稳定同位素非衍生化UPLC-MS/MS法快速测定重症肺炎患者血清氨基酸的变化  

Simultaneous determination of serum amino acids changes in patients with severe pneumonia by underivatized UPLC-MS/MS with stable isotope

作　　者：时正媛[1,2,3] 王淑梅 杨欣妤[1,2,3] 鄢丹 SHI Zheng-yuan;WANG Shu-mei;YANG Xin-yu;YAN Dan(Department of Pharmacy,Beijing Shijitan Hospital Affiliated to Capital Medical University,Beijing 100038,China;Beijing Key Laboratory of Bio-characteristic Profiling for Evaluation of Rational Drug Use,Beijing Shijitan Hospital Affiliated to Capital Medical University,Beijing 100038,China;Joint Laboratory for International Cooperation of Bio-characteristic Profiling for Evaluation of Clinical Rational Drug Use,Beijing Shijitan Hospital Affiliated to Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院药剂科,北京100038 [2]首都医科大学附属北京世纪坛医院临床合理用药生物特征谱学评价北京市重点实验室,北京100038 [3]首都医科大学附属北京世纪坛医院临床合理用药生物特征谱学评价国际合作联合实验室,北京100038

出　　处：《中国临床药理学杂志》2020年第1期76-79,共4页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金面上基金资助项目(81773891);国家科技部“重大新药创制”科技重大专项基金资助项目(2017ZX09301040);首都医科大学附属北京世纪坛医院院基金资助项目(2017-C03);北京市自然科学基金青年基金资助项目(7194280)

摘　　要：目的建立快速测定人血清中非衍生化的20种氨基酸的超高效液相色谱串联三重四极杆质谱(UPLC-MS/MS)分析方法,并用于检测重症肺炎患者血清氨基酸变化。方法用Acquity BEH Amide色谱柱(2.1 mm×100 mm, 1.7μm),以稳定同位素标记的氨基酸作为内标,流动相为乙腈-水-20 mmol·L^-1乙酸铵-0.5%甲酸溶液,梯度洗脱。选择重症肺炎者16例与健康者14例进行检测。结果在所设定的色谱条件下,基质效应不影响测定结果,各氨基酸在标准曲线范围内线性关系良好,相关系数均r2>0.99。准确度、批内和批间精密度均RSD<15%,在测定过程中稳定性符合要求。重症肺炎组与健康组相比,谷氨酸、苯丙氨酸、酪氨酸、丝氨酸和组氨酸水平显著升高,谷氨酰胺、苏氨酸和赖氨酸水平显著降低,其中这2组的谷氨酸含量分别为(177.49±65.38)和(77.43±31.83)μmol·L^-1;这2组的谷氨酰胺含量分别为(222.49±100.36)和(896.09±173.93)μmol·L^-1,组间比较差异均有统计学意义(均P<0.05)。结论该方法可用于重症肺炎患者血清中氨基酸的含量测定。Objective To develop an underivatized ultra-high perfor-mance liquid chromatography tandem mass spectrometric(UPLC-MS/MS) method for the simultaneous determination of 20 amino acids in human serum. Methods The chromatographic separation was performed on Acquity BEH Amide column(2.1 mm×100 mm,1.7 μm). The 20 corresponding stable isotope-labeled amino acids were used as the internal standards. The gradient elution mobile phase was consisted of acetonitrile-H2O-20 mmol·L^-1 ammonium acetate-0.5%formic acid solution. Sixteen cases in severe pneumonia group and 14 cases in healthy group were selected for detection. Results Assays were validated according to current bioanalytical guidelines, with good matrix effect, linearity(all r2 > 0.99), extraction recovery, intraday and interday precision(all RSD<15%), and stability. Comparison between severe pneumonia patient group and healthy group,the serum levels of glutamine, phenyla-lanine, tyrosine, serine and histidine were significantly higher in the severe pneumonia patient group(all P<0.05). Besides, glutamic acid, threonine and lysine were declined in the severe pneumonia patient group. The glutamic acid content in severe pneumonia group and healthy group group was( 177. 49 ± 65. 38),( 77. 43 ± 31. 83) μmol·L^-1,and the glutamine content of the two groups was( 222. 49 ± 100. 36),( 896. 09 ± 173. 93) μmol · L^-1,respectively,the difference were significantly( all P < 0. 05). Conclusion The quantitative analysis method of amino acids is simple,accurate and table,which was successfully applicable for amino acid analysis in the severe pneumonia patient.

关 键 词：氨基酸 超高效液相串联质谱法 非衍生化 稳定同位素标记 

分 类 号：R97[医药卫生—药品]