Journal of Pharmaceutical and Biomedical Analysis 142 (2017) 125–135 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Development, validation and application of a novel liquid chromatography tandem mass spectrometry assay measuring uracil, 5,6-dihydrouracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, -fluoro--ureidopropionic acid and -fluoro--alanine in human plasma Ottiniel Chavani a,∗ , Berit Packert Jensen a , R. Matthew Strother b , Christopher M. Florkowski a , Peter M. George a a Canterbury Health Laboratories, Christchurch, New Zealand b Oncology, Christchurch Hospital, Christchurch, New Zealand a r t i c l e i n f o Article history: Received 24 November 2016 Received in revised form 23 April 2017 Accepted 24 April 2017 Available online 6 May 2017 a b s t r a c t The plasma 5,6-dihydrouracil/uracil (UH 2 /U) ratio is a possible phenotypic marker of dihydropyrimidine dehydrogenase (DPD) activity, hence an index of 5-fluorouracil (5-FU) response and toxicity. Studies have re-affirmed the value of 5-FU and 5,6-dihydro-5-fluorouracil (FUH 2 ) for therapeutic drug monitoring (TDM). However, FUH 2 has limited stability in plasma, necessitating expedited plasma separation and freezing, where routine compliance may not be easy. The metabolites -fluoro--ureidopropionic acid (FUPA) and -fluoro--alanine (FAL) are stable in plasma and are probable candidates for TDM. This paper describes development, validation and application of an LC–MS/MS assay quantifying U, UH 2 , 5-FU, FUH 2 , FUPA and FAL in human plasma. © 2017 Elsevier B.V. All rights reserved. Extraction was by salt-assisted liquid–liquid extraction (LLE) in two-stages with pH adjustment. The supernatants were mixed, dried and reconstituted. Analytes were resolved on the Luna PFP (2) (150 × 2.00 mm, 3 ) column by gradient elution and analyzed by tandem mass spectrometry via electrospray ionisation in positive polarity. 1. Introduction 5-FU, a pyrimidine analogue, is a commonly used chemother- apeutic in solid tumours of the digestive tract, breast plus head and neck [1]. Its administration is typically intravenous bolus or continuous infusion. Most current dosing algorithms use body sur- face area to normalize exposure. However, >50% of patients have plasma 5-FU values outside the optimal range [2] and up to 100- fold individual variability in pharmacokinetic parameters have been observed [3–5]. An association between 5-FU pharmacoki- ∗ Corresponding author at: Toxicology, Canterbury Health Laboratories, P.O. Box 151, Christchurch 8140, New Zealand. E-mail address: Ottiniel.Chavani@cdhb.health.nz (O. Chavani). netic parameters and efficacy plus toxicity has been reported and optimal therapeutic windows have been proposed [6–8]. Variability in 5-FU exposure has been associated with dif- ferences in the three-step sequential metabolic pathway which rapidly degrades approximately 80% of systemic 5-FU [9] (Fig. 1) and is partially attributable to polymorphisms, environmental exposures or comorbidity. Diminished metabolism augments sys- temic 5-FU exposure which compromises efficacy and occasionally leads to toxicity. DPD is the initial, rate-limiting and saturable enzyme but variability in DHPase and -alanine synthase may also modify toxicity risk [10–14]. To minimize toxicity, a two-step strategy is needed: prediction of first dose, followed by TDM for subsequent doses. Response and toxicity are known to be improved by pharmacokinetic adjustment of 5-FU dose [7,15,16]. However, generally the first dose is deter- mined using body surface area, thus possibly not achieving optimal therapeutic concentration and is potentially toxic . At present, there is no universally applied a priori test for identification of patients with an increased risk of toxicity or treatment efficacy. The endogenous plasma UH 2 /U ratio is a reasonable index of response and toxicity. A high degree of concordance between ele- vated 5-FU and low endogenous UH 2 /U is known [17]. Hence it http://dx.doi.org/10.1016/j.jpba.2017.04.055 0731-7085/© 2017 Elsevier B.V. All rights reserved.