LC-MS/MS quantification of trimethylamine N-oxide in plasma and volumetric absorptive microsamples: analytical...

Trimethylamine N-oxide (TMAO) is a gut microbiome-related metabolite that may be associated with kidney injury and cardiovascular risk. Measuring TMAO in children after kidney transplantation may support investigation of the gut-kidney-cardiovascular axis; however, repeated venous sampling is often impractical in pediatric longitudinal studies. This study aimed to validate a rapid LC-MS/MS method for measuring TMAO in plasma and 10 μL dried capillary blood samples collected using volumetric absorptive microsampling (VAMS). TMAO was quantified using its stable-isotope-labelled internal standard, TMAO-d9, by hydrophilic interaction chromatography (HILIC) with positive electrospray ionization. For TMAO, an endogenous analyte, the calibration curves were determined using a surrogate matrix of 5% bovine serum albumin in phosphate-buffered saline (PBS) for plasma and a surrogate whole-blood matrix for VAMS. Quality control samples were prepared in authentic matrices. The validation process involved examining the calibration curve, LLOQ, accuracy, precision, recovery, matrix effect, stability, incurred sample reanalysis, matrix parallelism, and comparability of plasma-VAMS results. The assay calibration curve was linear from 0.1 to 20 μmol/L. The LLOQ was 0.1 μmol/L for both matrices. The mean calibration R² values were 0.9989 ± 0.0007 for plasma and 0.9981 ± 0.0011 for VAMS. The surrogate and true matrices exhibited similar parallelism (slopes of 0.97 and 0.93 for plasma and VAMS, respectively). In the pediatric kidney transplant population, plasma TMAO was highly correlated with serum creatinine (r = 0.964) and inversely correlated with eGFR (r = -0.788). Similar associations were observed between VAMS plasma-estimated TMAO and creatinine (r = 0.951) and eGFR (r = -0.784). The validated method enables reliable quantification of TMAO in both plasma and VAMS microsamples and demonstrates analytical feasibility for future longitudinal biomarker studies in pediatric kidney transplantation. However, these clinical findings should be considered exploratory rather than evidence of clinical utility.