Plasma and serum metabolic analysis of healthy adults shows characteristic profiles by subjects’ sex and age

Pre-analytical factors like sex, age, and blood processing methods introduce variability and bias, compromising data integrity, and thus deserve close attention. This study aimed to explore the influence of participant characteristics (age and sex) and blood processing methods on the metabolic profile. A Thermo UPLC-TSQ-Quantiva-QQQ Mass Spectrometer was used to analyze 175 metabolites across 9 classes in 208 paired serum and lithium heparin plasma samples from 51 females and 53 males. Comparing paired serum and plasma samples from the same cohort, out of the 13 metabolites that showed significant changes, 4 compounds related to amino acids and derivatives had lower levels in plasma, and 5 other compounds had higher levels in plasma. Sex-based analysis revealed 12 significantly different metabolites, among which most amino acids and derivatives and nitrogen-containing compounds were higher in males, and other compounds were elevated in females. Interestingly, the volcano plot also confirms the similar patterns of amino acids and derivatives higher in males. The age-based analysis suggested that metabolites may undergo substantial alterations during the 25-35-year age range, indicating a potential metabolic turning point associated with the age group. Moreover, a more distinct difference between the 25–35 and above 35 age groups compared to the below 25 and 25–35 age groups was observed, with the most significant compound decreased in the above 35 age groups. These findings may contribute to the development of comprehensive metabolomics analyses with confounding factor-based adjustment and enhance the reliability and interpretability of future large-scale investigations.