Methotrexate AssayAccurately screen for MTX with low-end sensitivity.

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.

Proven homogeneous enzyme immunoassay similar to EMIT® technology

Applications available for ADVIA® Chemistry Systems, Dimension® and Dimension Vista® Integrated Systems, and Viva™ Drug Testing Systems

Convenient, liquid-stable, ready-to-use reagents

10X more sensitive than Syva Emit assay—can provide physicians with results when needed, especially at 72-hour mark post-dose, when levels are expected to be <0.05

Very low cross-reactivity to 7-hydroxymethotrexate (<0.07%)

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 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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Methotrexate AssayAccurately screen for MTX with low-end sensitivity.

Methotrexate Assay

Features & Benefits

Technical Specifications

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