A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children

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

Methotrexate polyglutamates as a potential marker of adherence to long-term therapy in children with juvenile idiopathic arthritis and juvenile dermatomyositis:an observational, cross-sectional study

Introduction: Methotrexate (MTX) is a cornerstone of treatment in a wide variety of inflammatory conditions, including juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM). However, owing to its narrow therapeutic index and the considerable interpatient variability in clinical response, monitoring of adherence to MTX is important. The present study demonstrates the feasibility of using methotrexate polyglutamates (MTXPGs) as a biomarker to measure adherence to MTX treatment in children with JIA and JDM. Methods: Data were collected prospectively from a cohort of 48 children (median age 11.5 years) who received oral or subcutaneous (SC) MTX therapy for JIA or JDM. Dried blood spot samples were obtained from children by finger pick at the clinic or via self- or parent-led sampling at home, and they were analysed to determine the variability in MTXPG concentrations and assess adherence to MTX therapy. Results: Wide fluctuations in MTXPG total concentrations (>2.0-fold variations) were found in 17 patients receiving stable weekly doses of MTX, which is indicative of nonadherence or partial adherence to MTX therapy. Age (P = 0.026) and route of administration (P = 0.005) were the most important predictors of nonadherence to MTX treatment. In addition, the study showed that MTX dose and route of administration were significantly associated with variations in the distribution of MTXPG subtypes. Higher doses and SC administration of MTX produced higher levels of total MTXPGs and selective accumulation of longer-chain MTXPGs (P < 0.001 and P < 0.0001, respectively). Conclusions: Nonadherence to MTX therapy is a significant problem in children with JIA and JDM. The present study suggests that patients with inadequate adherence and/or intolerance to oral MTX may benefit from SC administration of the drug. The clinical utility of MTXPG levels to monitor and optimise adherence to MTX in children has been demonstrated. Trial registration: ISRCTN Registry identifier: ISRCTN93945409. Registered 2 December 2011

Individual MRM traces obtained upon injection of processed DBS with blank blood into a column post-infused with MTXPG solutions; chromatograms indicated no matrix effect over the time of analysis.

<p>Individual MRM traces obtained upon injection of processed DBS with blank blood into a column post-infused with MTXPG solutions; chromatograms indicated no matrix effect over the time of analysis.</p

Results of within day (intra-day) and between days (inter-day) accuracy and precision of MTXPG analysis (n = 5).

a<p>Relative standard deviation (RSD)% = [standard deviation (SD)/mean]×100.</p>b<p>Relative error (RE)% = [(measured−nominal)/nominal]×100.</p

Bland-Altman plots comparing MTXPG_1–5 and MTXPG_total concentrations in DBS versus RBCs in all patient samples investigated (n = 94).

<p>Bland-Altman plots comparing MTXPG_1–5 and MTXPG_total concentrations in DBS versus RBCs in all patient samples investigated (n = 94).</p

Results of the five calibration curves for MTXPGs; calibration points (nmol/L)^a, slope±SD, intercept and correlation coefficient of the standard curves.

a<p>Spiked concentrations of calibration standards [measured concentrations (mean±SD) are shown within brackets].</p

Representative MRM chromatograms of a blank DBS sample (A), DBS spiked with MTXPGs (final total concentration of 100 nM) subjected to enzymatic conversion to MTXPG1 (B), and DBS sample spiked with MTXPG1–5 measured individually at a final concentration of 100 nM each (C).

<p>Representative MRM chromatograms of a blank DBS sample (A), DBS spiked with MTXPGs (final total concentration of 100 nM) subjected to enzymatic conversion to MTXPG1 (B), and DBS sample spiked with MTXPG1–5 measured individually at a final concentration of 100 nM each (C).</p

Representative chromatograms of an extracted DBS sample obtained from JIA patient treated with MTX; (pg1–5): MRM chromatograms of individual MTXPGs; concentrations found were MTXPG₁, 9 nmol/L; MTXPG₂, 10 nmol/L; MTXP₃, 15 nmol/L; MTXPG₄, 10 nmol/L and MTXPG₅, 7 nmol/L, respectively.

<p>(MTX-total): Chromatogram after conversion of total MTXPGs to MTXPG₁; total concentration found was 49 nmol/L.</p

MRM parameters for the analysis of MTXPGs.

<p>MRM parameters for the analysis of MTXPGs.</p

Effect of varying blood haematocrit on the measured area of DBS spot formed (mean area ± SD; n = 8).

<p>Effect of varying blood haematocrit on the measured area of DBS spot formed (mean area ± SD; n = 8).</p