Amber Gray (Presenter)
Mayo Clinic Foundation
Authorship: Amber Gray, Alan Lueke, Fernando G. Cosio, Leslie J Donato, Allan S. Jaffe and Jeff W. Meeusen
Mayo Clinic, Rochester, MN
| Short Abstract ADMA has been previously implicated in all-cause mortality in patients with ESRD and is useful as a biomarker to predict adverse outcomes in a high risk population. Current methods for measuring ADMA concentrations are not optimized for clinical workflows as they often involve cumbersome sample preparation or lengthy liquid chromatography to separate the symmetric dimethyl arginine (SDMA) isomer. We developed a facile, rapid and reproducible UPLC-MS/MS method for ADMA that is ideal for implementation in a clinical lab. Detection of ADMA and the C13-ADMA internal standard was achieved in ESI mode using an API 3200 (AB Sciex) coupled with a Dionex Ultimate 3000 RS UHPLC system (ThermoScientific). Total analysis time was 7.17 minutes per sample with only 2 minutes sent to the mass spectrometer allowing for multiplexing of the assay to further reduce analysis time. |
Long Abstract
Background: Asymmetric dimethyl arginine (ADMA) functions as a competitive inhibitor with arginine for nitric oxide synthase. The inhibition of the nitric oxide pathway has been associated with vascular endothelial dysfunction, which is also a key element in the progression of end-stage renal disease (ESRD) and atherothrombosis. ADMA has been previously implicated in all-cause mortality in patients with ESRD and is useful as a biomarker to predict adverse outcomes in a high risk population. Current methods for measuring ADMA concentrations are not optimized for clinical workflows as they often involve cumbersome sample preparation or lengthy liquid chromatography to separate the symmetric dimethyl arginine (SDMA) isomer.
Objective: We sought to develop a facile, rapid and reproducible UPLC-MS/MS method for ADMA that was ideal for implementation in a clinical lab.
Methods: Detection of ADMA and the C13-ADMA internal standard was achieved in ESI mode using an API 3200 (AB Sciex) coupled with a Dionex Ultimate 3000 RS UHPLC system (ThermoScientific). ADMA was monitored at 46.1/203.1 m/z, while SDMA and the C13-labeled ADMA internal standard were monitored at 172.2/203.1, 162.2/208.2 respectively. C13-internal standard is added to EDTA plasma followed by protein precipitation using acentonitrile. The plasma is then filtered to remove the precipitate and 45 µL of the filtrate is injected onto a Cyclone MCX cation exchange turboflow column (0.5x50mm, ThermoScientific) equilibrated with methanol + 0.1% formic acid running at 2 mL/min. The analytes are subsequently eluted at 0.1 mL/min using water containing 5% ammonium hydroxide onto a Agilent Poroshell 120 HILIC column (2.1 x50mm, 2.7um, Agilent) equilibrated with 5:95 water:acetonitrile containing 1% acetic acid at 0.7 mL/min. Final elution of analytes is then achieved by reducing the flow rate to 0.5 mL/min and increasing the aqueous component to 40%.
Assay precision was assessed via day to day monitoring of two concentrations of quality control plasma samples. Assay accuracy was confirmed using three plasma pools originating from waste specimens and spiking with 50, 100, and 300 ng/mL ADMA each. Spiked concentrations were compared to the mathematically expected concentrations. Mixing studies were used for verification of the analytical measurement range. A low sample (38 ng/mL) was mixed with a high concentration sample (612 ng/mL) in several ratios (100:0, 75:25, 50:50, 25:75, and 0:100). The neat high (100:0) and low (0:100) samples were used to determine the theoretical values for the mixed samples. The reference interval was confirmed by analyzing samples from 20 healthy donors with no history of cardiovascular disease, cancer, bleeding disorders or kidney disease. Clinical utility was evaluated by measuring plasma ADMA concentrations in 1,172 patients with a diagnosis of ESRD evaluated for potential kidney transplant. Study participants were followed for a median of 3.2 years and the primary outcomes of coronary revascularization or death (any cause) were recorded.
Results: The UPLC-MS/MS method described had a total analysis time of 7.17 minutes per sample with only 2 minutes sent to the mass spectrometer. This allows for multiplexing of the assay to further reduce analysis time to 3.58 minutes per sample. Inter-assay precision (n=7) for ADMA was ≤5% at 95 and 373 ng/mL. Spike and recovery experiments generated assay recoveries ranging between 97-114%. A linear regression between measured concentrations and theoretically calculated concentrations produced a linear regression with a slope of 0.9936 and R2 0.9975. Reference range for healthy donors was confirmed at 63-137 ng/mL; all 20 patients assessed fell within this reference range. There were 171 events (42 revascularizations and 129 deaths) among 163 patients during an average follow-up of 1.7±1.2 years. ADMA was significantly higher among patients with events (158 ng/mL vs. 143 ng/mL; p<0.0001). The hazard ratio for an ADMA greater than the upper reference limit of 137 ng/mL was 2.24 (95CI 1.54-3.31).
Conclusions: We have developed a rapid and reproducible UHPLC-MS/MS method to measure ADMA with demonstrated clinical utility among patients with ESRD.
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