Identification—
A: Thin-Layer Chromatographic Identification Test 
201
—
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture containing a fluorescent indicator having optimal intensity at 254 nm.
Test solution:
5000 µg per mL in water.
Application volume:
1 µL.
Developing solvent system:
a mixture of ethyl acetate, pyridine, water, acetic acid, and propionic acid (50:50:10:5:5).
Procedure—
Separately apply 1 µL each of the Standard solution and the Test solution to the thin-layer chromatographic plate, and thoroughly dry the starting points in warm air. Develop over 10 cm using the Developing solvent system, dry the plate in a current of hot air, and dip for 3 seconds in a 1 g per L solution of sodium periodate. Dip the plate for 3 seconds in a mixture of dehydrated alcohol, sulfuric acid, acetic acid, and anisaldehyde (90:5:1:1). Dry the plate in a current of hot air until colored spots become visible. The background color may be brightened by exposure to warm steam. Examine in daylight. The chromatogram obtained from the Standard solution shows two blue-grey spots with RF values of about 0.13 (1,6-GPS) and 0.16 (1,1-GPM). The chromatogram obtained from the Test solution shows principal spots similar in position and color to the chromatogram obtained from the Standard solution.
B:
The retention times of the two principal peaks in the chromatogram of the Assay preparation correspond to those in the chromatogram of the Standard preparation, as obtained in the Assay.
Limit of nickel—
[NOTE—The purity of the reagents and the water used must be suitable for trace analysis, and the reagents and water must be free of nickel.
]
Nickel standard solution—
Transfer 1 mL of nickel standard solution TS into a 100-mL volumetric flask, add 1 mL of nitric acid, dilute with water to volume, and mix. This solution contains the equivalent of 0.1 µg of nickel per mL.
Test solution—
Accurately weigh about 8 g of Isomalt into a 50-mL volumetric flask, add 8 mL of water and 3 mL of 65% nitric acid solution, and incubate at 95
for 1 hour. Allow the solution to cool to room temperature, add another 3 mL of 65% nitric acid solution, and incubate at 95
until all brown vapors have dissipated (about 1–1.5 hours). Allow the solution to cool to room temperature, carefully add 3 mL of 30 percent hydrogen peroxide, and keep the solution at 95
until the evolution of gas has ceased (about 1–2 hours). Allow the solution to cool to room temperature. Repeat the procedure two more times, i.e., adding 30 percent hydrogen peroxide, heating to 95
, and cooling to room temperature. Dilute the resulting solution with water to 50 mL.
Blank—
Prepare as directed for the Test solution, except to omit the addition of Isomalt.
Standard solutions—
Into seven identical 10-mL volumetric flasks, introduce respectively 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of Nickel standard solution equivalent to 0, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 µg of nickel. To each flask, add a 2.0-mL portion of the Test solution, and dilute with water to volume.
Blank solutions—
Prepare as directed for Standard solutions except to replace 2 mL of the Test solution with 2 mL of the Blank.
Procedure—
Concomitantly determine the absorbances of the
Standard solutions and the
Blank solutions at the wavelength of maximum absorbance at 232.0 nm, with a suitable atomic absorption spectrophotometer (see
Spectrophotometry and Light-Scattering 851) equipped with a graphite furnace and a nickel hollow-cathode lamp. Record the average of the steady readings for each of the
Standard solutions and the
Blank solutions. Plot the absorbances of the
Standard solutions versus the quantity of nickel, in µg, in the portion of
Nickel standard solution added to each
Standard solution flask. Extrapolate the line joining the points on the graph until it meets the concentration axis. The distance between this point and the intersection of the axes represents the amount of nickel (
AT), in µg, present in the portion of
Test solution that was added to each of the
Standard solution flasks. Similarly, plot the absorbance of the
Blank solutions versus the quantity of nickel, in µg, in the portion of
Nickel standard solution added to each of the
Blank solution flasks, to determine the quantity of nickel (
AB) in the portion of
Blank added to each of the
Blank solution flasks. Calculate the quantity, in µg, of nickel in the portion of Isomalt taken by the formula:
25(AT – AB)
Not more than 1 µg per g, calculated on the anhydrous basis, is found.
Related compounds—
Mobile phase—
Prepare as directed in the Assay.
Resolution solution—
Dissolve accurately weighed quantities of USP Isomalt RS,
USP Mannitol RS, and
USP Sorbitol RS, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having known concentrations of about 20 mg per mL, 0.1 mg per mL, and 0.1 mg per mL, respectively.
Standard solution—
Dissolve an accurately weighed quantity of
USP Sorbitol RS and
USP Mannitol RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.1 mg of each per mL.
Test solution—
Use the Assay preparation.
Chromatographic system—
Prepare as directed in the Assay. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the typical retention time of 1,1-GPM is about 12.3 minutes; the relative retention times are about 1.2 for 1,6-GPS, about 1.6 for mannitol, about 2.0 for sorbitol, and 1.0 for 1,1-GPM; and the resolution, R, between 1,1-GPM and 1,6-GPS is not less than 2.0.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of mannitol or sorbitol in the portion of Isomalt taken by the formula:
5000C/W(rU / rS)
in which
C is the concentration, in mg per mL, of
USP Mannitol RS or
USP Sorbitol RS in the
Standard solution; W is the weight, in mg, of Isomalt used to prepare the
Test solution; and
rU and
rS are the individual peak responses of mannitol or sorbitol obtained from the
Test solution and the
Standard solution, respectively: not more than 0.5% of mannitol and not more than 0.5% of sorbitol is found. Calculate the percentage of any other impurity in the portion of Isomalt taken by the formula:
100(ri / rs)
in which
ri is the peak response for each impurity, and
rs is the sum of the responses of all the peaks obtained from the
Test solution: not more than 0.5% of any individual impurity is found; and not more than 2.0% of total impurities, including mannitol and sorbitol, is found. Disregard any impurity peak that is less than 0.1%.
Assay—
Mobile phase—
Use degassed water.
Standard preparation—
Dissolve an accurately weighed quantity of USP Isomalt RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 20 mg per mL.
Assay preparation—
Transfer about 1000 mg of Isomalt, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a refractive index detector maintained at a constant temperature, a 7.8-mm × 30-cm column that contains packing L19, and a 4.6-mm × 3-cm guard column that contains packing L19. The flow rate is about 0.5 mL per minute. The column temperature is maintained at 80 ± 1
. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 1.2 for 1,6-GPS and 1.0 for 1,1-GPM; the resolution,
R, between 1,1-GPM and 1,6-GPS is not less than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%, determined from the 1,6-GPS and 1,1-GPM peak responses.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the 1,6-GPS and 1,1-GPM peaks. Calculate the quantity, in mg, of 1,6-GPS in the portion of Isomalt taken by the formula:
50C(rU / rS)
in which
C is the concentration, in mg per mL, of 1,6-GPS in the
Standard preparation, with calculation based on the declared 1,6-GPS content of USP Isomalt RS; and
rU and
rS are the 1,6-GPS peak responses obtained from the
Assay preparation and the
Standard preparation, respectively. Similarly, calculate the quantity, in mg, of 1,1-GPM in the portion of Isomalt taken.
NF24
--Glucopyranosyl-D-sorbitol.