Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay for the simultaneous quantification of 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol in human plasma.

18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones.

Simultaneous determination of 18 tetrahydrocorticosteroid sulfates in human urine by liquid chromatography/electrospray ionization-tandem mass spectrometry.

A liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of eighteen tetrahydrocorticosteroid sulfates in human urine has been developed. The analytes were 3- and 21-monosulfates and 3,21-disulfates of tetrahydrocortisol (THF), tetrahydrocortisone (THE), tetrahydro-11-deoxycortisol (THS), and their corresponding 5α-H stereoisomers. The mass spectrometric behavior of these sulfates in negative-ion ESI-MS/MS revealed the production of intense structure specific product ions within the same group of sulfates and permitted distinction between regioisomeric sulfates by collision-induced fragmentation with the MS/MS technique using a linear ion-trap instrument. For the quantitative analysis, selected reaction monitoring analysis in the negative-ion detection mode using triple-stage quadrupole mass spectrometer was performed by monitoring transitions from [M-H](-) to the most abundant product ion of each tetrahydrocorticosteroid sulfate. After addition of 3- and 21-monosulfates of [2,2,3ß,4,4-d5]-THF, -THE, and -THS as internal standards, urine sample was applied to a solid phase extraction using a lipophilic-weak anion exchange cartridge column, and then analyzed by LC/ESI-MS/MS. The method had satisfactory performance in terms of intra- and inter-assay precision (less than 9.7% and 9.6%, respectively), and accuracy (91.2-108.2%). The limit of quantification was lower than 2.5 ng/mL for all sulfates examined. We applied this method to determine the concentration of eighteen tetrahydrocorticosteroid sulfates in the urine of healthy subjects. Thus, we have developed a sensitive, precise and accurate assay for urinary tetrahydrocorticosteroid sulfates that should be useful for clinical and biological studies.

Charged chelate-capillary electrophoresis of endogenous corticosteroids.

Separation of endogenous 17- or 18-hydroxylated corticosteroids (of the 21-hydroxylated 4-pregnen series) as charged chelates in capillary electrophoresis with borate as the ligand is demonstrated. Aldosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone, cortisone, cortisol, and 11-deoxycortisol are separated and resolved by 400 mM borate buffer at pH 9.0. Separation characteristics of the corticosteroid charged chelates were examined by varying the separation buffer borate concentration, pH, ionic strength, and addition of organic modifiers. The borate ion [B(OH)4]- is identified as the critical buffer component. Corticosteroids chelate borate with proximal hydroxyls composed of either the 17- or 18-hydroxyl in combination with the 21-position hydroxyl. Corticosteroid/borate chelation as indicated by CE results is corroborated with 11B-nuclear magnetic resonance (11B-NMR) spectra. Chelation is a readily reversible process, with the strength of the resultant chelate, as opposed to the charge-to-mass ratio, predominantly determining analyte mobility in charged chelate - capillary electrophoresis (CC-CE).

18, 19-Dihydroxycorticosterone: a new metabolite in human urine.

Urines of patients with primary aldosteronism were extracted, the extract repeatedly chromatographed with reversed phase HPLC. The fractions immunoactive against 18-hydroxycorticosterone antiserum and being more polar than the 18-hydroxycorticosterone were further purified, derivatized and investigated by GC/MS. In this manner the natural occurrence of the 18, 19-dihydroxycorticosterone, which was lately synthetized, in human urine was confirmed.