Faecal amino acids stability: investigating optimal sampling conditions for analysis.
Author(s): Opperman RCM, Vermeer E, Bosch S, de Meij TGJ, de Boer NKH, Struys EA
Publication: Metabolomics, 2025, Vol. 21, Page 81
PubMed ID: 40515879 PubMed Review Paper? No
Purpose of Paper
This paper investigated the stability of 20 amino acids in fecal specimens stored at 4°C and 20°C for up to 7 days, at -20°C for up to 90 days, in an OMNImetGUT sampling device at room temperature for up to 7 days, and over the course of three freeze-thaw cycles. Inter- and intra-day variability in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, as well as variability attributable to the sampling location, were also investigated.
Conclusion of Paper
The inter-day coefficient of variance (CV) in LC-MS/MS measurements was high for valine (18%), α-aminoadipic acid (20%), tyrosine (35%) and taurine (69%) but was <15% for the other 16 amino acids. The intra-day CV of the assay was high for tyrosine (19%) and taurine (137%) but the variability in taurine was partially explained by lack of detection in 3 of 8 measurements. Taurine was consequently excluded from all other comparisons. Generally, most amino acid concentrations varied <30% among the sampling locations, but there were outliers. When feces were stored at 4°C or 20°C for ≥1 day, changes >30% were observed in at least one specimen for most amino acids. Apart from lysine and tyrosine levels in the feces of one of three volunteers, changes in amino acid levels were <30% when the fecal specimen was stored at -20°C for up to 90 days. In both fecal specimens stored in OMNImetGUT sampling devices at room temperature, most amino acids were stable for 7 days, but ≥30% decreases were found in isoleucine and valine levels after ≥1 day, in histidine after ≥3 days and threonine after 7 days. Levels of amino acids were generally stable (<30% change) after three freeze-thaw cycles; however, larger declines in most amino acids were observed in one specimen after two cycles, which the authors state may reflect a processing effect since levels then increased toward baseline after 3 cycles.
Studies
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Study Purpose
This paper investigated the stability of 20 amino acids in fecal specimens stored at 4°C and 20°C for up to 7 days, at -20°C for up to 90 days, in an OMNImetGUT sampling device at room temperature for up to 7 days, and over the course of three freeze-thaw cycles. Inter- and intra-day variability in the assay, as well as variability attributed to the sampling location, were also investigated. Multiple fecal specimens from three healthy volunteers were collected into seven different containers. The reference specimen was homogenized with distilled water and vortexed. The remaining six specimens were subsampled within 2 h of collection. To assess the intra-specimen variability, specimens were obtained from six different sites, including superficial and deep sites. Inter-day and Intra-day variability in measurements were assessed by performing 5 replicate measurements in a single run and 4 runs on different days. To analyze the effects of storage, specimens were stored at -20°C for 7, 30 and 90 days; at 4°C for 1, 3 and 7 days; and at 20°C for 1, 3 and 7 days. To evaluate the effect of freeze-thaw cycling, specimens were subjected to 1, 2 or 3 additional cycles of thawing for 10 min at room temperature, followed by freezing at -80°C for 10 min. To evaluate amino acid stability in OMNImet®·GUT sampling devices, matched devices were stored immediately at -80°C and stored at room temperature for 3 or 7 days before freezing. After storage, all specimens were frozen at -80°C, homogenized with water and freeze-dried. Specimens were then mixed with water at a ratio of 100 mg/5 mL and filtered before quantification of amino acids by LC-MS/MS.
Summary of Findings:
The inter-day coefficient of variance (CV) in LC-MS/MS was high for valine (18%), α-aminoadipic acid (20%), tyrosine (35%) and taurine (69%) but was <15% for the other 16 amino acids. The intra-day CV of the assay was high for tyrosine (19%) and taurine (137%) but the variability in taurine was partially explained by lack of detection in 3 of 8 measurements. Taurine was consequently excluded from all other comparisons. Generally, most amino acid concentrations varied <30% among the sampling locations, but there were outliers. When feces were stored at 4°C or 20°C for ≥1 day, changes >30% were observed in at least one specimen for most amino acids. Apart from lysine and tyrosine levels in the feces of one of three volunteers, changes in amino acid levels were <30% when the fecal specimen was stored at -20°C for up to 90 days. In both fecal specimens stored in OMNImetGUT sampling devices at room temperature, most amino acids were stable for 7 days, but ≥30% decreases were found in isoleucine and valine levels after ≥1 day, in histidine after ≥3 days and threonine after 7 days. Levels of amino acids were generally stable (<30% change) after three freeze-thaw cycles; however, larger declines in most amino acids were observed in one specimen after two cycles, which the authors state may reflect a processing effect since levels then increased toward baseline after 3 cycles.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Superficial and deep locations compared
Storage Storage temperature -80°C
-20°C
4°C
20°C
Storage Storage duration < 2h
1 day
3 days
7 days
30 days
90 days
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
Biospecimen Preservation Type of fixation/preservation OMNImet-GUT
Frozen
None (fresh)