title: A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children.
abstract: 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, 2.1 × 150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization.
  
  
  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
  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.
authors.affiliation: Clinical and Practice Research Group, School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom ; Aston Pharmacy School, Aston University, Birmingham, United Kingdom.
authors.forename: Ahmed F
authors.initials: AF
authors.lastname: Hawwa
authors.affiliation: Clinical and Practice Research Group, School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.
authors.forename: Abdelqader
authors.initials: A
authors.lastname: Albawab
authors.affiliation: School of Medicine, Queen's University Belfast and Musgrave Park Hospital, Belfast, United Kingdom.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Rooney
authors.affiliation: Institute of Child Health, University College London, London, United Kingdom ; Arthritis Research UK Centre for Adolescent Rheumatology at University College London, University College London Hospital and Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom.
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Wedderburn
authors.affiliation: Department of Women's and Children's Health, University of Liverpool, The Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Beresford
authors.affiliation: Clinical and Practice Research Group, School of Pharmacy, Queen's University Belfast, Belfast, United Kingdom.
authors.forename: James C
authors.initials: JC
authors.lastname: McElnay
mesh_terms: D018501:Antirheumatic Agents / Q000097:blood / Q000302:isolation & purification*; D015415:Biomarkers / Q000097:blood*; D002648:Child; D002853:Chromatography, Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D006801:Humans; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000097:blood / Q000302:isolation & purification*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D018501:Antirheumatic Agents; D015415:Biomarkers; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate