Evaluation of the separation of steroids in combined forms by RP HPLC with UV-detection and gas chromatography

The aim of the current study is the evaluation of the separation of steroids from accompanying substances in drug products as follows: estradiol hemihydrate from didrogesterone (in Femoston tabl.) and estradiol valerate from levonorgestrel (in Climonorm tabl.) and ciproterone acetate (in Climen tabl.). Reversed phase (RP) HPLC with UV-detection and gas chromatography were applied. For the RP HPLC method with UV-detection the following conditions were used: a) column: Nova Pack C-18; isocratic elution with mobile phase: acetonitrile : methanol : water = 40 : 5 : 55 v/v/v; flow rate: 1 ml/min; UV-detection at lambda = 204 nm; b) column Nova Pack C-18; gradient elution with: 0-10 min: acetonitrile : methanol : water = 35 : 5 : 60 v/v/v; 10-20 min; acetonitrile : methanol : water = 70 : 5 : 25 v/v/v, flow rate: 1 ml/min, UV-detection at lambda = 230 nm. Although RP-HPLC separation at isocratic conditions allows determination of estradiol hemihydrate with high reproducibility with the highest sensitivity at lambda= 204 nm, the analysis in medicinal products requires additional time for elution of components more non-polar than estradiol hemihydrate, which are present in the sample: in Femoston tabl.: t(R) = 13.42 min for didrogesterone; t(R) = 4.85 min for estradiol hemihydrate. The experimental results showed that RP HPLC separation with gradient elution is characterized by higher selectivity. It is found that the detection wavelength lambda = 230 nm is optimal for the achievement of high sensitivity and it is universal for the identification of other active principles and for obtaning of a stable base line with gradient elution. For the GC method: a column HP-35 (30 m x 0.25 mm x 0.25 mu m), temperature program from 100 degrees C to 330 degrees C and mass detection were used. Degradation of analytes at high temperature, their different degree of ionization and the different sensitivity of their detection lead to uncertainty in the GC/MS analysis, therefore, HPLC is the more suitable method for analysis of steroid components.