Assay and Identification of Betamethasone Sodium Phosphate by HPTLC

Dissolve 10 mg of betamethasone sodium phosphate CRS in 10 mL methanol.

Weigh and crush 10 tablets of betamethasone sodium phosphate to a fine powder. Transfer an equivalent of 10 mg betamethasone of the powdered tablets to a 10 mL volumetric flask. Add methanol with intermittent swirling and sonicate for 5 mins before topping up to volume in methanol. Filter solution through 0.45 µm PVDF syringe filter.

Silica Gel 60 F₂₅₄, 20 x 10 cm, HPTLC plate (1.05642)

Prewash the plate with methanol and dry at 120 °C for 30 min. Cool plate in desiccator before use.

5 μL

Camag Linomat 5 using band length = 4 mm, track distance = 20 mm, distance from left edge = 20 mm, distance from lower edge = 8 mm. Allow to dry completely before plate development.

Glacial acetic acid:water:butanol 20:20:60 (v:v:v) 

Unsaturated chamber for over 3/4 of the HPTLC plate. Dry in air after removal from chamber.

Detection A - Examine in UV light at 254 nm

Detection B - Weigh and transfer 0.25 g of 2,4-dihydroxybenzaldehyde into a 100 mL volumetric flask. Add 50 mL of glacial acetic acid to dissolve followed by 12.5 mL of sulfuric acid. Make up to mark with glacial acetic acid. Allow the solution to cool before use. Spray the HPTLC plate with this solution and heat plate at 90 °C for 35 min or until the spots appear. Allow the plate to cool and examine in day lightand under UV at 365 nm (viewing at 365 nm not done for this study)

Camag Scanner 4 in absorption mode, scan speed = 20 mm/s, slit = 3 x 0.2 mm, spectral recording = 200 to 380 nm;

Measurements done at wavelength 254 nm.

Reference 1

0.02504

0.701

Reference 2

0.02466

0.709

Reference 3

0.02508

0.704

Reference 4

0.02447

0.701

Reference 5

0.02531

0.706

% RSD Area Under Peak

1.36%

% RSD Retardation Factor

0.49%

% Assay Betamethasone sodium phosphate (sample)

99.71%

1

LOQ

2.8

2.84

101.4

2

80%

4.0

3.86

96.5

3

100%

5.0

5.22

104.4

4

120%

6.0

6.16

102.7