LC-MS/MS is a technique which can reduce analytical specificity issues associated with steroid analysis using immunoassays, while providing analytical sensitivity for the steroid of interest. Aldosterone is a mineralocorticoid steroid hormone that is present at very low physiological concentrations. Traditionally, aldosterone has been analysed by radioimmunoassay (RIA) methods. However, these methods can suffer from a lack of specificity due to the cross reactivity of structurally similar steroid hormones. This results in greater imprecision and inaccuracy, most notably at the lower concentrations found in plasma or serum samples. In this investigation, the performance of UPLC coupled to a newly developed tandem quadrupole mass spectrometer, has been shown to provide quantification down to 2pg/mL aldosterone using only 200µL of a serum sample.
Aldosterone certified reference material (Cerilliant, Round Rock TX) was used to create calibrators in stripped serum (Golden West Biologicals, Temecula CA). QCs were prepared in pooled plasma (SeraLab, Haywards Heath UK). Unadulterated plasma samples were used to perform a system comparison between the Xevo® TQ-S and prototype mass spectrometers. Samples were precipitated and SPE was performed using a Waters® Oasis® MAX µElution 96 well plate. Automated extraction was performed using the Tecan Freedom Evo 100/4 Liquid Handler. An ACQUITY UPLC® I-Class system was used to inject samples onto a Waters CORTECS® UPLC C18 column using a water/methanol gradient and analysed using multiple reaction mode (MRM) on the tandem mass spectrometers operated in ESI negative mode.
Injection of a 2.8 pmol/L (1 pg/mL) solvent standard was performed on both the Xevo TQ-S and prototype systems. This demonstrated approximately a 7 fold increase in peak area using the prototype system and more importantly, a 3 fold increase in S/N. This increase is also reflected in extracted matrix samples, where a 3-5 fold increase in S/N was observed for the low calibrator (42 pmol/L, 15pg/mL), when assessed over three occasions. On each occasion, precision assessments using QC samples (n = 10) were performed at the following concentrations; 58, 117, 482 and 1856 pmol/L (20, 40, 170 and 660 pg/mL) spanning the calibration range of 42 – 4161 pmol/L (15 – 1500 pg/mL). Total imprecision of the QC samples (n=30) was ≤ 8.3% CV for both mass spectrometers. In addition, comparison of aldosterone plasma samples (n=45) quantified using both mass spectrometers was shown to provide a Deming fit of y = 0.98x – 5.90, demonstrating no statistically significant proportional or constant bias (p>0.05). Altman Bland analysis demonstrated a mean negative bias of 4.2% using the prototype MS compared to the Xevo TQ-S system and linear regression analysis demonstrated an excellent correlation between the two mass spectrometers (r2 0.999). In conclusion, it has been demonstrated that the analytical performance of the prototype mass spectrometer for the analysis of aldosterone provides a 3 fold increase in analytical sensitivity over the Xevo TQ-S, providing greater confidence in quantification of low level physiological samples for clinical research. We have shown the methodology can be transferred from the Xevo TQ-S, whilst maintaining excellent levels of precision performance.
A new prototype tandem quadrupole mass spectrometer has shown to provide 3 fold improvement in S/N for aldosterone analysis.
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