Skip to main content

Advertisement

SpringerLink
Log in

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

An analytical method coupling online solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed to quantify 16 endogenous nucleoside mono- and triphosphates in cellular samples. Separation was achieved on a porous graphitic carbon (PGC) column without ion-pairing agent in the mobile phase. Low levels of the ion-pairing agent diethylamine (DEA) added to the reconstitution solution were necessary to prevent peak tailing of nucleoside triphosphates. The mass spectrometer, a triple quadrupole with an electrospray ionisation source, was operated in positive mode. Two multiple reaction monitoring (MRM) segments were programmed, each an internal standard. Extraction and separation of nucleoside mono- and triphosphates were obtained within 20 min. The total duration of a single run was 37 min. Calibration curves, performed with labelled nucleotides added to the sample matrix, ranged from 0.29 to 18.8 pmol injected for deoxyribonucleotides and from 3.9 to 3,156 pmol for ribonucleotides. Accuracy did not deviate more than −14.6 and 10.2 % from nominal values for all compounds at all levels. CV results were all lower than 17.0 % for the LLOQ level and 14.6 % for the other levels. Quality control (QC) samples were also in agreement with acceptance criteria, except for the lower QC of GMP. Ion suppression, matrix effect, extraction recoveries and stability were assessed. After validation, the method was applied to the evaluation of the effects of gemcitabine and hydroxyurea on nucleotide pools in Messa cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B (2011) Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell 145:435–446

    Article  CAS  Google Scholar 

  2. Jansen RS, Rosing H, de Wolf CJ, Beijnen JH (2007) Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry. Rapid Commun Mass Spectrom 21:4049–4059

    Article  CAS  Google Scholar 

  3. Cohen S, Jordheim LP, Megherbi M, Dumontet C, Guitton J (2010) Liquid chromatography methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: a review. J Chrom B 878:1912–1928

    Article  CAS  Google Scholar 

  4. Zhang W, Tan S, Paintsil E, Dutschman GE, Gullen EA, Chu E, Cheng YC (2011) Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique. Biochem Pharmacol 82:411–417

    Article  CAS  Google Scholar 

  5. Yamaoka N, Kudo Y, Inazawa K, Inagawa S, Yasuda M, Mawatari KI, Nakagomi K, Kaneko K (2010) Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry. J Chrom B 878:2054–2060

    Article  CAS  Google Scholar 

  6. Zhao Y, Liu G, Liu Y, Yuan L, Hawthorne D, Shen JX, Guha M, Aubry A (2013) Improved ruggedness of an ion-pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells. Rapid Commun Mass Spectrom 27:481–488

    Article  CAS  Google Scholar 

  7. Jansen RS, Rosing H, Schellens JHM, Beijnen JH (2009) Retention studies of 2′-2′-difluorodeoxycytidine and 2′-2′-difluorodeoxyuridine nucleosides and nucleotides on porous graphitic carbon: development of a liquid chromatography–tandem mass spectrometry method. J Chromatogr A 1216:3168–3174

    Article  CAS  Google Scholar 

  8. Peifer S, Schneider K, Nürenberg G, Volmer DA, Heinzle E (2012) Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS. Anal Bioanal Chem 404:2295–2305

    Article  CAS  Google Scholar 

  9. Xing J, Apedo A, Tymiak A, Zhao N (2004) Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry. Rapid Commun Mass Spectrom 18:1599–1606

    Article  CAS  Google Scholar 

  10. Tuytten R, Lemière F, Van Dongen W, Esmans EL, Slegers H (2002) Short capillary ion-pair high performance liquid chromatography coupled with electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono-, di- and triphosphates. Rapid Commun Mass Spectrom 16:1205–1215

    Article  CAS  Google Scholar 

  11. Wang J, Lin T, Lai J, Cai Z, Yang MS (2009) Analysis of adenosine phosphates in HepG-2 cell by a HPLC–ESI-MS system with porous graphitic carbon as stationary phase. J Chrom B 877:2019–2024

    Article  CAS  Google Scholar 

  12. Cordell RL, Hill SJ, Ortori CA, Barrett DA (2008) Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry. J Chrom B 871:115–124

    Article  CAS  Google Scholar 

  13. Cohen S, Megherbi M, Jordheim LP, Lefebvre I, Perigaud C, Dumontet C, Guitton J (2009) Simultaneous analysis of eight nucleoside triphosphates in cell lines by liquid chromatography coupled with tandem mass spectrometry. J Chrom B 877:3831–3840

    Article  CAS  Google Scholar 

  14. Darque A, Valette G, Rousseau F, Wang LH, Sommadossi JP, Zhou XJ (1999) Quantitation of intracellular triphosphate of emtricitabine in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 43(9):2245–2250

    CAS  Google Scholar 

  15. Font E, Rosario O, Santana J, García H, Sommadossi JP, Rodriguez JF (1999) Determination of zidovudine triphosphate intracellular concentrations in peripheral blood mononuclear cells from human immunodeficiency virus-infected individuals by tandem mass spectrometry. Antimicrob Agents Chemother 43(12):2964–2968

    CAS  Google Scholar 

  16. Robbins BL, Waibel BH, Fridland A (1996) Quantitation of intracellular zidovudine phosphates by use of combined cartridge-radioimmunoassay methodology. Antimicrob Agents Chemother 40(11):2651–2654

    CAS  Google Scholar 

  17. Chen L, Wang H, Zeng Q, Xu Y, Sun L, Xu H, Ding L (2009) Online coupling of solid-phase extraction to liquid chromatography—a review. J Chromatogr Sci 47:614–623

    Article  CAS  Google Scholar 

  18. Shah VP, Midha KK, Findlay JWA, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A (2000) Bioanalytical method validation—a revisit with a decade of progress. Pharm Res 17(12):1551–1557

    Article  CAS  Google Scholar 

  19. Kuklenyik Z, Martin A, Pau CP, Holder A, Youngpairoj AS, Zheng Q, Cong ME, Garcia-Lerma JG, Heneine W, Pirkle JL, Barr JR (2009) On-line coupling of anion exchange and ion-pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors. J Chrom B 877:3659–3666

    Article  CAS  Google Scholar 

  20. Vela JE, Olson LY, Huang A, Fridland A, Ray AS (2007) Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2-deoxyadenosine triphosphate by ion-pairing LC/MS/MS. J Chrom B 848:335–343

    Article  CAS  Google Scholar 

  21. Liu DY, Cojocaru L (2010) The determination of a tetra-phosphate compound in rat plasma. LC/MS/MS AAPS Annual Meeting and Exposition, New Orleans, LA, November 2010

    Google Scholar 

  22. Tuytten R, Lemière F, Witters E, Van Dongena W, Slegers H, Newton RP, Van Onckelen H, Esmans EL (2006) Stainless steel electrospray probe: a dead end for phosphorylated organic compounds? J Chromatogr A 1104:209–221

    Article  CAS  Google Scholar 

  23. Jansen RS, Rosing H, Schellens JHM, Beijnen JH (2011) Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs. Mass Spec Rev 30:321–343

    Article  CAS  Google Scholar 

  24. Wakamatsu A, Morimoto K, Shimizu M, Kudoh S (2005) A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry. J Sep Sci 28(14):1823–1830

    Article  CAS  Google Scholar 

  25. Antonopoulos A, Favetta P, Helbert W, Lafosse J (2007) On-line liquid chromatography–electrospray ionisation mass spectrometry for κ-carrageenan oligosaccharides with a porous graphitic carbon column. J Chromatogr A 1147:37–41

    Article  CAS  Google Scholar 

  26. Vainchtein LD, Rosing H, Schellens JHM, Beijnen JH (2010) A new validated HMPL-MS/MS method for the simultaneous determination of the anti-cancer agent capecitabine and its metabolites: 5′-deoxy-5-fluorouridine, 5-fluorouracil and 5′-fluorodihydrouracil, in human plasma. Biomed Chromatogr 24:374–386

    CAS  Google Scholar 

  27. Yamaoka N, Inazawa K, Inagawa S, Yasuda M, Mawatari K, Nakagomi K, Fujimori S, Yamada Y, Kaneko K (2011) Simultaneous determination of purine and pyrimidine metabolites in hprt-deficient cell lines. Nucleosides Nucleotides Nucleic Acids 30:1256–1259

    Article  CAS  Google Scholar 

  28. Petroselli G, Erra-Balsells R, Cabrerizo FM, Lorente C, Capparelli AL, Braun AM, Oliveros E, Thomas AH (2007) Photosensitization of 2′-deoxyadenosine-5′-monophosphate by pterin. Org Biomol Chem 5:2792–2799

    Article  CAS  Google Scholar 

  29. Yang FQ, Li DQ, Feng K, Hu DJ, Li SP (2010) Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography–mass spectrometry. J Chromatogr A 1217:5501–5510

    Article  CAS  Google Scholar 

  30. Coulier L, Gerritsen H, van Kampen JJA, Reedijk ML, Luider TM, Osterhaus ADME, Gruters RA, Brüll L (2011) Comprehensive analysis of the intracellular metabolism of antiretroviral nucleosides and nucleotides using liquid chromatography–tandem mass spectrometry and method improvement by using ultra performance liquid chromatography. J Chrom B 879:2772–2782

    Article  CAS  Google Scholar 

  31. Fung EN, Cai Z, Burnette TC, Sinhababu AK (2001) Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography–tandem mass spectrometry. J Chrom B 754:285–295

    Article  CAS  Google Scholar 

  32. Pruvost A, Théodoro F, Agrofoglio L, Negredo E, Bénech H (2008) Specificity enhancement with LC-positive ESI-MS/MS for the measurement of nucleotides: application to the quantitative determination of carbovir triphosphate, lamivudine triphosphate and tenofovir diphosphate in human peripheral blood mononuclear cells. J Mass Spectrom 43:224–233

    Article  CAS  Google Scholar 

  33. Quinn R, Basanta-Sanchez M, Rose RE, Fabris D (2013) Direct infusion analysis of nucleotide mixtures of very similar or identical elemental composition. J Mass Spectrom 48:703–712

    Article  CAS  Google Scholar 

  34. Shi G, Wu JT, Li Y, Geleziunas R, Gallagher K, Emm T, Olah T, Unger S (2002) Novel direct detection method for quantitative determination of intracellular nucleoside triphosphates using weak anion exchange liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 16:1092–1099

    Article  CAS  Google Scholar 

  35. Van Moorsel CJA, Bergman AM, Veerman G, Voorn DA, Ruiz van Haperen VWT, Kroep JR, Pinedo HM, Peters GJ (2000) Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumor and leukaemia cell lines. Biochim Biophys Acta 1474:5–12

    Article  Google Scholar 

  36. Golkar SO, Czene S, Gokarakonda A, Haghdoost S (2013) Intracellular deoxyribonucleotide pool imbalance and DNA damage in cells treated with hydroxyurea, an inhibitor of ribonucleotide reductase. Mutagenesis 28:653–660

    Article  CAS  Google Scholar 

  37. Hakansson P, Hofer A, Thelander L (2006) Regulation of mammalian ribonucleotides reduction and dNTP pools after DNA damage and in resting cells. J Biol Chem 281:7834–7841

    Article  CAS  Google Scholar 

  38. Bianchi V, Pontis E, Reichard P (1986) Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis. J Biol Chem 261(34):16037–16042

    CAS  Google Scholar 

  39. Collins A, Oates D (1987) Hydroxyurea: effects on deoxyribonucleotide pool size correlated with effects on DNA repair on mammalian cells. Eur J Biochem 169:299–305

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank E. Cros for providing the Messa cells, and B. Vignal and S. Coste for their technical support.

Author information

Authors and Affiliations

  1. Laboratoire de Biochimie et Toxicologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, 69495, Pierre-Bénite, France

    Christelle Machon & Jérôme Guitton

  2. Laboratoire de Chimie Analytique, Université de Lyon, Université Lyon 1, ISPB Faculté de pharmacie, 69008, Lyon, France

    Christelle Machon & Lars Petter Jordheim

  3. INSERM U1052 – CNRS 5286, Centre de Recherche en Cancérologie de Lyon, 69000, Lyon, France

    Lars Petter Jordheim & Charles Dumontet

  4. Institut des Biomolécules Max Mousseron, UMR 5247 CNRS-UM1-UM2, Université Montpellier 2, 34095, Montpellier Cedex 05, France

    Jean-Yves Puy & Isabelle Lefebvre

  5. Laboratoire de Toxicologie, Université Lyon 1, ISPB, Faculté de pharmacie, 69008, Lyon, France

    Jérôme Guitton

Authors
  1. Christelle Machon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lars Petter Jordheim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Yves Puy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Isabelle Lefebvre
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Charles Dumontet
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jérôme Guitton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jérôme Guitton.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Machon, C., Jordheim, L.P., Puy, JY. et al. Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 406, 2925–2941 (2014). https://doi.org/10.1007/s00216-014-7711-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-7711-1

Keywords

Search

Navigation