A: Infrared Absorption 197K.

B: Proceed as directed in Identification test B under Lactose Monohydrate, except to use USP Anhydrous Lactose RS instead of USP Lactose Monohydrate RS in Standard solution A and B and to use Anhydrous Lactose in the Test solution.

C: Proceed as directed in Identification test C under Lactose Monohydrate.

Silylation reagent— Prepare a mixture of pyridine and trimethylsilylimidazole (72:28).

Resolution mixture— Prepare a mixture of alpha lactose monohydrate and beta lactose having an anomeric ratio of about 1:1 based on the labeled anomeric contents of the alpha lactose monohydrate and the beta lactose.

Chromatographic system (see Chromatography 621)—The gas chromatograph is equipped with a flame-ionization detector and a 4-mm × 0.9-m glass column packed with 3% liquid phase G19 on support S1A. The column temperature is maintained at about 215, and the injection port and the detector temperatures are maintained at about 275. The carrier gas is helium, flowing at a rate of about 40 mL per minute.

Derivatization procedure— Transfer about 1 mg of Anhydrous Lactose to a 5-mL reaction vial equipped with a screw cap, add 0.45 mL of dimethyl sulfoxide, seal the vial tightly with a screw cap, and mix on a vortex mixer to dissolve. Add 1.8 mL of Silylation reagent, seal the vial tightly with a screw cap, and mix gently. Transfer about 1 mg of Resolution mixture to a second 5-mL reaction vial equipped with a screw cap, add 0.45 mL of dimethyl sulfoxide, seal the vial tightly with a screw cap, and mix on a vortex mixer to dissolve. Add 1.8 mL of Silylation reagent, seal the vial tightly with a screw cap, and mix gently. Maintain both vials at room temperature for 20 minutes before using.

Procedure— Inject a 2.0-µL portion of the derivatized Resolution mixture into the chromatograph, and record the peak areas for the major peaks: the relative retention times are about 0.7 for the silyl derivative of alpha lactose and 1.0 for the silyl derivative of beta lactose, and the resolution, R, between the two peaks is not less than 3.0. Similarly inject a 2.0-µL portion of the derivatized Anhydrous Lactose into the chromatograph, and record the peak areas for the major peaks. Determine the percentage of alpha anomer in the Anhydrous Lactose by the formula: in which ra is the response of the alpha anomer silyl derivative peak and rb is the response of the beta anomer silyl derivative peak. Determine the percentage of beta anomer in the Anhydrous Lactose by the formula:

(Official January 1, 2007)