Abstract:

The drug budesonide exists as 22R and 22S enantiomers. However, the drug activity of 22R-budesonide is 2-3 times stronger than that of 22S-budesonide. The development of enantiomeric separation and quantitative analysis methods for budesonide can provide an important basis for its drug development and quality control. At present, the enantiomers of budesonide are separated on a reversed C₁₈ solid phase column. However, chiral stationary phases are rarely reported for the separation of the enantiomers of budesonide. In this study, a high performance liquid chromatography (HPLC) method with a chiral stationary phase was developed for the rapid separation and determination of budesonide enantiomers. The effects of the type of chiral stationary phase, mobile phase additives, and column temperature on the resolution of the budesonide enantiomers were also investigated. The results showed that the chiral stationary phase amylose-tris-[(S)-1-phenylethyl carbamate] was more suitable for the separation of budesonide enantiomers. The mobile phase additives used in the experiment had no significant effect on the chromatographic parameters (peak height, peak width, and resolution) of the budesonide enantiomers. However, with an increase in the column temperature, the peak width of the budesonide enantiomers decreased, while the peak height and resolution increased. The optimized HPLC conditions were as follows: column, Chiralpak AS-RH (150 mm×4.6 mm, 5.0 μm); mobile phase, acetonitrile-water (45∶55, v/v); column temperature, 40 ℃; flow rate, 1.0 mL/min; detector, diode array detector (DAD); detection wavelength, 246 nm; injection volume, 10 μL. The external standard method was used to quantify the budesonide enantiomers. Under the optimized conditions, the enantiomers were well separated, and the retention times of 22R-budesonide and 22S-budesonide were 6.40 min and 7.77 min, respectively. The resolution of the enantiomers was 4.64. The linear ranges of 22R-budesonide and 22S-budesonide were 0.16-1000 μg/mL and 0.20-1000 μg/mL, respectively. The peak area of the enantiomers showed a good linear relationship with the corresponding concentration, and the correlation coefficients (R²) were 0.9999. The limits of detection (LODs) of 22R-budesonide and 22S-budesonide were 0.05 μg/mL and 0.07 μg/mL, respectively, based on a signal-to-noise ratio of 3. The limits of quantification (LOQs) were calculated to be 0.16 μg/mL and 0.20 μg/mL, respectively, based on a signal-to-noise ratio of 10. The recoveries at four spiked levels were in the range of 102.63% to 104.17%, with the relative standard deviations (RSDs) of 0.08% to 0.57% (n=6). The budesonide solution was stored in dark at 4 ℃ for 24 h, and no obvious degradation was observed. Finally, the method was applied to determine four actual samples of budesonide suspension for inhalation in a batch. The samples were dissolved in methanol, filtered through a 0.45 μm microporous membrane, and then analyzed. The amounts of 22R-budesonide and 22S-budesonide in the samples were in the ranges of 283.15-284.63 μg/mL and 259.86-261.51 μg/mL, respectively. This method is simple and rapid, in addition to having good repeatability and high accuracy. It can be used for the resolution of budesonide enantiomers and for quality control in budesonide preparations.

Key words: chiral stationary phase, high performance liquid chromatography (HPLC), enantiomers, budesonide