A: To 1 drop of a solution (1 in 10), add 4 drops of 5% phenol solution, then rapidly add 15 drops of sulfuric acid TS: a deep yellow to orange color is produced.

B: With vigorous swirling, add 1 mL of acetone to 1 mL of a solution (1 in 10): the solution remains clear.

C: With vigorous swirling, add 2 mL of acetone to the solution obtained in Identification test B: a heavy, milky turbidity develops immediately.

D: To 1 mL of a solution (1 in 50), add 4 mL of alkaline cupric citrate TS. Boil vigorously for 2 to 4 minutes. Remove from heat, and allow the precipitate (if any) to settle: the supernatant is blue or blue-green.

Matrix modifier solution— Prepare a solution in water containing 100.0 mg of dibasic ammonium phosphate per 10 mL of solution.

Lead nitrate stock solution— Dissolve 159.8 mg of lead nitrate in 100 mL of water to which has been added 1 mL of nitric acid, then dilute with water to 1000 mL. Prepare and store this solution in glass containers free from soluble lead salts.

Standard lead solution— On the day of use, dilute 10.0 mL of Lead nitrate stock solution with water to 100.0 mL, and mix. Each mL of Standard lead solution contains the equivalent of 10 µg of lead.

Standard solutions— Into five separate 100-mL volumetric flasks, pipet 0.2, 0.5, 1, 2, and 5 mL, respectively, of Standard lead solution, dilute with water, and mix. The Standard solutions contain 0.02, 0.05, 0.1, 0.2, and 0.5 µg of lead per mL, respectively.

Test solution— Transfer about 1.0 g of Polydextrose, accurately weighed and calculated on the anhydrous and ash-free basis, into a 10-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Spiked test solution— Transfer about 1.0 g of Polydextrose, accurately weighed and calculated on the anhydrous and ash-free basis, into a 10-mL volumetric flask, and dissolve in water. Add 100 µL of the Standard lead solution, dilute with water to volume, and mix. This solution contains 0.1 µg of added lead per mL.

Procedure— Concomitantly determine the absorbances of 10-µL aliquots of the five Standard solutions, a mixture of 10 µL of the Matrix modifier solution and 10 µL of the Test solution, and a mixture of 10 µL of the Matrix modifier solution and 10 µL of the Spiked test solution at the lead emission line at 283.3 nm, with a suitable graphite furnace atomic absorption spectrophotometer (see Spectrophotometry and Light-scattering 851). The spectrophotometer is equipped with a pyrolytic tube with a platform and a lead hollow-cathode lamp, using a slit width of 0.7 mm (set low) and a deuterium arc lamp for background correction. The drying temperature of the furnace is maintained at 130 for 40 seconds after a 20-second ramp time using an argon flow rate of 300 mL per minute; the ashing temperature is maintained at 800 for 40 seconds after a 20-second ramp time using an argon flow rate of 300 mL per minute; and the atomization temperature is maintained at 2400 for 6 seconds using an argon flow rate of 50 mL per minute. Clean the graphite furnace at 2600 for 5 seconds after a 1-second ramp time using an argon flow rate of 300 mL per minute, and recharge the graphite furnace at 20 for 20 seconds after a 2-second ramp time using an argon flow rate of 300 mL per minute. Plot the absorbance of each Standard solution, compensated for background correction, versus its content of lead, in µg per mL, and draw the best straight line fitting the five points. From this plot, determine the concentrations, CT and CST, in µg per mL, of lead in the Test solution and the Spiked test solution, respectively. Calculate the percentage recovery taken by the formula: in which 0.1 is the amount of lead, in µg per mL, added to the Spiked test solution. Calculate the content, in µg per g, of lead in Polydextrose taken by the formula: in which W is the weight, in g, of Polydextrose taken to prepare the Test solution: not more than 0.5 µg of lead per g is found.

Test solution— Transfer about 1.0 g of Polydextrose, accurately weighed and calculated on the anhydrous and ash-free basis, into a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Procedure— Determine the absorbance of the Test solution in a 1-cm quartz cell at 283 nm, with a suitable spectrophotometer, using water as the blank. Calculate the percentage of 5-hydroxymethylfurfural and related compounds in the Polydextrose taken by the formula: in which 0.1 is the volume, in L, of the Test solution; 126 is the molecular weight, in g per mol, of 5-hydroxymethylfurfural; A is the absorbance of the Test solution; 16,830 is the molar extinction coefficient, in L per mol per cm, of 5-hydroxymethylfurfural at a wavelength of 283 nm; L is the path length, in cm, of the spectrophotometer cell; and W is the weight, in g, of Polydextrose taken to prepare the Test solution: not more than 0.1% is found.

Mobile phase— Dissolve 35.0 g of sodium nitrate and 1.0 g of sodium azide in 100 mL of water. Dilute with water to 4 L, and mix. Pass through a filter having a 0.45-µm or finer porosity, and degas by applying an aspirator vacuum for 30 minutes. The resulting Mobile phase is 0.1 N sodium nitrate containing 0.025% sodium azide.

Standard solution— Transfer about 20 mg each of USP Dextrose RS, stachyose, and 5800-, 23,700-, and 100,000-molecular weight (MW) pullulan standards, accurately weighed, into a 10-mL volumetric flask. Dissolve in and dilute with Mobile phase to volume, and mix. Pass through a syringe filter having a 0.45-µm or finer porosity into a suitable autosampler vial, and seal.

Test solution— Transfer about 50 mg of Polydextrose, accurately weighed, into a 10-mL volumetric flask. Dissolve in and dilute with Mobile phase to volume, and mix. Pass through a syringe filter having a 0.45-µm or finer porosity into a suitable autosampler vial, and seal.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a refractive index detector set at a sensitivity of 4 × 106 refractive index units full scale and maintained at a temperature of 35 ± 0.1 and a 7.8-mm × 30-cm column that contains packing L25 and is maintained at a temperature of 45. The flow rate is about 0.8 mL per minute. [note—After installation of a new column, pump Mobile phase through the column overnight at a rate of about 0.3 mL per minute. Before calibration or analysis, increase the flow slowly over a 1-minute period to 0.8 mL per minute. Continue to pump Mobile phase through the column at this flow rate for at least 1 hour before the first injection. Check the flow gravimetrically, and adjust it if necessary. Reduce the flow rate to about 0.1 mL per minute when the system is not in use.] Chromatograph replicate injections of the Standard solution, allowing 15 minutes between injections, and record the retention times of the components of the Standard solution as directed for Procedure: the retention times for each component determined on replicate injections agree within ±2 seconds; and dextrose and stachyose are baseline resolved from one another and from the 5800-MW pullulan standard. [note—Elevated valleys are usually observed between the peaks for the three pullulan standards.]

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the retention times for the major peaks. Plot the average retention time, in seconds, of each component in the Standard solution versus its molecular weight, in g per mol; draw the best cubic line fitting the five points; and calculate the correlation coefficient for the line. A suitable system is one that yields a line having a correlation coefficient of not less than 0.99. From the molecular weight distribution graph, generate an MW distribution profile of Polydextrose: no measurable peak above a molecular weight of 22,000 is found.

Internal standard solution— Transfer about 50 mg of n-octadecane, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with pyridine to volume, and mix.

Standard stock solution— Dissolve accurately weighed quantities of USP Dextrose RS, USP Sorbitol RS, and USP 1,6-Anhydro-d-glucose RS in pyridine to obtain a solution having a known concentration of about 0.5 mg of dextrose, about 0.4 mg of sorbitol, and about 0.35 mg of 1,6-anhydro-d-glucose per mL.

Standard solution— Transfer 1.0 mL of Standard stock solution to a reaction vial equipped with a screw cap. Add 1.0 mL of Internal standard solution and 0.5 mL of N-(trimethylsilyl)imidazole to the reaction vial, seal the vial with the screw cap, and mix. Place the vial in an ultrasonic bath at 70 for 60 minutes, remove, and allow the contents to cool.

Test solution— Transfer 20 mg of Polydextrose, accurately weighed and calculated on the anhydrous and ash-free basis, into a reaction vial equipped with a screw cap. Add 1.0 mL of Internal standard solution, 1.0 mL of pyridine, and 0.5 mL of N-(trimethylsilyl)imidazole to the reaction vial, seal the vial with the screw cap, and mix. Place the vial in an ultrasonic bath at 70 for 60 minutes, remove, and allow the contents to cool.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and 2-mm × 250-cm glass column packed with 3% phase G2 on support S1AB. The detector temperature is maintained at about 230. The column temperature is maintained at about 175, and the injection port temperature is maintained at about 210. Helium is used as the carrier gas, flowing at a rate of about 30 mL per minute. Splitless mode is used. Chromatograph the Standard solution, and record the peak response as directed for Procedure. [note—For peak identification purposes, relative retention times are 0.3, 0.4, 0.45, 0.8, 1.0, and 1.2 for d-anhydroglucose (levoglucosan) pyranose form, d-anhydroglucose furanose form (not present in the Standard), n-octadecane, alpha-d-glucose; d-sorbitol, beta-d-glucose, respectively.]

Procedure— Separately inject equal volumes (about 1µL) of the Standard solution and the Test solution into the chromatograph. Record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of each monomer in Polydextrose taken by the formula: in which C is the concentration, in mg per mL, of the respective monomer in the Standard stock solution; W is the weight, in mg, of Polydextrose taken to prepare the Test solution; RU and RS are the ratios of the peak of the respective monomer silyl derivative peak to the peak area of the internal standard peak obtained from the Test solution and the Standard solution, respectively: not more than 4.0% of 1,6-anhydrous-d-glucose is found, and not more than 6.0% of dextrose and sorbitol is found. [note—In the case of glucose, the peak areas for the alpha- and beta-epimers are combined, and in the case of 1,6-anhydro-d-glucose, the peak areas for the pyranose and furanose forms are combined.]

Phenol solution— Add 20 mL of water to 80 g of phenol, and mix.

Dextrose stock standard solution— Transfer 100 mg of USP Dextrose RS, accurately weighed, to a 500-mL volumetric flask, dilute with water to volume, and mix.

Standard preparations— Quantitatively dilute the Dextrose stock standard solution with water to obtain five Standard preparations containing 5, 10, 20, 40, and 50 µg of dextrose per mL, respectively.

Assay preparation— Transfer approximately 10 mg of Polydextrose, accurately weighed and calculated on the anhydrous and ash-free basis, to a 250-mL volumetric flask, dilute with water to volume, and mix.

Procedure— Freshly pipet 2.0 mL of each of the Standard preparations into a separate acetone-free, 15-mL vial equipped with a screw-cap. Transfer 2.0 mL of the Assay preparation to a separate acetone-free, 15-mL vial equipped with a screw-cap, and transfer 2.0 mL of water to a separate acetone-free, 15-mL vial equipped with a screw-cap to provide a blank. To each vial, add 0.12 mL of the Phenol solution, cap the vial, and mix gently. Uncap each vial, and rapidly add 5.0 mL of sulfuric acid. Immediately recap each vial, and shake vigorously. [Caution—Wear rubber gloves and a safety shield while adding sulfuric acid. ] Allow the vials to stand at room temperature for 45 minutes. Concomitantly determine the absorbances of the solutions in 1-cm cells at 490 nm with a suitable spectrophotometer, using the blank to set the instrument. Prepare a standard curve by plotting the absorbances of the solutions from the Standard preparations versus their contents of dextrose, in µg per mL. Draw the straight line best fitting the five points, and extrapolate the line until it intercepts the absorbance axis (y axis). From the curve, determine the concentration CD, in µg per mL, of dextrose in the Assay preparation. Calculate the percentage of dextrose polymer units in the Polydextrose taken by the formula: in which 1.05 is an experimentally derived correction factor to account for the polymer (which also contains a small amount of sorbitol) not giving the exact amount of color given by an equivalent amount of glucose monomers; WT is the weight, in mg, of the Polydextrose taken to prepare the Assay preparation; PG and PL are the percentages of dextrose and 1,6-anhydrous-d-glucose (levoglucosan), respectively, determined in the test for Limit of monomers; and 1.11 is a conversion factor from 1,6-anhydrous-d-glucose, which gives an equivalent amount of color to an equivalent weight of glucose.