A liquid chromatography-tandem mass spectrometry method for the determination of 5-Fluorouracil degradation rate by intact peripheral blood mononuclear cells.
5-Fluorouracil (5-FU) is a major chemotherapy drug used for the treatment of tumors. It is catabolized mainly by dihydropyrimidine dehydrogenase, and patients with a complete or partial deficiency of dihydropyrimidine dehydrogenase activity are at risk of developing severe 5-FU-associated toxicity. The aim of this study was to demonstrate that intact peripheral blood mononuclear cells (PBMCs) can be an effective model to evaluate the degradation rate of 5-FU. We developed a sensitive and specific liquid chromatography-tandem mass spectrometry method to measure in vitro the rate of 5-FU degradation by intact PBMC. 5-FU degradation rate was determined by measuring the decrease of a fixed amount of 5-FU (10 microg/mL) added to a solution of PBMC, after 2 hours incubation, expressed as nanogram per milliliter of 5-FU degraded per minute x 10(6) cells. Freshly prepared intact PBMC can degrade efficiently in vitro-added 5-FU. The assay consists of 3 steps: (1) PBMC isolation from peripheral blood, (2) PBMC incubation with 5-FU in vitro for different times, and (3) determination of 5-FU amount to calculate the degradation rate. 5-FU was analyzed by a Q Trap 2000 triple quadrupole/ion trap mass spectrometer in the multiple-reaction-monitoring modes. The chromatographic separation was accomplished using a C18 column with a run time of 16 minutes. By analyzing samples from 39 patients with no 5-FU toxicity, the mean 5-FU degradation rate was 1.85 +/- 0.50 ng x mL(-1) x min(-1) x 10(6) cells. The assessment of a test to measure 5-FU degradation rate in PBMC of patients before 5-FU administration could represent a prescreening method for evaluating the possible toxicity of this drug as an aid to set up a personalized medicine approach for each patient.
Lostia
Alfonso M
AM
Dipartimento di Scienze Biochimiche, UOD Diagnostica Molecolare Avanzata, Azienda Ospedaliera Sant'Andrea, II Facoltà di Medicina e Chirurgia, Sapienza Università di Roma, Rome, Italy.
Lionetto
Luana
L
Ialongo
Cristiano
C
Gentile
Giovanna
G
Viterbo
Antonella
A
Malaguti
Paola
P
Paris
Ida
I
Marchetti
Luca
L
Marchetti
Paolo
P
De Blasi
Antonio
A
Simmaco
Maurizio
M
D000368:Aged; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005472:Fluorouracil / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D012033:Refractory Period, Psychological; D053719:Tandem Mass Spectrometry
D005472:Fluorouracil