standards for calibrating and facilitating the identification of one or more cyanobacterial toxins by mass spectroscopy. The kit, preferably, comprises sample preparation devices for forming sample extract, a column for separating the compounds of the sample extract and upon application of a gradient releasing the cyanobacterial toxins, if present, such that the cyanobacterial toxins are released to a mass spectrometer for identification. 
     A preferred column has a packing of particles having an average size of 1-3 microns. A preferred particle has a chromatographic surface selected from the group consisting of a bridged ethyl hybrid and high strength silica. A preferred column has an operating pressure of 6,000 to 15,000 psi. 
     Other features and advantages of the present invention will be apparent to individuals skilled in the arts upon viewing the figures and the detailed description that follow. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  depicts in schematic form an instrument for performing the method of the present invention; 
         FIG. 2  depicts a kit embodying features of the present invention; and 
         FIG. 3  depicts a separation and mass chromatogram of cyanobacterial toxins. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Embodiments of the present invention will be described in detail as methods and kits for the detection of cyanobacterial toxins. Embodiments of the present invention have utility for monitoring water supplies, protecting public health and fisheries, as well as food testing. The descriptions that follow are preferred embodiments reflecting what the inventors now consider is the best mode to practice their invention. Such descriptions are capable of modification and alteration by those skilled in the art without departing from the teaching hereof. 
     One embodiment of the present invention, directed to a method for detecting the presence or absence of cyanobacterial toxins in a sample is depicted in schematic form in  FIG. 1  with respect to an instrument generally designated by the numeral  11 . The instrument, generally designated by the numeral  11 , has the following major elements: sample preparation means  13 , a chromatography system  15 , a column  17  and a mass spectrometer  19 . 
     The method comprises the step of preparing a water sample potentially comprising cyanobacterial toxins with respect to sample preparation means  13 . As depicted, sample preparation means  13  comprises at least one sample extraction cartridge  21   a  and, preferably two sample extraction cartridges  21   a  and  21   b.    
     Sample extraction cartridges  21   a  and  21   b  are depicted in cross section. Each sample extraction cartridge  21   a  and  21   b  has a solid phase  23   a  and  23   b . A solid phase may comprise a bed of particles or a porous monolith resin. A preferred solid phase is particular and has a surface chemistry of poly(divinylbenzene-co-N-vinylpyrrolidone). That is, the particles may totally comprise the polymer or such polymer is carries as a surface layer on a substrate that is selected from a different material. Common materials which may be used as a substrate include, by way of example, without limitation, silica, aluminium and titanium oxides and other polymeric compounds. The surface chemistry of poly(divinylbenzene-co-N-vinylpyrrolidone) allows the particles of the sample extraction column to retain cyanobacterial toxins on a water wettable surface or allow the surface to be functionalized in a manner to capture cyanobacterial toxins more selectively or efficiently. 
     Sample extraction cartridges having a surface chemistry of poly(divinylbenzene-co-N-vinylpyrrolidone) are sold by Waters Corporation (Milford, Mass., USA) under the trademark OASIS®. Sample extraction cartridges without a surface chemistry of poly(divinylbenzene-co-N-vinylpyrrolidone) may also be used. Such sample extraction cartridges are sold by Waters Corporation (Milford, Mass., USA) under the trademark SEP-PAK. 
     Particles having a surface functionalized are preferably functionalized as weak cation exchange resins and or weak anion exchange resins for the preferential or efficient capture of cyanobacterial toxins. For example, without limitation, the sample extract is formed by extracting on weak anion exchange resin to favor alkaloid cyanobacterial toxins and formed on weak cation exchange resins to favor cyclic peptide cyanobacterial toxins. 
     Thus, as depicted, the sample is divided into two or more parts with one part directed to a first sample extraction cartridge  21   a  having a particle bed  23   a  comprising a weak anion exchange resin to favor alkaloid cyanobacterial toxins. A second sample extraction cartridge  21   b  has a particle bed  23   b  comprising a weak cation exchange resin to favor cyclic peptide cyanobacterial toxins. Such sample extraction cartridges  21   a  and  21   b  having weak cation or weak anion exchange resins are sold by several vendors including those sold by Waters Corporation (Milford, Mass.) under the trademark OASIS® WCX and OASIS® WAX. 
     The sample preparation means is depicted as sample extraction cartridges  21   a  and  21   b  as single well type devices with the understanding that such devices may have many forms comprising, by way of example, without limitation, single columns, cartridges and well devices as well as multiple well devices, such as 96 well plates and the like. 
     Most preferably, the method comprises the step of forming at least one sample extract with both a weak cation exchange resin and a weak anion exchange resin. The sample extract from sample extraction cartridge  21   a  is eluted and placed in a vial  25   a  and the sample extract from sample extraction cartridge  21   b  is eluted placed in vial  25   b . The sample extraction cartridges  21   a  and  21   b  retain the cyanobacterial toxin and release the cyanobacterial toxin upon elution with a mobile phase in a more concentrated form. This sample extract is placed and held in vials  25   a  and  25   b.    
     The vials  25   a  and  25   b  are placed in a chromatography system  15  autosampler, depicted in schematic form as a circular tray  27  holding vials  25 ′,  25 ″, and  25 ′″. Chromatography systems are well known in the art. A preferred chromatography system  15  has an operating pressure of 6,000 to 15,000 psi. Such chromatography systems  15  are sold by Waters Corporation (Milford, Mass., USA) under the trademark ACQUITY®. 
     Next, as depicted in  FIG. 2 , the method comprises the step of placing the sample extract on the head of chromatographic column  17 . Chromatographic column  17  is packed with particles having a mean particle size of 1 to 3 microns. Chromatographic column  17  has an operating pressure of 6,000 to 15,000 psi. A preferred column has particles with a chromatographic surface of a bridged ethyl hybrid composition or a high strength silica. columns  17 , having a 1.7 micron particle size, are sold by Waters Corporation (Milford, Mass., USA) under the trademark ACQUITY® with a BEH designation with respect to a bridged ethyl hybrid chemistry and a HSS designation with respect to high strength silica chemistry. In the event the sample extract held in vials  25 ′,  25 ″ or  25 ′″ has one or more cyanobacterial toxin, a retained cyanobacterial toxin is held on the particles until eluted under gradient conditions. The compounds of the sample are retained on the column to form one or more retained compounds. 
     Next, the one or more retained compounds are eluted under a gradient of organic solvent to form an eluted compound. And, in the event said sample extract contained such cyanobacterial toxins, such eluted compound is a toxin. A preferred gradient comprises a first solvent comprising 0.1% Formic Acid (H 2 O) and a second solvent comprising 0.1% Formic Acid (acetonitrile). The gradient is applied at a flow rate of 0.1 to 1.0 ml/min, and more preferably, at about 0.45 ml/min over a period of approximately six minutes moving from 2% of the first solvent to 80% of the second solvent. 
     This eluted cyanobacterial toxin, if present, is placed in a mass spectrometer  19  to form a mass spectra. The presence or absence of the cyanobacterial toxin is determined from the mass spectra. 
     Preferably, the mass spectrometer  19  forms one or more fragments of the cyanobacterial toxin. The formation of fragments in mass spectroscopy is sometimes denoted as MS/MS and is known to those skilled in the art. The spectra of the fragments are used to identify and determine the presence or absence of an cyanobacterial toxin. Mass spectrometers are sold by several venders including Waters Corporation (Milford, Mass., USA) under the trademark MICROMASS® TQD. 
     The identification of the cyanobacterial toxin, if present, is facilitated by placing one or more standards comprising a known labeled cyanobacterial toxin or closely related compound on the head of a column to be retained and eluted in the manner of sample cyanobacterial toxin. The eluted standard cyanobacterial toxin is placed in a mass spectrometer  19  to form a known spectra of the standard cyanobacterial toxin to which sample spectra are compared. Such labeled cyanobacterial toxin or closely related compound is used in a deuterated form known to individuals skilled in the art. The examples feature Cyclo (Arg-Ala-Asp-D-Phe-Val) and [Leu5]-Enkephalin. 
     The small particle column and high pressure performance of the chromatography system allow the method steps of placing the sample extract on the head of a chromatographic column, eluting and placing the cyanobacterial toxin in a mass spectrometer to be performed in a time period of three to fifteen minutes, and routinely in a period of approximately eight to nine minutes. 
     Preferably, the mass spectrometer forms one or more fragments of the cyanobacterial toxin and the spectra of the fragments are used to identify and determine the presence or absence of the cyanobacterial toxin. Preferably, the method comprises comparing the spectra from the parent ions and fragments to those obtained with standards. 
     Turning now to  FIG. 2 , a kit embodying features of the present invention, generally designated by the numeral  51 , is illustrated. The kit is a collection of parts and reagents bundled together with suitable packaging and instructions for their use in the method described above. Kit  51  comprises one or more standard vials, of which three are depicted designated  55 ′,  55 ″ and  55 ′″, containing standard solutions for calibrating and facilitating the identification of one or more cyanobacterial toxins by mass spectroscopy. The kit  51  further comprises one or sample preparation devices in the form of extraction cartridges, of which three are depicted  21 ′,  21 ″, and  21 ′″ for forming sample extract. The kit further comprises a column  17  for separating the compounds of the sample extract and upon application of a gradient releasing the cyanobacterial toxins, if present, such that the cyanobacterial toxins are released to a mass spectrometer for identification. The kit  51  further comprises instructions  57  for the use of these parts and reagents in the method as previously described. The kit is depicted with suitable packaging, which is known in the art, and may comprise plastic wraps and bubble shells, boxes, wrapping and the like. 
     Further features of the present invention are described with respect to the following examples. 
     Example 1 
     Multi Column Solid Phase Extraction (SPE) Using Mixed Mode Cartridges 
     This discussion is focused on an analysis of cylindrospermopsin, anatoxin-a and microcystins from lake or process water or water extracts from filters or other surfaces. 
     Method Summary: 
     Cartridges are initially conditioned, equilibrated and loaded in series (OASIS®WCX on top followed by OASIS® WAX cartridge with a union connecting the two). Once loaded they are separated and processed individually and run as two separate runs 
     Protocol: 
     WAX (6 cc 150 mg Waters part number 186002493)—This is for cylindrospermopsin enrichment (this is on the bottom) 
     Condition: 3 mL MeOH 
     Equilibrate: 5 mL H2O 
     Load: Up to 500 mL of sample water (pH the water to pH 5.5 with formic acid) 
     Separate Columns and Continue on Each Separately 
     Wash: 2 mL 1% Formic in DI H2O 
     Wash 2: 2 mL MeOH 
     Elute: 3 mL 1% ammonium hydroxide in DI H2O 
     Run “as is” or evaporate to dryness and reconstitute in mobile phase 
     WCX (6 cc 150 mg Waters part number 186002498)—This is for anatoxin-a, and microcystins enrichment (this is on the top) 
     Condition: 3 mL MeOH 
     Equilibrate: 5 mL H2O 
     Load: Up to 500 mL of sample water (pH the water to pH 5.5 with formic acid) 
     Separate Columns and Continue on Each Separately 
     Wash: 2 mL pH 9 ammonium hydroxide or ammonium bicarbonate in DI H2O 
     Wash 2: 2 mL MeOH 
     Elute: 3 mL 1% Formic acid in DI H2O 
     These cartridges are run “as is” or evaporated to dryness and reconstituted in mobile phase. 
     Example 2 
     Anatoxin-a, Cylindrospermopsin, and Microcystins by Extreme Pressure High Performance Liquid Chromatography/MS/MS 
     This example features the separation and mass spectral analysis of anatoxin-a, cylindrospermopsin, and several microcystins by high performance liquid chromatography and mass spectrometry. 
     Column Used: HSS T3 2.1×100 mm @35 C
         OR BEH C18 2.1×100 mm @35 C       

     Solvent A: 0.1% Formic Acid in H2O 
     Solvent B: 0.1% Formic in Acid in Acetonitrile 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 [Gradient Table] 
               
             
          
           
               
                 Time (min) 
                 Flow Rate 
                 % A 
                 % B 
                 Curve 
               
               
                   
               
             
          
           
               
                 Initial 
                 0.450 
                 98.0 
                 2.0 
                 — 
               
               
                 0.80 
                 0.450 
                 98.0 
                 2.0 
                 6 
               
               
                 9.00 
                 0.450 
                 30.0 
                 70.0 
                 6 
               
               
                 9.05 
                 0.450 
                 20.0 
                 80.0 
                 6 
               
               
                 9.90 
                 0.450 
                 20.0 
                 80.0 
                 6 
               
               
                 9.91 
                 0.450 
                 98.0 
                 2.0 
                 6 
               
               
                 12.00 
                 0.450 
                 98.0 
                 2.0 
                 6 
               
               
                   
               
             
          
         
       
     
     These results are set forth in  FIG. 3 . These results suggest that the compounds identified can be separated and identified in less than nine minutes. 
     TQD Conditions (MS/MS) 
     Function 1—Cylindrospermopsin 
     Retention window (mins): 1.000 to 2.220 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 3 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 416.20 &gt; 176.23   0.060   45.0   39.0   Auto   Cylindro (C1)       2: 416.20 &gt; 194.25   0.060   45.0   37.0   Auto   Cylindro (Q)       3: 416.20 &gt; 416.20   0.060   45.0   5.0   Auto   Cylindro (C2)                    
Function 2—Anatoxin-a
 
     Retention window (mins): 2.220 to 3.000 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 3 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 166.13 &gt; 43.03   0.060   20.0   23.0   Auto   Anatoxin (Q)       2: 166.13 &gt; 149.14   0.060   20.0   15.0   Auto   Anatoxin (C1)       3: 166.13 &gt; 166.13   0.060   25.0   5.0   Auto   Anatoxin (C2)                    
Function 3—Cyclo (Arg-Ala-Asp-D-Phe-Val)—Used as an Internal Standard
 
     Retention window (mins): 4.150 to 4.550 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 3 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 589.47 &gt; 72.77   0.050   55.0   73.0   Auto   Cyclo (IS)-C2       2: 589.47 &gt; 120.16   0.050   55.0   57.0   Auto   Cyclo (IS)       3: 589.47 &gt; 589.47   0.050   55.0   5.0   Auto   Cyclo (IS)-C1                    
Function 4—=[Leu5]-Enkephalin (Used as an Internal Standard)
 
     Retention window (mins): 4.500 to 5.000 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 3 channels 
                                                                               Dwell   Cone       Delay           Chan Reaction   (secs)   Volt.   Col. Energy   (secs)   Compound                                1: 556.42 &gt; 120.16   0.050   35.0   53.0   Auto   Enk (IS)-C2       2: 556.42 &gt; 136.17   0.050   35.0   53.0   Auto   Enk (IS)       3: 556.42 &gt; 556.42   0.050   35.0   5.0   Auto   Enk (IS)-C1                    
Function 5—Microcystin RR
 
     Retention window (mins): 5.400 to 6.000 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 3 channels 
                                                                               Dwell   Cone       Delay           Chan Reaction   (secs)   Volt.   Col. Energy   (secs)   Compound                                1: 519.99 &gt; 135.10   0.070   45.0   32.0   Auto   RR (Q)       2: 519.99 &gt; 519.99    0.070   45.0   5.0   Auto   RR (C1)       3: 1038.69 &gt; 135.10   0.070   90.0   80.0   Auto   RR (C2)                    
Function 6—Microcystin YR
 
     Retention window (mins): 6.100 to 6.400 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 1045.60 &gt; 135.18   0.050   95.0   50.0   Auto   YR (Q)       2: 1045.60 &gt; 1045.60   0.050   95.0   5.0   Auto   YR (C1)                    
Function 7—Microcystin LR
 
     Retention window (mins): 6.150 to 6.550 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
                                                                               Dwell   Cone       Delay           Chan Reaction   (secs)   Volt.   Col. Energy   (secs)   Compound                                1: 995.66 &gt; 135.11   0.050   85.0   50.0   Auto   LR (Q)       2: 995.66 &gt; 995.66   0.050   50.0   5.0   Auto   LR (C1)                    
Function 8—Microcystin LA
 
     Retention window (mins): 7.400 to 7.750 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
                                                                               Dwell   Cone       Delay           Chan Reaction   (secs)   Volt.   Col. Energy   (secs)   Compound                                1: 910.57 &gt; 135.11   0.050   45.0   50.0   Auto   LA (Q)       2: 910.57 &gt; 910.57   0.050   45.0   5.0   Auto   LA (C1)                    
Function 9—Microcystin LY
 
     Retention window (mins): 7.600 to 8.000 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 1002.63 &gt; 135.18   0.050   50.0   50.0   Auto   LY (Q)       2: 1002.63 &gt; 1002.63   0.050   50.0   5.0   Auto   LY (C)                    
Function 10—Microcystin LW
 
     Retention window (mins): 8.200 to 8.700 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
                                                                               Dwell   Cone   Col.   Delay           Chan Reaction   (secs)   Volt.   Energy   (secs)   Compound                                1: 1025.63 &gt; 135.05   0.070   45.0   42.0   Auto   LW (Q)       2: 1025.63 &gt; 1025.63   0.070   45.0   5.0   Auto   LW (C1)                    
Function 11—Microcystin LF
 
     Retention window (mins): 8.430 to 8.700 
     Ionization mode: ES+ 
     Data type: MRM data 
     Function type: MRM of 2 channels 
     
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Dwell 
                 Cone 
                   
                 Delay 
                   
               
               
                 Chan Reaction 
                 (secs) 
                 Volt. 
                 Col. Energy 
                 (secs) 
                 Compound 
               
               
                   
               
             
             
               
                 1: 986.63 &gt; 135.05 
                 0.050 
                 40.0 
                 50.0 
                 Auto 
                 LF (Q) 
               
               
                 2: 986.63 &gt;