A: Solvent system—Mix 60 mL of butyl alcohol with 40 mL of pyridine and 30 mL of water.

Standard preparation— Prepare a solution of USP Xylose RS in water to obtain a solution having a concentration of 100 mg per mL.

Test preparation— Dissolve 1 g of Xylose in water, and add water to make 10 mL.

Spray reagent— Dissolve 1.66 g of phthalic acid and 0.93 g of freshly distilled aniline in 100 mL of water-saturated butyl alcohol. The solution may be stored in a brown glass bottle in a cold place, but is to be discarded if darkening becomes marked.

Chromatographic sheet— Use filter paper (Whatman No. 1 or equivalent). Draw a spotting line 6 cm from one edge of the sheet.

Procedure— Line a suitable chromatographic chamber, prepared for descending chromatography (see Chromatography 621), with blotting paper. Fill the solvent trough with Solvent system, and place a sufficient amount of Solvent system in the bottom of the chamber to permit the lining to be in contact with it. Allow the chamber to equilibrate for not less than 16 hours. To the spotting line apply 2 µL of the Standard preparation stepwise so that the spot is not more than 3 mm in diameter. Similarly apply 2 µL of the Test preparation to the spotting line and 4 cm from the Standard preparation spot. Expose the sheet to the atmosphere of the Solvent system in the closed chamber for 4 hours, then dip the edge of the sheet into the Solvent system in the trough, and develop until the liquid front has reached about 2.5 cm from the end of the sheet. Remove the sheet from the chamber, dry it with the aid of a gentle current of air, apply the Spray reagent, and dry the sheet at 105 to 110 for 5 to 10 minutes. If the spots are faint, respray and redry, and if necessary view under UV light: the RF value of the spot from the Test preparation corresponds to that from the Standard preparation.

B: Standard preparation—Transfer 10 mg of USP Xylose RS to a suitable vial, and add 1 mL of pyridine, 0.2 mL of hexamethyldisilazane, and 0.1 mL of chlorotrimethylsilane. Cap the vial, shake vigorously for 30 seconds, and allow to stand for 5 minutes.

Test preparation— Using 10 mg of Xylose, proceed as directed under Standard preparation.

Procedure— Use a gas chromatograph equipped with a flame-ionization detector and a 3-mm × 1.8-m stainless steel column packed with 10% phase G2 on support S1A. Under typical conditions, nitrogen being used as the carrier gas, the column temperature is operated at 170, and the injector block and detector temperatures at 300. Inject 0.5 µL each of the Test preparation and the Standard preparation: the retention times correspond.

Test solution: 100 mg per mL, in 0.012 N ammonium hydroxide.

p-Bromoaniline solution— Dissolve 2 g of p-bromoaniline in 100 mL of thiourea-saturated glacial acetic acid. Store in an amber glass bottle, and prepare weekly.

Standard preparation— Dissolve a suitable quantity of USP Xylose RS, accurately weighed, in saturated benzoic acid solution to obtain a solution having a known concentration of about 100 µg per mL.

Assay preparation— Dissolve about 1000 mg of Xylose, accurately weighed, in saturated benzoic acid solution in a 100-mL volumetric flask, and dilute with saturated benzoic acid solution to volume. Pipet 1 mL of this solution into a second 100-mL volumetric flask, dilute with saturated benzoic acid solution to volume, and mix.

Procedure— [note—In this procedure, keep strict control of time between steps.] Pipet 1-mL portions of the Standard preparation into each of two test tubes, and pipet 1-mL portions of the Assay preparation into each of two other test tubes. Into each tube pipet 5 mL of p-Bromoaniline solution, and mix. Loosely stopper one tube from each pair, place in a water bath at 70 for 10 minutes, remove, cool rapidly to room temperature, and mix. Set the tubes in the dark for 70 minutes. Concomitantly determine the absorbances of the treated solutions at the wavelength of maximum absorbance at 520 nm, with a suitable spectrophotometer, using the respective untreated solutions as blanks. Calculate the quantity, in mg, of C5H10O5 in the portion of Xylose taken by the formula: in which C is the concentration, in µg per mL, of USP Xylose RS in the Standard preparation; and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.