Short communicationQuantification of l-arginine, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma: A step improvement in precision by stable isotope dilution mass spectrometry
Highlights
► A quantification method for l-arginine and its metabolites ADMA and SDMA is presented. ► A very easy and fast sample preparation is proposed. ► Stable isotope labeled I.S.(s) are used for each l-arginine, ADMA and SDMA. ► Very good performance regarding precision and accuracy is achieved due to the I.S.(s). ► Matrix independency is achieved due to the I.S.(s).
Introduction
The amino acid l-arginine (ARG) and its metabolites asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are involved in many physiological processes in living cells. Of special interest is the transformation of ARG to the cell signaling substance nitric oxide (NO) and citrulline. In neuronal tissue, NO is involved in the processes of learning and memory [1] and in the vascular endothelium it is essential for the regulation of vascular tone and endothelial function [2]. The enzymatic synthesis of NO from ARG is competitively inhibited by ADMA. Thus, elevated levels of ADMA, or even more pronounced, reduced ratios of ARG and ADMA [3], in plasma are considered as prognostic markers for various vascular diseases, such as hypertension, diabetes mellitus, hypercholesterolemia, atherosclerosis and congestive heart failure [4]. SDMA, on the other hand, is not directly involved in the NO synthesis, but can serve as an early marker for impaired glomerular filtration rate (GFR) and the onset of kidney disease, which may have advantages in comparison to the established GFR markers creatinine and cystatin C [5], [6]. For both ADMA and SDMA together, elevated levels in plasma can be predictive for adverse outcome of stroke in humans [7].
To facilitate scientific research in these fields, precise, fast, and easy to handle analytical methods for the quantification of ARG, ADMA and SDMA are of constant need. HPLC coupled to tandem mass spectrometry (LC–MS/MS) can be considered as the gold standard for this task because of its unmatched selectivity and sensitivity [8]. However, a very high degree in precision and accuracy in the quantification of ADMA and SDMA is needed to achieve relevant results from clinical studies [9]. In this regard, LC–MS/MS is less favorable because of the susceptibility of current atmospheric pressure ionization sources for matrix effects, which can introduce severe quantification errors if no measures are taken to control them [10]. The most convenient way to overcome the detrimental effects of the matrix on the quantification results is the introduction of stable isotope labeled analogs as I.S.(s) for every analyte to be quantified [8]. Unfortunately, even in recent publications [11], [12], [13], no dedicated stable isotope labeled I.S. for SDMA was used. Since in these publications the focus was more on speed than on precision, quite unsatisfying results regarding intra-day and inter-day precisions for ADMA and especially for SDMA were reported.
In this work, we report a fast, easy, precise and accurate method for the quantification of ARG, ADMA and SDMA in human plasma by LC–MS/MS, utilizing the commercially available I.S.(s) 13C6-arginine for ARG, D7-ADMA for ADMA and, for the first time in LC–MS/MS, D6-SDMA for SDMA. Applying this isotope dilution technique for all analytes led to non-matrix-dependent, precise and robust quantification results. Sample preparation required only protein precipitation and separation of the analytes was achieved on a silica column by the HILIC separation process.
Section snippets
Chemicals
ARG, ADMA (as hydrochloride salts) and SDMA (as di(p-hydroxyazobenzene-p′-sulfonate) salt) were purchased from Sigma–Aldrich (Munich, Germany). The stable isotope labeled I.S.(s) 13C6-arginine hydrochloride (isotopic purity 99%) and D7-ADMA hydrochloride (isotopic purity 98%) were obtained from Cambridge Isotope Laboratories (Andover, MA, USA) whereas D6-SDMA (isotopic purity 99%) was purchased from Toronto Research Chemicals (New York, ON, Canada). All other chemicals were of analytical grade
Sample preparation
A main feature of the current method is the very simple sample preparation. It consisted only of the addition of the I.S., buffering and protein precipitation by the addition of acetonitrile. No cumbersome extraction of the analytes with, for example, solid phase ion exchange columns, was necessary. Thus, the procedure was fast, inexpensive and robust. The utilization of acetonitrile as protein precipitation agent fulfilled the additional purpose of yielding a sample composition very similar to
Conclusion
The method presented here for the determination of ARG, ADMA and SDMA in human plasma is reliable, precise and accurate. The relatively easy, fast and low cost sample preparation procedure makes this approach suitable for large studies. The small required plasma volume of 50 μl enables the measurement even in situations where the total sample size is restricted. If necessary, we believe that the plasma volumes used within the sample preparation procedure could be further downsized without loss
Conflict of interest statement
The authors declare to have no conflicts of interests.
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