Skip to main content
SpringerLink
Log in

Certification of nitrate in spinach powder reference material SPIN-1 by high-precision isotope dilution GC–MS

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

Abstract

A high-precision exact-matching quadruple isotope dilution method (ID4MS) was employed for the quantitation of nitrate in an air-dried spinach powder Certified Reference Material (CRM). The analyte was extracted in hot water following addition of 15NO\({}_{3}^{-}\) internal standard. The blend was then treated with sulfamic acid to remove nitrite and with triethyloxonium tetrafluoroborate to promote aqueous conversion of nitrate into volatile EtONO2. The derivative was analyzed by headspace GC–MS with 3-min elution time. The method performance was validated with a series of tests which demonstrated adequate selectivity and ruggedness. This method supported the development of novel SPIN-1 CRM giving a modest contribution to its uncertainty (uchar = 0.85%). With respect to previous attempts, the SPIN-1 was proven stable, homogeneous (uhom = 0.44%), and suitable for spinach monitoring under EU regulations. On dried basis, the nitrate content of SPIN-1 was found to be 22.53 ± 0.43 mg/g (Uc = 1.9%, k = 2). The material was also used in an inter-laboratory study where four laboratories employed a total of ten measurement methods.

SPIN-1 Certified Reference Material for nitrate in spinach powder

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

Similar content being viewed by others

References

  1. IARC. IARC Monographs On the Evaluation of Carcinogenic Risks to Humans Vol 94. Ingested nitrate and nitrite, and cyanobacterial peptide toxins. International Agency for Research on Cancer. Lyon: Distributed by WHO Press; 2010.

    Google Scholar 

  2. Weitzberg E, Lundberg JO. Novel aspects of dietary nitrate and human health. Annu Rev Nutr 2013;33: 129–159. https://doi.org/10.1146/annurev-nutr-071812-161159.

    Article  CAS  PubMed  Google Scholar 

  3. Hord NG. Dietary nitrates, nitrites, and cardiovascular disease. Curr Atheroscler Rep 2011;13:484–492. https://doi.org/10.1007/s11883-011-0209-9.

    Article  CAS  PubMed  Google Scholar 

  4. Lidder S, Webb AJ. Vascular effects of dietary nitrate (as found in green leafy vegetables and beetroot) via the nitrate-nitrite-nitric oxide pathway. Brit J Clin Pharmaco 2013;75:677–696. https://doi.org/10.1111/j.1365-2125.2012.04420.x.

    Article  CAS  Google Scholar 

  5. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 2008;51:784–790. https://doi.org/10.1161/hypertensionaha.107.103523.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jones AM. Dietary nitrate supplementation and exercise performance. Sports Med 2014;44:S35–S45. https://doi.org/10.1007/s40279-014-0149-y.

    Article  Google Scholar 

  7. Vermeer ITM, Pachen DMFA, Dallinga JW, Kleinjans JCS, Maanen JMS. Volatile N-nitrosamine formation after intake of nitrate at the ADI level in combination with an amine-rich diet. Environ Health Persp 1998; 106:459–463.

    CAS  Google Scholar 

  8. Ward MH, Kilfoy BA, Weyer PJ, Anderson KE, Folsom AR, Cerhan JR. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epidemiology 2010;21:389–395. https://doi.org/10.1097/ede.0b013e3181d6201d.

    Article  PubMed  PubMed Central  Google Scholar 

  9. DellaValle CT, Xiao Q, Yang G, Shu XO, Aschebrook-Kilfoy B, Zheng W, Li HL, Ji BT, Rothman N, Chow WH, Gao YT, Ward MH. Dietary nitrate and nitrite intake and risk of colorectal cancer in the Shanghai Women’s Health Study. Int J Cancer 2013;134:2917–2926. https://doi.org/10.1002/ijc.28612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. European Commission Commission Regulation (EU) No 1258/2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for nitrates in foodstuffs, Official Journal of the European Union, L 320/15.

  11. Santamaria P. Nitrate in vegetables: toxicity, content, intake and EC regulation. J Sci Food Agr 2006;86:10–17. https://doi.org/10.1002/jsfa.2351.

    Article  CAS  Google Scholar 

  12. Wang QH, Yu LJ, Liu Y, Lin L, gang Lu R, ping Zhu J, He L, Lu ZL. Methods for the detection and determination of nitrite and nitrate: A review. Talanta 2017;165:709–720. https://doi.org/10.1016/j.talanta.2016.12.044.

    Article  CAS  PubMed  Google Scholar 

  13. Farrington D, Damant AP, Powell K, Ridsdale J, Walker M, Wood R. A comparison of the extraction methods used in the UK nitrate residues monitoring program. J Assoc Public Anal 2006;34:1–11.

    Google Scholar 

  14. Merino L. Development and validation of a method for determination of residual nitrite/nitrate in foodstuffs and water after zinc reduction. Food Anal Method 2009;2:212–220. https://doi.org/10.1007/s12161-008-9052-1.

    Article  Google Scholar 

  15. Chetty AA, Prasad S. Flow injection analysis of nitrate-N determination in root vegetables: Study of the effects of cooking. Food Chem 2009;116:561–566. https://doi.org/10.1016/j.foodchem.2009.03.006.

    Article  CAS  Google Scholar 

  16. Özdestan O, Üren A. Development of a cost-effective method for nitrate and nitrite determination in leafy plants and nitrate and nitrite contents of some green leafy vegetables grown in the Aegean region of Turkey. J Agr Food Chem 2010;58:5235–5240. https://doi.org/10.1021/jf904558c.

    Article  CAS  Google Scholar 

  17. Kong Thoo Lin P, Araujo AN, Montenegro MCBSM, Pėrez-Olmos R. New PVC nitrate-selective electrode: application to vegetables and mineral waters. J Agr Food Chem 2005;53:211–215. https://doi.org/10.1021/jf049227u.

    Article  CAS  Google Scholar 

  18. Santos JR, Santos JLM, Lima JLFC. Single interface flow system with potentiometric detection for the determination of nitrate in water and vegetables. Talanta 2010;80:1326–1332. https://doi.org/10.1016/j.talanta.2009.09.031.

    Article  CAS  PubMed  Google Scholar 

  19. Szabo Z, Böddi K, Mark L, Szabo LG, Ohmacht R. Analysis of nitrate ion in nettle (Urtica dioica L) by ion-pair chromatographic method on a C30 stationary phase. J Agr Food Chem 2006;54:4082–4086. https://doi.org/10.1021/jf0524628.

    Article  CAS  Google Scholar 

  20. Croitoru MD. Nitrite and nitrate can be accurately measured in samples of vegetal and animal origin using an HPLC-UV/VIS technique. J Chromatogr B 2012;911:154–161. https://doi.org/10.1016/j.jchromb.2012.11.006.

    Article  CAS  Google Scholar 

  21. Iammarino M, Di Taranto A, Cristino M. Monitoring of nitrites and nitrates levels in leafy vegetables (spinach and lettuce): a contribution to risk assessment. J Sci Food Agr 2014;94:773–778. https://doi.org/10.1002/jsfa.6439.

    Article  CAS  Google Scholar 

  22. Iammarino M, Di Taranto A, Cristino M. Endogenous levels of nitrites and nitrates in wide consumption foodstuffs: results of five years of official controls and monitoring. Food Chem 2013;140:763–771. https://doi.org/10.1016/j.foodchem.2012.10.094.

    Article  CAS  PubMed  Google Scholar 

  23. D’Imperio M, Renna M, Cardinali A, Buttaro D, Serio F, Santamaria P. Calcium biofortification and bioaccessibility in soilless “baby leaf” vegetable production. Food Chem 2016;213:149–156. https://doi.org/10.1016/j.foodchem.2016.06.071.

    Article  CAS  PubMed  Google Scholar 

  24. Jimidar M, Hartmann C, Cousement N, Massart D. Determination of nitrate and nitrite in vegetables by capillary electrophoresis with indirect detection. J Chromatogr A 1995;706:479–492. https://doi.org/10.1016/0021-9673(94)01290-u.

    Article  CAS  Google Scholar 

  25. Merusi C, Corradini C, Cavazza A, Borromei C, Salvadeo P. Determination of nitrates, nitrites and oxalates in food products by capillary electrophoresis with pH-dependent electroosmotic flow reversal. Food Chem 2010;120:615–620. https://doi.org/10.1016/j.foodchem.2009.10.035.

    Article  CAS  Google Scholar 

  26. Campanella B, Onor M, Pagliano E. Rapid determination of nitrate in vegetables by gas chromatography mass spectrometry. Anal Chim Acta 2017;980:33–40. https://doi.org/10.1016/j.aca.2017.04.053.

    Article  CAS  PubMed  Google Scholar 

  27. Pagliano E, Meija J, Nadeau K, Campanella B, Onor M, Iammarino M, D’Amore T, Berardi G, D’Imperio M, Parente A, Mihai O, Mester Z. 2019. SPIN-1: Air-dried spinach powder Certified Reference Material for nitrate and trace metals. Ottawa: National Research Council Canada. Available from: https://doi.org/10.4224/crm.2019.spin-1.

  28. Pagliano E, Mester Z, Meija J. Reduction of measurement uncertainty by experimental design in high-order (double, triple, and quadruple) isotope dilution mass spectrometry: application to GC-MS measurement of bromide. Anal Bioanal Chem 2013;405:2879–2887. https://doi.org/10.1007/s00216-013-6724-5.

    Article  CAS  PubMed  Google Scholar 

  29. Pagliano E, Mester Z, Meija J. Calibration graphs in isotope dilution mass spectrometry. Anal Chim Acta 2015;896:63–67. https://doi.org/10.1016/j.aca.2015.09.020.

    Article  CAS  PubMed  Google Scholar 

  30. Castanheira I, Oliveira L, Valente A, Alvito P, Costa HS, Alink A. The need for reference materials when monitoring nitrate intake. Anal and Bioanal Chem 2004;378:1232–1238. https://doi.org/10.1007/s00216-003-2382-3.

    Article  CAS  Google Scholar 

  31. van der Veen AMH. Bayesian analysis of homogeneity studies in the production of reference materials. Accred Qual Assur 2017;22:307–319. https://doi.org/10.1007/s00769-017-1292-6.

    Article  Google Scholar 

  32. Doymaz I. Thin-layer drying of spinach leaves in a convective dryer. J Food Process Eng 2009;32(1):112–125. https://doi.org/10.1111/j.1745-4530.2007.00205.x.

    Article  Google Scholar 

  33. Pagliano E, Mester Z. Determination of elevated levels of nitrate in vegetable powders by high-precision isotope dilution GC–MS. Food Chem 2019;286:710–714. https://doi.org/10.1016/j.foodchem.2019.02.048.

    Article  CAS  PubMed  Google Scholar 

  34. Koepke A, Lafarge T, Possolo A, Toman B. Consensus building for interlaboratory studies, key comparisons, and meta-analysis. Metrologia 2017;54:S34–S62. https://doi.org/10.1088/1681-7575/aa6c0e.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Research Council Canada, 1200 Montreal Road, Ottawa, Ontario, K1A 0R6, Canada

    Enea Pagliano, Juris Meija, Ovidiu Mihai & Zoltán Mester

  2. Consiglio Nazionale delle Ricerche, Istituto di Chimica dei Composti Organometallici, UOS di Pisa, Via Moruzzi 1, 56124, Pisa, Italy

    Beatrice Campanella & Massimo Onor

  3. Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121, Foggia, Italy

    Marco Iammarino, Teresa D’Amore & Giovanna Berardi

  4. Consiglio Nazionale delle Ricerche, Istituto di Scienze delle Produzioni Alimentari, Via Giovanni Amendola 122/O, 70126, Bari, Italy

    Massimiliano D’Imperio & Angelo Parente

Authors
  1. Enea Pagliano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juris Meija
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beatrice Campanella
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Massimo Onor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marco Iammarino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Teresa D’Amore
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Giovanna Berardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Massimiliano D’Imperio
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Angelo Parente
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ovidiu Mihai
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Zoltán Mester
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Enea Pagliano.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(PDF 2.66 MB)

(PDF 228 KB)

(XLSM 39.9 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pagliano, E., Meija, J., Campanella, B. et al. Certification of nitrate in spinach powder reference material SPIN-1 by high-precision isotope dilution GC–MS. Anal Bioanal Chem 411, 3435–3445 (2019). https://doi.org/10.1007/s00216-019-01803-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-01803-4

Keywords

Search

Navigation