Validação de parâmetros de mérito para quantificação de microcistina-LR por meio de cromatografia líquida de alta eficiência acoplado a detector de arranjo de diodos

Abstract

Cyanobacteria are microorganisms that, in certain environmental conditions, can produce and release different metabolites, such as cyanotoxins. One hepatotoxic cyanotoxin of great importance is microcystin-LR (MC-LR) that commonly appears in cyanobacterial blooms, mainly of Microcystis genera, with severe effects to the human body and animals if ingested. The detection of this metabolite in water is usually performed using chromatographic techniques, which require previous steps of optimization and validation of the chosen method. The objective of this study was to optimize and validate a chromatographic method for quantification of microcystin-LR, and also analyse a cyanobacterial extract containing the toxin by chromatography. As a consequence of the low sensitivity of the technique in detecting this compound, recovery tests of this analite using the solid phase extraction (SPE) technique were performed. The analysis were conducted in a high performance liquid chromatograph coupled with a diode array detector (HPLC-DAD) (Agilent – 1260 Infinity). Based on published data, the chosen method consisted of an isocratic elution using acetonitrile (ACN) with 0,05% of trifluoracetic acid (TFA) (phase A) and water with 0,05% TFA (phase B) as a mobile phase and a C18 chromatographic column (250 mm x 4,6 mm x 5 μm). The best conditions of analysis were established, including 238 nm for monitoring, 60:40 H2O:ACN for mobile phase proportion, 20 µL for injection volume, and 1,0 mL min-1 as flow rate. With the validation in ultra pure water, were established that the method is linear within the working range tested, with a limit of detection (LOD) of 0,06 mg L-1 and a limit of quantification (LOQ) of 0,17 mg L-1. A method for the concentration of microcystin-LR was also established, using the solid phase extraction technique, with CHROMABOND® C-18 end-capped cartridges, with recovery of 98,51% ± 2,74%. Tests of exctract samples obtained from a cyanobacterial culture, prepared in previous studies, were also performed. The previous established condition to analyse the standard in ultrapure water and methanol was not suitable to quantify the extract matrix toxin, due to co-elution of other compounds with the MC-LR. Therefore, gradient elutions were tested, being established 30:70 ACN:H2O until 50:50 ACN:H2O in 70 minutes as the best condition to separate the co-eluting peaks. The gradient elution method was linear for the concentration range tested, with LOD of 0,03 mg L- 1 and LOQ of 0,09 mg L-1. One of the extracts, called extract 2, didn’t show any sign of MCLR, while the other two extracts (1 and 3), showed concentrations of 7,10 mg L-1 and 0,68 mg L-1, respectively.