King's College London, London, United Kingdom.
Anal Chem. 2013 Aug 6;85(15):7257-63. doi: 10.1021/ac401149w. Epub 2013 Jul 25.
High-throughput multiplatform metabolomics experiments are becoming an integral part of clinical and systems biology research. Such methods call for the adoption of robust sample storage and transport formats for small volumes of biofluids. One such format is the dried biofluid spot, which combines small volume requirements with easy portability. Here, we describe ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics of dried rodent serum, urine, and cerebrospinal fluid spots. An in-vial extraction and UHPLC-MS analysis method was first developed and validated by fingerprinting two test fluids, rat serum and RPMI cell nutrient medium. Data for these extracts were compared in terms of (i) peak area measurements of selected features to assess reproducibility and (ii) total fingerprint variation after data pretreatment. Results showed that percentage peak area variation was found to range between 1.4 and 9.4% relative standard deviation (RSD) for a representative set of molecular features. Upon application of the method to spots bearing serum, urine or cerebrospinal fluid (CSF) from healthy rats and mice, a total of 1,182 and 2,309 reproducible molecular features were obtained in positive and negative ionization modes, respectively, of which 610 (positive) and 991 (negative) were found in both rats and mice. Feature matching was used to detect similarities and differences between biofluids, with the biggest overlap found between fingerprints obtained in urine and CSF. Our results thus demonstrate the potential of such direct fingerprinting of dried biofluid spots as a viable alternative to the use of small (10-15 μL) volumes of neat biofluids in animal studies.
高通量多平台代谢组学实验正在成为临床和系统生物学研究不可或缺的一部分。此类方法需要采用可靠的样本储存和运输方式来处理小体积的生物流体。其中一种方法是干燥生物流体斑，它结合了小体积要求和易于携带的特点。在此，我们描述了干燥啮齿动物血清、尿液和脑脊液斑的超高效液相色谱-质谱（UHPLC-MS）代谢组学。首先通过对两种测试液（大鼠血清和 RPMI 细胞营养培养基）进行指纹分析，开发并验证了一种管内提取和 UHPLC-MS 分析方法。这些提取物的数据是通过（i）选择特征的峰面积测量来评估重现性和（ii）数据预处理后的总指纹变化来比较的。结果表明，对于一组代表性的分子特征，峰面积百分比变化的范围在 1.4%到 9.4%的相对标准偏差（RSD）之间。将该方法应用于来自健康大鼠和小鼠的血清、尿液或脑脊液（CSF）斑上，分别在正离子和负离子模式下获得了 1182 和 2309 个可重现的分子特征，其中 610 个（正离子）和 991 个（负离子）在大鼠和小鼠中均有发现。特征匹配用于检测生物流体之间的相似性和差异性，其中在尿液和 CSF 指纹之间发现了最大的重叠。因此，我们的研究结果表明，直接对干燥生物流体斑进行指纹分析具有很大的潜力，可以作为替代使用小体积（10-15 μL）生物流体进行动物研究的可行方法。
