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(2020) 14:32 Mukota et al. BMC Chemistry https://doi.org/10.1186/s13065-020-00684-4 RESEARCH ARTICLE BMC Chemistry Open Access Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish Aziz Kimera Mukota1, Melanie Flore Kamini Gondam2, Julie Judith Takadong Tsafack2, James Sasanya3, Wim Reybroeck4, Muhammad Ntale5, Steven Allan Nyanzi5 and Emmanuel Tebandeke5* Abstract The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antimicrobials including sulfonamides, β-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation liquid and those read 14 h after addition of the scintillation liquid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%. The Charm II tests were found to be 100% group specific, as none of the antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at maximum residue limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2 MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes. Keywords: Charm II tests, Antimicrobial residues, Rapid screening, Method validation, Aquaculture fish, Maximum residue limit *Correspondence: emmanuel@cns.mak.ac.ug 5 Department of Chemistry, College of Natural Sciences, Makerere University, P.O. Box, 7062, Kampala, Uganda Full list of author information is available at the end of the article Introduction Fish farming is a fast emerging industry that besides creating employment, is a source of good quality animal protein and essential macronutrients in the diet. Fish and fish related products provide income and livelihoods for numerous communities across the world besides playing © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mukota et al. BMC Chemistry (2020) 14:32 a crucial role in assuring sufficient availability of safe and healthy food [1, 2]. The increased demand for fish for the growing international population, especially in the developing world, has continued to deplete the sustainable yields from lakes, rivers, swamps, seas and other natural water bodies. Aquaculture is growing rapidly and is seen as a remedy to address and supplement the dwindling quantities and shortfall in wild catch [3]. However, big numbers of fish in a confined volume of space tend to increase incidences of bacterial infections and other diseases; which greatly affects yield in the aquaculture business. Productivity in aquaculture may be enhanced by use of antimicrobials such as tetracyclines, macrolides, beta-lactams, sulfonamides, and streptomycins, for the prevention and treatment of opportunistic infections in fish [4, 5]. Antimicrobials are used to control ectoparasitic, fungal and bacterial diseases of the body and gills of fish [6–8]. Tetracyclines in particular are frequently employed in aquaculture due to their broad spectrum of activity as well as their low cost, compared to other antibiotics. The tetracyclines are used to combat bacterial hemorrhagic septicemia in catfish as well as diseases caused by Pseudomonas liquefaciens [9]. Currently, there are over 20 tetracyclines available; although, tetracycline, chlortetracycline, oxytetracycline, and doxycycline are the most common ones in veterinary medicine and aquaculture [10, 11]. The aforementioned antibiotics are the only tetracyclines with registration within the European Union (EU) for use as veterinary medicinal products in food producing animals; with established maximum residue limits (MRLs) in different food matrices [12]. Other antimicrobials such as sulfonamides, beta-lactams, macrolides and aminoglycosides also have a wide spectrum of activities against most Gram positive and Gram negative organisms and are used for the prevention and treatment of bacterial infections in livestock and aquaculture. The antimicrobials are typically administered in the water, often as components of fish feed, and are occasionally injected [13, 14]. The extensive use and misuse of antimicrobials in farm animals as growth promoters or as nonspecific means of infection prevention has been reported to lead to accumulation of residues in edible tissue [7, 15, 16]; which may cause allergic and toxic effects in consumers as well as contributing to the development of antimicrobial resistant bacteria [17–20]. In this respect, residues in foodstuffs create public health concerns, consumer perception problems and trade disputes that have enormous negative impacts on the food industry. In order to protect human health, regulatory authorities like the EU, established maximum residue limits (MRLs) for some pharmaceutical compounds in fish and other foodstuffs Page 2 of 15 of animal origin [12]. The safety concerns regarding drug residues in various food products, calls for development and validation of rapid and reliable techniques for detection of these compounds. Such rapid techniques can facilitate fast decision making to minimize technical barriers to trade and also enhance routine monitoring in order to protect consumer health. The Charm II radio receptor assay technique developed by Charm Sciences Inc, is one of the rapid screening techniques for detection of residues of antimicrobials such as beta-lactams, sulfonamides, tetracyclines, chloramphenicol, quinolones, macrolides and aminoglycosides in various food products including fish, meat, eggs, honey, and milk, as well as non-food matrices including water, feed and urine. This technique utilizes a microbial cell with receptor sites that bind the specific antimicrobial drug. The analytical process involves a binder being added to a sample extract along with an amount of 3H or 14C labeled antimicrobial tracer. Any antimicrobial in the sample extract competes for the binding sites with the tracer. The amount of tracer that binds to the receptor sites is measured and compared to a previously determined control point. Therefore, the more radiolabelled antimicrobial detected in the mixture, the lower the concentration of antimicrobial in the sample. The smaller the amount of tracer measured, the greater the drug concentration in the sample [21, 22]. The Charm II technique has very limited validation data for the detection of antimicrobials in different fish species. Thus, this study conducted a primary validation of the Charm II tests in order to generate comprehensive analytical data to prove the validity, applicability and also address potential limitations of the Cham II assays particularly for the screening of antimicrobials in different aquaculture fish species. Materials and methods 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 streptomycin (STMSU-023A). Consumables and equipment used for the tests included: M2 Buffer, zero and positive control standards, MSU extraction buffer, 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. Mukota et al. BMC Chemistry (2020) 14:32 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 A, 10,000 µg/kg; sulfamethazine, 1000 µg/ kg; chlortetracycline, 4000 µg/kg; and streptomycin, 10,000 µg/kg. Other analytical standards were purchased from Sigma Aldrich, Pfizer Inc., US Pharmacopeia Convention and Acros Organics (Additional file 1: Table S1a). These standards were appropriately diluted with deionized water to make working standard solutions 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 to 300 µg/kg. 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]. Fish samples selected for the study The fish materials used in the study were obtained from dead fish purchased from Melle and Ghent fish 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. 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. Preparation of control samples All fish samples were first tested with the different Charm II kits and only used in case no veterinary drug Page 3 of 15 residues was detected. Absence of residual antibiotics in the fish samples was confirmed through evaluation of their counts per minute in comparison with results obtained using the negative control extraction buffers supplied with the Charm II kits. The control buffers are contaminant free and are used to qualify the matrix as negative when a known negative is not available. The tolerance considered for the fish matrix to qualify as negative and selected for use in subsequent test was for counts within ± 20% of the average result obtained with the respective negative control extraction buffer. Samples with counts beyond the tolerance limits were discarded while those meeting the criteria were selected for the study. The selected 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 in the study is shown in Additional file 1: Table S1a. 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 0.1 M hydrochloric acid for high pH. Determination of tetracyclines 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 solution was mixed for about 10 s and the mixture was incubated at 35 °C for 5 min; and then centrifuged for another 5 min on a IEC Centra CL-3 centrifuge. The supernatant was poured off carefully, deterring 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 Mukota et al. BMC Chemistry (2020) 14:32 Page 4 of 15 water, the scintillation liquid (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. 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. Determination of macrolides in the fish samples In the determination of streptomycin, the white tablet from the kit containing the binding reagent (STMSU023A) 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 °C 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 liquid (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. During the detection of macrolides, the white tablet from the Charm II kit containing the binding reagent (EMSU023A) 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 and the contents mixed on a vortex for 10 s. The resultant was incubated at 55 °C 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 °C 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 the formed pellet. After suspension of the pellet in water, the scintillation liquid (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. 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 (SMMSU022C) 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 solution was mixed by swirling the contents up and down for about 15 s. The mixture was incubated at 65 °C for 3 min, and then centrifuged for another 3 min. The supernatant was poured off carefully, deterring 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 liquid (3.0 ml) was added and test tube Determination of aminoglycoside‑streptomycin in the fish samples Determination of β‑lactams in the fish samples In the determination of β-lactams, the green tablet from the Charm II kit containing the binding reagent (PMSU050A) was introduced into a 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 °C 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 liquid (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 Mukota et al. BMC Chemistry (2020) 14:32 liquid scintillation counter for 60 s on the [­14C] channel. The cpm of the sample was compared with the control point. 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. Detection capability The CCβ was examined by spiking blank fish matrices with different antimicrobials including tetracyclines, macrolides, β-lactams, aminoglycosides, and sulfonamides. The number of samples analyzed for each individual antimicrobial agent ranged from 20 to 30 as indicated in Table 3. The spiking concentrations varied around the recommended maximum residue limit (MRL), including 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 MRL and MRL, for the respective antimicrobial. The CCβ was then determined as the lowest concentration of the antimicrobial that could be detected in the sample giving at least 95% positive results. Repeatability The repeatability of the technique was studied by analysis of selected fish samples spiked with different antimicrobials including tetracyclines, macrolides, β-lactams, aminoglycosides, and sulfonamides. The total number of samples analyzed for each individual antimicrobial compound ranged from 20 to 30, and n ≥ 6 for the same fish species. The spiking concentrations varied around the MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 MRL and MRL, for the respective antimicrobial. The analysis was performed within a short interval, by a single researcher using the same method and scintillation fluid counter equipment. Reproducibility The reproducibility of the method was studied by repeat analysis of selected fish samples spiked with different antimicrobials including tetracyclines, macrolides, β-lactams, aminoglycosides, and sulfonamides. The number of samples analyzed for each individual antimicrobial ranged from 20 to 30, with n ≥ 6 for the same fish species. The spiking concentrations varied around the recommended MRL, including 0.05 MRL, 0.25 MRL, 0.5 MRL, 0.75 MRL and MRL, for the respective antimicrobial. The analysis was performed on different days by two different Page 5 of 15 researchers using the same method and a scintillation fluid counter equipment. Robustness The robustness of the techniques was tested by deliberately varying the experimental time indicated in the Charm II analytical protocol. This was intended to study the effect of variation in reading time interval for a large batch of processed samples. Reading of the cpm for the samples spiked with 50 µg/kg amoxicillin was done immediately after the addition of the scintillation liquid and then after 14 h on the same batch of extracted sample. The samples after the first reading were stored overnight in the fridge at 4 °C, removed and allowed to attain room temperature, and then read the second time after vortexing. Cross reaction activity Cross reactivity was investigated by spiking residue-free blank fish samples with high concentrations (up to 10 MRL) of the respective antimicrobial belonging to other antimicrobial groups and the samples run on targeted channels to investigate false identification. Data Analysis All data generated was statistically analyzed using oneway analysis of variance (ANOVA) to examine any significant differences between the observed results under different experimental setups. Results and discussion Counts per minute for blank samples The blank samples used in the study were those fish tissue matrices which were carried through the complete analytical procedure, and no antimicrobial residues were detected in them using the respective Charm II assay kits [21]. The blank fish samples to which the binder and tracer had been added but without addition of an antimicrobial agent were extracted with the different kits and read on the respective channels. The results of the cpm for the blank fish samples are summarized in Table 1. From Table 1, the cpm for tilapia, trout, salmon, pangasius, seabass, dorate, catfish, and lingue fish species were statistically evaluated using ANOVA and it was found that the overall F-calculated (0.22) was less than F-critical (2.5), which implied that there was no significant difference between results for the blank fish samples of the aforementioned species when using antimicrobial test kits for β-lactams, tetracyclines, macrolides and streptomycins. However significant difference in cpm values was observed with the sulfonamides extraction kit while testing catfish, lingue and pangasius. The cpm for Mukota et al. BMC Chemistry (2020) 14:32 Page 6 of 15 Table 1 Blank counts per minute for the different fish species obtained using the Charm II technique Scintillation counter results (cpm) Charm II test β-lactams kit Fish species Mean SD Sulfonamides kit Tetracyclines kit Macrolides kit Streptomycins kit Mean Mean Mean Mean SD SD SD SD Tilapia 2704 0.9 2596 736 3027 0.7 2448 191.1 5103 346 Trout 2506 192.0 2332 1184 2830 260 2799 87.1 4799 259.9 Salmon 2571 207.4 2472 541.2 2939 165.0 2110 117.2 3085 133.4 Pangasius 2469 195.7 5625 1254 2931 221.4 2893 110 4796 437.7 Seabass 2432 232.1 2144 672.1 2971 252.2 2700 153.6 4805 594.7 Dorate 2512 171.1 1977 621.4 2864 93.4 2803 167.3 4967 485.8 Catfish 2493 312.7 5872 774.3 4454 650.1 Lingue cpm counts per minute, SD standard deviation these species were almost double those of the other types of fish and their F-calculated (15.1) was greater than F-critical (2.4). The big variation in cpm for the catfish, lingue and pangasius fish species as compared to the rest could be attributed to the high fish fat content extracted by the sulfonamide kit protocol. In this respect, the three fish species (catfish, lingue and pangasius) need to be handled separately when calculating control points to minimize chances of getting false negative or false positive results. For the rest of the fish species, the blank cpm results were used to derive the respective control points for the different residues. Evaluation of the Control Points for the different drug residues The control point (CP) of a sample is the cut-off point between a negative or positive result. Any antimicrobial agent present in the sample extract competes for the binding sites with the tracer, thus, the greater the cpm measured, the lower the antimicrobial drug concentration in the sample and vice versa. Samples with high counts are considered negative (tracer antimicrobials are largely bound to the binder) while those with low counts are considered positive (tracer antimicrobials are largely free in solution). The CP for the different antimicrobials were determined independently; and with the exception of tetracyclines, the MRL value for each drug was spiked to the respective blank fish sample. In order to cater for the deviations in the different fish matrices, a percentage tolerance was added to or subtracted from the obtained average cpm value of the spiked blank fish sample. The CP evaluation was performed according to the Charm II protocol, and the percentages added to the mean value of spiked samples at detection capability or subtracted from the mean value of blanks serve to minimise occurrence of false positive or negative readings [21, 24, 25]. In this respect, the CP for the β-lactams was evaluated from averaging the results of 6 negative samples spiked with penicillin G at 25 µg/kg (0.5 MRL) and adding 20% of the obtained average cpm value. Whereas, for the sulfonamides, the CP was evaluated by averaging results of negative samples spiked at 50 µg/kg with sulfamethazine and adding 30% of the average obtained cpm value. A control point of 1530 was calculated for the β-lactams. On the other hand, the CP for tetracyclines was calculated by averaging cpm results of negative control standards provided in the tetracyclines test kit and subtracting 40% of the obtained average cpm value (Table 2). For macrolides, the CP was derived from averaging the results of 6 negative samples spiked with erythromycin A at 100 µg/kg (0.5 MRL) and adding 20% of the obtained average cpm value. Using a similar approach, the CP for streptomycin was derived from averaging results of negative samples spiked at 25 µg/kg with streptomycin and adding 30% of the average obtained cpm value. During the analysis of antimicrobial residues in fish samples, results less than or equal to each respective CP were interpreted as positive while those greater than the CP, as negative. Blank sample readings below the set CP were considered false positive. The results in Table 2, show that the false positive rate was 0% for tetracyclines, β-lactams and sulfonamides; 3.6% for macrolides, and 5% for streptomycin; this proved the validity of the obtained data since it met the acceptance criteria of being within 5%. A comparison of the CP for the different antimicrobials obtained using the Charm II assay with the corresponding cut-off points (Fm) and technical threshold (T) values, calculated following Annex II of the EU guideline for Community Reference Laboratories Residues for validation of screening methods [26], is shown in Table 3. According to the EU guideline, the cut off factor (Fm), refers to the response or signal from a screening Mukota et al. BMC Chemistry (2020) 14:32 Page 7 of 15 Table 2 Control points for the different antimicrobials in blank fish samples Antimicrobial family Spiked samples Blank samples Level of analyte Mean cpm spiking (µg/kg) of spiked samples Allowance for matrix effect Control point cpm Mean blank cpm Range of blank No. of false cpm readings positives/no. of samples False positive rate (%) β-lactams 25 µg/kg penicil- 1275 lin G 1530 2502 2160–2907 0/30 0 Sulfonamides 50 µg/kg sulfamethazine 1096 Spiked cpm + 20% 1424 3162 1431–6995 0/30 0 Tetracyclines 0 µg/kg tetracycline 2524 Spiked cpm + 30% 1514 2524 2451–3269 0/30 0 Macrolides 100 µg/kg eryth- 1765 romycin A Blank cpm − 40% 2118 2587 1906–2952 1/28 3.6 Streptomycin 25 µg/kg streptomycin Spiked cpm + 20% Spiked cpm + 30% 3346 4605 2942–5488 1/20 5.0 2574 Number of samples used per parameter Ns ≥ 20 Table 3 Comparison of control points by Charm II protocol, cut-off points and technical threshold values calculated according to the EU guideline [26, 27] Antimicrobial family Tetracyclines Macrolides β-Lactams Compound Spiked concentration (μg/kg) B average response of blank samples Calculated T value as per EU guideline T = B − 1.64 * SDb [26, 27] Calculated Fm Calculated value as per EU control point CP guideline as per Charm II assay Fm = M + 1.64 * SDs [26, 27] Tetracycline 25 2958 2776 816 Chlortetracycline 25 3050 2780 1417 Oxytetracycline 100 2891 2579 1427 Erythromycin A 100 2814 2564 1904 Tilmicosin 100 2486 2164 2002 Tylosin A 100 2512 2115 1740 Penicillin G 25 2523 2176 1438 Ampicillin 50 2455 2042 1341 50 2702 2536 1487 Oxacillin Amoxicillin 300 2398 2171 1478 Dicloxacillin 300 2524 2384 1489 Cloxacillin 300 2500 2368 1413 1514 2118 1530 Aminoglycosides Streptomycin 25 4822 3867 2592 3346 Sulfonamides Sulfamethazine 25 2593 1428 1379 1424 Sulfadimethoxine 25 2266 1129 972 Sulfamerazine 25 2210 1095 930 Sulfadiazine 25 2297 1296 1184 Sulfathiazole 25 2266 1643 1485 Cut-off factor (Fm) = M + 1.64 * SDs; Technical threshold (T) = B − 1.64 * SDb; M, mean response of spiked samples; B, mean response of blank samples; SDs, standard deviation of the spiked sample readings; SDb, standard deviation of blank readings test which indicates that a sample contains an analyte at or above the screening target concentration [26], while the Charm II protocol CP is the cut-off point between a negative or positive result [21]. On the other hand, the technical threshold (T), refers to the limit for positivity [26]. For the Charm II technique the readings of the blank samples are greater than those for spiked samples, because the responses are inversely proportional to concentrations of the antimicrobials. In this respect, the assay is considered valid only when Fm < T and the CCβ is validated when Fm < B. Accordingly, the number of spiked samples with mean responses below Mukota et al. BMC Chemistry (2020) 14:32 Page 8 of 15 the cut-off level (deemed positive) is identified and the false positive rate determined. If T < Fm < B, the falsepositive rate is greater than 5%. In the case Fm < T the false positive rate is below 5%. If more than 5% of the spiked samples at the screening target concentration gave a response greater than the cut-off level (deemed false negative), the concentration chosen for the spiking is considered too low for validation and a higher concentration is tested [26, 27]. From the results presented in Table 3, the Fm values obtained using the EU guideline and the respective calculated CP according to the Charm II protocol are comparable. For all antimicrobials, the respective CCβ, presented in Table 3 are valid since in all cases the Fm < B. In addition, for all antibiotics involved in the study the Fm < T, which implies that the Charm II techniques is validated for the detection of antimicrobial residues in fish matrix, with a false positive rate of less than 5%. In comparison with the Charm II protocol, it should be noted that in all cases the CP value for a particular family of antibiotics is slightly higher the corresponding Fm readings, with the exception of sulfathiazole. This suggests that there will be less incidences of false negative readings in the detection of the different antimicrobial compounds in fish matrix based on CP values, although this may increase incidences of false positive readings. Detection capability for the different antimicrobials in selected fish species The CCβ is the lowest concentration of the analyte that could be detected in the sample giving at least 95% positive results. In the CCβ studies, blank negative fish tissue samples were spiked with different antimicrobials at various concentrations. Spiked samples that exhibited readings above the set CP value, were interpreted as false negatives. In case more than 5% of the spiked samples at a target concentration gave false negative readings, the concerned concentration was deemed too low for validation and a higher concentration was considered. A summary of the CCβ for the different drugs involved in the study is presented in Table 4. Results show that the Charm II technique can detect tetracycline and chlortetracycline spiked at 25 µg/kg (0.25 MRL) and oxytetracycline at 100 µg/kg (MRL) for the different fish species (cat fish, trout, salmon, seabass, tilapia, lingue, dorade, and pangasius) with 100% detection. However, the batch of the multiantimicrobial standard, provided in the Charm II kit was not sensitive enough for chlortetracycline to be detected at 100 µg/kg (MRL) level. This could be attributed to the deterioration of the chlortetracycline in the standard due to poor handling, probably during transportation. In this respect, a Sigma Aldrich standard was used and chlortetracycline detected at a concentration as low as 0.25 MRL. Interestingly, it was observed that the technique is also capable of detecting other antimicrobials belonging to the Table 4 Detection capability for the selected antimicrobials Family Tetracyclines (CP = 1514 cpm) Macrolides (CP = 2118 cpm) β-Lactams (CP = 1530 cpm) Compound Counter results (cpm) Mean Min 650 Max % Detection of each antimicrobial Tetracycline 100 25 20 20 724 Chlortetracycline 100 25 21 21 1200 942 1421 100 Oxytetracycline 100 100 31 31 1269 1074 1460 100 Erythromycin A 200 100 30 30 1669 954 1955 100 Tilmicosin 50 100 21 21 1565 1221 2078 100 Tylosin A 825 100 100 100 21 21 1440 1103 1742 100 Penicillin G 50 25 22 22 1175 921 1421 100 Ampicillin 50 50 21 21 1055 837 1451 100 Amoxicillin 50 50 22 22 1132 908 1409 100 Oxacillin 300 300 24 24 1286 1082 1459 100 Dicloxacillin 300 300 22 21 1186 827 1839 95.5 Cloxacillin 300 300 20 19 1143 681 1547 95.0 500 25 22 22 2424 1642 3074 100 813 1831 96.6 Aminoglycosides (CP = 3346 cpm) Streptomycin Sulfonamides (CP = 1424 cpm) EU-MRL CCβ (μg/kg) No of samples No (μg/kg) of positive samples Sulfamethazine 100 25 29 28 1240 Sulfadimethoxine 100 25 20 20 968 737 923 100 Sulfamerazine 100 25 21 21 842 716 960 100 Sulfadiazine 100 25 20 20 948 735 1361 100 Sulfathiazole 100 25 20 19 989 698 1782 95.0 Mukota et al. BMC Chemistry (2020) 14:32 tetracycline family (tetracycline, oxytetracycline) and not limited to the chlortetracycline provided for in the Charm II test kit. The sulfa drugs including, sulfadimethoxine, sulfadiazine, sulfamerazine were detected at 25 µg/kg (0.25 MRL) for the different fish species (trout, salmon, seabass, tilapia and dorade) at 100% detection; sulfamethazine was detected at 25 µg/kg (0.25 MRL) at 96.6% detection (3.4% false negatives), and sulfathiazole was detected at 25 µg/ kg (0.25 MRL) at 95.0% detection (5.0% false negatives). The results also show that the technique can detect other antimicrobials belonging to the sulfonamides group (sulfamethazine, sulfadimethoxine, sulfamerazine, sulfadiazine and sulfathiazole), which are not included in the MSU multi-antimicrobial standard mix, provided in the Charm II test kit. For the macrolides; erythromycin A, tilmicosin, and tylosin A were detected at 100 µg/kg, for the different fish species (cat fish, trout, salmon, seabass, tilapia, lingue, dorade, and pangasius) with 100% detection. Whereas, results for the β-lactams show that Page 9 of 15 penicillin G, ampicillin, amoxicillin, oxacillin, dicloxacillin and cloxacillin were detected at 25 µg/kg, 50 µg/kg, 50 µg/kg, 300 µg/kg, 300 µg/kg and 300 µg/kg respectively, for all fish species involved in the study. Thus, penicillin G is detected at 0.5 MRL, whereas ampicillin, amoxicillin, oxacillin, dicloxacillin and cloxacillin are all detected at their respective MRL. However, 4.5 and 5% of the results for dicloxacillin and cloxacillin respectively, were false negatives (Table 4). Further more, the Charm II technique is capable of detecting streptomycin at 25 µg/kg (0.05 MRL) for all fish species involved in the study at 100% detection. A comparison of the CCβ and MRL for the different antimicrobials is shown in Fig. 1. The results show that, CCβ for the validated antimicrobials were below or equal to the MRL for all drug residues in this study, with the exception of tilmicosin which was detected at 2 MRL. Most of the drug residues exhibited CCβ in the range 0.05 MRL to 0.5 MRL, with 100% detection. Moreover, the incidences of false negative results observed for all Fig. 1 The detection capabilities and maximum residue limits for the different antimicrobials Mukota et al. BMC Chemistry (2020) 14:32 Page 10 of 15 antimicrobials involved in the study were within the 5% requirement of the EU decision 2002/657, and therefore the validation results are satisfactory. The Charm II technique exhibited better CCβ for tetracyclines at 25 ppb (0.25 MRL) compared to other rapid screening techniques such as the ELISA kit of R-Biopharm for screening tetracycline antibiotic residues in the muscle of chicken, beef, and shrimp, which detected the same at 100 ppb (MRL) [27]. In another study, results of the revolutionary Biochip Array Technology showed better detectability for tylosin A and oxytetracycline at 0.10 and 0.5 of the respective MRL in samples [28]. The limits of detection (LOD) obtained using the Charm Test II assays, and the limits of quantitation (LOQ) for selected literature chemical methods are presented in Additional file 1: Table S1b. The LOD results for fish matrix obtained in this validation using the Charm II kits, are comparable to the manufacturer’s claims for the tissue matrix. However, some antimicrobial compounds could be detected in fish tissue at levels lower than the manufacturer’s claim (Additional file 1: Table S1b). The LOD results were also compared with the LC–MS/MS analysis of sulfadimethoxine [29], HPLC–MS/MS analyses of tetracyclines, chlortetracycline, oxytetracycline, sulfadimethoxine, sulfamerazine and sulfadiazine [30]; and LC–ESI–MS/MS analyses of a range of tetracyclines, β-lactams, aminoglycosides and sulfonamides [31]. Generally, the rigorous chemical techniques, as expected, offer lower LOQ values compared to the respective LOD obtained with the Charm II tests. Nonetheless, the Charm II test demonstrated ability to detect a wider range of antimicrobials belonging to different classes including tetracyclines, macrolides, β-lactams, aminoglycosides and sulfonamides at MRL or lower levels, but it requires use of different antimicrobial test kits in parallel; unlike some of the chemical techniques that can simultaneously detect numerous antimicrobials [30, 31]. Repeatability of the method Repeatability analysis was performed using the same Charm II protocol for a specific antimicrobial on different fish species performed by the same researcher. The analysis was evaluated by means of the intra-day coefficient of variations and the results are presented in Table 5. Results of the repeatability study characterized by the relative standard deviation (%RSD) were satisfactory with a precision of less than 12% for the different antimicrobial drugs including tetracyclines, macrolides, β-lactamss, aminoglycosides, and sulfonamides; spiked in blank fish samples at MRL, 0.5 MRL or concentration less than 0.5 MRL and analysed under repeatability conditions (n ≥ 6). The coefficient of variation expressed as percentage relative standard deviation (­RSDr) ranged from 7.8 to 9.8% for tetracyclines (chlortetracycline and oxytetracycline), 2.8 to 6.3% for macrolides (erythromycin A), 6.9 to 9.7% for β-lactams (penicillin G), 10.01 to 11.5% for aminoglycosides (streptomycin); and for sulfonamides (sulfathiazole) it was from 1.2 to 8.7%. These results, ably demonstrate the protocol’s repeatability when used for testing different antimicrobial residues in fish tissue matrix. A closer look at results obtained under repeatability conditions in the analysis of different fish samples spiked with 25 µg/kg sulfathiazole is presented in Table 6. The results showed that there was no significant difference in cpm readings for the same fish species, and amongst different fish species including dorade, salmon and seabass, spiked with sulfathiazole at the same concentration level (ANOVA, overall F-critical 3.35 > F-calculated 1.99) with RSD < 10%. Similar observations were made for the other Table 5 Repeatability study at MRL, 0.5 MRL or concentration < 0.5 MRL Family Compound Spiking concentration (µg/kg) Mean cpm SDr Tetracyclines Chlortetracycline 25 µg/kg (0.25 MRL) 1207.0 118.2 Oxytetracycline 100 µg/kg (MRL) 1270.06 Macrolides Erythromycin A 100 µg/kg (0.5 MRL) 1762.4 110.4 6.3 200 µg/kg (MRL) 1478.1 41.2 2.8 β-Lactams Penicillin G 25 µg/kg (0.5 MRL) 1285.6 89.3 6.9 648.5 62.7 9.7 50 µg/kg (MRL) 98.41 RSDr (%) 9.8 7.75 Aminoglycosides Streptomycin 250 µg/kg (0.5 MRL) 1125.8 112.7 10.01 500 µg/kg (MRL) 1110.5 127.2 11.5 Sulfonamides Sulfathiazole 25 µg/kg (0.25 MRL) 922.2 80.1 8.7 100 µg/kg (MRL) 706.5 8.6 1.2 SDr, standard deviation under repeatability conditions, RSDr, relative standard deviation under repeatability conditions, Mean cpm average of counts per minute under reproducibility conditions