A new quantitative method for pseudouridine and uridine in human serum and its clinical application in acute myeloid leukemia

Pseudouridine, a C-C glycosidic isomer of uridine, is derived from uridine via isomerization, and pseudouridylation is the most common post-transcriptional modification. Our previous study shows pseudouridine may serve an important role in acute myeloid leukemia (AML). The clinical value of pseudouridine and uridine is hampered by the lack of a quantitative methods with high sensitivity, specificity, and stability. Here, we established a supercritical fluid chromatography-tandem triple quadrupole mass spectrometry (SFC-TQ-MS)-based method to quantitate serum pseudouridine and uridine simultaneously. The procedure involves protein precipitation of sample, extraction with solid phase extraction (SPE) plate, 5-min SFC separation by applying gradient elution on a Acquity UPC2 Torus DIOL column, and analysis by TQ-MS using well-characterized calibration standards. After validation, the method was used to measure pseudouridine and uridine concentrations in 143 serum samples from healthy controls (HCs) and AML patients to evaluate their prognostic potential. The successfully validated assay had a linear range of 5–5000 ng/mL, accuracies between 97 % and 102 %, and intra- and inter-assay imprecision <10 %. Compared to HCs, pseudouridine was raised significantly, while uridine was curtailed severely in patients with AML. With a median concentration of 671.4 ng/mL as the prognostic cut-off value, high level pseudouridine independently predicted poor survival of AML patients. Quantification of serum pseudouridine and uridine by SFC-TQ-MS provides an analytically sensitive and reproducible method for clinical diagnosis, and high concentration of pseudouridine is an independent prognostic factor for patients with AML.