Methotrexate Assay
Accurately screen for MTX with low-end sensitivity.
Precision
Precision was determined as described in CLSI/NCCLS Protocol EP5-A2. The six-level ARK Methotrexate Control and pooled human specimens containing methotrexate were used in the study. Each level was assayed in quadruplicate twice a day for 20 days. Each of the runs per day was separated by at least two hours. The within run, between day, total SD, and percent CVs were calculated.
| Within Run | Between Day | Total | ||||||
| Sample | N | Mean (µg/mL) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| ARK Methotrexate Control | ||||||||
| LOW | 160 | 0.06 | 0.005 | 8.2 | 0.005 | 7.3 | 0.007 | 10.7 |
| MID | 160 | 0.37 | 0.011 | 3.0 | 0.008 | 2.1 | 0.014 | 3.8 |
| HIGH | 160 | 0.76 | 0.032 | 4.3 | 0.030 | 4.0 | 0.045 | 5.9 |
| 5* | 160 | 4.8 | 0.15 | 3.1 | 0.13 | 2.8 | 0.20 | 4.2 |
| 50* | 160 | 49 | 1.36 | 2.8 | 2.32 | 4.8 | 2.72 | 5.6 |
| 500* | 160 | 476 | 15.17 | 3.2 | 30.75 | 6.5 | 34.66 | 7.3 |
| Patient Pool | ||||||||
| LOW | 160 | 0.07 | 0.006 | 9.1 | 0.005 | 7.5 | 0.008 | 11.7 |
| MID | 160 | 0.41 | 0.013 | 3.3 | 0.026 | 6.4 | 0.029 | 7.2 |
| HIGH | 160 | 0.82 | 0.037 | 4.5 | 0.043 | 5.2 | 0.057 | 7.0 |
| 5* | 160 | 4.6 | 0.14 | 3.1 | 0.183 | 4.0 | 0.24 | 5.3 |
| 50* | 160 | 45 | 1.33 | 3.0 | 2.63 | 5.9 | 2.93 | 6.6 |
| 500* | 160 | 461 | 11.84 | 2.6 | 27.04 | 5.9 | 29.60 | 6.4 |
*Samples were diluted in ARK Methotrexate Dilution Bufer.
Mean result and SD were multiplied by the dilution factor.
Specificity
Cross-reactivity to 7-Hydroxymethotrexate, the major metabolite
The ARK Methotrexate Assay did not crossreact with the major metabolite 7-OH-MTX.
| Reagent Lot | 7-OH MTX (µmol/L) | Serum Control (µmol/L) | MTX (µmol/L) | Cross Reactivity (%) |
| Cross Reactivity 7-OH MTX in the presence of 0.05 µmol/L MTX | ||||
| D2 | 5.0 | 0.06 | 0.06 | 0.01 |
| D3 | 5.0 | 0.04 | 0.05 | 0.02 |
| Cross Reactivity 7-OH MTX in the presence of 0.05 µmol/L MTX | ||||
| D2 | 50.0 | 0.44 | 0.47 | 0.07 |
| D3 | 50.0 | 0.46 | 0.48 | 0.05 |
Cross-reactivity to the minor, inactive metabolite 2,4-diam ino-N10-methylpteroic acid (DAMPA)
The ARK Methotrexate Assay crossreacts substantially with the minor metabolite DAMPA. Tests were performed in the absence of the parent drug methotrexate. Crossreactivity to DAMPA ranged 64.3 to 100%. The assay should not be used during possible compassionate therapy with glucarpidase (carboxypeptidase G2) that rapidly converts circulating methotrexate to DAMPA.
Drugs that crossreact
The ARK Methotrexate Assay crossreacts slightly with triamterene and trimethoprim, however these drugs may be contraindicated for MTX cancer treatment due to additional adverse effects if co-administered. The structures of these compounds closely match the pteridine ring moiety of methotrexate.
| Compound | Tested (µmol/L) | Apparent MTX (µmol/L) | Cross Reactivity (%) |
| MTX Absent | |||
| Triamterene | 25 | 0.46 | 1.85 |
| Trimethoprim | 100 | 0.17 | 0.17 |
| MTX Present 0.05 (µmol/L) | |||
| Triamterene | 25 | 0.89 | 3.32 |
| Trimethoprim | 100 | 0.16 | 0.12 |
| MTX Present 0.05 (µmol/L) | |||
| Triamterene | 25 | 1.04 | 2.31 |
| Trimethoprim | 100 | 0.99 | 0.54 |
Cross-reactivity to folate analogs and other compounds
The ARK Methotrexate Assay did not cross-react (≤ 0.01%) with folate analogs or other compounds at ≥ 1000 μmol/L as tested.
| Compound | Texted (μmol/L) |
| Adriamycin | 1000 |
| Cyclophosphamide | 1500 |
| Cytosine | 1000 |
| Dihydrofolic Acid | 1000 |
| DL-6-Methyl-5,6,7,8-Tetrahydropterine | 1000 |
| Folic Acid | 1000 |
| Folinic Acid (leucovorin) | 1000 |
| 5-Fluorouracil | 3000 |
| 6-Mercaptopurine | 1000 |
| 5-Methyltetrahydrofolic acid | 1000 |
| Prednisolone | 1000 |
| Pyrimethamine | 1000 |
| Sulfamethoxazole | 1600 |
| Tetrahydrofolic Acid | 1000 |
| Vinblastine | 1000 |
| Vincristine | 1000 |
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