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Hawwa, A. F.; AlBawab, A.; Rooney, M.; Wedderburn, L. R.; Beresford, M. W.; McElnay, J. C. (2014)
Journal: PLoS ONE
Languages: English
Types: Article
Subjects: Chemistry, Research Article, Biology, Diagnostic Medicine, Test Evaluation, Rheumatology, Medicine, Analytical Chemistry, Pediatric Epidemiology, Q, Epidemiology, Epidemiological Methods, R, Population Biology, Science, Pediatrics, JUVENILE IDIOPATHIC ARTHRITIS, ACUTE LYMPHOBLASTIC-LEUKEMIA, RHEUMATOID-ARTHRITIS, LIQUID-CHROMATOGRAPHY, HPLC DETERMINATION, PHARMACOKINETICS, THERAPY, DERMATOMYOSITIS, SPECTROMETRY, IMMUNOASSAY
Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS).

Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization.

Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique.

Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.
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    • 1. Ravelli A, Martini A (2007) Juvenile idiopathic arthritis. Lancet 369 (9563):767- 778.
    • 2. Ramanan AV, Campbell-Webster N, Ota S, Parker S, Tran D, et al. (2005) The effectiveness of treating juvenile dermatomyositis with methotrexate and aggressively tapered corticosteroids. Arthritis and rheumatism 52 (11):3570- 3578.
    • 3. Murray KJ, Lovell DJ (2002) Advanced therapy for juvenile arthritis. Best practice & research Clinical rheumatology 16 (3):361-378.
    • 4. Choy EH, Isenberg DA (2002) Treatment of dermatomyositis and polymyositis. Rheumatology 41 (1):7-13.
    • 5. Pilkington CA, Wedderburn LR (2005) Paediatric idiopathic inflammatory muscle disease: recognition and management. Drugs 65 (10):1355-1365.
    • 6. Cutolo M, Sulli A, Pizzorni C, Seriolo B, Straub RH (2001) Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis. Annals of the rheumatic diseases 60 (8):729-735.
    • 7. Bannwarth B, Pehourcq F, Schaeverbeke T, Dehais J (1996) Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clinical pharmacokinetics 30 (3):194-210.
    • 8. Dalrymple JM, Stamp LK, O'Donnell JL, Chapman PT, Zhang M, et al. (2008) Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis. Arthritis and rheumatism 58 (11):3299-3308.
    • 9. Schmiegelow K, Bjork O, Glomstein A, Gustafsson G, Keiding N, et al. (2003) Intensification of mercaptopurine/methotrexate maintenance chemotherapy may increase the risk of relapse for some children with acute lymphoblastic leukemia. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 21 (7):1332-1339.
    • 10. Dervieux T, Hancock M, Evans W, Pui CH, Relling MV (2002) Effect of methotrexate polyglutamates on thioguanine nucleotide concentrations during continuation therapy of acute lymphoblastic leukemia with mercaptopurine. Leukemia 16 (2):209-212.
    • 11. Bostrom BC, Erdmann GR, Kamen BA (2003) Systemic methotrexate exposure is greater after intrathecal than after oral administration. Journal of pediatric hematology/oncology 25 (2):114-117.
    • 12. Buice RG, Evans WE, Karas J, Nicholas CA, Sidhu P, et al. (1980) Evaluation of enzyme immunoassay, radioassay, and radioimmunoassay of serum methotrexate, as compared with liquid chromatography. Clinical chemistry 26 (13):1902- 1904.
    • 13. Hayashi H, Fujimaki C, Tsuboi S, Matsuyama T, Daimon T, et al. (2008) Application of fluorescence polarization immunoassay for determination of methotrexate-polyglutamates in rheumatoid arthritis patients. The Tohoku journal of experimental medicine 215 (1):95-101.
    • 14. Kamen BA, Takach PL, Vatev R, Caston JD (1976) A rapid, radiochemicalligand binding assay for methotrexate. Analytical biochemistry 70 (1):54-63.
    • 15. Emara S, Razee S, Khedr A, Masujima T (1997) On-line precolumn derivatization for HPLC determination of methotrexate using a column packed oxidant. Biomedical chromatography: BMC 11 (1):42-46.
    • 16. Chladek J, Martinkova J, Simkova M, Vaneckova J, Koudelkova V, et al. (1998) Pharmacokinetics of low doses of methotrexate in patients with psoriasis over the early period of treatment. European journal of clinical pharmacology 53 (6):437- 444.
    • 17. Dervieux T, Orentas Lein D, Marcelletti J, Pischel K, Smith K, et al. (2003) HPLC determination of erythrocyte methotrexate polyglutamates after low-dose methotrexate therapy in patients with rheumatoid arthritis. Clinical chemistry 49 (10):1632-1641.
    • 18. Brooks AJ, Begg EJ, Zhang M, Frampton CM, Barclay ML (2007) Red blood cell methotrexate polyglutamate concentrations in inflammatory bowel disease. Therapeutic drug monitoring 29 (5):619-625.
    • 19. Chen G, Fawcett JP, Mikov M, Tucker IG (2009) Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography-tandem mass spectrometry. Journal of pharmaceutical and biomedical analysis 50 (2):262-266.
    • 20. van Haandel L, Becker ML, Leeder JS, Williams TD, Stobaugh JF (2009) A novel high-performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells. Rapid communications in mass spectrometry: RCM 23 (23):3693-3702.
    • 21. 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).
    • 22. FDA (2001) Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration. Available: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegu latoryInformation/Guidances/ucm070107.pdf. Accessed 2013 May.
    • 23. Annesley TM (2003) Ion suppression in mass spectrometry. Clinical chemistry 49 (7):1041-1044.
    • 24. Chambers E, Wagrowski-Diehl DM, Lu Z, Mazzeo JR (2007) Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 852 (1-2):22-34.
    • 25. Bland JM, Altman DG (1995) Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 852 (8982):1085-1087.
    • 26. Shabir GA (2003) Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. Journal of chromatography A 987 (1-2):57-66.
    • 27. Suyagh MF, Iheagwaram G, Kole PL, Millership J, Collier P, et al. (2010) Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples. Analytical and bioanalytical chemistry 397 (2):687-693.
    • 28. Becker ML, van Haandel L, Gaedigk R, Lasky A, Hoeltzel M, et al. (2010) Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations. Arthritis and rheumatism 62 (6):1803-1812.
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