Haiqing Ding (Presenter)
Cleveland HeartLab, Inc.
Bio: Haiqing Ding, a senior scientist working at Cleveland HeartLab, Inc. with over 15 years of Mass Spec experience.
Authorship: Haiqing Ding, Cory Bystrom
Cleveland HeartLab, Inc.
| Short Abstract Here we report a reliable LC-MS/MS method for the determination of ADMA and SDMA in human serum by ion-pair chromatography-mass spectrometry. With heptafluorobutyric acid as an ion-pair additive to mobile phases, polar molecules ADMA and SDMA can be well retained on a reversed-phase column. This eliminates the need for pre-column derivatization which is time consuming. Baseline separation of ADMA from SDMA can be easily achieved compared to normal-phase chromatography. The method was validated on an Agilent multi-stream LC-MS/MS System. The method provides not only good linearity, accuracy, and precision, but also increased productivity with the use of multi-stream LC system. |
Long Abstract
Introduction
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) and was assessed as a cardiovascular biomarker [1] while symmetric dimethylarginine (SDMA) possesses only a weak potency towards NOS [2]. Due to their high polarity and difficult to retain on reversed-phase columns, most LC-MS/MS assays employ pre-column derivatization to increase their retention on a reversed-phase column, or hydrophilic interaction with normal-phase chromatography [3]. Here we report a reliable LC-MS/MS method for the determination of ADMA and SDMA in human serum by ion-pair chromatography-mass spectrometry.
Methods
Sample extraction: Sample extraction was carried out on a Tecan automation system. An aliquot of human serum was transferred to a 96-well plate and treated with internal standard working solution. The sample plate was vortexed, centrifuged and supernatant was transferred to another 96-well plate and diluted with 200 µL of 0.5% heptafluorobutyric acid (HFBA) in water. 3 µL was injected onto LC-MS/MS system.
Instrumentation and methodology: The extracted samples were analyzed using an Agilent 1290 HTC autosampler and an Agilent 1260 4-stream binary pump system interfaced with an Agilent 6490 triple quad mass spectrometer. ADMA, SDMA, and internal standard ADMA-d6 were monitored using jet stream electrospray ionization in positive-ion mode with multiple reaction monitoring (MRM). C18 columns (2.1 x 50 mm, 2.6 micron) were developed with a gradient of 0.05% HFBA in water and B 0.05% HFBA in methanol at 0.4 mL/min.
Results
Baseline separation of ADMA and SDMA was achieved and calibration was linear over the range of 10.0 – 500 ng/ml with correlation coefficients were greater than 0.999 for both ADMA and SDMA with a weighted 1/x fit. The intra- and inter-day accuracy (%bias) from all 4 streams ranged from -6.2% to 11.3% for both ADMA and SDMA. The intra- and inter-day precisions (%CV) from all 4 streams ranged from 0.4% to 3.0% for both ADMA and SDMA. The 4-stream HPLC system displayed excellent agreement when comparing quantitative results with correlation coefficients greater than 0.999 for each stream and a deviation of slops of calibration curves less than 5% RSD between the 4 streams. Deviations in retention time between the 4 streams were also minimal, well below 5% RSD when over 600 injections were made to the 4-stream system.
References & Acknowledgements:
References
[1] Kielstein J, Cooke J. Should We Measure Asymmetric Dimethylarginine in Patients with Coronary Artery Disease? Clin Chem 2007; 53:161-163.
[2] Servillo L, Giovane A, D’Onofrio N, Casale R, Cautela D, Castaldo D, Balestrieri M. Determination of Homoarginine, Arginine, NMMA, ADMA, and SDMA in Biological Samples by HPLC-ESI-Mass Spectrometry. Int J Mol Sci. 2013; 14: 20131–20138
[3] Schwedhelm E. Quantification of ADMA: analytical approaches. Vasc Med. 2005; 10: Suppl 1:S89-95
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