Analytical and Bioanalytical Chemistry

, Volume 397, Issue 2, pp 687–693 | Cite as

Development and validation of a dried blood spot–HPLC assay for the determination of metronidazole in neonatal whole blood samples

  • Maysa Faisal Suyagh
  • Godwill Iheagwaram
  • Prashant Laxman Kole
  • Jeff Millership
  • Paul Collier
  • Henry Halliday
  • James C. McElnay
Original Paper


A selective and sensitive high-performance liquid chromatography method with UV detection for the determination of metronidazole in dried blood spots (DBS) has been developed and validated. DBS samples [spiked or patient samples] were prepared by applying blood (30 µL) to Guthrie cards. Discs (6 mm diameter) were punched from the cards and extracted using water containing the internal standard, tinidazole. The extracted sample was chromatographed without further treatment using a reversed phase system involving a Symmetry® C18 (5 µm, 3.9 × 150 mm) preceded by a Symmetry® guard column of matching chemistry and a detection wavelength of 317 nm. The mobile phase comprised acetonitrile/0.01 M phosphate solution (KH2PO4), pH 4.7, 15:85, v/v, with a flow rate of 1 mL/min. The calibration was linear over the range 2.5–50 mg/mL. The limits of detection and quantification were 0.6 and 1.8 µg/mL, respectively. The method has been applied to the determination of 203 DBS samples from neonatal patients for a phamacokinetic/pharmacodynamic study.


Dried blood spots Guthrie card Metronidazole HPLC 


  1. 1.
    Paediatric Formulary Committee (2006) BNF for children. BMJ, LondonGoogle Scholar
  2. 2.
    Jager-Roman E, Doyle PE, Baird-Lambert J, Cvejic M, Buchanan N (1982) J Pediatr 100:651–654CrossRefGoogle Scholar
  3. 3.
    Hall P, Kaye CM, McIntosh N, Steele J (1983) Arch Dis Child 58:529–531CrossRefGoogle Scholar
  4. 4.
    Rubenson A, Rosetzsky A (1986) Eur J Clin Pharmacol 29:625–628CrossRefGoogle Scholar
  5. 5.
    Upadhyaya P, Bhatnagar V, Basu N (1988) J Pediatr Surg 23:263–265CrossRefGoogle Scholar
  6. 6.
    Sandall JM, Millership JS, Collier PS (2006) McElnay JC J Chromatogr B 839:36–44CrossRefGoogle Scholar
  7. 7.
    Smyth JM, Darwish M, Collier PS, Millership JS, Petersen S, Halliday HL, McElnay JC (2004) Br J Clin Pharmacol 58:249–258CrossRefGoogle Scholar
  8. 8.
    Wilcken B, Wiley V (2008) Pathology 40:104–115CrossRefGoogle Scholar
  9. 9.
    AbuRuz S, Millership JS, McElnay JC (2006) J Chromatogr B 832:202–207CrossRefGoogle Scholar
  10. 10.
    Millership JS, Collier PS, McElnay JC (2003) J Pharm Pharmacol 55:S65Google Scholar
  11. 11.
    Oliveira EJ, Watson DG, Morton NS (2004) J Pharm Biomed Anal 29:803–809CrossRefGoogle Scholar
  12. 12.
    Kaye CM, Sankey MG, Thomas LA (1980) Br J Clin Pharmacol 9:528–529Google Scholar
  13. 13.
    Gibson RA, Lattanzio L, McGee H (1984) Clin Chem 30:784–787Google Scholar
  14. 14.
    Okonkwo PO, Eta EI (1988) Life Sci 42:539–545CrossRefGoogle Scholar
  15. 15.
    Venkateshwaran TG, Stewart JT (1995) J Chromatogr B 672:300–304CrossRefGoogle Scholar
  16. 16.
    Jessa MJ, Barrett DA, Shaw PN, Spiller RC (1996) J Chromatogr B 677:374–379CrossRefGoogle Scholar
  17. 17.
    Yeung PK, Little R, Jiang Y, Buckley SJ, Pollak PT, Kapoor H, Veldhuyzen van Zanten SJ (1998) J Pharm Biomed Anal 17:1393–1398CrossRefGoogle Scholar
  18. 18.
    Menelaou A, Somogyi AA, Barclay ML, Bochner F (1999) J Chromatogr B 731:261–266CrossRefGoogle Scholar
  19. 19.
    Galmier MJ, Frasey AM, Bastide M, Beyssac E, Petit J, Aiache JM, Lartigue-Mattei C (1998) J Chromatogr B 720:239–243CrossRefGoogle Scholar
  20. 20.
    Stambaugh JE, Feo LG, Manthel RW (1968) J Pharmacol Exp Ther 161:373–381Google Scholar
  21. 21.
    Pahkla ER, Koppel T, Saag M, Pahkla R (2005) J Clin Peridontol 32:163–166CrossRefGoogle Scholar
  22. 22.
    do Nascimento TG, de Jesus Oliveira E, Macedo RO (2005) J Pharm Biomed Anal 37:777–783CrossRefGoogle Scholar
  23. 23.
    Mustapha KB, Odunola MT, Garba M, Obodozie O (2006) Afr J Biotechnol 5:1188Google Scholar
  24. 24.
    Emami J, Ghassami N, Hamishehkar H (2006) DARU 14:15–21Google Scholar
  25. 25.
    Sagan C, Salvador A, Dubreuil D, Poulet PP, Duffaut D, Brumpt I (2005) J Pharm Biomed Anal 38:298–306CrossRefGoogle Scholar
  26. 26..
    U.S. Department of Health and Human Services, Food and Drug Administration (2001) Guidance for industry: bioanalytical method validation. U.S. Department of Health and Human Services, Food and Drug Administration, Silver Spring, MayGoogle Scholar
  27. 27.
    The European Agency for the Evaluation of Medicinal Products (1996) ICH harmonised tripartite guideline: validation of analytical procedures: methodology. ICH Topic Q2B (CPMP/ICH/281/95). The European Agency for the Evaluation of Medicinal Products, London.Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Maysa Faisal Suyagh
    • 1
  • Godwill Iheagwaram
    • 1
  • Prashant Laxman Kole
    • 1
  • Jeff Millership
    • 1
  • Paul Collier
    • 1
  • Henry Halliday
    • 2
  • James C. McElnay
    • 1
  1. 1.Clinical and Practice Research Group, School of Pharmacy, Medical Biology CentreQueen’s University BelfastBelfastUK
  2. 2.Perinatal Medicine, Royal Maternity Hospital, and Department of Child HealthQueen’s University BelfastBelfastUK

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