Simultaneous determination of homocysteine and asymmetric dimethylarginine in human urine by liquid chromatography–tandem mass spectrometry

• LC–MS/MS determination of homocysteine and asymmetric dimethylarginine in urine.
• Use of HILIC column in isocratic conditions without derivatization.
• Urine samples of healthy volunteers and CAD patients were analyzed.
• A simple approach to aid in diagnosis of CAD.

Increased circulating concentrations of homocysteine (HCY) and asymmetric dimethylarginine (ADMA) are associated with vascular disease and vascular risk factors. HCY has been shown to inhibit the activity of endothelial dimethylaminohydrolase (DDAH), causing the accumulation of ADMA and the inhibition of nitric oxide synthesis. The concentrations of HCY and ADMA in biological fluids are used in the clinical diagnosis of cardiovascular diseases and this necessitates the development of a rapid and sensitive method for simultaneous determination of HCY and ADMA. A rapid, simple and sensitive method for simultaneous determination of HCY and ADMA by liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with electro spray ionization (ESI) in human urine was reported here. The methodology designed here was used to estimate these molecules in urine samples collected from patients reported to Cardiology Department of our hospital. Chromatographic separation was performed on Atlantis HILIC silica (100 mm × 2.1 mm, 5 μm, Waters). Positive multiple reactions monitoring (MRM) mode was chosen for quantification of each analyte and cystamine dihydrochloride (CYA) was used as the internal standard (IS) for the assay. The intra-assay precision and accuracy were in the range of 2.4–4.8 and −1.8% to 3.1%, respectively. The inter-assay precision and accuracy were in the range of 3.0–4.2% and −1.2% to 3.2%, respectively. The recoveries were between 94.9% and 101.4%. Our approach is simple, rapid and could be extended to routine urine assay.