Therapeutic Drug-Monitoring of Methotrexate-Polyglutamates in Rheumatoid Arthritis

__Abstract__ Rheumatoid Arthritis (RA) is a chronic autoimmune disease, characterized by the swelling of joints, uncontrolled proliferation of synovial tissue and multisystem co-morbidities. RA mainly affects the joints of the hands, feet, knees, wrist and elbows, with joint damage occurring early in the disease course. RA affects an estimated 1% of the general population and women have a higher risk of developing RA than men. Despite the fact that treatment strategy has changed considerably over the years, reflected by a much improved disease outcome, there is still no cure for RA. Early initiation of therapy is effective in prevention of joint damage and results in milder medication regimes while maintaining disease remission. Early in the disease, the inflammation is less self-perpetuating and easier to suppress, therefore it is important to start treatment as early as possible in order to optimize outcome, minimize medical costs, improve quality of life, and improve medical decision making

A U-HPLC-ESI-MS/MS-based stable isotope dilution method for the detection and quantitation of methotrexate in plasma

INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 μm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 μM (r > 0.99), and the coefficient of variation was 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 x FPIA - 7.3). CONLUSIONS: The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS. Copyrigh

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization–triple quadrupole–tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Using fluorescence polarization immunoassay for determination of erythrocyte methotrexate polyglutamates, a quick and easy test?

BACKGROUND:: The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTX-PGs). We aimed to validate an immunoassay for the measurement of MTX-PG in erythrocytes. METHODS:: Samples were analyzed by an adapted fluorescence polarization immune assay (FPIA) method on the FLx analyzer (Abbott). Cross-reactivity was determined in both plasma and erythrocyte pellet. In erythrocyte pellet, the imprecision, linearity, and lower limit of quantitation were determined. The method was compared with our in-house liquid chromatography tandem mass spectrometry (LC-MS/MS) method for total MTX-PG. RESULTS:: For the adapted FPIA method, a linear range of 25-1000 nmol/L (R = 0.993) was obtained for total MTX-PG in erythrocytes. A coefficient of variation of <17% for interday and <8% for intraday imprecision was found and average recovery was 91%. Lower limit of quantitation was determined at 50 nmol/L total MTX-PG with a coefficient of variation of 15%. There was no significant proportional bias of the FPIA assay compared with our in-house LC-MS/MS method, but a (nonsignificant) constant positive bias was present [FPIA = 1.00 (95% confidence interval: 0.60-1.95) × LC-MS/MS + 31.00 nmol/L (95% confidence interval: -11.83 to 61.00)]. Results could be very different for individual patients as reflected in the poor R of 0.419. CONCLUSIONS:: The FPIA method can be used to measure total MTX-PG in erythrocytes. Although there was no significant bias detected compared with the LC-MS/MS method, the FPIA method showed constant positive bias, probably because of interference from folates and MTX metabolites 2,4-diamino-N10-methylpteroic acid and 7-hydroxy-MTX. The correlation between both methods was average and resulted in large differences in individual patients, most likely because of problems during sample preparation

Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in juvenile idiopathic arthritis patients

Objective: To determine association of erythrocyte methotrexate polyglutamates (MTX-PG) with disease activity and adverse effects in a prospective juvenile idiopathic arthritis (JIA) cohort. Methods: One hundred and thirteen JIA patients were followed from MTX start until 12 months. Erythrocyte MTX-PGs with 1-5 glutamate residues were measured at 3 months with tandem mass spectrometry. The outcomes were Juvenile Arthritis Disease Activity Score (JADAS)-27 and adverse effects. To determine associations of MTX-PGs with JADAS-27 at 3 months and during 1 year of MTX treatment, linear regression and linear mixed-model analyses were used. To determine associations of MTX-PGs with adverse effects during 1 year of MTX treatment, logistic regression was used. Analyses were corrected for JADAS-27 at baseline and co-medication. Results: Median JADAS-27 decreased from 12.7 (IQR: 7.8-18.2) at baseline to 2.9 (IQR: 0.1-6.5) at 12 months. Higher concentrations of MTX-PG3 (β: -0.006, p=0.005), MTX-PG4 (β: -0.015, p=0.004), MTX-PG5 (β: -0.051, p=0.011) and MTX-PG3-5 (β: -0.004, p=0.003) were associated with lower disease activity at 3 months. Higher concentrations of MTX-PG3 (β: -0.005, p=0.028), MTX-PG4 (β: -0.014, p=0.014), MTX-PG5 (β: -0.049, p=0.023) and MTX-PG3-5 (β: -0.004, p=0.018) were associated with lower disease activity over 1 year. None of the MTX-PGs was associated with adverse effects. Conclusions: In the first prospective study in JIA, long-chain MTX-PGs were associated with lower JADAS-27 at 3 months and during 1 year of MTX treatment. Erythrocyte MTX-PG could be a plausible candidate for therapeutic drug monitoring of MTX in JIA