Global fish production reached more than 171 million tonnes by 2016, of which 88% were directly used for human consumption and 12% (20 million tonnes) were used for the production of fishmeal and fish oil in aquaculture [1
]. Fish and fishery products are an important source of essential nutrients in the human diet, and demand is growing in line with the increasing world population [2
]. Aquaculture is the fastest-growing food industry and the intensification of the production depends on the utilisation of other resources for aquafeeds than fishmeal, for which exploitation is reaching an unsustainable level. Therefore, agricultural crops, mainly legumes, cereal grains and oilseeds, have been introduced in steadily increasing amounts into fish feeds, completely or partially replacing marine protein sources [3
Plant protein sources mainly include soy, pea, lupine, alfalfa, wheat, corn, rape seeds, sunflower seeds, cotton seeds, sesame seeds, mustard oil cake, and white leadtree leaves [4
]. Moreover, proteins from insects, microalgae, krill and single-cell proteins have been explored as replacements for fishmeal, but plant proteins are by far the most used ingredients in feed in aquaculture. The considerable changes in the diet composition of farmed fish include ingredients with physicochemical properties that potentially could lead to challenges regarding fish health and welfare, and product quality [5
]. However, new processing technologies for plant protein extraction of undesirable components such as fertilisers, pesticides, persistent organic pollutants and heavy metals have allowed the transition from marine to agricultural sources [6
]. The growth performance of plant-fed fish has been found to be adequate in short feeding studies [7
], but concern about potential negative health effects from natural toxins and anti-nutritional factors including phytoestrogens remains [4
]. Some anti-nutritional factors are considerably resistant against heat and digestion and have the potential for carry-over into the food chain. Several studies have shown that bioactive compounds may affect physiological functions in animals and humans including negative effects on intestinal health [9
]; however, information for fish is limited [4
]. The potential transfer of undesirable substances from new sources of aquafeeds might thus lead to potential health risks for consumers of fish products [10
]. The assessment of transmissibility requires analytical methods that can be reliably applied for the detection of relevant natural contaminants in agricultural crops, and the considerable prevalence of mycotoxins and phytoestrogens makes them priority target analytes. However, only a few recent studies have surveyed mycotoxin levels in fish feed or farmed fish [11
], and phytoestrogens are even less investigated [17
There is a risk of mycotoxicosis in farmed fish due to the presence of mycotoxins in plant feed ingredients, but information on effects in fish is limited [11
]. Mycotoxins comprise a large variety of secondary metabolites produced by fungi such as Fusarium
spp. and Pencillium
spp. that infect agricultural crops both in the field and during storage, depending on their preferred growth conditions [20
]. The presence of mycotoxins in practically all feed- and foodstuffs worldwide, although at different levels, is critical for nutritional security and safety, and important for animal and human health and welfare [21
]. In moderate climate zones, major mycotoxin classes associated with Fusarium
crop infections are trichothecenes, zearalenones and enniatins. The most important trichothecenes (polycyclic sesquiterpenoids) are A-type HT-2 toxin (HT-2) and T-2 toxin (T-2) and B-type deoxynivalenol (DON), including the acetylated and glucosidated derivatives 3-acetyl-deoxynivalneol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and deoxynivalenol-3-glucoside (DON-3G), as well as nivalenol (NIV). Furthermore, the mycoestrogen zearalenone (ZEN) shows considerable occurrence and toxicity. The ionophoric enniatins (ENN) B, B1, A, and A1 are detectable in almost all grain samples and considered an emerging threat [22
]. In contrast, toxicity caused by ergot alkaloids such as ergosine, ergonovine, ergotamine, ergocristin, ergocornine and α-ergocryptine in Claviceps purpurea
-infected cereals has been known as ergotism for centuries. Ergot contamination is a sporadic issue but appears to have increased in recent years. The storage mycotoxin of main concern in Nordic countries is ochratoxin A (OTA), a pentaketidic isocoumarin produced by Penicillium
sp. In contrast, aflatoxins and fumonisins normally do not occur in Norwegian feed commodities [23
]. The European Commission has recommended maximum levels for important mycotoxins in different feed commodities [24
]. Fish ingredients and composite fish feed are not specifically mentioned but the guidance levels for DON (5 mg/kg); ZEN (2 mg/kg) and OTA (0.25 mg/kg) also apply to aquaculture. Additionally, an indicative value for the sum of T-2 and HT-2 (250 µg/kg) in compound feed is provided by the EU Commission recommendation [25
]. Comparable values have not been established for NIV, enniatins or ergot alkaloids because of the limited occurrence and toxicity data.
Phytoestrogens are plant-derived polyphenolic non-steroidal compounds with structural and functional similarity to animal oestrogens, which can bind to oestrogen receptors and activate oestrogen receptor-dependent pathways in mammals and fish [26
]. Thus, they have the potential to disrupt the endocrine system by competing with endogenous hormones. Phytoestrogens can be broadly differentiated into isoflavones, coumestans and lignans, depending on the alkylation pattern in the basic isoflavone molecule structure [27
]. Legumes, especially soy, are rich in isoflavones, which occur in plants mainly in glucosidated form, whereas the unconjugated molecules are prevalent after uptake. Important representatives of this substance class are the glucosides daidzin, genistin, glycitin and their respective free counterpart’s daidzein, genistein and glycitein [28
]. They are also potential substrates for metabolic glucuronidation or sulphatation reactions in the liver and kidneys due to the hydroxyl groups in the molecule and could be excreted as conjugates [29
]. Processed soy protein concentrates have an increased aglycon content, which results in improved phytoestrogen absorption from the diet [30
]. Exposure of fish to phytoestrogens in feed has been shown to cause reproductive effects and to affect growth and metabolism [31
], but the levels in the edible tissue of soy-fed fish and potential human exposure have not been investigated so far.
The assessment of possible health risks from the consumption of fish fed with plant-derived feed requires the development of appropriate analytical methods for the detection of transferred contaminants and bioactive compounds. Mycotoxins are usually analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with different multi-toxin methods and in various matrices such as bulk cereals, flour, nuts, food products and hay bales [32
]. Advanced sampling schemes and extraction protocols have been developed, resulting in improved homogeneity and recovery so that method validation can be performed [41
]. Sample preparation often includes single-step solvent extraction using acidic acetonitrile/water mixtures, followed by solid-phase extraction (SPE) or immunoaffinity purification [39
]. Matrix effects can be controlled by using matrix-matched calibration and isotope-labelled internal standards (ISTD), which are available for trichothecenes but not for enniatins and ergot alkaloids [32
]. Notably, fewer LC-MS/MS methods have been described for ergot alkaloids than for Fusarium
toxins, focussing on rye, feed and seeds as typical matrices [34
]. In contrast, phytoestrogens are mostly measured in physiological samples including human and animal plasma, milk and urine in connection with monitoring of dietary exposure [42
]. The LC-MS/MS methods developed for the detection of phytoestrogens in soy and food items use methanol‒water extraction and reversed-phase (RP) chromatography [44
Earlier studies have measured several mycotoxins in feed ingredients, aquafeeds and fish fillets [11
] but ergot alkaloids were not among the analytes. In addition, we have found one report of the occurrence of phytoestrogens in foods of animal origin, including a few fish samples [47
]. Considering the potential consumer health risk resulting from the extensive introduction of agricultural crops into fish feed and contaminant carry-over, analytical methods for the reliable detection of natural toxins and bioactive compounds are required. The present study was thus intended to fill this gap by developing a multiplexed LC-MS/MS method for the simultaneous quantification of 25 relevant feed-borne mycotoxins and phytoestrogens in feed and fish.
4. Materials and Methods
LC-MS grade acetonitrile (MeCN), methanol (MeOH) and water (Optima, LC/MS grade,) were provided by Fisher Scientific (Loughborough, Leics., UK), and ethanol (EtOH) was obtained from VWR International (Lutterworth, Leics., UK). Acetic acid (CH3COOH) (>99.8%), formic acid (HCOOH) (>98%) and ammonium acetate (CH3COONH4) (>98%) were purchased from Merck KGaA (Darmstadt, Germany).
The mycotoxins deoxynivalenol (DON), 3-actetyl-deoxynivalenol (3-ADON), nivalenol (NIV), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEN), deoxynivalenol-3-glucoside (DON-3G), 15-acetyl-deoxynivalenol (15-ADON), ochratoxin A (OTA), ergosine, α-ergocryptine and ergocristine as well as the stable isotope-labelled analogues U-[13C-15]-NIV, U-[13C-15]-DON, U-[13C-21]-DON-3G, U-[13C-17]-3ADON, U-[13C-17]-15ADON, U-[13C-22]-HT-2, U-[13C-24]-T-2, U-[13C-20]-OTA, U-[13C-18]-ZEN were provided by Romer labs (Tulln, Austria) as solutions in MeCN, ranging from 10 to 100 mg/L. Intermediate standard solutions at 10 mg/L were prepared for DON-3G and 15-ADON by dilution of stock solutions with MeCN. The enniatins A, A1, B, and B1 (ENN A, A1, B, B1), ergonovine, ergotamine, ergocornine, methysergide maleate salt (MetErg) and bromocriptine mesylate (BromCri) were provided as solids by Sigma-Aldrich (St. Louis, MO, USA). Stock solutions in MeOH or MeCN were prepared for ergot alkaloids in the range of 100 to 500 mg/L, and for enniatins with 200 mg/L in MeOH. A combined intermediate standard solution with 10 mg/L was prepared for both enniatins and ergot alkaloids by combining appropriate aliquots of stock standard solutions, evaporating the mixture with a gentle stream of nitrogen and re-dissolving in MeCN/water (50:50). Finally, a combined standard solution containing all mycotoxins (Set A) was prepared by combining aliquots of stock or intermediate standard solutions, evaporating the solvent and re-dissolving in the appropriate volume MeCN/water (50:50) to obtain final concentrations of about 200 µg/L (200.0–200.12 µg/L, depending on the stock solution provided by the manufacturer).
The phytoestrogens daidzin, genistin, glycitin, daidzein, genistein, and glycitein were bought in crystalline form from Sigma-Aldrich (St. Louis, MO, USA), and stock solutions were prepared in MeOH or DMSO (glycitein) ranging from 500 to 1000 mg/L. Individual intermediate standard solutions at a concentration of 5 mg/L were prepared by dilution with MeOH. A combined standard solution (Set B; 200 µg/L) containing all phytoestrogens was prepared by further dilution in MeCN/water (50:50). The finished Set A and Set B solutions were stable at −20 °C for several months and used for the preparation of standard calibration curves.
Additionally, a 25-in-1 multi-analyte mixture was prepared and used in spiking experiments. All analytes were combined with regard to the concentrations of their respective stocks or intermediate standard solutions so that a final concentration of 25 µg/L per analyte was reached after spiking into feed, zebrafish and salmon samples. The multi-analyte mixture was evaporated and re-dissolved in MeCN/water (50:50). It was stable at −20 °C for about a month.
A combined internal standard (ISTD) solution for 15 mycotoxins, containing stable isotope-labelled analogues and the ergot homologues MetErg and BromCri, was prepared in MeCN/water (50:50) to reach final concentrations of 251 µg/L U-[13C-18]-ZEN, 500 µg/L U-[13C-22]-HT-2, 443 µg/L U-[13C-22]-T-2, 506 µg/L U-[13C-15]-DON, 502 µg/L U-[13C-17]-3ADON, 500 µg/L U-[13C-17]-15ADON, 500 µg/L U-[13C-20]-OTA, 530 µg/L U-[13C-15]-NIV, 530 µg/L U-[13C-21]-DON-3G, 624 µg/L BromCri and 500 µg/L MetErg. The different concentrations were chosen with regard to the respective measurement sensitivities in the developed multi-analyte LC-HRMS/MS method. The ISTD solution was stored at −20 °C, adjusted to room temperature (RT) and mixed thoroughly prior to use. It was added in a ratio of 1:5 to the study samples.
4.2. Preparation of Fish Diets
Diets with definite amounts of wheat gluten, soy protein concentrate or pea protein concentrate were produced at Nofima Feed Technology Centre, Fyllingsdalen, Norway. The diets were based on fishmeal (FM) as main protein source, which was replaced by 15% or 30% plant proteins. All diets contained 12% wheat that was required for binding in the extrusion process, in addition to minor inclusion of wheat as carrier for some of the additives used (Table 1
). In total seven diets were produced: (1) control feed (FM), (2) 15% soy protein concentrate (SPC15), (3) 30% soy protein concentrate (SPC30), (4) 15% wheat gluten (WG15), (5) 30% wheat gluten (WG30), (6) 15% pea protein concentrate (PPC15), and (7) 30% pea protein concentrate (PPC30). The ingredients used for the preparation of diets included FM Norsildmel AS (Bergen, Norway), SPC from Agilia A/S (Videbæk, Denmark), PPC from AM Nutrition AS (Stavanger, Norway) and WG from Tereos Syral (Marckolsheim, France). All diets had an inclusion of 4% fish oil at extrusion. The feed were produced on a pilot scale twin-screw, co-rotating Wenger TX 52 extruder (Wenger, Sabetha, KS., USA) with a die of 2.5 mm diameter. After extrusion, the diets were dried for 40–70 min in a carousel dryer (Paul Klöckner, Verfahrenstechnik GmbH, Hachenburg, Germany) at 65 °C to a water content of 7–8%. The salmon diets 1 to 5 were, in addition, oil-coated with 16% fish oil after extrusion by vacuum-coating (Dinnissen, Sevenum, Netherlands) to meet the standard dietary inclusion of oil for the fish size studied. The salmon feed had a pellet size of 3.5 mm, while the zebrafish feed were ground and sieved to a pellet size of 0.6–0.8 mm.
4.3. Feeding Studies in Zebrafish and On-Growing Salmon
Four-month-old zebrafish (Danio rerio) (AB strain) with a mean weight of 0.214 g were distributed into 28 tanks (n = 16) and were maintained in a flow-through system with 20 % water exchange per hour (ZebTEC Stand-Alone Toxicology Rack, Techniplast, London, UK) under daily-monitored standard husbandry conditions, including a stable temperature of 28 ± 0.5 °C, pH 7.5, water conductivity of 1500 µS/cm and photoperiod of 12 h light:12 h dark at the Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway. The feeding study included 336 fish that were distributed into the system’s 3.5-litre tanks according to the seven experimental diets. Four replicate groups per diet, each consisting of 12 fish (six per gender) in one tank (and an additional four fish to compensate for potential losses during the study period), were hand-fed twice daily with a total feed amount equal to 2.5% of their body weight over a period of 46 days. The feeding behaviour and health and welfare of the fish were regularly controlled. At the end of the study, the fish were not fed for 24 h prior to sampling. They were separated by gender and euthanised individually by transfer into a tank containing a lethal dose of 200 mg/L tricaine methanesulfonate (MS222) (Sigma-Aldrich, St. Louis, MO, USA), buffered with an equal amount of sodium bicarbonate. The liver, spleen and intestines were carefully dissected under a light microscope and immediately frozen in liquid nitrogen along with the rest of the carcass. All samples were stored at −80 °C for further analyses.
The zebrafish feeding study was conducted in compliance with the guidelines provided by the Norwegian Animal Research Authority (FOTS ID 12581, 27 July 2017) and approved by the Nord University (Norway) ethics committee.
One-year-old post-smolt Atlantic salmon (Salmo salar; salmo breed strain) with a mean weight of 223 g were randomly distributed into 15 experimental tanks (1 m3; n = 32) filled with seawater at the Nofima Research Station, Sunndalsøra, Norway. The oil-coated diets 1–5 were given to randomised triplicate tanks by automatic disc feeders. Excess feed was collected once daily for calculation of feed intake. The water temperature was maintained at an average of 10.6 (±0.6) °C. The oxygen level at the tank outlets was higher than 90% at study start and about 80% at the study’s end. The water flow in each tank was set to 20 L/min.
The feeding was conducted for nine weeks. At the start of the experiment, 15 fish were sampled, and the muscle, liver and intestine were collected. After five weeks, muscle was sampled from one fish from each tank of the FM, SPC30 and WG30 groups. At the termination of the study, five fish from each tank were collected and weighed. The sampled fish were anaesthetised with 60–80 mg/L MS222, transferred and euthanised with a double dose (120–160 mg/L) MS222. Blood was drawn from the caudal vein using 2.5-mL vacutainers (VACUETTE® 2.5 mL Z serum separator clot activator; Greiner Bio-One, Kremsmünster, Austria) and centrifuged at 2500× g for 15 min at 4 °C (Allegra 6R Centrifuge, Beckman, Indianapolis, IN, USA), and sera were stored at −20 °C. The livers and intestines of the fish were removed, and tissue samples were frozen with liquid nitrogen and stored at −80 °C. Fillets were stored at −20 °C. The remaining fish in each tank were weighed in bulk, and their mean weight was calculated, including the sampled fish.
The salmon feeding study was performed in compliance with the national regulations for the use of animals in experiments [57
]. The experiment was classified as not requiring a specific license [58
] as none of the planned experimental treatments were expected to cause any distress or discomfort for the fish.
4.4. Extraction of Fish Feed, Zebrafish and Salmon Samples
4.4.1. Fish Feed
Fish feed pellets were homogenised with a grinding mill (Retsch, Haan, Germany), and 2.5 g were weighed into 50-mL polypropylene tubes. After the addition of 20 mL extraction solvent, the samples were vortexed for 1 min, extracted on a horizontal shaker (Edmund Bühler, Tübingen, Germany) with 200 min−1 at room temperature (RT) for 30 min, and centrifuged with 2000× g for 10 min at 4 °C (Beckman Coulter, Brea, CT, USA). The supernatants were transferred into fresh 50-mL tubes and let to settle overnight (ON) at 4 °C. Subsequently, 0.5 mL of the supernatants were centrifuged for 1 min at 20,000× g through 0.22 µm nylon filters (Costar Spin-X; Corning, Inc., Corning, NY, USA) and 40 µL of the filtrates were transferred into LCMS vials. Finally, 10 µL ISTD solution were added to each vial. Samples were store refrigerated until analysis by LC-HRMS/MS.
The composition of the extraction solvent was optimised during method development in spiking experiments. Multi-analyte mixture (50 µL) was added to 2.5 g ground feed, which was then kept under a laminar hood for 30 min, allowing the solvent to evaporate. Extractions were performed either in one step with 20 mL acidic (0.1% formic acid (FA)) MeCN/water mixtures of different compositions (50:50; 60:40; 70:30; or 80:20) or in two steps with acidic MeCN/water (I: 80:20; II: 20:80). Based on the best recovery rates for mycotoxins and phytoestrogens, MeCN/water (70:30; 0.1% FA) was selected for all further experiments.
Three frozen, gutted zebrafish, for each replicate and diet, were thawed and, after separation of the heads, ground to a fine powder with pestle and mortar in liquid nitrogen. The powdered tissue (0.1 g) was weighed and extracted with 1 mL extraction solvent (MeCN/water 70:30; 0.1% FA). The mixture was homogenised by ultra-sonication (Branson, Danbury, CT, USA) for 10 min at 30 °C, centrifuged at 4000× g for 10 min at 4 °C (Thermo Scientific, Waltham, MA, USA), and the supernatant was transferred into fresh 5-mL tubes. An aliquot (0.5 mL) was filtered as described before, and 40 µL of the filtrates were transferred into LCMS vials, mixed with 10 µL of the ISTD solution, and analysed by LC-HRMS/MS.
The recoveries of mycotoxins and phytoestrogens from the zebrafish matrix was investigated during method development by different acidic MeCN/water extraction solvents in spiking experiments with multi-analyte mixture.
The salmon fillets were half-thawed. Tissue pieces of equal size were sampled from four different areas using a steel puncher (0.5 cm in diameter) (Figure 2
). The tissue samples were ground with a pestle and mortar, combined, and 2.5 g were transferred into a 50-mL tube, extracted with 20 mL extraction solvent (MeCN/water 70:30; 0.1% FA) and thoroughly homogenised for 40 s by ultra-turrax (Janke & Kunkel, IKA-Werke, Staufen, Germany). To avoid cross-contamination, the ultra-turrax was washed with water for 20 s between samples from the same fish tank and with water and MeOH for 40 s between samples from different tanks. The samples were vortexed for 30 s and extracted using a horizontal shaker (Edmund Bühler) with 200 min−1
at RT for 1 h. Subsequently, they were centrifuged with 2000× g
for 10 min at 4 °C (Beckman Coulter), and the supernatants were transferred into fresh 50-mL tubes and let to settle overnight at 4 °C. Subsequently, 0.5-mL aliquots were filtered as described before, and 40 µL of the filtrates were transferred into LCMS vials, mixed with 10 µL of the ISTD solution, and analysed by LC-HRMS/MS. The recovery of mycotoxins and phytoestrogens from the salmon matrix was investigated as described for zebrafish.
4.5. Preparation of Matrix-Assisted Standard Calibration Curves
Calibration curves in solvent were prepared by evaporating 200 µL Set A solution with nitrogen and re-dissolving with 200 µL Set B, resulting in a standard solution with 200 µg/L for all 25 analytes included in this study. The standard solution was serially diluted with MeCN/water (50:50) to produce calibrants with 200, 100, 50, 10, 5 and 1 µg/L. For the preparation of the matrix-assisted standard calibration curves, 40 µL aliquots of the calibrants were transferred into LCMS vials and 10 µL ISTD solution was added. They were evaporated with nitrogen at 40 °C and re-dissolved in the same volume of blank matrix extract that had been prepared either from control feed or from zebrafish or salmon in the respective FM-control groups by pooling equal volumes of replicates. The calibration standards were transferred into LCMS vials and analysed by LC-HRMS/MS.
4.6. Development of the Multi-Analyte Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS/MS) Method
Multi-analyte analysis was performed on a Q-Exactive™ Hybrid Quadrupole-Orbitrap HRMS/MS equipped with a heated electrospray ion source (HESI-II) and coupled to a Vanquish UHPLC system (Thermo Scientific). The instrument setup was similar to that described in a previous study [36
]; however, there were several modifications and different analytes were included. The HESI-II interface was operated at 300 °C, alternatively in positive and negative mode during one run. The parameters were adjusted as follows: spray voltage 3.2 and 2.5 kV (positive and negative mode, respectively), capillary temperature 280 °C, sheath gas flow rate 35 L/min, auxiliary gas flow rate 10 L/min, and S-lens RF level 55.
The Q-Exactive HRMS/MS was operated in full scan (FS) mode with the inclusion of targeted fragmentation (data-dependent MS/MS: dd-MS2
). For full scans, the mass ranges were set to m
90–900 and 200–900 in negative and positive mode, respectively. FS data were acquired at a mass resolution of 70,000 full width half-maximum (FWHM) at m
200, while mass resolution was set to 17,500 FWHM at m
200 during dd-MS2. The automated gain control (AGC) target was set to 5 × 105
ions for a maximum injection time (IT) of 250 ms in the FS mode, whereas for dd-MS2
mode the AGC target was 1 × 105
and the IT was 100 ms. The inclusion list for the targeted analysis contained the m
, retention times (RT), and normalised collision energies (NCE) (Table 2
). NCE values were determined by direct infusion of standard solutions in the mobile phase (MeCN/water (50:50), containing 5 mM ammonium acetate and 0.1% acetic acid) by using a syringe pump at a flow rate of 5 μL/min. The quadrupole mass filter was operated with an isolation window of m
3. External mass calibration of the Q-Exactive HRMS/MS was performed every three days over the mass range m
90–2000, in accordance with the manufacturer’s instructions. The identification of the 25 mycotoxins and phytoestrogens included in the multi-analyte method was supported by the determination of specific retention times, fragmentation patterns and accurate masses, which were obtained using a mass accuracy window of ±5 ppm with respect to the theoretical accurate masses (Table S1
). Chromatographic separation was achieved at 30 °C on a 150 × 2.1 mm Kinetex reversed-phase F5 column (2.6 µm, 100Å; Phenomenex, Torrance, CA, USA) with a 0.5 μm × 0.004” ID, HPLC KrudKatcher Ultra Column In-Line filter. The flow rate of the mobile phase was 0.25 mL/min, and the injection volume was 1 μL. Eluent A was water and eluent B was MeOH (both containing 5 mM ammonium acetate and 0.1% acetic acid). Since the solubility of ammonium acetate in MeOH is limited, it was first dissolved in 25 mL water before MeOH was added. The total run time was 43 min, and gradient elution was employed starting at 3% B for 1 min, linearly increasing to 15% B in 15 min, to 79% B in 10 min, and finally, to 100% B in 13 min. After washing the column for 2 min with 100% B, the mobile phase was returned to the initial conditions and the column was eluted isocratically for 2.5 min. The column was regularly washed with 70% methanol to prevent cross-contamination. Calibration standards and samples were analysed in randomised order and intercepted with blank solvent samples to minimise analytical bias from sample positions and to reduce sample-to-sample carry-over.
4.7. Validation of the Multi-Analyte LC-HRMS/MS Method
The method was validated with regard to the guidelines established by the International Organization for Standardization [49
]. The analytical selectivity was determined by the combination of LC retention time and high-resolution mass detection including dd-MS2
product ion qualifying of the different analytes. Measured peak areas were used for quantification. Sensitivity for the different analytes was expressed, by the slope of the respective six-point standard calibration curves (mean of three to four independent experiments) that were calculated by linear regression analysis in both solvent (MeCN 50:50) and the different matrices. The linear range was defined as the concentration interval, in which the regression coefficient R2
was ≥ 0.96. Although internal standard calibrations were used for 15 of the analytes for the compensation of matrix interferences, potential suppression and enhancement (SSE%) of signals from the co-eluting matrix were estimated for all analytes as the ratio of the slope of the matrix-assisted standard calibration curve to the calibration curve in MeCN/water (50:50). If SSE values were above or below 100%, signal enhancement or suppression by the matrix could be assumed.
Considering the negligible noise in the extracted high-resolution mass chromatograms, the limits of detection (LOD) and limits of quantification (LOQ) of the 25 analytes were calculated based on the standard deviation of the y-intercept of the respective calibration curves and their corresponding slopes (m) as
]. The accuracy of the method was assessed by determining recovery by spiking experiments and precision in terms of total within laboratory precision (RSiR
) considering intra- and interday variabilities together [60
]. Furthermore, coefficients of variation (% CV) were determined for all concentration points in the solvent and matrix-assisted standard calibration curves. Recovery rates were calculated for all analytes as the mean of three to four experiments at a spiking level of 25 µg/L. In a few cases, where the matrix-assisted standard curves in feed or fish matrices did not pass through the origin but showed a positive signal on the ordinate due to background noise, this was corrected by virtually moving the curve with parallel shift on the abscissa. The corresponding concentration difference was added to the spike concentration used in the recovery experiments according to Recovery(spike corrected)
= (measured concentration − blank)/(spiked concentration + concentration difference to origin).
Measured results for fish feed and fish study samples were converted from concentrations (µg/L) into content in the respective matrix (µg/kg) by using the factors 0.1 for zebrafish and 0.125 for salmon and feed.
4.8. Data Analysis
The Q-Exactive was calibrated using Xcalibur software, version 2.2 (Thermo Scientific). The molecular formulas and exact masses of the target analytes were calculated using the built-in Qualbrowser of the Xcalibur 2.2 software, which was also applied for signal quantification. Microsoft Excel (Version 2016, Microsoft Corporation, Redmond, WA, USA) was used for basic statistics (e.g., calculation of mean, minimum and maximum values, regression and relative standard deviation).