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

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, 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.

Development and validation of an assay for the measurement of gentamicin concentrations in dried blood spots using UHPLC-MS/MS.

Gentamicin sulfate (GEN) is an aminoglycoside antibiotic with a narrow therapeutic range of plasma concentrations. The collection of venous blood represents a significant burden for patients, especially in neonatology. Dried blood spots (DBS) obtained from capillary blood can be an alternative for drug measurements in this particular population. This study aimed to develop and validate an assay for the quantification of GEN in DBS using UHPLC-MS/MS. Total GEN concentrations were obtained by adding the individual concentrations of the GEN forms C1, C1a, and C2. The assay used a DBS disk containing approximately 17 µL of blood for GEN quantitation in the range of 0.1-40 mg L-1. Measurement accuracy for total GEN was in the range of 102.6-108.6%, inter-assay precision was 11.3-13.1% and intra-assay precision was 9.1-12.8.% GEN was stable for 21 days at - 20 and 8 °C, but only for 24 h at room temperature. Blood Hct affected the accuracy within acceptable limits (93.8-95% at Hct% of 30, 104.3-113% at Hct% of 50). Blood spotted volume did not affect GEN measurement accuracy. Concentrations of GEN in DBS obtained after heel pricks were correlated to plasma levels in a small cohort of neonatal patients. However, percentual differences between estimated plasma concentrations and actual plasma levels presented values between - 64-35.3% (average difference of - 1.9%). The use of DBS for the measurement of GEN concentrations can increase access to TDM of this antibiotic due to the ease of sample collection and the facilitated specimen transportation logistics when testing is not available onsite.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis.

BACKGROUND: Therapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. OBJECTIVE: This study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. RESULTS: The use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2-40 mg L-1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8-10.0%, intra-assay CV of 10.2-11.0%, and accuracy of 96.8-104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15-3.57 mg L-1 for GEN C1, 0.12-3.55 mg L-1 for GEN C1a, 0.20-5.77 mg L-1 for GEN C2, and 0.47-12.88 mg L-1 for the GEN complex, all within the linear range of the assay. CONCLUSION: A sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.

Vancomycin and creatinine determination in dried blood spots: Analytical validation and clinical assessment.

This study aims to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for vancomycin and creatinine measurement in dried blood spots (DBS) and to evaluate its clinical application. The analytes were extracted from DBS and analyzed by LC-MS/MS. Vancomycin and creatinine DBS and plasma concentrations were compared in 54 and 35 samples, respectively, from 29 patients. Accuracy was 94.4-102.6%, intra-assay precision was 2.1-5.6%, and inter-assay precision was 3.5-7.0%. Patients vancomycin plasma to DBS concentration ratios were highly variable (1.148-5.022), differently from creatinine (0.800-1.283). The assay has adequate analytical performance. Plasma concentrations can be satisfactorily predicted from DBS measurements for creatinine, but not for vancomycin, which limits its clinical application.