OBJECTIVE:To establish a method for simultaneous determination of 5 effective components in Luohua zizhu dry extract. METHODS:UPLC-MS/MS was conducted. The separation was performed on an Zorbax Eclipse Plus C18 column with mo-bile phase of acetonitrile-water(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃and sam-ple size was 2 μL. The analytes were detected in the multiple reaction monitoring(MRM)mode. Nitrogen was used as drying gas and atomized gas. The temperature and flow rate of drying gas were 325 ℃ and 6 L/min. The pressure of atomized gas was 45 psi. The temperature and flow rate of sheath gas were 350℃and 12 L/min. The voltage of capillary were 4000 V(+)and 3500 V(-). The voltage of nozzle was 500 V. RESULTS:The linear ranges of luteoloside,acteoside,quercetin,luteolin and rutin were 0.5048-252.4 ng/mL(r=0.9999),0.7124-356.2 ng/mL(r=0.9990),0.5094-254.7 ng/mL(r=0.9962),0.3030-151.5 ng/mL(r=0.9998) and 0.6022-301.1 ng/mL(r=0.9996),respectively. RSDs of precision,stability and reproducibility tests were all less than 3.0%. The limit of quantitation were 0.42,0.87,0.33,0.12,0.76 ng/mL. The recoveries were 97.99%-101.20%(RSD=1.3%,n=6), 96.50%-101.20%(RSD=1.7%,n=6), 94.81%-99.34%(RSD=1.7%,n=6), 97.54%-100.51%(RSD=1.2%,n=6), 93.37%-98.70%(RSD=1.9%,n=6),respectively. CONCLUSIONS:The method is simple,precise,stable and reproducible, and can be used for simultaneous determination of 5 effective components in Luohua zizhu dry extract.

Objective To investigate the adrenal steroidogenic function in genotype-proven heterozygotes carrying mutations in CYP17A1 gene in vivo. Methods Eight patients and 14 family members from 5 families with 17-hydroxylase/17,20-lyase deficiency (17OHD) were recruited. The mutations of the CYP17A1 gene in these individuals were screened by direct sequencing of PCR products. The hormonal response to ACTH was evaluated in the 14 genotype-proven carriers and 45 age- and sex-matched normal subjects. Results Three mutations were found in 5 unrelated families. 14 carriers with CYP17A1 mutation were identified, including 7 heterozygotes with D487_F489del, 6 with Y329fs, and 1 for H373L. Compared to the normal subjects, the carriers exhibited lower basal and ACTH-stimulated cortisol levels, but higher ACTH-stimulated corticosterone level. The ratios of corticosterone to cortisol in the genotype-proven heterozygotes were higher than those of normal individuals at baseline and following ACTH-stimulation. Similarly, progesterone level and ratios of progesterone to 17-hydroxyprogesterone in the male heterozygotes were also higher than that of normal individuals before and after stimulation. No significant differences were observed in the hormone levels between two genotypes (D487_F489del vs Y329fs). Conclusions Genotype-proven carriers of 17OHD without apparent clinical symptoms exhibit decreased enzyme activity,analogous to mildly impaired adrenal 21-hydroxylase activity in the carriers of CYP21 A2 gene mutation.