Determination of Serum Aldosterone
Determination of Serum Aldosterone
Aims Accurate serum aldosterone determination is critical to the screening and diagnosis of primary aldosteronism, the localisation of aldosterone producing tumours, and the investigation of other disorders of the renin-angiotensin system. Mass spectrometry offers a means to overcome problems with method-dependent bias between competitive immunoassays for aldosterone. The authors have developed a simple, sensitive and precise liquid–liquid extraction aldosterone method for the ABSCIEX API-5000 liquid chromatography and tandem mass spectrometry (LC-MS/MS) system.
Methods Using d7-aldosterone internal standard, 500 μl of sample is extracted with 2500 μl of methyl tertbutyl ether followed by dry-down, reconstitution and LC-MS/MS analysis in ESI negative mode. Method validation was undertaken using standard approaches and comparison made against a commercial radioimmunoassay. Accuracy was assessed using EQA material with assigned aldosterone concentrations.
Results The assay was linear up to 3420 pmol/l (LOQ=50 pmol/l, LOD<22 pmol/l). Total CVs were ≤5% for concentrations ≥120 pmol/l and 10% at the LOQ. Mean accuracy was 98.5% against GCMS assigned material.
Conclusion The authors present a precise, sensitive and simple aldosterone method suitable for routine clinical use that requires no solid phase extraction or specialised ion sources.
Accurate determination of serum aldosterone is essential for screening, diagnosis and subtype evaluation of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis and renal tubular channelopathies.
Owing to its low plasma concentration, aldosterone has traditionally been determined by radioimmunoassay or competitive chemiluminescent immunoassay, though a number of liquid chromatography and tandem mass spectrometry (LC-MS/MS) approaches are described, all using negative ionisation mode. Methods employing liquid–liquid extraction (LLE) propose the use of a dichloromethane/diethylether mixture followed by either atmospheric pressure chemical ionisation or electrospray ionisation (ESI). Alternatively, methods employing protein precipitation and solid phase extraction (SPE) use either atmospheric pressure photoionisation (APPI) or ESI.
Although the simplicity of LLE is appealing, a dichloromethane/diethylether mixture is undesirable due to the toxicity of dichloromethane and high vapour pressure of both solvents. The cost and complexity of SPE cleanup and/or APPI-based ionisation may render the alternative unfavourable as well. We have therefore developed an accurate, precise and sensitive LLE aldosterone method using methyl tertbutyl-ether (MTBE) for the ABSCIEX API-5000 instrument in ESI negative ion mode. Since MTBE has lower Hg at 25°C) vapour pressure (249 mm Hg at 25°C) or dichloromethane than either of diethylether (537 mm Hg at 25°C) (435 mm and is not implicated in cancer development as is dichloromethane, it is a much safer alternative.
This project was approved by the Research Ethics Board of St. Paul's Hospital and the University of British Columbia.
Abstract and Introduction
Abstract
Aims Accurate serum aldosterone determination is critical to the screening and diagnosis of primary aldosteronism, the localisation of aldosterone producing tumours, and the investigation of other disorders of the renin-angiotensin system. Mass spectrometry offers a means to overcome problems with method-dependent bias between competitive immunoassays for aldosterone. The authors have developed a simple, sensitive and precise liquid–liquid extraction aldosterone method for the ABSCIEX API-5000 liquid chromatography and tandem mass spectrometry (LC-MS/MS) system.
Methods Using d7-aldosterone internal standard, 500 μl of sample is extracted with 2500 μl of methyl tertbutyl ether followed by dry-down, reconstitution and LC-MS/MS analysis in ESI negative mode. Method validation was undertaken using standard approaches and comparison made against a commercial radioimmunoassay. Accuracy was assessed using EQA material with assigned aldosterone concentrations.
Results The assay was linear up to 3420 pmol/l (LOQ=50 pmol/l, LOD<22 pmol/l). Total CVs were ≤5% for concentrations ≥120 pmol/l and 10% at the LOQ. Mean accuracy was 98.5% against GCMS assigned material.
Conclusion The authors present a precise, sensitive and simple aldosterone method suitable for routine clinical use that requires no solid phase extraction or specialised ion sources.
Introduction
Accurate determination of serum aldosterone is essential for screening, diagnosis and subtype evaluation of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis and renal tubular channelopathies.
Owing to its low plasma concentration, aldosterone has traditionally been determined by radioimmunoassay or competitive chemiluminescent immunoassay, though a number of liquid chromatography and tandem mass spectrometry (LC-MS/MS) approaches are described, all using negative ionisation mode. Methods employing liquid–liquid extraction (LLE) propose the use of a dichloromethane/diethylether mixture followed by either atmospheric pressure chemical ionisation or electrospray ionisation (ESI). Alternatively, methods employing protein precipitation and solid phase extraction (SPE) use either atmospheric pressure photoionisation (APPI) or ESI.
Although the simplicity of LLE is appealing, a dichloromethane/diethylether mixture is undesirable due to the toxicity of dichloromethane and high vapour pressure of both solvents. The cost and complexity of SPE cleanup and/or APPI-based ionisation may render the alternative unfavourable as well. We have therefore developed an accurate, precise and sensitive LLE aldosterone method using methyl tertbutyl-ether (MTBE) for the ABSCIEX API-5000 instrument in ESI negative ion mode. Since MTBE has lower Hg at 25°C) vapour pressure (249 mm Hg at 25°C) or dichloromethane than either of diethylether (537 mm Hg at 25°C) (435 mm and is not implicated in cancer development as is dichloromethane, it is a much safer alternative.
This project was approved by the Research Ethics Board of St. Paul's Hospital and the University of British Columbia.
