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Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

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Abstract

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

LC-MS/MS MRM chromatogram of mixed nucleoside and nucleotide standard

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References

  1. Hess JR, Greenberg NA (2012) The role of nucleotides in the immune and gastrointestinal systems: potential clinical applications. Nutr Clin Pract 27:281–294

    Article  Google Scholar 

  2. Cosgrove M (1998) Perinatal and infant nutrition. Nucleotides Nutrition 14:748–752

    Article  CAS  Google Scholar 

  3. Carver JD, Walker WA (1995) The role of nucleotides in human nutrition. J Nutr Biochem 6:58–72

    Article  CAS  Google Scholar 

  4. Commission of the European Communities (1991) Second addendum to the reports of the Scientific Committee for Food concerning the essential requirements of infant formulae and follow-up milks based on cow’s milk proteins and the minimum requirements for soya-based infant formulae and follow-up milks. http://ec.europa.eu/food/fs/sc/scf/reports/scf_reports_28.pdf. Accessed 16 June 2010

  5. Gill BD, Indyk HE (2007) Development and application of a liquid chromatographic method for analysis of nucleotides and nucleosides in milk and infant formulas. Int Dairy J 17:596–605

    Article  CAS  Google Scholar 

  6. Gill BD, Indyk HE (2007) Determination of nucleotides and nucleosides in milks and pediatric formulas: a review. J AOAC Int 90:1354–1364

    CAS  Google Scholar 

  7. Perrin C, Meyer L, Mujahid C, Blake CJ (2001) The analysis of 5′-mononucleotides in infant formulae by HPLC. Food Chem 74:245–253

    Article  CAS  Google Scholar 

  8. Inoue K, Obara R, Hino T, Oka H (2010) Development and application of an HILIC-MS/MS method for the quantitation of nucleotides in infant formula. J Agric Food Chem 58:9918–9924

    Article  CAS  Google Scholar 

  9. Gill BD, Indyk HE, Manley-Harris M (2012) Determination of total potentially available nucleosides in bovine, caprine, and ovine milk. Int Dairy J 24:40–43

    Article  CAS  Google Scholar 

  10. Leach JL, Baxter JH, Molitor BE, Ramstack MB, Masor ML (1995) Total potentially available nucleosides of human milk by stage of lactation. Am J Clin Nutr 61:1224–1230

    CAS  Google Scholar 

  11. Gill BD, Indyk HE, Manley-Harris M (2011) Determination of total potentially available nucleosides in bovine milk. Int Dairy J 21:34–41

    Article  CAS  Google Scholar 

  12. Inoue K, Obara R, Akiba T, Hino T, Oka H (2008) Determination of nucleotides in infant formula by ion-exchange liquid chromatography. J Agric Food Chem 56:6863–6867

    Article  CAS  Google Scholar 

  13. Ferreira IMPLVO, Mendes E, Gomes AMP, Faria MA, Ferreira MA (2001) The determination and distribution of nucleotides in dairy products using HPLC and diode array detection. Food Chem 74:239–244

    Article  CAS  Google Scholar 

  14. Oliveira C, Ferreira IMPLVO, Mendes E, Ferreira M (1999) Development and application of an HPLC/diode array methodology for determination of nucleotides in infant formulae and follow-up milks. J Liq Chromatogr Relat Technol 22:571–578

    Article  CAS  Google Scholar 

  15. Gill BD, Indyk HE, Kumar MC, Sievwright NK, Manley-Harris M (2010) A liquid chromatographic method for routine analysis of 5′-mononucleotides in pediatric formulas. J AOAC Int 93:966–973

    CAS  Google Scholar 

  16. Ren Y, Zhang J, Song X, Chen X, Li D (2011) Simultaneous determination of 5′-monophosphate nucleotides in infant formulas by HPLC-MS. J Chromatogr Sci 49(4):332–337

    Article  CAS  Google Scholar 

  17. Viñas P, Campillo N, Melgarejo GF, Vasallo MI, López-García I, Hernández-Córdoba M (2010) Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods. J Chromatogr A 1217(32):5197–5203

    Article  Google Scholar 

  18. Inoue K, Dowell D (2012) HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21. J AOAC Int 95(3):603–605

    Article  CAS  Google Scholar 

  19. EPA (1999) Guidelines establishing test procedures for the analysis of pollutants (40 CFR Appendix B to Part 136, Definition and procedure for the determination of the method detection limit). Environmental Protection Agency, Washington, DC

    Google Scholar 

  20. Youden WJ, Steiner EH (1975) Statistical manual of the AOAC. AOAC International, Arlington, VA

    Google Scholar 

  21. Gill BD, Indyk HE, Kumar MC, Sievwright NK, Manley-Harris M, Dowell D (2012) Analysis of 5′-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First Action 2011.20. J AOAC Int 95:599–602

    Article  CAS  Google Scholar 

  22. Asakawa Y, Tokida N, Ozawa C, Ishiba M, Tagaya O, Asakawa N (2008) Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry. J Chromatogr A 1198–1199:80–86

    Google Scholar 

  23. De Vijlder T, Boschmans J, Witters E, Lemière F (2011) Study on the loss of nucleoside mono-, di- and triphosphates and phosphorylated peptides to a metal-free LC-MS hardware. Int J Mass Spectrom 304:83–90

    Article  Google Scholar 

  24. Kim J, Camp DG, Smith RD (2004) Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry. J Mass Spectrom 39:208–215

    Article  CAS  Google Scholar 

  25. Liu S, Zhang C, Campbell JL, Zhang H, Yeung KKC, Han VKM, Lajoie GA (2005) Formation of phosphopeptide–metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection. Rapid Commun Mass Spectrom 19:2747–2756

    Article  CAS  Google Scholar 

  26. 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 28:1823–1830

    Article  CAS  Google Scholar 

  27. Yamaoka N, Kudo Y, Inazawa K, Inagawa S, Yasuda M, Mawatari K, 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 Chromatogr B 878:2054–2060

    Article  CAS  Google Scholar 

  28. Tuytten R, Lemière F, Witters E, Van Dongen 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 

  29. Applied Biosystems (2006) Degradation and impurity product profiling with LC/MS/MS: the use of phosphate-containing mobile phases and fast data analysis using LightSight™ software. http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_042526.pdf. Accessed 10 January 2013

  30. Dionex Corporation (2001) Extending the performance of LC/MS to nonvolatile buffers with the AQA. http://www.dionex.com/en-us/webdocs/4740-TN519_LPN1274.pdf. Accessed 10 January 2013

  31. Agilent Technologies (1998) The effect of nonvolatile buffers on the Agilent 1100 series LC/MSD. http://www.chem.agilent.com/Library/applications/5968-2690_016655.pdf. Accessed 10 January 2013

  32. Waters Corporation (1998) A phosphate buffer study: performance comparison of various LC-MS API interfaces using phosphate buffer containing mobile phase. http://www.waters.com/waters/library.htm?cid=511436&lid=1526095. Accessed 10 January 2013

  33. St Claire RL (2000) Positive ion electrospray ionization tandem mass spectrometry coupled to ion-pairing high-performance liquid chromatography with a phosphate buffer for the quantitative analysis of intracellular nucleotides. Rapid Commun Mass Spectrom 14:1625–1634

    Article  CAS  Google Scholar 

  34. Curtis M, Minier MA, Chitranshi P, Sparkman OD, Jones PR, Xue L (2010) Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C5H5O]+ and its in-source adducts. J Am Soc Mass Spectrom 21:1371–1381

    Article  CAS  Google Scholar 

  35. Sullivan D (2012) Infant formula and adult/pediatric nutritional methods approved First Action using the AOAC voluntary consensus standards process. J AOAC Int 95:287–290

    Article  CAS  Google Scholar 

  36. Horwitz W (2002) AOAC requirements for single laboratory validation of chemical methods. Draft 2002-11-07. AOAC International, Gaithersburg, MD

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Acknowledgments

The financial assistance of Fonterra Co-operative Group Limited and the Tertiary Education Commission in providing an Enterprise Scholarship is gratefully acknowledged.

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Authors and Affiliations

  1. Fonterra Co-operative Group Ltd., P.O. Box 7, Waitoa, 3380, New Zealand

    Brendon D. Gill & Harvey E. Indyk

  2. University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand

    Brendon D. Gill & Merilyn Manley-Harris

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  1. Brendon D. Gill
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  2. Harvey E. Indyk
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  3. Merilyn Manley-Harris
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Correspondence to Brendon D. Gill.

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Gill, B.D., Indyk, H.E. & Manley-Harris, M. Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 405, 5311–5319 (2013). https://doi.org/10.1007/s00216-013-6935-9

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  • DOI: https://doi.org/10.1007/s00216-013-6935-9

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