The limits of detection (LODs) and quantification (LOQs) under the present chromatographic conditions were determined

by diluting the standard solution when the signal-to-noise ratios (S/N) of analytes were almost 3 and 10, respectively. The S/N was calculated as the peak height divided by the background noise value. The background noise was measured from the background start to background end time. The selectivity and specificity of the analytical method were assessed in relation to interference peaks by comparing their retention times with those of steroids standard of the respective extracted and aqueous lower limit Enzalutamide concentration of quality control samples. The sensitivity was evaluated by calculating the precision and accuracy of lower limit of quality control sample in each of the at least three acceptable precision and accuracy batches individually and in total. For ELSD applications, nevertheless, selection of operational parameters is essential and should be paid careful attention; the obtained results were showed in Table 2. S/N was used as the key criteria for optimization ERK inhibition of two principal parameters, drift tube temperature and nebulizing gas flow rate. The drift tube temperature and nebulizing gas flow were used as 60 °C,

and from 2.5 to 3.0 L/min, respectively. The previous chromatographic conditions for determination of steroids by HPLC–ELSD were used as the why basis for mobile phase selection and optimization. The gradient elution program was carefully adjusted and after several trials the new gradient program was selected until it permitted the best separation ability for all the analytes investigated. For the purpose of correct identification, a HPLC–ELSD analysis was performed on sample solutions under the LCMS-dual ESI-MS conditions. The mass spectra data of steroids in positive ion mode and it’s adducts were listed in Table 3. In positive ion mode, the compounds

of interest exhibited mainly protonated ions and sodium adduct ions. Finally, the identified steroids by comparing their retention times and MS data with those of reference compounds (Fig. 1). As shown in Table 4, acceptable results of the regression analysis, the correlation coefficients (r2), LODs and LOQs were obtained for all the analytes: all calibration curves showed good linear regression (r2 > 0.9909, 0.9983, 0.9905) within the test ranges and pictorial representation showed in Table 1; the LODs and LOQs of the three steroids were in the range of 88–292 μg/ml, 68–225 μg/ml and 347–1157 μg/ml, respectively. The intra- and inter-day variations were less than 5% and the percentage recoveries were in the range of 97–105% with R.S.D. less than 5%. The results of the repeatability test shown in Table 5 for intraday and Table 6 for interday demonstrated that the developed assay was reproducible (R.S.D. < 5%).