Establishment Of An Analytical Method For Simultaneous Determination Of Three Amino Acids In Human Serum Based On LC-MS/MS And Its Application

Objective: High levels of homocysteine(Hcy)are closely associated with coronary heart disease,cerebrovascular disease,and peripheral vascular diseases.Consequently,the level of homocysteine in human is a crucial health indicator.Simultaneously measuring concentrations of homocysteine and methionine can help to explain the potential mechanisms of hyperhomocysteinemia.To provide an efficient and reliable detection method for large-scale clinical detection,this study was based on the characteristics of homocysteine and its related metabolites,and established a method for the simultaneous determination of homocysteine,cysteine,and methionine using liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods: A validated LC-MS/MS technique was utilized to simultaneously determine homocysteine,cysteine,and methionine concentrations in human serum.Optimization was conducted using a step-wise approach,beginning with tuning mass spectrometry conditions(mass-to-charge ratio and energy parameters).Subsequently,chromatographic conditions were then optimized,including mobile phase,column temperature,and flow rate.Pretreatment methods were optimized,including the use of a reductant and a precipitant.Following method development,methodological verification was conducted including specificity,standard curve linearity,and precision assays.Finally,400 serum samples were quantified using the validated method,and 80 samples were randomly chosen to compare with the results of Outgoing inspection.The method applied was as follows: the chromatographic conditions used comprised an Infinity Lab Poroshell 120 EC-C18 column(2.7μm,4.6×50 mm),mobile phase A,0.1% formic acid water,and mobile phase B,and acetonitrile.The column temperature was set at 40℃ with a detection time of 2 mins,a flow rate of 0.60 m L/min,and an injection volume of 5 μL,with gradient elution utilized.Positive-ion scanning,MRM scanning format and ESI ion source were applied for mass spectrometry conditions.For sample pre-treatment,disulfides were reduced using dithiothreitol,which was then followed by protein precipitation with0.2% formic acid acetonitrile.The revised paragraph was improved in several ways.Results: This study validated the detection method by examining its specificity and precision.The method showed good specificity as the intra-day and inter-day precision did not exceed 15%,and retention time was relatively short.The linear correlation coefficient was between 0.9953 and 0.9998 indicating excellent linearity.The extraction recovery rate did not have a significant difference and the matrix effect was stable.The stability results indicated that the intra-day and inter-day precision of serum did not exceed 15% under various storage conditions,which implies the stability of detection conditions is high.Serum samples from 400 subjects were measured and compared with the external delivery method,showing good correlation and verifying the accuracy and effectiveness of this method.Consequently,this technique is recommended for clinical testing.Conclusion: A method for simultaneous detection of homocysteine,cysteine,and methionine using LC-MS/MS has been developed.The method is characterized by its low reagent cost,simple operation,and short detection time.This technique is suitable for both research and clinical samples,and has the potential for the development of relevant kits.