3.1 Reagents, materials and equipment
The antimicrobial test assay kit was obtained from Charm Sciences Inc., Lawrence, MA; and included items for the detection of beta-lactams (PMSU-050A); sulfonamides (SMMSU-022C), macrolides (EMSU-023A); tetracyclines (TMSU-025); and streptomycins (STMSU-023A). Consumables and equipment used for the tests included: M2 Buffer, zero and positive control standards, MSU extraction buffer, and radioactive labelled tablets; scintillation fluid (Opti-Fluor O, PerkinElmer), Intronic incubator (Charm Sciences Inc), Wallac 1409 scintillator counter, refrigerated centrifuge Sigma 4K15c (Sigma-Aldrich), R2 blender (Robot-Coupe) and a water bath (Julabo MB13). In addition, scintillation vials, AES mix masticator stomacher and IEC Centra CL-3 centrifuge were also used.
3.2 Preparation of standard reference material and stock solutions
The multi antimicrobial concentrate standard (MSU, Charm Sciences Inc.) was prepared fresh on the day of use and diluted with 10 ml of deionized water, shaken well and allowed to stand on ice for 15 min. The reconstituted stock solution contained; penicillin G, 1000 µg/kg; erythromycin, 10,000 µg/kg; sulfamethazine,1000 µg/kg; chlortetracycline, 4,000 µg/kg; and streptomycin, 10,000µg/kg. Other analytical standards were purchased from Sigma Aldrich, Pfizer Inc. US Pharmacopeia Convention and Acros Organics (Supporting information, Table 1a). These standards were similarly diluted with deionized water to make stock solutions of 10,000 µg/kg of the respective antimicrobial and kept below 4°C. The working standards were used for spiking fish samples at different concentration levels ranging from 25 µg/kg to 300 µg/kg.
3.3 Methods
The study carried out a primary validation of the Charm II tests for the detection of antimicrobial residues in aquaculture fish. The Validation was performed according to Commission Decision 2002/657/EC [23] and all methods of analysis used were adopted from the general Charm II protocols [21].
3.4 Fish samples selected for the study
The fish materials used in the study were obtained from dead fish purchased from Melle and Ghensh shops and supermarkets in Belgium. Aquaculture fish species including cat fish (Siluriformes), trout (Oncorhynchus mykiss), salmon, (Salmo salar), seabass (Dicentrarchus labrax), tilapia (Oreochromis niloticus), lingue (Molva molva), dorade (Sparus aurata) and pangasius (Pangasius bocourti), were selected for the study. Fish sample materials were taken by carefully removing the muscle tissue from the side of each fish taking precaution to exclude scales and skin. The fish samples that were not used immediately were stored below -18°C for a maximum of 2 months.
3.5 Sample preparation
The fresh fish sample was weighed in a centrifuge tube and stored at -18°C until further processing. The frozen fish samples were thawed at 4°C overnight and cut into small pieces before blending in a high speed blender. The blended fish material (10 g) was transferred into a polypropylene centrifuge tube and used immediately.
3.5.1 Preparation of control samples
All fish samples were first tested with the different Charm II kits and only used in case no veterinary drug residues was detected. The blank fish materials after blending were spiked with antimicrobial standards of known concentrations and used as control samples for the establishment of the control point counts per minute (cpm). A list of control standards used is shown in the supporting information (Table 1a).
- Extraction of drugs from the fish materials
The MSU extraction buffer (30 ml) was added to blended fish material (10 g) in a polypropylene centrifuge tube. The mixture was homogenized using a stomacher for 2 min and returned to the centrifuge tube. The homogenate was incubated in water bath at 80°C for 30 min, during the determination of streptomycin, macrolides, or beta- lactams; and 45 min, when determining tetracyclines or sulfa drugs. After incubation, the tube was cooled on ice water for 10 min and then centrifuged at 3300 rpm for 10 min, using a refrigerated centrifuge 4K15C (Sigma-Aldrich). The resulting supernatant solution was collected and used for the required tests. The pH of the supernatant was where necessary adjusted to pH 7.5 using reconstituted Charm II kit M2 buffer for low pH, or 0.1M hydrochloric acid for high pH.
3.7 Determination of tetracycline in the fish samples
In the detection of tetracycline, the white tablet from the kit containing the binding reagent (TMSU-025) was introduced into a test tube, and water (300 μl) was added. The contents of the tube were mixed for at least 10 s to ensure breakup of the tablet. The sample extract or control sample (4 ml) was added to the tube, followed by addition of the orange tablet containing the tracer reagent from the kit (TMSU-025). The resultant was mixed for about 10 s and the mixture was incubated at 35 OC for 5 min; and then centrifuged for another 5 min on a IEC Centra CL-3 centrifuge. The supernatant was poured off carefully, avoiding the formed pellet from sliding out of test tube. Deionized water (300 μl) was added to the tube and the contents mixed thoroughly to break up the pellet. After suspension of the pellet in water, the scintillation fluid (3.0 ml) was added and test tube capped. The tube was shaken until the mixture had a uniform cloudy appearance. The glass tube contents were transferred completely into a scintillation vial and the mixture counted using a Wallac liquid scintillation counter for 60 s on the [3H] channel. The results for the sample was compared with the control point counts per minute.
3.8 Determination of macrolides in the fish samples
During the detection of macrolides, the white tablet from the Charm II kit containing the binding reagent (EMSU-023A) was introduced into test tube, and water (300 μl) was added. The contents of the tube were mixed for at least 10 s to ensure breakup of the tablet. The sample extract or control sample (4 ml) was added to the tube and the contents mixed on a vortex for 10 s. The resultant was incubated at 55 OC for 2 min, followed by addition of a green tablet containing the tracer reagent from the kit (EMSU-023A). The resultant was mixed on a vortex for 10 s. The mixture was incubated at 55 OC for 2 min, and then centrifuged for 5 min. The supernatant was poured off carefully and the edge of tube blotted on absorbent paper. Deionized water (300 μl) was added to the tube and the contents mixed thoroughly to break up pellet. After suspension of the pellet in water, the scintillation fluid (3.0 ml) was added and the test tube capped. The contents were mixed on a vortex until the mixture had a uniform cloudy appearance. The content of the glass tube was transferred completely into a scintillation vial and the mixture counted using a Wallac liquid scintillation counter for 60 s on the [14C] channel. The counts per minute (cpm) of the sample was compared with the control point.
3.9 Determination of sulfa drugs in the fish samples
In the detection of sulfa drugs, the white tablet from the Charm II kit containing the binding reagent (SMMSU-022C) was introduced into a test tube, and water (300 μl) added. The contents of the tube were mixed for at least 10 s to ensure breakup of the tablet. The sample extract or control sample (4 ml) was added to the tube, followed by addition of the pink tablet containing the tracer reagent (SMMSU-022C) from the kit. The resultant was mixed by swirling the contents up and down for about 15 s. The mixture was incubated at 65 OC for 3 min, and then centrifuged for another 3 min. The supernatant was poured off carefully, avoiding the formed pellet from sliding out of test tube; and the edge of tube was blotted on absorbent paper. Deionized water (300 μl) was added to the tube and the contents mixed thoroughly to break up the pellet. After suspension of the pellet in water, the scintillation fluid (3.0 ml) was added and test tube capped. The tube was shaken until the mixture had a uniform cloudy appearance. The glass tube contents were transferred completely into a scintillation vial and the mixture counted using a Wallac liquid scintillation counter for 60 s on the [3H] channel. The cpm results of the sample were compared with the control point.
3.10 Determination ofaminoglycoside- streptomycins in the fish samples
In the determination of streptomycins, the white tablet from the kit containing the binding reagent (STMSU-023A) was introduced into a test tube, and water (300 μl) added. The contents of the tube were mixed for at least 10 s to ensure breakup of the tablet. The sample extract or control sample (2 ml) was added to the tube and mixed. This was followed by addition of the green tablet containing the tracer reagent (STMSU-023A). The resultant was mixed by swirling the contents up and down for about 10 s. The mixture was incubated at 35 OC for 2 min, and then centrifuged for another 3 min. The supernatant was poured off carefully and the edge of tube was blotted with absorbent paper. Deionized water (300 μl) was added to the tube and the contents mixed thoroughly. After suspension of the pellet in water, the scintillation fluid (3.0 ml) was added and test tube capped. The tube was shaken until the mixture had a uniform cloudy appearance. The glass tube contents were transferred completely into a scintillation vial and the mixture counted using a Wallac liquid scintillation counter for 60 s on the [3H] channel. The cpm results for the sample were compared with the control point.
3.11 Determination of beta-lactams in the fish samples
In the determination of macrolides, the green tablet from the Charm II kit containing the binding reagent (PMSU-050A) was introduced into test tube, and water (300 μl) was added. The contents of the tube were mixed to ensure breakup of the tablet. The sample extract or control (2 ml) was added to the tube and the contents mixed on a vortex for 10 s. The resultant was incubated at 55 OC for 2 min, followed by addition of a yellow tablet containing the tracer reagent (PMSU-050A) from the kit. The resultant was mixed on a vortex for 10 s. The mixture was incubated at 55 OC for 2 min, and then centrifuged for 5 min at 1750 G. The supernatant was poured off carefully and the edge of tube blotted on absorbent paper. Deionized water (300 μl) was added to the tube and the contents mixed thoroughly to break up the pellet. After suspension of the pellet in water, the scintillation fluid (3.0 ml) was added and test tube capped. The contents were mixed on a vortex until the mixture had a uniform cloudy appearance. The mixture was transferred completely into a scintillation vial and counted using a Wallac liquid scintillation counter for 60 s on the [14C] channel. The cpm of the sample was compared with the control point.
3.12 Method validation
The method validation was done according to the criteria of the European Commission Decision 2002/657/EC [23]. The validation parameters performed included; detection capability (CCβ), repeatability, reproducibility, robustness and cross reaction activity.
3.12.1 Detection capability
The detection capability (CCβ) was examined by spiking blank fish matrices with different antimicrobials including tetracyclines, macrolides, β-lactam, aminoglycosides, and sulfonamides. The spiking concentrations varied around the recommended maximum residue limit (MRL), including 0.05MRL, 0.25MRL, 0.5MRL, 0.75MRL and MRL, for the respective antimicrobial.
3.12.2 Repeatability
The repeatability of the technique was studied by analysis of fish samples spiked with 25 µg/kg of sulfathiazole for the selected species of dorade, salmon and sea bass. The analysis was performed within a short interval, by a single researcher using the same method and scintillation fluid counter equipment.
3.12.3 Reproducibility
The reproducibility of the method was studied by repeat analysis fish samples spiked at the same concentration level of 100 µg/kg with oxytetracycline for the selected species of pangassius, salmon and seabass. The analysis was performed on different days by two different researchers using the same method and a scintillation fluid counter equipment.
3.12.4 Robustness
The robustness of the techniques was tested by deliberately varying the experimental time indicated in the Charm II analytical protocol. Reading of the cpm for the samples spiked with 50 µg/kg amoxicillin was done immediately after the addition of the scintillation fluid and then after 14 hours on the same batch of extracted sample. The samples after the first reading were stored overnight in the fridge at 4 0C, removed and allowed to attain room temperature, and then read the second time after vortexing.
3.12.5 Cross reaction activity
Cross reactivity was investigated by spiking residue-free blank fish samples with high concentrations, above 100 MRLs of the respective antimicrobial belonging to other antimicrobials groups and the samples run on targeted channels to investigate false identification.
3.13 Data Analysis
All data generated was statistically analyzed using one-way analysis of variance (ANOVA) to examine any significant differences between the observed results under different experimental setups.