De novo synthesis of trideuteromethyl esters of amino acids for use in GC–MS and GC-tandem MS exemplified for ADMA in human plasma and urine: Standardization, validation, comparison and proof of evidence for their aptitude as internal standards

Abstract

Asymmetric dimethylarginine (ADMA, N G, N G-dimethyl- l-arginine) is an endogenous inhibitor of nitric oxide (NO) synthesis, a potential risk factor for cardiovascular diseases and a powerful biochemical parameter in clinical studies. In our previous work we have reported on a GC-tandem MS method for the accurate and precise quantification of ADMA in biological fluids using de novo synthesized [ 2H 3]-methyl ester ADMA (d 3Me-ADMA) as internal standard (IS). This method provides basal ADMA concentrations in biological fluids that agree with those obtained by other groups using other validated methods for ADMA. Unanimously, de novo synthesized stable-isotope labeled analogues are considered not ideal IS, because they must be prepared in a matrix different from the biological sample. Recently, [2,3,3,4,4,5,5- 2H 7]-ADMA (d 7-ADMA) has become commercially available and we took this opportunity to test the reliability of the de novo synthesized d 3Me-ADMA as an IS for ADMA in GC-tandem MS. In this article, we report on the re-validation of the previously reported GC-tandem MS method for ADMA in human plasma and urine using d 7-ADMA as IS, and on comparative quantitative analyses of ADMA by GC-tandem MS using d 7-ADMA and d 3Me-ADMA. After thorough standardization of d 7-ADMA and methods validation, we obtained by GC-tandem MS very similar ADMA concentrations in plasma and urine using d 7-ADMA and d 3Me-ADMA. The present study gives a proof of evidence for the aptitude of 2H 3-ADMA as IS in GC-tandem MS and suggests that de novo synthesis of stable-isotope labeled alkyl esters of amino acids and amino acid derivates may be a generally applicable method in mass spectrometry-based methods for amino acids. This approach is especially useful for amino acids for which no stable-isotope labeled analogues are commercially available.