TY - JOUR
AU - Marley, Elaine
AB - Abstract Background Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. Objective A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. Method AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. Results The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81–112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4–12% RSD and intermediate precisions of 5–13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. Conclusions A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. Highlights An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products. The most abundant aflatoxins found in plant-based foods are B1, B2, G1, and G2; they are secondary metabolites produced by species of Aspergillus fungi, particularly A. flavus and A. parasiticus (1). Cows that are fed with stored grain-based feed spoilt by Aspergillus fungi hepatically hydroxylate approximately 5% of the aflatoxin B1 to produce aflatoxin M1 (AFM1, Figure 1), the major aflatoxin metabolite expressed in milk (2–4). Because AFM1 is heat stable, it is potentially found in pasteurized milk and other dairy products derived from contaminated raw milk, and countries therefore require dairy products to be tested for and certified free of AFM1 as a requirement for international trade (5, 6). AFM1 is currently not found in New Zealand as the local climate does not favor Aspergillus growth (pre harvest) and dairy cows in New Zealand are predominantly fed on local pasture or pasture-based silage (7, 8). Figure 1. Open in new tabDownload slide Structure of aflatoxin M1. Figure 1. Open in new tabDownload slide Structure of aflatoxin M1. AFM1 has been detected in milk and other dairy products produced in many areas around the world, including countries from Africa, Europe, the Middle East, Asia, and North and South America (9–13). This compound has been shown to cause hepatocellular carcinomas and renal damage, and is cytotoxic as well as mutagenic (14–17). Because of these adverse health effects, the level of AFM1 allowed in milk and other dairy products is regulated in many countries (6, 11, 13). In the past, AFM1 was analyzed using HPTLC, but is now more commonly analyzed using chromatography with fluorescence detection or MS, or by immunoassay techniques such as enzyme-linked immunosorbent assay, thin film electrochemical biosensors, and optical biosensors (18–23). As AFM1 is usually regulated and present at very low concentrations (ng/L), extracts need to be concentrated using either immunoaffinity or C18 solid phase extraction cartridges before chromatographic analysis (19, 24, 25). For the high-throughput analysis of aflatoxins, R-Biopharm has developed an automated immunoaffinity cartridge system (RIDA®CREST ICE) to replace the manual operation required with conventional immunoaffinity columns (26). This device allows for a higher sample throughput and improved precision, and is less prone to blockage compared with manual immunoaffinity columns. The purpose of this study was to develop and validate a high-throughput method for the routine analysis of AFM1 in whey protein concentrate (WPC), whey protein isolate (WPI), whey powder (WP), milk protein concentrate (MPC), liquid milk, and cheese using RIDACREST ICE coupled to HPLC‒fluorescence. METHOD Apparatus Pipettes.—Eppendorf Research® Plus 2–20, 20–200, 100–1000, 1000–10 000 microlitres (Eppendorf, Hauppauge, NY). Centrifuge.—Heraeus Multifuge X3 centrifuge (ThermoFisher, Waltham, MA). Centrifuge tubes.—Polypropylene 15 and 50 mL (ThermoFisher). Vortex mixer.—Genius 3 (IKA, Wilmington, NC). Analytical balance.—AE 260 analytical delta range (±0.1 mg) or equivalent (Mettler-Toledo, Columbus, OH), calibrated with National Institute of Standards and Technology (Gaithersburg, MD) traceable calibration weights. HPLC column.—Kinetex Phenyl-Hexyl 2.6 µm, 4.6 × 150 mm (Phenomenex, Torrance, CA). HPLC system.—Prominence HPLC system consisting of two LC-20AT pumps, an SIL-20AC autosampler, a CTO-20AC column oven, a CBM-20A control module, an RF-20AX fluorescence detector, a DGU-20A5R degasser unit, and data processing with Lab Solutions software version 5.73 (Shimadzu, Kyoto, Japan). RIDACREST ICE controlled by Clarity software version 8.2 (R-Biopharm, Darmstadt, Germany). Immunoaffinity cartridges.—IMMUNOPREP® ONLINE AFM1 cartridges (R-Biopharm Rhône, Glasgow, Scotland). AFLAPREP® M WIDE immunoaffinity columns.—(R-Biopharm Rhône). Graduated cylinders.—100, 250, and 1000 mL. Volumetric flasks.—500 and 1000 mL. HPLC injection vials.—Amber, 2 mL with Teflon-coated caps. Conical flasks.—150 mL. Linear shaker.—HS 501 digital (Ika-Werke, Staufen, Germany). pH meter.—S220 pH/Ion meter (Mettler Toledo). Evaporator.—Techne sample concentrator (Cole-Palmer, Vernon Hills, IL). Glass test tubes.—15 mL. Nylon syringe filters.—0.2 µm (Phenex, Phenomenex). Disposable plastic syringes.—6 mL (Electrolube, Brookvale, NSW, Australia). Reagents Acetic acid (CH3COOH).—Reagent grade (Mallinckrodt, Staines, UK). Ammonium acetate (NH4CH3COO).—Reagent grade (Sigma Aldrich, St Louis, MO). Sodium hydroxide (NaOH).—Reagent grade (Merck, Kenilworth, NJ). Methanol (CH3OH).—HPLC grade (Mallinckrodt). Water (H2O).—HPLC grade (Genpure18.2 MΩ/cm Barnstead, Waltham, MA). Formic acid (HCOOH).—HPLC grade (Fisher, Waltham, MA). Acetonitrile (CH3CN).—HPLC grade (Mallinckrodt). Nitric acid (HNO3).—Reagent grade (Mallinckrodt). Triton X-100.—Reagent grade (Mallinckrodt). Supel™QuE citrate extraction tube salts.—Reagent grade (Sigma Aldrich). Tris(hydroxmethyl)aminoethane.—Reagent grade (Sigma Aldrich). AFM1 standard.—0.5 μg/mL (R-Biopharm Rhône). Isopropanol.—HPLC grade (Fisher). Solutions (n) Extraction solution.—5 mL acetic acid, 495 mL acetonitrile. (o) 1 M NaOH solution.—Dissolve 4 g NaOH pellets in 100 mL water. (p) Loading buffer.—Dissolve 1.54 g ammonium acetate in 1 L water, and adjust the pH to 6.8–7.0 using 1 M NaOH solution. (q) Reconstitution buffer.—Mix 450 mL Loading buffer with 50 mL methanol. (r) Elution buffer.—Dissolve 3.85 g ammonium acetate in 640 mL water. Then add 100 mL acetonitrile and 260 mL methanol and adjust the pH to 2.0 using concentrated nitric acid. (s) Cartridge wash buffer.—Dissolve 1.54 g ammonium acetate and 3.02 g tris(hydroxymethyl)aminomethane in 875 mL water, add 125 mL methanol, and pH adjust to 8.0 with nitric acid. (t) Mobile phase A.—980 mL water, 20 mL methanol, and 1 mL formic acid. (u) Mobile phase B.—900 mL acetonitrile, 100 mL water, and 1 mL formic acid. (v) Autosampler wash.—250 mL water and 250 mL acetonitrile. (w) Pump seal wash.—800 mL, 200 mL isopropanol. Standards Prepare an intermediate standard solution (10 ng/mL) by diluting 20 µL of the 500 ng/mL stock standard with 0.980 mL acetonitrile. Prepare standards of 0.025, 0.050, 0.100, and 0.150 ng/mL to validate detector linearity. Samples A sample each of WPC, WPI, WP, and MPC known to be free from AFM1 was used to carry out the AFM1 spiking experiments. A permeate-free, full cream homogenized milk and grated Edam cheese purchased retail were also used to carry out spiking experiments, and an Food Analysis Performance Assessment Scheme (FAPAS) reference cheese sample was used (Fera Science Limited, UK). Sample Preparation Milk protein powders Add milk protein powder (6 ± 0.05 g) to a 125 mL conical flask with 50 mL water. Place the flask on a hot plate stirrer at 50°C and mix with a stir bar for 30 min; then cool to room temperature. Weigh milk protein solution (10 ± 0.05 g) into a 50 mL centrifuge tube and spike at 50 and 100 ng/L. Liquid milk Add milk (10 ± 0.01 g) at ambient temperature directly to a 50 mL centrifuge tube and spike at 50 and 100 ng/L. Cheese Add cheese (5 ± 0.05 g) at ambient temperature to a 50 mL centrifuge tube. Add water (50°C, 10 mL), cool the sample to room temperature, and spike at 50 and 100 ng/L. Sample Extraction Add extraction solution (20 mL) and the salts in one extraction tube to the sample and vortex mix. Shake (200 rpm, 90 min) the sample tubes and centrifuge (3400 rcf, 10 min). Transfer the top acetonitrile layer (2 mL) to a test tube, add one drop of Triton X-100, and dry the sample under N2 at 60°C until a small viscous residue remains. Add reconstitution buffer (2 mL) and vortex mix the test tube. Use a syringe filter if the sample extract is cloudy, prior to transfer of a minimum of 1.5 mL to an autosampler vial. HPLC Conditions (a) Column temperature.—40°C. (b) Injection volume.—1000 μL. (c) Binary gradient.—Settings in Table 1. (d) Fluorescence detector.—Excitation: 355 nm, emission: 430 nm. (e) RIDACREST ICE conditions—Settings in Table 2. Carry out external standard calibration using a single-level 50 ng/L aqueous aflatoxin standard subjected to the entire procedure. Table 1. HPLC gradient conditions used following automated cartridge cleanup Time, min . Flow rate, mL/min . Mobile phase composition, % . Aa . Bb . 0.0 1.0 100 0 5.0 1.0 100 0 5.3 0.1 100 0 8.0 0.1 100 0 8.3 0.8 60 40 15.7 0.8 60 40 16.0 0.8 0 100 28.0 0.8 0 100 28.3 1.0 100 0 29.0 1.0 100 0 30.0 0.8 100 0 Time, min . Flow rate, mL/min . Mobile phase composition, % . Aa . Bb . 0.0 1.0 100 0 5.0 1.0 100 0 5.3 0.1 100 0 8.0 0.1 100 0 8.3 0.8 60 40 15.7 0.8 60 40 16.0 0.8 0 100 28.0 0.8 0 100 28.3 1.0 100 0 29.0 1.0 100 0 30.0 0.8 100 0 a Mobile phase A = 2% methanol, 0.1% formic acid. b Mobile phase B = 90% acetonitrile, 0.1% formic acid. Open in new tab Table 1. HPLC gradient conditions used following automated cartridge cleanup Time, min . Flow rate, mL/min . Mobile phase composition, % . Aa . Bb . 0.0 1.0 100 0 5.0 1.0 100 0 5.3 0.1 100 0 8.0 0.1 100 0 8.3 0.8 60 40 15.7 0.8 60 40 16.0 0.8 0 100 28.0 0.8 0 100 28.3 1.0 100 0 29.0 1.0 100 0 30.0 0.8 100 0 Time, min . Flow rate, mL/min . Mobile phase composition, % . Aa . Bb . 0.0 1.0 100 0 5.0 1.0 100 0 5.3 0.1 100 0 8.0 0.1 100 0 8.3 0.8 60 40 15.7 0.8 60 40 16.0 0.8 0 100 28.0 0.8 0 100 28.3 1.0 100 0 29.0 1.0 100 0 30.0 0.8 100 0 a Mobile phase A = 2% methanol, 0.1% formic acid. b Mobile phase B = 90% acetonitrile, 0.1% formic acid. Open in new tab Table 2. RIDA®CREST ICE conditionsa Step . High pressure dispenser flow, mL/min . Solution . Volume, mL . Conditioning 5.0 Loading buffer 2.0 Sample extract 1.0 Loading buffer 1.0 Cartridge wash 2.0 Cartridge wash buffer 6.0 Elution 0.3 Elution buffer 0.6 Clamp wash 5.0 Loading buffer 2.0 Step . High pressure dispenser flow, mL/min . Solution . Volume, mL . Conditioning 5.0 Loading buffer 2.0 Sample extract 1.0 Loading buffer 1.0 Cartridge wash 2.0 Cartridge wash buffer 6.0 Elution 0.3 Elution buffer 0.6 Clamp wash 5.0 Loading buffer 2.0 a Run time = 24 min, configured to operate in single-cartridge mode only. Open in new tab Table 2. RIDA®CREST ICE conditionsa Step . High pressure dispenser flow, mL/min . Solution . Volume, mL . Conditioning 5.0 Loading buffer 2.0 Sample extract 1.0 Loading buffer 1.0 Cartridge wash 2.0 Cartridge wash buffer 6.0 Elution 0.3 Elution buffer 0.6 Clamp wash 5.0 Loading buffer 2.0 Step . High pressure dispenser flow, mL/min . Solution . Volume, mL . Conditioning 5.0 Loading buffer 2.0 Sample extract 1.0 Loading buffer 1.0 Cartridge wash 2.0 Cartridge wash buffer 6.0 Elution 0.3 Elution buffer 0.6 Clamp wash 5.0 Loading buffer 2.0 a Run time = 24 min, configured to operate in single-cartridge mode only. Open in new tab Results and Discussion Method Optimization The experimental method followed the protocol supplied by the manufacturer, apart from the following modifications: (1) addition of extraction salts before the 90 min shaking period followed by centrifugation; (2) the cartridge wash buffer pH = 8; (3) extension of the automated immunoaffinity cycle time to 24 min; (4) an increase in the chromatographic run time to 30 min, as seen in Figure 2. Figure 2. Open in new tabDownload slide Chromatogram of aflatoxin M1 in whey powder. Figure 2. Open in new tabDownload slide Chromatogram of aflatoxin M1 in whey powder. Method Validation Detector linearity was demonstrated through analysis of multi-level AFM1 standard solutions (n = 4) covering an analyte range from 25 to 150 ng/L and was performed by direct injection bypassing the RIDACREST ICE. Linearity was evaluated by least-squares regression analysis of peak area versus concentration, with acceptable values for r2 of 0.997 being obtained, as shown in Figure 3. Figure 3. Open in new tabDownload slide Calibration curve for aflatoxin M1 showing linear detector response. Figure 3. Open in new tabDownload slide Calibration curve for aflatoxin M1 showing linear detector response. Method recovery was determined by spiking samples of WP, WPC, WPI, MPC, liquid milk, and cheese with 50 and 100 ng/L AFM1, as shown in Table 3. The recovery results fit within the AOAC guidelines of between 70–125% recovery for analytes present at 10 µg/L or less (27). Table 3. Recovery of aflatoxin M1 from a range of spiked matrices Sample . Average recovery, % . 50 ng/L . 100 ng/L . Whey powder 90 102 Whey protein isolate 97 103 Whey protein concentrate 88 101 Milk protein concentrate 112 108 Milk 99 85 Cheese 80 81 Sample . Average recovery, % . 50 ng/L . 100 ng/L . Whey powder 90 102 Whey protein isolate 97 103 Whey protein concentrate 88 101 Milk protein concentrate 112 108 Milk 99 85 Cheese 80 81 Open in new tab Table 3. Recovery of aflatoxin M1 from a range of spiked matrices Sample . Average recovery, % . 50 ng/L . 100 ng/L . Whey powder 90 102 Whey protein isolate 97 103 Whey protein concentrate 88 101 Milk protein concentrate 112 108 Milk 99 85 Cheese 80 81 Sample . Average recovery, % . 50 ng/L . 100 ng/L . Whey powder 90 102 Whey protein isolate 97 103 Whey protein concentrate 88 101 Milk protein concentrate 112 108 Milk 99 85 Cheese 80 81 Open in new tab Because of the absence of appropriate certified reference material, a cheese sample containing AFM1 from the FAPAS proficiency scheme with assigned mean and range values was analyzed. The FAPAS cheese sample had an assigned value of 2.005 ± 0.510 µg/kg (uncertainty at 95% confidence interval) with duplicate results obtained using this method of 1.705 and 2.005 µg/kg, yielding a z-score of −1.42, below the limit of |2| for outlying results. This method was also compared with duplicate 50 ng/L spiked WPI and WPC samples, which were prepared and analyzed using the manual immunoaffinity column method. The results of this study are shown in Table 4. The manual immunoaffinity and HPLC method was based on reference methods (AOAC Offiical Method 2000.08 and IDF 171, 28, 29) with minor modifications: (1) the milk samples were dissolved in 25 mL water; (2) aflatoxin was extracted from the cartridges using 7.5 mL acetonitrile with back flushing; (3) analytical chromatography conditions were isocratic elution with 30% acetonitrile at 0.7 mL/min on a 5 µm, 150 × 4.6 mm Prodigy column (Phenomenex). Table 4. Comparison of aflatoxin M1 results measured by automated online (cartridge) and manual (column) cleanup in whey protein concentrate and whey protein isolate spiked at 50 ng/L Sample . RIDA®CREST ICE,ng/L . Manual column,ng/L . Whey protein concentrate 43–55 46–48 (48)a (47)b Whey protein isolate 49–64 49–66 (55)c (58)b Sample . RIDA®CREST ICE,ng/L . Manual column,ng/L . Whey protein concentrate 43–55 46–48 (48)a (47)b Whey protein isolate 49–64 49–66 (55)c (58)b a Range and mean of 12 replicates. b Range and mean of 2 replicates. c Range and mean of 11 replicates. Open in new tab Table 4. Comparison of aflatoxin M1 results measured by automated online (cartridge) and manual (column) cleanup in whey protein concentrate and whey protein isolate spiked at 50 ng/L Sample . RIDA®CREST ICE,ng/L . Manual column,ng/L . Whey protein concentrate 43–55 46–48 (48)a (47)b Whey protein isolate 49–64 49–66 (55)c (58)b Sample . RIDA®CREST ICE,ng/L . Manual column,ng/L . Whey protein concentrate 43–55 46–48 (48)a (47)b Whey protein isolate 49–64 49–66 (55)c (58)b a Range and mean of 12 replicates. b Range and mean of 2 replicates. c Range and mean of 11 replicates. Open in new tab Method precision was evaluated by the analysis of duplicates of WP, WPI, WPC, MPC, liquid milk, and cheese samples spiked with 50 ng/L AFM1. Acceptable precision was demonstrated, with a repeatability of between 3.7 and 11.7% RSD, an intermediate precision of between 4.5 and 12.8% RSD, and calculated HorRat values between 0.3 and 1.2 (30). The full precision results are shown in Table 5. Table 5. Precision results from aflatoxin M1 samples spiked at 50 ng/L Sample . Repeatability, RSD, % (HorRat) . Intermediate precision, RSD, % . Whey powder 9.6 (1.2) 12.6 Whey protein isolate 5.6 (1.1) 12.0 Whey protein concentrate 3.7 (0.7) 7.3 Milk protein concentrate 11.7 (0.9) 10.9 Milk 4.8 (0.3) 4.5 Cheese 9.8 (0.3) 12.8 Sample . Repeatability, RSD, % (HorRat) . Intermediate precision, RSD, % . Whey powder 9.6 (1.2) 12.6 Whey protein isolate 5.6 (1.1) 12.0 Whey protein concentrate 3.7 (0.7) 7.3 Milk protein concentrate 11.7 (0.9) 10.9 Milk 4.8 (0.3) 4.5 Cheese 9.8 (0.3) 12.8 Open in new tab Table 5. Precision results from aflatoxin M1 samples spiked at 50 ng/L Sample . Repeatability, RSD, % (HorRat) . Intermediate precision, RSD, % . Whey powder 9.6 (1.2) 12.6 Whey protein isolate 5.6 (1.1) 12.0 Whey protein concentrate 3.7 (0.7) 7.3 Milk protein concentrate 11.7 (0.9) 10.9 Milk 4.8 (0.3) 4.5 Cheese 9.8 (0.3) 12.8 Sample . Repeatability, RSD, % (HorRat) . Intermediate precision, RSD, % . Whey powder 9.6 (1.2) 12.6 Whey protein isolate 5.6 (1.1) 12.0 Whey protein concentrate 3.7 (0.7) 7.3 Milk protein concentrate 11.7 (0.9) 10.9 Milk 4.8 (0.3) 4.5 Cheese 9.8 (0.3) 12.8 Open in new tab The instrumental LOD was estimated by serial dilution of the AFM1 until a signal-to-noise ratio of three was obtained, yielding a measured value for the LOD of 0.010 ng/mL in diluent. Method robustness was investigated by performing a Plackett–Burman trial (31, 32), as previously described (33). The seven factors assessed were: extract volume (9 and 11 mL), volume of extraction solvent (18 and 22 mL), shaking time (105 and 75 min), centrifuge speed (3450 and 3350 × g), centrifuge time (12 and 8 min), Triton X-100 drops (2 drops and ½ normal drop), and a dummy factor (G, g). The method was found to be robust for the parameters evaluated, and the results obtained were normally distributed, with variances conforming to those expected by chance (Figure 4). As with similar external-standard-based methods, critical method parameters included accurate measurement of the sample weight, extract volume, and aliquot volume. Figure 4. Open in new tabDownload slide Half-normal plot of method ruggedness evaluation for aflatoxin M1 in whey protein isolate. Figure 4. Open in new tabDownload slide Half-normal plot of method ruggedness evaluation for aflatoxin M1 in whey protein isolate. The RIDACREST ICE offers a practical solution to the requirement for the high sample throughput (up to 42 samples in a single run per day) needed for measuring AFM1 for global compliance with maximum regulatory limits for AFM1 in milk and milk-based products (34, 35). Conclusions A method that is intended for use in high-throughput laboratories as part of routine product compliance release testing, to show that these dairy products contain less than the maximum regulatory levels of AFM1, was developed. This method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. Acknowledgments The authors would like to thank Iain McGrail for technical assistance with instrumentation. We would also like to acknowledge the assistance of Elizabeth Manning of R-Biopharm Rhône for facilitating the collaboration between the authors and proof reading the manuscript and Claire Woodhall for proofreading the manuscript. Conflict of interest The authors declares that they have no conflict of interest. References 1 Pitt J.I. ( 2000 ) Br. Med. Bull . 56 , 184 – 192 . doi:10.1258/0007142001902888 Crossref Search ADS PubMed 2 Du X. , Schrunk D.E. , Imerman P.M. , Smith L. , Francis K. , Tahara J. , Tkachenko A. , Reimschuessel R. , Rumbeiha W.K. ( 2019 ) J. AOAC Int. 102 , 1530 – 1534 . doi:10.5740/jaoacint.18-0355 Crossref Search ADS PubMed 3 Fink-Gremmels J. ( 2008 ) Food Addit. Contam. A 25 , 172 – 180 . doi:10.1080/02652030701823142 Crossref Search ADS 4 Marchese S. , Polo A. , Ariano A. , Velotto S. , Costantini S. , Severino L. ( 2018 ) Toxins 10 , 214 – 233 . doi:10.3390/toxins10060214 Crossref Search ADS 5 Polzhoffer K.-P. ( 1977 ) Z. Lebensm. Unters. Forsch . 164 , 80 – 81 . doi:10.1007/BF01354305 Crossref Search ADS PubMed 6 Campagnollo F.B. , Ganev K.C. , Khaneghah A.M. , Portela J.B. , Cruz A.G. , Granato D. , Corassin C.H. , Oliveira C.A.F. , Sant'Ana A.S. ( 2016 ) Food Control 68 , 310 – 329 . doi:10.1016/j.foodcont.2016.04.007 Crossref Search ADS 7 Pitt J.I. ( 2012 ) in International Agency for Research on Cancer , Pitt J.I. , Wild C.P. , Baan R.A. , Gelderblom W.C.A. , Miller J.D. , Riley R.T. , Wu F. (Eds), IARC Scientific Publications, Geneva, Switzerland , pp 1 – 28 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 8 The New Zealand Mycotoxin Surveillance Program 06-14 Report Series. MPI Technical Report – Paper No. 2016/21. https://www.fisheries.govt.nz/dmsdocument/12924/direct (accessed July 31, 2020 ) 9 Mulanda M. , Ngoma L. , Nyirenda M. , Motsei L. , Bakunzi F. ( 2013 ) in Mycotoxin and Food Safety in Developing Countries Makun H.A. (Ed.), InTech , London , pp 39 – 60 . doi:10.5772/53286 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 10 Galvano F. , Galofaro V. , Galvano G. ( 1996 ) J. Food Prot . 59 , 1079 – 1090 . doi:10.4315/0362-028X-59.10.1079 Crossref Search ADS PubMed 11 Benkerroum N. ( 2016 ) Int. Dairy J . 62 , 63 – 75 . doi:10.1016/j.idairyj.2016.07.002 Crossref Search ADS 12 Rahmani J. , Alipour S. , Miri A. , Fakhri Y. , Riahi S.-M. , Keramati H. , Moradi M. , Amanidaz N. , Pouya R.H. , Bahmani Z. , Khaneghah M. ( 2018 ) Food Chem. Toxicol . 118 , 653 – 666 . doi:10.1016/j.fct.2018.06.016 Crossref Search ADS PubMed 13 Iqbal S.Z. , Jinap S. , Pirouz A.A. , Ahmad Faizal A.R. ( 2015 ) Trends Food Sci. Technol . 46 , 110 – 119 . doi:10.1016/j.tifs.2015.08.005 Crossref Search ADS 14 Cullen J.M. , Ruebner B.H. , Hsieh L.S. , Hyde D.M. , Hsieh D.P. ( 1987 ) Cancer Res. . 47 , 1913 – 1917 PubMed 15 Purchase I.F.H. ( 1967 ) Toxicology 5 , 339 – 342 doi: 10.1016/S0015-6264(67)83060-8 16 Neal G.E. , Eaton D.L. , Judah D.J. , Verma A. ( 1998 ) Toxicol. Appl. Pharmacol . 151 , 152 – 158 doi: 10.1006/taap.1998.8440 Crossref Search ADS PubMed 17 Wong J.J. , Hsieh D.P. ( 1976 ) Proc. Natl. Acad. Sci. USA 73 , 2241 – 2244 . doi:10.1073/pnas.73.7.2241 Crossref Search ADS 18 Mwanza M. , Abdel-Hadi A. , Ali A.M. , Egbuta M. ( 2015 ) J. Dairy Sci . 98 , 6660 – 6667 . doi:10.3168/jds.2014-9220 Crossref Search ADS PubMed 19 Shephard G.S. ( 2016 ) J. AOAC Int . 99 , 842 – 848 . doi:10.5740/jaoacint.16-0111 Crossref Search ADS PubMed 20 Zheng M.Z. , Richard J.L. , Binder J. ( 2006 ) Mycopathologia 161 , 261 – 273 . doi:10.1007/s11046-006-0215-6 Crossref Search ADS PubMed 21 Zhang K. , Wong J.W. , Hayward D.G. , Vaclavikova M. , Liao C.-D. , Trucksess M.W. ( 2013 ) J. Agric. Food Chem. 61 , 6265 – 6273 . doi:10.1021/jf4018838 Crossref Search ADS PubMed 22 Santos A.O. , Vaz A. , Rodrigues P. , Veloso A.C.A. , Venâncio A. , Peres A.M. ( 2019 ) Chemosensors 7 , 3 .doi: 10.3390/chemosensors7010003 Crossref Search ADS 23 Indyk H.E. , Chetikam S. , Gill B.D. , Wood J.E. , Woollard D.C. ( 2019 ) Food Anal. Methods 12 , 2630 – 2637 . doi:10.1007/s12161-019-01621-5 Crossref Search ADS 24 Scott P.M. , Trucksess M.W. ( 1997 ) J. AOAC Int . 80 , 941 – 950 Crossref Search ADS PubMed 25 Romero-González R. , Martínez Vidal J.L. , Aguilera-Luiz M.M. , Garrido Frenich A. , ( 2009 ) J. Agric. Food Chem. 57 , 9385 – 9392 . doi: 10.1021/jf903154a Crossref Search ADS PubMed 26 Rhemrev R. , Pazdanska M. , Marley E. , Biselli S. , Staiger S. ( 2015 ) J. AOAC Int . 98 , 1585 – 1590 . doi:10.5740/jaoacint.15-124 Crossref Search ADS PubMed 27 Official Methods of Analysis ( 2019 ) 21st Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Appendix K 28 Official Methods of Analysis ( 2019 ) 21st Ed., AOAC INTERNATIONAL, Gaithersburg, MD, Method 2000.08 29 ISO 14501:2007: Milk and milk powder―Determination of aflatoxin M1 content; Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography, including IDF 171:2007, International Organization for Standardization, Brussels, Belgium 30 Horwitz W. , Albert R. ( 2006 ) J. AOAC Int . 89 , 1095 – 1109 Crossref Search ADS PubMed 31 Plackett R.L. , Burman J.P. ( 1946 ) Biometrika 33 , 305 – 325 doi: 10.1093/biomet/33.4.305 Crossref Search ADS 32 Youden W.J. , Steiner E.H. ( 1975 ) Statistical Manual of the AOAC , AOAC INTERNATIONAL , Gaithersburg, MD Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 33 Gill B.D. , Indyk H.E. , Kumar M.C. , Sievwright N.K. , Manley-Harris M. ( 2010 ) J. AOAC Int . 93 , 966 – 973 Crossref Search ADS PubMed 34 European Union ( 2010 ), Off. J. EU L50 , 8 – 12 35 Codex Alimentarius ( 1995 ) Codex General Standard for Contaminants and Toxins in Food and Feed (Codex Stan. 193–1995) Schedule I ‒ Mycotoxins, p 30 © AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection
JF - Journal of AOAC INTERNATIONAL
DO - 10.1093/jaoacint/qsaa164
DA - 2020-12-18
UR - https://www.deepdyve.com/lp/oxford-university-press/determination-of-aflatoxin-m1-in-liquid-milk-cheese-and-selected-milk-RkM1ADhJvD
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -