Methotrexate Assay
Accurately screen for MTX with low-end sensitivity.

Methotrexate Assay
 
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The ARK Methotrexate Assay is the only liquid methotrexate assay on the market. It provides a broad assay range of 0.04–1.20 µmol/L for accurate measurements of Methotrexate. The measurements obtained are used in monitoring levels of methotrexate to help ensure appropriate therapy. Methotrexate is an antimetabolite used in the treatment of specific cancers, psoriasis, rheumatoid arthritis, and other autoimmune conditions.

Methotrexate Assay

Methotrexate Assay

Features & Benefits

The ARK Methotrexate assay is the only liquid methotrexate assay on the market. It provides an assay range of 0.04–1.20 µmol/L that meets customers’ needs for low-end sensitivity.
 

  • Methotrexate is an antineoplastic drug that is used in the treatment of specific cancers (especially pediatric), psoriasis, rheumatoid arthritis, and other autoimmune conditions.

 

  • Methotrexate is extremely potent and has the potential to be very toxic. It inhibits a key enzyme and affects DNA/RNA synthesis in the cells. Thus, it prevents the growth of cells that grow rapidly, such as cancer, bone marrow, and skin cells.

 

  • Methotrexate serum levels are dependent on many variables, including dose, mode of administration, individual metabolism, and individual pharmacokinetics. Leucovorin is usually administered intravenously as a rescue (counteracting) therapy.


Guidelines for methotrexate therapy with leucovorin rescue usually recommend continuance of leucovorin until the methotrexate level falls below 0.05 μmol/L, although ≤0.10 μmol/L is sometimes followed. This is why low-level sensitivity is so important, especially for pediatric patients.

Technical Specifications

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 RunBetween DayTotal
SampleNMean (µg/mL)SDCV (%)SDCV (%)SDCV (%)
ARK Methotrexate Control
LOW1600.060.0058.20.0057.30.00710.7
MID1600.370.0113.00.0082.10.0143.8
HIGH1600.760.0324.30.0304.00.0455.9
5*1604.80.153.10.132.80.204.2
50*160491.362.82.324.82.725.6
500*16047615.173.230.756.534.667.3
Patient Pool
LOW1600.070.0069.10.0057.50.00811.7
MID1600.410.0133.30.0266.40.0297.2
HIGH1600.820.0374.50.0435.20.0577.0
5*1604.60.143.10.1834.00.245.3
50*160451.333.02.635.92.936.6
500*16046111.842.627.045.929.606.4

 

*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 Lot7-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
D25.00.060.060.01
D35.00.040.050.02
Cross Reactivity 7-OH MTX in the presence of 0.05 µmol/L MTX
D250.00.440.470.07
D350.00.460.480.05

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.

CompoundTested (µmol/L)Apparent MTX (µmol/L)Cross Reactivity (%)
MTX Absent
Triamterene250.461.85
Trimethoprim1000.170.17
MTX Present 0.05 (µmol/L)
Triamterene250.893.32
Trimethoprim1000.160.12
MTX Present 0.05 (µmol/L)
Triamterene251.042.31
Trimethoprim1000.990.54

 

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.

CompoundTexted (μmol/L)
Adriamycin1000
Cyclophosphamide1500
Cytosine1000
Dihydrofolic Acid1000
DL-6-Methyl-5,6,7,8-Tetrahydropterine1000
Folic Acid1000
Folinic Acid (leucovorin)1000
5-Fluorouracil3000
6-Mercaptopurine1000
5-Methyltetrahydrofolic acid1000
Prednisolone1000
Pyrimethamine1000
Sulfamethoxazole1600
Tetrahydrofolic Acid1000
Vinblastine1000
Vincristine1000

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