4.1 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 antibiotic 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 counts per minute (cpm) for the blank fish samples are summarized in Table 1.
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
|
Sulfonamides
Kit
|
Tetracyclines
kit
|
Macrolides
Kit
|
Streptomycins
Kit
|
Fish species
|
Mean
|
SD
|
Mean
|
SD
|
Mean
|
SD
|
Mean
|
SD
|
Mean
|
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
|
|
|
|
|
|
|
Lingue
|
|
|
4454
|
650.1
|
|
|
|
|
|
|
cpm, counts per minute; SD, standard deviation
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 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.
4.2 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 beta-lactams was evaluated from averaging the results of 6 negative samples spiked with benzyl penicillin 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 beta-lactam. On the other hand, the CP for tetracyclines was calculated by averaging cpm results of negative control standards provided in the tetracycline test kit and subtracting 40% of the obtained average cpm value (Table 2).
Table 2: Control points for the different antimicrobials in blank fish samples
Antimicrobial
family
|
Spiked samples
|
Blank samples
|
Level of analyte spiking (µg/kg)
|
Mean cpm of Spiked Samples
|
Variation allowance for matrix effect
|
Control point
cpm
|
Mean blank
cpm
|
Range of blank cpm readings
|
No. of false positives/No. of samples
|
False positive
rate (%)
|
β-lactams
|
25 µg/kg Benzyl Penicillin
|
1275
|
Spiked cpm +20%
|
1530
|
2502
|
2160-2907
|
0/30
|
0
|
Sulfonamides
|
50 µg/kg Sulfamethazine
|
1096
|
Spiked cpm +30%
|
1424
|
3162
|
1431-6995
|
0/30
|
0
|
Tetracyclines
|
0 µg/kg Tetracycline at
|
2524
|
Blank cpm - 40%
|
1514
|
2524
|
2451-3269
|
0/30
|
0
|
Macrolides
|
100 µg/kg Erythromycin A
|
1765
|
Spiked cpm +20%
|
2118
|
2587
|
1906-2952
|
1/28
|
3.6
|
Streptomycins
|
25 µg/kg Streptomycin
|
2574
|
Spiked cpm +30%
|
3346
|
4605
|
2942-5488
|
1/20
|
5.0
|
Number of samples used per parameter Ns ≥ 20
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 streptomycins 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-lactam and sulfonamides; 3.6% for macrolides, and 5% for streptomycins; this proved the validity of the obtained data since it met the acceptance criteria of being within 5%. A comparison of the control points (CP) for the different antibiotics 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.
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
|
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 value as per EU guideline
Fm = M + 1.64 * SDs [26, 27]
|
Calculated control point CP as per Charm II assay
|
Tetracyclines
|
Tetracycline
|
25
|
2958
|
2776
|
816
|
1514
|
Chlortetracycline
|
25
|
3050
|
2780
|
1417
|
Oxytetracycline
|
100
|
2891
|
2579
|
1427
|
Macrolides
|
Erythromycin
|
100
|
2814
|
2564
|
1904
|
2118
|
Tilmicosin
|
100
|
2486
|
2164
|
2002
|
Tylosin
|
100
|
2512
|
2115
|
1740
|
β-lactam
|
Penicillin G
|
25
|
2523
|
2176
|
1438
|
1530
|
Ampicillin
|
50
|
2455
|
2042
|
1341
|
Amoxicillin
|
50
|
2702
|
2536
|
1487
|
Oxacillin
|
300
|
2398
|
2171
|
1478
|
Dicloxacillin
|
300
|
2524
|
2384
|
1489
|
Cloxacillin
|
300
|
2500
|
2368
|
1413
|
Aminoglycosides
|
Streptomycin
|
25
|
4822
|
3867
|
2592
|
3346
|
Sulfonamides
|
Sulfamethazine
|
25
|
2593
|
1428
|
1379
|
1424
|
Sulfadimethoxine
|
25
|
2266
|
1129
|
972
|
Sulfamerasine
|
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
According to the EU guideline, the cut off factor (Fm), refers to the response or signal from a screening test which indicates that a sample contains an analyte at or above the screening target concentration [26], while the Charm II protocol control point (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 antibiotics. In this respect, the assay is considered valid only when Fm < T and the detection capability CCβ is validated when Fm < B. Accordingly, the number of spiked samples with mean responses below the cut-off level (deemed positive) is identified and the false positive rate determined. If T< Fm < B, the false-positive 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 cutoff 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 antibiotics, the respective detection capabilities 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.
4.3 Detection capability for the different antimicrobials in selected fish species
The detection capability (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 detection capabilities 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 pangassius) with 100% detection. However, the batch of the multi antimicrobial 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 0.25MRL. Interestingly, it was observed that the technique is also capable of detecting other antimicrobials belonging to the tetracycline family (tetracycline, oxytetracycline) and not limited to the chlortetracycline provided for in the Charm II test kit.
Table 4: Detection capability for the selected antibiotics
Family
|
Compound
|
EU-MRL
(μg kg-1)
|
CCβ
(μg kg-1)
|
No of samples
|
No of positive samples
|
Counter results (cpm)
|
% Detection of each antibiotic
|
Mean
|
Min
|
Max
|
Tetracyclines
(CP= 1514 cpm)
|
Tetracycline
|
100
|
25
|
20
|
20
|
724
|
650
|
825
|
100
|
Chlortetracycline
|
100
|
25
|
21
|
21
|
1200
|
942
|
1421
|
100
|
Oxytetracycline
|
100
|
100
|
31
|
31
|
1269
|
1074
|
1460
|
100
|
Macrolides
(CP= 2118 cpm)
|
Erythromycin
|
200
|
100
|
30
|
30
|
1669
|
954
|
1955
|
100
|
Tilmicosin
|
50
|
100
|
21
|
21
|
1565
|
1221
|
2078
|
100
|
Tylosin
|
100
|
100
|
21
|
21
|
1440
|
1103
|
1742
|
100
|
β-lactams
(CP= 1530 cpm)
|
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
|
Aminoglycosides (CP= 3346cpm)
|
Streptomycin
|
500
|
25
|
22
|
22
|
2424
|
1642
|
3074
|
100
|
Sulfonamides
(CP= 1424 cpm)
|
Sulfamethazine
|
100
|
25
|
29
|
28
|
1240
|
813
|
1831
|
96.6
|
Sulfadimethoxine
|
100
|
25
|
20
|
20
|
968
|
737
|
923
|
100
|
Sulfamerasine
|
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
|
The sulfa drugs including, sulfadimethoxine, sulfadiazine, sulfamerasine 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 pangassius) with 100% detection. Whereas, results for the beta-lactams show that benzyl penicillin, 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, benzylpenicillin is detected at 0.5MRL, 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 on, the Charm II technique is capable of detecting streptomycin at 25 µg/kg (0.05MRL) for all fish species involved in the study at 100% detection.
A comparison of the detection capabilities (CCβ) and maximum residue limits (MRL) for the different antimicrobials is shown in Figure 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 tilmcosin which was detected at 2 MRL. Most of the drug residues exhibited detection capabilities in the range 0.05 MRL to 0.5 MRL, with 100% detection. Moreover, the incidences of false negative results observed for all antibiotics 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 detection capability 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 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 Table 1b (Supporting information). 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 (Table 1b, Supporting Information). The LOD results were also compared with the LC-MS/MS analysis of sulfadimethoxine [29], HPLC-MS/MS analyses of tetracyclines, chlortetracycline, oxytetracycline, sulfadimethoxine, sulfamerasine 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 antibiotics 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].
4.4 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 intraday 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, beta-lactam, aminoglycosides, and sulfonamides; spiked in blank fish samples at MRL, 0.5MRL or concentration less than 0.5MRL 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), 6.9 to 9.7% for beta-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.
Table 5: Repeatability study at MRL, 0.5MRL or Concentration < 0.5MRL
Family
|
Compound
|
Spiking concentration (µg/kg)
|
Mean cpm
|
SDr
|
RSDr
|
Tetracyclines
|
Chlortetracycline
|
25µg/kg(0.25MRL)
|
1207.0
|
118.2
|
9.8%
|
Oxytetracycline
|
100 µg/kg (MRL)
|
1270.06
|
98.41
|
7.75%
|
Macrolides
|
Erythromycin
|
100 µg/kg (0.5MRL)
|
1762.4
|
110.4
|
6.3%
|
200 µg/kg (MRL)
|
1478.1
|
41.2
|
2.8%
|
β-lactam
|
Penicillin G
|
25 µg/kg(0.5MRL)
|
1285.6
|
89.3
|
6.9%
|
50 µg/kg (MRL)
|
648.5
|
62.7
|
9.7%
|
Aminoglycosides
|
Streptomycin
|
250 µg/kg (0.5MRL)
|
1125.8
|
112.7
|
10.01%
|
500 µg/kg (MRL)
|
1110.5
|
127.2
|
11.5%
|
Sulfonamides
|
Sulfathiazole
|
25 µg/kg(0.25MRL)
|
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
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 antimicrobial agents, whose summarized results are presented in Table 5.
Table 6: Repeatability in the detection of sulfathiazole at 25 µg/kg for selected fish samples
Parameter
|
Fish samples spiked with sulfathiazole at 25 µg/kg, cpm
|
Dorade
|
Salmon
|
Seabass
|
|
989
|
914
|
735
|
|
815
|
969
|
780
|
|
862
|
886
|
976
|
|
876
|
1015
|
896
|
|
782
|
932
|
890
|
|
934
|
1075
|
978
|
|
985
|
930
|
976
|
|
877
|
1070
|
898
|
|
863
|
935
|
975
|
|
986
|
890
|
976
|
Average
|
896.9
|
961.6
|
908
|
SD
|
73.6
|
69.3
|
88.3
|
RSD
|
0.082
|
0.07
|
0.097
|
4.5 Reproducibility of the method
The reproducibility studies were performed by two different researchers following the same Charm II protocol on selected fish species, spiked with different antimicrobial agents and evaluated by means of intra-day and inter-day coefficient of variations. The reproducibility study characterized by the relative standard deviation (%RSD) was satisfactory with a precision of less than 15.3% for the different antimicrobial drugs (tetracyclines, macrolides, beta-lactam, aminoglycosides, and sulfonamides) spiked in blank fish samples at MRL, 0.5MRL or concentration less than 0.5MRL and studied under reproducibility conditions (n ≥ 6). The coefficient of variation calculated as percentage relative standard deviation (%RSD) for tetracyclines (chlortetracycline and oxytetracycline) was 7.2 to 11.4%; macrolides (erythromycin) ranged from 5.8 to 8.9%; beta-lactam (penicillin G) from 10.4 to 11.2%; aminoglycosides from 8.9 to 15.1% and sulfonamides (sulfathiazole) from 2.8 to 8.3% as indicated in Table 7.
Table 7: Reproducibility study at MRL, 0.5MRL or Concentration < 0.5MRL
Family
|
Compound
|
Spiking concentration (µg/kg)
|
Mean cpm
|
SDR
|
RSDR
|
Tetracyclines
|
Chlortetracycline
|
25µg/kg(0.25MRL)
|
1224.5
|
139.2
|
11.4%
|
Oxytetracycline
|
100 µg/kg (MRL)
|
1277
|
92.6
|
7.2%
|
Macrolides
|
Erythromycin
|
100 µg/kg (0.5MRL)
|
1748.5
|
156.1
|
8.9%
|
200 µg/kg (MRL)
|
1456.5
|
83.9
|
5.8%
|
β-lactam
|
Penicillin G
|
25 µg/kg(0.5MRL)
|
1204.9
|
135.0
|
11.2%
|
50 µg/kg (MRL)
|
702.1
|
73.0
|
10.4%
|
Aminoglycosides
|
Streptomycin
|
250 µg/kg (0.5MRL)
|
1110.6
|
98.7
|
8.9%
|
500 µg/kg (MRL)
|
1132.6
|
171.4
|
15.1%
|
Sulfonamides
|
Sulfathiazole
|
25 µg/kg(0.25MRL)
|
943.7
|
78.1
|
8.3%
|
100 µg/kg(MRL)
|
647.1
|
18.1
|
2.8%
|
SDr, Standard deviation under reproducibility conditions; RSDr, Relative standard deviation under reproducibility conditions
An elaborate presentation of some results of the reproducibility studies performed by two different researchers following the same Charm II protocol on selected fish species, spiked with oxytetracycline at a concentration level of 100 µg/kg, is presented in Table 8. A comparison of the results obtained by the two researchers for the same fish species showed no significant difference; and the overall analysis showed no significant difference in the cpm results for the different fish species including seabas, pangasius and salmon (ANOVA, F-critical 4.1 > F-calculated 0.64), with RSD < 10%, which further demonstrates the technique’s reproducibility with little matrices interference. Similar observations were made for the other antimicrobial compounds, whose summarized results are presented in Table 7.
Table 8: Reproducibility in the detection of oxytetracycline at 100 µg/kg for selected fish samples
Fish sp.
|
Researcher 1, cpm
|
Researcher 2, cpm
|
Seabass
|
1176
|
1296
|
1177
|
1265
|
1295
|
1177
|
1334
|
1281
|
1127
|
1166
|
1341
|
1417
|
1170
|
1460
|
1261
|
1074
|
1371
|
1361
|
1201
|
1335
|
Pangassius
|
1225
|
1185
|
1166
|
1223
|
1094
|
1224
|
1408
|
1407
|
1408
|
1405
|
Salmon
|
1307
|
1307
|
1378
|
1299
|
1298
|
1310
|
1274
|
1311
|
1313
|
1279
|
1266.2
|
1289.1
|
Average
|
1176
|
1296
|
Standard deviation
|
94.6
|
96.2
|
RSD
|
0.07
|
0.07
|
4.6 Robustness of the method
Analysis of batches of many samples often require a couple of hours before completion; and there is likely to be a time interval between the first and last analysis of the processed samples. In the robustness testing of the Charm II assay, the effect of variation in reading time interval for processed samples was studied. Robustness testing was performed on samples spiked with 50 µg/kg amoxicillin and analysed on the beta-lactam channel immediately after mixing (0 hour) and after 14 hours. The control point for beta-lactam was set at 1530, and the robustness results are presented in Table 9.
Table 9: Robustness testing using amoxicillin spiked at 50 µg/kg for selected fish samples
Time
|
Run
|
Spiked at 50 µg/kg
|
Non Spiked
|
Pangasiuss, cpm
|
Dorade, cpm
|
Blanks, cpm
|
Fish species
|
Results at 0 hrs
|
1
|
1019
|
1069
|
2433
|
Pangasius
|
2
|
1024
|
959
|
2398
|
Pangasius
|
3
|
1017
|
1019
|
2399
|
Pangasius
|
4
|
1201
|
1069
|
2064
|
Dorade
|
5
|
1155
|
1033
|
2200
|
Dorade
|
6
|
1020
|
1067
|
2109
|
Dorade
|
Results after 14 hrs
|
7
|
1120
|
1120
|
2399
|
Pangasius
|
8
|
1059
|
1080
|
2064
|
Pangasius
|
9
|
1260
|
1195
|
2399
|
Pangasius
|
10
|
1011
|
1113
|
2068
|
Dorade
|
11
|
1089
|
1089
|
2210
|
Dorade
|
12
|
1099
|
1092
|
2205
|
Dorade
|
Average
|
|
1089.5
|
1075.4
|
2245.7
|
|
SD
|
|
81.6
|
57.9
|
150.8
|
|
RSD
|
|
0.07
|
0.05
|
0.07
|
|
From Table 9, it is evident that there was no significant difference in the cpm for both pangasius and dorade spiked with 50 µg/kg of beta-lactams and read after 0 or 14 hours (ANOVA, F-critical 4.3 > F- calculated 0.2) confirming the robustness of the method, in regard to variation in reading time intervals of the processed samples. A comparison of cpm for blank fish samples for both pangasius and dorade after 0 and 14 hours, also showed that there was no significant difference between counts since ANOVA F-critical 4.9 > F-calculated 0.2. The combined results of these studies demonstrate that the Charm II technique is quite robust for the analysis of antimicrobials in fish.
4.7 Specificity and cross reactivity of the technique
The cross reactivity analysis was carried out in order to determine whether the presence of non-target drugs may lead to false identification of the target drug; or whether the identification of the target analyte may be hindered by the presence of one or more interferences. Representative blank fish samples were spiked with different antimicrobial drugs at known concentration levels higher than those likely to interfere with the identification of the analyte of interest, and then analysed using the respective Charm II protocol for the target drug. The aminoglycosides (spectinomycin, neomycin and paromomycin) were analysed using the macrolide channel (meant for erythromycin, tilmicosin and tylosin). A standard mix containing aminoglycosides (spectinomycin, neomycin and paromomycin) was used to spike different fish samples at 150, 300 and 500 µg/kg level, and the results are presented in Table 10.
Table 10: Specificity and cross reactivity tests using the Macrolides kit
Blanks
cpm
|
AMGL spiked at 150 µg/kg,
Salmon cpm
|
AMGL spiked at 300 µg/kg,
Salmon cpm
|
AMGL spiked at 500 µg/kg,
Pangassius cpm
|
AMGL spiked at 150 µg/kg,
Catfish cpm
|
AMGL spiked at 300 µg/kg,
Catfish cpm
|
AMGL spiked at 500 µg/kg,
Trout cpm
|
5377
|
5097
|
5239
|
5239
|
5536
|
5511
|
4694
|
5538
|
4860
|
4931
|
4931
|
5224
|
5393
|
5262
|
5538
|
5106
|
4800
|
5034
|
5223
|
5803
|
5412
|
5076
|
4703
|
4900
|
5351
|
4966
|
5558
|
5686
|
5571
|
5039
|
4950
|
5121
|
5236
|
5807
|
5728
|
Macrolide calculated control point cpm= 2118
|
AMGL, Aminoglycosides standard mix containing spectinomycin, neomycin and paromomycin
Results show that although the macrolides which were the targeted antimicrobials tested positive (samples spiked with erythromycin at 200 µg/kg, gave 1478 cpm), the non-target aminoglycosides intentionally analyzed on the same channel, tested negative since in all cases the observed cpm were above the set control point of the macrolides of 2118. In similar studies, cross reactivity was further investigated by spiking residue-free, blank fish samples with high concentrations (10 MRL) of antimicrobial substances belonging to other groups (sulfonamides, beta-lactams, macrolides, and tetracyclines) and were analysed on the aminoglycosides channel; and the results are presented in Table 11.
Table 11: Specificity and cross reactivity tests with mixed standards of different antimicrobial using
the Aminoglycosides kit
Antibiotics used and their respective MRL
|
Mixed standard and respective spiking level, µg/kg
|
Fish Species
|
Spiked fish samples cpm
|
Tetracycline
(MRL 100 µg/kg)
Penicilin G
(MRL 50 µg/kg)
Sulfamethazine
(MRL 100 µg/kg)
Tilmicosin
(50MRL µg/kg)
|
Tetracycline spiked at 1000 µg/kg
Penicilin G spiked at 500 µg/kg
Sulfamethazine spiked at 1000 µg/kg
Tilmicosin spiked at 500 µg/kg
|
Cat fish
|
5742
|
Cat fish
|
6286
|
Cat fish
|
5780
|
Cat fish
|
5776
|
salmon
|
5418
|
salmon
|
5700
|
salmon
|
5584
|
Trout
|
5776
|
Trout
|
5584
|
Trout
|
6155
|
Trout
|
5962
|
Trout
|
5777
|
Trout
|
5800
|
Trout
|
5671
|
Trout
|
6010
|
Trout
|
5699
|
Trout
|
5700
|
Trout
|
5800
|
Trout
|
5810
|
Trout
|
5156
|
Aminoglycosides calculated control point cpm= 3346
|
The results of these studies also showed that no residues of the non-target drugs (tetracycline, penicillin G, sulfamethazine and tilmicosin) could be detected using the aminoglycosides channel as shown in Table 11; whereas samples spiked with spectromycin at 500 µg/kg tested positive with 1110 cpm when analysed under the same channel. All spiked samples tested negative and the non-target compounds could not be detected even at high concentration (10 MRL). Similar observations were made when utilizing the Biochip Array Technology assay, where none of the tested antibiotics could be detected under cross-reactivity studies [28].