Simultaneous Quantification of Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum via Coated Blade Spray-Tandem MS

Measurement of methotrexate (MTX) in patients receiving high doses of MTX (HDMTX) is important due to the risk of toxicity and wide variations in clearance between different patients receiving the same dose. Coated Blade Spray (CBS) is a sample preparation technology that can be directly interfaced with MS instrumentation for rapid screening and quantitation. As a proof-of-concept, we have developed a CBS method for the quantitation of MTX and its metabolites 7-hydroxy Methotrexate (7OH-MTX) and DAMPA in human serum via tandem mass spectrometry. Our preliminary results revealed that CBS could deliver performance comparable, or better, than those typically achieved with technologies generally used in the clinical laboratory, such as immunoassay and liquid chromatography-MS. Glucarpidase therapy is recommended when clearance of MTX is delayed. Typically, this enzyme inactivates MTX by cleaving it into two nontoxic metabolites: DAMPA and 7OH-MTX. It is well known that these metabolites interfere with commonly used MTX immunoassays, which means the assays can offer inaccurate and potentially misleading results. Therefore, more specific methods, such as those based on mass spectrometry, are preferred for the determination of MTX in patients under Glucarpidase therapy. Whilst several LC-MS/MS methods for MTX have been previously developed, these tend to require long chromatography times or complex sample prep workflows. Thus, having faster and specific assays for MTX and metabolites is ideal. In this study, we demonstrate the quantitation capabilities of CBS-MS/MS towards MTX and metabolites in serum. A split plot design of experiments was used to optimize the collection and elution conditions via CBS-MS/MS. Our results revealed that the optimized CBS-tandem MS method could deliver a LLOQ of 1 ng/mL for MTX and metabolites based on a mean CV and bias <20% at this concentration. Linearity experiments suggested the assay was linear to approximately 5µg/mL. Mean bias and CV for sixteen IQC material spiked in four different samples of serum (i.e., 4 each) was <15% and <5% respectively at all concentrations. A method comparison of the CBS-MS/MS method to a LC-MS/MS method was performed using 64 spiked samples across the range of 25 ng/mL to 4.8 µg/mL. Passing-Bablok analysis showed a linear relationship between the two methods. Finally, our CBS method has proven to be very robust with at least 250 consecutive injections (CV < 3.7% with IS correction) that can be completed by an analyst standing in front of the MS in less than 2 hrs and without having to replace/clean the mass spectrometer inlet.