LC-MS/MS Analysis of Angiotensin I for Assessment of Plasma Renin Activity in Clinical Research
Presentation Number: SUN 516
Date of Presentation: April 2nd, 2017
Dominic Foley*1, Andrew J Peck2 and Lisa Calton1
1Waters Corporation, Wilmslow, United Kingdom, 2Waters Corporation, Milford, MA
Background: An LC-MS/MS method for the measurement of angiotensin I (Ang1) enabling investigation of Plasma Renin Activity (PRA) for clinical research activities testing biomarkers of hypertension is presented. Renin converts angiotensinogen to Ang1, which is subsequently converted to Angiotensin II (Ang2). Ang2 is a potent vasopressor and plays a central role in regulation of blood pressure. Measurement of Ang2 is challenging due to its low circulating levels and short half life. Ang1 is a more stable peptide and is therefore a more suitable candidate to evaluate PRA. An analytical method was developed using µElution Solid Phase Extraction (SPE) in 96-well plate format, reducing sample preparation time and optimizing analytical sensitivity.
Methods: Ang1 purchased from Cambridge Biosciences (Cambridge, UK) was used to create calibrators and QC materials using Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS). Precision of the method for analyzing Ang1 was performed using BSA/PBS Ang1 QCs. Precision of the method for analyzing PRA was performed using high and low value assigned K2EDTA plasma pools and PRA controls (PN:600, Bio-Rad, UK). Samples were treated with buffer containing; 0.5M sodium acetate pH 5.5, 18mM EDTA, 4mM PMSF and 0.02mM SBTI. Samples were incubated for 3 hours at 37°C to generate Ang1. Samples were diluted with Ammonium Hydroxide and Ang1-13C515N internal standard (Cambridge Biosciences, UK) was added. SPE was carried out with a Waters® Oasis®MAX µElution 96-well plate, which negated the need for evaporation and allowed direct injection of the SPE eluate. Offline automated extraction was performed using a Tecan Freedom Evo 100. Using an ACQUITY UPLC I-Class system, samples were injected onto a 2.1 x 50 mm Waters ACQUITY UPLC HSS T3 column with pre-column VanGuard T3, using a water/acetronitrile/formic gradient and quantified with a Waters Xevo TQ-S mass spectrometer.
Results:The method was linear from 0.15 – 116 nmol/L for Ang1 (0.05 – 38.5 nmol/L/hr). Coefficients of variation (CV) for total precision and repeatability on five separate days for low, mid and high Ang1 BSA/PBS QC samples were all ≤ 10.0% (n = 30) for Ang1 at 1.5, 7.7 and 77.1 nmol/L. PRA low and high plasma pools and Bio-Rad controls were ≤ 15.0% over the five occasions. Analytical sensitivity investigations demonstrate a CV < 20% at 0.15 nmol/L (0.05 nmol/L/hr) for Ang1 with S/N >10:1. Both extraction efficiency and matrix effects were demonstrated to be reproducible and the internal standard was shown to compensate for any ion suppression observed.
Conclusions:We have developed an analytical method for clinical research to quantify Ang1 and evaluate the PRA in plasma utilizing SPE with LC-MS/MS. This offline automated method demonstrates good linearity, precision and accuracy, while providing high sample throughput capabilities.
For Research Use Only, Not for use in diagnostic procedures.
Nothing to Disclose: DF, AJP, LC