Related Experiment Video
Updated: Jan 13, 2026
08:38
Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS
Published on: November 8, 2015
17.4K
Analytical Validation of a Second-Generation Methotrexate Immunoassay
Rebecca Wilson1,2, Diane Yamaguchi3, Jamie Garrett3
1Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital, Cincinnati, OH, United States.
The Journal of Applied Laboratory Medicine
|October 28, 2025
Summary
This study validates a new methotrexate immunoassay, finding it accurate for most clinical uses. However, interference from DAMPA necessitates careful interpretation, especially in glucarpidase-treated patients.
Area of Science:
- Clinical Chemistry
- Analytical Toxicology
- Pharmacology
Background:
- Methotrexate is a vital antifolate therapy for cancers, autoimmune diseases, and transplant recipients.
- Accurate monitoring of methotrexate is crucial due to potential toxicity.
- Plasma methotrexate measurement is complicated by metabolite interference, making immunoassays and LC-MS/MS common methods.
Purpose of the Study:
- To evaluate the analytical performance of a second-generation methotrexate immunoassay.
- To compare the immunoassay's accuracy and reliability against LC-MS/MS.
- To assess potential interference from key methotrexate metabolites.
Main Methods:
- Analytical performance was assessed via precision, accuracy, linearity, and interference testing.
- Method comparison was conducted against LC-MS/MS.
- Cross-reactivity with 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA), 7-OH methotrexate, and folic acid was measured.
Main Results:
- The immunoassay demonstrated good precision (%CV 2.6%–4.4%) and linearity (0.03–1.3 µM).
- Significant interference was observed with DAMPA above 1 µM, impacting correlation with LC-MS/MS (R2 = 0.3241).
- No significant interference was noted for 7-OH methotrexate or folic acid within tested ranges.
Conclusions:
- The validated immunoassay is suitable for most clinical methotrexate monitoring applications.
- Interference from DAMPA requires careful consideration during assay selection and result interpretation.
- Particular caution is advised for glucarpidase-treated patients due to potential DAMPA interference.
Related Concept Videos
01:15Data Validation
568
Method validation is a crucial process in analytical chemistry designed to confirm that a given method consistently produces reliable and high-quality results. This process is essential when a method is applied to different sample matrices or when procedural modifications are made, ensuring that the results meet acceptable standards across various applications.
Key parameters for method validation include:
Key parameters for method validation include:
568
01:26Therapeutic Drug Monitoring: Drug Analysis Methods
172
Therapeutic Drug Monitoring (TDM) is a clinical practice that measures specific drug levels in a patient's blood or body tissues to tailor drug therapy effectively. This monitoring is critical for managing drugs with narrow therapeutic indices like digoxin and phenytoin, ensuring they are both safe and effective. For instance, monitoring theophylline levels in asthma patients involves precision and sensitivity to adjust doses according to individual responses to therapy, ensuring efficacy and...
172
01:29Therapeutic Drug Monitoring: Affecting Factors
196
Therapeutic Drug Monitoring (TDM) is the clinical practice of measuring specific drug levels in a patient's blood or body tissues to manage and optimize therapy. TDM is crucial for drugs with narrow therapeutic windows, like warfarin and phenytoin, where incorrect doses can lead to treatment failure or severe side effects. This monitoring ensures the dosage administered is within a safe and effective range. The factors affecting therapeutic drug monitoring include:Patient-Specific Factors:a.
196