Determination of Endogenous Concentrations of Uracil and Dihydrouracil in Dried Saliva Spots by LC-MS/MS: Method Development, Validation, and Clinical Application

Abstract

Background: The aim of this study was to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the measurement of uracil (U) and dihydrouracil (UH2) concentrations in dried saliva spots (DSSs), for the evaluation of dihydropyrimidine dehydrogenase (DPD) enzyme activity. Results: Nine 18-mm diameter DSS discs were extracted with acetate:isopropyl alcohol (85:15, vol/vol) and analyzed by LC-MS/MS. The assay was linear in the range of 10–1000 ng·mL−1, with accuracy between 89% and 112% and precision between 5.7% and 13%. The metabolic ratio [UH2]/[U] was stable in DSS for up to 9 days at 45°C. Concentrations of U and UH2, as well as the metabolic ratio, were highly concordant between matrices. Using a metabolic ratio classification cutoff of 1.16 for the identification of slow DPD metabolizers, 98.7% concordance was achieved between SS and saliva. Conclusions: DSS samples could be a useful alternative for DPD activity screening, particularly in locations with limited access to highly equipped laboratories.

A Simple and Rapid UPLC‐UV Method for Detecting DPD Deficiency in Patients With Cancer

“…[21][22][23] Recently, several groups have proposed to assay U and UH2 from saliva samples, so as to facilitate large-scale screening from an easily accessible tissue, but these assays are still based on HPLC-UV or LC-MS/MS analyses. [24][25][26] Similarly, alternate DPD testing using a loading dose of exogenous U subsequently required MS. 27 With respect to the increasing number of patients who require screening, developing a simple, rapid, and affordable bioanalytical method that is easily transposable in any laboratory is challenging. In particular, shifting from HPLC to UPLC is critical so as to decrease analytical run-times.…”

Potential use of a dried saliva spot (DSS) in therapeutic drug monitoring and disease diagnosis

“…To date, DSS has been widely used in diagnosis of several diseases [ [11] , [12] , [13] , [14] , [15] ], for the bio-detection of several drugs [ [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] ] and in metabolomics analysis [ 25 , 26 ]. Seven years (June 2014–March 2021) of literature research on DSS as a sampling technology are summarized ( Table 1 ).…”

“…Furthermore, it has been used to detect drug metabolites in human saliva, including antiepileptic drugs, antipsychotic drugs, methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine [ 16 , 17 , 22 ]. However, the recovery of analytes is lower than that of biological metabolites [ 24 , 25 ]. This could be due to either the high protein binding rate of some drugs or the absence of denaturation of the protein in saliva.…”

“…Table 1 shows published articles on the DSS methods [ [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] ], in which an IS was added to the extraction solvent instead of dropping it onto the filter paper. Zheng et al.…”

Pharmacogenomic tests in Oncology - finding the right dose

“…Further studies are needed to validate the strategies mentioned above of DPD function measurements in a larger patient population. (8) Irinotecan (IRI) is a prodrug converted in the liver by carboxylesterases (CES) to 7-ethyl-10hydroxycamptothecin (SN-38), which is much more active and cytotoxic than its parent drug. Treatment with irinotecan is usually associated with doselimiting toxicities, mainly diarrhea and neutropenia/ leukopenia.…”