MSACL 2022 Abstract

INTRODUCTION: Measurement of methotrexate (MTX) in patients receiving high doses of MTX is important due to the risk of toxicity and wide variations in clearance between different patients receiving the same dose. Glucarpidase therapy is recommended when clearance of MTX is delayed. Typically, this enzyme inactivates MTX by cleaving it into two non-toxic 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. Coated Blade Spray (CBS) is an open-bed SPE technology that can be directly interfaced with mass spectrometry (MS) instrumentation for rapid screening and quantitation of drugs and metabolites in biofluids.

OBJECTIVES: In this work, as a proof-of-concept, we present for the first time a fully automated CBS workflow for the quantitation of MTX and its metabolites 7-Hydroxy Methotrexate (7OH-MTX) and DAMPA in human serum via tandem mass spectrometry.

METHODS: Coated Blade Spray (CBS) devices coated with HLB particles were purchased from Restek Corporation. Devices were modified to have a pipette-like back end (hereinafter refer as CBS 2.0). Instrumental analysis via tandem mass spectrometry were performed on either a Thermo TSQ Altis or an LTQ-XL. An Opentrons OT-2 liquid handler comprising two single-channel pipettors was used in this work. One of the pipettors was modified to pivot the CBS 2.0 devices from the sample preparation position (vertical) to the sample injection position (horizontal). Liquids/samples handling, CBS location/movement, coating wetting, MS start triggering, elution timing, high voltage application timing, and MS-inlet clean-up were fully automated and controlled by a PCB and a custom-made program in Python.

RESULTS: The automated CBS-tandem MS method delivered an 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 an LC-MS/MS method was performed using 64 spiked samples across the range of 25 ng/mL to 4.8µg/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 480 consecutive injections (CV < 3.7% with IS correction) that can be completed by the robot in less than 4h and without having to replace/clean the mass spectrometer inlet.

CONCLUSIONS: Our results revealed that the robotic-CBS delivered a performance equivalent to the manual workflow. Furthermore, the results are comparable, or better, than those typically achieved with clinically accepted technologies such as immunoassay and liquid chromatography-MS/MS.