Veratox® for Fluoroquinolone Technical Product Information

Validation of the Veratox® for Fluoroquinolone Screening Immunoassay for Semi-Quantitative Determination of Fluoroquinolone in Shrimp and Avian Muscle June 2016

Table of contents Description of Test...... 2

Description of Validation Study...... 2

Detalied Description of the Method...... 2

Validation Study...... 3

References...... 4

Appendices...... 5 Veratox® for Fluoroquinolone

DESCRIPTION OF TEST II. ELISA Procedure Fluoroquinolones are a class of antibiotics that act by inhibiting bacterial DNA 1. Add 75 µL of each Enrofloxacin Standard in duplicate to different wells gyrase, and enzyme essential for DNA synthesis. The fluoroquinolone group using a new pipette tip for each well. Add standards to wells only in includes norfloxacin, enrofloxacin, ciprofloxacin, danofloxacin, pipemidic acid, the order from low concentration to high concentration (0, 0.05, 0.1, and others. Fluoroquinolones are used in poultry production and in aquaculture. 0.2, 0.4 and 0.8 ng/mL). 2. Add 75 µL of each sample in duplicate to different wells using a new Veratox for Fluoroquinolone is a competitive enzyme-linked immunoassay (ELISA) pipette tip for each well. screening test for semi-quantitative determination of fluoroquinolone in shrimp 3. Add 75 µL of Fluoroquinolone-HRP Conjugate to each well and mix by and avian muscle. The test contains microtiter wells coated with anti-fluoroquino- gently rocking the plate manually for 1 minute. lone antibody. Sample is added to the wells along with a fluoroquinolone-horse- 4. Incubate the plate for 30 minutes at room temperature (20–25°C/ radish peroxidase conjugate. If fluoroquinolone is present in the sample, it will 68–77°F). compete with the conjugate for antibody binding sites. Following washing of the 5. Thoroughly decant or aspirate solution from the wells and discard the wells and addition of substrate-chromogen solution, color intensity is inversely liquid. Wash the plate 3 times with 250 µL 1X Wash Solution per well. proportional to the amount of fluoroquinolone in the sample. A standard curve is After the addition of wash buffer to each well, let the plate incubate at constructed using a series of fluoroquinolone standards provided with the test kit. room temperature for 20 seconds to allow equilibration of the buffer. After the last wash, invert the plate and gently tap the plate dry on DESCRIPTION OF VALIDATION STUDY paper towels. A single-laboratory validation study was conducted in accordance with Guidelines Note: Perform the next step immediately after drying the plate. Do not for the Validation of Screening Methods for Residues of Veterinary Medicine (Com- allow the plate to air dry between working steps. munity Reference Laboratories Residues CRLs 20/1/2010 (“validation guidelines” 6. Add 100 µL of TMB Substrate to each well. Incubate the plate for 20 [1]) following the criteria set forth in European Commission Decision 2002/657/EC minutes at room temperature in the dark. Time the reaction immediately (“commission decision”[2]). As the test is intended for use as a semi-quantitative after adding the substrate. Mix by sliding the plate back and forth on a screening assay, parameters selected for evaluation included detection capability flat surface for 1 minute while incubating. (CCß), precision (coefficient of variation or CV), and test applicability and rugged- Note: To avoid contamination, do not put remaining substrate back into ness. In addition, information regarding concentration range, matrices and species, the original container. Any substrate solution exhibiting coloration is matrix and laboratory conditions, and cross-reactivity is provided as required [2]. indicative of deterioration or contamination and should be discarded. Detailed Description of the Method Covering the microtiter plate during incubation is recommended. Assay Range: 0.5–80 ng/mL (parts per billion, ppb) 7. After incubation, add 100 uL Stop Buffer to each well to stop the Matrices: Muscle enzyme reaction. Species: Shrimp, avian 8. Read the plate as soon as possible following the addition of stop buffer Matrix Conditions: Controlled room temperature (20–25°C). Samples may be using a microtiter plate reader with 450 nm wavelength. stored frozen at -20oC and thawed prior to analysis. Note: Before reading, use a lint-free wipe on the bottom of the plate to Laboratory Conditions: Controlled room temperature (20–25°C/68–77°F). remove any moisture or fingerprints that may interfere with the readings. Assay Procedure III. Data Analysis Details regarding kit contents, reagent preparation instruction, storage conditions, A standard curve is constructed by plotting the mean relative absorbance and precautions are provided in the kit insert (Appendix I). (%) obtained from each reference standard against its concentration in I. Sample Extraction for Shrimp and Avian ng/mL on a logarithmic scale. 1. Remove fat from the sample. Homogenize the sample using a suitable Relative absorbance (%) = absorbance standard (or sample) X 100 mixer. absorbance zero standard 2. Weigh out 1.0 g (+ 0.05 g) of the homogenized sample into a 15 mL Use the mean relative absorbance values for each sample to determine the corre- plastic conical tube. sponding concentration of fluoroquinolone in ng/mL (ppb) from the standard curve. 3. Add 4.0 mL of a 70:30 (% v/v) mixture of methanol and deionized water. 4. Vortex the sample for 10 minutes at maximum speed using a multi-vor- The following figure represents a typical fluoroquinolone standard curve. texer, or 5 minutes manually. 5. Centrifuge the sample for 10 minutes at 4,000 x g at room temperature Fluoroquinolone Standard Curve (20–25°C/68–77°F). 100% 6. Transfer 0.5 mL of the supernatant to a new tube containing 0.5 mL of 90% 80% 1X Sample Extraction Buffer. 70% 7. Vortex the sample thoroughly for 1 minute. Incubate the sample for 10 60% 50% minutes at room temperature, then vortex the sample for an additional Absorbance 40% 30 seconds. 30% 8. Use 75 µL for the assay. Relative 20% NOTE: Dilution factor is 10. 10% 0% 0.010.1 1

Standard (ng/mL) (LN)

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VALIDATION STUDY Figure 2. Results of testing of 20 avian muscle samples 1. Determination of Assay Detection Capability (CCß, 5%) (blank and spiked at 1 ppb enrofloxacin) with the Materials and Methods Veratox for Fluoroquinolone assay Shrimp and avian muscle samples were from a North American source. 1.80 Additional information about sample source is available upon request. Shrimp 1.60 Assay detection capability in shrimp was determined through testing of 60 1.40 samples, both unspiked and spiked at a screening target concentration of pp b 1.20 1 ppb enrofloxacin. Analyses were performed by 3 different operators using two different lots of Veratox test kits. Samples were tested in duplicate and 1.00 the results averaged. The screening concentration chosen is below the 0.80 concentration, 100 ppb European Union (EU) Maximum Residue Limit (MRL) for fluoro- 0.60 quinolone in shrimp. In accordance with the validation guidelines [1], at 0.40 least 20 samples are required when the screening concentration is 50% Enrofloxacin of the MRL. 60 samples are required when the screening concentration is 0.20 between 90 and 100% of the MRL. 0.00 Avian 0510 15 20 Sample Procedures were as described above except that 20 samples were tested. Blank Samples Threshold Value (T) Mean Blank The screening concentration was set at 1 ppb enrofloxacin, again below Spiked Samples Cut-off Factor (Fm) Mean Spike the 100 ppb EU the MRL for fluoroquinolone in avian muscle. Testing of at least 20 samples is indicated when validating a second matrix or species. Results Values for calculation of detection capability CCß (5%) and determination of Results of testing of blank and spiked shrimp and avian muscle samples are guideline compliance are shown in Table 1 (see Appendices I and II for raw data): presented as Figures 1 and 2, respectively. Raw data for shrimp and avian Table 1. Performance characteristic calculations for the samples are included as Appendices I and II, respectively. Veratox for Fluoroquinolone assay

Figure 1. Results of testing of 60 shrimp samples Result (ppb) (blank and spiked at 1 ppb enrofloxacin) with the Parameter Shrimp Avian Veratox for Fluoroquinolone assay Mean of blank sample values (B) 0.15 0.50 Standard deviation of blank sample values (SDb) 0.09 0.13 1.40 Threshold value (T = B + 1.64*SDb) 0.30 0.71

1.20 Mean of spiked sample values (M) 0.90 1.16 pp b

1.00 Standard deviation of spiked sample values (SD) 0.22 0.25

0.80 Cutoff factor (Fm = M – 1.64*SD) 0.54 0.75

concentration, Detection capability is at or below spiking level (CCß) 1 1 0.60 Based on these results, the detection capability (CCß, 5%) of the Veratox for 0.40 Fluoroquinolone test in shrimp is determined to be 1 ppb, below the EU MRL of 0.20 Enrofloxacin 100 ppb. CCß for the test in avian muscle is calculated to be 1 ppb, below the 0.00 EU MRL of 100 ppb for fluoroquinolones in this matrix. 0102030405060 For shrimp, the highest response in the blank sample population was 0.41 ppb. Sample The lowest response in the spiked population was 0.46 ppb. There was 1 false Blank Samples Threshold Value (T) Mean Blank compliant result. Up to 3 are allowed in a 60-sample data set, thus results are Spiked Samples Cut-off Factor (Fm) Mean Spike compliant with the criteria of Annex I of the validation guidelines [1]. For avian, the highest response in the blank sample population was 0.77 ppb, and the lowest response in the spiked population was also 0.77 ppb. There were no false compliant results. Results are compliant with the Annex I criteria. Further, according to Annex II of the validation guidelines, the detection capability is validated when Fm > B. This is true for both matrices. Also according to Annex II, the false positive rate of the Veratox assay is determined to be below 5% for both matrices, as Fm > T in both cases.

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With respect to the determination of detection capability, results meet all criteria Table 2. Cross-reactivity testing results for the of Annex I and Annex II of the validation guidelines for shrimp and avian. Veratox for Fluoroquinolone assay

2. Determination of Assay Precision Compound Cross-Reactivity (%) Materials and Methods Enrofloxacin 100 Precision testing was conducted using 20 shrimp samples, both blank and spiked with 1 ppb enrofloxacin as for the detection capability trials. Samples Ciprofloxacin 100 were tested in duplicate and the results averaged. Testing was conducted Difloxacin 100 in 1 day by 1 operator using a single Veratox kit lot. Sarafloxacin 100 Results Norfloxacin 143 Results of precision testing are shown in Figure 3. Raw data is included as Appendix III. The mean fluoroquinolone result was 0.78 ppb, with SD of 0.17 Oxolinic acid 112 ppb and CV of 22%. The commission decision discusses using the Horwitz Pipemidic acid 102 Equation for determination of acceptable coefficient of variation values for Ofloxacin 97 both intra-laboratory and inter-laboratory data. However, it is noted in the Danofloxacin 73 decision that concentrations lower than 100 µg/kg (100,000 ppb) produce unacceptably high values. In this case, relevant to this study, CVs should Flumequin 62 be “as low as possible”. Discussion and Conclusions Figure 3. Precision testing results for the Veratox for Fluoroquinolone Results of the validation study described here demonstrate that the Veratox assay using 20 shrimp samples (blank and spiked at 1 ppb enrofloxacin) for Fluoroquinolone assay is an accurate and robust screening method for Precision results for avian can be derived from the detection capability data (see semi-quantitative determination of fluoroquinolone concentration in shrimp and Appendix III): avian muscle. In experiments following guidelines according to European Com- mission Decision 2002/657/EC, the detection capability (CCß, 5%) of the assay 1.20 was determined to be 1 ppb in shrimp (below the EU MRL of 100 ppb), and 1

1.00 ppb in avian (below the EU MRL of 100). The assay showed very good precision, with CVs of 22% or less. Specificity testing showed strong reactivity with 10 0.80 fluoroquinolone-class antibiotics.

0.60 The Veratox assay is an accurate and reliable tool that can enable regulatory agencies and food producers to detect fluoroquinolone in shrimp and avian 0.40 samples in response to customer food quality and safety concerns. The assay is

Enrofloxacin cocentration, ppb a cost effective and easy-to-use diagnostic tool compared to instrument based 0.20 methods such as HPLC and GC. 0.00 1234567891011121314151617181920 REFERENCES Sample 1. Community Reference Laboratories Residues (CRLs) (2010) Guidelines for the validation of screening methods for residues of veterinary medicines Mean = 0.78 ppb 20/1/2010 SD = 0.17 ppb 2. European Commission (2002) Commission decision 2002/657/EC (imple- CV = 22% menting council directive 96/23/EC concerning the performance of analytical methods and the interpretation of results) 3. Specificity Assay( Cross-Reactivity) Materials and Methods Specificity of the Veratox assay was assessed by measuring cross-reactivity of the assay with a number of antibiotics belonging to the fluoroquinolone class. Varying concentrations of the drugs were tested in the assay and standard curves constructed. Reactivity of the target drug is compared to

that of enrofloxacin. Cross-reactivity is expressed as CRTar = IC50Ref/IC50Tar) x 100 where CR = cross-reactivity (%), Tar = target drug, Ref = reference drug

(enrofloxacin), and IC50 = analyte concentration that results in B/B0 of 50%. Results Results are shown in Table 2. All fluoroquinolone drugs tested were strongly reactive in the assay, with cross-reactivity ranging from 62% to 143% relative to reactivity with enrofloxacin.

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Appendix I. Results of testing of 60 shrimp samples (blank and spiked at 1 ppb enrofloxacin) with the Veratox for Fluoroquinolone assay

Shrimp Unspiked Samples Shrimp Samples Spiked at 1.0 ppb Enrofloxacin

Abs Measured Abs Measured Sample Abs1 Abs2 SD %CV Abs1 Abs2 SD %CV % Recovery Avg ppb Avg ppb

1 1 2.604 2.547 2.576 0.040 1.6 0.00 1.922 1.844 1.883 0.056 2.9 0.86 85.8 2 2 2.610 2.632 2.621 0.016 0.6 0.00 1.981 1.868 1.924 0.080 4.1 0.80 79.7 3 3 2.412 2.386 2.399 0.018 0.8 0.18 2.098 2.012 2.055 0.061 3.0 0.62 61.6 4 5 2.426 2.446 2.436 0.014 0.6 0.14 1.757 1.732 1.744 0.018 1.0 1.08 107.7 5 8 2.457 2.429 2.443 0.019 0.8 0.13 1.829 1.794 1.812 0.025 1.4 0.97 96.8 6 9 2.452 2.361 2.406 0.064 2.7 0.18 2.145 2.205 2.175 0.042 1.9 0.46 45.9 7 15 2.492 2.485 2.488 0.004 0.2 0.07 2.097 2.043 2.070 0.038 1.8 0.60 59.6 8 4 2.444 2.431 2.437 0.009 0.4 0.17 1.964 1.995 1.980 0.022 1.1 0.83 82.6 9 7 2.454 2.393 2.424 0.043 1.8 0.18 1.886 1.905 1.895 0.013 0.7 0.97 97.2 10 10 2.485 2.462 2.474 0.016 0.7 0.12 1.989 1.952 1.970 0.027 1.3 0.84 84.2 11 11 2.388 2.417 2.403 0.020 0.9 0.21 2.252 2.081 2.167 0.121 5.6 0.54 53.6 12 13 2.388 2.402 2.395 0.010 0.4 0.22 1.979 2.003 1.991 0.017 0.9 0.81 80.7 13 16 2.439 2.450 2.444 0.008 0.3 0.16 1.967 2.098 2.033 0.093 4.6 0.74 74.0 14 17 2.456 2.437 2.447 0.014 0.6 0.15 2.024 1.953 1.989 0.050 2.5 0.81 81.2 15 6 2.601 2.602 2.602 0.000 0.0 0.05 2.029 2.109 2.069 0.056 2.7 0.68 67.5 16 12 2.568 2.593 2.581 0.018 0.7 0.07 1.929 1.914 1.922 0.010 0.5 0.91 90.6 17 14 2.579 2.485 2.532 0.067 2.6 0.12 1.853 1.919 1.886 0.047 2.5 0.97 96.7 18 18 2.642 2.645 2.644 0.002 0.1 0.01 2.126 2.021 2.074 0.075 3.6 0.67 66.8 19 19 2.590 2.548 2.569 0.030 1.2 0.08 1.868 1.963 1.915 0.067 3.5 0.92 91.7 20 20 2.620 2.702 2.661 0.058 2.2 0.00 2.167 2.161 2.164 0.004 0.2 0.54 54.2 21 2 2.439 2.391 2.415 0.034 1.4 0.32 1.800 1.880 1.840 0.057 3.1 1.05 104.7 22 5 2.653 2.588 2.620 0.046 1.7 0.08 1.898 1.850 1.874 0.034 1.8 1.00 99.7 23 6 2.541 2.583 2.562 0.030 1.2 0.15 1.861 1.886 1.874 0.018 0.9 1.00 99.8 24 10 2.509 2.550 2.530 0.029 1.2 0.19 1.879 1.897 1.888 0.013 0.7 0.98 97.7 25 11 2.553 2.608 2.580 0.039 1.5 0.13 2.042 1.996 2.019 0.033 1.6 0.80 79.7 26 12 2.615 2.612 2.613 0.002 0.1 0.09 1.635 1.657 1.646 0.016 1.0 1.36 136.2 27 13 2.536 2.591 2.563 0.039 1.5 0.15 1.901 2.062 1.982 0.114 5.7 0.85 84.7 28 1 2.595 2.476 2.535 0.084 3.3 0.06 1.674 1.776 1.725 0.072 4.2 1.12 112.1 29 3 2.551 2.504 2.527 0.033 1.3 0.07 1.827 1.771 1.799 0.040 2.2 1.00 99.8 30 4 2.565 2.543 2.554 0.016 0.6 0.04 1.804 1.845 1.824 0.029 1.6 0.96 95.8 31 7 2.470 2.498 2.484 0.019 0.8 0.12 1.616 1.758 1.687 0.100 5.9 1.19 118.7 32 8 2.520 2.383 2.451 0.097 3.9 0.16 1.811 1.898 1.855 0.062 3.3 0.91 91.1 33 9 2.327 2.358 2.343 0.022 0.9 0.28 1.587 1.567 1.577 0.015 0.9 1.39 139.3

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Appendix I. (continued)

34 23 2.610 2.518 2.564 0.065 2.5 0.03 1.870 1.876 1.873 0.004 0.2 0.88 88.3 35 15 2.576 2.521 2.548 0.039 1.5 0.15 1.660 1.462 1.561 0.141 9.0 1.37 137.4 36 16 2.624 2.585 2.605 0.027 1.1 0.09 1.684 1.630 1.657 0.038 2.3 1.22 121.9 37 17 2.508 2.418 2.463 0.064 2.6 0.25 1.861 1.664 1.762 0.140 7.9 1.06 106.3 38 19 2.446 2.452 2.449 0.005 0.2 0.26 1.964 1.892 1.928 0.051 2.6 0.84 84.3 39 20 2.469 2.394 2.431 0.053 2.2 0.28 2.019 1.956 1.987 0.045 2.2 0.77 77.0 40 21 2.405 2.276 2.341 0.091 3.9 0.38 1.911 1.900 1.905 0.007 0.4 0.87 87.1 41 1 2.507 2.513 2.510 0.004 0.2 0.17 1.857 1.929 1.893 0.051 2.7 1.18 117.9 42 6 2.476 2.419 2.448 0.040 1.7 0.27 1.890 1.775 1.833 0.081 4.4 1.29 129.3 43 8 2.619 2.551 2.585 0.048 1.9 0.03 2.113 2.073 2.093 0.028 1.3 0.83 82.9 44 10 2.533 2.497 2.515 0.025 1.0 0.16 1.911 1.825 1.868 0.061 3.3 1.23 122.5 45 13 2.565 2.511 2.538 0.038 1.5 0.12 2.104 2.168 2.136 0.046 2.1 0.76 75.9 46 23 2.531 2.584 2.558 0.037 1.5 0.09 2.079 1.952 2.016 0.090 4.5 0.96 96.0 47 4 2.365 2.352 2.358 0.009 0.4 0.18 2.103 2.091 2.097 0.009 0.4 0.83 82.8 48 5 2.418 2.351 2.384 0.048 2.0 0.13 2.082 1.974 2.028 0.076 3.8 1.02 102.0 49 7 2.334 2.374 2.354 0.028 1.2 0.19 2.054 1.983 2.018 0.051 2.5 1.05 104.7 50 12 2.321 2.307 2.314 0.010 0.4 0.29 1.981 1.978 1.980 0.002 0.1 1.16 116.0 51 14 2.312 2.292 2.302 0.014 0.6 0.31 2.010 2.139 2.074 0.091 4.4 0.89 89.0 52 15 2.262 2.265 2.263 0.002 0.1 0.41 2.059 2.108 2.084 0.034 1.7 0.86 86.4 53 24 2.389 2.414 2.402 0.018 0.7 0.09 2.200 2.197 2.199 0.002 0.1 0.56 56.5 54 2 2.294 2.338 2.316 0.031 1.3 0.16 1.639 1.618 1.628 0.015 0.9 0.86 85.8 55 3 2.354 2.322 2.338 0.023 1.0 0.15 1.810 1.848 1.829 0.026 1.4 0.60 60.4 56 9 2.147 2.195 2.171 0.034 1.6 0.28 1.516 1.598 1.557 0.058 3.7 0.96 96.2 57 17 2.470 2.456 2.463 0.010 0.4 0.06 1.573 1.831 1.702 0.183 10.7 0.76 75.8 58 19 2.442 2.420 2.431 0.015 0.6 0.08 1.831 1.765 1.798 0.047 2.6 0.64 64.0 59 22 2.504 2.490 2.497 0.010 0.4 0.04 1.827 1.738 1.782 0.063 3.5 0.66 65.9 60 23 2.419 2.414 2.417 0.003 0.1 0.09 1.733 1.947 1.840 0.151 8.2 0.59 59.3 Mean 2.472 0.15 Global Ave Abs 1.907 0.90 Standard Dev 0.103 0.09 Standard Dev 0.159 0.22 %CV 4.2 NM %CV 8.4 24.2 Minimum Lowest 0.0 Lowest 0.46 Maximum Highest 0.41 Highest 1.4 110.0

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Appendix II. Results of testing of 20 avian muscle samples (blank and spiked at 1 ppb enrofloxacin) with the Veratox for Fluoroquinolone assay

Avian Unspiked Samples Avian Samples Spiked at 1.0 ppb Enrofloxacin

Abs Measured Abs Measured Sample Abs1 Abs2 SD %CV Abs1 Abs2 SD %CV % Recovery Avg ppb Avg ppb

1 1 1.920 1.987 1.954 0.047 2.4 0.77 1.475 1.468 1.471 0.005 0.3 1.73 173.4 2 2 2.134 2.125 2.129 0.006 0.3 0.49 1.517 1.557 1.537 0.028 1.8 1.58 157.8 3 3 2.014 2.085 2.050 0.050 2.4 0.62 1.633 1.867 1.750 0.166 9.5 1.13 113.4 4 4 2.028 2.122 2.075 0.066 3.2 0.58 1.667 1.636 1.652 0.022 1.3 1.33 132.8 5 5 1.995 2.162 2.078 0.118 5.7 0.57 1.696 1.559 1.628 0.097 5.9 1.38 137.8 6 6 2.051 2.091 2.071 0.028 1.4 0.58 1.499 1.663 1.581 0.116 7.3 1.48 147.9 7 7 2.031 2.025 2.028 0.004 0.2 0.65 1.583 1.702 1.643 0.084 5.1 1.35 134.6 8 8 2.158 2.211 2.184 0.038 1.7 0.37 1.730 1.838 1.784 0.076 4.3 1.03 102.8 9 9 2.221 2.188 2.204 0.024 1.1 0.34 1.642 1.796 1.719 0.108 6.3 1.16 115.8 10 10 2.067 2.088 2.077 0.015 0.7 0.53 1.696 1.777 1.737 0.057 3.3 1.12 112.1 11 11 2.080 2.134 2.107 0.038 1.8 0.48 1.739 1.780 1.760 0.029 1.7 1.07 107.5 12 12 2.078 2.165 2.121 0.061 2.9 0.46 1.770 1.724 1.747 0.033 1.9 1.10 110.1 13 13 2.126 2.252 2.189 0.089 4.1 0.36 1.856 1.879 1.867 0.017 0.9 0.87 87.1 14 14 2.055 2.154 2.104 0.070 3.3 0.49 1.841 1.766 1.803 0.053 2.9 0.99 99.0 15 15 1.960 2.010 1.985 0.036 1.8 0.46 1.542 1.518 1.530 0.017 1.1 1.21 120.9 16 16 2.212 2.235 2.223 0.016 0.7 0.19 1.764 1.550 1.657 0.152 9.1 0.96 96.1 17 17 1.923 1.808 1.865 0.081 4.4 0.63 1.722 1.720 1.721 0.002 0.1 0.85 85.1 18 18 1.891 1.873 1.882 0.013 0.7 0.60 1.516 1.585 1.551 0.049 3.2 1.17 116.6 19 19 1.977 1.942 1.960 0.025 1.3 0.50 1.614 1.644 1.629 0.021 1.3 1.01 101.3 20 20 2.084 1.969 2.026 0.082 4.0 0.41 1.710 1.833 1.772 0.087 4.9 0.77 76.8 Mean 2.066 0.50 Global Ave Abs 1.677 1.16 Standard Dev 0.100 0.13 Standard Dev 0.106 0.25 %CV 4.9 NM %CV 6.3 21.3 Minimum Lowest 0.2 Lowest 0.8 Maximum Highest 0.8 Highest 1.7 110.0

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Appendix III. Results of precision testing for the Veratox for Fluoroquinolone assay (shrimp samples blank and spiked with 1 ppb enrofloxacin)

Shrimp Unspiked Samples Shrimp Samples Spiked at 1.0 ppb Enrofloxacin

Measured Measured Sample Abs1 Abs2 Abs Avg SD %CV Abs1 Abs2 Abs Avg SD %CV % Recovery ppb ppb

1 2.604 2.547 2.576 0.040 1.6 0.00 1.922 1.844 1.883 0.056 2.9 0.86 85.8 2 2.610 2.632 2.621 0.016 0.6 0.00 1.981 1.868 1.924 0.080 4.1 0.80 79.7 3 2.412 2.386 2.399 0.018 0.8 0.18 2.098 2.012 2.055 0.061 3.0 0.62 61.6 4 2.426 2.446 2.436 0.014 0.6 0.14 1.757 1.732 1.744 0.018 1.0 1.08 107.7 5 2.457 2.429 2.443 0.019 0.8 0.13 1.829 1.794 1.812 0.025 1.4 0.97 96.8 6 2.452 2.361 2.406 0.064 2.7 0.18 2.145 2.205 2.175 0.042 1.9 0.46 45.9 7 2.492 2.485 2.488 0.004 0.2 0.07 2.097 2.043 2.070 0.038 1.8 0.60 59.6 8 2.444 2.431 2.437 0.009 0.4 0.17 1.964 1.995 1.980 0.022 1.1 0.83 82.6 9 2.454 2.393 2.424 0.043 1.8 0.18 1.886 1.905 1.895 0.013 0.7 0.97 97.2 10 2.485 2.462 2.474 0.016 0.7 0.12 1.989 1.952 1.970 0.027 1.3 0.84 84.2 11 2.388 2.417 2.403 0.020 0.9 0.21 2.252 2.081 2.167 0.121 5.6 0.54 53.6 12 2.388 2.402 2.395 0.010 0.4 0.22 1.979 2.003 1.991 0.017 0.9 0.81 80.7 13 2.439 2.450 2.444 0.008 0.3 0.16 1.967 2.098 2.033 0.093 4.6 0.74 74.0 14 2.456 2.437 2.447 0.014 0.6 0.15 2.024 1.953 1.989 0.050 2.5 0.81 81.2 15 2.601 2.602 2.602 0.000 0.0 0.05 2.029 2.109 2.069 0.056 2.7 0.68 67.5 16 2.568 2.593 2.581 0.018 0.7 0.07 1.929 1.914 1.922 0.010 0.5 0.91 90.6 17 2.579 2.485 2.532 0.067 2.6 0.12 1.853 1.919 1.886 0.047 2.5 0.97 96.7 18 2.642 2.645 2.644 0.002 0.1 0.01 2.126 2.021 2.074 0.075 3.6 0.67 66.8 19 2.590 2.548 2.569 0.030 1.2 0.08 1.868 1.963 1.915 0.067 3.5 0.92 91.7 20 2.620 2.702 2.661 0.058 2.2 0.00 2.167 2.161 2.164 0.004 0.2 0.54 54.2 2.499 0.11 Global Ave Abs 1.986 0.78 0.090 0.07 Standard Dev 0.117 0.17 3.6 NM %CV 5.9 21.8 Lowest 0.0 Lowest 0.5 Highest 0.2 Highest 1.1 110.0

Appendix IV. Veratox for Fluoroquinolone kit insert

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