[Applicable to the determination of total aflatoxins (AF; sum of AFB1
, and AFG2
) in powdered ginseng and ginger at 2−16 μg/kg and ochratoxin A (OTA) in the matrixes at 1−8 μg/kg.]
Caution: The International Agency for Research on Cancer (IARC) has classified AF as a human carcinogen (group 1 A) and OTA as a possible human carcinogen (group 2B; 1). Wear protective clothing, gloves, and safety glasses at all times, and perform all standard and sample preparation stages inside the fume hood. Clean accidental spills of AF and OTA with diluted bleach (1 to 10 dilution), and let stand 10 min. After wiping off the bleach solution of the area, wipe with 5% aqueous acetone. Rinse all glassware with diluted bleach before washing. Methanol and acetonitrile are hazardous and must be poured in a fume hood. All analyses should be carried out inside the fume hood. Dispose of waste solvents according to applicable environmental rules and regulations.
See and for the results of the interlaboratory study supporting acceptance of the method.
Interlaboratory study results for aflatoxins and ochratoxin A in powdered ginseng by liquid chromatography after immunoaffinity column cleanup
Interlaboratory study results for aflatoxins and ochratoxin A in powdered ginger by liquid chromatography after immunoaffinity column cleanup
A test portion is extracted with methanol and 0.5% aqueous sodium bicarbonate solution (700 + 300, v/v). The extract is centrifuged, diluted with phosphate buffer (PB; 0.1 M, pH 7.4) containing 1% Tween 20, filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing, the toxin is eluted from the column with methanol and determined and quantified by high-performance liquid chromatography (LC) with fluorescence detection. For AF post-column derivatization, PHRED or Kobra cell is used.
B. Plant Material
Finely ground (<125 μm) ginseng (Panax quinquefolius) for the recovery study was purchased from Schumacher Ginseng (Marathon, WI). The ground ginger (the dried, ground rhizomes of Zingiber officinale Roscoe) for the recovery study was purchased from McCormick (Baltimore, MD). Naturally contaminated ginger (Zingiber officinale) was purchased from Penn Herb (Philadelphia, PA).
(a) Orbital shaker.—VWR DS-500E (VWR International, Bridgeport, NJ), or equivalent, capable of 400 rpm.
(b) Centrifuge tube.—50 mL, polypropylene.
(c) Centrifuge.—Allegra X-22R (VWR International).
(d) Glass microfiber filter paper.—Whatman No. 934AH, 11 cm (Whatman, Inc., Clifton, NJ).
(e) Immunoaffinity column.—AflaOchraTest column (G1017; Vicam, Watertown, MA). Aflatoxin/OTA immunoaffinity columns contain monoclonal antibodies cross reactive toward aflatoxins B1, B2, G1, and G2, and monoclonal antibodies cross reactive toward OTA. The immunoaffinity columns should have a minimum capacity of not <100 ng total aflatoxin and 100 ng OTA and should give a recovery of not <80% for aflatoxins B1, B2, G1, and G2, and OTA when 5 ng of each aflatoxins B1, B2, G1, and G2, and OTA is applied in 10 mL of 10% methanol:phosphate buffered saline (PBS; v/v). The columns should have a shelf life of 18 months at 4°C or 12 months at room temperature. The use by date was September 19, 2008.
(f) Column reservoir.—50 mL polypropylene (Alltech Associates, Deerfield, IL) with luer tip or 25 mL glass syringe with luer tip, or equivalent.
(g) Column manifold.—Vicam G1104 12-position stand, or equivalent.
(h) LC system.—Waters Model 2690 Alliance Separation System (Waters, Milford, MA), Waters Model 2475 fluorescence detector; or equivalent LC system. Operating conditions.—Flow rate, for AF 0.8 mL/min; for OTA 1.0 mL/min. Detector, for AF set at excitation wavelength (Ex) 362 nm and emission wavelength (Em) 440 nm; for OTA set at Ex 333 nm and Em 460 nm. Column, for AF, Waters, Cat. No. AQ12S031546WT, YMC ODS-AQ S-3, 4.6 × 150 mm, or equivalent; for OTA, Beckman, Cat. No. 235332, Ultrasphere, 4.6 × 250 mm, 5 μm (Beckman Instruments, Inc., Fullerton, CA), or equivalent.
(i) Post-column derivatization (PCD) systems for AF.—(1) PHRED cell.—Post-column photochemical derivatization cell (AURA Industries, New York, NY), or equivalent. (Caution: Avoid looking at the UV lamp.) (2) Kobra cell.—Electrochemical cell, post-column bromination derivatization cell (R-Biopharm Inc., Marshall, MI). (Caution: Set at 100 μA, do not turn on current until LC pump is operating to avoid overheating the cell membrane.)
(a) Solvent and reagents.—LC grade methanol, acetonitrile, ACS grade acetic acid, sodium chloride (NaCl), monosodium phosphate, disodium phosphate, PBS (pH 7.4; Sigma P-3813; Sigma-Aldrich, St. Louis, MO), Tween 20 (Sigma P-5927), and Milli-Q water (ultrapure water).
(b) Extraction solvent.—Methanol–0.5% sodium bicarbonate (700 + 300, v/v); mix and equilibrate to room temperature. Prepare fresh daily.
(c) PBS solution.—10 mM. Dissolve 1 package Sigma P-3813 PBS powder in 1 L water.
(d) PB solution.—0.1 M. Dissolve 8.69 g disodium phosphate (anhydrous) and 4.66 g monosodium phosphate (anhydrous) or 5.36 g monosodium phosphate monohydrate in 800 mL water, adjust to pH 7.4 with 2 M sodium hydroxide, add 10 mL Tween 20, and dilute to 1 L.
(e) Aflatoxins.—Cat. Nos. A 6636, A 9887, A 0138, and A 0263 for AFB1, AFB2, AFG1, and AFG2, respectively (Sigma Aldrich). Prepare stock standard solutions of each of the 4 AF at 10 μg/mL in acetonitrile according to revised AOAC Method 971.22.
(1) Preparation of 10 μg/mL first individual aflatoxin stock standard solution.—Weigh 10 mg of each aflatoxin into a separate 100 mL volumetric flask. Add 50 mL acetonitrile, mix, dilute to mark with additional acetonitrile, and mix. Then pipet 10 mL of this solution into another 100 mL volumetric flask and dilute to mark with acetonitrile. Record UV spectrum of each aflatoxin solution. Determine concentration of aflatoxin solution by measuring absorbance (A) at wavelength of maximum absorption close to 360 nm and using equation in 971.22 [μg Aflatoxin/mL = (A × MW × 1000)/ε], where MW is molecular weight and ε is molecular absorptivity. The concentration should be approximately 10 μg/mL.
(2) Preparation of 400 ng/mL AF second stock standard solution (mixture of the 4 AFB1, B2, G1, and G2 at 200, 50, 100, and 50 ng/mL).—Add appropriate amount of each AF stock standard to the same volumetric flask and dilute to volume with acetonitrile. Use the 400 ng/mL AF second stock standard as the spiking solution for recovery study. Store stock standard solution at −18°C. Equilibrate to room temperature before use.
(3) Preparation of working AF calibrant solution.—Prepare 6 calibrants in separate 10 mL volumetric flasks according to . Dilute to volume with methanol–water (1 + 1, v/v). Store in refrigerator and equilibrate to room temperature before use. Prepare working calibrant solutions daily.
(f) OTA standard solutions.—O1877 (Sigma Aldrich).
(1) Preparation of a 30 μg/mL first OTA stock standard solution.—Weigh 10 mg OTA into a 100 mL volumetric flask. Add 50 mL methanol, mix, dilute to mark with additional methanol, and mix. Pipet 1 mL of the 100 μg/mL OTA solution into a 3 mL volumetric flask and dilute to volume with methanol. Determine absorbance at 333 nm. Use molar absorptivity of 6330 to calculate concentration. The stock standard solution should be approximately 30 μg/mL. Store stock standard solution at −18°C. Equilibrate to room temperature before use.
(2) Preparation of 200 ng/mL second OTA stock standard solution.—Add appropriate amount of stock standard to a 25 mL volumetric flask and dilute to volume with methanol.
(3) Preparation of working OTA calibrant solution.—Prepare 6 calibrants in separate 10 mL volumetric flasks according to . Dilute to volume with methanol–water (1 + 1, v/v). Store in refrigerator and equilibrate to room temperature before use. Prepare working calibrant solutions daily.
(g) Mobile phase.—Isocratic, 0.8 mL/min for AF and 1 mL/min for OTA.
(1) For AF post-column derivatization with PHRED cell.—Water–methanol–acetonitrile (600 + 250 +150, v/v/v).
(2) For AF post-column derivatization with Kobra cell.—1 L water–methanol–acetonitrile (600 + 250 + 150, v/v/v) + 350 μL 4 M nitric acid (M of concentrated nitric acid is 15.9) + 120 mg potassium bromide, and mix.
(3) For OTA.—Acetonitrile–water–acetic acid (500 + 500 + 10, v/v/v).
Weigh 5 g test sample in a 50 mL centrifuge tube. Add 1 g NaCl and 25 mL MeOH–0.5% NaHCO3 (700 + 300, v/v). Mix on a Vortex mixer until sample particles and extract solvent are well mixed. Shake at 400 rpm for 10 min. Centrifuge for 10 min at 7000 rpm (g value = 5323 mm/s2) or at speed that can result a firm pellet of residues. Immediately pipet 7 mL into a 50 mL centrifuge, add 28 mL 0.1 M PB containing 1% Tween 20, mix, and filter through glass microfiber paper. Collect 25 mL filtrate (equivalent to 1 g test sample) into a 25 mL graduate cylinder and proceed immediately with immunoaffinity column (IAC) chromatography.
F. Immunoaffinity Column Isolation
(Caution: For immunoaffinity column cleanup, columns must be kept at room temperature for at least 15 min before use.)
Remove top cap from column and connect with reservoir. Remove end cap from column and attach to column manifold (the fit must be tight). Let liquid in column pass through until liquid is about 2−3 mm above column bed. Pass 25 mL filtrate into reservoir. Let filtrate flow through column by gravity force. Let column run dry. In order to start flow easily again, remove column from manifold, add about 2 mL 10 mM PBS into column, reattach column to reservoir, wash column with additional 3 mL 10 mM PBS and then with 5 mL water (the 5 mL 10 mM PBS can be added directly to column reservoir if other techniques are used to dislodge the air bubble at the end of the column and to start flow easily again). Let column run dry, then force 3 mL air through column with a syringe. Place a 3 mL volumetric flask under column. Elute and collect AF and OTA in the 3 mL volumetric flask with 1 mL HPLC grade methanol; let drip freely. Let column run dry. Let stand for 1 min, then elute with additional 1 mL methanol and collect into the same volumetric flask. Let column run dry and force 10 mL air through column. Dilute eluate to volume with water and perform LC analysis for AF immediately. Perform LC analysis for OTA next.
G. LC Analysis
(a) Aflatoxins.—Post-column derivatization for AF (use UV cell or Kobra cell).—Inject 50 μL reagent blank (calibrant 1), AF working standards, or test sample into LC column. Identify AF peaks in test sample extract by comparing retention time with those of standards. AF elute in the order of G2, G1, B2, and B1. After passing through the PHRED cell or Kobra cell, the AFG1 and AFB1 have been derivatized to form AFG2a (derivative of G1) and AFB2a (derivative of B1). The chemical structures of the derivatives resulted from electrochemical bromination and photolysis are not the same. The structures of AFB1 and AFG1 photolysis products have not been established. The retention times of AFG2, AFG2a, AFB2, and AFB2a are between about 14 and 27 min using the PHRED cell (); retention times are shorter using the Kobra cell. The peaks should be baseline resolved. Construct standard curves of each AF. Determine concentration of each AF in test solution from calibration curve.
Liquid chromatograms of aflatoxin standard and aflatoxins in ginger product.
Aflatoxins calibration curves.—Calibration curves should be prepared for each of the aflatoxin using the working calibrant solutions containing the 4 aflatoxins described. These solutions cover the range of 0.25−4 ng/mL for AFB1, 0.0625−1 ng/mL for AFB2, 0.125−2 ng/mL for AFG1, and 0.0625−1 ng/mL for AFG2. Make the calibration curves prior to analysis according to and check the plot for linearity. If test portion area response is outside (higher) the calibration range, then the purified test extract should be diluted with methanol–water (1 + 1, v/v) and reinjected into the LC column.
(b) OTA.—Inject 50 μL reagent blank (calibrant 1), OTA working standards, or test sample into LC column. Identify OTA peaks in test sample extracts by comparing retention time with those of standards. OTA elutes with retention times of between 10−11 min (). There is a small peak with a retention time about 0.5 min ahead the OTA peak in ginseng. Construct standard curves of OTA. Determine concentration of OTA in test solution from calibration curve.
Liquid chromatograms of ochratoxin A standard and ochratoxin A in ginger product.
OTA calibration curve.—Calibration curves should be prepared for OTA using the working calibrant solutions. These solutions cover the range of 0.25−4 ng/mL for OTA. Make the calibration curves prior to analysis according to and check the plot for linearity. If test portion area response is outside (higher) the calibration range, then the purified test extract should be diluted with methanol–water (1 + 1, v/v) and reinjected into the LC column.
(c) Quantitation of aflatoxins and OTA.—Quantitation of AF and OTA should be performed by measuring peak areas at each AF and OTA retention time and comparing them with the corresponding calibration curve.
Plot peak area (response, Y
-axis) of each toxin (OTA and each AF) standards against concentration (ng/mL, X
-axis) and determine slope (S) and Y
-intercept (a). Calculate level of toxin in sample with the following formula, where R is the test solution peak area, V is the final volume (mL) of the injected test solution, and F is the dilution factor. F is 1 when V is 3 mL. W is 1 g test sample passed through the immunoaffinity column. The total AF is the sum of the AFG2
, and B1
) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (1993) Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, Vol. 56, International Agency for Research on Cancer, Lyon, France, pp 489−521.
) J. AOAC Int