All chemicals were reagent grade or higher in quality. Formic acid (FA) and ammonium hydroxide solution (NH4OH) were purchased from Sigma-Aldrich (Milan, Italy). HPLC gradient grade methanol (MeOH), acetonitrile (ACN) and ultrapure water (H2O) were supplied by Carlo Erba (Cornaredo, Italy). Strata™-X cartridges (200 mg, 3 mL) were purchased from Phenomenex (Castel Maggiore, Italy). DA (≥90%), OA sodium salt (MQ 100) and analytical grade standard MC-RR, MC-LR, MC-YR and MC-LW solutions were supplied by Sigma-Aldrich (Milan, Italy). Individual stock standard solutions of all analytes (1 μg mL−1) were prepared in MeOH, stored in the dark (−20 °C) and renewed weekly.
Mc yr
Manufactured by Merck Group
Sourced in Italy, United States
The MC-YR is a piece of laboratory equipment manufactured by Merck Group. It is designed to perform a specific function within a controlled laboratory setting. The core function of the MC-YR is to [core function description]. No further details or interpretations about the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Mc rr, by Merck Group (6 mentions)
Mc lr, by Merck Group (5 mentions)
Mc lw, by Merck Group (2 mentions)
Nodularin nod, by Merck Group (2 mentions)
Formic acid, by Merck Group (1 mentions)
Ammonium hydroxide solution nh4oh, by Merck Group (1 mentions)
Strata x cartridge, by Phenomenex (1 mentions)
Agilent 1290 infinity 2, by Agilent Technologies (1 mentions)
Qtof 6550, by Agilent Technologies (1 mentions)
Eclipse xdb c18, by Agilent Technologies (1 mentions)
Mc lf, by Enzo Life Sciences (1 mentions)
Mc hilr, by Enzo Life Sciences (1 mentions)
Mc ly, by Enzo Life Sciences (1 mentions)
Mc lw, by Enzo Life Sciences (1 mentions)
Mc wr, by Enzo Life Sciences (1 mentions)
Mc htyr, by Enzo Life Sciences (1 mentions)
7 protocols using mc yr
1
Microcystin Quantification in Water
2
Cyanotoxin and Nutrient Analysis Protocol
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Three 100 ml aliquots from each dialysis bag were immediately placed on ice and returned to the lab for filtration on a 0.7 μm Whatman GF/F glass microfiber filter (Fisher Scientific cat # 09-874-64) and stored at −20 °C for cyanotoxin analysis. According to Fastner et al.63 and Dyble et al.64 (link), toxin samples were lyophilized first and then sonicated in 75% aqueous methanol. MC analogues (MC-LR, MC-RR, MC-YR, MC-LA; Sigma-Aldrich) and cylindrospermopsin (CYN) (Sigma-Aldrich) analysis was performed by High-Performance Liquid Chromatography coupled Mass Spectrometry (HPLC/MS) using a Thermo Surveyor MSQ Single Quadrupole Mass Selective Detector and Thermo Spectrasystem gradient chromatographic system according to a method described by Barco et al.65 (link). Total MC concentrations were reported as the sum of all congeners (HPLC/MS-Total).
Total Kjeldahl nitrogen (TKN-N) and ammonia (NH3-N) were analyzed on a BRAN+LUEBBE Autoanalyzer66 . Nitrate (NO3-N), total phosphorus (TP-P), and soluble reactive phosphorus (SRP-P) were analyzed on an ion chromatograph (detections limit: 0.005 mg/L, Standard Methods 4100 C)67 .
Total Kjeldahl nitrogen (TKN-N) and ammonia (NH3-N) were analyzed on a BRAN+LUEBBE Autoanalyzer66 . Nitrate (NO3-N), total phosphorus (TP-P), and soluble reactive phosphorus (SRP-P) were analyzed on an ion chromatograph (detections limit: 0.005 mg/L, Standard Methods 4100 C)67 .
3
Methylation of Microcystin Variants
4
Quantification of Cyanobacterial Metabolites
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Fifteen cyanobacterial metabolites were analyzed as part of this study, including six hepatotoxins, six bioactive cyanopeptides, and three neurotoxins. For the hepatotoxins, certified reference material for microcystin-LR (MCLR) was purchased from the National Research Council of Canada Biotoxins program (Halifax, NS, Canada). Nodularin (NOD) (purity >94%), MCLA (>95%), MCYR (>90%), and MCRR (>90%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA), and cylindrospermopsin (CYL) (>95%) was purchased from Abraxis (Warminster, PA, USA). For the peptides, anabaenopeptin B (Apt B) (>95%) and AptF (>95%), cyanopeptolin 1007 (Cpt1007) (>95%), Cpt 1021 (>95%), and Cpt 1040 (>95%), and microginin 690 (Mgn690) (>95%) were purchased from MARBIONC (Wilmington, NC, USA). Additionally, the neurotoxins anatoxin-a (ATX), homoanatoxin-a (hATX), and saxitoxin (SXT) were also targeted in this study. ATX fumarate (96%) was purchased from Tocris Bioscience (Minneapolis, MN, USA) as a racemic mixture, hATX (>95%) was purchased from Abraxis, and SXT was purchased from the National Research Council of Canada Biotoxins program (Halifax, NS, Canada).
5
Quantification of Cyanotoxins by HPLC-QTOF
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All the extracts were later analyzed by HPLC Agilent 1290 Infinity II coupled to a hybrid mass spectrophotometer Agilent Q-TOF 6550, with an ionization source JetStream electrospray + i-Funnel. Samples were analyzed for eight MC variants ([Dhb7]-MC-LR, MC-RR, MC-YR, MC-LR, MC-LW, MC-LF, anatoxin-a (ANA) and nodularin (NOD) from SIGMA-ALDRICH). Compounds were separated in an Agilent Eclipse XDB-C18 4.6 × 150 mm, 5 mm column by Millipore water with 0.1 % formic acid (v/v, eluent A) and acetonitrile with 0.1% formic acid (v/v, eluent B). The elution program was 0–2 min 30% B, 6–12 min 90% B, with a linear increase of B between 2 and 6 min, and a 5-min post run at 30% B. The injection volume, flow and column temperature were 20 mL, 0.5 mL/min and 40 °C, respectively. MS operated in the positive mode and nitrogen was used as the drying and collision gas. The quadrupole was operated in the unit mode and four spectra/sec were recorded. Samples were quantified against a calibration curve and subsequently corrected for recovery (Table 7 ). Two replicas were obtained per sample and each replica was injected once.
The presence of phenylalanine was discarded because its theoretical m/z was 166.0863 and the employed method could separate it easily from anatoxin [58 (link)].
The presence of phenylalanine was discarded because its theoretical m/z was 166.0863 and the employed method could separate it easily from anatoxin [58 (link)].
6
Accurate Quantification of Cyanotoxins
7
Quantifying Microcystin Variants in Crocodile and Chicken Samples
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Standards of MC-LR, MC-RR and MC-YR were purchased from Sigma. A combined standard was prepared in methanol containing 1000 ng ml -1 of each of the microcystins.
Serial dilutions were made to obtain a range between 500 and 62.5 ng ml -1 . The liver sample of the slaughtered crocodile and chicken egg samples were spiked with MC-LR, MC-RR and MC-YR (Abraxis) and were analysed by LC-HRMS to determine the percentage recovery of the three microcystin variants.
Serial dilutions were made to obtain a range between 500 and 62.5 ng ml -1 . The liver sample of the slaughtered crocodile and chicken egg samples were spiked with MC-LR, MC-RR and MC-YR (Abraxis) and were analysed by LC-HRMS to determine the percentage recovery of the three microcystin variants.
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