| 李熙拓,陈孝各,邢 求,王 彦,闫 超.超高效液相色谱-质谱联用法测定人尿液中5-羟吲哚乙酸浓度方法学验证[J].,2018,(15):2841-2846 |
| 超高效液相色谱-质谱联用法测定人尿液中5-羟吲哚乙酸浓度方法学验证 |
| Method Validation for Determination of 5-Hydroxyindoleacetic Acid in Human Urine with Ultra-performance Liquid Chromatography-tandem Mass Spectrometry |
| 投稿时间：2017-12-27 修订日期：2018-01-23 |
| DOI：10.13241/j.cnki.pmb.2018.15.008 |
| 中文关键词: 超高效液相色谱 三重四级杆质谱 5-羟吲哚乙酸 人工尿液 |
| 英文关键词: Words performance liquid chromatography (HPLC) Triple quadrupole mass spectrometry 5-Hydroxyindoleacetic acid Artificial urine |
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| 中文摘要: |
| 摘要 目的：采用超高效液相色谱-质谱联用法(UPLC-MS/MS)建立人尿液中5-羟吲哚乙酸（5-HIAA）的检测方法。方法：采用人工尿液UR-N-CONTROL LEVEL 2 NP配制标准曲线，质控样品采用人工尿液和天然尿液配制而成，以5-HIAA的同位素氘标记物5-HIAA-d5为内标，尿液样品采用乙腈进行稀释处理后，采用UPLC-MS/MS检测。色谱柱为Waters HSS T3 （100×2.1 mm, 1.8 μm），流动相A为含0.02 %乙酸的2 mM醋酸铵水溶液，B相为：含0.02 %乙酸的乙腈溶液，在2.5 min内使用5 % 的B相至100 % 的B相进行梯度洗脱，流速为0.5 mL/min，进样量3.0 μL，柱温50 ℃；采用电喷雾负离子模式电离，5-HIAA和5-HIAA-d5内标的监测离子分别为m/z 190.1→146.2和m/z 195.1→151.0。结果：该方法测定的5-HIAA在0.0500 至50.0 μg/mL范围内线性良好，r ≥0.9948，最低定量限（LLOQ）为0.0500 μg/mL。5-HIAA批内、批间准确度在89.3 %~99.8 %之间，精密度 （CV）≤ 8.4 %。平均回收率为100.3 %~102.3 %。结论：该方法专属性强、灵敏度高，快速。人工尿液和天然尿液在定量分析时无差异，适合于人尿液中5-HIAA的浓度测定。 |
| 英文摘要: |
| ABSTRACT Objective: An efficient analytical method for the quantitative determination of 5-hydroxyindoleacetic acid (5-HIAA) in human urine by ultra-performance liquid chromatography (UPLC) - tandem mass spectrometry (MS/MS) was developed. Methods: The artificial human urine UR-N-CONTROL LEVEL 2 NP was used for calibration curve preparation, artificial urine and natural urine were used for quality control samples preparation. 5-Hydroxyindoleacetic acid-d5 (5-HIAA-d5) was used as internal standard, urine samples were diluted with acetonitrile. The supernatants were detected by UPLC-MS/MS. Separation was carried out on a Waters HSS T3 analyti- cal column (100×2.1 mm, 1.8 μm). Mobile phase consisting A: 2 mM ammonium acetate (containing 0.02 % acetic acid ) and B: ace- tonitrile (containing 0.02 % acetic acid). Gradient elution using the proportion of mobile phase B ranged from 5 % to 100 % in 2.5 min at a flow rate of 0.5 mL/min. The column temperature was 50 ℃ and the injection volume was 3.0 μL. The electrospray source and nega- tive ion scan were used. 5-HIAA and the internal standard (5-HIAA-d5) were monitored at m/z 190.1 146.2 and m/z 195.1 151.0, respec- tively. Results: The linear calibration curve was observed in the concentration range from 0.0500 to 50.0 μg/mL, with the r ≥ 0.9948. The lower limit of quantification was 0.0500 μg/mL. The accuracy (intra-assay and inter-assay) for 5-HIAA was between 89.3 % and 99.8 %, with the precision (CV) ≤ 8.4 %. The mean recoveries ranged from 100.3 % to 102.3 %. Conclusion: The method is proved to be highly selective, sensitive and rapid. Artificial urine and natural urine are no differences in quantitative analysis. They are suitable for determination of 5-HIAA in human urine. |
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