Assay and Identification of Betamethasone Sodium Phosphate by HPTLC

Dr. Ajay Kaparwan, Application Scientist, R&D

Abstract

This article introduces a highly efficient thin-layer chromatography (HPTLC) method for accurately measuring and identifying betamethasone, a glucocorticoid commonly used in different therapeutic applications. The method utilizes a Silica Gel 60 F₂₅₄ HPTLC plate and involves two detection approaches, UV light at 254 nm and derivatization with 2,4-dihydroxybenzaldehyde. The method exhibits linearity across a concentration range of 1 to 7 µg/spot. The detection limit (LOD) and quantification limit (LOQ) were established as 0.91 µg/spot and 2.74 µg/spot, respectively. The method was validated with an assay range of 96.0 to 103%, confirming its suitability for quality control of betamethasone in pharmaceutical applications.

Section Overview

• Introduction
• Experimental
• Results
• Conclusion
• References

Introduction

Betamethasone sodium phosphate

Betamethasone Sodium Phosphate is the disodium salt of the 21-phosphate ester of betamethasone, a synthetic glucocorticoid with metabolic, immunosuppressive and anti-inflammatory actions.¹

The European Pharmacopoeia monograph for betamethasone sodium phosphate employs high-performance thin layer chromatography (HPTLC) technique for the identification and spectrophotometric method for assay.^2,3

Here we show a method for identification and assay of betamethasone sodium phosphate using 2 detection approaches and a densitometric measurement.

Experimental

Table 1. HPTLC conditions for identification and assay of betamethasone sodium phosphate

Samples
Reference solution:	Dissolve 10 mg of betamethasone sodium phosphate CRS in 10 mL methanol.
Sample solution:	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.
Chromatographic Conditions
TLC plate:	Silica Gel 60 F254, 20 x 10 cm, HPTLC plate (1.05642)
Plate pretreatment:	Prewash the plate with methanol and dry at 120 °C for 30 min. Cool plate in desiccator before use.
Sample application:	5 μL
Auto spotter:	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.
Mobile phase:	Glacial acetic acid:water:butanol 20:20:60 (v:v:v)
Development:	Unsaturated chamber for over 3/4 of the HPTLC plate. Dry in air after removal from chamber.
Detection and Derivatization:*	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)
Scanning Parameters:	Camag Scanner 4 in absorption mode, scan speed = 20 mm/s, slit = 3 x 0.2 mm, spectral recording = 200 to 380 nm;
Densitometry:	Measurements done at wavelength 254 nm.
* Ph. Eur. 10.0 described detection A&B, Ph. Eur. 11.0 describes only detection B.

Results

Identification

Figure 1. Determination of Repeatability of betamethasone spots for reference solution and sample on chromatogram measured at wavelength 254nm (Detection A).

Figure 2. The principal spot of reference and sample in the chromatogram obtained with the test solution in day light after derivatization (Detection B).

According to Ph. Eur. 11.0, the principal spot in the chromatogram obtained with the test solution should be similar in position, color, and size to the principal spot in the chromatogram obtained with the reference solution. (Figure 2).

Reproducibility

Figure 3. Densitogram for Repeatability and Area under the peak measured at wavelength 254nm.

Table 2. System suitability and repeatability betamethasone reference solution repeatability (5 μg per spot) measured at 254 nm

Reference Sol	Area under the peak	Retardation factor
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
Mean	0.02491	0.704
Standard Deviation	0.00034	0.0034
% RSD	1.36	0.49

Table 3. System suitability test results observed.

Component	Observed Value
% RSD Area Under Peak	1.36%
% RSD Retardation Factor	0.49%
% Assay Betamethasone sodium phosphate (sample)	99.71%

Assay for Betamethasone Sodium Phosphate by HPTLC

Linearity LOD and LOQ

Linearity of betamethasone sodium phosphate peak area measurements by densitometry at 254 nm for a calibration range of 1 to 7 µg/spot.

Figure 4. Determination of Linearity of betamethasone sodium phosphate measured at wavelength 254nm.

Figure 5. Densitogram for Linearity of betamethasone sodium phosphate measured at wavelength 254 nm.

Figure 6. Linearity scan of betamethasone sodium phosphate.

Table 4. Calibration data for betamethasone sodium phosphate measured at 254 nm

Linearity conc (μg per spot)	Area under the peak
1	0.00214
2	0.00678
3	0.00969
4	0.01298
5	0.01569
6	0.01733
7	0.02096
Intercept	0.000287
Slope	0.002984
STYX	0.000819
R2	0.9867
LOD (μg per spot)	0.91
LOQ (μg per spot)	2.74

Figure 7. Calibration curve for betamethasone sodium phosphate by HPTLC at 254 nm.

Accuracy and Recovery

Figure 8. Determination of accuracy of betamethasone sodium phosphate with spiking over sample on chromatogram at measured wavelength 254nm.

Figure 9. Densitogram for accuracy of betamethasone sodium phosphate with spiking over sample on plate/chromatogram measured at wavelength 254nm.

Table 5. Accuracy and recovery determination for betamethasone sodium phosphate spiked samples

No.	Spike Level	μg per spot Added	μg per spot Found	% Recovery
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

Conclusion

The presented instrumental HPTLC method is simple, linear, and selective over a concentration range from 1 to 7 µg/spot. The RSD for repeatability of area under the curve and retardation factor is < 2%. The limit of detection (LOD) was 0.91 µg/spot, and the Limit of Quantification (LOQ) was 2.74 µg/spot. The percentage recovery of betamethasone ranged from 96.5 to 104.4%.

References

1. Betamethasone Sodium Phosphate. Nih.gov. . [Internet]. PubChem.[updated 10 Jul 2024]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Betamethasone-Sodium-Phosphate

2. Betamethasone Sodium Phosphate. Monograph 01/2008:0810. 10.0, 1970 (expired). [Internet]. Eur. Pharmacopeia. Available from: https://www.edqm.eu/en/-/shutdown-of-european-pharmacopoeia-10th-edition

3. Betamethasone Sodium Phosphate Monograph 07/2021:0810. 11.0, 2089. [Internet]. Eur. Pharmacopeia. Available from: https://pheur.edqm.eu/