Cécile Meunier1, Dominique Blondelle2, Patrice Faure3, Jean-Philippe Baguet4, Caroline Le Goff5, Olivier Chabre6, Véronique Ducros7. 1. Unit of Hormone and Nutrition, Department of Biochemistry, Toxicology and Pharmacology, University Hospital, Grenoble, France. Electronic address: CMeunier@chu-grenoble.fr. 2. Unit of Hormone and Nutrition, Department of Biochemistry, Toxicology and Pharmacology, University Hospital, Grenoble, France. Electronic address: DBlondelle@chu-grenoble.fr. 3. Unit of Hormone and Nutrition, Department of Biochemistry, Toxicology and Pharmacology, University Hospital, Grenoble, France; HP2 Laboratory (INSERM U1042), The Joseph Fourier University, Grenoble, France. Electronic address: PFaure@chu-grenoble.fr. 4. Unit of Hypertension, Department of Cardiology, University Hospital, Grenoble, France; INSERM U1039, The Joseph Fourier University, Grenoble, France. Electronic address: jp.baguet@ghm-grenoble.fr. 5. Department of Clinical Chemistry, University of Liège, CHU Sart-Tilman, Liège, Belgium. Electronic address: c.legoff@chu.ulg.ac.be. 6. Department of Endocrinology, University Hospital, Grenoble, France. Electronic address: OChabre@chu-grenoble.fr. 7. Unit of Hormone and Nutrition, Department of Biochemistry, Toxicology and Pharmacology, University Hospital, Grenoble, France. Electronic address: VDucros@chu-grenoble.fr.
Abstract
BACKGROUND: Accurate quantitation of aldosterone is essential for screening, diagnosis and subtype classification in primary aldosteronism. A simple, sensitive method for aldosterone in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated. METHODS: Plasma samples were diluted with water containing d7-aldosterone as internal standard. The samples were extracted with methyl-tert-butyl-ether (MTBE) on SLE cartridges. Separation was carried out on a Luna C18 (2) column using a methanol-water gradient. Detection was performed in the negative electrospray multiple reaction monitoring (MRM) quantitation. The use of water-based calibrators was evaluated against calibrators prepared in steroid-free serum. RESULTS: The assay was linear up to 3265pmol/L with an LOQ of approximately 40pmol/L. Within-run and between-run precision for plasma aldosterone were less than 10% except at low level near LOQ but were still less than 14.7% (Westgard's desirable specification). The mean recovery of the analyte added to plasma was greater than 97.7% and matrix effects were less than 4%. Comparison with another LC-MS/MS method was performed on a more sensitive instrument (ABSciex TQ 5500) and gave the equation API 3000=0.957×TQ 5500+12.6, linear regression r(2)=0.974 (n=43). An estimation of the reference interval for adults was established on a group of healthy volunteers (n=53). Calibration with water-based calibrators was validated and can be used for measurement of aldosterone by LC-MS/MS. CONCLUSIONS: This method is reliable, easy to perform on plasma specimens in a clinical environment and is attractive because of its simplicity.
BACKGROUND: Accurate quantitation of aldosterone is essential for screening, diagnosis and subtype classification in primary aldosteronism. A simple, sensitive method for aldosterone in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated. METHODS: Plasma samples were diluted with water containing d7-aldosterone as internal standard. The samples were extracted with methyl-tert-butyl-ether (MTBE) on SLE cartridges. Separation was carried out on a Luna C18 (2) column using a methanol-water gradient. Detection was performed in the negative electrospray multiple reaction monitoring (MRM) quantitation. The use of water-based calibrators was evaluated against calibrators prepared in steroid-free serum. RESULTS: The assay was linear up to 3265pmol/L with an LOQ of approximately 40pmol/L. Within-run and between-run precision for plasma aldosterone were less than 10% except at low level near LOQ but were still less than 14.7% (Westgard's desirable specification). The mean recovery of the analyte added to plasma was greater than 97.7% and matrix effects were less than 4%. Comparison with another LC-MS/MS method was performed on a more sensitive instrument (ABSciex TQ 5500) and gave the equation API 3000=0.957×TQ 5500+12.6, linear regression r(2)=0.974 (n=43). An estimation of the reference interval for adults was established on a group of healthy volunteers (n=53). Calibration with water-based calibrators was validated and can be used for measurement of aldosterone by LC-MS/MS. CONCLUSIONS: This method is reliable, easy to perform on plasma specimens in a clinical environment and is attractive because of its simplicity.
Authors: Graeme Eisenhofer; Mirko Peitzsch; Denise Kaden; Katharina Langton; Christina Pamporaki; Jimmy Masjkur; George Tsatsaronis; Anastasios Mangelis; Tracy A Williams; Martin Reincke; Jacques W M Lenders; Stefan R Bornstein Journal: Clin Chim Acta Date: 2017-05-04 Impact factor: 3.786