Carole Miossec
email: carole.miossec@univ-pau.fr

Tiphaine Mille
email: tiphaine.mille@univ-pau.fr

Laurent Lanceleur
email: laurent.lanceleur@univ-pau.fr

Mathilde Monperrus
email: mathilde.monperrus@univ-pau.fr

Simultaneous determination of 42 pharmaceuticals in seafood samples by solvent extraction coupled to liquid chromatography-tandem mass spectrometry

Keywords: pharmaceuticals, emerging pollutants, seafood, fish tissue, multi-residue method, liquid chromatography, tandem mass spectrometry

diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pharmaceuticals are a large group of chemicals that are daily used for human and veterinary medicine. This group of anthropogenic chemicals is among the ones with the largest input into the environment [START_REF] Petrovic | Analysis, Removal, Effects and Risk of Pharmaceuticals in the Water Cycle[END_REF]. As pharmaceuticals consumption is continuously increasing [START_REF] Nikolaou | Occurrence patterns of pharmaceuticals in water and wastewater environments[END_REF], it raises concerns about their impact on the environment and undesired physiological effects they can cause to aquatic organisms [START_REF] Althakafy | Determination of selected emerging contaminants in freshwater invertebrates using a universal extraction technique and liquid chromatography accurate mass spectrometry[END_REF][START_REF] Besse | Human pharmaceuticals in surface waters. Implementation of a prioritization methodology and application to the French situation[END_REF][START_REF] Fent | Ecotoxicology of human pharmaceuticals[END_REF][START_REF] Länge | Environmental risk assessment of pharmaceutical drug substancesconceptual considerations[END_REF][START_REF] Ramirez | Occurrence of pharmaceuticals and personal care products in fish: results of a national pilot study in the United States[END_REF][START_REF] Zeilinger | Effects of synthetic gestagens on fish reproduction[END_REF]. Residues of several pharmaceuticals have been found in surface and ground waters, soils and animal tissues across the world at concentrations depending upon the pharmaceutical and the nature and proximity of sources [START_REF] Álvarez-Muñoz | Occurrence of pharmaceuticals and endocrine disrupting compounds in macroalgaes, bivalves, and fish from coastal areas in Europe[END_REF][START_REF] Gaw | Sources, impacts and trends of pharmaceuticals in the marine and coastal environment[END_REF]. Certain painkillers, antimicrobials, antidepressants, contraceptives and antiparasitics are commonly found (European Commission, 2019). Some antibiotic residues detected in food can have negative effects on consumer health and safety [START_REF] Chiesa | Validated multiclass targeted determination of antibiotics in fish with high performance liquid chromatographybenchtop quadrupole orbitrap hybrid mass spectrometry[END_REF]. In addition, several pharmaceuticals, such as diclofenac (anti-inflammatory), have been identified as priority substances for regulation at EU level via the Water Framework Directive (Official Journal of the European Union, 2013).

Consequently, there is a growing need to develop reliable analytical methods that enable rapid, robust, sensitive and selective determination of these emerging pollutants at trace levels in seafood.

In recent decades, pressurized liquid extraction (PLE) [START_REF] Huerta | Analysis of multi-class pharmaceuticals in fish tissues by ultra-high-performance liquid chromatography tandem mass spectrometry[END_REF], ultrasonic-assisted extraction (UAE) [START_REF] Liu | Simultaneous determination of 29 pharmaceuticals in fish muscle and plasma by ultrasonic extraction followed by SPE-UHPLC-MS/MS[END_REF], solvent-based extraction (SLE) [START_REF] Bayen | Direct injection of tissue extracts in liquid chromatography/tandem mass spectrometry for the determination of pharmaceuticals and other contaminants of emerging concern in mollusks[END_REF], microwave-assisted extraction (MAE) (Guedes-Alonso, [START_REF] Guedes-Alonso | Determination of steroid hormones in fish tissues by microwave-assisted extraction coupled to ultra-high performance liquid chromatography tandem mass spectrometry[END_REF], matrix solidphase dispersion (MSPD) [START_REF] Hertzog | Study of vortex-assisted MSPD and LC-MS/MS using alternative solid supports for pharmaceutical extraction from marketed fish[END_REF] or QuEChERS [START_REF] Lopes | Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry[END_REF] have been used for the extraction of organic pollutants from solid complex samples. After extraction, pharmaceuticals are commonly analysed by liquid chromatography -tandem mass spectrometry (LC-MS/MS) methods using an electrospray ionization source (ESI) for the simultaneous determination of a wide-range of polar compounds.

Previous studies used protocols which allowed to characterize pharmaceuticals with method detection limits (MDL) reaching the low ng/g range [START_REF] Guidi | Quinolones and tetracyclines in aquaculture fish by a simple and rapid LC-MS/MS method[END_REF][START_REF] Luo | Air-assisted liquid-liquid microextraction integrated with QuEChERS for determining endocrine-disrupting compounds in fish by highperformance liquid chromatography-tandem mass spectrometry[END_REF].

However, most of the published methods only focused on a short range of compounds, antibiotics being the most frequent class reported. So, there is still a need to develop universal methods applicable to different matrices and able to cover a large range of compounds with different physicochemical properties, which is challenging as it generally requires a compromise in the selection of experimental conditions [START_REF] Petrovic | Recent trends in the liquid chromatography-mass spectrometry analysis of organic contaminants in environmental samples[END_REF].

The presence of undesirable sample components that co-elute with the analytes, altering the ionization process and thus the signal is the main drawback associated to LC-MS/MS methods [START_REF] Gracia-Lor | Multi-class determination of around 50 pharmaceuticals, including 26 antibiotics, in environmental and wastewater samples by ultrahigh performance liquid chromatography-tandem mass spectrometry[END_REF]. Matrix effects may lead to a suppression or an enhancement of the signal, which can result in a wrong analytes quantification. Matrix effects depend on each analyte/matrix combination, but also on the sample preparation, the chromatographic separation, mass spectrometry instrumentation and the ionization conditions [START_REF] Gosetti | Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry[END_REF]. The evaluation of matrix effects should be included in the validation process of the method considering the different matrices studied. Several strategies were proposed to solve matrix effects, including modifications of sample pre-treatment, chromatographic or MS conditions and calibration techniques [START_REF] Gosetti | Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry[END_REF]. The use of deuterated internal standards is, by far, the most used in the pharmaceutical residues analysis field [START_REF] Gracia-Lor | Multi-class determination of around 50 pharmaceuticals, including 26 antibiotics, in environmental and wastewater samples by ultrahigh performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Gros | Development of a multi-residue analytical methodology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening and trace level determination of pharmaceuticals in surface and wastewaters[END_REF].

The objective of the present study was to develop a multi-residue analytical methodology based on a simple solvent extraction protocol followed by LC-MS/MS detection for the simultaneous analysis of 42 pharmaceuticals commonly used for human and veterinary purposes (including antibiotics, analgesics, anti-inflammatories, cardiovascular agents, anxiolytics and human indicators) in seafood, in order to provide a routine method for the monitoring of these emerging contaminants. The sample preparation was optimized by testing different extraction solvent and studying target compounds retrieval after several evaporation techniques and filtration of the final extract with different kinds of filters. Matrix effects and analytical performances of the optimal procedure were evaluated. Finally, the developed method was successfully applied to investigate occurrence of target pharmaceuticals in muscles of four seafood species with commercial interest collected from the Bay of Biscay (Southern France). Since several compounds were detected in the water leaving the local wastewater treatment plant [START_REF] Miossec | Multi-residue analysis of 44 pharmaceutical compounds in environmental water samples by solid phase extraction coupled to liquid chromatography -tandem mass spectrometry[END_REF], the present study allowed to investigate the potential bioaccumulation of these pharmaceuticals in the marine biota.

Materials and Methods

Reagents and materials

Samples collection and pre-treatment

Three fish species hake (Merluccius merluccius), red mullet (Mullus surmuletus) and sole (Solea solea) and one crustacean species shrimp (Palaemon serratus) were collected from the coastal area of the Bay of Biscay (Southern France). For fish, the white dorsal muscle was separated (skin and bone excluded) and for shrimp, the abdomen muscle was separated.

Muscles were then freeze-dried with a VaCo2 lyophilizer (Zirbus, Bad Grund, Germany), grinded to a homogeneous powder using a glass mortar and stored at -80 °C prior to extraction and analysis.

Sample preparation

An aliquot of 0.2 g of freeze-dried sample was weighed into a 50 mL polypropylene tube.

Internal standards atenolol-d7, carbamazepine-d10, ibuprofen-d3, nordiazepam-d5 and ofloxacin-d3 were added at concentrations of 400, 400, 10,000, 400, and 2000 ng/g, respectively, to each sample, by adding 40 µL of the multicomponent solution. After addition of 10 mL of extraction solvent (MeOH +1% acetic acid), the tube was shaken by vortex for 1 min. The tube was then centrifuged for 5 min at 4,500 rpm and the supernatant was transferred into 10 mL glass tubes, evaporated to dryness under a gentle air stream using a TurboVap LV Evaporator system (Zymark, Hopkinton, USA), and dissolved in 1mL MeOH/water (5/95 v/v). Finally, extracts were vortexed a few seconds, filtered through 0.2 µm polytetrafluoroethylene (PTFE) syringe filters, transferred into vials and kept at -20°C until analysis (Fig. S1).

LC-MS/MS analysis

Analysis were performed by LC-MS/MS using an Acquity UPLC system (Waters) connected to a Xevo TQ MS triple quadrupole with an electrospray source (ESI) interface (Waters). A C18 Acquity UPLC HSS T3 (1.8µm particle size, 50mm x 2.1mm i.d.,) column (Waters) preceded by a guard column (1.8µm particle size, 5mm x 2.1mm i.d.,) of the same packing material was used at a flow rate of the mobile phase 0.4 mL/min. The column temperature was fixed at 40°C and sample manager was maintained at 15°C. Sample injection volume was 5 µL. Two injections were used for the quantification of all compounds both in positive and negative ionization mode. The analysis in positive mode was performed using ultrapure water with 0.1% formic acid as eluent (A) and ACN as eluent (B). In negative mode, eluent (A) was ultrapure water with 0.01% formic acid and eluent (B) was ACN. For both modes, the initial composition was 2% (B) during 2 min and increased linearly to reach 60% at 4 min and 100% at 6 min. It held 1 min before returning to the initial composition (2% B) at 7.1 min and held for 3 min. The total analysis run time was then 10 min (See example of chromatograms in Supporting Information Fig. S2).

For the mass spectrometer, cone gas and desolvation gas flows were set at 10 and 600 L/h, respectively. Drying gas, as well as nebulizing gas were nitrogen, generated by pressurized air in a Nitrocraft nitrogen generator (Air Liquide). Source temperature was set to 150°C and desolvation temperature to 600°C. Capillary voltages of 0.5 kV (positive ionization mode) and -1.0 kV (negative ionization mode) were applied. Collision gas was Argon with a purity > 99.999% (Linde). Waters MassLynx software was used for the instrument control, data acquisition and data treatment. Quantification was carried out in Multiple Reaction Monitoring (MRM) mode, selecting two characteristics transitions for each compound. Table 1 presents MRM transitions, retention times, ion ratios and internal standards for each compound.

Quantification and quality control

Matrix-matched calibrations with deuterated analogs of the target analytes were used to quantify target compounds. Six-point calibration curves were performed with 0.2 g of different seafood species spiked with increasing pollutants concentration levels ranging from 0 to 500 ng/g as well as internal standards at various concentration (described in 2.3), and the optimized procedure was carried out.

Analytical performances and quality control were evaluated for each run by solvent blanks (MeOH/water 5/95 v/v) and procedural blanks to evaluate contamination and detection limits. Accuracy was also evaluated using matrix spikes at 2 levels (20 and 200 ng/g) to determine compound recoveries.

Results and discussion

Sample preparation optimization

Extraction solvent

The choice of the appropriate solvent is a crucial step in the sample pretreatment procedure to extract the desired analytes with minimum coextraction of matrix interferences [START_REF] Kung | A generic and rapid strategy for determining trace multiresidues of sulfonamides in aquatic products by using an improved QuEChERS method and liquid chromatography-electrospray quadrupole tandem mass spectrometry[END_REF]. To date, various extraction solvents have been used for the extraction of pharmaceuticals: MeOH [START_REF] Hertzog | Study of vortex-assisted MSPD and LC-MS/MS using alternative solid supports for pharmaceutical extraction from marketed fish[END_REF], ACN [START_REF] Freitas | Multiresidue and multi-class determination of antibiotics in gilthead sea bream (Sparus aurata) by ultra high-performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Saxena | Simultaneous determination of multi-residue and multi-class antibiotics in aquaculture shrimps by UPLC-MS/MS[END_REF], MeOH/ACN mixture [START_REF] Bayen | Direct injection of tissue extracts in liquid chromatography/tandem mass spectrometry for the determination of pharmaceuticals and other contaminants of emerging concern in mollusks[END_REF][START_REF] Kim | Human and veterinary pharmaceuticals in the marine environment including fish farms in Korea[END_REF][START_REF] Ondarza | Pharmaceuticals, illicit drugs and their metabolites in fish from Argentina: Implications for protected areas influenced by urbanization[END_REF] and same organic solvents with acetic acid acidification [START_REF] Mu | Simultaneous determination of 14 antiviral drugs and relevant metabolites in chicken muscle by UPLC-MS/MS after QuEChERS preparation[END_REF][START_REF] Yao | Simultaneous determination of 24 personal care products in fish muscle and liver tissues using QuEChERS extraction coupled with ultra pressure liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometer analyses[END_REF]. The present approach aims to develop a method for the extraction of a maximum of target compounds (which includes weakly basic and weakly acidic molecules) in one single step.

Effects of several extraction solvents such as ACN, MeOH and MeOH + 1% acetic acid on extraction efficiencies were evaluated. A lyophilized hake muscle was spiked with 200 ng/g of all target compounds (by adding 40 µL of a 1 mg/L multicomponent solution) prior to the extraction step. Comparison of peak areas indicated that extraction with ACN led to poor recoveries for some compounds, and especially for the least polar, in accordance with the fact that ACN is more polar than MeOH (Fig. 1). Recoveries of target molecules were higher for 34 compounds out of 42 when extracted with MeOH compared to extraction with ACN. Among MeOH and acidified MeOH, even if extraction rates were globally similar, acidification provided slightly higher responses. This result was expected, as the extraction of polar compounds is pH dependent, and compounds which are carrying a carboxylic group (i.e. ampicillin, norfloxacin, ofloxacin, ciprofloxacin…) are expected to present best recoveries in acidic conditions. Consequently, MeOH + 1% acetic acid was selected as the optimal solvent to extract all target analytes in one single step.

Evaporation and filtration

The evaporation stage could be a critical step, especially for the most volatile compounds, which may be lost. Retrievals of our target compounds after evaporation under different conditions has previously been investigated [START_REF] Miossec | Multi-residue analysis of 44 pharmaceutical compounds in environmental water samples by solid phase extraction coupled to liquid chromatography -tandem mass spectrometry[END_REF], and evaporation to dryness under air stream at room temperature was chosen as a gentle evaporation method in order to concentrate while preserving molecules as much as possible. The quantification with a matrix-matched calibration using internal standards allows to correct for losses observed for some analytes.

When injecting tissues extracts into an HPLC column, suspended particles may affect chromatographic performances, by creating interferences with target compounds, or by fouling even clogging the column. Therefore, the filtration of the final extract is an essential step. To date, different filtration materials have been indifferently used : PTFE [START_REF] Bayen | Direct injection of tissue extracts in liquid chromatography/tandem mass spectrometry for the determination of pharmaceuticals and other contaminants of emerging concern in mollusks[END_REF][START_REF] Rodrigues | Development of a Multiresidue Method for the Determination of 24 Pharmaceuticals in Clams by QuEChERS and Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry[END_REF][START_REF] Saxena | Simultaneous determination of multi-residue and multi-class antibiotics in aquaculture shrimps by UPLC-MS/MS[END_REF], polyvinylidene difluoride (PVDF) [START_REF] Carmona | Multi-residue determination of 47 organic compounds in water, soil, sediment and fish-Turia River as case study[END_REF][START_REF] Freitas | Multiresidue and multi-class determination of antibiotics in gilthead sea bream (Sparus aurata) by ultra high-performance liquid chromatography-tandem mass spectrometry[END_REF] and nylon [START_REF] Lopes | Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high performance liquid chromatography coupled to tandem mass spectrometry[END_REF][START_REF] Zhao | Development of a simple multiresidue determination method of 80 veterinary drugs in Oplegnathus punctatus by liquid chromatography coupled to quadrupole Orbitrap mass spectrometry[END_REF].

Various types of syringe filters were tested in order to study the potential loss by adsorption of target compounds, which may occur during the filtration step (and eventually leading to an underestimation of final results). A solution containing all the target compounds at 100 µg/L in MeOH/water (5/95 v/v) was filtered through 0.2 µm PTFE, PVDF and nylon syringe filters and then analysed following the above described LC-MS/MS method. Responses obtained for pharmaceutical compounds were compared with those obtained from the analysis of the same spiked non-filtered solution (Fig. 2).

It appeared that six compounds (losartan, ketoprofen, amiodarone, niflumic acid, ibuprofen and gemfibrozil) were completely retained by the nylon syringe filter, and that three compounds (spiramycin, azithromycin and amiodarone) were totally retained by the PVDF syringe filter, meanwhile none of them was fully retained when using the PTFE filter.

Antibiotics presented global low recoveries after PVDF filtration (ofloxacin, clarithromycin, roxithromycin and josamycin below 20%). In summary, PTFE (0.2 µm) syringe filters seems to be the best choice for the filtration of these final extracts intended for the analysis of this kind of pharmaceuticals, showing satisfactory results for 30 out of the 42 tested compounds (recovery >50%).

Method validation

Matrix effects evaluation

Matrix effects causing signal suppression or enhancement are mainly due to matrix compounds eluted with the same retention time as the target compounds [START_REF] Rogatsky | Evaluation of matrix effect and chromatography efficiency: new parameters for validation of method development[END_REF][START_REF] Stüber | Evaluation of three calibration methods to compensate matrix effects in environmental analysis with LC-ESI-MS[END_REF]. They also depend on the matrix nature, the efficiency of the sample preparation step, the detection response and the chromatographic behaviour.

Therefore, interfering compounds should be eliminated during the sample preparation while the analytes should be conserved. In this work, in order to evaluate the matrix effects, different seafood matrices (hake, red mullet, sole and shrimp) were spiked by adding 200 ng/g of the target compounds and subjected to the optimized procedure.

Matrix effects (ME, %) were calculated according to:

MEx % = Ax -Ax Ax -1 100
Where Ax(matrix) is the area of the compound x in the spiked matrix, Ax(blank) is the area of the compound x in the non-spiked matrix and Ax(solvent) is the area of the compound x in the spiked procedural blank.

Table 2 gives the results for all the target analytes. An enhancement of the signal leading to a positive value indicates a positive matrix effect. A signal suppression leading to a negative value corresponds to a negative matrix effect.

Matrix effects were different within the evaluated compounds and also within the different types of seafood. ME ranged from -83%, the highest signal suppression (acetaminophen and acetylsalicylic acid in shrimp), to +2,191%, the highest signal enhancement (amiodarone in hake). As expected, signal suppression has been observed to be higher in red mullet muscle than in the three other matrices, probably because of higher fat content level. As the 5 selected internal standards didn't cover the behaviour of the 42 molecules, both the addition of deuterated internal standards and the realization of matrix-matched calibrations are therefore mandatory to balance matrix effects and correctly quantify all the molecules.

Method performances

Analytical performances of the optimized method are reported in Table 3. Concerning linearity, equations and R 2 were calculated in the range 0-500 ng/g. Limits of quantification (LOQ) were determined as lowest injected compound concentrations in matrix that yielded a signal-to-noise (S/N) ratio of 10.

Recoveries (R, %) were also determined at two concentration levels according to:

R % = C -C C ! 100
Where C(spiked) is the concentration in the spiked matrix, C(blank) is the concentration in the non-spiked matrix and C(ref) is the theoretical added concentration. Precision was expressed as the relative standard deviation (RSD) of 3 replicates.

Coefficients of determination were higher than 0.99 for all compounds except amoxicillin in red mullet, acetylsalicylic acid and ibuprofen in sole, amoxicillin and acetylsalicylic acid in shrimp, demonstrating that the method is linear in the range assayed.

Limits of quantification ranged from 0.1 to 40.2 ng/g with most of the molecules between 0.1 and 5.0 ng/g. Amoxicillin, acetylsalicylic acid and ibuprofen exhibited lower sensitivities in the four biologic matrices related to their lower MS detection sensitivities.

Recoveries achieved for all target compounds at spiking level 20 ng/g ranged from 29% to 164% in hake muscle, from 33% to 128% in red mullet muscle, from 28% to 188% in sole muscle and from 26% to 132% in shrimp muscle. Recoveries achieved for all target compounds at spiking level 200 ng/g ranged from 69% to 131% in hake muscle, from 51% to 114% in red mullet muscle, from 45% to 119% in sole muscle and from 64% to 117% in shrimp muscle.

RSDs at spiking level 20 ng/g ranged from 1.4% (atenolol) to 62.9% (piperacillin) in hake, from As a result, a sensitive, reliable and repeatable analytical method for the quantitative determination of pharmaceutical residues in seafood samples was established and validated.

Application to real seafood samples

The optimized and validated methodology was applied to analyse seafood muscles (hake, red mullet, sole and shrimp) collected from the coastal area of the Bay of Biscay (Southern France).

Four of the 42 molecules (azithromycin, clarithromycin, acetaminophen and caffeine) were detected at concentrations above the LOQ at least once (Table 4). Caffeine (human indicator)

was found in all samples, which is in accordance with literature [START_REF] Álvarez-Muñoz | Occurrence of pharmaceuticals and endocrine disrupting compounds in macroalgaes, bivalves, and fish from coastal areas in Europe[END_REF].

Concentrations ranged from the LOQ to 11.4 ng/g (caffeine in shrimp muscle). The two antibiotics (azithromycin and clarithromycin) were only measured in red mullet and sole at concentrations below 1.0 ng/g. The analgesic (acetaminophen) was measured once in hake with a very low concentration (1.4 ng/g).

As seafood species considered in this study were caught in the open sea, these low occurrences and concentrations (low ng/g range) were expected and are in agreement with previous studies [START_REF] Chiesa | Validated multiclass targeted determination of antibiotics in fish with high performance liquid chromatographybenchtop quadrupole orbitrap hybrid mass spectrometry[END_REF][START_REF] Hertzog | Study of vortex-assisted MSPD and LC-MS/MS using alternative solid supports for pharmaceutical extraction from marketed fish[END_REF][START_REF] Wang | Analysis of selected pharmaceuticals in fish and the fresh water bodies directly affected by reclaimed water using liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Zhao | Development of a simple multiresidue determination method of 80 veterinary drugs in Oplegnathus punctatus by liquid chromatography coupled to quadrupole Orbitrap mass spectrometry[END_REF]. In addition, several pharmaceuticals such as diclofenac and ibuprofen undergo an efficient biotransformation into glucuronide metabolites in the fish bile before being excreted.

Therefore, muscle is not the target organ for bioconcentration and metabolism of these contaminants [START_REF] Lahti | Uptake from water, biotransformation, and biliary excretion of pharmaceuticals by rainbow trout[END_REF] that coupled with the high dilution effect in the open sea, could explain these obtained results.

Conclusions

An efficient and sensitive LC-MS/MS method was successfully developed for the simultaneous quantification of 42 pharmaceutical compounds in seafood. Sample preparation is easy and fast, making it a perfect routine method for the monitoring of those emerging contaminants in seafood samples.

Matrix effects were calculated for hake, red mullet, sole and shrimp muscles and resulted in a higher signal suppression in red mullet, the matrix with the highest fat content. Matrixmatched calibrations using deuterated internal standards were used for quantification, which allowed to correct for the matrix effects and obtain acceptable recoveries. Analysis by LC-MS/MS in positive and negative ionization modes provided good sensitivity and selectivity, with limits of quantification ranging from 0.1 to 40.2 ng/g. This method was successfully applied to the determination of target compounds in seafood samples collected from the Bay of Biscay (Southern France). We also suggest that this analytical method could be used as routine method for future environmental and safety monitoring.

Figure captions

Fig. 1 :

 1 Fig. 1: Influence of extraction solvent (ACN, MeOH, MeOH + 1% acetic acid) on measured areas (matrix: hake muscle, spiking level 200 ng/g, n=3).

Fig. 2 :

 2 Fig. 2: Recovery (n=3) of the target compounds after filtration of a spiked solution of MeOH/water (5/95) at 100 µg/L.

Table 1 :

 1 MS/MS parameters for the analysis of target analytes by MRM in negative and positive ionization modes.

	Compounds	Internal standards	RT (min)	Ionization modes	MRM 1	MRM 2	Ion Ratios (Quant/Qual)
	Atenolol-d7	-	3.54	ESI +	274.4 > 145.1	274.4 > 190.3	3.0
	Ofloxacin-d3	-	3.91	ESI +	365.4 > 261.1	365.4 > 321.2	1.3
	Carbamazepine-d10	-	4.70	ESI +	247.4 > 204.2	247.4 > 186.9	10.7
	Nordiazepam-d5	-	4.91	ESI +	276.0 > 140.2	276.0 > 165.1	0.8
	Metronidazole	Carbamazepine-d10	2.49	ESI +	172.3 > 128.1	172.3 > 82.0	2.4
	Acetaminophen	Carbamazepine-d10	2.69	ESI +	152.0 > 110.1	152.0 > 92.8	4.8
	Amoxicillin	Carbamazepine-d10	2.78	ESI +	366.4 > 114.0	366.4 > 349.2	1.3
	Acetazolamide	Carbamazepine-d10	3.36	ESI +	223.3 > 180.9	223.3 > 164.0	1.1
	Sulfadiazine	Carbamazepine-d10	3.51	ESI +	251.3 > 156.0	251.3 > 92.0	0.8
	Atenolol	Atenolol-d7	3.55	ESI +	267.5 > 145.1	267.5 > 190.1	1.8
	Caffeine	Carbamazepine-d10	3.83	ESI +	195.4 > 138.1	195.4 > 110.1	3.5
	Ampicillin	Carbamazepine-d10	3.84	ESI +	350.4 > 106.0	350.4 > 160.0	2.4
	Trimethoprim	Carbamazepine-d10	3.88	ESI +	291.4 > 230.1	291.4 > 123.1	0.9
	Norfloxacin	Ofloxacin-d3	3.90	ESI +	320.4 > 276.2	320.4 > 233.1	1.3
	Ofloxacin	Ofloxacin-d3	3.91	ESI +	362.4 > 261.2	362.4 > 318.2	1.3
	Ciprofloxacin	Ofloxacin-d3	3.93	ESI +	332.4 > 288.1	332.4 > 245.2	1.2
	Sulfamethazine	Carbamazepine-d10	4.06	ESI +	279.3 > 186.0	279.3 > 92.0	1.3
	Tetracycline	Ofloxacin-d3	4.07	ESI +	445.4 > 410.1	445.4 > 154.0	1.3
	Phenazone	Carbamazepine-d10	4.09	ESI +	189.4 > 131.0	189.4 > 55.4	26.1
	Metoprolol	Carbamazepine-d10	4.09	ESI +	268.4 > 116.1	268.4 > 121.1	2.7
	Spiramycin	Ofloxacin-d3	4.11	ESI +	843.7 > 174.1	843.7 > 142.1	3.4
	Azithromycin	Ofloxacin-d3	4.14	ESI +	749.7 > 158.1	749.7 > 591.5	1.1
	Sulfamethoxazole	Carbamazepine-d10	4.31	ESI +	254.3 > 92.0	254.3 > 156.0	1.1
	Oxolinic acid	Carbamazepine-d10	4.44	ESI +	262.4 > 244.1	262.4 > 216.0	4.7
	Erythromycin A	Carbamazepine-d10	4.50	ESI +	734.7 > 158.2	734.7 > 576.5	5.6
	Piperacillin	Carbamazepine-d10	4.53	ESI +	518.4 > 143.1	518.4 > 114.7	5.1
	Cyclophosphamide	Carbamazepine-d10	4.53	ESI +	261.3 > 140.0	261.3 > 106.0	2.5
	Tylosine	Ofloxacin-d3	4.57	ESI +	916.7 > 174.1	916.7 > 100.9	5.1
	Carbamazepine	Carbamazepine-d10	4.72	ESI +	237.4 > 194.1	237.4 > 179.1	8.7
	Flumequine	Carbamazepine-d10	4.78	ESI +	262.4 > 202.0	262.4 > 244.1	0.9
	Oxazepam	Nordiazepam-d5	4.79	ESI +	287.0 > 241.1	287.0 > 269.1	1.2
	Clarithromycin	Carbamazepine-d10	4.80	ESI +	748.7 > 158.1	748.7 > 590.4	6.6
	Roxithromycin	Ofloxacin-d3	4.82	ESI +	837.8 > 158.1	837.8 > 679.4	2.6
	Lorazepam	Nordiazepam-d5	4.84	ESI +	323.1 > 277.0	323.1 > 305.0	2.2
	Losartan	Carbamazepine-d10	4.86	ESI +	423.4 > 207.1	423.4 > 405.2	2.1
	Nordiazepam	Nordiazepam-d5	4.92	ESI +	271.0 > 140.0	271.0 > 165.0	1.7
	Josamycin	Ofloxacin-d3	4.93	ESI +	828.7 > 109.1	828.7 > 174.1	1.3
	Ketoprofen	Carbamazepine-d10	5.06	ESI +	255.4 > 105.1	255.4 > 209.1	1.3
	19-Norethindrone	Carbamazepine-d10	5.15	ESI +	299.4 > 109.0	299.4 > 91.0	1.8
	Amiodarone	Carbamazepine-d10	5.45	ESI +	646.3 > 100.1	646.3 > 86.1	1.2
	Ibuprofen-d3	-	5.53	ESI -	207.9 > 163.8	-	-
	Hydrochlorothiazide	Ibuprofen-d3	3.68	ESI -	296.1 > 269.0	296.1 > 205.0	1.2
	Acetylsalicylic acid	Ibuprofen-d3	4.30	ESI -	179.1 > 137.0	179.1 > 93.0	3.5
	Niflumic acid	Ibuprofen-d3	5.46	ESI -	281.2 > 237.0	281.2 > 177.0	5.4
	Diclofenac	Ibuprofen-d3	5.47	ESI -	294.1 > 250.0	294.1 > 214.1	18.5
	Ibuprofen	Ibuprofen-d3	5.53	ESI -	205.2 > 161.1	-	-
	Gemfibrozil	Ibuprofen-d3	5.78	ESI -	249.2 > 121.0	249.2 > 127.1	17.9
	MRM 1: transition used for quantification
	MRM 2: transition used for confirmation

Table 2 :

 2 Matrix effects (%) calculated for all target analytes in different seafood matrices. an intense signal suppression due to the presence of the matrix, ME (%) is close to -100 If there is an intense signal enhancement due to the presence of the matrix, ME (%) is close to 100 <LOQ

	ME (%)

Acknowledgments

This work was financially supported by ERDF (European Regional Development Fund) and AEAG (Agence de l'Eau Adour-Garonne) grants in the framework of the MICROPOLIT project.

Authors are grateful to people who helped in seafood samples collection and preparation.

Conflict of interest statement

Authors have declared no conflict of interest.


Jean-Sébastien Hulot
email: jean-sebastien.hulot@aphp.fr

COVID-19 in patients with cardiovascular diseases

Keywords: Acute cardiac injury, Hypertension, Heart failure, Myocardium

A novel coronavirus of zoonotic origin emerged in China at the end of December 2019, spreading rapidly throughout the country [START_REF] Zhu | A Novel Coronavirus from Patients with Pneumonia in China[END_REF][START_REF] Li | Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia[END_REF], and is affecting a large number of countries at the time this editorial was written (early March 2020). The infection, called Coronavirus Disease 2019 (COVID-19), is caused by a virus officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the World Health Organization. SARS-CoV-2 is an enveloped RNA beta-coronavirus with a phylogenetic similarity to another known coronavirus, SARS-CoV, which caused an outbreak of SARS in 2003 [START_REF] Wu | A new coronavirus associated with human respiratory disease in China[END_REF]. While the epidemiological and clinical characteristics of are not yet fully determined, first evidence indicates important consequences for the cardiovascular system and implications for cardiologists [START_REF] Madjid | ACC Clinical Bulletin: Cardiac Implications of Novel Coronavirus (COVID-19)[END_REF]. First, early case reports suggest that patients with previous or underlying cardiovascular diseases are at higher risk for developing severe symptoms if infected with SARS-CoV-2. In a report on 138 patients with COVID-19 hospitalized in Wuhan (Hubei province, China) [START_REF] Wang | Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China[END_REF], 64 (46.4%) had one or more coexisting medical conditions, mostly cardiovascular or cerebrovascular. Hypertension was present in 31.2%, diabetes in 10.1%, and cardiovascular disease in 14.5% of patients [START_REF] Wang | Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China[END_REF].

Interestingly, these proportions were significantly higher in patients with the most severe forms of COVID-19 (i.e. requiring hospitalization in an intensive care unit), with hypertension in 58.3%, diabetes in 22.2%, cardiovascular disease in 25.0% and cerebrovascular disease in 16.7% [START_REF] Wang | Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China[END_REF]. Similarly, while the true overall mortality rate of COVID-19 is still undetermined, and is estimated (based on a crude mortality ratio) at between 3% and 4%, it could be higher in older patients (>60 years) or patients with pre-existing comorbid conditions such as cardiovascular disease (10.5%), diabetes (7.3%) or hypertension (6.0%) [START_REF] Wu | Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention[END_REF]. While the disease can present as a pulmonary disease, the case-fatality rate for patients with underlying cardiovascular disease is greater (10.5%) than in patients with underlying chronic respiratory disease (6.3%) [START_REF] Madjid | ACC Clinical Bulletin: Cardiac Implications of Novel Coronavirus (COVID-19)[END_REF].

Second, whereas COVID-19 typically presents with symptoms of lower tract respiratory infection, a significant proportion of patients experience cardiovascular symptoms at initial presentation [START_REF] Zheng | COVID-19 and the cardiovascular system[END_REF]. These symptoms notably include palpitations and chest tightness. In addition, it is likely that SARS-CoV-2 can cause myocardial damage. In different reports, an increase in highsensitivity cardiac troponin I (cTnI) was noted in 10-20% of patients infected with COVID-19 [START_REF] Wang | Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China[END_REF][START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF]. In China, an estimated 11.8% of patients who died from COVID-19 presented substantial heart damage, with elevated levels of cTnI or cardiac arrest during hospitalization, without having any pre-existing cardiovascular disease [START_REF] Zheng | COVID-19 and the cardiovascular system[END_REF]. The exact conditions leading to acute myocardial injury are still unclear, but could be linked to either acute myocarditis or acute coronary syndrome, as previously experienced with the Middle East respiratory syndrome-related coronavirus (MERS-CoV) [START_REF] Zheng | COVID-19 and the cardiovascular system[END_REF]. It was notable that classical symptoms and presentation of acute myocardial infarction can be overshadowed in the context of COVID-19, leading to a potential delay in diagnosis [START_REF] Madjid | ACC Clinical Bulletin: Cardiac Implications of Novel Coronavirus (COVID-19)[END_REF].

Overall, these first data indicate a level of interaction between SARS-CoV-2 and the cardiovascular system, either by a direct or an indirect mechanism. SARS-CoV and SARS-CoV-2 infections are triggered by binding of the virus' spike protein to angiotensin-converting enzyme 2 (ACE2) [START_REF] Hoffmann | SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor[END_REF]. ACE2 is a membrane-bound zinc metallopeptidase involved in the cleavage of angiotensin. ACE2 is highly expressed in the lungs but also in the heart, where it is localized to macrophages, vascular endothelium, smooth muscle and myocytes [START_REF] Burrell | Myocardial infarction increases ACE2 expression in rat and humans[END_REF]. Intriguingly, ACE2 expression is increased post-myocardial infarction [START_REF] Burrell | Myocardial infarction increases ACE2 expression in rat and humans[END_REF] and in patients with diabetes [START_REF] Soro-Paavonen | Circulating ACE2 activity is increased in patients with type 1 diabetes and vascular complications[END_REF], two populations that are at higher risk of developing severe forms of COVID-19. ACE2 also regulates important cardiovascular and metabolic functions, including regulation of blood pressure and glycaemia [START_REF] Bindom | The sweeter side of ACE2: physiological evidence for a role in diabetes[END_REF][START_REF] Jiang | Angiotensin-converting enzyme 2 and angiotensin 1-7: novel therapeutic targets[END_REF]. Whether SARS-CoV-2 directly affects the cardiovascular system by targeting ACE2-expressing cells remains to be explored. Another possibility involves an indirect effect of the immune response to SARS-CoV-2 on the myocardium and the vessels. It is likely that severe forms of COVID-19 involve a cytokine storm, which may play a role in coronary plaque instability, as previously observed with SARS-CoV [START_REF] Channappanavar | Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology[END_REF][START_REF] Tsui | Coronary plaque instability in severe acute respiratory syndrome[END_REF]. In addition, patients infected with SARS-CoV or SARS-Cov-2 typically present with lymphopaenia [START_REF] Wang | Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China[END_REF][START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF], a condition that has been associated with the development of atherosclerosis and adverse cardiovascular outcomes [START_REF] Nunez | Relationship between low lymphocyte count and major cardiac events in patients with acute chest pain, a non-diagnostic electrocardiogram and normal troponin levels[END_REF].

The situation of the COVID-19 outbreak is rapidly evolving, with uncertain clinical and physiopathological profiles. However, the first case reports indicate that COVID-19 has important effects on the heart and the vessels, which should be known by the cardiovascular community.

Sources of funding

J

  .S.H is supported by INSERM, the French National Research Agency (NADHeart ANR-17-CE17-0015-02, PACIFIC ANR-18-CE14-0032-01, CORRECT_LMNA ANR-19-CE17-0013-02), BPIFrance (2018-PSPC-07), the ERA-Net-CVD (ANR-16-ECVD-0011-03, Clarify project), Fédération Française

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.


Immunopathogenesis of Idiopathic

While genetic studies have elucidated rare forms of inherited nephrotic syndrome, they failed to identify a genetic defect in INS with relapse, except an association with some variants of class II-MHC antigens [START_REF] Lane | Genetics of Childhood Steroid Sensitive Nephrotic Syndrome: An Update[END_REF]. Studies on genetic polymorphisms in the variable region of the Tcell receptor (TCR) ß-chain have revealed a selective recruitment of some variable  gene families in peripheral CD8 + T cells from nephrotic patients with frequent relapses [START_REF] Frank | Dominant T cells in idiopathic nephrotic syndrome of childhood[END_REF].

The hypothesis that INS results from a systemic disorder of immune system is supported by clinical observations such as its sensitivity to steroid therapy and immunosuppressive drugs, as well as the rapid occurrence of relapses upon antigen challenge (infections or vaccination) particularly in children. [START_REF] Kemper | Serum levels of immunoglobulins and IgG subclasses in steroid sensitive nephrotic syndrome[END_REF][START_REF] Yang | Circulating follicular T helper cells are possibly associated with low levels of serum immunoglobulin G due to impaired immunoglobulin class-switch recombination of B cells in children with primary nephrotic syndrome[END_REF]. In another study, it has been shown that T cells of patients who respond to Rituximab display a weaker response to PMA/ionomycin activation, characterized by a lower percentage of CD3 + CD4 + CD154 + , IFN + CD3 + and IL-2 + CD3 + T-cell subsets compared with non-responders, suggesting that hyporesponsiveness to T-cell stimulation could be used to identify patients susceptible to respond to Rituximab [START_REF] Chan | T Lymphocyte Activation Markers as Predictors of Responsiveness to Rituximab among Patients with FSGS[END_REF].

Several disorders of T-cell subpopulations have been reported during relapses (3). The

The role of B cells in INS pathogenesis remains to be clarified. The frequency of B-cell subpopulations before and after Rituximab therapy has been investigated in pediatric patients,

showing that memory B cell recovery is faster in patients who relapse compared to nonrelapsers [START_REF] Colucci | B Cell Reconstitution after Rituximab Treatment in Idiopathic Nephrotic Syndrome[END_REF]. Rituximab is thought to erase germinal center B cells in human lymph nodes, without affecting the TFH cell population [START_REF] Wallin | Human Tfollicular helper and T-follicular regulatory cell maintenance is independent of germinal centers[END_REF]. This raises the possibility that the delayed reconstitution of switched memory B cells could result from qualitative alterations of TFH cells induced by B-cell depletion. Indeed, expansion of switched memory B cells requires cognate TFH cells since it is abrogated in the absence of IL-21 receptor or CD40 ligand [START_REF] Zabel | Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation[END_REF].

Remission following B-cell depletion suggests that glomerular disease could be induced by some B lymphocyte subsets such as autoreactive B-cells but some arguments advocate a more intricate mechanism: (i) immunofluorescence studies on renal biopsies consistently show the absence of Ig deposits in INS relapse and (ii) remission can be maintained despite complete recovery of peripheral B-cell compartment, while relapses can occur in presence of sustained CMIP (CMaf-inducing protein) is a poorly described gene, initially identified in T cells of patients with INS [START_REF] Oniszczuk | CMIP is a negative regulator of T cell signaling[END_REF]. In normal lymphoid tissue, CMIP is found selectively expressed into lymphoid follicles, mainly at the interface of B-cell and T-cell zones [START_REF] Audard | Occurrence of minimal change nephrotic syndrome in classical Hodgkin lymphoma is closely related to the induction of c-mip in Hodgkin-Reed Sternberg cells and podocytes[END_REF], while in peripheral T cells CMIP is scarcely detected in resting conditions [START_REF] Oniszczuk | CMIP is a negative regulator of T cell signaling[END_REF], which suggests that in basic conditions, CMIP acts primarily in secondary lymphoid organs. On the other hand, CMIP was found highly expressed by subset of as yet uncharacterized T cells. Selective expression of CMIP in peripheral T cells by targeted transgenesis in mice results in T cell dysfunction.

CMIP interferes with the early events of T-cell signaling by inhibiting lipid raft clustering, the activation of Src kinases (Fyn and Lck) and ZAP-70 (Figure ). Transgenic T-cells exhibit a lower proliferative capacity and are less prone to producing cytokines, notably IL2, after stimulation [START_REF] Oniszczuk | CMIP is a negative regulator of T cell signaling[END_REF]. Moreover, CMIP binds to the P85 subunit of PI3 kinase and prevents its dissociation from the p110 catalytic subunit, which results in inhibition of PI3-kinase activation and downstream signaling molecules, notably Akt. It is interesting to note that Akt activation is inhibited in vitro and in vivo in T cells and podocytes overexpressing CMIP, as well as in INS disease ((15) and manuscript in preparation). These observations may account for the lower reactivity of T lymphocytes to mitogens and the decrease of delayed hypersensitivity reported in patients with INS. However, a clear identification of CMIP function in the immune system is mandatory for a better understanding of its role in INS pathogenesis.

  Nephrotic Syndrome B. Savas, F. Fofana, S. Le Gouvello, A Pawlak, D. Sahali*, M. Ollero, Université Paris Est (UPEC), Faculté de Santé, UMRS 955, Créteil F-94010, France and AP-HP, Groupe Henri-Mondor Albert-Chenevier, Service de Néphrologie, Créteil F-94010, France. (*corresponding author) Idiopathic nephrotic syndrome (INS) defines a group of rare glomerular diseases characterized by massive proteinuria and hypoalbuminemia in the absence of glomerular inflammatory lesions or immunoglobulin deposits. INS includes two main entities based on kidney biopsy findings: minimal-change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS). The treatment of INS relies on steroids and/or immunosuppressive drugs (calcineurin inhibitors, cyclophosphamide, mycophenolate mofetil, rituximab). Distinct clinical patterns can be observed such as steroid sensitive with frequent relapses (in 70-80% of cases), and primary or secondary steroid resistant forms. INS is considered as a chronic medical condition interfering in childhood as in adulthood with wellbeing and health-related quality of life.

  frequency of Treg is consistently reduced[START_REF] Liu | Th17/Treg imbalance in adult patients with minimal change nephrotic syndrome[END_REF], and is restored after response to therapy[START_REF] Boumediene | NEPHRUTIX: A randomized, double-blind, placebo vs Rituximab-controlled trial assessing T-cell subset changes in Minimal Change Nephrotic Syndrome[END_REF]. The downregulation of Treg cells in relapse is correlated with a significant decrease in IL-2, which is crucial to the development and maintenance of Treg cells. Experimental evidence suggests that Treg cells contribute to control humoral response by limiting expansion of T follicular helper (TFH) and B cells. Depletion of Treg cells increases switched B cells, while adoptive transfer of Treg cells suppresses in vivo B-cell response. Interestingly, Treg cells prevent expansion of autoreactive B cells and induce their apoptosis (5), which suggests that downregulation of Treg may lead to escapement of autoreactive, potentially switched memory B, which may contribute to the development of autoimmune diseases. Indeed, relapses are found to be associated with more rapid reconstitution of switched memory B cells (6). The finding that Rituximab, a B-cell-depleting agent, maintains long-lasting INS remission points out a potential role for B lymphocyte in the mechanisms of INS pathogenesis. In a multicenter, double-blind, randomized, placebo vs Rituximab-controlled trial, we have analyzed the modifications on T-cell subsets induced by B-cell depletion (3). Our results suggest that Rituximab does not interfere with CD4 + T-cell, CD8 + T-cell or CD45RO + T-cell frequencies, while relapses are associated with downregulation of CD8 + T-cell subset and an increase in the frequency of CD4 + CD45RO + CD30 + circulating memory T-cell subset, which are involved in recall memory antibody responses. On the other hand, Rituximab reduces specifically the frequency of TFH cells. Interestingly, recent studies showed an expansion of TFH in relapses, associated with a defect in immunoglobulin switching, accounting for the increase in IgM class and low production of IgG affecting some subclasses, according to unknown mechanisms

B

  cell -depletion (12). Beside their role in antibody-mediated mechanisms, B cells are also involved in antigen presentation, T-cell activation/regulation and production of cytokines and growth factors. Thus, B cells may facilitate disease activity by sustaining pathogenic T-cell responses through antibody-independent mechanisms. Therefore, although Rituximab may be considered as an innovative therapeutic agent in frequent relapsing, steroid-dependent INS, the mechanisms by which it interferes with T-cell disorders remain unclear.

signaling cascades, ultimately resulting in activation of transcription factors such as NF-kB, NFAT, and AP-1. (B), overexpression of CMIP inhibits the activation of Src kinases and the activation of ZAP-70, suggesting that CMIP interferes with the T-cell proximal signaling and prevents the clustering of lipids rafts, the cytoskeleton reorganization and the activation of downstream signaling cascades.