A LC-MS/MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine.

Aldosterone (ALD), its precursor 18-hydroxycorticosterone (18-OHB) and its metabolite tetrahydroaldosterone (TH-ALD) are important biomarkers for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in the detection of small molecules of hormones because it has advantages in terms of specificity and sensitivity. The objective of this study is to develop a new LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB, and TH-ALD in human urine and attempt to diagnose primary aldosteronism using different indicators. The urine samples were treated with a solid-phase extraction pretreatment technique and the three analytes were separated on a reversed-phase column and detected on a triple quadrupole mass spectrometer. The established method was validated according to CLSI C62-A standard guidelines. The calibration ranges from 25 pg/mL to 5000 pg/mL for aldosterone (free), 18-hydroxycorticosterone and tetrahydroaldosterone, and the lower limit of quantification for these three analytes was 25 pg/mL. The matrix effects and recoveries of these three analytes ranged from 85.1 % to 115 % and from 86.3 % to 114 %, respectively. The intra-day and inter-day precision ranged from 1.29 % to 6.78 % and from 1.77 % to 8.64 %, respectively. The performance of the method met the requirements of the guidelines. 40 clinical urine samples including 22 PA patients and 18 non-PA patients were detected, and the ROC curves of three diagnostic indicators were established. The area under the curve (AUC) of ALD (free) is the biggest, so ALD (free) was the best compound to be used as a diagnostic indicator in this study. When the cut-off point was taken as 141 ng/24-h, the sensitivity was 72.7 % and the specificity was 88.9 %. We developed and validated an LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB and TH-ALD in human urine. Our study provides a reference for the use of new biomarkers for the diagnosis of primary aldosteronism.

Simultaneous quantitation of urine aldosterone and tetrahydroaldosterone in healthy Chinese subjects using a validated LC-MS/MS method.

Aldosterone (ALD) is excreted in urine mainly as glucuronide conjugates of ALD and tetrahydroaldosterone. Measuring these urinary metabolites might be an alternative screening test to plasma ALD for primary aldosteronism. We report a validated LC-MS/MS method to measure both analytes simultaneously. Urine samples underwent enzymatic hydrolysis to release the analytes from their glucuronide conjugates followed by organic solvent extraction and LC-MS/MS. The analytical performance of this method was evaluated. The within-batch and between-batch coefficients of variation for urine ALD and urine THA were all ≤5.2 and ≤3.7%. The lower limit of quantification was 0.5 nmol/L, and the linearity was up to at least 2770 nmol/L for both analytes. No significant matrix interference and carryover were observed. Both analytes in urine were stable for at least 48 h at 10°C and at least 18 months at -80°C. Local reference intervals were established from 126 healthy normotensive Chinese subjects (53% women, age: 20-65 years). Reference intervals for urine ALD and tetrahydroaldosterone were 2-38 and 9-139 nmol/day, respectively. This validated method can be applied to screening and diagnosing primary aldosteronism.

Urinary tetrahydroaldosterone is associated with circulating FGF23 in kidney stone formers.

The spectrum of diseases with overactive renin-angiotensin-aldosterone system (RAS) or elevated circulating FGF23 overlaps, but the relationship between aldosterone and FGF23 remains unclarified. Here, we report that systemic RAS activation sensitively assessed by urinary tetrahydroaldosterone excretion is associated with circulating C-terminal FGF23. We performed a retrospective analysis in the Bern Kidney Stone Registry, a single-center observational cohort of kidney stone formers. Urinary excretion of the main aldosterone metabolite tetrahydroaldosterone was measured by gas chromatography-mass spectrometry. Plasma FGF23 concentrations were measured using a C-terminal assay. Regression models were calculated to assess the association of plasma FGF23 with 24 h urinary tetrahydroaldosterone excretion. We included 625 participants in the analysis. Mean age was 47 ± 14 years and 71% were male. Mean estimated GFR was 94 ml/min per 1.73 m2. In unadjusted analyses, we found a positive association between plasma FGF23 and 24 h urinary tetrahydroaldosterone excretion (ß: 0.0027; p = 4.2 × 10-7). In multivariable regression models adjusting for age, sex, body mass index and GFR, this association remained robust (ß: 0.0022; p = 2.1 × 10-5). Mineralotropic hormones, 24 h urinary sodium and potassium excretion as surrogates for sodium and potassium intake or antihypertensive drugs did not affect this association. Our data reveal a robust association of RAS activity with circulating FGF23 levels in kidney stone formers. These findings are in line with previous studies in rodents and suggest a physiological link between RAS system activation and FGF23 secretion.

Enabling insights into the maturation of the renin-angiotensin-aldosterone system in children-Development of a low-volume LC-MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite.

INTRODUCTION: As part of the renin-angiotensin-aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. METHODS: A HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight-mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. RESULTS: Using the Sciex TripleTOF 6600, a reliable determination in 50 µL serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between -1.21 and -6.99 % and 2.07 and -10.22 %, respectively, confirming compliance with international guidelines. CONCLUSION: A simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 µL serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.