Criteria for Quality Control Protocols for Various Algal Toxin Methods [Project #2942]

Transcription

1 Criteria for Quality Control Protocols for Various Algal Toxin Methods [Project #2942] ORDER NUMBER: 2942 DATE AVAILABLE: April 2012 PRINCIPAL INVESTIGATORS: John Papageorgiou, Brenton Nicholson, Wasa Wickramasinghe, Glen Shaw, Geoff Eaglesham, Wayne Carmichael, and Jeanette Frey BACKGROUND: Cyanobacterial or bluegreen algal blooms are known to occur in freshwater sources worldwide. The fact many species of cyanobacteria produce highly toxic compounds is of particular concern to water utilities, health regulatory bodies, and obviously users (drinking and recreational) of freshwater supplies. In terms of geographical occurrences of toxic cyanobacteria, the United States is mostly affected by microcystin (hepatotoxic), anatoxin (neurotoxic), and cylindrospermopsin (hepatotoxic) producing cyanobacteria. In contrast, Australia is affected by saxitoxin (neurotoxic), microcystin, and cylindrospermopsin producing cyanobacteria but not anatoxin producing strains. The first line of action in managing episodes of algal blooms in freshwater sources involves their taxonomic identification and analysis of their intracellular content for the presence of toxic metabolites. Subsequent management decisions are usually influenced by informal cyanotoxin guideline values. Currently significant efforts are being made by the World Health Organization (WHO), United States, and Australian authorities to develop and implement mandatory cyanotoxin drinking water guidelines in order to expedite and refine current water quality management practices associated with toxic algal bloom occurrences. Consequently, this has escalated the requirement for precise and robust quantitative analytical cyanotoxin methods. Toxicological data to date suggest formal WHO drinking water guidelines for all of the common cyanotoxins, cylindrospermopsin, anatoxins, saxitoxins, and microcystins (current WHO guideline for -LR variant is 1.0 g/l) will ultimately be in the low microgram per litre (g/l) range. Based on results of a previous Water Research Foundation funded project (#2789: Determination and Significance of Emerging Algal Toxins) and a literature review (see chapter 6 of this report), high performance liquid chromatographic (HPLC) methods and to a lesser extent, enzyme linked immunosorbent assays (ELISA) can detect and quantitate toxins present in water and cyanobacterial extracts in low g/l levels. This suggests that such analytical methods can be utilized as water quality management tools when making decisions regarding assessment of guideline compliance, selection of water treatment strategies, and activation of appropriate health alerts. However, inclusion of appropriate quality assurance (QA) plans when undertaking cyanotoxin analyses are essential in order to minimize any ambiguity currently experienced by water utility managers when determining the validity of analytical data.

2 OBJECTIVES: The Water Research Foundation and the Cooperative Centre for Water Quality and Treatment funded this study to develop QA protocols for the quantitative analysis of microcystins, saxitoxins, cylindrospermopsin, and anatoxin-a present in water and cyanobacterial extracts. The sub-objectives of this project involved the evaluation of the key precision influencing (quality control or QC) elements listed below for the analysis of microcystins by ELISA and HPLC coupled to photodiode array detection (HPLC-PDA); saxitoxins by high performance ion exchange chromatography coupled to fluorescence detection (HPIC-FD) and hydrophilic interaction liquid chromatography coupled to mass spectroscopic detection (HILIC-MS); cylindrospermopsin by HPLC-PDA, HPLC-MS, and HILIC-MS; and anatoxin-a by HILIC-MS and HPLC-MS. Reproducibility/variability of analytical method Linearity of detector response and limit of detection/quantitation Sample concentration/extraction performance (recovery and precision) Production and/or acquisition of certifiable cyanotoxin analytical standards Preservation of samples/sample stability CONCLUSIONS: Microcystins Evaluation of QC Parameters for Envirologix ELISA The Envirologix diagnostics kit is capable of detecting microcystins present in water down to 0.01 g/l. The average concentration of a 1.0 g/l microcystins-lr (MC-LR) standard tested was determined to be 0.77 ± 0.23 g/l. While this concentration is accurate when taking into account the standard deviation (SD), within plate and plate to plate variability (high CV values) was poor for replicate analyses of this standard MC-LR solution. Even poorer reproducibility was observed for replicate analysis of MC-LR extracted from two cyanobacterial strains representing an approximate intracellular toxin concentration range of 1 to 10 g/l. Evaluation of QC Parameters for HPLC-PDA Limit of quantitation (LOQ) of all microcystin variants by HPLC-PDA was found to be 0.5 g/ml. While this value exceeds the WHO guideline value of 1 g/l, concentrations of 1 g/l can be achieved provided samples are concentrated 500 fold during sample preparation. Poor reproducibility was observed for HPLC-PDA analysis of poor quality (high dissolved organic carbon content or DOC) raw waters containing microcystins in concentrations less than 0.5 g/l levels. Nevertheless, relatively high quality raw waters not requiring initial sample clean-up and pre-concentration (waters containing microcystin variants at levels of 0.5 g/ml and higher) can be analyzed with adequate precision using an external standard approach. The cost of a HPLC-PDA system is within the range of US$ 80, ,000.

3 Extraction of Microcystins from Cyanobacteria Intracellular Microcystin-LR can be efficiently extracted from toxic Microcystis aeruginosa by subjecting the cells to three minutes of sonication or five freeze-thaw cycles. Extraction/Concentration of Microcystins from Water Waters HLB Oasis solid phase extraction (SPE) cartridges out-performed Waters C 18 SPE cartridges for the concentration of microcystins in water of variable quality. Production of Certifiable Microcystin-LR and -LA Analytical Standards Microcystin standards of high quality can be prepared in the laboratory, provided respective microcystin producing cyanobacterial cultures are available. Intracellular microcystin containing extracts can be purified to homogeneity by repeated preparative C 18 reverse phase HPLC. HPLC-MS, proton nuclear magnetic resonance ( 1 HNMR) spectroscopy and amino acid compositional analysis can be used to confirm the identity and purity of microcystin isolates and therefore to certify them as analytical standards. Preservation of Microcystin Containing Water Samples Microcystin containing solutions degrade over time, but when kept at low temperatures in the dark degradation is minimal. Microcystins present in dam water show the highest decomposition as compared to microcystins in ultra-high purity water probably due to more microbiological activity in the former case. Saxitoxins Preliminary Evaluation of QC Parameters for HPIC-FD HPIC-FD coupled to chemical post-column derivatisation is a sensitive, rapid and precise HPLC method for the direct (no sample pre-concentration) determination of typical cyanobacterial PSPs (mainly C1, C2, GTX2, GTX3, NeoSTX, STX) present in natural fresh waters and cyanobacteria in greater than 20 g/l levels. However, HPIC-FD analysis of water samples containing STXs in levels lower than 20 g/l (3 g/l is the Australian drinking water guideline for STX equivalents) is possible provided a suitable pre-concentration step is included in the analytical protocol. Validation of carbon based SPE cartridges for the concentration of STXs in water is required prior to establishing HPIC-FD as a water industry method for the analysis of STXs. The cost of a HPLC-FD system is within the range of US$ 80, ,000. Evaluation of QC Parameters for HILIC-MS HILIC-MS/MS incorporating SPE sample pre-concentration and the use of a HPLC-MS/MS 4000Q trap system (or equivalent) is capable of quantitating STX s present in waters in concentrations well below the Australian 3.0 g/l STX guideline with adequate precision.

4 HILIC-MS/MS would be a high powered effective water utility management/monitoring tool for STXs, however, it would be expected that such an analytical capability would be financially cost prohibitive for small water utilities. The cost of a state-of-the-art HILC-MS/MS system is within the range of US$ 350, ,000. Extraction of Saxitoxins from Cyanobacteria Saxitoxins can be efficiently and rapidly extracted from toxic Anabaena circinalis by subjecting freeze-dried cells that are suspended in a solvent system composed of 50% MeOH in 50 mm aqueous acetic acid to sonication. Extraction/Concentration of Saxitoxins from Water Carbograph SPE cartridges appear to be effective for concentrating STXs from water. Further validation of these cartridges using a selection of waters of variable quality is required prior to establishing this SPE cartridge as the choice means of concentrating STXs in water. Production of Certifiable Saxitoxins Analytical Standards Saxitoxin standards of high quality can be prepared in the laboratory. Adequate quantities of STXs can be readily obtained by extracting toxic strains of the cyanobacteria, Aphanizomenon flos-aquae, and Anabaena circinalis. STX containing extracts are purified to homogeneity using a combination of size exclusion liquid chromatography and ion exchange chromatography. HILIC-MS/MS and 500 MHz 1 HNMR spectroscopy is required to confirm the identity and purity of saxitoxin isolates and therefore certify them as analytical standards. Preservation of Saxitoxin Containing Water Samples Cyanobacterial PSP variants such as C1/2, GTX2/3, and STX are relatively stable when present in waters of varying quality containing 5 mm Acetic Acid (AcOH) (spiked) for a period of up to six months and within a temperature range of 4 to ~25 0 C. Cylindrospermopsin Evaluation of QC Parameters for HILIC-MS/MS Water samples containing CYN present in levels as low as 20 ng/l can be analysed by HILIC- MS/MS using a state-of-the-art MS detector (API4000Q Trap or equivalent) provided the sample is pre-concentrated 100 fold prior to analysis. The precision of HILIC-MS (using 4000Q Trap MS or equivalent) is adequate for the direct (no pre-concentration) analysis of CYN present in waters at levels between 10 and 100 g/l. Evaluation of QC Parameters for HPLC-MS/MS CYN can be quantitatively determined by HPLC-MS/MS even when present at 0.2 g/l. The LOQ or lowest concentration minimum reporting level (LCMRL) determined for the analysis of

5 CYN by this method was 0.37 g/l. Excellent linearity of detector response was noted for CYN present in waters in the range of g/l using an AB Sciex API300 MS based HPLC-MS system. However, the reproducibility of this method using this HPLC-MS system is suspect for direct analysis of CYN present in waters in less than 5 g/l levels. It is envisaged that analysis of CYN present in waters in sub 1 g/l levels could be directly (without pre-concentration) carried out by this HPLC-MS method with far greater precision providing a highly sensitive modern instrument such as an API4000Q trap based HPLC-MS or its equivalent be used. Evaluation of QC Parameters for HPLC-PDA In general, CYN present in water of variable quality in levels as low as 0.05 mg/l can be determined by HPLC-PDA utilizing an external standard quantitation approach with adequate precision. However, poor precision achieved in an interlaboratory exercise for the HPLC-PDA analysis of some CYN containing water samples reflects the temperamental nature of this analytical method and the need for the development of an internal standard based HPLC-PDA protocol especially for waters samples requiring pre-concentration (sub 0.05 mg/l). Extraction of Cylindrospermopsin from Cyanobacteria Simple freeze-drying of a water sample containing a toxic strain of Cylindrospermopsis raciborskii is sufficiently effective for the release of intracellular CYN content. Additional extraction steps involving freeze-thawing or sonication in the presence of aqueous formic acid systems do not enhance the recovery of intracellular CYN. Extraction/Concentration of Cylindrospermopsin from Water The combined use of C 18 modified silica and carbon based SPE cartridges in-conjunction with an elution system comprising an aqueous formic acid - methylene chloride mixture is optimal for the concentration and recovery of CYN from poor quality freshwater sources. Production of Certifiable Cylindrospermopsin Analytical Standard Cylindrospermopsin for the purpose of producing analytical standards can be sourced from toxic strains of Cylindrospermopsis raciborskii. Initial confirmation of the presence of CYN in extracts can be carried out using HPLC-PDA. CYN containing extracts are concentrated and purified to homogeneity using carbon SPE and semi-preparative reverse phase HPLC, respectively. UV spectrophotometric analysis, HILIC-MS/MS, and 500 MHz 1 HNMR spectroscopy is required to confirm the identity and purity of purified CYN isolates and therefore certify them as analytical standards. Preservation of Cylindrospermopsin Containing Water Samples Both analytical standard CYN solutions and natural water samples containing CYN in concentrations of approximately 5 mg/l can be safely stored with in a temperature range of 4 to 25 C in the dark for a period of at least six months. The excellent stability noted for CYN in poor reservoir quality water indicates that CYN present in natural waters even in the low mg/l

6 range would most likely be stable for a substantial length of time. However, to further safeguard against possible microbial-induced degradation, CYN-containing natural water samples (reservoir waters) and analytical standard solutions should be stored under 10 C in the dark. Anatoxin-a Evaluation of QC Parameters for HILIC-MS HILIC-MS/MS incorporating SPE sample concentration (100 fold concentration is achieved) and the use of a HPLC-MS/MS 4000Q trap system or equivalent is capable of quantitating anatoxin-a (ANTX-a) present in waters in concentrations well below the proposed 1.0 g/l with good precision. However, as concluded for the concomitant analysis of STXs and CYN, it would be expected that purchase of a HILIC-MS/MS system would be financially cost-prohibitive for small water utilities. Evaluation of QC Parameters for HPLC-MS ANTX-a can be quantitated in waters with adequate precision by HPLC-MS/MS without preconcentration even when present in waters at 1 g/l. However, as concluded for CYN, it is envisaged that accurate HPLC-MS analysis of ANTX-a present in waters in sub 1 g/l levels without pre-concentration could be realized providing a highly sensitive modern instrument such as an API4000Q Trap based HPLC-MS or its equivalent be used. Extraction of Anatoxin-a from Cyanobacteria Currently little detailed information on the efficacy of extraction of ANTX-a from cyanobacteria (Anabaena) is present in the literature. The efficacy of freeze-drying, freeze-thawing, and sonication has not been examined due to a lack of available ANTX-a producing cyanobacteria strains during this project. However, based on the chemical structure of ANTX-a, the extraction of ANTX-a from Anabaena is likely to be similar to that used for CYN from C. Raciborskii. Extraction/Concentration of Anatoxin-a from Water Waters Oasis HLB and Waters Oasis WCX SPE cartridges did not produce the 90% or greater recovery yield desired for the pre-concentration/treatment of ANTX-a from water. The retention of ANTX-a may be influenced by the ph and organic solvent concentration used in the concentration process. It is advisable to assess both columns at lower ph values as well as a higher range of MeOH concentrations. Production of Certifiable Anatoxin-a Analytical Standard Like all cyanobacterial toxins, analytical anatoxin-a (ANTX-a) standard material can be prepared in-house providing an ANTX-a producing cyanobacterial culture can be sourced together with chromatographic materials and chemicals necessary for its extraction and purification. ANTX-a containing extracts can also be purified by reverse phase HPLC. Once again, HPLC-MS/MS and

7 500 MHz 1 HNMR spectroscopy is required to confirm the identity and purity of purified ANTXa isolates and therefore certify them as analytical standards. Preservation of Anatoxin-a Containing Water Samples Neither DOC concentration nor temperature has an effect on the recovery of ANTX-a from water samples stored over a 6 month period. The only factor affecting the recovery of the toxin is time. However, in order to safeguard against degradation promoted by any means, ANTX-a samples should be analyzed within the first four months after collection. While DOC concentration does not affect the stability of ANTX-a, when possible, ANTX-a solutions should be prepared in ddih 2 O prior to storage. RECOMMENDATIONS: Frequent occurrences of toxic cyanobacteria in surface waters in both Australia and the United States present a potential health risk in drinking water supplies. Therefore, significant pressure by regulatory authorities and consumers is exerted on water utilities in the United States and Australia to produce safe drinking water. The first level of action during cyanobacteria occurrences should always be the taxonomic identification of the species contaminating the freshwater supply. It is then recommended that water utility authorities use the appropriate chromatographic based detection method to determine whether the particular species is toxic, and if so, the concentration of the intra-cellular and extracellular toxin contributions as outlined in Table R.1. In the remote case where taxonomic identification is not possible, it is recommended that the sample be screened for the presence of all known cyanotoxins using the appropriate methods as shown in Table R.1. For this purpose ELISA based methods as recommended in WaterRF project report #2789 can be used to rapidly screen water supplies for the presence of cyanotoxins; however, samples that return a positive result should be re-analysed by a chromatographic method for confirmation. Recommended specific QA plans/operating guidelines for water utility analysts for the quantitative analysis of cyanotoxins (determination of both intracellular and extracellular or dissolved concentrations) using chromatographic methods are presented on Tables 2.6 and 2.7 (microcystins), Tables 3.10 and 3.11 (saxitoxins), Tables 4.21 and 4.22 (cylindrospermopsin), and Tables 5.6 and 5.7 (anatoxin-a) of this report. It is envisaged that utilization of such protocols will lead to more informed decisions by water utilities when managing cyanobacteria occurrences in freshwater supplies.

8 Table R.1 Recommended chromatographic based methods for the quantitative analysis of cyanotoxins relevant to North American and Australian water supplies* Cyanobacterial species (to be identified by microscopy) Probable toxin associated with cyanobacterial species Probable location for cyanobacterial occurrence Analytical method (for intra and extracellular concentration determination) Planktonic species Anabaena bergii Cylindrospermopsin Australia HPLC-PDA or HILIC- MS/MS or HPLC-MS Anabaena circinalis Saxitoxins Australia HPIC-FD or HILIC- MS/MS Anabaena flos-aquae Anatoxin-a Canada HILIC-MS/MS or HPLC-MS Anabaena flos-aquae Anatoxin-a(s) Canada HILIC-MS/MS or HPLC-MS Anabaena flos-aquae Microcystins Canada HPLC-PDA Aphanizomenon flosaquae MS/MS HPIC-FD or HILIC- Saxitoxins USA Aphanizomenon HPLC-PDA or HILIC- Cylindrospermopsin Australia ovalisporum MS/MS or HPLC-MS Cylindrospermopsis HPLC-PDA or HILIC- Cylindrospermopsin Australia, USA raciborskii MS/MS or HPLC-MS Lyngbya wollei Saxitoxins USA HPIC-FD or HILIC- MS/MS Microcystis aeruginosa Microcystins Australia, USA HPLC-PDA Nodularia spumigena Nodularin Australia HPLC-PDA Benthic species Hapalosiphon Microcystins USA HPLC-PDA hibernicus Phormidium sp. not known Australia *Some information present in this table has been extracted from the Executive Summary, WaterRF project report #2789, Determination and Significance of Emerging Algal Toxins FUTURE WORK: Overall, results of this project justify the development of internal standard based HPLC methods for the quantitative analysis of cyanotoxins (microcystins, saxitoxins, cylindrospermopsin, and anatoxin-a) requiring their pre-concentration from water using solid phase extraction (SPE) cartridges. The inclusion of an appropriate internal standard (IS) in the water sample prior to its concentration using SPE cartridges will ultimately correct errors in the concentration values caused by sample matrix effects, poor SPE performance, poor analytical instrument performance, and/or inexperienced analysts. Such methods would lead to the establishment of less complicated and more robust cyanotoxin analytical quality assurance (QA) plans for water utilities.