An Analytical Strategy for Reliable Metabolome Analysis of Clinical Leftover Sera Using Timed Aliquoting

Accurate metabolome analysis depends not only on advanced
analytical
techniques but also on strict control of preanalytical variables.
This study presents an analytical strategy for reliable untargeted
metabolomics using clinical leftover sera, focusing on the impact
of timed aliquoting during short-term storage. Leftover serum samples
from routine clinical testing, typically stored at 4 °C for up
to 7 days, offer a valuable and accessible resource for biomarker
discovery. However, variable delays in sample aliquoting and storage
can compromise metabolite stability. We used high-coverage 12C-/13C-dansylation LC–MS to profile the amine/phenol
submetabolome in serum samples collected from healthy individuals
at multiple time points postdraw. The study included 630 LC–MS
runs (105 subjects × 6 time points) in the discovery set and
280 runs (70 subjects × 4 time points) in the validation set,
quantifying 1382 and 1352 metabolites, respectively. Although time-dependent
changes in metabolite abundances were observed, these shifts were
relatively small between adjacent time points. Notably, clear sex-based
metabolic separation was observed when using samples aliquoted at
the same time or within 24-h intervals, whereas discriminatory power
diminished when samples with longer storage time differences were
combined. These findings demonstrate that, with carefully timed aliquoting,
clinical leftover sera can be reliably used for metabolomics. Our
study establishes a practical and scalable workflow to control preanalytical
variationspecifically by minimizing storage time differencesthereby
enabling broader use of clinical samples in biomarker discovery and
population-scale metabolomics.

精准的代谢组学分析不仅依赖于先进的分析技术，还需严格控制分析前变量（preanalytical variables）。本研究提出了一种利用临床剩余血清开展可靠非靶向代谢组学（untargeted metabolomics）分析的策略，重点关注短期储存过程中定时分装的影响。常规临床检测剩余的血清样本通常可在4℃下储存长达7天，是极具价值且易于获取的生物标志物发现资源。然而，样本分装与储存环节的可变延迟会损害代谢物的稳定性。本研究采用高覆盖度12C-/13C-丹磺酰化液相色谱-质谱联用（LC–MS）技术，对健康个体采血后多个时间点采集的血清样本中的胺/酚类亚代谢组进行代谢谱分析。本研究的发现集包含630次LC–MS运行（105名受试者×6个时间点），验证集包含280次LC–MS运行（70名受试者×4个时间点），分别定量了1382和1352种代谢物。尽管观察到代谢物丰度存在时间依赖性变化，但相邻时间点间的丰度偏移相对较小。值得注意的是，当使用同一时间点或24小时内分装的样本时，可观察到清晰的基于性别的代谢分离现象；而当合并储存时间差异较大的样本时，区分能力会显著下降。上述研究结果表明，只要严格控制分装时间，临床剩余血清即可可靠地用于代谢组学研究。本研究建立了一套实用且可扩展的分析前变异控制流程——尤其是通过最小化储存时间差异——从而能够更广泛地将临床样本应用于生物标志物发现与人群规模代谢组学研究。