couplée à une ionisation à pression atmosphérique et à la
spectrométrie de masse à haute résolution (LDTD-APCI-
HRMS)
Une version de ce chapitre se trouve dans l’article intitulé :
Total microcystins analysis in fish tissue using laser thermal desorption-atmospheric pressure chemical ionization-high resolution mass spectrometry (LDTD-APCI-HRMS)
Audrey Roy-Lachapelle, Morgan Solliec, Marc Sinotte, Christian Deblois et Sébastien Sauvé, Journal of Agricultural and Food Chemistry, 2015. 63(33); p. 7440-7449.
Note sur ma contribution
Ma participation aux travaux de recherche: J’ai conçu le design expérimental en collaboration avec le Prof. Sauvé et Morgan Solliec et j’ai réalisé les manipulations, l’analyse, l’interprétation des résultats.
Rédaction : J’ai rédigé l’article en m’appuyant sur les commentaires du Prof. Sauvé, mon directeur de thèse.
Collaboration des co-auteurs: Morgan Solliec m’a assisté dans le développement du projet et la rédaction de l’article, Marc Sinotte a contribué à l’écriture de l’article et Christian Deblois m’a fourni des résultats présentés dans l’article.
Abstract
Microcystins (MCs) are cyanobacterial toxins encountered in aquatic environments worldwide. Over 100 MC variants have been identified and have the capacity to covalently bind to animal tissue. This study presents a new approach for cell-bound and free microcystins analysis in fish tissue using sodium hydroxide as a digestion agent and Lemieux oxidation to obtain the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) moiety, common to all microcystin congeners. The use of laser diode thermal desorption-atmospheric pressure chemical ionization coupled to with Q-Exactive mass spectrometer (LDTD-APCI-HRMS) led to an analysis time of approximately 10 seconds per sample and high-resolution detection. Digestion/oxidation and solid phase extraction recoveries ranged from 70 to 75% and 86 to 103%, respectively. Method detection and quantification limits values were 2.7 and 8.2 µg kg- 1, respectively. Fish samples from cyanobacteria-contaminated lakes were analyzed and
concentrations ranging from 2.9 to 13.2 µg kg-1 were reported.
6.1 Introduction
Cyanobacteria—commonly known as blue green algae—are encountered worldwide, mostly in eutrophic water bodies [190]. Although they are essentially harmless in small numbers, some species generate harmful algal blooms (HABs) that are linked to the production of many types of potent toxins associated with human and animal health concerns
[280]. Some cyanotoxins are known for their bioaccumulation and potential for biomagnification in the food chain [281]. Knowing that cyanobacteria in surface waters are part of the diet of aquatic animals, the associated toxins are ultimately available to upper trophic levels, possiblyincreasing exposure and health threats to higher organisms [281, 282]. Microcystins (MCs) are the most known and widespread cyanotoxins and are generated by at least 20 cyanobacteria genera [283]. The cyclic structure of MCs (Figure 6-1) includes
uncommon amino acids such as the β-amino acid Adda (3-amino-9-methoxy-2,6,8-trimethyl- 10-phenyldeca-4(E),6(E)-dienoic acid), which is responsible for the toxic activities . In the polypeptide cycle, two amino acids (X and Z) have multiple combinations, causing the MCs to be found in multiple variants. More recently, new MCs were identified, indicating that all the sites from the peptide ring can be areas of adenylation, and over 100 different structures have been identified [32, 194, 284]. They all have hepatotoxic properties, meaning that they will
primarily accumulate in the liver when ingested at high doses. While bioaccumulating in the liver, the toxins inhibit protein phosphatases PP1 and 2A through covalent and non-covalent binding, ultimately leading to liver necrosis and death at high doses. Tumour-promoting effects have been demonstrated at lower exposure, and studies have shown links between MCs and hepatic cancer in human populations [24, 285, 286]. The World Health Organization (WHO) proposed a maximum lifetime tolerable daily intake (TDI) for humans of 0.04 μg/kg/day for MC-LR [6, 287].
Figure 6-1. Lemieux oxidation applied to Microcystin-LR releasing the Adda moiety, the 2-
methyl-3-methoxy-4-phenylbutyric acid (MMPB) for total microcystins analysis. Microcystin structure includes (1) Adda, (2) D-glutamic acid, (3) N-methyldehydroalanine, (4) D-alanine, (5) variable L-amino acid, (6) D-methylaspartic acid and (7) variable L-amino acid.
MCs are present in fish tissue in two different forms: 1) the free fraction consisting of the dissolved and reversibly bound MCs to tissues and 2) the covalently bound fraction associated with PP or cysteine-containing peptides within tissues. Extracting the bound fraction has been a challenge for tissue extraction procedures in fish exposed to MCs [130]. Conventional extraction methods involve organic solvents using aqueous or acidified methanol (MeOH). However, solvent extraction yielded poor recovery values since only the free fraction of MCs was recovered and detected [129]. A different approach was explored to analyse the total MCs in fish tissue via Lemieux oxidation (Figure 6-1) in order to release the
oxidative product of Adda, the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) [130, 131, 268]. Common to all congeners, MMPB makes it possible to quantify the total MCs and has been successfully used for analyses in water by our research group and other laboratories [104, 107, 288, 289]. As for fish tissue analysis, a method was proposed using trypsinization
followed by Lemieux oxidation and SPE cleanup and MMPB was detected using liquid chromatography coupled with mass spectrometry (LC-MS) [130, 131, 268]. However, the results from oxidation recoveries, SPE extraction efficiency and signal recovery from matrix effects gave poor values. Moreover, the global procedures were laborious: up to 20 hours of sample preparation required for the trypsinization step and long LC-MS analysis times with over 40-minute runs [130].
We have developed a new approach to detect total MCs (free and protein-bound) in fish tissues. First, the tissue was digested with sodium hydroxide followed by Lemieux oxidation. A different SPE procedure was proposed to enhance recovery values and decrease the amount of salts in the extracts. Finally, the MMPB moiety was analyzed using a technology known as laser diode thermal desorption (LDTD) coupled with atmospheric pressure chemical ionisation (APCI), and the detection was done with a high-resolution mass spectrometer, the Q-Exactive (HRMS). The LDTD has ultra-fast analysis times (<15 seconds/sample), works with small amount of samples (2-10 µL) per analysis and requires only a small volume of solvents since there is no liquid chromatography. In a previous study, the LDTD was successfully used to analyze MMPB in water samples with a triple quadrupole mass spectrometer detector (MS/MS) [288]. Anatoxin-a was also detected with this technology for its direct determination in water using both MS/MS and HRMS [159, 250]. The LDTD was also successfully applied to small molecules in different matrices, including wastewater, sludge, sediment, soil and biological matrices [155, 160, 162-164, 290]. For this method, the Q-Exactive was chosen for two reasons: the sensitivity is higher than a standard triple quadrupole, thus giving better limits of detection and quantification, and the selectivity is better since the mass spectrometer enables precursor fragmentation and exact mass detection. Indeed, this hybrid Orbitrap detector combines a quadrupole precursor selection with a high energy collisional dissociation cell (HCD) making it possible to fragment selected precursor ions. Also, with a resolving power of up to 140,000 full width half maximum (FWHM) at m/z 200, the mass accuracy obtained with the Q-Exactive is between 1 and 3 ppm [248].