Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectronmetry with very straightforward sample preparation

摘要

Nitric oxide (NO) is synthesized by NO synthase from L-arginine, which can be competitively blocked by endogenous inhibitors such as asymmetric dimethylarginine (ADMA), but not by symmetric dimethylarginine (SDMA). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH) to dimethylamine and citrulline. A growing number of published clinical studies documented a strong correlation between increased ADMA blood levels and cardiovascular morbidity and mortality. We present here a highly sensitive method for the determination of this compounds in plasma and urine by means of HPLC-MS. The sample preparation is very simple and comprises only protein precipitation and concentration in the case of plasma samples and dilution in the case of urine. The samples are derivatized automatically with orthophthaldialdehyde and 2-mercaptoethanol, are separated on a 250 mm * 4 mm RP18 column by gradient elution with formate buffer/methanol and are detected by ESI-MS. The calibration functions are linear and cover the range from normal to pathologic concentration values of the analytes. The intra-day relative standard deviation (R.S.D.) of the assay for ADMA in plasma is 7.5% and the corresponding inter-day R.S.D. is 5.7%. In urine, these values for ADMA are 3.8 and 6.4%, respectively. All other analytes in plasma as well as in urine exhibit intra-day R.S.D. below 8%. The corresponding inter-day R.S.D. are all below 13%.