超高效液相色谱-串联质谱法测定人血浆中精氨酸及衍生物含量

Determination of Amino Acid Biomarkers in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

建立了超高效液相色谱-串联质谱（UPLC-MS/MS）法同时测定使用艾普拉唑后人血浆中二甲基精氨酸（ADMA）、对称二甲基精氨酸（SDMA）、单甲基精氨酸（NMMA）、瓜氨酸（Cit）和L-精氨酸（L-Arg）的浓度。采用HILIC亲水相互作用色谱和非衍生化的蛋白沉淀法进行分离分析,色谱柱选取Waters Atlantic HILIC柱（2.1 mm×50 mm×3μm）,流动相由乙腈（含0.5%乙酸和0.025%三氟乙酸）-水（含0.5%乙酸和0.025%三氟乙酸）（85∶15,V/V）组成,流速0.25mL/min。采用多反应离子监测（MRM）模式,以电喷雾离子源（ESI）正离子方式检测。结果显示,ADMA、SDMA、NMMA、L-Arg和Cit的线性关系良好,相关系数r均大于0.994 0;ADMA、SDMA和NMMA的线性范围为0.1~5mmol/L,L-Arg和Cit的线性范围为10~250mmol/L;5种氨基酸的日内、日间精密度均小于15%,准确度在85%~115%之间。该方法快速、简便、灵敏,可为相关疾病的临床诊断提供一种高效的检测手段。

Anultra high per formance liquid chromatography- tandem mass spect romet ry （UPLC-MS/MS） method was developed for the simultaneous determination of ADMA, SDMA, NMMA, L-Arg and Cit in human plasma after administration of ilaprazole. The plasma samples were prepared using protein precipitation by acetonitrile after addition of deuterated internal standard. The analytical column was Waters Atlantic HILIC （2. 1 mm× 50 mm× 3 μm） , and th e mobile p h ase was composed of acetonitrile （contai -ning 0. 5% acetic acid and 0. 025% trifluoroacetic acid ） -water （containing 0. 5% acetic acid and 0. 025% tr if luoroacetic acid） （85 ： 15, V /V ） . The flow rate was 0. 25 mL /m in and the sample run time was 3. 0 min. A tan d em mass sp ectrometer coupled with p o si-tive electro-spray ionization （ESI） source was used for detection. The quantitative anal-ysis was performed on selective ion chromatograms acquired by a multiple reaction moni-toring （MRM） mode of following transitions： ADMA （m/z 203.2→46.0）, SDMA （m /z 203. 2 → 172. 1）,NMMA （m /z 189. 0 → 70. 0 ）,Cit （m /z 176. 0 →70. 0）,L -A rg （m/z 175. 1→60. 071） , and D7-ADMA （m/z 210. 0→77. 1）. T h e sp ecif ic ity , accu ra cy , linearity, intra-day and inter-day precision, recovery and matrix effect were investigated in this study according to the guidance issued by the Food and Drug Administration （FDA） of USA. The method was validated over the concentration range of 0.1-5 mmol/L for ADMA, SDMA and NMMA, 10-250 mmol/L for L -Arg and Cit, respec -tively. Inter-day and intra-day precision were less than 15 % and accuracy was within 85 %-115 %. The lin ea r regre ssion （weighed by 1/x 2） was applied to e s tab l ish th e re la -tionship between plasma concentration and peak area ratio of each analyte （minus that of the blank） to its IS. Topical equations of ADMA, SDMA, NMMA, L-Arg and Cit were as follows： ：y = 0. 385 lx ＋ 0. 207 7 （r =0. 996 7）, 3; = 0. 418 0x ＋ 0. 200 7 （r =0. 998 3）, 3; = 0. 366 2x ＋ 0. 048 31（r=0. 998 5）, 3; = 0. 011 53x ＋ 1 . 204 9 （ r = 0 . 998 0） ,and 3; =0 . 043 21x ＋ 8 . 315 4 （r= 0 . 994 1）,resp ect ive ly . The extraction reco v eries of A DM A , SDMA,NMMA,Cit and L-Arg were （92. 5 ± 8 . 6 ） % ,（ 8 8 . 5 ± 6. 5 ） % ,（ 50. 6 ± 3.1）% ,（27. 0±0 . 3） % and （33. 9% ± 2. 1） % , resp ect ive ly . The matrix effects about ADMA, SDMA, NMMA,Cit and L-Arg were （90. 5±6 .3 ） % ,（ 83. 0±3. 5 ）% ,（ 50. 6± 2.5）% ,（41. 0 ± 0. 1） % , and （32. 9 ± 0 . 2） % , resp ect ive ly . Therefore , this method is rapid,simple and sensitive, and which can provide a highly efficient detection means to the future clinical disease diagnosis.