Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 1, 48–53, http://dx.doi.org/10.1080/19440049.2014.990995
Mini-column screening assay for tetracyclines in chicken Ali R. Shalaby* Food Science and Technology Department, National Research Centre, Dokki, Cairo, Egypt (Received 17 August 2014; accepted 17 November 2014) A simple, rapid, reliable and economical mini-column (MC) method for the detection of tetracyclines (TCs) residues in chicken meat was developed. The method employs a commonly available Pasteur pipette which is tightly packed with silica gel and anhydrous sodium sulfate. Clean-up and detection of illegal levels can be achieved on the same column. Viewing the developed MC under an ultraviolet lamp revealed that TCs can be detected as a compact golden yellow fluorescent band at the junction between the anhydrous sodium sulfate and silica gel layers. Comparing the yellow band of control extracts with those fortified (100 ng ml−1) showed no overlap between analyte and impurities. The limit of detection (LOD) of the MC assay was 1 ng, indicating that the chicken sample containing 10 µg TCs kg−1 sample could be easily detected. Moreover, the intensity of the yellow band increased whenever TCs levels in the extract increased. Evaluation utility of the method with blind samples as controls or samples fortified with total TCs at various levels indicated that the total blank and spiked samples at levels equal or below the permissible limits were assessed as accepted. The method can provide an alternative to microbial screening assays and could be used as an effective pre-screening technique in public health laboratories. Keywords: chicken; tetracyclines; mini-column; chromatography
Introduction Broad-spectrum antibiotics are a class of drugs widely used in veterinary medicine to treat and control a variety of bacterial infections and as animal growth promoters (De Ruyck et al. 1999). Tetracyclines (TCs), which are licensed for use in a wide range of livestock species, are the main antibiotics used in poultry production because they are the cheapest class of antibiotic available and their cost in real terms is declining due to improved manufacturing technology (Chopra & Roberts 2001). The widespread misuse of TCs residues in poultry production results in the presence of their residues in edible tissues at levels above the MRL in various countries such as Mexico (Vázquez-Moreno et al. 1990), Kenya (Omija et al. 1994), Belgium (De Wasch et al. 1998), Saudi Arabia (Al-Ghamdi et al. 1999), Iran (Salehzadeh et al. 2006), Pakistan (Shahid et al. 2007), Bulgaria (Pavlov et al. 2008) and Egypt (Salama et al. 2011). Such high levels led to the presence of TCs residues in edible tissues. Cooking procedures do not completely eliminate such residues and so potentially create a health hazard for consumers if the MRL is exceeded (Abu-Raiia et al. 2013). In order to guarantee the safety of animal-derived foods, the joint FAO/WHO Expert Committee on Food Additives (2004) recommended limits of 200 and 600 µg kg−1 for chicken tissues and liver, respectively, expressed as the sum of the TCs group. The corresponding
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
limits set by the European Union (Council Regulation No. 2377/90) were 100 and 300 µg kg−1. Public health concerns associated with residues and contaminants have been the impetus for determining such hazards in the food supply. Most of analytical methods for TCs residues are highly sensitive and give good precision and accuracy. However, they are costly to acquire, run and maintain and require relatively sophisticated laboratory facilities and specialist operators. So, there is an expanding need for a simple, cost-effective and rapid screening technique as an easy tool that can be used in the field for quick inspection. Questionable samples (higher than a certain value) can be selected to be sent for confirmatory analysis by a more sensitive and specific method. The most common approach is the use of microbial assays as pre-screening method (Mitema & Omija 1992; Kurittu et al. 2000; Tsai & Kondo 2001; Kabir et al. 2004; Cantwell & O’Keeffe, 2006; Pikkemaat 2009; Pikkemaat et al. 2009; Idowu et al. 2010), despite their disadvantages, like poor sensitivity and selectivity, false-positive results and long analytical time. Therefore, a quick and reliable method that overcomes most of these disadvantages should be developed. The MC technique was previously successful at determining aflatoxins in Copra meal (Arim & Aguinaldo 1999), the detection of sterigmatocystin (Ramakrishna & Bhat 1990) and deoxynivalenol (Ramakrishna et al. 1989) in agricultural
Food Additives & Contaminants: Part A commodities, and for rapid detection of argemone oil in rapeseed/mustard oils (Sashidhar et al. 1990). It seems that the MC technique is suitable as an alternative to the microbial assay for field tests at least because it has the advantages of a short time, low cost, is very simple, high speed and can be applied in situ as a screening alert method for TCs residues in chicken food samples before being accepted for human consumption. The aim of this work is to establish a rapid, simple and economic MC assay for total TCs residues in chicken meat.
Materials and methods Chemicals and reagents Standards of oxytetracycline (OTC), tetracycline (TTC), chlortetracycline (CTC) and doxycycline (DOC) were supplied by Sigma (St. Louis, MO, USA). Benzene (BNZ), acetone, chloroform, methanol (MeOH) and acetonitrile (ACN) were of HPLC grade. Citric acid monohydrate, ethylenediaminetetraacetic acid disodium salt (Na2EDTA) and anhydrous sodium sulfate were of analytical grade. Water was purified before use in a Milli-Q system (Millipore, Bedford, MA, USA). Silica gel (63–200 mesh) was from Merck Chemicals (Merck, Poole, Dorset, UK; activated at 110°C for 1 h and cooled in a dessicator). Glass wool and a disposable Pastier pipette were also used.
Samples Three individual chicken samples were purchased from each of three different suppliers. Each chicken was individually filleted and homogenised in a food processor, and the tissue was then stored at –20°C until use. Samples were analysed for TCs residues by an HPLC method according to Shalaby et al. (2011) to ensure that they were free of these residues.
Standard solution preparation A stock standard solution of a TCs mixture was prepared by dissolving 10 mg (2.5 mg each of OTC, TTC, CTC and DOC) in 10 ml of methanol to obtain a final concentration of 1 mg ml−1. The stock standard solution was put in amber glass to prevent photo-degradation and stored at −20°C; it is stable in this condition for at least 4 weeks. Stock solutions were diluted with methanol to give a series of working standard solutions which were prepared weekly. Other series of working standard solution were prepared using ACN.
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spiking. Spiked samples were allowed to stand in the dark inside a refrigerator at 4°C for 1 h before analysis. TCs extraction Four extraction methods were trialled as follows: ● First method: 2 ml of an EDTA solution (5%, pH 9) and 8 ml of MeOH were added to the tube containing spiked sample (1 g), the mixture was vigorously shaken for 1 min and then filtered on Whatman No. 4 to provide a filtrate suitable for MC application. ● Second method: 2 ml of an EDTA solution (5%, pH 9) and 10 ml of MeOH were added to the tube containing spiked sample (1 g), the mixture was vigorously shaken for 1 min. Thereafter, about 4.0 g anhydrous sodium sulfate were added, vigorously shaken and filtered on Whatman No. 4 containing anhydrous sodium sulfate to provide a filtrate suitable for MC application. ● Third method: as described for the first method, but the MeOH was replaced with ACN and EDTA was replaced with citric acid (0.3 N). ● Fourth method: as described for the second method, but the MeOH was replaced with ACN and EDTA was replaced with citric acid (0.3 N). TCs-negative samples were extracted by the four extraction methods. The extracts obtained were spiked with TCs to provide the levels of 10 and 20 ng ml−1. Mini-column preparation Commercially available Pasteur pipettes (about 10 × 0.5 cm) were adapted for use by inserting a small glass wool plug in the restricted end and packed with silica gel (about 20 mm height). EDTA and sodium sulfate anhydrous layers were added to the column and tested for suitable amounts of chemicals and their arrangement on the column which help to obtain a clear band separated from the impurities. Each layer was tightly packed by frequent tapping of the column while adding the various chemicals. Several trials were conducted to select the most suitable solvent system for visualising the TCs, where various composites of two or more solvents were tested. The most effective solvent system that makes the silica gel transparent under an ultraviolet (UV) lamp (where the background will be dark and the TCs band will be more distinct) will be used as a “clarifier”. The suitable volume of the “clarifier” was determined to be 1 ml.
Spiking samples Homogenised chicken sample (1 ± 0.01 g) was weighed into a 25-ml culture tube and spiked by the addition of a TCs standard solution to provide the desired level of
Preparation of spiked blind sample The ability of the method to detect the rejected (TCs > 100 ng g−1) and the accepted (TCs ≤ 100 ng g−1) samples,
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according to European Union legal limits, was tested in a blind system. A template was prepared by independent colleagues to fortify 60 chicken extracts at levels above or below 100 ng g−1. The fortified extracts were subjected to the MC assay. The template was then randomly assigned a number from 1 to 60, and the blind assay was performed in this order, using 1 ml extract. Further, a 1 ml extract containing 10 ng TCs (that match the European Union recommendation) was assayed to be used as a reference MC as a guide for reviewers. The results were then reviewed by the independent colleagues with respect to the different levels of TCs. A similar experiment was performed using fortification levels above or below 200 ng g−1, to match the US recommendation. The reviewer reference MC was obtained using 1 ml extract containing 20 ng TCs (to match the US recommendation).
TCs
Figure 1. More distinct tetracyclines (TCs) residues’ fluorescent band.
yellow fluorescence band. The small diameter (about 4 mm i.d.) of the MC along with using the “clarifier” led visually to the easy detection of the yellow band (Figure 1).
Results and discussion Optimisation the MC assay Detection and clarity of TCs TCs on TLC gave a characteristic golden yellow fluorescence under a UV lamp at 365 nm. This property was used to detect TCs on silica gel MC. At the beginning, MC packed with silica gel and 1 ml of MeOH standard solutions (10 ng ml−1) was loaded into the MC, eluted by gravity and viewed under UV lamp. Silica gel layer became cloudy and detection of the TCs’ yellow band was difficult due to blurring. Using a standard solution prepared by ACN, a similar observation was obtained. So, several trials were conducted to select the most suitable solvent system for visualising the TCs’ band, where various composites of two or more solvents were tested. Five systems (Table 1) gave a bright and sharp yellow band on the silica MC. The visually sharpest band was obtained with the composite number 4 (benzene: MeOH:ACN, 6:3:1). So, composite number 4 was used as a “clarifier”. The suitable volume of the “clarifier” was determined to be 1 ml and did not cause any dispersion of the TCs’ band. So, 1 ml of the “clarifier” was chosen to visualise the TCs band. It was found that TCs were selectively adsorbed at the top of the silica gel, giving a single compact and intense
MC composition A TC standard solution when applied into the column gave a compact yellow fluorescent band at the surface of silica gel (Figure 2A). When the negative extract was applied to the MC, a white fluorescent band (impurities) was observed at the surface of the silica gel (Figure 2B). The extracts of TCs when applied to the MC failed to separate from confounding fluorescent impurities. Some trials using anhydrous sodium sulfate in the extracted solution and on the MC were tried in order to try to separate the
A
B
Table 1. Effective elution systems tested. Number 1 2 3 4 5
Composition Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile
Ratio 5: 5: 6: 6: 8:
5: 5: 3: 2: 4:
2 1 1 1 1
Figure 2.
(A) TCs residues’ band and (B) impurities band.
Food Additives & Contaminants: Part A
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Impurities
A
B
TCs
Figure 3.
Dispersion of the TCs residues’ fluorescent band.
Figure 5. A total of 1 ml of spiked extract at 100 ng g−1 loaded into the mini-column: (A) MeOH extract and (B) ACN extract. Na2SO4 anhydrous Compact TCs band
second extract gave a band greater than that given by the fourth method (Figure 5). So the second extraction method was chosen as the extraction step for the MC assay.
Silica gel
Figure 4.
Compact TCs residues’ fluorescent band.
TCs from non-TCs fluorescing impurities. No combination of solvents, eluents or clarifiers enables the certain identification of the TCs on the unmodified MC, due to the dispersion of TCs throughout the MC (Figure 3). However, when a 10 mm capping of anhydrous sodium sulfate was layered on top of the silica MC, this led to unequivocal separation of fluorescing impurities, which were retained on the sodium sulfate layer, and the TCs, which were retained as a sharp band on top of the silica. The second extraction system gave the sharpest band (Figure 4), so this was incorporated into the method.
Volume of extract A total of 1 ml of this extract was found to be sufficient to give effective separation and visualisation of the TCs as a compact yellow fluorescent band while minimising band dispersion.
Suggested MC assay Sample extraction A total of 1 g of homogenised chicken sample was placed into a 25-ml culture tube. A total of 2 ml of EDTA solution (5%, pH 9) and 10 ml of MeOH were added into the sample and mixture shaken vigorously for 1 min. Thereafter, about 4.0 g anhydrous sodium sulfate were added, shaken vigorously and filtered on Whatman number 4 containing anhydrous sodium sulfate. The obtained filtrate was suitable for MC application.
Sample extraction
MC preparation
Both the second and the fourth extraction methods were evaluated. Both extractions were clear and suitable for the application into the MC. Therefore, 1 ml of each spiked extract (10 ng ml−1) was separately loaded into the MCs, drained by gravity and viewed under a UV lamp. The
A small glass wool plug was inserted into the restricted end of the commercially available Pastier pipette (about 10 × 0.4 cm) and packed with anhydrous sodium sulfate (about 2 mm), silica gel (about 20 mm) and anhydrous sodium sulfate (about 10 mm). Each layer should be
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tightly packed by frequent tapping of the column while adding the various chemicals.
Table 2. Evaluation of the blind samples according to European Commission (2010) recommendation (100 ng g−1). Sample tested
Development and detection of TCs A total of 1 ml sample extract was loaded into the column and allowed to drain by gravity. A total of 1 ml of the “clarifier” solvent was added into the column and allowed to drain by gravity. The column is viewed under a UV source for the presence of a yellow fluorescence band of TCs. The fluorescent band will appear at the junction of the anhydrous sodium sulfate and silica gel layers.
10 10 10 10 10 10
Spiked levels
TCs applied
Evaluation
0 ng g−1 50 ng g−1 80 ng g−1 90 ng g−1 100 ng g−1 110 ng g−1
0 ng 5 ng 8 ng 9 ng 10 ng 11 ng
Accepted Accepted Accepted Accepted Accepted Rejected
Table 3. Evaluation of the blind samples according to FAO/ WHO (2004) recommendation (200 ng g−1). Samples tested
Spiked levels
Suitability of the assay Limits of detection (LODs) The levels of TCs spiked in the present study were based on the permissible limits of both the United States and European Union, i.e. 200 and 100 ng g−1, respectively. The control samples showed no yellow band and the LOD was 1 ng (which matches 10 ng g−1). The lower LOD obtained proved that the suggested assay is highly acceptable to be used as a preliminary assay for differentiating between the positive and negative samples. It was observed that the intensity of the yellow fluorescence band on the MC increased with increasing TCs level in the extract. This observation suggests that the MC assay could be used for the semi-quantitative determination of TCs residues in chicken samples. In such a case, MCs of known concentrations should be run with the unknown sample extracts for comparison. However, the MC technique should not be used for quantitative purposes where accurate quantitative data are required.
Selectivity The selectivity of an assay is a measure of the extent to which the method can be used to detect a particular compound in the matrices analysed without interference from matrix components. The proposed assay is selective and can be easily used to detect 1 ng of TCs in the presence of matrix materials.
Assessment It is of importance to determine whether the suggested MC assay could be successful in identifying contaminated samples that are at or above the permissible level. So, reference MCs were prepared by spiked samples at levels equal to the permissible limits (10 or 20 ng ml−1 for the European Union and United States respectively) to be used as a guide for evaluation. In order to test the proposed procedure, two groups of blind samples were prepared. Samples of the first group were spiked with TCs at
10 10 10 10 10 10
100 150 180 190 200 220
ng ng ng ng ng ng
g−1 g−1 g−1 g−1 g−1 g−1
TCs applied 10 15 18 19 20 22
ng ng ng ng ng ng
Evaluation Accepted Accepted Accepted Accepted Accepted Rejected
levels above or below the European Union permissible limit, whilst the second group of samples were spiked at levels above or below the US permissible limit. All samples were subjected to assay by the proposed procedure. Comparing MCs of the blind samples with the reference MCs obtained gave the results shown in Tables 2 and 3. All samples spiked at levels above the permissible limits were assessed as rejected. Also, the total blank and spiked samples at levels equal or below the permissible limits were evaluated as acceptable. However, to decrease the false-positive samples, a MC with a smaller diameter, i.e. 3 mm instead of 4 mm, should be used. In this case the TCs bands will be clearer and the comparison easier and more precise, and accordingly the error will be reduced.
Conclusions The proposed method is rapid, reliable and economical. It requires a much lower level of expertise than conventional analytical methods. With experience the working time required to test one sample is less than 20 min, and with batch analysis the working time is reduced to approximately 10 min per sample. The method seems to be eminently suitable for use by public health laboratories for TCs screening purposes, provided that positive samples are confirmed by more elaborate methods. The advantage of the present assay is that it can be successfully used at a field level for the rapid screening of chicken for the presence of TCs (a quality tool). Moreover, the assay can provide an alternative to microbial screening assays and could be used as an effective pre-screening technique in public health laboratories.
Food Additives & Contaminants: Part A References Abu-Raiia SH, Shalaby AR, Salama NA, Emam WH, Mehaya FM. 2013. Effect of ordinary cooking procedures on tetracycline residues in chicken meat. J Food Drug Anal. 21:80–86. Al-Ghamdi M, El-Morsy SF, Al-Mustafa ZH, Al-Ramadhan M, Hanif M. 1999. Antibiotic resistance of Escherichia coli isolated from poultry workers, patients and chicken in the eastern province of Saudi Arabia. Trop Med Int Health. 4:278–283. Arim RH, Aguinaldo AR. 1999. Optimization and validation of a minicolumn method for determining aflatoxins in copra meal. J AOAC Int. 82:877–882. Cantwell H, O’Keeffe M. 2006. Evaluation of the premi test and comparison with the one-plate test for the detection of antimicrobials in kidney. Food Addit Contam. 23:120–125. Chopra I, Roberts M. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 65:232–260. De Ruyck H, De Ridder H, Van Renterghem R, Van Wambeke F. 1999. Validation of HPLC method of analysis of tetracycline residues in eggs and broiler meat and its application to a feeding trial. Food Addit Contam. 16:47–56. De Wasch K, Okerman L, Croubels S, De Brabander H, Van Hoof J, De Backer P. 1998. Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory test. Analyst. 123:2737–2741. European Commission. 2010. On pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Commission Regulation (EC) 37-2010. FAO/WHO. 2004 Residues of some veterinary drugs in animals and foods. Sixty-second report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, FAO FNP 41/16. Rome: FAO. Idowu F, Junaid K, Paul A, Gabriel O, Paul A, Sati N, Maryam M, Jarlath U. 2010. Antimicrobial screening of commercial eggs and determination of tetracycline residue using two microbiological methods. Int J Poult Sci. 9:959–962. Kabir J, Umoh VJ, Audu-okoh E, Umoh JU, Kwaga JKP. 2004. Veterinary drug use in poultry farms and determination of antimicrobial drug residues in commercial eggs and slaughtered chicken in Kaduna State, Nigeria. Food Control. 15:99–105. Kurittu J, Lönnberg S, Virta M, Karp M. 2000. A group-specific microbiological test for the detection of tetracycline residues in raw milk. J Agric Food Chem. 48:3372–3377. Mitema ES, Omija B. 1992. Determination of oxytetracycline residues in chicken eggs using microbiological methods. Proceedings of the Third World Congress on Food
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Borne Infections and Intoxications; June 16–19. Berlin: Robert-von-Ostertag Institute of the Federal Health Office; p. 780–783. Omija B, Mitema ES, Maitho TE. 1994. Oxytetracycline residue levels in chicken eggs after oral administration of medicated drinking water to laying chickens. Food Addit Contam. 11:641–647. Pavlov A, Lashev L, Vachin I, Rusev V. 2008. Residues of antimicrobial drugs in chicken meat and offals. Trakia J Sci. 6:23–25. Pikkemaat MG. 2009. Microbial screening methods for detection of antibiotic residues in slaughter animals. Anal Bioanal Chem. 395:893–905. Pikkemaat MG, Rapallini M, Oostra S, Elferink JWA. 2009. Comparison of three microbial screening methods for antibiotics using routine monitoring samples. Anal Chim Acta. 637:298–304. Ramakrishna Y, Bhat RV. 1990. Minicolumn chromatography for the detection of sterigmatocystin in agricultural commodities. Mycopathologia. 110:153–155. Ramakrishna Y, Sashidhar RB, Bhat RV. 1989. Minicolumn technique for the detection of deoxynivalenol in agricultural commodities. Bull Environ Contam Toxicol. 42:167–171. Salama NA, Abou-Raya SH, Shalaby AR, Emam WH, Mehaya FM. 2011. Incidence of tetracycline residues in chicken meat and liver retailed to consumers. Food Addit Contam: Part B. 4:88–93. Salehzadeh F, Madani R, Salehzadeh A, Rokni N, Golchinefar F. 2006. Oxytetracycline residue in chicken tissues from Tehran slaughterhouses in Iran. Pak J Nutr. 5:377–381. Sashidhar RB, Sudershan RV, Bhat RV. 1990. Rapid detection of argemone oil in rapeseed/mustard oils with a pressurized mini-column. Jaocs. 67:338–339. Shahid MA, Muhammad S, Muhammad A, Muhammad JA, Muhammad A, Arfan A. 2007. Status of oxytetracycline residues in chicken meat in Rawalpindi/Islamabad area of Pakistan. Asian J Poult Sci. 1:8–15. Shalaby AR, Salama NA, Abu-Raiia SH, Emam WH, Mehaya FM. 2011. Validation of HPLC method for determination of tetracycline residues in chicken meat and liver. Food Chem. 124:1660–1666. Tsai CE, Kondo F. 2001. Improved agar diffusion method for detecting residual antimicrobial agents. J Food Protect. 64:361–366. Vázquez-Moreno L, Bermúdez A. MC, Languré A, HigueraCiapara I, Aguayo M, Flores E. 1990. Antibiotic residues and drug resistant bacteria in beef and chicken tissues. J Food Sci. 55:632–634.
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Mini-column screening assay for tetracyclines in chicken Ali R. Shalaby* Food Science and Technology Department, National Research Centre, Dokki, Cairo, Egypt (Received 17 August 2014; accepted 17 November 2014) A simple, rapid, reliable and economical mini-column (MC) method for the detection of tetracyclines (TCs) residues in chicken meat was developed. The method employs a commonly available Pasteur pipette which is tightly packed with silica gel and anhydrous sodium sulfate. Clean-up and detection of illegal levels can be achieved on the same column. Viewing the developed MC under an ultraviolet lamp revealed that TCs can be detected as a compact golden yellow fluorescent band at the junction between the anhydrous sodium sulfate and silica gel layers. Comparing the yellow band of control extracts with those fortified (100 ng ml−1) showed no overlap between analyte and impurities. The limit of detection (LOD) of the MC assay was 1 ng, indicating that the chicken sample containing 10 µg TCs kg−1 sample could be easily detected. Moreover, the intensity of the yellow band increased whenever TCs levels in the extract increased. Evaluation utility of the method with blind samples as controls or samples fortified with total TCs at various levels indicated that the total blank and spiked samples at levels equal or below the permissible limits were assessed as accepted. The method can provide an alternative to microbial screening assays and could be used as an effective pre-screening technique in public health laboratories. Keywords: chicken; tetracyclines; mini-column; chromatography
Introduction Broad-spectrum antibiotics are a class of drugs widely used in veterinary medicine to treat and control a variety of bacterial infections and as animal growth promoters (De Ruyck et al. 1999). Tetracyclines (TCs), which are licensed for use in a wide range of livestock species, are the main antibiotics used in poultry production because they are the cheapest class of antibiotic available and their cost in real terms is declining due to improved manufacturing technology (Chopra & Roberts 2001). The widespread misuse of TCs residues in poultry production results in the presence of their residues in edible tissues at levels above the MRL in various countries such as Mexico (Vázquez-Moreno et al. 1990), Kenya (Omija et al. 1994), Belgium (De Wasch et al. 1998), Saudi Arabia (Al-Ghamdi et al. 1999), Iran (Salehzadeh et al. 2006), Pakistan (Shahid et al. 2007), Bulgaria (Pavlov et al. 2008) and Egypt (Salama et al. 2011). Such high levels led to the presence of TCs residues in edible tissues. Cooking procedures do not completely eliminate such residues and so potentially create a health hazard for consumers if the MRL is exceeded (Abu-Raiia et al. 2013). In order to guarantee the safety of animal-derived foods, the joint FAO/WHO Expert Committee on Food Additives (2004) recommended limits of 200 and 600 µg kg−1 for chicken tissues and liver, respectively, expressed as the sum of the TCs group. The corresponding
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
limits set by the European Union (Council Regulation No. 2377/90) were 100 and 300 µg kg−1. Public health concerns associated with residues and contaminants have been the impetus for determining such hazards in the food supply. Most of analytical methods for TCs residues are highly sensitive and give good precision and accuracy. However, they are costly to acquire, run and maintain and require relatively sophisticated laboratory facilities and specialist operators. So, there is an expanding need for a simple, cost-effective and rapid screening technique as an easy tool that can be used in the field for quick inspection. Questionable samples (higher than a certain value) can be selected to be sent for confirmatory analysis by a more sensitive and specific method. The most common approach is the use of microbial assays as pre-screening method (Mitema & Omija 1992; Kurittu et al. 2000; Tsai & Kondo 2001; Kabir et al. 2004; Cantwell & O’Keeffe, 2006; Pikkemaat 2009; Pikkemaat et al. 2009; Idowu et al. 2010), despite their disadvantages, like poor sensitivity and selectivity, false-positive results and long analytical time. Therefore, a quick and reliable method that overcomes most of these disadvantages should be developed. The MC technique was previously successful at determining aflatoxins in Copra meal (Arim & Aguinaldo 1999), the detection of sterigmatocystin (Ramakrishna & Bhat 1990) and deoxynivalenol (Ramakrishna et al. 1989) in agricultural
Food Additives & Contaminants: Part A commodities, and for rapid detection of argemone oil in rapeseed/mustard oils (Sashidhar et al. 1990). It seems that the MC technique is suitable as an alternative to the microbial assay for field tests at least because it has the advantages of a short time, low cost, is very simple, high speed and can be applied in situ as a screening alert method for TCs residues in chicken food samples before being accepted for human consumption. The aim of this work is to establish a rapid, simple and economic MC assay for total TCs residues in chicken meat.
Materials and methods Chemicals and reagents Standards of oxytetracycline (OTC), tetracycline (TTC), chlortetracycline (CTC) and doxycycline (DOC) were supplied by Sigma (St. Louis, MO, USA). Benzene (BNZ), acetone, chloroform, methanol (MeOH) and acetonitrile (ACN) were of HPLC grade. Citric acid monohydrate, ethylenediaminetetraacetic acid disodium salt (Na2EDTA) and anhydrous sodium sulfate were of analytical grade. Water was purified before use in a Milli-Q system (Millipore, Bedford, MA, USA). Silica gel (63–200 mesh) was from Merck Chemicals (Merck, Poole, Dorset, UK; activated at 110°C for 1 h and cooled in a dessicator). Glass wool and a disposable Pastier pipette were also used.
Samples Three individual chicken samples were purchased from each of three different suppliers. Each chicken was individually filleted and homogenised in a food processor, and the tissue was then stored at –20°C until use. Samples were analysed for TCs residues by an HPLC method according to Shalaby et al. (2011) to ensure that they were free of these residues.
Standard solution preparation A stock standard solution of a TCs mixture was prepared by dissolving 10 mg (2.5 mg each of OTC, TTC, CTC and DOC) in 10 ml of methanol to obtain a final concentration of 1 mg ml−1. The stock standard solution was put in amber glass to prevent photo-degradation and stored at −20°C; it is stable in this condition for at least 4 weeks. Stock solutions were diluted with methanol to give a series of working standard solutions which were prepared weekly. Other series of working standard solution were prepared using ACN.
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spiking. Spiked samples were allowed to stand in the dark inside a refrigerator at 4°C for 1 h before analysis. TCs extraction Four extraction methods were trialled as follows: ● First method: 2 ml of an EDTA solution (5%, pH 9) and 8 ml of MeOH were added to the tube containing spiked sample (1 g), the mixture was vigorously shaken for 1 min and then filtered on Whatman No. 4 to provide a filtrate suitable for MC application. ● Second method: 2 ml of an EDTA solution (5%, pH 9) and 10 ml of MeOH were added to the tube containing spiked sample (1 g), the mixture was vigorously shaken for 1 min. Thereafter, about 4.0 g anhydrous sodium sulfate were added, vigorously shaken and filtered on Whatman No. 4 containing anhydrous sodium sulfate to provide a filtrate suitable for MC application. ● Third method: as described for the first method, but the MeOH was replaced with ACN and EDTA was replaced with citric acid (0.3 N). ● Fourth method: as described for the second method, but the MeOH was replaced with ACN and EDTA was replaced with citric acid (0.3 N). TCs-negative samples were extracted by the four extraction methods. The extracts obtained were spiked with TCs to provide the levels of 10 and 20 ng ml−1. Mini-column preparation Commercially available Pasteur pipettes (about 10 × 0.5 cm) were adapted for use by inserting a small glass wool plug in the restricted end and packed with silica gel (about 20 mm height). EDTA and sodium sulfate anhydrous layers were added to the column and tested for suitable amounts of chemicals and their arrangement on the column which help to obtain a clear band separated from the impurities. Each layer was tightly packed by frequent tapping of the column while adding the various chemicals. Several trials were conducted to select the most suitable solvent system for visualising the TCs, where various composites of two or more solvents were tested. The most effective solvent system that makes the silica gel transparent under an ultraviolet (UV) lamp (where the background will be dark and the TCs band will be more distinct) will be used as a “clarifier”. The suitable volume of the “clarifier” was determined to be 1 ml.
Spiking samples Homogenised chicken sample (1 ± 0.01 g) was weighed into a 25-ml culture tube and spiked by the addition of a TCs standard solution to provide the desired level of
Preparation of spiked blind sample The ability of the method to detect the rejected (TCs > 100 ng g−1) and the accepted (TCs ≤ 100 ng g−1) samples,
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according to European Union legal limits, was tested in a blind system. A template was prepared by independent colleagues to fortify 60 chicken extracts at levels above or below 100 ng g−1. The fortified extracts were subjected to the MC assay. The template was then randomly assigned a number from 1 to 60, and the blind assay was performed in this order, using 1 ml extract. Further, a 1 ml extract containing 10 ng TCs (that match the European Union recommendation) was assayed to be used as a reference MC as a guide for reviewers. The results were then reviewed by the independent colleagues with respect to the different levels of TCs. A similar experiment was performed using fortification levels above or below 200 ng g−1, to match the US recommendation. The reviewer reference MC was obtained using 1 ml extract containing 20 ng TCs (to match the US recommendation).
TCs
Figure 1. More distinct tetracyclines (TCs) residues’ fluorescent band.
yellow fluorescence band. The small diameter (about 4 mm i.d.) of the MC along with using the “clarifier” led visually to the easy detection of the yellow band (Figure 1).
Results and discussion Optimisation the MC assay Detection and clarity of TCs TCs on TLC gave a characteristic golden yellow fluorescence under a UV lamp at 365 nm. This property was used to detect TCs on silica gel MC. At the beginning, MC packed with silica gel and 1 ml of MeOH standard solutions (10 ng ml−1) was loaded into the MC, eluted by gravity and viewed under UV lamp. Silica gel layer became cloudy and detection of the TCs’ yellow band was difficult due to blurring. Using a standard solution prepared by ACN, a similar observation was obtained. So, several trials were conducted to select the most suitable solvent system for visualising the TCs’ band, where various composites of two or more solvents were tested. Five systems (Table 1) gave a bright and sharp yellow band on the silica MC. The visually sharpest band was obtained with the composite number 4 (benzene: MeOH:ACN, 6:3:1). So, composite number 4 was used as a “clarifier”. The suitable volume of the “clarifier” was determined to be 1 ml and did not cause any dispersion of the TCs’ band. So, 1 ml of the “clarifier” was chosen to visualise the TCs band. It was found that TCs were selectively adsorbed at the top of the silica gel, giving a single compact and intense
MC composition A TC standard solution when applied into the column gave a compact yellow fluorescent band at the surface of silica gel (Figure 2A). When the negative extract was applied to the MC, a white fluorescent band (impurities) was observed at the surface of the silica gel (Figure 2B). The extracts of TCs when applied to the MC failed to separate from confounding fluorescent impurities. Some trials using anhydrous sodium sulfate in the extracted solution and on the MC were tried in order to try to separate the
A
B
Table 1. Effective elution systems tested. Number 1 2 3 4 5
Composition Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile Benzene–methanol–acetonitrile
Ratio 5: 5: 6: 6: 8:
5: 5: 3: 2: 4:
2 1 1 1 1
Figure 2.
(A) TCs residues’ band and (B) impurities band.
Food Additives & Contaminants: Part A
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Impurities
A
B
TCs
Figure 3.
Dispersion of the TCs residues’ fluorescent band.
Figure 5. A total of 1 ml of spiked extract at 100 ng g−1 loaded into the mini-column: (A) MeOH extract and (B) ACN extract. Na2SO4 anhydrous Compact TCs band
second extract gave a band greater than that given by the fourth method (Figure 5). So the second extraction method was chosen as the extraction step for the MC assay.
Silica gel
Figure 4.
Compact TCs residues’ fluorescent band.
TCs from non-TCs fluorescing impurities. No combination of solvents, eluents or clarifiers enables the certain identification of the TCs on the unmodified MC, due to the dispersion of TCs throughout the MC (Figure 3). However, when a 10 mm capping of anhydrous sodium sulfate was layered on top of the silica MC, this led to unequivocal separation of fluorescing impurities, which were retained on the sodium sulfate layer, and the TCs, which were retained as a sharp band on top of the silica. The second extraction system gave the sharpest band (Figure 4), so this was incorporated into the method.
Volume of extract A total of 1 ml of this extract was found to be sufficient to give effective separation and visualisation of the TCs as a compact yellow fluorescent band while minimising band dispersion.
Suggested MC assay Sample extraction A total of 1 g of homogenised chicken sample was placed into a 25-ml culture tube. A total of 2 ml of EDTA solution (5%, pH 9) and 10 ml of MeOH were added into the sample and mixture shaken vigorously for 1 min. Thereafter, about 4.0 g anhydrous sodium sulfate were added, shaken vigorously and filtered on Whatman number 4 containing anhydrous sodium sulfate. The obtained filtrate was suitable for MC application.
Sample extraction
MC preparation
Both the second and the fourth extraction methods were evaluated. Both extractions were clear and suitable for the application into the MC. Therefore, 1 ml of each spiked extract (10 ng ml−1) was separately loaded into the MCs, drained by gravity and viewed under a UV lamp. The
A small glass wool plug was inserted into the restricted end of the commercially available Pastier pipette (about 10 × 0.4 cm) and packed with anhydrous sodium sulfate (about 2 mm), silica gel (about 20 mm) and anhydrous sodium sulfate (about 10 mm). Each layer should be
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tightly packed by frequent tapping of the column while adding the various chemicals.
Table 2. Evaluation of the blind samples according to European Commission (2010) recommendation (100 ng g−1). Sample tested
Development and detection of TCs A total of 1 ml sample extract was loaded into the column and allowed to drain by gravity. A total of 1 ml of the “clarifier” solvent was added into the column and allowed to drain by gravity. The column is viewed under a UV source for the presence of a yellow fluorescence band of TCs. The fluorescent band will appear at the junction of the anhydrous sodium sulfate and silica gel layers.
10 10 10 10 10 10
Spiked levels
TCs applied
Evaluation
0 ng g−1 50 ng g−1 80 ng g−1 90 ng g−1 100 ng g−1 110 ng g−1
0 ng 5 ng 8 ng 9 ng 10 ng 11 ng
Accepted Accepted Accepted Accepted Accepted Rejected
Table 3. Evaluation of the blind samples according to FAO/ WHO (2004) recommendation (200 ng g−1). Samples tested
Spiked levels
Suitability of the assay Limits of detection (LODs) The levels of TCs spiked in the present study were based on the permissible limits of both the United States and European Union, i.e. 200 and 100 ng g−1, respectively. The control samples showed no yellow band and the LOD was 1 ng (which matches 10 ng g−1). The lower LOD obtained proved that the suggested assay is highly acceptable to be used as a preliminary assay for differentiating between the positive and negative samples. It was observed that the intensity of the yellow fluorescence band on the MC increased with increasing TCs level in the extract. This observation suggests that the MC assay could be used for the semi-quantitative determination of TCs residues in chicken samples. In such a case, MCs of known concentrations should be run with the unknown sample extracts for comparison. However, the MC technique should not be used for quantitative purposes where accurate quantitative data are required.
Selectivity The selectivity of an assay is a measure of the extent to which the method can be used to detect a particular compound in the matrices analysed without interference from matrix components. The proposed assay is selective and can be easily used to detect 1 ng of TCs in the presence of matrix materials.
Assessment It is of importance to determine whether the suggested MC assay could be successful in identifying contaminated samples that are at or above the permissible level. So, reference MCs were prepared by spiked samples at levels equal to the permissible limits (10 or 20 ng ml−1 for the European Union and United States respectively) to be used as a guide for evaluation. In order to test the proposed procedure, two groups of blind samples were prepared. Samples of the first group were spiked with TCs at
10 10 10 10 10 10
100 150 180 190 200 220
ng ng ng ng ng ng
g−1 g−1 g−1 g−1 g−1 g−1
TCs applied 10 15 18 19 20 22
ng ng ng ng ng ng
Evaluation Accepted Accepted Accepted Accepted Accepted Rejected
levels above or below the European Union permissible limit, whilst the second group of samples were spiked at levels above or below the US permissible limit. All samples were subjected to assay by the proposed procedure. Comparing MCs of the blind samples with the reference MCs obtained gave the results shown in Tables 2 and 3. All samples spiked at levels above the permissible limits were assessed as rejected. Also, the total blank and spiked samples at levels equal or below the permissible limits were evaluated as acceptable. However, to decrease the false-positive samples, a MC with a smaller diameter, i.e. 3 mm instead of 4 mm, should be used. In this case the TCs bands will be clearer and the comparison easier and more precise, and accordingly the error will be reduced.
Conclusions The proposed method is rapid, reliable and economical. It requires a much lower level of expertise than conventional analytical methods. With experience the working time required to test one sample is less than 20 min, and with batch analysis the working time is reduced to approximately 10 min per sample. The method seems to be eminently suitable for use by public health laboratories for TCs screening purposes, provided that positive samples are confirmed by more elaborate methods. The advantage of the present assay is that it can be successfully used at a field level for the rapid screening of chicken for the presence of TCs (a quality tool). Moreover, the assay can provide an alternative to microbial screening assays and could be used as an effective pre-screening technique in public health laboratories.
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