一种用于定量检测生物体液中碱基和核苷的在线 SPE-LC-MS/MS 方法。
An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.
机构信息
Department of Chemistry, Physics, and Atmospheric Science, Jackson State University, Jackson, MS 39217, USA.
Department of Biology, Tougaloo College, Tougaloo, MS 39174, USA.
出版信息
Anal Methods. 2024 Apr 25;16(16):2505-2512. doi: 10.1039/d4ay00100a.
Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 μM (cytidine) in urine and from 0.015 (guanosine) to 0.79 μM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 μM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 μM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.
由于这些化合物的高亲水性，固相萃取（SPE）和液相色谱（LC）分离核苷酸碱基是具有挑战性的。在此，我们报告了一种新的在线 SPE-LC-MS/MS 方法，用于在柱前衍生化氯乙醛（CAA）后对其进行定量。所提出的方法具有选择性和灵敏度，检测限达到纳摩尔水平。通过使用该方法对尿液和唾液样本进行了分析。在尿液中，腺嘌呤、鸟嘌呤、胞嘧啶、腺苷、鸟苷和胞苷的浓度范围为 0.19（鸟苷）至 1.83 μM（胞苷），在唾液中，浓度范围为 0.015（鸟苷）至 0.79 μM（腺嘌呤）。有趣的是，发现唾液中胞苷的甲基化与尿液中的明显不同。虽然 5-羟甲基胞苷在唾液中的含量非常低（<0.05 μM），但它在尿液中是最主要的甲基化胞苷，含量高达 3.33 μM。由于采用在线 SPE，因此所提出的 LC-MS/MS 定量分析方法方便进行，并具有良好的分析准确性和重复性。
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