Indian Journal of Clinical Biochemistry

, Volume 31, Issue 1, pp 3–12 | Cite as

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

  • Khushaboo PandeyEmail author
  • Rama Shankar Dubey
  • Bhim Bali Prasad
Review Article


The most important objectives that are frequently found in bio-analytical chemistry involve applying tools to relevant medical/biological problems and refining these applications. Developing a reliable sample preparation step, for the medical and biological fields is another primary objective in analytical chemistry, in order to extract and isolate the analytes of interest from complex biological matrices. Since, main inborn errors of metabolism (IEM) diagnosable through uracil analysis and the therapeutic monitoring of toxic 5-fluoruracil (an important anti-cancerous drug) in dihydropyrimidine dehydrogenase deficient patients, require an ultra-sensitive, reproducible, selective, and accurate analytical techniques for their measurements. Therefore, keeping in view, the diagnostic value of uracil and 5-fluoruracil measurements, this article refines several analytical techniques involved in selective recognition and quantification of uracil and 5-fluoruracil from biological and pharmaceutical samples. The prospective study revealed that implementation of molecularly imprinted polymer as a solid-phase material for sample preparation and preconcentration of uracil and 5-fluoruracil had proven to be effective as it could obviates problems related to tedious separation techniques, owing to protein binding and drastic interferences, from the complex matrices in real samples such as blood plasma, serum samples.


Bio-analytical applications Dihydropyrimidine dehydrogenase (DPD) 5-Fluorouracil Separation techniques Therapeutic drug monitoring Uracil 



The author greatly acknowledged to her funding Agency University Grant Commission-Dr. D. S. Kothari Post Doctoral Fellowship, New Delhi (Grant No. S-25/24198 DSK-PDF) for providing financial support, Prof. S. S. Pandey, for providing valuable suggestions, and the Head of Department of Biochemistry, Faculty of Sciences, BHU, Varanasi, for pursuing the project.

Conflict of interest

The author declared no competing interests.


  1. 1.
    Coudore F, Roche D, Lefeuvre S, Faussot D, Billaud EM, Loriot MA, et al. Validation of an ultra-high performance liquid chromatography tandem mass spectrometric method for quantifying uracil and 5, 6-dihydrouracil in human plasma. J Chromatogr Sci. 2012;00:1–8.Google Scholar
  2. 2.
    Déporte R, Amiand M, Moreau A, Charbonnel C, Campion L. High performance liquid chromatographic assay with UV detection for measurement of dihydrouracil/uracil ratio in plasma. J Chromatogr B. 2006;834:170–7.CrossRefGoogle Scholar
  3. 3.
    Dolegowska B, Ostapowicz A, Stanczyk-Dunaj M, Blogowski W. Spectrophotometric methods as a novel screening approach for analysis of dihydropyrimidine dehydrogenase activity before treatment with 5-fluorouracil chemotherapy. J Physiol Pharmacol. 2012;63:411–21.PubMedGoogle Scholar
  4. 4.
    Hall AJ, Manesiotis P, Mossing JT, Sellergren B. Molecularly imprinted polymers (MIPs) against uracils: functional monomer design, monomer-template interactions in solution and MIP performance in chromatography. Mater Res Soc Symp Proc. 2002;723:11–5.Google Scholar
  5. 5.
    Takeuchi T, Hishiya T. Molecular imprinting of proteins emerging as a tool for protein recognition. Org Biomol Chem. 2008;6:2459–67.CrossRefPubMedGoogle Scholar
  6. 6.
    Chen L, Xu S, Li J. Recent advances in molecular imprinting technology: current status, challenges and highlighted applications. Chem Soc Rev. 2011;40:2922–42.CrossRefPubMedGoogle Scholar
  7. 7.
    Cervini P, Cavalheiro ÉTG. Strategies for preparation of molecularly imprinted polymers modified electrodes and their application in electroanalysis: a review. Anal Lett. 2012;45:297–313.CrossRefGoogle Scholar
  8. 8.
    Jiang H, Lu J, Ji J. Circadian rhythm of dihydrouracil/uracil ratios in biological fluids: a potential biomarker for dihydropyrimidine dehydrogenase levels. Br J Pharmacol. 2004;141:616–23.PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Bates CD, Watson DG, Willmott N, Logan H, Goldberg J. The analysis of 5-fluorouracil in human plasma by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS) with stable isotope dilution. J Pharm Biomed Anal. 1991;9:19–21.CrossRefPubMedGoogle Scholar
  10. 10.
    Kubo M, Sasabe H, Shimizu T. Highly sensitive method for the determination of 5-fluorouracil in biological samples in the presence of 2′-deoxy-5-fluorouridine by gas chromatography-mass spectrometry. J Chromatogr. 1991;564:137–45.CrossRefPubMedGoogle Scholar
  11. 11.
    Prasad BB, Tiwari K, Singh M, Sharma PS, Patel AK, Srivastava S. Ultratrace analysis of uracil and 5-fluorouracil by molecularly imprinted polymer brushes grafted to silylated solid-phase microextraction fiber in combination with complementary molecularly imprinted polymer-based sensor. Biomed Chromatogr. 2009;23:499–509.CrossRefPubMedGoogle Scholar
  12. 12.
    Jiang H, Jiang J, Hu P, Hu Y. Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 2002;769:169–76.CrossRefGoogle Scholar
  13. 13.
    Svobaite R, Solassol I, Pinguet F, Ivanauskas L, Brès J, Bressolle FM. HPLC with UV or mass spectrometric detection for quantifying endogenous uracil and dihydrouracil in human plasma. Clin Chem. 2008;54:1463–72.CrossRefPubMedGoogle Scholar
  14. 14.
    César IC, Cunha-Júnior GF, Duarte Byrro RM, Vaz Coelho LG, Pianetti GA. A rapid HPLC-ESI-MS/MS method for determination of dihydrouracil/uracil ratio in plasma: evaluation of toxicity to 5-flurouracil in patients with gastrointestinal cancer. Ther Drug Monit. 2012;34:59–66.CrossRefPubMedGoogle Scholar
  15. 15.
    Lacassai E, Marquet P, Gaulier JM, Dreyfuss M-F, Lachatre F. Sensitive and specific multiresidue methods for the determination of pesticides of various classes in clinical and forensic toxicology. Forensic Sci Int. 2001;121:116–25.CrossRefGoogle Scholar
  16. 16.
    Smith RA, Tibbels TS, Smith TE, Cohen SM. Quantification of uracil in rodent diet. Anal Biochem. 1991;195:375–7.CrossRefPubMedGoogle Scholar
  17. 17.
    Paw B, Misztal G. Chromatographic analysis (TLC) of uracil arabinoside, uracil, and cytosine in human plasma. Acta Pol Pharma. 1995;52:455–7.Google Scholar
  18. 18.
    Beltran J, Lopez FJ, Hernandez F. Solid-phase microextraction in pesticide residue analysis. J Chromatogr A. 2000;885:389–404.CrossRefPubMedGoogle Scholar
  19. 19.
    Krutz LJ, Senseman SA, Sciumbato AS. Solid-phase microextraction for herbicide determination in environmental samples. J Chromatogr A. 2003;999:103–21.CrossRefPubMedGoogle Scholar
  20. 20.
    O’Mahony J, Molinelli A, Nolan K, Smyth MR, Mizaikoff B. Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity. Biosens Bioelectron. 2005;20:1884–93.CrossRefPubMedGoogle Scholar
  21. 21.
    Yano K, Tanabe K, Takeuchi T, Matsui J, Ikebukoro K, Karube I. Molecularly imprinted polymers which mimic multiple hydrogen bonds between nucleotide bases. Anal Chim Acta. 1998;363:111–7.CrossRefGoogle Scholar
  22. 22.
    Sellegren B. Imprinted polymers with memory for small molecules, proteins, or crystals. Angew Chem Int Ed. 2000;39:1031–7.CrossRefGoogle Scholar
  23. 23.
    Xia SL, Wang HY, Sun H, Liu YK, Cao SK. Preparation of molecularly imprinted copolymer membrane for uracil recognition. Chin Chem Lett. 2003;14:793–6.Google Scholar
  24. 24.
    Xia SL, Wang HY, Kobayashi T. Phase inversion molecularly imprinting of uracil targeted membranes made of polyacrylonitrile copolymers having methacrylic acid and acrylic acid segments for recognition and permselective binding. Mater Res Soc Symp Proc. 2004;787:103–7.Google Scholar
  25. 25.
    Lin CI, Joseph AK, Chang CK, Lee YD. Synthesis and photoluminescence of molecularly imprinted polymers appended onto CdSe/ZnS core-shells. Biosens Bioelectron. 2004;20:127–31.CrossRefPubMedGoogle Scholar
  26. 26.
    Wang HY, Xia SL, Sun H, Liu YK, Cao SK, Kobayashi T. Molecularly imprinted copolymer membranes functionalized by phase inversion imprinting for uracil recognition and permselective binding. J Chromatogr B. 2004;804:127–34.CrossRefGoogle Scholar
  27. 27.
    Sreenivasan K. Synthesis and evaluation of molecularly imprinted polymers for nucleic acid bases using aniline as monomer. React Funct Polym. 2007;67:859–64.CrossRefGoogle Scholar
  28. 28.
    Kobayashi T, Leong SS, Zhang Q. Using polystyrene-co-maleic acid molecularly imprinted membranes prepared in supercritical carbon dioxide. J Appl Polym Sci. 2008;108:757–68.CrossRefGoogle Scholar
  29. 29.
    Prasad BB, Srivastava S, Tiwari K, Sharma PS. Development of uracil and 5-fluorouracil sensors based on molecularly imprinted polymer-modified hanging mercury drop electrode. Sens Mater. 2009;21:291–306.Google Scholar
  30. 30.
    Abraham J, Mathew B. Facile synthesis and characterization of vinyl functionalized multiwalled carbon nanotubes based molecular imprinted polymer for uracil. Int J Chem Tech Res. 2014;6:1462–71.Google Scholar
  31. 31.
    Ackland SP, Garg MB, Dunstan RH. Simultaneous determination of dihydrofluorouracil and 5-fluorouracil in plasma by high-performance liquid chromatography. Anal Biochem. 1997;246:79–85.CrossRefPubMedGoogle Scholar
  32. 32.
    Joulia JM, Pinguet F, Grosse PY, Astre C, Bressolle F. 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;692:427–35.CrossRefPubMedGoogle Scholar
  33. 33.
    Micoli G, Turci R, Arpellini M, Minoia C. Determination of 5-fluorouracil in environmental samples. J Chromatogr B. 2001;750:25–32.CrossRefGoogle Scholar
  34. 34.
    Nassim MA, Shirazi FH, Cripps CM, Veerasinghan S, Molepo MJ, Obrocea M, et al. 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;10:513–6.PubMedGoogle Scholar
  35. 35.
    Mahnik SN, Rizovski B, Fuerhacker M, Mader RM. Determination of 5-fluorouracil in hospital effluents. Anal Bioanal Chem. 2004;380:31–5.CrossRefPubMedGoogle Scholar
  36. 36.
    Alsarra IA, Alarifi MN. Validated liquid chromatographic determination of 5-fluorouracil in human plasma. J Chromatogr B. 2004;804:435–9.CrossRefGoogle Scholar
  37. 37.
    Stashkevich MA, Khomutov EV, Shatova OP, Dumanskiĭ IV, Zinkovich II. Distribution of 5-fluorouracil between lymphocytes and blood plasma. Ukr Biokhim Zh. 2013;85:94–7.Google Scholar
  38. 38.
    Nakayama Y, Matsumoto K, Inoue Y, Katsuk T, Kadowaki K, Shibao K, et al. Correlation between the urinary dihydrouracil-uracil ratio and the 5-FU plasma concentration in patients treated with oral 5-FU analogs. Anticancer Res. 2006;26:3983–8.PubMedGoogle Scholar
  39. 39.
    Mullot JU, Karolak S, Fontova A, Huart B, Levi Y. Development and validation of a sensitive and selective method using GC/MS-MS for quantification of 5-fluorouracil in hospital wastewater. Anal Bioanal Chem. 2009;394:2203–12.CrossRefPubMedGoogle Scholar
  40. 40.
    Ciccolini J, Mercier C, Blachon MF, Favre R, Durand A, Lacarelle B. A simple and rapid high-performance liquid chromatographic (HPLC) method for 5-fluorouracil (5-FU) assay in plasma and possible detection of patients with impaired dihydropyrimidine dehydrogenase (DPD) activity. J Clin Pharm Ther. 2004;29:307–15.CrossRefPubMedGoogle Scholar
  41. 41.
    Remaud G, Boisdron-Celle M, Morel A, Gamelin A. 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;824:153–60.CrossRefGoogle Scholar
  42. 42.
    Salvador A, Millerioux L, Renou A. 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;63:609–15.CrossRefGoogle Scholar
  43. 43.
    Kosovec JE, Egorin MJ, Gjurich S, Beumer JH. Quantitation of 5-fluorouracil (5-FU) in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2008;22:224–30.CrossRefPubMedGoogle Scholar
  44. 44.
    Kovalova L, Mc Ardell CS, Hollender J. Challenge of high polarity and low concentrations in analysis of cytostatics and metabolites in wastewater by hydrophilic interaction chromatography/tandem mass spectrometry. J Chromatogra A. 2009;1216:1100–8.CrossRefGoogle Scholar
  45. 45.
    Licea-Perez H, Wang S, Bowen C. 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 Analyt Technol Biomed Life Sci. 2009;877:1040–6.CrossRefPubMedGoogle Scholar
  46. 46.
    Carli D, Honorat M, Cohen S, Megherbi M, Vignal B, Dumontet C, et al. Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:2937–44.CrossRefPubMedGoogle Scholar
  47. 47.
    Alanazi FK, Yassin AE, El-Badry M, Mowafy HA, Alsarra IA. Validated high-performance liquid chromatographic technique for determination of 5-fluorouracil: applications to stability studies and simulated colonic media. J Chromatogr Sci. 2009;47:558–63.CrossRefPubMedGoogle Scholar
  48. 48.
    Montange D, Bérard M, Demarchi M, Muret P, Piédoux S, Kantelip JP, et al. An APCI LC-MS/MS method for routine determination of capecitabine and its metabolites in human plasma. J Mass Spectrom. 2010;45:670–7.PubMedGoogle Scholar
  49. 49.
    Chen J, Zhou M. Determination of eniluracil and 5-fluorouracil in human plasma by LC-MS/MS. Bioanalysis. 2010;2:2011–7.CrossRefPubMedGoogle Scholar
  50. 50.
    Liu K, Zhong D, Zou H, Chen X. Determination of tegafur, 5-fluorouracil, gimeracil and oxonic acid in human plasma using liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2010;52:550–6.CrossRefPubMedGoogle Scholar
  51. 51.
    Tsume Y, Provoda CJ, Amidon GL. The achievement of mass balance by simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil, 5-dihydrouracil, α-fluoro-β-ureidopropionate, α-fluoro-β-alanine using LC-MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879:915–20.PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Serdar MA, Sertoğlu E, Uyanık M, Tapan S, Akın O, Cihan M. Determination of 5-fluorouracil and dihydrofluorouracil levels by using a liquidchromatography-tandem mass spectrometry method for evaluation of dihydropyrimidine dehydrogenase enzyme activity. Cancer Chemother Pharmacol. 2011;68:525–9.CrossRefPubMedGoogle Scholar
  53. 53.
    Yang CG, Ciccolini J, Blesius A, Dahan L, Bagarry-Liegey D, Brunet C, et al. DPD-based adaptive dosing of 5-FU in patients with head and neck cancer: impact on treatment efficacy and toxicity. Cancer Chemother Pharmacol. 2011;67:49–56.CrossRefPubMedGoogle Scholar
  54. 54.
    Peer CJ, McManus TJ, Hurwitz HI, Petros WP. Development and utilization of a combined LC-UV and LC-MS/MS method for the simultaneous analysis of tegafur and 5-fluorouracil in human plasma to support a phase I clinical study of oral UFT®/leucovorin. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;898:32–7.CrossRefPubMedGoogle Scholar
  55. 55.
    Deenen MJ, Rosing H, Hillebrand MJ, Schellens JH, Beijnen JH. Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;913–914:30–40.CrossRefPubMedGoogle Scholar
  56. 56.
    Büchel B, Rhyn P, Schürch S, Bühr C, Amstutz U, Largiadèr CR. LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients. Biomed Chromatogr. 2013;27:7–16.CrossRefPubMedGoogle Scholar
  57. 57.
    Büchel B, Sistonen J, Joerger M, Aebi Y, Schürch S, Largiadèr CR. Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients. Clin Chem Lab Med. 2013;51:1681–8.CrossRefPubMedGoogle Scholar
  58. 58.
    Cavrini V, Bonazzi D, Di Pietra AM. Analysis of flucytosine dosage forms by derivative UV spectroscopy and liquid chromatography. J Pharm Biomed Anal. 1991;9:401–7.CrossRefPubMedGoogle Scholar
  59. 59.
    Guerrieri A, Palmisano F, Zambonin PG, De Lena M, Lorusso V. 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;617:71–7.CrossRefPubMedGoogle Scholar
  60. 60.
    Bonazzi D, Andrisano V, Gatti R, Cavrini V. Analysis of pharmaceutical creams: a useful approach based on solid-phase extraction (SPE) and UV spectrophotometry. J Pharm Biomed Anal. 1995;13:1321–9.CrossRefPubMedGoogle Scholar
  61. 61.
    Kugimiya A, Mukawa T, Takeuchi T. Synthesis of 5-fluorouracil imprinted polymers with multiple hydrogen-bonding interactions. Analyst. 2001;126:772–4.CrossRefPubMedGoogle Scholar
  62. 62.
    Puoci F, Iemma F, Cirillo G, Picci N, Matricardi P, Alhaique F. Molecularly imprinted polymers for 5-fluorouracil release in biological fluids. Molecules. 2007;12:805–14.CrossRefPubMedGoogle Scholar
  63. 63.
    Singh B, Chauhan N. Preliminary evaluation of molecular imprinting of 5-fluorouracil within hydrogels within hydrogels for use as drug delivery systems. Acta Biomater. 2008;4:1244–54.CrossRefPubMedGoogle Scholar
  64. 64.
    Cirillo G, Iemma F, Puoci F, Parisi DI, Curcio M, Spizziri UG, et al. Imprinted hydrophilic nanospheres as drug delivery systems for 5-fluorouracil sustained release. J Drug Targeting. 2009;17:72–7.CrossRefGoogle Scholar
  65. 65.
    Huynh TP, Pieta P. D’ Souza F, Kutner W. Molecularly imprinted polymer for recognition of 5-fluorouracil by RNA-type nucleobase pairing. Anal Chem. 2013;85:8304–12.CrossRefPubMedGoogle Scholar

Copyright information

© Association of Clinical Biochemists of India 2015

Authors and Affiliations

  • Khushaboo Pandey
    • 1
    Email author
  • Rama Shankar Dubey
    • 2
  • Bhim Bali Prasad
    • 3
  1. 1.Department of Biochemistry, Faculty of ScienceBanaras Hindu UniversityVaranasiIndia
  2. 2.Tilkamanjhi Bhagalpur UniversityBhagalpurIndia
  3. 3.Analytical Division, Chemistry Department, Faculty of ScienceBanaras Hindu UniversityVaranasiIndia

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