Measuring total midazolam and measuring the amount unbound to plasma proteins present different analytical challenges. In a crossover study of nine healthy volunteers, Lammers and colleagues measured both after an overnight fast and after a 36-hour fast.
For midazolam, only the samples taken at the observed time of maximum concentration were included in the free-fraction analysis. Other unbound readings fell below the lower limit of quantification. Even at the peak sampling time, 55.6% of unbound midazolam readings were below that limit, while all total concentrations at that time could be quantified. The researchers assigned half the quantification limit to the below-limit values when calculating the free fraction.
The study reported no statistically significant difference in midazolam’s free fraction between fasting conditions. Its post hoc calculation estimated only 7% power to detect a 15% difference in mean free fraction with nine participants. The total concentrations and the assigned values therefore cannot resolve whether a change in protein binding occurred. In a laboratory review, distinguish measured concentrations from values substituted for calculation.
Lammers et al., European Journal of Drug Metabolism and Pharmacokinetics (2018), full text · DOI