Validation of an Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometric Method for Quantifying Uracil and 5,6-Dihydrouracil in Human Plasma

Abstract

A simple, rapid, sensitive and specific ultra-high-performance liquid chromatography-tandem mass spectrometry method (Waters UPLC-MS-MS) is developed and validated for the quantification of uracil (U) and 5,6-dihydrouracil (UH2) levels in human plasma. Analytes are extracted using ethyl acetate and isopropanol after deproteination, and separated by high-performance liquid chromatography (HPLC) (Acquity UPLC BEH C18 column) in a binary mobile phase system under gradient elution conditions at a flow rate of 0.4 mL/min. 5-Bromo-uracil (UBr) is used as the internal standard. The detection is performed on a triple-quadrupole mass spectrometer via electrospray positive ionization. Multiple reaction monitoring mode using the transitions m/z 112.82 → 70.05, m/z 114.88 → 55.04 and m/z 190.83 → 117.86 is used to quantify U, UH2 and UBr, respectively. The method is linear in the concentration range of 0.625-160.0 ng/mL. The total run time is 4.5 min per injection. Nine-point calibration curve and four-points quality controls are used. Excellent linearity and precision are observed with correlation coefficient (r(2)) > 0.9999. The intra-batch and inter-batch precisions are ≤ 7.3% and ≤ 8.6%, and accuracy is ≤ 17%. The developed method is shown to be suitable for routine quantitative determination of U, UH2 and 5,6-dihydrouracil-to-uracil ratio in clinical practice.

A new sample preparation and separation combination for the precise, accurate, and simultaneous determination of uracil and dihydrouracil in human plasma by reversed‐phase HPLC

“…To modify the sample protein precipitation, silver nitrate as a precipitant of protein was developed. Compared with ammonium sulfate and the mixture of methanol and acetonitrile , the use of 50% w/v silver nitrate solution was found to be more effective for the removal of protein, subsequently saturated sodium chloride solution was used to precipitate silver nitrate. The final result showed it was a simple and more economical method that can be used routinely.…”

A Critical Review on Clinical Application of Separation Techniques for Selective Recognition of Uracil and 5-Fluorouracil

“…Another sensitive and specific ultra-high-performance liquid chromatography-MS/MS method (waters UPLC-MS-MS) have been developed and validated for the quantification of Ura and (UH2) levels in human plasma. In this method, analytes were extracted by deproteinization and separated out using Acquity UPLC BEH C18 column for simultaneous quantification of Ura, UH 2 , 5-FU and 5,6-dihydrofluorouracil (5-FUH2) in human plasma [1].…”

“…Being a molecule of biological importance, uracil (Ura) and its laboratory derivative i.e., 5-fluorouracil (5-FU) have attracted the attention of several research groups who have attempted to selectively recognize these analytes by means of molecular imprinting and hybrid techniques. Ura is a naturally occurring pyrimidine derivative and it is used for drug delivery in pharmaceuticals [1]. Dihydropyrimidine dehydrogenase (DPD) deficiency with a defect of the pyrimidine degradation pathway is critically important in the clinical diagnosis of Ura concentration because this is directly associated with several inborn errors of metabolism (IEM) processes [2].…”

“…Around 80 % of administered dose of 5-FU is catabolized by DPD enzyme. Patients suffering with DPD deficiency (a pharmacogenetics syndrome) or insufficient activity of this enzyme are at great risk of the development of severe 5-FU toxicity [1,3]. Knowledge of 5-FU concentration levels in body fluids, such as blood and urine, allows pharmacotherapy to be optimized and provides the basis for studies on patient compliance, bioavailability, and pharmacokinetics, in addition to the influences of co-medication.…”

“…Many opportunities for applications particularly molecularly imprinted polymer (MIP) as chromatographic, LLE, SPE, and SPME materials for Ura and 5-FU analysis are still to be exploited. Depending on the origins of samples and analytical objectives, Ura and 5-FU analysis have been carried out using various analytical instruments in many situations such as clinical control for diagnosis and treatment of diseases, pharmacology, and toxicology [1][2][3][4][8][9][10]. In the next section, we would like to contemplate on the current chromatographic methods developed for the purposes of separating the Ura and 5-FU from complex samples containing interferents and detecting them once separation is complete (Fig.…”