Total Analysis of Microcystins in Fish Tissue Using Laser Thermal

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Journal of Agricultural and Food Chemistry

Total Analysis of Microcystins 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§ and Sébastien Sauvé†* †

Department of Chemistry, Université de Montréal, Montréal, QC, Canada

‡

Direction du suivi de l’état de l’environnement (DSEE), Ministère du Développement durable, de

l’Environnement et de la Lutte contre les changements climatiques (MDDELCC), Québec, QC, Canada §

Centre d’expertise en analyse environnementale (CEAEQ), Ministère du Développement durable, de

l’Environnement et de lutte aux changements climatiques (MDDELCC), Québec, QC, Canada

Telephone: 514 343-6749, fax: 514 343-7586, e-mail: [email protected] Université de Montréal, Departement of Chemistry, CP 6128 Centre-Ville, Montréal, QC, H3C 3J7, Canada

ACS Paragon Plus Environment


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ABSTRACT

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Microcystins (MCs) are cyanobacterial toxins encountered in aquatic environments worldwide. Over

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100 MC variants have been identified and have the capacity to covalently bind to animal tissue. This

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study presents a new approach for cell-bound and free microcystins analysis in fish tissue using sodium

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hydroxide as a digestion agent and Lemieux oxidation to obtain the 2-methyl-3-methoxy-4-

7

phenylbutyric acid (MMPB) moiety, common to all microcystin congeners. The use of laser diode

8

thermal desorption-atmospheric pressure chemical ionization coupled to with Q-Exactive mass

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spectrometer (LDTD-APCI-HRMS) led to an analysis time of approximately 10 seconds per sample

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and high-resolution detection. Digestion/oxidation and solid phase extraction recoveries ranged from 70

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to 75% and 86 to 103%, respectively. Method detection and quantification limits values were 2.7 and

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8.2 µg kg-1, respectively. Fish samples from cyanobacteria-contaminated lakes were analyzed and

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concentrations ranging from 2.9 to 13.2 µg kg-1 were reported.

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Keywords: Microcystins, fish, cyanotoxins, LDTD-APCI, HRMS

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INTRODUCTION

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Cyanobacteria—commonly known as blue green algae—are encountered worldwide, mostly in

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eutrophic water bodies.1 Although they are essentially harmless in small numbers, some species

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generate harmful algal blooms (HABs) that are linked to the production of many types of potent toxins

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associated with human and animal health concerns.2 Some cyanotoxins are known for their

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bioaccumulation and potential for biomagnification in the food chain.3 Knowing that cyanobacteria in

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surface waters are part of the diet of aquatic animals, the associated toxins are ultimately available to

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upper trophic levels, possiblyincreasing exposure and health threats to higher organisms.3,4

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Microcystins (MCs) are the most known and widespread cyanotoxins and are generated by at least 20

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cyanobacteria genera.5 The cyclic structure of MCs (Fig. 1) includes uncommon amino acids such as

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the β-amino acid Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid),

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which is responsible for the toxic activities.6 In the polypeptide cycle, two amino acids (X and Z) have

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multiple combinations, causing the MCs to be found in multiple variants. More recently, new MCs

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were identified, indicating that all the sites from the peptide ring can be areas of adenylation, and over

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100 different structures have been identified.7-9 They all have hepatotoxic properties, meaning that they

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will primarily accumulate in the liver when ingested at high doses. While bioaccumulating in the liver,

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the toxins inhibit protein phosphatases PP1 and 2A through covalent and non-covalent binding,

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ultimately leading to liver necrosis and death at high doses. Tumour-promoting effects have been

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demonstrated at lower exposure, and studies have shown links between MCs and hepatic cancer in

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human populations.10-12 The World Health Organization (WHO) proposed a maximum lifetime

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tolerable daily intake (TDI) for humans of 0.04 µg/kg/day for MC-LR.13,14

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MCs are present in fish tissue in two different forms: 1) the free fraction consisting of the dissolved and

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reversibly bound MCs to tissues and 2) the covalently bound fraction associated with PP or cysteine-

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containing peptides within tissues. Extracting the bound fraction has been a challenge for tissue ACS Paragon Plus Environment


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extraction procedures in fish exposed to MCs.15 Conventional extraction methods involve organic

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solvents using aqueous or acidified methanol (MeOH). However, solvent extraction yielded poor

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recovery values since only the free fraction of MCs was recovered and detected.16 A different approach

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was explored to analyse the total MCs in fish tissue via Lemieux oxidation (Fig. 1) in order to release

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the oxidative product of Adda, the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB).15,17,18 Common

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to all congeners, MMPB makes it possible to quantify the total MCs and has been successfully used for

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analyses in water by our research group and other laboratories.19-22 As for fish tissue analysis, a method

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was proposed using trypsinization followed by Lemieux oxidation and SPE cleanup and MMPB was

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detected using liquid chromatography coupled with mass spectrometry (LC-MS).15,17,18 However, the

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results from oxidation recoveries, SPE extraction efficiency and signal recovery from matrix effects

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gave poor values. Moreover, the global procedures were laborious: up to 20 hours of sample

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preparation required for the trypsinization step and long LC-MS analysis times with over 40-minute

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runs.15

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We have developed a new approach to detect total MCs (free and protein-bound) in fish tissues. First,

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the tissue was digested with sodium hydroxide followed by Lemieux oxidation. A different SPE

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procedure was proposed to enhance recovery values and decrease the amount of salts in the extracts.

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Finally, the MMPB moiety was analyzed using a technology known as laser diode thermal desorption

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(LDTD) coupled with atmospheric pressure chemical ionisation (APCI), and the detection was done

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with a high-resolution mass spectrometer, the Q-Exactive (HRMS). The LDTD has ultra-fast analysis

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times ( 0.05).

301 302

Method validation. The evaluation of the LDTD-APCI-HRMS method was done according to these

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parameters: linearity, sensitivity, accuracy, precision, selectivity and matrix effects, which were ACS Paragon Plus Environment

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previously discussed in the sample clean-up and elution section. Due to the unavailability of

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isotopically-labeled MMPB, 4-phenylbutyric acid (4-PB) was used as the internal standard based on

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previous studies.17,20 All validation parameters were evaluated using spiked blank fish tissue to take

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matrix effects into account. A seven-point standard addition calibration curve spiked prior to the SPE

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procedure was used with a linear dynamic range between 15 and 2 500 µg kg-1 analyzed in triplicate.

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The optimal concentration of 4-PB used as signal variation correction was set at 500 µg kg-1 depending

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on the lowest variability throughout the calibration linearity range (data not shown). Ultimately,

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correlation coefficients showed a good linearity with R2 > 0.999. The method detection limit and

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method quantification limit (MDL and MQL) were 2.7 and 8.2 µg kg-1, which is comparable to

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previous work. Fig. S5 shows a sample LDTD peak signal obtained around the MQL (8 µg kg-1).17

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Table 2 summarizes the validation parameters determined at three concentrations from the linearity

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range (25, 250 and 1 000 µg kg-1). Values from the accuracy expressed as the relative errors (RE-%)

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and from intraday and interday precisions expressed as relative standard deviations (RSD-%) were

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equal to or lower than 10%, which means that the method was both selective and precise.

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Fish tissue samples. The method was applied to determine the total MCs present in 49 samples of fish

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tissue caught in 3 different lakes of the province of Québec, Canada, where cyanobacterial blooms

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occurred during the growing season. Tables 3 and A-1 present the results obtained using three different

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analytical methods. The results from the method developed in this study are presented first. Then, the

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same samples were submitted to LDTD-APCI-MS/MS analysis using a previously developed method

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for total MCs in lake water.20 The purpose here was to compare the sensitivity and selectivity of

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MS/MS detection with HRMS detection using LDTD-APCI technology. Finally, CEAEQ results using

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a standard analytical method for MCs in fish tissue are presented for the same samples. Of the 49

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samples (Table S1), 24 had a positive response to MCs, including 22 that were above the detection limit

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(Table 3). Concentrations varied between 2.9 and 13.2 µg kg-1 of fresh fish tissues weight. Four



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samples (1 to 4) had relatively higher detected concentrations, which could be explained by the type of

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fish tissue. Indeed, three of these samples included the fish viscera, which are known to bioaccumulate

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higher amounts of MCs. These three samples were the only results higher than the estimated MQL

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values. Of the 24 samples with positive MC detection, only 5 were detected using the LDTD-APCI-

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MS/MS technique with relative differences in values of -13% on average. This difference was

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explained by the different response of matrix effects using MS/MS, for which the signal recovery was

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82%. The detection and quantification limits of the method were 6.8 and 20.4 µg kg-1, respectively.

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While these values are comparable with those obtained in previous studies, the use of HRMS offered

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better sensitivity (i.e. 2.7 and 8.2 µg kg-1, respectively), which supports the choice of our detection

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method.

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Using the CEAEQ’s standard analytical method, only one sample showed the positive detection of MCs

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at 2.0 µg kg-1 of MC-YR congener. This value represents 15% of the recovered total MCs from the

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same sample. The detection limit of each studied MC variant was 1 µg kg-1, and detection method

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sensitivity therefore cannot explain this discrepancy. Two reasons may explain the substantial

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underestimation of MCs. First it is possible that the extraction method may not be able to release the

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covalently bound MCs from the tissue. Williams, D.E. et al. provided experimental evidence, using

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Lemieux oxidation, MMPB-GC/MS, radiolabeled MMPB and MeOH extraction-PPase to demonstrate

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that a substantial amount of MCs were covalently bound in vivo.38,39 MeOH extraction actually

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recovered ≈24% of the total MC-LR measured with MMPB in liver from salmons intraperitoneally

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injected. This underestimation is similar to what this study obtained for whole fish in the only sample

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with a detection using the conventional extraction method. Other authors estimated that as much as 60

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to 90% of total MCs could be covalently bound.38-41 Second, other variants and variant isomers present

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in the samples could have been missed altogether. Indeed, some tissue samples from the same lake and

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date had MC detections suspected by CEAEQ to be YR isomer (data not shown). Due to the absence of

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appropriate standards for LC-MS/MS, the CEAEQ analytical laboratory could not certify the


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identification and concentration of the suspected isomer, although its characteristics were so close to the

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available standard that it was deemed with a very high probability to be an isomer of MC-YR. As was

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recently demonstrated with total MCs in water,20 this study also illustrates the inherent risk of relying

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on a limited number of standards when trying to assess total MC contamination in tissue. Our results

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show the importance of analyzing total MCs in fish tissue using proper sample preparation and

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extraction methods along with selective and sensitive detection methods.

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Risk assessment. The maximum lifetime TDI for humans (i.e. 0.04 µg/kg/day) set out by the WHO has

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been used in several exposure scenarios to estimate the potential risk for fish consumers. Scenarios

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aiming to protect at-risk groups (e.g. children42 or important consumers of locally caught fish43) are

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expected to be the most stringent. Assuming that no exposure would come from drinking water or

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recreational exposure, the TDI would transfer into ≈8 µg kg-1 of fish for a 60-kg adult who consumes

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300 g of fish per day on average,43,44 or to ≈24 µg kg-1 for those who consume 100 g per day.45 Some of

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the results presented in Table 3 would then exceed the TDI if fish consumption is high enough. These

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results are noteworthy since only one of the 49 samples was over the detection limit (i.e. 1 µg kg-1) of

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the initial LC-MS/MS analysis. However, it was observed that only three samples were detected above

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the estimated MQL (8.2 µg kg-1). Though further studies are required to improve sensitivity, the

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findings here were satisfactory for MCs detection around the WHO’s TDI.

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Likewise, assuming that 15% of the total MCs were detected in studies analyzing only unbound

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concentrations, several studies including this one would have MC levels above the WHO’s TDI using

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our method.45-49 Overall these results suggest that certain groups may be at a higher risk than previously

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estimated and that more research on the toxicity of total MCs is needed.

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Moreover, the WHO has classified MCs as a “possible human carcinogenic” (Class II-B).50 It is

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therefore important to enhance our ability to estimate the total concentration from all sources of



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exposure to this class of pollutants since cancer may develop after a long latency period and possibly at

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relatively low exposure levels.

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Although the variants, isomers, conjugates and covalently-bound MCs probably all have different

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toxicological properties that have yet to be understood, a method to analyze quickly total MCs is

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nevertheless essential to make prudent exposure measurements while research progress. Furthermore,

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this new method for total MCs in tissue would be helpful in a tiered approach to screen out samples that

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do not contain these toxins, enabling resource allocation where other variants could be playing an

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unnoticed role in toxicity. Though many studies have explored the availability of MCs in aquatic

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animal consumption after cooking and digestion, the bioavailability and toxicity of bound MCs are still

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poorly understood.51-53 However, findings showed that serious risk to animal and human health is most

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likely possible and that contamination monitoring could prevent eventual poisoning cases. It would

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therefore be beneficial to understand the fate, transportation, and accumulation of MCs in the food web,

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with or without other analyses and assays.54

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In conclusion, a new approach to detect of total microcystins (MCs) in fish tissue has been

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demonstrated and validated using LDTD-APCI-HRMS. MCs are known to covalently bind with animal

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tissue, and effective sample preparation is therefore necessary to analyze both free and bound fractions

394

and the possible MCs congeners. We explored the possibility of using sodium hydroxide as a digestive

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agent for fish tissue prior to a well-known oxidation step, Lemieux oxidation, for total MCs analysis. A

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new approach for sample clean-up was also explored using an SDB-L SPE cartridge for recovery

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enhancement and eliminating the salts present in solution. The use of LDTD-APCI has already been

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reported for the analysis of MMPB in water but HRMS detection with the Q-Exactive was chosen for

399

better sensitivity and selectivity. The use of LDTD-APCI-HRMS enabled ultra-fast analysis times at 10

400

s per sample. Digestion and oxidation were optimized and recoveries ranged from 70 to 75%

401

respectively. SPE recoveries ranged from 86 to 103%, and signal recoveries from matrix effects ranged

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from 90 to 93%. An internal calibration was used with 4-PB and the validated method gave linear ACS Paragon Plus Environment

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correlation coefficients (R2) of 0.999, MDL and MQL were 2.7 and 8.2 µg kg-1, respectively. Accuracy

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and interday/intraday variation coefficients for target compounds were below 10%. Interday and

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intraday precision was evaluated below 10%. Of a total of 49 fish samples from lakes with HABs, 25

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(51%) had a positive response for total microcystins with concentrations ranging from 2.9 to 13.2 µg

407

kg-1 fresh weight in tissue. Using standardized extraction and LC-MS/MS methods, only one sample

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(2%) had a microcystin response with a concentration of 2 µg kg-1 of MC-YR. For that sample, the total

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MCs were seven times higher than the initial value. These results suggest that MC concentration in fish

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tissues could be significantly underestimated using standard analytical methods and that a suitable

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extraction method combined with total MC detection is required for prudent risk management.

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ACKNOWLEDGEMENTS

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This research was carried out with the financial support of the the Fond de recherche Québec–Nature et

416

technologies and the Natural Sciences and Engineering Research Council of Canada (NSERC). The

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authors would also like to thank Thermo Fisher Scientific and Phytronix Technologies for their support

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as well as Paul B. Fayad and Sung Vo Duy for their technical and scientific expertise.

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SUPPORTING INFORMATION

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Supporting information is available free of charge via the Internet at http://pubs.acs.org.

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FIGURE CAPTIONS

Figure 1. Lemieux oxidation applied to Microcystin-LR releasing the Adda moiety, the 2-methyl-3methoxy-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.

Figure 2. Optimization of (a) the digestion and (b) the oxidation by varying the reaction time (first xaxis) and the reagents concentration (second x-axis). KMnO4 and NaIO4 concentrations were identical during the oxidation. Vertical error bars represent standard deviations from the mean (n = 3).

Figure 3. Effect of different solvents used for post SPE samples reconstitution and LDTD desorption after sample deposition in wells on MMPB mean peak area. Vertical error bars represent standard deviations from the mean (n = 6).

Figure 4. Number of data acquisition points for LDTD peaks of MMPB with different laser ramp times.



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



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Table 1. Parameters of HRMS detection.

Compound MMPB

4-PB

Chemical Formula C12H16O3-Ha C9H11Ob C7H7c C10H12O2-H C7H7

Calculated exact mass (m/z) 207.10212 135.08099 91.05478 163.07590 91.05478

Experimental exact mass (m/z) 207.1019 135.0808 91.0547 163.0756 91.0546

a

Precursor ion Quantification ion (most abundant MS/MS transition) c Confirmation ion (second most abundant MS/MS transition) d Fragmentation energy for precursor ion in HCD cell e Accuracy of average measured mass f Second abundant ion in isotopic pattern b


Mass NCEd Accuracye (%) (ppm) 25 1.2 1.5 1.1 30 1.7 1.5

Confirmation Ionf (m/z) 208.1042 135.0829 91.0571 163.0780 91.0568

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Table 2. Validation parameters using internal calibration with three different concentrations (n = 6). Concentrations (µg kg-1) 25 250 1000

Accuracy RE (%) 10 6 5

Intraday RSD (%) 6 4 5

Interday RSD (%) 7 5 9

Digestion/oxidation recovery ± SD (%)

SPE recovery ± SD (%)

70 ± 7 75 ± 9 72 ± 6

91 ± 6 95 ± 9 98 ± 7


Signal recovery from matrix effects ± SD (%) 92 ± 7 90 ± 10 93 ± 9


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Table 3. Comparison of results from fish samples with detected MCs above method detection limit using three detection methods.

No. Sample

Location

Fish specie

Fish part

Total MC via MMPB and HRMS (µg kg-1)a (RSD - %)

1 2 3 4 6 7 9 11 14 17 18 20 22 24 27 33 35 38 40 41 44 45 46 49

Lac Vert Lac Vert Lac Vert Lac Vert Lac Vert Lac Vert Lac Roxton Lac Roxton Lac Roxton Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir Lac Noir

Rainbow smelt Mullet Mullet Mullet Brook trout Brook trout Yellow perch Yellow perch Yellow perch Yellow perch Yellow perch Yellow perch Walleye Walleye Walleye Brown bullhead Brown bullhead Lake whitefish Lake whitefish Lake whitefish Lake whitefish Lake whitefish Lake whitefish Yellow perch

No head/viscera Whole Whole Whole Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle

11.9 (7) 8.7 (8) 13.2 (6) 9.5 (10) 5.0 (10) 4.5 (9) 4.0 (10) 3.1 (11) 5.2 (9) 4.5 (7) 7.3 (11) 2.7 (12) 4.4 (8) 2.9 (10) 3.4 (9) 3.8 (9) 5.2 (8) 7,1 (8) 6,2 (9) 5.9 (7) 7.3 (10) 4.5 (11) 3.0 (9) 3.7 (11)

Total MC via MMPB and MS/MS (µg kg-1)b (RSD - %) 10.3 (9) 7.5 (12) 11.9 (11) 8.1 (11) ND ND ND ND ND ND 6.9 (12) ND ND ND ND ND ND ND ND ND ND ND ND ND

Total MC with standards (µg kg-1)c ND ND 2.0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

ND – Not detected a Total microcystins determined via MMPB using LDTD-APCI-HRMS. b Total microcystins determined via MMPB using LDTD-APCI-MS/MS. c Microcystins determined via the summation of all microcystins for which standards allowed detection and quantification (i.e. MC-LA, MC-LR, MC-RR and MC-YR) using LC-MS/MS. d Values in bold represent concentrations above the MQL.


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TOC Graphic



TOC Graphic 266x153mm (300 x 300 DPI)


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Total Analysis of Microcystins in Fish Tissue Using Laser Thermal

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