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.