Retention mechanisms and selectivity in internal-surface reversed-phase liquid chromatography. Studies with cyanobacterial peptide toxins

Summary Microcystins-LA,-LR,-RR,-YR and nodularin, cyanobacterial peptide toxins, were separated by internal-surface reversed-phase (ISRP), high-performance liquid chromatography. The capacity factors of the toxins were measured in the range pH 2–8 using acetonitrile, isopropanol or tetrahydrofuran in potassium dihydrogenphosphate mobile phase. The main retention mechanism of the ISRP column was reversed-phase interaction but cation-exchange offered additional selectivity at neutral and slightly acidic pH. At neutral pH (10% modifier, 0.1 M buffer) the elution order was microcystin-LA (two nonpolar residues leucine and alanine as the variable amino acids), nodularin, microcystin-LR,-YR and-RR (two basic arginines as the variable amino acids). The retention times of all toxins except microcystin-RR were substantially longer at acidic pH. At pH 2 (10% modifier, 0.1 M buffer) where the cation-exchange mechanism was inoperative the elution order was changed to microcystin-RR, nodularin, microcystin-LR,-YR and-LA. The best separation was achieved at pH 2 where even two desmethylated microcystin-RR analogs could be separated from microcystin-RR.     

Redox Mechanisms of Nodularin and Chemically Degraded Nodularin

The electrochemical behaviour of Nodularin (NOD), a hepatotoxic cyclic pentapeptide, was studied at a glassy carbon electrode. NOD electrochemical oxidation is an irreversible, pH‐independent process, involving the transfer of one electron. Upon incubation in different pH electrolytes, chemical degradation of NOD was electrochemically detected by the appearance of a new oxidation peak. The chemically degraded NOD (cdNOD), undergoes an irreversible, pH‐dependent oxidation, and its redox products are reversibly oxidised. The charge transfer properties of cdNOD as well as of its redox metabolites were investigated. Mechanisms for NOD oxidation, NOD chemical degradation and oxidation of cdNOD and its metabolites were proposed.     

Micellar electrokinetic capillary chromatography of algal toxins

Recent methods employed for the analysis of algal toxins have focused on high performance liquid chromatography. However these methods suffer from poor resolution, poor efficiency, and long analysis times. This study involves the investigation of a number of toxins including nodularin, microcystin LR, YR, and RR which are cyclic peptides produced by strains of blue-green algae. The electroseparation mode was micellar electrokinetic capillary chromatography (MECC) using a borate buffer containing sodium dodecyl sulfate (SDS) as the surgactant of choice. The method was optimized with standard toxin compounds and employed for the screening opf toxins in supercritical fluid extracts (SFE) of freeze-thawed algal scum samples.     

Chromatographic and spectral behaviour and detection of hepatotoxic nodularin in fish, clam, mussel and mouse tissues using HPLC analysis

Summary High-performance liquid chromatography (HPLC) was applied in the analysis of nodularin (NODLN), a potent, bioaccumulable hepatotoxin. The behaviour of NODLN in biological matrices and possibility to analyse biota samples for NODLN content was examined using a conventional HPLC/diode array detector method that uses C₁₈ solid-phase cartridge clean up. Tissues of European flounder, blue mussel (spiked and naturally contaminated), clam (exposed to NODLN in an aquariuml and mouse (subjected to i. p. administration of NODLN) were analysed. UV detection was 5 times more sensitive than electrochemical detection. Recovery of NODLN from spiked tissues was 59% for mussel, 53% for flounder, and 44–75% for mouse tissues. NODLN was detected in clams exposed with NODLN, but not in naturally contaminated mussels where NODLN conjugation occurs. Through the use of spectral processing, free NODLN was unambiguously identified from tissue samples. The HPLC method showed limits of quantification between 90 and 150 μg NODLN kg⁻¹ dw. The method proved applicable for routine tissue analysis and can be used in the monitoring of acutely toxic NODLN levels.     

Determination of cyanobacterial cyclic peptide hepatotoxins in drinking water using CE

Four cyanobacteria hepatotoxins microcystin LR, microcystin RR, microcystin YR, and nodularin were simultaneously determined in drinking water using CZE and MEKC coupled with UV detection. The toxins were satisfactorily separated in both CZE and MEKC modes. Detection limits were in the range of 0.82–4.81 μg/mL, with R² values of 0.994–0.999. The linearity range tested for the standards was 5–100 μg/mL and RSD percentages were in the range of 1.0–2.5% for retention time and 3.0–10.2% for peak area. When a known amount of standard was spiked into a known volume of water and extracted, recoveries were 90.3% (RR), 101.5% (nodularin), 90.6% (YR), and 88.2% (LR). The use of SPE enabled cleanup and pre‐concentration of a real sample to achieve a 100‐fold concentration factor. Detection limits after SPE of the real sample spiked with microcystins were 0.090 μg/L (RR), 0.076 μg/L (YR), and 0.110 μg/L (LR), with RSD percentage values of 9.9–11.7% for peak area and 2.2–3.3% for retention time. The technique developed provides an alternative method for determining microcystins at levels of concentration that will be able to meet WHO drinking water guidelines for microcystins.     

Detection of microcystins with protein phosphatase inhibition assay, high-performance liquid chromatography-UV detection and enzyme-linked immunosorbent assay: Comparison of methods

A colorimetric protein phosphatase inhibition assay (PPI assay), a commercial enzyme-linked immunosorbent assay (ELISA) test and different HPLC methods using UV detection were compared for the detection of cyanobacterial hepatotoxins, microcystins (MCYST) and nodularin. The suitability of the methods to detect different toxin variants was evaluated by using pure toxins and laboratory cultures as well as water and bloom samples of toxic cyanobacteria. The emphasis of the study was on the analysis of polar demethyl microcystin variants that are common in nature but for which there exist no commercial standards. The IC₅₀ values of MCYST-LR for the PPI assay and the ELISA test were 2.2-2.5 and 0.26-0.38 μg l⁻¹, respectively. The most important factors that decreased toxin recovery in sample treatment were the use of C₁₈ cartridges and polypropylene containers. Good recoveries of toxins were obtained by using hydrophilic-lipophilic balanced (Oasis HLB, Waters) cartridges for concentrating the samples. The results obtained with the PPI assay, the ELISA test and HPLC correlated quantitatively well with the exception of [d-Asp³] microcystins. Concentrations of [d-Asp³]MCYST-RR measured with the PPI assay were only 5% of those obtained by the ELISA test and HPLC. Concentrations of hydrophobic microcystin variants were lower when analysed with ELISA than with the other methods. The World Health Organisation (WHO) has set a guideline value of 1 μg l⁻¹ for the world-wide most common microcystin variant, MCYST-LR in drinking water. Since the quantitative ranges of the PPI assay and the ELISA test are within microcystin concentrations in natural waters, and both tests are easy to perform, they show potential for routine use in the screening and monitoring of microcystins from drinking water supplies and from recreational waters.     

Screening for cyanobacterial hepatotoxins, microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography-electrospray ionisation mass spectrometry

Water samples taken from 93 freshwater and brackish water locations in Aland (SW Finland) in 2001 were analysed for biomass-bound microcystins and nodularin, cyanobacterial peptide hepatotoxins, by liquid chromatography-mass spectrometry (LC-MS) in selected ion recording (SIR) and multiple reaction monitoring modes, HPLC-UV, and enzyme-linked immunosorbent assay (ELISA). The extracted toxins were separated on a short C18 column with a gradient of acetonitrile and 0.5% formic acid, and quantified on a Micromass Quattro Micro triple-quadrupole mass spectrometer with an electrospray ion source operated in the positive SIR or scan mode. An injection of 50 pg of microcystin-LR, m/z 995.5, on column gave a signal-to-noise ratio of 17 (peak-to-peak) at the chosen SIR conditions. In-source or MS-MS fragmentation to m/z 135.1, a fragment common to most microcystins and nodularin, was used for confirmatory purposes. Microcystins with a total toxin concentration equal to or higher than 0.2 microg l(-1) were confirmed by all three methods in water samples from 14 locations. The highest toxin concentration in a water sample was 42 microg l(-1). The most common toxins found were microcystins RR, LR and YR with different degrees of demethylation (non-, mono- or didemethylated). Parallel results achieved with ELISA and HPLC-UV were generally in good agreement with the LC-MS SIR results.     

超高效液相色谱-四极杆-飞行时间质谱法快速测定水产品中微囊藻毒素和节球藻毒素

通过超声提取、固相萃取纯化、超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)联用技术快速测定水产品中的微囊藻毒素-RR、-YR、-LR和节球藻毒素.分别采用选择离子监测质荷比(m/z)为519.84、1045.66、995.67、825.54分子离子峰进行定量分析.该法检出限为5.0～10.0μg/kg,在浓度0.02～5mg/kg的范围内,峰面积与样品浓度呈良好线性关系;4种藻毒素的回收率为76.2％～93.7％,相对标准偏差为2.0％～7.1％.采用上述方法对45个太湖水产品样品进行测定,发现有少量水产品中存在藻毒素污染,其中微囊藻毒素-RR最高含量为15.2μg/kg,微囊藻毒素-LR最高含量为0.84μg/kg,MC-YR、节球藻毒素均未检出.此方法可作为监测水产品体内蓄积藻毒素的分析方法.