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

Abstract

Dried blood spot (DBS) sampling is a minimally invasive method used to collect blood samples of any population for personalized medicine. We aimed to develop a sensitive and reliable analytical method for measuring 6β-hydroxycortisol (6β-OHF) and cortisol concentrations in DBS by liquid chromatography/tandem mass spectrometry so as to utilize DBS as a less invasive blood sampling method for calculating the ratio of 6β-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 μl for 6β-OHF and 1.01 pg/50 μl for cortisol. The 6β-OHF and cortisol in DBS were stable for 28 days at room temperature. The intraday and interday accuracy and precision of the method was <12%. Additionally, the 6β-OHF and cortisol in DBS were measured before, during, and after 3 days of clarithromycin administration to two of the subjects. Then, their concentration was compared in the plasma and whole blood collected simultaneously. The concentrations of 6β-OHF and cortisol in four DBS ranged from 0.007 to 0.079 ng/50 μl and from 1.15 to 6.66 ng/50 μl, respectively. The 6β-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6β-OHF/cortisol ratio in DBS also showed a strong correlation with that in whole blood (r = 0.9694) and plasma (r = 0.9383). This method provides high accuracy and precision for measuring 6β-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6β-OHF/cortisol ratio. The 6β-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting.

Development of a Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analytical Method for Urinary Endogenous Substrates and Metabolites for Predicting Cytochrome P450 3A4 Activity

“…To date, several biomarkers of CYP3A4 activity have been identified in plasma and urine; however, the most suitable CYP3A4 biomarkers for evaluating its activity have not been conclusively identified. [19][20][21][22][23][24][25][26][27][28] Therefore, a clinically available high-throughput method is required to investigate the utility of CYP3A4 biomarkers.…”

“…19,[21][22][23][24][25][26]43) C and 6β-OHC can be detected in both the positive-and negative-ion modes, although 6β-OHC requires derivatization owing to the low sensitivity of LC-MS/MS and GC/MS. 26,27,43) Therefore, studies on 6β-OHC detection have primarily focused on the establishment of a validated method. Recently, the negative-ion detection mode was used for 6β-OHC quantification.…”

“…Recently, the negative-ion detection mode was used for 6β-OHC quantification. 27,28) Moreover, various compounds, including hormones, have been added to an aqueous ammonium solution as a basic condition, resulting in an ethylene-crosslinked hybrid silica gel column that has been used as the analytical separation column. [46][47][48][49] Overall, the LC-MS/MS conditions for the simultaneous analysis of endogenous urinary CYP3A4 biomarkers were successfully optimized.…”

“…To date, the concentration ratios of 4β-hydroxycholesterol to cholesterol, 6β-hydroxycortisol (6β-OHC) to cortisol (C), and 1β-hydroxydeoxycholic acid (1β-OHDCA) to deoxycholic acid (DCA) in the plasma or urine have been correlated with the concentration of drugs metabolized by CYP3A4. [19][20][21][22][23][24][25][26][27][28] Notably, 1β-OHDCA has been reported as a CYP3A4-specific biomarker, although CYP3A4 and CYP3A5 often exhibit an overlap in substrate specificity. 22) As 4β-hydroxycholesterol has a relatively long half-life (approximately 17 d) compared with that of other biomarkers, 29) 6β-OHC and 1β-OHDCA may represent better indicators of the actual CYP3A4 activity at the time of sample collection.…”

Effects of digital processing on repeatability assessment of a multiple reaction monitoring liquid chromatography–tandem mass spectrometry system by ISO 11843‐7

“…A liquid chromatography system hyphenated with tandem mass spectrometry (LC-MS/MS) system is widely used for the quantification analyses of chemical substances and biological components in various real samples. [1][2][3][4][5][6] In addition, by utilizing a multiple reaction monitoring (MRM) scan mode, an LC-MS/MS method is considered the gold standard for quantification analyses of known compounds. [7][8][9] To obtain precise quantified results from MRM chromatograms, it is important that repeatability in an LC-MS/MS system is suitably assessed.…”