Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed
Abstract
:1. Introduction
2. Results and Discussion
2.1. Highlights of Major Modification of the Improved Aflatoxin Test Procedure
- (a)
- Primary sampling or sample selection (number and size of incremental samples, type of sampling tools for open and closed sub-lots, random procedure, size of incremental and aggregate samples, Section 4.1.1)
- (b)
- Secondary sampling (size of laboratory sample determined employing FAO Mycotoxin Sampling Tool, coning and quartering method improved by performing all coning and shoveling procedures under a steadfast funnel for mass-reduction of aggregate sample to 2 kg laboratory sample, Section 4.1.2)
- (c)
- Tertiary sampling (size of test portion determined employing FAO Mycotoxin Sampling Tool, the improved coning and quartering method in Point (b) for mass-reduction of laboratory sample to 25 g test portion, Section 4.1.3)
- (d)
- Quaternary sampling (homogenization and splitting of test portion by water slurring at matrix/water, 25:37.5, w/w; optimal matrix to organic solvent ratio for solid–liquid extraction, slurry/extraction solvent, 1.3:86.5, w/v, Section 4.1.4)
- (e)
- Quantification of AFB1 (optimal organic solvent to aqueous buffer ratio for AFB1 extraction back to aqueous phase-modified extract to aqueous buffer mixture was modified to 80% acetonitrile extraction solvent: PBS-T mixture, 100:650, v/v, Section 4.1.5)
2.2. Enzyme-Linked Immunosorbent Assay of Prepared Standards for Determination of Aflatoxin Content in Chicken Feed Samples
2.3. Validation Results of the Improved Aflatoxin Test Procedure
2.3.1. Method Accuracy and Precision
2.3.2. Limits of Detection (LOD) and Quantification (LOQ) Values
2.3.3. Linearity
2.3.4. Robustness and Ruggedness of the Aflatoxin Extraction Procedure
2.3.5. Evaluation of the Improved Aflatoxin Test Procedure
3. Conclusions
4. Materials and Methods
4.1. Description of Improved Aflatoxin Test Procedure
4.1.1. Sample Selection (Primary Sampling)
4.1.2. Comminution and Mass Reduction of Samples (Secondary Sampling)
4.1.3. Preparation of Test Portions (Tertiary Sampling)
4.1.4. Sample Extraction (Quaternary Sampling)
4.1.5. Quantification of AFB1 in Feed Samples
4.2. In-House Validation of Improved Aflatoxin Test Procedure
4.2.1. Materials
4.2.2. Study Design
4.2.3. Aflatoxin Spike and Recovery Studies
Comparison of Conventional (Dry) and Novel Water Slurry (Wet) Milling Procedure
Comparison of Different Extraction Conditions Associated with Wet Milling Procedure
4.2.4. Replication Studies
Estimation of Intra-Laboratory Variability (Repeatability)
Estimation of Intermediate Variability Associated with Wet Milling Procedure
4.2.5. Determination of Limits of Detection (LOD) and Quantification (LOQ)
4.2.6. Linearity Studies
4.2.7. Robustness and Ruggedness Studies
4.2.8. Evaluation of the Improved Aflatoxin Test Procedure
4.3. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameters Values of 4-Parameter Logistic Curve | |||||
---|---|---|---|---|---|
Maximum signal intensity (a) | Slope at inflection point (b) | Concentration at inflection point ((50% B/B0), IC50 (c) | Minimum signal intensity (d) | Coefficients of correlation (r) | Coefficients of determination (r2) |
98.9 | 1.58 | 0.072 ng/mL | 5.6 | 0.998 | 0.997 |
Feed Aliquot Group | Extraction Conditions ε | Study |
---|---|---|
I (R = 6) * | (i) dry milling (ii) matrix/ACN a (25:133, w/v); (iii) ACN a extract/PBS-T b (10.6:989.4, v/v), (iv) final dilution factor = 500 | Spike and recovery (comparison of dry and wet milling) |
II (R = 6) * | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (2:133, w/v); (iii) ACN a extract/PBS-T b (100:650, v/v), (iv) final dilution factor = 1247 | |
III (R = 10) * | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:130, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v), (iv) final dilution factor = 2500 | Spike and recovery studies (comparison of three extraction conditions used in wet milling) |
IV (R = 10) * | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:65, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v), (iv) final dilution factor = 1250 | |
V (R = 10) * | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:86.5, w/v); (iii) ACN a extract/PBS-T b (100:650, v/v), (iv) final dilution factor = 1247 | |
VI (R = 10) | (i) dry milling (ii) matrix/ACN a (25:130, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v), (iv) final dilution factor = 52 | Intra-laboratory variability (Repeatability) |
VII (R = 10) | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:130, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v), (iv) final dilution factor = 2500 | |
VIII (R = 10) | (i) dry milling (ii) matrix/ACN a (1.3:130, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v), (iv) final dilution factor = 1000 | |
IX (R = 10) | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:130, w/v); (iii) ACN a extract/PBS-T b (100:900, v/v) (iv) final dilution factor = 2500 | |
X (R = 15) | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry n/ACN a, (1.3:130, w/v); (iii) ACN a extract: PBS-T b (100:900, v/v); (iv) final dilution factor = 2500 | Intermediate variability (within-laboratory reproducibility) |
XI (R = 15) | (i) wet milling @ matrix/water (25:37.5, w/w); (ii) slurry/ACN a, (1.3:86.5, w/v); (iii) ACN a extract/PBS-T b (100:650, v/v) (iv) Final dilution factor = 1247 | |
XII (R = 4) | (i) dry milling (ii) ACN a extract/PBS-T b (1:1000, v/v) (iii) final dilution factor = 1000 | Determination of LOD and LOQ |
XIII (R = 5) | (i) dry milling ACN a extract/ACN a/PBS-T b (1:5:100, v/v/v) (ii) final dilution factor = 500 | |
XIV (R = 5) | (i) wet milling (ii) ACN a extract/PBS-T b (1:1247, v/v) (iii) final dilution factor = 1247 |
Milling Method | Group | Aflatoxin Extraction Conditions | * Mean AFB1 % Recovery | % Bias | Measurement Variability | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Expected Concentration | Size of Analytical Sample | DF of Water Slurry | DF in ACN * | DF in PBS-T b | Final DF | StDev. | CV | CV Effect | HorRat ** Value | ||||
Dry | I (R = 6) | 20 ppb (R = 3) | 25 g | - | 5.3 | 94.3 | 500 | 57 | ±6 | 10.9 | 0.57 | ||
100 ppb (R = 3) | 103 | ±13 | 12.4 | 0.82 | |||||||||
All replicates | 80 a | −20 | ±26 | 33.1 | 9.5 R4 | 0.57–0.82 | |||||||
Wet | II (R = 6) | 20 ppb (R = 3) | 2 g | 2.5 | 66.5 | 7.5 | 1247 | 119 | ±25 | 21.2 | 0.95 | ||
100 ppb (R = 3) | 123 | ±9 | 7.4 | 0.8 | |||||||||
All replicates | 121 a | 21 | ±17 | 14.1 h | −9.5 R2 | 0.8–0.95 | |||||||
Wet | III (R = 10) | 20 ppb (R = 5) | 1.3 g | 2.5 | 100 | 10 | 2500 | 145 | ±19 | 12.8 | 0.58 | ||
100 ppb (R = 5) | 112 | ±9 | 17.4 | 0.44 | |||||||||
All replicates | 129 b,c | 29 | ±22 | 17.2 | −3.8 R3 | 0.44–0.58 | |||||||
IV (R = 10) | 20 ppb (R = 5) | 1.3 g | 2.5 | 50 | 10 | 1250 | 49 | ±12 | 23.2 | 1.04 | |||
100 ppb (R = 5) | 85 | ±15 | 18.1 | 1.04 | |||||||||
All replicates | 67 b | −33 | ±23 | 34.2 | 13.3 R5 | 1.04 | |||||||
V (R = 10) | 20 ppb (R = 5) | 1.3 g | 2.5 | 66.5 | 7.5 | 1247 | 80 | ±13 | 15.8 | 0.71 | |||
100 ppb (R = 5) | 80 | ±6 | 6.9 | 0.39 | |||||||||
All replicates | 80 c | −20 | ±9 | 11.5 | −9.5 R1 | 0.39–0.71 | |||||||
Groups II and V replicates analyzed together (R = 16) | 20 ppb (R = 8) | - | 2.5 | 66.5 | 7.5 | 1247 | 94 | ±26 | 27.6 | 1.24 | |||
100 ppb (R = 8) | 96 | ±23 | 24.1 | 1.38 | |||||||||
All replicates | 95 | −5 | ±24 | 25.0 | 1.24–1.38 |
Sampling Stage | Intra-Laboratory Assay Precision (Within-Laboratory Repeatability) | Intermediate Precision (Within-Laboratory Reproducibility) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Group Number/ Mass Reduction Method | Milling Method and Size of Analytical Sample | EC | AFB1 Level (ppb) Mean ± Sd. | Variability | Group Number/ Mass Reduction Method | Milling Method and Size of Analytical Sample | EC | AFB1 Level (ppb) Mean ± Sd | Variability | |||
RSDr | HorRat ValueEffect | RSDR | HorRat Value Effect | |||||||||
Secondary | Group VI Coning and quartering (R = 10) | Dry 25 g | 1 | 15 ± 1 | 6.5 | 0.3 H1 | - | |||||
Tertiary | Group VII Water slurry (R = 10) | Wet 1.3 g | 2 | 68 ± 17 | 24.5 | 1.5 H5 | Group X Water slurry (R = 15) | Wet 1.3 g | 2 | 69 ± 21 | 30.3 | 1.3 H4 |
Tertiary | Group VIII Coning and quartering (R = 10) | Dry 1.3 g | 3 | 73 ± 67 | 91.5 | 5.8 H6 | Group XI Water slurry (R = 15) | Wet 1.3 g | 4 | 19 ± 5 | 26.9 | 0.9 H2 |
Tertiary | Group IX Water slurry (R = 10) | Wet 1.3 g | 2 | 50 ± 10 | 20.6 | 1.2 H3 | - |
Baseline Response (OD) Statistics | Limits of Detection and Quantification in µg AFB1 Per kg of Chicken Feed | ||
---|---|---|---|
Dry Milling | Wet Milling | ||
Group XII (R = 4) (Final Dilution Factor = 1000) | Group XIII (R = 5) (Final Dilution Factor = 500) | Group XIV (R = 5) (Final Dilution Factor = 1247) | |
Mean-2StDev (Limit of detection) | 16.3 | 10.6 | 7.5 |
Mean-5StDev (Limit of quantification) | 31 | 22.3 | 16 |
Baseline response (OD) statistics | Magnitude of signal, noise, their ratio and precision (absorbance) | ||
Group XII | Group XIII | Group XIV | |
B0 | 1.773 | 1.752 | 1.871 |
StDev (blank) | 0.0399 | 0.0485 | 0.0339 |
Signal/noise ratio | 44.44 | 36.12 | 55.19 |
Coefficient of variance | 2.34 | 2.99 | 1.97 |
Concentration and Optical Density of AFB1 Standard Solutions | ||||||
---|---|---|---|---|---|---|
Solution 1 | Solution 2 | Solution 3 | Solution 4 | Solution 5 | Solution 6 | |
Spiked AFB1 level (ng/mL) | 0.02 | 0.05 | 0.1 | 0.2 | 0.4 | 0.5 |
Measured level (ng/mL) | 0.03 | 0.06 | 0.12 | 0.28 | 0.36 | >0.4 |
Measured optical density | 1.429 | 1.106 | 0.663 | 0.297 | 0.226 | 0.199 |
Number of Replicates Required to Attain Acceptable Intra-Assay Precision | |||||
---|---|---|---|---|---|
Two Replicates Required | Three Replicates Required | Four Replicates Required | Five Replicates Required | Six Replicates Required | |
Number and percentage of samples analyzed | 188 (74.9%) | 51 (20.3%) | 8 (3.2%) | 2 (0.8%) | 2 (0.8%) |
Study Reference | Characteristics of Aflatoxin Test Procedures | ||||
---|---|---|---|---|---|
Homogenization Method | Aggregate Sample (kg) | Laboratory Sample (kg) | Size of Test Portion | Analytical Method | |
[15] | Dry milling | 4 | Not given | 5 g | LC-MS/MS |
[16] | Dry milling | 1–2 | 1 | 5 g | ELISA |
[19] | Dry milling | 1 | 1 | 5 g | LC-MS/MS |
[32] | Dry milling | Not given | Not given | Not given | HPLC/ELISA |
[18] | Dry milling | Not given | Not given | 50 g | VICAM Fluorimeter |
[13] | Not given | Not given | Not given | Not given | Not given |
[14] | Not given | 1 | Not given | Not given | TLC |
Novel method | Wet milling (water slurring) | >2 | 2 | at least 25 g × 2 | ELISA or any other quantification method |
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Kibugu, J.; Mdachi, R.; Munga, L.; Mburu, D.; Whitaker, T.; Huynh, T.P.; Grace, D.; Lindahl, J.F. Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed. Toxins 2021, 13, 216. https://doi.org/10.3390/toxins13030216
Kibugu J, Mdachi R, Munga L, Mburu D, Whitaker T, Huynh TP, Grace D, Lindahl JF. Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed. Toxins. 2021; 13(3):216. https://doi.org/10.3390/toxins13030216
Chicago/Turabian StyleKibugu, James, Raymond Mdachi, Leonard Munga, David Mburu, Thomas Whitaker, Thu P. Huynh, Delia Grace, and Johanna F. Lindahl. 2021. "Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed" Toxins 13, no. 3: 216. https://doi.org/10.3390/toxins13030216
APA StyleKibugu, J., Mdachi, R., Munga, L., Mburu, D., Whitaker, T., Huynh, T. P., Grace, D., & Lindahl, J. F. (2021). Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed. Toxins, 13(3), 216. https://doi.org/10.3390/toxins13030216