Stability of targeted metabolite profiles of urine samples under different storage conditions

Abstract

Introduction: Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives: We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods: Pooled, fasting urine samples were collected and stored at -80 °C (biobank standard), -20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ™ p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results: The concentrations of 63 investigated metabolites were stable at -20 and 4 °C for up to 24 h when compared to samples immediately stored at -80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E-04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion: Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

Keywords: Amino acids; Pre-analytics; Storage conditions; Targeted metabolomics; Urine.

Fig. 1

Overview of the study design. Urine from six female volunteers was pooled and aliquoted before being stored at −80, −20, 4, 9, and 20 °C for 0, 2, 8, and 24 h. For freeze and thaw cycles, samples were frozen for 24 h and thawed for 2 h per cycle. Each sample was measured four times

Fig. 2

Concentrations of two metabolites over time for various storage conditions. Influence of storage conditions on the concentrations of Valine and C6:1. The concentration of C6:1 at 9 °C increased, whereas the concentration of valine decreased at 20 and 9 °C in urine over the course of 24 h