A mass spectrometry-based unique fragment approach for the identification of microcystinsby P. I. Benke, M. C. S. Vinay Kumar, D. Pan, S. Swarup

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Cite this: Analyst, 2015, 140, 1198

Received 18th September 2014,

Accepted 3rd December 2014

DOI: 10.1039/c4an01702a www.rsc.org/analyst

A mass spectrometry-based unique fragment approach for the identification of microcystins†

P. I. Benke,‡a,b M. C. S. Vinay Kumar,‡c D. Panc and S. Swarup*a,b,c

Both qualitative and quantitative methods for the analysis of microcystin variants have been established.

The qualitative method involves a unique fragment approach, specific for each variant in identifying microcystins, while the quantitative method involves the quantification of microcystins in cellular matrices of cyanobacteria and reservoir water samples. For the identification of fragments associated with their respective microcystin (MC) variants, theoretically obtained fragments of microcystins were compared with tandem mass spectrometric (MS/MS) fragments obtained from an ESI-Orbitrap mass spectrometer.

Here, we present the first report of MC variants produced by the model algal strain M. aeruginosa

NIES-843. A robust comparative study between the unique fragments and the conventional product ions for quantitative measurements of microcystin has also been carried out. The method with high robustness was further validated by determining the MC level changes in the intracellular matrices of M. aeruginosa samples, grown under high and low nitrogen conditions, and by testing the amount of MC in environmental water samples.

Introduction

Microcystins are non-ribosomal peptides present in toxin producing cyanobacteria, which are common in aquatic habitats such as lakes, water canals, and reservoirs. These toxins have low lethal doses and can also cause substantial liver damage.

Microcystins are known to be produced by many microorganisms, such as Microcystis, Planktothrix, Anabaena and Nostoc1,2 and are released into the water sources during algal bloom.

Toxic algal blooms have been studied worldwide, ever since the beginning of the twentieth century.3–5

Of the many classes of toxins such as nephrotoxins, haematotoxins, neurotoxins, myotoxins etc. which are detrimental to specific organs, microcystins belong to the liver damaging hepatotoxins. Microcystins, form a group of cyclicpeptides consisting of a unique group (2S,3S,8S,9S)-3-amino9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) and N-methyldehydroalanine (Mdha) or dehydroalanine (dha) along with five amino acids (Fig. 1). Among these amino acids, three are ‘D’ isoforms and remain constant or with minor modifications such as demethylation (dm) or methylation, while the two ‘L’ isoforms6 contribute to the structural variations.

Microcystins are named after the amino acids that contribute to structural variations,7 for example, MC-LR is named after the amino acids leucine (L) and arginine (R) that contribute to the change. Similarly other MC variants are named based on other amino acids which can be found in Table 1. The variants of MC differ from each other in their toxicity,8 with the MC-LR variant being the most toxic. Since the biological functions of the different variants are not known, the study of these variants and an analytical method for the same, becomes very important.

Studies on the structure and identification of microcystins have been evolving from as early as 1960’s;9 several identification, isolation and purification methods have been established on various analytical platforms (e.g. Ultra Violet (UV),

Fig. 1 The general structure of microcystin (compositions of the substituents are described in Table 1). †Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4an01702a ‡These authors contributed equally to this work. aNUS Environmental Research Institute (NERI), T-Lab Building, National University of Singapore, 5A Engineering Drive 1, Singapore. E-mail: sanjay@nus.edu.sg bSingapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang

Technological University, Singapore cMetabolites Biology Lab, Department of Biological Sciences, National University of

Singapore, 14 Science Drive 4, Singapore 1198 | Analyst, 2015, 140, 1198–1206 This journal is © The Royal Society of Chemistry 2015

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Mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR)).10–13 Due to its lower detection sensitivity, the MS approach is generally preferred over NMR methods. However, small variations in the toxin structure make liquid chromatographic (LC) separations rather difficult for microcystins.14 In addition, the coupling of LC with MS is not adequate, since most of the high intensity MS/MS fragments happen to be common among the microcystin variants.

Environmental conditions, including nutrient availability, have been reported to play a major role in increasing the biomass of microalgae during algal bloom, which in turn increases the intracellular levels of the microcystins.15–17 The methods currently used for measuring the intracellular concentrations of microcystins are LC-UV/diode array detector (DAD) and ELISA.18,19 However, both have limitations. LC-UV/

DAD is not very sensitive and not specific enough as compared to the LC-MS methods, on the other hand, ELISA though a sensitive method, has a very narrow range (0.1 ppb to 10 ppb) for quantifications, which is 3 to 4 orders of magnitude below the intracellular toxin levels (i.e. µM) of cyanobacterial algae.

Hence to bring the intracellular concentration of algal samples in the quantitation range, very high levels of sample dilutions are required, which incorporate high variability in the quantitative results.18,20

In order to overcome the aforementioned analytical problems in identification and quantification, a novel method based on unique fragments, specific for each variant, has been established. While there are more than eighty variants of microcystins, the identification method focuses on those ten variants which are most frequently reported in toxin producing cyanobacteria.21,22 Also, a microcystin quantitation method for both intracellular algal extracts and extra cellular water samples is demonstrated, after a thorough comparison between the fragments related to structural variations and conventional fragments for quantitation purpose. Due to its high robustness, a multiple reaction monitoring-based triple quadrupole (MRM-QQQ) – MS method for conventional fragments was chosen for further studies. A validation study was performed on the commercially available M. aeruginosa PCC 7806 strain, under different nitrogen conditions during the growth stages and with environmental samples from freshwater reservoirs.