Development of an HPLC-DAD Method for the Extraction and Quantification of 5-Fluorouracil, Uracil, and 5-Fluorodeoxyuridin Monophosphate in Cells and Culture Media of Lactococcus lactis

Petros Mavromatis; Kyriaki Stampouli; Angeliki Vliora; Anna Mayilyan; Victoria Samanidou; Maria Touraki

doi:10.3390/separations9110376

journal article Open Access Nov 17, 2022

Development of an HPLC-DAD Method for the Extraction and Quantification of 5-Fluorouracil, Uracil, and 5-Fluorodeoxyuridin Monophosphate in Cells and Culture Media of Lactococcus lactis

Petros Mavromatis Kyriaki Stampouli Angeliki Vliora Anna Mayilyan Victoria Samanidou

Maria Touraki

Separations Vol. 9 No. 11 pp. 376 · MDPI AG

View at Publisher Save 10.3390/separations9110376

Abstract

The drug 5-fluorouracil (5-FU) is a common cancer chemotherapeutic, presenting toxicity. Mild toxicity is treated with administration of probiotics. The interaction of these probiotics with the drug may have a crucial effect on its therapeutic efficacy. In the present work, a method for the quantification of uracil, 5-FU, and its active metabolite 5-fluorodeoxyuridin monophosphate in cells and culture medium of the probiotic L. lactis is presented. Extraction using H2O containing 0.05% v/v formic acid (1:5 v/v) was followed by ammonium sulphate protein precipitation and SPE. Analysis was conducted in a Nucleosil column using a gradient of water, formic acid, and acetonitrile. Calibration curves were constructed for 5-FU (5–100 μg/mL), uracil (5–20 μg/mL), and 5-fluorodeoxyuridin monophosphate (5–20 μg/mL) using 5-bromouracil as the internal standard (R2 ≥ 0.999). The photodegradation of 5-FU amounted to 36.2% at 96 h. An administration experiment in the dark revealed a decline in 5-FU concentration in the culture media (88.3%) and uptake by the cells, while the uracil and FdUMP levels increased in the cells. The inactive metabolite 5,6 dihydrofluorouracil was detected in the medium. Our results demonstrate that uptake and metabolism of 5-FU in L. lactis cells leads to a decline in the drug levels and in the formation of both the active and the inactive metabolites of the drug.

Topics

No keywords indexed for this article. Browse by subject →

References

58

[1]

DeVita "A history of cancer chemotherapy" Cancer Res. (2008) 10.1158/0008-5472.can-07-6611

[2]

Dushinsky "The synthesis of 5-fluoropyrimidines" J. Am. Chem. Soc. (1957) 10.1021/ja01573a087

[3]

Shirasaka "Development history and concept of an oral anticancer agent S-1 (TS-1): Its clinical usefulness and future vistas" Jpn. J. Clin. Oncol. (2009) 10.1093/jjco/hyn127

[4]

5-Fluorouracil: mechanisms of action and clinical strategies

Daniel B. Longley, D. Paul Harkin, Patrick G. Johnston

Nature Reviews Cancer 2003 10.1038/nrc1074

[5]

5-FU promotes stemness of colorectal cancer via p53-mediated WNT/β-catenin pathway activation

Yong-Hee Cho, Eun Ji Ro, Jeong-Su Yoon et al.

Nature Communications 2020 10.1038/s41467-020-19173-2

[6]

Houghton "Thymineless death in colon carcinoma cells is mediated via fas signaling" Proc. Natl. Acad. Sci. USA (1997) 10.1073/pnas.94.15.8144

[7]

Diasio "Clinical Pharmacology of 5-Fluorouracil" Clin. Pharmacokinet. (1989) 10.2165/00003088-198916040-00002

[8]

Danesi "Improved analysis of 5-Fluorouracil and 5,6-dihydro-5-Fluorouracil by HPLC with diode array detection for determination of cellular dihydropyrimidine dehydrogenase activity and pharmacokinetic profiling" Ther. Drug Monit. (2005) 10.1097/01.ftd.0000162016.11148.1b

[9]

Lampropoulou "Fluoropyrimidine-induced toxicity and DPD deficiency. A case report of early onset, lethal capecitabine-induced toxicity and mini review of the literature. Uridine triacetate: Efficacy and safety as an antidote. Is it accessible outside USA?" J. Oncol. Pharm. Pract. (2020) 10.1177/1078155219865597

[10]

Ezzeldin "Dihydropyrimidine dehydrogenase deficiency, a pharmacogenetic syndrome associated with potentially life-threatening toxicity following 5-fluorouracil administration" Clin. Color. Cancer (2004) 10.3816/ccc.2004.n.018

[11]

Saif "Pharmacokinetically guided dose adjustment of 5-fluorouracil: A rational approach to improving therapeutic outcomes" J. Natl. Cancer Inst. (2009) 10.1093/jnci/djp328

[12]

Zhang "5-Fluorouracil induced dysregulation of the microbiome-gut-brain axis manifesting as depressive like behaviors in rats" Biochim. Et Biophys. Acta Mol. Basis Dis. (2020) 10.1016/j.bbadis.2020.165884

[13]

Ruotsalainen "Lactobacillus supplementation for diarrhoea related to chemotherapy of colorectal cancer: A randomised study" Br. J. Cancer (2007) 10.1038/sj.bjc.6603990

[14]

Rodriguez-Arrastia, M., Martinez-Ortigosa, A., Rueda-Ruzafa, L., Folch Ayora, A., and Ropero-Padilla, C. (2021). Probiotic Supplements on Oncology Patients’ Treatment-Related Side Effects: A Systematic Review of Randomized Controlled Trials. Int. J. Environ. Res. Public Health, 18. 10.3390/ijerph18084265

[15]

Feng "Oral Administration of Probiotics Reduces Chemotherapy-Induced Diarrhea and Oral Mucositis: A Systematic Review and Meta-Analysis" Front. Nutr. (2022) 10.3389/fnut.2022.823288

[16]

Baldwin "Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis" Nutr. Cancer (2010) 10.1080/01635580903407197

[17]

Darbandi "The effects of probiotics on reducing the colorectal cancer surgery complications: A periodic review during 2007–2017" Clin. Nutr. (2020) 10.1016/j.clnu.2019.11.008

[18]

Agraib "The effect of probiotics supplementation on the side effects of chemo radiotherapy for colorectal cancer: A literature review" Oncol. Radiother. (2020)

[19]

Singh "The complex mechanism of antimycobacterial action of 5-fluorouracil" Chem. Biol. (2015) 10.1016/j.chembiol.2014.11.006

[20]

Kyrila "Bisphenol A removal and degradation pathways in microorganisms with probiotic properties" J. Hazard. Mater. (2021) 10.1016/j.jhazmat.2021.125363

[21]

Multimodal interactions of drugs, natural compounds and pollutants with the gut microbiota

Anna E. Lindell, Maria Zimmermann-Kogadeeva, Kiran R. Patil

Nature Reviews Microbiology 2022 10.1038/s41579-022-00681-5

[22]

Sousa "The gastrointestinal microbiota as a site for the biotransformation of drugs" Int. J. Pharm. (2008) 10.1016/j.ijpharm.2008.07.009

[23]

Ritter "Bacterial Metabolism Affects the C. elegans Response to Cancer Chemotherapeutics" Cell (2017) 10.1016/j.cell.2017.03.046

[24]

Martinussen "Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis" J. Bacteriol. (1994) 10.1128/jb.176.21.6457-6463.1994

[25]

Rowland "Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis" Biochemistry (1997) 10.1021/bi971628y

[26]

Dobritzsch "Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil" EMBO J. (2001) 10.1093/emboj/20.4.650

[27]

Breda "A review of analytical methods for the determination of 5-fluorouracil in biological matrices" Anal. Bioanal. Chem. (2010) 10.1007/s00216-010-3633-8

[28]

Escoriaza "Simple and sensitive determination of 5-fluorouracil in plasma by high-performance liquid chromatography. Application to clinical pharmacokinetic studies" J. Chromatogr. B Biomed. Sci. Appl. (1999) 10.1016/s0378-4347(99)00446-6

[29]

Casale "Simultaneous HPLC determination of 5-fluorouracil and its metabolites in plasma of cancer patients" Biomedical chromatography: BMC (2002) 10.1002/bmc.181

[30]

Ackland "Simultaneous Determination of Dihydrofluorouracil and 5-Fluorouracil in Plasma by High-Performance Liquid Chromatography" Anal. Biochem. (1997) 10.1006/abio.1996.9943

[31]

Maring "A simple and sensitive fully validated HPLC-UV method for the determination of 5-fluorouracil and its metabolite 5,6-dihydrofluorouracil in plasma" Ther. Drug Monit. (2005) 10.1097/00007691-200502000-00006

[32]

Merlin "Determination of 5-Fluorouracil and Its Main Metabolites in Plasma by High-Performance Liquid Chromatography" J. Chromatogr. (1992)

[33]

Joulia "Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study" J. Chromatogr. B Biomed. Sci. Appl. (1997) 10.1016/s0378-4347(96)00518-x

[34]

Guerrieri "Solid-phase extraction of fluoropyrimidine derivatives on a copper-modified strong cation exchanger: Determination of doxifluridine, 5-fluorouracil and its main metabolites in serum by high-performance liquid chromatography with ultraviolet detection" J. Chromatogr. (1993) 10.1016/0378-4347(93)80423-2

[35]

Nassim "An HPLC method for the measurement of 5-fluorouracil in human plasma with a low detection limit and a high extraction yield" Int. J. Mol. Med. (2002)

[36]

Wickremsinhe "High sensitive assay employing column switching chromatography to enable simultaneous quantification of an amide prodrug of gemcitabine (LY2334737), gemcitabine, and its metabolite dFdU in human plasma by LC-MS/MS" J. Chromatogr. B Biomed. Sci. Appl. (2013)

[37]

Salvador "Simultaneous LC-MS-MS Analysis of Capecitabine and its Metabolites (5′-deoxy-5-fluorocytidine, 5′-deoxy-5-fluorouridine, 5-fluorouracil) After Off-Line SPE from Human Plasma" Chromatographia (2006) 10.1365/s10337-006-0799-5

[38]

Pandey "A Critical Review on Clinical Application of Separation Techniques for Selective Recognition of Uracil and 5-Fluorouracil" Indian J. Clin. Biochem. IJCB (2016) 10.1007/s12291-015-0482-4

[39]

Analytical method development and validation of anticancer agent, 5-fluorouracil, and its metabolites in biological matrices: An updated review

Nadhiratul-Farihin Semail, Aemi Syazwani Abdul Keyon, Bahruddin Saad et al.

Journal of Liquid Chromatography & Related Tec... 2020 10.1080/10826076.2020.1781654

[40]

Anderson "Gas Chromatographic—Mass Spectrometric Method for Routine Monitoring of 5-Fluorouracil in Plasma of Patients Receiving Low-Level Protracted Infusions" J. Chromatogr. B Biomed. Sci. Appl. (1992) 10.1016/0378-4347(92)80272-r

[41]

Marunaka "Determination of 5-Fluorouracil and Pyrimidine Bases in Plasma by Gas Chromatography-Chemical Ionization-Mass Fragmentography" J. Chromatogr. B Biomed. Sci. Appl. (1980) 10.1016/s0378-4347(00)84326-1

[42]

Remaud "Sensitive MS/MS-liquid chromatography assay for simultaneous determination of tegafur, 5-fluorouracil and 5-fluorodihydrouracil in plasma" J. Chromatogr. B Anal. Technol. Biomed. Life Sci. (2005) 10.1016/j.jchromb.2005.07.023

[43]

Wang "Development of a sensitive and selective LC-MS/MS method for the determination of alpha-fluoro-beta-alanine, 5-fluorouracil and capecitabine in human plasma" J. Chromatogr. B Biomed. Sci. Appl. (2009)

[44]

Holleran "LC–MS/MS Assay for the Quantitation of FdCyd and Its Metabolites FdUrd and FU in Human Plasma" J. Pharm. Biomed. Anal. (2016) 10.1016/j.jpba.2016.07.027

[45]

Bernal "Extraction Procedures for the HPLC determination of 5-fluorouracil in biological samples" J. Liq. Chromatogr. Relat. Technol. (1994) 10.1080/10826079408013184

[46]

Odagiri "Determination of 5-Fluorouracil in Plasma and Liver after Oral Administration of 5’-Deoxy-5-Fluorouridine using Gas Chromatography-Mass Spectrometry" J. Pharmacobio-Dyn. (1988) 10.1248/bpb1978.11.234

[47]

Ozawa "Cytosolic and microsomal activation of doxifluridine and tegafur to produce 5-fluorouracil in human liver" Cancer Chemother. Pharm. (2002) 10.1007/s00280-002-0528-1

[48]

Aldaz "Determination of 5-fluorouracil and its prodrug tegafur in plasma and tissue by high-performance liquid chromatography in a single injection: Validation for application in clinical pharmacokinetic studies" Ther. Drug Monit. (2003) 10.1097/00007691-200304000-00013

[49]

Wrightson "HPLC analysis of 5-FU and FdUMP in tissue and serum" Biochem. Biophys. Res. Commun. (1995) 10.1006/bbrc.1995.2693

[50]

Petrilli "Quantification of 5-FU in skin samples for the development of new delivery systems for topical cancer treatment" Die Pharm. (2018)

Showing 50 of 58 references

Metrics

7

Citations

58

References

Details

Published: Nov 17, 2022
Vol/Issue: 9(11)
Pages: 376
License: View

Authors

P

Petros Mavromatis