Anhydrous Lactose.
Page 2688
Change to:Anhydrous Lactose
Portions of the monograph text that are IP text, and are not part of the PDG harmonized text, are marked with
symbols (⧫⧫)
C12H22O11 Mol. Wt. 342.3
Anhydrous Lactose is O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranose (β-lactose), or a mixture of O-β-D-
galactopyranosyl-(1→4)-β-D-glucopyranose (β-lactose) and O-β-D-galactopyranosyl-(1→4)-α-D-glucopyranose
(α-lactose).
Category. Pharmaceutical aid
Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out.
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with
anhydrous lactose IPRS or with the reference spectrum of anhydrous lactose.
B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
Solvent mixture. 60 volumes of methanol and 40 volumes of water.
Mobile phase. A mixture of 50 volumes of ethylene dichloride, 25 volumes of glacial acetic acid, 15 volumes of
methanol and 10 volumes of water.
Spray reagent. Dissolve 0.5 g of thymol in 100 ml of a mixture of 95 volumes of ethanol and 5 volumes of
sulphuric acid.
Test solution. Dissolve 50 mg of the substance under examination in the solvent mixture and dilute to 100.0 ml
with the solvent mixture.
Reference solution (a). A 0.05 per cent w/v solution of anhydrous lactose IPRS in the solvent mixture.
Reference solution (b). A solution containing 0.05 per cent w/v, each of, dextrose IPRS, anhydrous lactose IPRS,
fructose IPRS and sucroseIPRS in the solvent mixture.
Apply to the plate 2 μl of each solution. Allow the spots to dry in a current of air and develop the plate in a
chamber using mobile phase saturated for about 1 hour prior to use. Develop the chromatogram until the mobile
phase has moved to three fourth length of the plate, dry the plate in air and develop the plate again using fresh
mobile phase. Remove the plate from the chamber, mark the solvent front and dry the plate in a warm air. Spray
the plate with spray reagent and heat the plate at 130º for 10 minutes. The principal spot in the chromatogram
obtained with the test solution corresponds to the spot observed in the chromatogram obtained with reference
solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows four clearly
separated spots. Ignore any spots remaining on the line of application.
C. Heat 5 ml of a 5.0 per cent w/v solution with 5 ml of 10 M ammonia in a water-bath at 80° for 10 minutes; a
red colour develops.
Tests
Appearance of solution. Dissolve 1.0 g in water by heating to 50°, dilute to 10 ml with water and allow to cool.
The solution is clear (2.4.1) and not more intensely coloured than reference solution BYS7 (2.4.1).
Acidity or alkalinity. Dissolve 6 g of the substance under examination in 25 ml of carbon dioxide-free water by
boiling, cool and add 0.3 ml of phenolphthalein solution. The solution is colourless and not more than 0.4 ml of
0.1 M sodium hydroxide is required to change the colour of the solution to pink or red.
Content of Alpha and Beta Anomers. Determine by gas chromatography (2.4.13).
Silyation reagent. A mixture of 19.5 volumes of dimethyl sulphoxide, 58.5 volumes of pyridine and 22 volumes
of trimethylsilylimidazole.
Test solution. Transfer 10 mg of Anhydrous Lactose into a vial with a screw cap, add 4 ml of silylation reagentand
dissolve with the aid of ultrasound for 20 minutes. Transfer 400 µl to an injection vial and add 1 ml of pyridine.
Close the vial and mix.
Reference solution. 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 beta-lactose. Transfer 10 mg of
the mixture into a vial with a screw cap, add 4 ml of silylation reagent and dissolve with the aid of ultrasound for
20 minutes. Transfer 400 µl to an injection vial and add 1 ml of pyridine. Close the vial and mix.
Chromatographic system
– Precolumn: a fused silica column 2 m × 0. 53 mm, packed with deactivated intermediate polarity (Such as
Restek),
– Column: a fused silica column 15 m × 0.25 mm, packed with 5 per cent phenyl and 95 per cent
methylpolysiloxane (film thickness 0.25 μm) (Such as Varian CP-Sil 8 CB),
– temperature:
column 80° for 1 minute, 80° to 150° @ 35° per minute, 150° to 300° @ 12° per minute and hold at 300° for 2
minutes,
inlet port 275° and detector at 325°,
– flame ionization detector,
– flow rate: 2.8 ml per minutes, using helium as carrier gas,
– injection volume: 0.5 μl.
The relative retention time with reference to beta-lactose for alpha-lactose (retention time is about 12 minutes) is
about 0.9.
Inject the reference solution. The test is not valid unless the resolution between the peaks due to alpha-lactose and
beta-lactose is not less than 3.
Inject the test solution. Calculate the content of alpha-lactose using the following expression:
Result = Sa /(Sa + Sb) × 100
Where, Sa = area of the peak due to alpha-lactose
Sb = area of the peak due to beta-lactose
Calculate the content of beta-lactose using the following expression:
Result = Sb/(Sa + Sb) × 100
Where, Sa = area of the peak due to alpha-lactose
Sb = area of the peak due to beta-lactose
Specific optical rotation (2.4.22). + 54.4°to + 55.9°, at 20º, determined in a solution prepared by dissolving 10 g
in 80 ml of water by heating at 50°, allow to cool and add 0.2 ml of 6 M ammonia. Allow to stand for 30 minutes
and dilute to 100.0 ml with water.
Light absorption (2.4.7). A 10.0 per cent w/v solution in water, shows an absorption maximum at about 400 nm
is not more than 0.04. Dilute 1.0 ml of the solution to 10.0 ml with water.When examined in the range 210 nm to
220 nm; absorbance is not more than 0.25 and in the range 270 nm to 300 nm; absorbance is not more than 0.07.
Heavy metals (2.3.13). Dissolve 4 g in 20.0 ml of water. 12 ml of the solution complies with the limit test for
heavy metals, Method D (5 ppm) using 10 ml of lead standard solution (1 ppm, Pb).
Sulphated ash (2.3.18). Not more than 0.1 per cent.
Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in oven at 80° for 2 hours.
Water (2.3.43). Not more than 1.0 per cent, determined on 1 g in a mixture of 1 volume of formamide and 2
volumes of methanol.
Microbial contamination (2.2.9). Total aerobic microbial count is not more than 100 CFU per g, the total
combined molds and yeasts count is not more than 50 CFU per g. 1 g is free from Escherichia coli.
Storage. Store protected from moisture.
Labelling. The label states (1) the relative quantities of alpha- and beta-lactose, determine compliance using
Content of Alpha and Beta Anomers (2) the particle size distribution, it also indicates the d 10, d50, and d90 values
and the range for each.
2.4.26. Solubility. Page264
Anhydrous lactose. Freely soluble in water, practically insoluble in ethanol (95 per cent).
