Dexamethasone HPLC Assay and Impurity Profiling Methods Following United States Pharmacopeia Monograph Guidelines Using a Titan C18 Column and UV Detection

Introduction

A simple, precise and sensitive Reverse-Phase High Pressure Liquid Chromatography gradient method was adapted for traceability, homogeneity and total chromatographic analysis of Dexamethasone. The given experimental conditions follow the USP43-NF38 monograph method for Dexamethasone Assay and Organic Impurity Profiling. Dexamethasone, Betamethasone, Dexamethasone acetate and Desoximetasone were baseline resolved within 20 minutes using a Titan C18 UHPLC column (2.1 x 100 mm, 1.9 µm). This column has the same packing, length and ID, but where the packing is bound to a slightly larger average particle size (1.7 µm) referenced in the monograph. The method was validated following the guidelines in USP General Chapters <621>, <1225>, and <1226>. Lower sample concentrations were used, but compensated by a larger injection volume (to maintain mass on column) to improve reproducibility. The chromatographic separation was achieved using a mixture of 3.4 g/L monobasic potassium phosphate solution (pH 3.0) and acetonitrile as the mobile phase with gradient elution and UV detection at 240 nm. Much shorter relative retention time (RRT) was seen for Dexamethasone acetate and Desoximetasone, but with excellent chromatographic resolution (Rs > 10) for both compounds. Under the applied conditions, system suitability requirements are met, and the method demonstrates good selectivity, reproducibility, sensitivity and accuracy.

Dexamethasone, Betamethasone, Dexamethasone Acetate, Desoxiametasone

 

 

Experimental Conditions
Column Titan™ C18 UHPLC
Column (100 x 2.1 mm, 1.9 µm)
Injection volume 5 µL
Detection UV @ 240 nm (analytical flow cell; 13 µL) Flow rate 0.4 mL/min
Mobile phase A Dissolve 3.40 g of potassium dihydrogen
phosphate in 1000 mL water and adjust to pH 3.0 using
phosphoric acid.
Temperature Column: 35°C
Mobile phase B Acetonitrile Pressure drop 320 - 470 bar
Diluent Acetonitrile and Water (56:44) v/v.
Time (min) % A % B
0 - 10 76 24
10 - 15 76 - 45 24 - 55
15 - 16 45 - 10 55 - 90
16 - 16.1 10 - 76 90 -24
16.1 - 20 76 24
Test solution Dissolve 0.04 g of Dexamethasone CRS
in 25 mL diluent (1.6 mg/mL).
System suitability solution Dissolve 12 mg of Dexamethasone and
8 mg of Betamethasone in 100 mL diluent
Standard solution Dissolve 1.6 mg of Dexamethasone, 2.4 mg of Betamethasone,
4.8 mg of Dexamethasone acetate using 10 mL
diluent further take 1.0 mL of this solution
and dilute to 100 mL using the diluent.
Reference solution Dilute 5 mg of EP dexamethasone for system
suitability in 5 mL diluent

Blank

Standard solution

System Suitability Solution

Dexamethasone (Test Solution)

Reference Solution

 

 

Chromatographic Data (System Suitability Solution)

Peaks Compound Retention Time (min) RRT RRT Reference per USP43-NF38 Resolution Requirement per USP43-NF38 Tailing Factor
1 Betamethasone 11.7 0.95 0.94     1.0
2 Dexamethasone 12.3 1.00 1.00 2.0 NLT 1.5 1.0

 

 

Chromatographic Data (Reference solution)

Peaks Compound Retention Time (min) RRT RRT Reference per USP43-NF38 Resolution Tailing Factor
1 Betamethasone 11.7 0.95 0.94 - 1.2
2 Dexamethasone 12.3 1.00 1.00 1.5 1.0
3 Desoximetasone 14.6 1.19 1.58 10.2 1.1
4 Dexamethasone Acetate 15.9 1.29 1.74 13.4 1.0

 

 

Repeatability (Reference solution)

Peaks Compound Area Response (n=3) Standard deviation RSD (%) RSD (%) Reference per USP43-NF-38
1 Betamethasone 39.7 0.48 1.2 5.0
2 Dexamethasone 25221.73 263.78 1.0 5.0
3 Desoximetasone 50.18 0.50 1.0 5.0
4 Dexamethasone Acetate 27.44 0.34 1.2 5.0

 

 

Materials