Elsevier

Journal of Chromatography A

Volume 1216, Issue 15, 10 April 2009, Pages 3147-3155
Journal of Chromatography A

Method validation of microcystins in water and tissue by enhanced liquid chromatography tandem mass spectrometry

https://doi.org/10.1016/j.chroma.2009.01.095Get rights and content

Abstract

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method has been developed and validated to identify and quantify trace levels of cyanotoxins or microcystins (MC) in water, bivalves and fish tissue with enhanced sensitivity and specificity. The method enables confirmation and quantification of six MCs (MC-LA, LF, LR, LW, RR and YR) with a single chromatographic run. The applied chromatography also allows determination of certain MC metabolites (Desmethyl-LR and -RR). By using LC-ESI–MS/MS in multiple reaction monitoring (MRM) mode, the limit of detection and quantitation for the microcystins studied, were determined to be between 0.2 and 1 pg on column (5:1 S/N ratio). These values are below the 2 pg detection limits found in the available literature.

Introduction

Cyanobacteria, also known as blue-green algae, are a small group of photosynthetic-planktonic bacteria whose evolution dates back more than 3.5 billion years. Cyanobacteria are widely distributed in eutrophic aquatic environments worldwide. Many of the common cyanobacterial species produce toxic metabolites which can be lethal to wildlife, domestic livestock and humans [1]. Contaminants associated with cyanobacteria are called cyanotoxins. The cyanotoxins are divided into three classes based on chemical structure: cyclic peptides, alkaloids and lipopolysaccharides.

Nodularins (NDLN) and microcystins (MC) are cyclic peptides containing five and seven amino acids, respectively. Microcystins, the most common and important cyanobacterial toxins, are cyclic heptapeptide hepatotoxins. More than 70 structural variants of microcystins, isolated primarily from the freshwater genera Microcystis, Planktothrix (Oscillatoria), Anabaena and Nostoc, have been described in the scientific literature [2]. Where as nodularins (less than 10 known variants) are cyclic pentapeptides and are produced mainly by Nodularia, found in brackish waters [3]. Toxic cyanobacterial blooms (microcystis) are an emerging issue in the United States and worldwide because of increasing amounts of nutrient pollutants (nitrogen and phosphorous) in surface waters and warmer weather patterns which favor the growth of cyanobacteria and lead to more microcystin outbreaks [4]. The increasing number of cyanobacteria infested surface waters used for drinking, irrigation and recreation water constitute a potential risk to public health, domestic animals and wildlife. These health hazards have led the World Health Organization (WHO) to establish a provisional guideline value of MC-LR of 1 μg/L for drinking water [5]. Health Canada calculated a tolerable daily intake (TDI) of 0.013 μg of MC-LR (kg of body weight)−1 day−1 (defined as a 60-kg adult consuming 1.5 L of water per day, with an MC-LR content of 0.5 ng/mL water) [6]. MCs are extremely stable in water because of their chemical structure, surviving in both warm and cold water and can tolerate radical changes in water chemistry, including pH. MCs can remain toxic even after being boiled [7].

The general structure of microcystins is cyclo(D-Ala-L-X-D-erythro-methylAsp (iso-linkage)-L-Z-Adda-D-Glu(iso-linkage)-N-methyldehy-droAla), where X and Z are variable l-amino acids (Fig. 1). By using amino acid single letter code classification, each microcystin is designated a name depending on the variable amino acids which complete their structure. For example, one of the most common toxins found in water supplies around the world, microcystin-LR contains the amino acids Leucine (L) and Arginine (R) in these variable positions.

The structures of NDLNs are similar to MCs but with a five-member amino acid ring instead of a seven-member one. The structure of nodularin-R (NDLN-R) is cyclo(D-erythro-methylAsp (iso-linkage)-L-Arg-Adda-D-Glu(iso-linkage)-2-(methyl-amino)-2-(Z)-dehydrobutyric acid). Adda stands for the amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid [2], [8].

The characteristic feature of both microcystins and nodularins is the presence of ADDA (4E, 6E-3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid). Structural variants of microcystins commonly contain other l-amino acids at two nonconserved sites in the peptide ring. Other structural variants arise from the presence or absence (desmethyl variants) of methyl groups at the β-Me-Asp (dm-MC-RR and dmMC-LR) and N-methyldehydroalanine (Mdha) (Nodularin) residues, but such changes have little effect on the toxicity of the molecules [9]. Isomerization of the ADDA moiety to form 6Z-ADDA microcystin analogues renders the molecule essentially non-toxic. Thus, 6E geometry in the ADDA moiety is considered a prerequisite for toxicity in microcystin congeners [10].

MCs are associated with freshwater environments and their bioaccumulation by aquatic animals, including zooplankton, fish and water filter feeders such as bivalves, has been reported by several authors [11], [12], [13], [14]. Liver, followed by kidney and intestine accumulate most of the MCs in exposed fish. Because these organisms are an important food source, not only for birds and fish but also for mammals, MCs can be transferred to higher trophic levels through the food chain leading to human toxicity.

Microcystins are known liver toxins. Carmichael suggests that “the extraordinarily high rates of liver cancer in humans in parts of China may be tied to the cyanobacterial toxins in water [15].” At a hemodialysis clinic in the town of Caruaru in north-east Brazil, an outbreak of severe hepatitis occurred where dialysis water contaminated with blue-green algal toxins caused the death of 50 patients from acute liver failure [16], [17]. Severe cases of gastroenteritis in North and South America [18] and Australia [19] have been linked to the consumption of drinking water contaminated by cyanobacteria. Toxic and non-toxic strains of the same cyanobacterial species show no predictable difference in appearance. Therefore, it is necessary to analyze the toxin content by physico-chemical, biochemical or biological methods [20], [21].

Various analytical techniques have been used to analyze these toxins, such as enzyme-linked immunosorbent assay (ELISA) [22], phosphatase inhibition assay [23], gas chromatography–mass spectrometry (GC–MS) [24], liquid chromatography with UV detection (HPLC-UV) [25], capillary electrophoresis (CE) [26] and, more recently, liquid chromatography–mass spectrometry (LC–MS) [27], [28], [29]. LC combined with different detectors, such as UV detection or mass spectrometry (MS), can identify and quantify MCs in freshwater, cyanobacterial blooms, fish, shellfish and other biological samples [30], [31], [32]. LC–MS offers the advantage of providing specificity and good sensitivity. For this reason LC–MS has increased in popularity [33], [34], [35]. HPLC is a powerful tool to separate specific toxins; however the typical detection technique (UV) does not get near the sensitivity and the selectivity of LC–MS without extensive sample preparation or enrichment prior to analysis. ELISA methods offer a fast screening tool but can suffer from false positives depending upon the matrix. In addition, ELISA can confirm the presence of microcystins but does not identify which specific toxin is present. While chromatographic methods are capable of detecting and identifying single congeners, routine quantification of all known congeners is almost impossible because new analogues, especially of microcystins, continue to be discovered [36], [37], [38]. The quantitative analysis by LC–MS of microcystins has usually involved separation on C18 sorbents followed by electrospray ionization and detection by MS instruments [39], [40], [41], [42], [43], [44]. Typical detection limits in recent work have been in the low pg range per injection. LC–MS has also been shown to provide valuable molecular weight information.

Because of the low provisional limits set by the WHO and Health Canada, effective consumer protection requires the sensitive and efficient detection of the whole spectrum of cyanobacterial cyclic peptide toxin congeners, many of which are as toxic as MC-LR, and regulation should not be restricted to MC-LR alone [45], [46]. This, however, requires that the present methods for cyclic peptide toxin analysis be able to quantify the individual congeners with similar sensitivities and at concentrations well below the proposed limits (because the toxic effects of the various congeners are expected to be additive).

The work presented in this article focused on the development of a simple, sensitive and selective LC–MS method to analyze as many of the target toxins as possible. Fig. 1 shows the list for the various toxins studied. One of the biggest challenges to monitoring these toxins is that very few of them are available as analytical standards. For this reason only six of the toxins, the only ones commercially available at the time, were analyzed. Four of the toxins (MC-LR, RR, YR and LA) are listed by the US EPA as the most important algal toxins in the United States, with MC-LR also listed by World Health Organization (WHO) as the most common toxin found.

Section snippets

Chemicals and reagents

Certified MC standards (LR, RR, LF, LW and NDLN-R) were purchased from Calbiochem (EMD Chemicals, La Jolla, CA) and LR, RR, YR, LA were purchased from Sigma–Aldrich (Allentown, PA). Burdick and Jackson HPLC grade solvents (acetonitrile, methanol, water), glass fiber filters (Type A/E, 90 mm, 1 μm) and Gelman Acrodisc® CR PTFE syringe filters (13 mm, 0.45 μm) were obtained from Pall Corp., Ann Arbor, MI, USA. Mobile phase additives, ACS grade formic acid (98%) and trifluoroacetic acid (99%) were

Fresh water solid phase extraction (SPE)

The method was first tested with several types of SPE cartridges (Waters Oasis® HLB {n-vinylpyrrolidone-divinylbenzene copolymer}, J.T. Baker C18 {octadecysilane} and Phenomenex Strata X {surface modified styrene divinylbenzene}). Acidified water samples (100 mL), fortified with MCs mixture at 5 μg/L, were extracted using the SPE procedure detailed earlier in Section 2.3.1. The study demonstrated that J.T. Baker C18 cartridges extract all the tested microcystins and Nodularin from the water with

Lake Success incident

In the middle of July 2007, US Army Corps of Engineers reported a major fish kill at Lake Success, Tulare County, California. Ten western grebes were also found dead around the lake. The number of bird and fish deaths increased to 5000 by August 3. Flavobacterium columnar (columnaris disease) was observed on the fish gills. It was unclear if the bacterial infection or cyanobacterial toxin was the direct cause of the deaths. A mixture of dead fish livers and fish guts were received, extracted

Conclusion

The method was developed and validated for measuring trace concentrations of microcystin toxins (MCs) including desmethyl microcystins (dm-MCs), in different matrices (water, mussels, fish fillet and liver) using LC–MS/MS. The limit of detection and quantitation for all microcystins were determined to be between 0.2 and 1 pg on column. These concentrations are below the 2 pg detection limits found in the available literature. This method enables the quantification of nodularin and microcystins in

Acknowledgements

We would like to thank Melissa Miller at Marine Wildlife Veterinary Care and Research Center, Russ Kanz with State Water Resource Control Board, Susan Corum at Department of Natural Resources and Andrew Gordus with California Department of Fish and Game for providing water and tissue field samples.

References (46)

  • K.I. Harada et al.

    Toxicon

    (1990)
  • A. Amorim et al.

    Toxicon

    (1999)
  • L. Xie et al.

    Environ. Pollut.

    (2004)
  • V.F. Magalhaes et al.

    Toxicon

    (2003)
  • C. Rivasseau et al.

    Anal. Chim. Acta

    (1999)
  • K. Kaya et al.

    Anal. Chim. Acta

    (1999)
  • L. Spoof et al.

    J Chromatogr. A

    (2003)
  • P.M. Ortea et al.

    Chemosphere

    (2004)
  • M. Yasin et al.

    J. Chromatogr. A

    (1996)
  • J. Dahlmann et al.

    J. Chromatogr. A

    (2003)
  • J. Meriluoto et al.

    Chromatographia

    (2004)
  • K.L. Rinehart et al.

    J. Am. Chem. Soc.

    (1988)
  • K. Sivonen, G. Jones, in: I. Chorus, J. Bertram (Eds.), Toxic Cyanobacteria in Water: a Guide to Public Health...
  • C. Rivasseau et al.

    J. Microcolumn Sep.

    (2000)
  • M. Namikoshi et al.

    Chem. Res. Toxicol.

    (1998)
  • K. Sivonen et al.

    Appl. Environ. Microbiol.

    (1992)
  • M. Krister et al.

    Environ. Toxicol.

    (2005)
  • W.W. Carmichael

    J. Appl. Bacteriol.

    (1992)
  • Z. Mohamed et al.

    Environ. Toxicol.

    (2003)
  • W.W. Carmichael

    Human Ecol. Risk Assess.

    (2001)
  • W.W. Carmichael et al.

    Environ. Health Persp.

    (2001)
  • E.M. Jochimsen et al.

    N. Engl. J. Med.

    (1998)
  • Cited by (79)

    • Stable isotope analysis reveals differences in domoic acid accumulation and feeding strategies of key vectors in a California hotspot for outbreaks

      2021, Harmful Algae
      Citation Excerpt :

      Collectively, these sea lions, the potential DA vectors, and Dungeness crabs are ‘key taxa’ that represent a subset of the Monterey Bay ecosystem. DA was measured primarily from viscera to obtain information from recently ingested prey (Lefebvre et al., 1999; Gulland, 2000) using standard protocols for liquid chromatography mass spectrometry (Mekebri et al., 2009; Peacock et al., 2018). Liver tissues from stranded sea lions were measured for DA, as these were the only available tissues that offer relatively recent dietary information (days to a couple of weeks) (Vander Zanden et al., 2015).

    • The tide turns: Episodic and localized cross-contamination of a California coastline with cyanotoxins

      2021, Harmful Algae
      Citation Excerpt :

      All extracts were analyzed at the University of California, Santa Cruz, with LC-MS with ESI using the same method described above for SPATT extracts. Extraction efficiency as reported by Mekebri et al. (2009) ranged from 79.9–104% for mussels, 102% for oysters, and 106% for fish fillet. Mussels were not analyzed for anatoxins or domoic acid due to the small amount of tissue available and because the current extraction protocol for microcystins was incompatible with the analytical method for anatoxins or domoic acid.

    • Marine algal toxins and their vectors in southern California cetaceans

      2021, Harmful Algae
      Citation Excerpt :

      In total, six fecal and two liver samples (~1 g wet wt.), collected between 2011 and 2018, were extracted using methods outlined in Mekebri et al. (2009) with modifications. Samples were homogenized for ~1 min in 10 mL of 90% MeOH/0.1% TFA, followed by 1 h of sonication and 30 min of centrifugation at 3400 x g. Extracts were filtered (0.45 μm) and further purified by SPE cleanup using Strata-X 30 mg polymeric columns.

    View all citing articles on Scopus
    View full text