Internal Standard Comparison
Initially, we used matched ISs to determine peak area ratios and calculate analyte concentrations. We then compared the suitability of IS substitutions by calculating analyte concentrations from peak area ratios created with other ISs in the method. For example, cortisol (F) concentrations were calculated using F-13C3 for ratios, then compared with concentrations calculated using each IS monitored in the C18 method (Ald-d 4, E-13C3, B-d 4, S-13C3, 11DOC-13C3). We repeated this for each analyte, resulting in six comparisons for each adrenal steroid and four for the gonadal steroids. To assess the performance of these substitutions, we used the equation above to calculate percent recovery and RSD for each substitution. While we considered substitutions with calibration curve slopes near 1.0 to represent better performance, that did not always mean they performed similarly to the matched IS. We compared endogenous concentrations between IS substitutions to determine whether substitutions substantially altered analyte concentrations calculated. Substitutions with calibration curves close to 1.0 were considered to perform more similarly to the matched IS and prioritized in identifying suitable IS substitutions.