Quantitation of Aldosterone in Serum at 1 pg/mL by LC-MS/MS

For Research Use Only. Not for Use in Diagnostic Procedures. The quantitative measurement of steroids by immunoassay suffers from non-specific reactions and cross-reactivity, which can ultimately result in the overestimation of the concentrations of these analytes in biological fluids such as serum. There is a trend to move towards LC-MS/MS due to its many advantages, which include sensitivity and selectivity. Nevertheless, the measurement of aldosterone in serum by LC-MS/MS poses analytical challenges owing to the low concentrations of this compound, interferences caused by endogenous steroids, and the relatively poor intrinsic ionization efficiency of this compound. In this work we present a method that has been developed on an ultra-sensitive tandem mass spectrometer to improve the limit of quantitation for aldosterone in serum compared to existing methods.

The analysis of aldosterone in serum was accomplished using UHPLC coupled to the AB SCIEX Triple Quad™ 6500 system. The method employs the Multiple Reaction Monitoring (MRM) scan mode for the detection of aldosterone, using negative electrospray ionization (ESI). Pre-analytical sample preparation consisted of liquid-liquid extraction in MTBE, followed by evaporation under N₂ gas, and reconstitution of the sample in the LC mobile phase. Chromatographic separation was accomplished using a Phenomenex Gemini-NX C18 analytical column (150x3.0mm, 5um). A gradient elution was employed, consisting of water + 2mM ammonium acetate (mobile phase A) and methanol + 2mM ammonium acetate (mobile phase B), at a flow rate of 500uL/minute. The total run-time for the method including re-equilibration was 10 minutes, to ensure adequate separation of the aldosterone analyte from endogenous interferences.

The method described here was used to analyze a series of human serum samples containing concentrations of aldosterone ranging from 14 pg/mL to 300 pg/mL. The limit of quantitation for aldosterone in serum was observed to be less than 1 pg/mL. In developing the method, great care was taken to ensure that the analyte of interest does not elute in a time window where potential interferences also elute. The method displayed excellent linearity over the concentration range from 1-1000 pg/mL (r=0.99971). Accuracies ranged from 89-118% over the entire concentration range from 1-1000 pg/mL of aldosterone, and the CV% ranges from 0.5-9.1%. The accuracy and CV% for the lowest calibrator, at 1 pg/mL, were 100% and 8.7%, respectively.