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HPLC Application Sheet
Methotrexate: Anti-Neoplastic, Anti-Tumor Drug, Analysis by LCMS
Click here to view
printable Application Sheet
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Note: Cogent TYPE-C™ columns require very little time for equilibration when using a gradient. ANP (Aqueous Normal Phase)
is a very MS friendly technique. By using ANP for analysis of polar compounds by LC-MS a 10-100 fold increase of sensitivity is
often observed. The sample can be dissolved in water or water + organic solvent mixture, which is advantageous over other analyses. |
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Method Conditions
| Column |
Cogent Bidentate C18™, 4µm, 100A |
| Catalog No. |
40018-25P |
| Dimensions |
4.6 x 250mm |
| Mobile Phase |
A: DI water + 0.5% formic acid
B: Acetonitrile
ANP gradient - gradient conditions
| Time (min.) |
%A |
%B |
| 0.00 |
10.0 |
90.0 |
| 1.00 |
10.0 |
90.0 |
| 5.00 |
80.0 |
20.0 |
| 10.00 |
80.0 |
20.0 |
| 10.01 |
10.0 |
90.0 |
| 12.00 |
10.0 |
90.0 |
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| Flow rate |
0.5 mL/minute |
| Injection Volume |
1 µL |
| Sample |
Methotrexate m/z 455
0.1 mg/mL in DI water + 0.5% formic acid
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| Detection |
APCI+ Single Ion Monitoring |
Discussion
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The powerful anticancer drug, methotrexate (4-amino-N10-methylpteroyl glutamic acid) acts as an antimetabolite and is used for
the treatment of many neoplastic diseases including acute leukemia, osteosarcoma, non-Hodgkins lymphoma, and breast cancer.
There is a great interest in pharmacological studies and clinical monitoring of methotrexate. A quadrupole mass spectrometer
operating in the positive – ion mode and an atmospheric pressure ionization (API) source was used for selective detection and
assured that no interfering peaks affect the quantitative results. A Cogent bidentate C18 column was the column of choice for
the ANP gradient analysis of the drug. The retention of the methotrexate is more than sufficient. The LC-MS method developed
assures both high specificity and sensitivity. |
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