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METHODS OF ANALYSIS
Foreign Organic Matter
Test Sample— Unless otherwise specified in the individual monograph, weigh the following quantities of the laboratory sample, taking care that the withdrawn portion is representative (quartering if necessary):
 Roots, rhizomes, bark, and herbs 500 g Leaves, flowers, seeds, and fruit 250 g Cut vegetable drugs (average weight of the pieces is less than 0.5 g) 50 g
Spread the sample out in a thin layer, and separate the foreign organic matter by hand as completely as possible. Weigh it, and determine the percentage of foreign organic matter in the weight of drug taken.
Total Ash
Accurately weigh a quantity of the Test Sample, representing 2 to 4 g of the air-dried material, in a tared crucible, and incinerate, gently at first, and gradually increase the temperature to 675 ± 25, until free from carbon, and determine the weight of the ash. If a carbon-free ash cannot be obtained in this way, extract the charred mass with hot water, collect the insoluble residue on an ashless filter paper, incinerate the residue and filter paper until the ash is white or nearly so, then add the filtrate, evaporate it to dryness, and heat the whole to a temperature of 675 ± 25. If a carbon-free ash cannot be obtained in this way, cool the crucible, add 15 mL of alcohol, break up the ash with a glass rod, burn off the alcohol, and again heat the whole to a temperature of 675 ± 25. Cool in a desiccator, weigh the ash, and calculate the percentage of total ash from the weight of the drug taken.
Acid-Insoluble Ash
Boil the ash obtained as directed under Total Ash, above, with 25 mL of 3 N hydrochloric acid for 5 minutes, collect the insoluble matter on a tared filtering crucible or ashless filter, wash with hot water, ignite, and weigh. Determine the percentage of acid-insoluble ash calculated from the weight of drug taken.
Water-Soluble Ash
Boil the ash obtained as directed for Total Ash with 25 mL of water for 5 minutes. Collect the insoluble matter in a sintered-glass crucible or on an ashless filter paper. Wash with hot water, and ignite for 15 minutes at a temperature not exceeding 450. Subtract the weight of this residue, in mg, obtained under Total Ash, and calculate the percentage of water-soluble ash with reference to the weight of sample as determined under Total Ash.
Alcohol-Soluble Extractives
Method 1 (hot extraction method)— Transfer about 4 g of air-dried, coarsely powdered material, accurately weighed, to a glass-stoppered conical flask. Add 100 mL of alcohol, and weigh the flask. Shake, and allow to stand for 1 hour. Attach a reflux condenser to the flask, and boil gently for 1 hour, cool, and weigh. Readjust to the original weight with alcohol. Shake, and filter rapidly through a dry filter. Transfer 25 mL of the filtrate to a tared flat-bottomed dish, and evaporate on a water bath to dryness. Dry at 105 for 6 hours, cool in a desiccator for 30 minutes, and weigh without delay. Calculate the content, in mg per g, of alcohol-extractable matter in the test specimen.
Method 2 (cold extraction method)— Transfer about 4 g of air-dried, coarsely powdered material, accurately weighed, to a glass-stoppered conical flask. Add 100 mL of alcohol, insert a stopper into the flask, and macerate for 24 hours, shaking frequently during the first 8 hours and then allowing to stand for 18 hours. Filter rapidly, taking precautions against loss of alcohol. Evaporate 25 mL of the filtrate to dryness in a tared, flat-bottomed, shallow dish, and dry at 105 to constant weight. Calculate the content, in mg per g, of alcohol-extractable matter in the test specimen.
Water-Soluble Extractives
Method 1 (hot extraction method)— Proceed as directed for Method 1 (hot extraction method) under Alcohol-soluble Extractives, except to use water in place of alcohol.
Method 2 (cold extraction method)— Proceed as directed for Method 2 (cold extraction method) under Alcohol-soluble Extractives, except to use water in place of alcohol.
Crude Fiber
Exhaust a weighed quantity of the Test Sample, representing about 2 g of the drug, with ether. Add 200 mL of boiling dilute sulfuric acid (1 in 78) to the ether-exhausted marc, in a 500-mL flask, and connect the flask to a reflux condenser. Reflux the mixture for 30 minutes, accurately timed, then filter through a linen or hardened-paper filter, and wash the residue on the filter with boiling water until the effluent washing is no longer acid. Rinse the residue back into the flask with 200 mL of boiling sodium hydroxide solution, adjusted to 1.25 percent by titration and free from sodium carbonate. Again reflux the mixture for 30 minutes, accurately timed, then rapidly filter through a tared filter, wash the residue with boiling water until the last washing is neutral, and dry it at 110 to constant weight. Incinerate the dried residue, ignite to constant weight, cool in a desiccator, and weigh the ash: the difference between the weight obtained by drying at 110 and that of the ash represents the weight of the crude fiber.
NOTE—The boiling with acid and alkali should continue for 30 minutes, accurately timed, from the time that the liquid (which is cooled below the boiling point by being added to the cold flask) again boils. After the solution has been brought to boiling, the heat should be turned low enough just to maintain boiling. During the boiling, the flask should be gently rotated from time to time to wash down any particles that may adhere to the walls of the flask. A slow current of air introduced into the flask during the boiling operation aids in preventing excessive frothing.
Starch Content
Method 1— The following is a general procedure for all reducing sugars and may be used to determine the starch content in botanical articles.
Malt Extract— Use clean new barley malt of known efficacy, and grind just before use. Prepare malt extract just prior to use. For every 80 mL of malt extract needed, digest 5 g of ground malt with 100 mL of water at room temperature for 2 hours. [NOTE—If an electric mixer is used, stir the mixture for 20 minutes.] Filter to obtain a clear extract, filtering again, if necessary, and mix the infusion well.
Test Solution— Extract about 5 g of the finely ground test specimen with five 10-mL portions of ether, using a filter that will completely retain the smallest starch granule. Allow the ether to evaporate from the residue, and wash with 250 mL of aqueous alcohol solution (10 in 100). Carefully wash the residue from the paper into a 500-mL beaker with about 100 mL of water. Heat to about 60 (avoiding, if possible, gelatinizing starch), and allow to stand for about 1 hour, stirring frequently to effect complete solution of sugars. Transfer to a wide-mouth bottle, rinse the beaker with a little warm water, and cool. Add an equal volume of alcohol, mix, and allow to stand for not less than 1 hour.
Centrifuge until the precipitate is closely packed on the bottom of the bottle, and decant the supernatant. Wash the precipitate with successive 50-mL portions of alcohol solution (50 in 100) by centrifuging and decanting through a suitable filter until the washings are sugar-free. [NOTE—To test for the presence of sugar, transfer a few drops of the washings to a test tube, add 3 or 4 drops of a 20% solution of 1-naphthol in alcohol, prepared by dissolving 200 mg of 1-naphthol in 1 mL of alcohol and 2 mL of water. Shake the test tube well to allow uniform mixing, allow 2 to 4 mL of sulfuric acid to flow down the sides of the test tube, and hold the test tube upright. If sugar is present, the interface of the two liquids is colored faint to deep violet, and on shaking, the whole solution becomes blue-violet.]
Transfer the residue from the bottle and hardened filter to a beaker with about 50 mL of water. Immerse the beaker in boiling water, and stir constantly for 15 minutes or until all of the starch is gelatinized. Cool the beaker to 55, add 20 mL of Malt Extract, and hold at this temperature for 1 hour. Heat again to boiling for a few minutes, cool to 55, add 20 mL of Malt Extract, and hold at this temperature for 1 hour or until the residue when treated with iodine TS shows no blue tinge upon microscopic examination. Cool, dilute with water to 250 mL, and filter.
General Procedure— Transfer 200 mL of the Test Solution to a flask fitted with a reflux condenser, add 20 mL of hydrochloric acid, and heat in a boiling water bath for 2½ hours. Cool, nearly neutralize with sodium hydroxide TS, complete neutralization with sodium carbonate TS, dilute with water to 500 mL, mix, and filter. The volume of aliquot taken depends on the starch content of the specimen under test (see Table 1). The aliquot should contain between 100 and 200 mg of dextrose. Transfer 50 mL of the filtrate to a 400-mL alkali-resistant glass beaker, add 50 mL of alkaline cupric tartarate TS, cover the beaker with a water glass, and heat. Adjust the flame in the burner so that the contents of the flask begin to boil in 4 minutes and continue boiling for exactly 2 minutes. Filter the hot solution at once through a sintered-glass filter. Wash the precipitate of cuprous oxide thoroughly with water at about 60, then with 10 mL of alcohol, and finally with 10 mL of ether.
Table 1. Determination of the Optimum Aliquot
 % of Expected Starch Content Aliquot in mL 60 25 50 35 40 50 30 50 20 50
For solutions of reducing sugars of comparatively high purity, proceed as directed under Method 1A to determine the amount of reduced copper obtained by weighing the dried cuprous oxide. For solutions of reducing sugars containing large amounts of organic impurities, including sucrose, proceed as directed under Method 1B to determine the amount of reduced copper obtained by titration with sodium thiosulfate.
METHOD 1A— Dry the precipitate obtained under General Procedure for 30 minutes in an oven at 110 ± 2, cool to room temperature in a desiccator, and weigh. Refer to Table 2 to find the quantity of dextrose, in mg, corresponding to the weight of cuprous oxide found. Determine the percentage of dextrose and then the content of starch by the following formula:
 Percentage of dextrose = wt. of dextrose in mg × 0.1 × 500 wt. of sample in g × aliquot in mL Content of starch = % dextrose × 0.9.
Table 2. Calculating Dextrose (Applicable when Cu2O is weighed directly) (Expressed in mg)
 Cuprous Oxide (Cu2O) Dextrose (D-Glucose) Cuprous Oxide (Cu2O) Dextrose (D-Glucose) Cuprous Oxide (Cu2O) Dextrose (D-Glucose) Cuprous Oxide (Cu2O) Dextrose (D-Glucose) Cuprous Oxide (Cu2O) Dextrose (D-Glucose) Cuprous Oxide (Cu2O) Dextrose (D-Glucose) 10 4.0 90 38.9 170 75.1 250 112.8 330 152.2 410 193.7 12 4.9 92 39.8 172 76.0 252 113.7 332 153.2 412 194.7 14 5.7 94 40.6 174 76.9 254 114.7 334 154.2 414 195.8 16 6.6 96 41.5 176 77.8 256 115.7 336 155.2 416 196.8 18 7.5 98 42.4 178 78.8 258 116.6 338 156.3 418 197.9 20 8.3 100 43.3 180 79.7 260 117.6 340 157.3 420 199.0 22 9.2 102 44.2 182 80.6 262 118.6 342 158.3 422 200.1 24 10.0 104 45.1 184 81.5 264 119.5 344 159.3 424 201.1 26 10.9 106 46.0 186 82.5 266 120.5 346 160.3 426 202.2 28 11.8 108 46.9 188 83.4 268 121.5 348 161.4 428 203.3 30 12.6 110 47.8 190 84.3 270 122.5 350 162.4 430 204.4 32 13.5 112 48.7 192 85.3 272 123.4 352 163.4 432 205.5 34 14.3 114 49.6 194 86.2 274 124.4 354 164.4 434 206.5 36 15.2 116 50.5 196 87.1 276 125.4 356 165.4 436 207.6 38 16.1 118 51.4 198 88.1 278 126.4 358 166.5 438 208.7 40 16.9 120 52.3 200 89.0 280 127.3 360 167.5 440 209.8 42 17.8 122 53.2 202 89.9 282 128.3 362 168.5 442 210.9 44 18.7 124 54.1 204 90.9 284 129.3 364 169.6 444 212.0 46 19.6 126 55.0 206 91.8 286 130.3 366 170.6 446 213.1 48 20.4 128 55.9 208 92.8 288 131.3 368 171.6 448 214.1 50 21.3 130 56.8 210 93.7 290 132.3 370 172.7 450 215.2 52 22.2 132 57.7 212 94.6 292 133.2 372 173.7 452 216.3 54 23.0 134 58.6 214 95.6 294 134.2 374 174.7 454 217.4 56 23.9 136 59.5 216 96.5 296 135.2 376 175.8 456 218.5 58 24.8 138 60.4 218 97.5 298 136.2 378 176.8 458 219.6 60 25.6 140 61.3 220 98.4 300 137.2 380 177.9 460 220.7 62 26.5 142 62.2 222 99.4 302 138.2 382 178.9 462 221.8 64 27.4 144 63.1 224 100.3 304 139.2 384 180.0 464 222.9 66 28.3 146 64.0 226 101.3 306 140.2 386 181.0 466 224.0 68 29.2 148 65.0 228 102.2 308 141.2 388 182.0 468 225.1 70 30.0 150 65.9 230 103.2 310 142.2 390 183.1 470 226.2 72 30.9 152 66.8 232 104.1 312 143.2 392 184.1 472 227.4 74 31.8 154 67.7 234 105.1 314 144.2 394 185.2 474 228.3 76 32.7 156 68.6 236 106.0 316 145.2 396 186.2 476 229.6 78 33.6 158 69.5 238 107.0 318 146.2 398 187.3 478 230.7 80 34.4 160 70.4 240 108.0 320 147.2 400 188.4 480 231.8 82 35.3 162 71.4 242 108.9 322 148.2 402 189.4 482 232.9 84 36.2 164 72.3 244 109.9 324 149.2 404 190.5 484 234.1 86 37.1 166 73.2 246 110.8 326 150.2 406 191.5 486 235.2 88 38.0 168 74.1 248 111.8 328 151.2 408 192.6 488 236.3
METHOD 1B—
Sodium Thiosulfate Solution— Transfer 3.9 g of sodium thiosulfate, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Potassium Iodide Solution— Dissolve 42 g of potassium iodide in 100 mL of water.
Sodium Acetate Solution— Dissolve 5.74 g of sodium acetate in 10 mL of water.
Traps for Volatile Oil Apparatus
Procedure— Wash the precipitated cuprous oxide obtained under General Procedure with water, cover this filter with a watch glass and dissolve the cuprous oxide with 5 mL of nitric acid directed under the watch glass with a pipet. Collect the filtrate in a 250-mL flask, wash the watch glass and the filter with water. Collect all the washings in the flask. Boil the contents of the flask to expel red fumes. Add about 5 mL of bromine TS, and boil until the bromine is completely removed. Cool, and proceed as directed under Copper Solution beginning with “add 10 mL of Sodium Acetate Solution.” From the volume of Sodium Thiosulfate Solution consumed, obtain the weight of copper, in mg, by multiplying by 1.1259 to obtain the weight, in mg, of cuprous oxide. From Table 2, find the quantity of dextrose, in mg, corresponding to the weight of cuprous oxide. The content of starch is equivalent to the weight, in mg, of dextrose obtained times 0.9. Conduct a blank determination, using 50 mL of alkaline cupric tartrate TS and 50 mL of Malt Extract. If the weight of the cuprous oxide so obtained exceeds 0.5 mg, correct the result of the determination accordingly. [NOTE—The alkaline cupric tartrate TS deteriorates on standing and the quantity of cuprous oxide obtained in the blank determination increases.]
Method 2— The following method is specific for dextrose (glucose), and because of its extreme sensitivity it may account for differences noted between values obtained from the same specimen. Duplicate determinations do not vary more than 2%.
Glucoamylase Solution— Prepare a solution of glucoamylase in water containing 30 International Units (IU) per mL. Use glucoamylase obtained preferably from Rhizopus delemar. The total glucoamylase activity of the test specimen being used should be not less than 150 IU.
Acetate Buffer Solution— Dissolve 16.4 g of sodium acetate in 100 mL of water, add 12.0 mL of glacial acetic acid, and mix. The pH of this solution is 4.8.
Phosphate Buffer— Dissolve 3.63 g of tris (hydroxymethyl) aminomethane and 5.0 g of monobasic sodium phosphate in 50.0 mL of water. At 37, adjust with phosphoric acid to a pH of 7.0, dilute with water to 100.0 mL, and mix. [NOTE—The pH of the buffer medium is sensitive to temperature and should be adjusted to the desired pH at the temperature to be used during incubation.]
Enzyme Solution— Dissolve 30 mg of glucose oxidase (Type II from Aspergillus niger), 3 mg of peroxidase (Type I from horseradish), and 10 mg of potassium ferrocyanide in 100 mL of Phosphate Buffer. [NOTE—This mixture can be stored in a refrigerator for up to 10 days.]
18 N Sulfuric Acid— Add slowly, while stirring, 54 mL of sulfuric acid to 102 mL of water, allow to cool to 25, and mix.
Standard Solutions— Dissolve an accurately weighed quantity of USP Dextrose RS in water to obtain a solution containing 1.0 mg of USP Dextrose RS per mL. Quantitatively dilute a known volume of this solution with water to obtain Standard Solutions A, B, C, D, and E, having known concentrations of 10, 20, 25, 40, and 50 µg per mL of USP Dextrose RS, respectively. [NOTE—Allow 4 hours for complete mutarotation before use.]
Control Solution— Transfer an accurately weighed quantity of about 0.4 g of starch to a previously tared flask and proceed as directed under Test Solutions beginning with “add 25 mL of water and, adjust the pH with phosphoric acid.”
Procedure— Concomitantly determine the absorbances of the Standard Solutions and the Test Solutions at the wavelength of maximum absorbance at about 540 nm, with a suitable spectrophotometer, using the Control Solution as the blank to set the instrument. Plot the absorbance values of the Standard Solutions versus concentration, in µg per mL, of dextrose, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of dextrose in each of the Test Solutions, calculate the average concentration, in µg per mL, of the solution under test. The percentage of starch content in the weight of the test specimen taken by the equation is calculated by the formula:
(0.9C / 106)(V1)(250 / V0)(100 / E)(100 / W) = 2.25CV1 / V0EW,
in which E is the weight, in g, of the test specimen taken; V0 is the volume, in mL, of the aliquot taken from the 250-mL volumetric flask; W is the percentage of dry weight of the test specimen; and V1 is the volume, in mL, if extra dilution is done (see Note 2 under Test Solutions). [NOTE—V0 is 1.0 when no extra dilution is done.]
Volatile Oil Determination
Set up a round-bottom, shortneck, 1-liter flask in a heating mantle set over a magnetic stirrer. Insert an egg-shaped stirring bar magnet in the flask, and attach a cold-finger condenser and an appropriate volatile oil trap of the type illustrated.
Coarsely comminute a sufficient quantity of the drug to yield from 1 to 3 mL of volatile oil. Small seeds, fruits, or broken leaves of herbs ordinarily do not need comminution. Very fine powders are to be avoided. If this is not possible, it may be necessary to mix them with purified sawdust or purified sand. Place a suitable quantity of the drug, accurately weighed, in the flask, and fill it one-half with water. Attach the condenser and the proper separator. Boil the contents of the flask, using a suitable amount of heat to maintain gentle boiling for 2 hours, or until the volatile oil has been completely separated from the drug and no longer collects in the graduated tube of the separator.
If a proper quantity of the volatile oil has been obtained in the graduated tube of the separator, it can be read to tenths of 1 mL, and the volume of volatile oil from each 100 g of drug can be calculated from the weight of the drug taken. The graduations on the separator “for oils heavier than water” are so placed that oil remains below the aqueous condensate that automatically flows back into the flask.
Water Content
For unground or unpowdered drugs, prepare about 10 g of the Laboratory Sample by cutting, granulating, or shredding, so that the parts are about 3 mm in thickness. Seeds or fruits smaller than 3 mm should be cracked. Avoid the use of high-speed mills in preparing the sample, and exercise care that no appreciable amount of moisture is lost during the preparation and that the portion taken is representative of the Laboratory Sample. Determine the water content as directed for Procedure for Articles of Botanical Origin in the Method III (Gravimetric) under Water Determination 921.
Test for Aflatoxins
CautionAflatoxins are highly dangerous, and extreme care should be exercised in handling aflatoxin materials.
This test is provided to detect the possible presence of aflatoxins B1, B2, G1, and G2 in any material of plant origin. Unless otherwise specified in the individual monograph, use the following method.
Zinc Acetate–Aluminum Chloride Reagent— Dissolve 20 g of zinc acetate and 5 g of aluminum chloride in sufficient water to make 100 mL.
Sodium Chloride Solution— Dissolve 5 g of sodium chloride in 50 mL of water.
Test Solution 1— Grind about 200 g of plant material to a fine powder. Transfer about 50 g of the powdered material, accurately weighed, to a glass-stoppered flask. Add 200 mL of a mixture of methanol and water (17:3). Shake vigorously by mechanical means for not less than 30 minutes, and filter. [NOTE—If the solution has interfering plant pigments, proceed as directed for Test Solution 2.] Discard the first 50 mL of the filtrate, and collect the next 40-mL portion. Transfer the filtrate to a separatory funnel. Add 40 mL of Sodium Chloride Solution and 25 mL of solvent hexane, and shake for 1 minute. Allow the layers to separate, and transfer the lower aqueous layer to a second separatory funnel. Extract the aqueous layer in the separatory funnel twice, each time with 25 mL of methylene chloride, by shaking for 1 minute. Allow the layers to separate each time, separate the lower organic layer, and collect the combined organic layers in a 125-mL conical flask. Evaporate the organic solvent to dryness on a water bath. Cool the residue. If interferences exist in the residue, proceed as directed for Cleanup Procedure; otherwise, dissolve the residue obtained above in 0.2 mL of a mixture of chloroform and acetonitrile (9.8:0.2), and shake by mechanical means if necessary.
Test Solution 2— Collect 100 mL of the filtrate from the start of the flow, and transfer to a 250-mL beaker. Add 20 mL of Zinc Acetate–Aluminum Chloride Reagent and 80 mL of water. Stir, and allow to stand for 5 minutes. Add 5 g of a suitable filtering aid, such as diatomaceous earth, mix, and filter. Discard the first 50 mL of the filtrate, and collect the next 80-mL portion. Proceed as directed for Test Solution 1, beginning with “Transfer the filtrate to a separatory funnel.”
Cleanup Procedure— Place a medium-porosity sintered-glass disk or a glass wool plug at the bottom of a 10-mm × 300-mm chromatographic tube. Prepare a slurry of 2 g of silica gel with a mixture of ethyl ether and solvent hexane (3:1), pour the slurry into the column, and wash with 5 mL of the same solvent mixture. Allow the absorbent to settle, and add to the top of the column a layer of 1.5 g of anhydrous sodium sulfate. Dissolve the residue obtained above in 3 mL of methylene chloride, and transfer it to the column. Rinse the flask twice with 1-mL portions of methylene chloride, transfer the rinses to the column, and elute at a rate not greater than 1 mL per minute. Add successively to the column 3 mL of solvent hexane, 3 mL of ethyl ether, and 3 mL of methylene chloride; elute at a rate not greater than 3 mL per minute; and discard the eluates. Add to the column 6 mL of a mixture of methylene chloride and acetone (9:1), and elute at a rate not greater than 1 mL per minute, preferably without the aid of vacuum. Collect this eluate in a small vial, add a boiling chip if necessary, and evaporate to dryness on a water bath. Dissolve the residue in 0.2 mL of a mixture of chloroform and acetonitrile (9.8:0.2), and shake by mechanical means if necessary.
Aflatoxin Solution— [CautionAflatoxins are highly toxic. Handle with care. ] Dissolve accurately weighed quantities of aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 in a mixture of chloroform and acetonitrile (9.8:0.2) to obtain a solution having concentrations of 0.5 µg per mL each of aflatoxin B1 and aflatoxin G1, and 0.1 µg per mL each of aflatoxin B2 and aflatoxin G2.
Procedure— Separately apply 2.5 µL, 5 µL, 7.5 µL, and 10 µL of the Aflatoxin Solution and three 10-µL applications of either Test Solution 1 or Test Solution 2 to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Superimpose 5 µL of the Aflatoxin Solution on one of the three 10-µL applications of the Test Solution. Allow the spots to dry, and develop the chromatogram in an unsaturated chamber containing a solvent system consisting of a mixture of chloroform, acetone, and isopropyl alcohol (85:10:5) until the solvent front has moved not less than 15 cm from the origin. Remove the plate from the developing chamber, mark the solvent front, and allow the plate to air-dry. Locate the spots on the plate by examination under UV light at 365 nm: the four applications of the Aflatoxin Solution appear as four clearly separated blue fluorescent spots; the spot obtained from the Test Solution that was superimposed on the Aflatoxin Solution is no more intense than that of the corresponding Aflatoxin Solution; and no spot from any of the other Test Solutions corresponds to any of the spots obtained from the applications of the Aflatoxin Solution. If any spot of aflatoxins is obtained in the Test Solution, match the position of each fluorescent spot of the Test Solution with those of the Aflatoxin Solution to identify the type of aflatoxin present. The intensity of the aflatoxin spot, if present in the Test Solution, when compared with that of the corresponding aflatoxin in the Aflatoxin Solution will give an approximate concentration of aflatoxin in the Test Solution.