Skip to main content
SpringerLink
Log in

Vitamina D: valutazione di un metodo cromatografico liquido ultra veloce (UPLC) e confronto con tecnica immunometrica e cromatografia liquida ad alta prestazione (HPLC) tradizionale

Vitamin D: evaluation of an ultrafast liquid chromatography method (UPLC) and its comparison with a traditional immunoassay and high-performance liquid chromatography (HPLC) technique

  • Articolo Originale
  • Published:
La Rivista Italiana della Medicina di Laboratorio - Italian Journal of Laboratory Medicine

Riassunto

Premesse

L’utilizzo di sistemi di cromatografia liquida ultra veloce, non accoppiata a un rivelatore di massa, consentirebbe il dosaggio accurato delle differenti forme di vitamina D, proprio dei metodi cromatografici, in tempi rapidi, con produttività simile a quella dei metodi immunometrici, senza richiedere investimenti iniziali elevati generalmente tipici dei sistemi LC/MS/MS. In questo studio, utilizzando plasma e siero come matrici, abbiamo confrontato un nuovo metodo cromatografico liquido ultraveloce (UPLC) con un metodo immunometrico e uno cromatografico tradizionale (HPLC).

Metodi

40 campioni di plasma e 50 campioni di siero sono stati valutati tramite kit ClinRep 25-OH-vitamina D2/D3® Recipe (BSN Srl, Castelleone, CR, Italia) su strumentazione UPLC Nexera Prominence LC20 (Shimadzu Italia Srl, Milano, Italia). I campioni di plasma sono stati analizzati con kit LIAISONsr 25-OH-Vitamin D Total (DiaSorin SpA, Saluggia, VC, Italia) su strumentazione Liaison Diagnostic System, mentre i campioni di siero sono stati misurati con kit 25-OH Vitamin D3/D2 by HPLC® Bio-Rad Laboratories Srl (Segrate, MI, Italia) su strumentazione Agilent Technologies (Santa Clara, CA, USA) HPLC serie 1200. Per la valutazione dell’imprecisione e dell’accuratezza sono stati utilizzati un controllo a bassa (LC) e alta concentrazione (HC) e un pool di plasma (PN).

Risultati

Il metodo UPLC presenta una buona precisione per entrambe le forme di vitamina D (5–7%). Sebbene per livelli bassi di vitamina D l’inaccuratezza si attesta a valori del 2%, per concentrazioni nell’intervallo di normalità (>30 ng/ml) essa è pari a 6,5-8,5%. Per il confronto DiaSorin-UPLC su plasma, l’analisi di Bland-Altman ha mostrato una differenza media percentuale pari a 74,5%, con valori circa doppi per il metodo UPLC rispetto al metodo DiaSorin, come confermato dall’analisi di regressione di Passing-Bablok (UPLC = DiaSorin*2.38). Per il confronto HPLC-UPLC su siero, l’analisi di Bland-Altman ha mostrato una differenza media percentuale pari a 11,6%, con valori più elevati per il metodo UPLC. L’analisi di regressione di Passing-Bablok ha evidenziato una relazione UPLC = HPLC*1,09+0,27. Il numero di previsioni non corrette, relativamente agli intervalli <10, 10-30, 30-100, >100 ng/ml, non supera il 20%.

Conclusioni

Il metodo UPLC presenta valori di vitamina D sostanzialmente più elevati rispetto al metodo DiaSorin, non risultando intercambiabile a meno di una modifica degli intervalli di riferimento. Pur presentando un’inaccuratezza superiore al limite desiderabile suggerito dalla recente letteratura, il metodo UPLC fornisce risultati di vitamina D sostanzialmente sovrapponibili al metodo HPLC tradizionale.

Summary

Background

The use of ultrafast liquid chromatography (UPLC) without a coupled mass spectrometer could allow accurate evaluation of the different forms of vitamin D by combining the advantages of both immunometric methods (rapid response time, high throughput) and traditional HPLC (better accuracy, precision and specificity) without high initial investment costs. In this study we compared a new UPLC method, an immunometric method and a traditional HPLC method for the evaluation of vitamin D levels in plasma and serum.

Methods

The vitamin D levels in 40 plasma samples and 50 serum samples were determined using a ClinRep® 25-OHvitamin D2/D3 Recipe kit (BSN Srl, Castelleone, CR, Italia) and a UPLC Nexera Prominence LC20 system (Shimadzu Italia Srl, Milano, Italia). The plasma samples were also analysed using a LIAISON® 25-OH-vitamin D Total kit (DiaSorin SpA, Saluggia, VC, Italia) and a Liaison diagnostic system, and the serum samples were also analysed using a 25-OH-vitamin D3/D2 Bio-Rad Laboratories Srl (Segrate, MI, Italy) HPLC kit and an Agilent Technologies (Santa Clara, CA, USA) series 1200 HPLC system. To verify repeatability and accuracy a plasma pool and low and high concentration controls were used.

Results

The UPLC method displayed good precision for both vitamin D forms (5–7 %). Although at low vitamin D levels inaccuracy was low (2 %), at higher concentrations (>30 ng/ml) inaccuracy ranged from 6.5 % to 8.5 %. For analysing plasma, UPLC indicated vitamin D levels twice those obtained with the DiaSorin kit, as confirmed by Passing-Bablok regression (UPLC va lues = DiaSorin values × 2.38), with a mean percentage difference of 74.5 %. For analysing serum, UPLC indicated higher vitamin D levels than HPLC, with a mean percentage difference of 11.6 %. The regression equation from Passing-Bablok analysis was: UPLC va lues = HPLC values × 1.09 + 0.27. The misclassification rate in relation to clinical decision points commonly used for the diagnosis of mild or moderate vitamin D deficiency (<10, 10-30, 30-100, >100 ng/ml) was lower than 20 %.

Conclusions

The UPLC method indicated higher vitamin D levels than those obtained using the DiaSorin kit and did not appear to be interchangeable without reference interval modification. Despite displaying inaccuracy close to or higher than the desirable limits suggested in the recent literature, the UPLC method indicated vitamin D levels compatible with those obtained by traditional HPLC.

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

Bibliografia

  1. Adami S, Romagnoli, E, Carnevale, V et al (2011) Linee guida su prevenzione e trattamento dell’ipovitaminosi D con colecalciferolo. Reumatismo 63:129–147

    Article  PubMed  CAS  Google Scholar 

  2. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357:266–281

    Article  PubMed  CAS  Google Scholar 

  3. Kulie T, Groff, A, Redmer, J et al (2009) Vitamin D: an evidence-based review. J Am Board Fam Med 22:698–706

    Article  PubMed  Google Scholar 

  4. Mithal A, Wahl, DA, Bonjour, JP et al (2009) Global vitamin D status and determinants of hypovitaminosis D. Osteoporos Int 20:1807–1820

    Article  PubMed  CAS  Google Scholar 

  5. Peterlik M, Grant, WB and Cross, HS (2009) Calcium, vitamin D and cancer. Anticancer Res 29:3687–3698

    PubMed  CAS  Google Scholar 

  6. Samuel S and Sitrin, MD (2008) Vitamin D’s role in cell proliferation and differentiation. Nutr Rev 66:S116–124

    Article  PubMed  Google Scholar 

  7. Souberbielle JC, Body, JJ, Lappe, JM et al (2010) Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice. Autoimmun Rev 9:709–715

    Article  PubMed  CAS  Google Scholar 

  8. van Etten E, Gysemans, C, Branisteanu, DD et al (2007) Novel insights in the immune function of the vitamin D system: synergism with interferon-beta. J Steroid Biochem Mol Biol 103:546–551

    Article  PubMed  Google Scholar 

  9. Jenab M, Bueno-de-Mesquita, HB, Ferrari, P et al (2010) Association between pre-diagnostic circulating vitamin D concentration and risk of colorectal cancer in European populations:a nested case-control study. BMJ 340:b5500

    Article  PubMed  Google Scholar 

  10. Lappe JM, Travers-Gustafson, D, Davies, KM et al (2007) Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin Nutr 85:1586–1591

    PubMed  CAS  Google Scholar 

  11. Roth CL, Elfers, C, Kratz, M et al (2011) Vitamin D Deficiency in Obese Children and Its Relationship to Insulin Resistance and Adipokines. Journal of Obesity doi:10.1155/2011/495101

  12. Gordon CM, DePeter, KC, Feldman, HA et al (2004) Prevalence of vitamin D deficiency among healthy adolescents. Arch Pediatr Adolesc Med 158:531–537

    Article  PubMed  Google Scholar 

  13. Cashman KD (2007) Vitamin D in childhood and adolescence. Postgrad Med J 83:230–235

    Article  PubMed  CAS  Google Scholar 

  14. Carter GD (2009) 25-Hydroxyvitamin D assays: the quest for accuracy. Clin Chem 55:1300–1302

    Article  PubMed  CAS  Google Scholar 

  15. de la Hunty A, Wallace, AM, Gibson, S et al (2010) UK Food Standards Agency Workshop Consensus Report: the choice of method for measuring 25-hydroxyvitamin D to estimate vitamin D status for the UK National Diet and Nutrition Survey. Br J Nutr 104:612–619

    Article  PubMed  Google Scholar 

  16. Shah I, James, R, Barker, J et al (2011) Misleading measures in Vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars. Nutr J 10:46

    Article  PubMed  CAS  Google Scholar 

  17. Singh RJ (2010) Quantitation of 25-OH-vitamin D (25OHD) using liquid tandem mass spectrometry (LC-MS-MS). Methods Mol Biol 603:509–517

    Article  PubMed  CAS  Google Scholar 

  18. Vogeser M (2010) Quantification of circulating 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry. J Steroid Biochem Mol Biol 121:565–573

    Article  PubMed  CAS  Google Scholar 

  19. Wallace AM, Gibson, S, de la Hunty, A et al (2010) Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedures, performance characteristics and limitations. Steroids 75:477–488

    Article  PubMed  CAS  Google Scholar 

  20. Wootton AM (2005) Improving the measurement of 25-hydroxyvitamin D. Clin Biochem Rev 26:33–36

    PubMed  Google Scholar 

  21. Stockl D, Sluss, PM and Thienpont, LM (2009) Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis. Clin Chim Acta 408:8–13

    Article  PubMed  CAS  Google Scholar 

  22. Farrell CJ, Martin, S, McWhinney, B et al (2012) State-of-the-Art Vitamin D Assays: A Comparison of Automated Immunoassays with Liquid Chromatography-Tandem Mass Spectrometry Methods. Clin Chem 58:531–542

    Article  PubMed  CAS  Google Scholar 

  23. Viljoen A, Singh, DK, Farrington, K et al (2011) Analytical quality goals for 25-vitamin D based on biological variation. J Clin Lab Anal 25:130–133

    Article  PubMed  CAS  Google Scholar 

  24. Westgard JO (1995) A method evaluation decision chart (MEDx chart) for judging method performance. Clin Lab Sci 8:277–283

    PubMed  CAS  Google Scholar 

  25. Sarafin K, Hidiroglou, N and Brooks, SPJ (2011) A Comparison of Two Immunoassays for Analysing Plasma 25-hydroxyvitamin D. The Open Clinical Chemistry Journal 4:45–49

    Article  CAS  Google Scholar 

  26. Wagner D, Hanwell, HE and Vieth, R (2009) An evaluation of automated methods for measurement of serum 25-hydroxyvitamin D. Clin Biochem 42:1549–1556

    Article  PubMed  CAS  Google Scholar 

  27. Moon HW, Cho, JH, Hur, M et al (2012) Comparison of four current 25-hydroxyvitamin D assays. Clin Biochem 45:326–330

    Article  PubMed  CAS  Google Scholar 

  28. Gowans EM, Hyltoft Petersen, P, Blaabjerg, O et al (1988) Analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. Scand J Clin Lab Invest 48:757–764

    Article  PubMed  CAS  Google Scholar 

  29. Harris EK (1979) Statistical principles underlying analytic goalsetting in clinical chemistry. Am J Clin Pathol 72:374–382

    PubMed  CAS  Google Scholar 

  30. Petersen PH, Fraser, CG, Westgard, JO et al (1992) Analytical goal-setting for monitoring patients when two analytical methods are used. Clin Chem 38:2256–2260

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. SC Laboratorio Analisi Azienda Ospedaliera S.S. Antonio e Biagio e C. Arrigo, Alessandria, Italy

    Vincenza Bianchi, Fabiola Martino, Silvana Piccinini, Antonietta Pinca, Graziano Sida & Carlo Arfini

  2. LABORAF Diagnostica e Ricerca San Raffaele S.p.A., Milano, Italy

    Massimo Locatelli

  3. Università del Piemonte Orientale Amedeo Avogadro, Novara, Italy

    Matteo Vidali

  4. Dipartimento di Scienze Mediche, Università del Piemonte Orientale “Amedeo Avogadro”, Via Solaroli 17, 28100, Novara, Italy

    Matteo Vidali

  5. Laboratorio Ricerche Chimico Cliniche, Ospedale “Maggiore della Carità”, C.so Mazzini 18, 28100, Novara, Italy

    Matteo Vidali

Authors
  1. Vincenza Bianchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Massimo Locatelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matteo Vidali
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fabiola Martino
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Silvana Piccinini
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Antonietta Pinca
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Graziano Sida
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Carlo Arfini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matteo Vidali.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bianchi, V., Locatelli, M., Vidali, M. et al. Vitamina D: valutazione di un metodo cromatografico liquido ultra veloce (UPLC) e confronto con tecnica immunometrica e cromatografia liquida ad alta prestazione (HPLC) tradizionale. Riv Ital Med Lab 8, 138–148 (2012). https://doi.org/10.1007/s13631-012-0054-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13631-012-0054-z

Parole chiave

Key words

Search

Navigation