Sensitive and simultaneous quantitation of 6β‐hydroxycortisol and cortisol in human plasma by LC‐MS/MS coupled with stable isotope dilution method

Abstract

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6β-hydroxycortisol (6β-OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6β-OHF and cortisol in human plasma using high-performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6β-[9,11,12,12- H ]hydroxycortisol and [1,2,4,19- C ]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6β-OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra-day and inter-day assay reproducibilities of the determined 6β-OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6β-OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6β-OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6β-OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.

Dried blood spots analysis of 6β‐hydroxycortisol and cortisol using liquid chromatography/tandem mass spectrometry for calculating 6β‐hydroxycortisol to cortisol ratio

“…Plasma measurement validation was performed in our previous study. 21 As observed, the recovery of 6β-OHF and cortisol from plasma was 86.4% and 94.0%, respectively. Their relative standard deviation (RSD) and accuracy were also within 5%.…”

“…However, because plasma 6β-OHF concentration is as low as 1 ng/ml, we have developed a sensitive and reliable quantification method to measure plasma concentration of 6β-OHF with a lower limit of quantification (LLOQ) of 38 pg/ml. 21 We applied this method to evaluate CYP3A activity using a one-point blood sample in a 2-year-old male patient who had higher midazolam plasma concentrations and confirmed that this patient had low CYP3A activity. 22 Instead of venous blood sampling, DBS sampling can also be applied to any populations and is easy to handle during measurement.…”

“…We used the optimized ionization conditions for 6β-OHF and cortisol in our previous study. 21 In ESI, ionization efficacy is known to be affected by organic base or organic acid concentration of the mobile phase. 24 First, using methanol as an organic mobile phase, seven concentrations of formic acid in the aqueous phase were evaluated.…”

“…The plasma concentration of 6β-OHF and cortisol ranged from 0.173 to 1.549 ng/ml and from 23.78 to 294.71 ng/ml in healthy subjects in our previous study. 21 Because the whole blood volume for four DBSs was approximately 50 μl and the hematocrit was 35% to 50%, calibration graphs were prepared in the range of 0.001 to 0.108 for 6β-OHF The concentration of 6β-OHF was 0.018 ng, and the concentration of cortisol was 2.10 ng C 4 . Good linearity (r > 0.9996) was observed between the peak area ratio (y) and molar ratio (x) (Figure 3).…”

Stochastic dynamic mass spectrometric quantification of steroids in mixture

“…The frequently employed radioimmunoassay cannot be utilized to determine mixtures of STs despite its sensitivity. Further, the labeled with stable isotopes internal standards of STs are very frequently not commercially available [7,8]. This requires effort on development of free of isotope labeling protocols for determination of such analytes.…”