Feasibility study on production of a matrix reference material for cyanobacterial toxins

Abstract

The worldwide increase in cyanobacterial contamination of freshwater lakes and rivers is of great concern as many cyanobacteria produce potent hepatotoxins and neurotoxins (cyanotoxins). Such toxins pose a threat to aquatic ecosystems, livestock, and drinking water supplies. In addition, dietary supplements prepared from cyanobacteria can pose a risk to consumers if they contain toxins. Analytical monitoring for toxins in the environment and in consumer products is essential for the protection of public health. Reference materials (RMs) are an essential tool for the development and validation of analytical methods and are necessary for ongoing quality control of monitoring operations. Since the availability of appropriate RMs for cyanotoxins has been very limited, the present study was undertaken to examine the feasibility of producing a cyanobacterial matrix RM containing various cyanotoxins. The first step was large-scale culturing of various cyanobacterial cultures that produce anatoxins, microcystins, and cylindrospermopsins. After harvesting, the biomass was lyophilized, blended, homogenized, milled, and bottled. The moisture content and physical characteristics were assessed in order to evaluate the effectiveness of the production process. Toxin levels were measured by liquid chromatography with tandem mass spectrometry and ultraviolet detection. The reference material was found to be homogeneous for toxin content. Stability studies showed no significant degradation of target toxins over a period of 310 days at temperatures up to +40 °C except for the anatoxin-a, which showed some degradation at +40 °C. These results show that a fit-for-purpose matrix RM for cyanotoxins can be prepared using the processes and techniques applied in this work.

Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

“…The fact both 1 and 3 are biosynthesized together by the MC synthetase of M. aeruginosa strain CPCC-464, and that 1 was subsequently found to have similar inhibitory potency to MC-LR (2) against protein phosphatase 2A (PP2A) (Figure 4), both indicate that 1 has the same absolute stereochemistry as 2 and 3 and that 1 is therefore [D-Leu 1 ]MC-LY ( Figure 1). Authentic 3 in a cyanobacterial bloom extract from Poplar Island, MD, USA, whose structure has been verified as [D-Leu 1 ]MC-LR by purification and NMR analysis [11], had identical retention time and product ion spectra to the peak for 3 in CPCC-464 when analyzed by LC-HRMS/MS, thus verifying its identity as proposed by Hollingdale et al [5]. LC-HRMS/MS also showed the presence of a minor microcystin with [M + H] + at m/z 1071.5556 (C51H79O13N10S + , Δ 1.2 ppm) consistent with [D-Leu 1 ]MC-M(O)R (6) tentatively identified in bloom samples from south-western Ontario, Canada [9] and Maryland, USA [11], by untargeted LC-HRMS/MS methods and selective chemical oxidation.…”

“…LC-HRMS chromatograms with and without mercaptoethanol derivatization are shown in Figures S1 and S2. Compounds 1 and 3 were hypothesized to be [D-Leu 1 ]MC-LY and [D-Leu 1 ]MC-LR, respectively [5], while the MS characteristics of 6 are consistent with [D-Leu 1 ]MC-M(O)R, tentatively identified recently by LC-HRMS/MS in cyanobacterial blooms in Canada and the USA [9,11]. Large scale culturing of CPCC-464 followed by centrifugation provided 188 g of biomass for purification of 1.…”

“…As part of feasibility studies for a cyanobacterial matrix reference material [5], a survey of cyanobacterial cultures from Canada was conducted using LC with UV and MS detection. Among the samples analyzed, two Microcystis aeruginosa cultures from Saskatchewan and Alberta, CPCC-464 and CPCC-299, showed the presence of a new microcystin tentatively identified as [D-Leu 1 ]MC-LY (1) [5] together with the previously reported [6,7] and well-characterized [8] [D-Leu 1 ]MC-LR (3). [D-Leu 1 ]MC-LY (1) was also tentatively identified recently by LC-HRMS/MS in a cyanobacterial bloom sample from southwestern Ontario, Canada [9], indicating that it may be a significant component of natural cyanobacterial blooms in this and other parts of the world.…”

Selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography–differential mobility spectrometry–tandem mass spectrometry (HILIC–DMS–MS/MS)

“…Other samples included an in-house positive-control reference material made from the homogenized tissue of a cycad plant (Cycas debaoensis), obtained from Jurassic Plants Nursery (Halfmoon Bay, BC, Canada) and a recently developed pilot-scale cyanobacterial reference material containing other cyanotoxins [32].…”

“…Mussel tissue reference materials and unprocessed mussel samples were all found to contain BMAA, BAMA, and 2,4-DAB. Interestingly, only 2,4-DAB was detected in the cyanobacterial reference material (RM-BGA [32]). Results from HILIC-DMS-MS/MS analysis of one of the mussel tissue reference materials, CRM-ASP-Mus, are shown in Fig.…”