Determination of trace amino acids in human serum by a selective and sensitive pre-column derivatization method using HPLC-FLD-MS/MS and derivatization optimization by response surface methodology.
Analysis of trace amino acids (AA) in physiological fluids has received more attention, because the analysis of these compounds could provide fundamental and important information for medical, biological, and clinical researches. More accurate method for the determination of those compounds is highly desirable and valuable. In the present study, we developed a selective and sensitive method for trace AA determination in biological samples using 2-[2-(7H-dibenzo [a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC) as labeling reagent by HPLC-FLD-MS/MS. Response surface methodology (RSM) was first employed to optimize the derivatization reaction between DBCEC and AA. Compared with traditional single-factor design, RSM was capable of lessening laborious, time and reagents consumption. The complete derivatization can be achieved within 6.3 min at room temperature. In conjunction with a gradient elution, a baseline resolution of 20 AA containing acidic, neutral, and basic AA was achieved on a reversed-phase Hypersil BDS C(18) column. This method showed excellent reproducibility and correlation coefficient, and offered the exciting detection limits of 0.19-1.17 fmol/μL. The developed method was successfully applied to determinate AA in human serum. The sensitive and prognostic index of serum AA for liver diseases has also been discussed.
Li
Guoliang
G
Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, People's Republic of China.
Cui
Yanyan
Y
You
Jinmao
J
Zhao
Xianen
X
Sun
Zhiwei
Z
Xia
Lian
L
Suo
Yourui
Y
Wang
Xiao
X
D024342:Amino Acids, Acidic / Q000097:blood* / Q000737:chemistry; D024361:Amino Acids, Basic / Q000097:blood* / Q000737:chemistry; D021542:Amino Acids, Neutral / Q000097:blood* / Q000737:chemistry; D001774:Blood Chemical Analysis; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D005019:Ethyl Ethers / Q000737:chemistry; D005453:Fluorescence; D005563:Formic Acid Esters / Q000737:chemistry; D006505:Hepatitis / Q000097:blood; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D008875:Middle Aged; D015233:Models, Statistical; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D021542:Amino Acids, Neutral; D005019:Ethyl Ethers; D005563:Formic Acid Esters; D007202:Indicators and Reagents; C007658:ethyl chloroformate