7-hydroxy Methotrexate
(Synonyms: 7-羟基甲氨蝶呤) 目录号 : GC49051
A metabolite of methotrexate
Cas No.:5939-37-7
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
7-hydroxy Methotrexate (7-hydroxy MTX) is the major metabolite of the folic acid derivative MTX .1 It is formed by the 7-oxidation of MTX by hepatic aldehyde oxidases. Unlike MTX, 7-hydroxy MTX does not inhibit thymidylate synthase in isolated human acute myeloid and acute lymphoblastic leukemia cells.2 7-hydroxy MTX has a maximum tolerated dose of 0.1 g/kg, which is 30- to 50-fold lower than MTX, in rats.3 Serum levels of 7-hydroxy MTX have been used as a marker of MTX toxicity during chemotherapy.4,5
1.Johns, D.G., Iannotti, A.T., Sartorelli, A.C., et al.The relative toxicites of methotrexate and aminopterinBiochem. Pharmacol.15(5)555-561(1966) 2.Lingg, R.M., Hempel, G., Rots, M.G., et al.Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assayCancer Chemother. Pharmacol.56(3)322-327(2005) 3.FuskevÅg, O.M., Kristiansen, C., Lindal, S., et al.Maximum tolerated doses of methotrexate and 7-hydroxy-methotrexate in a model of acute toxicity in ratsCancer Chemother. Pharmacol.46(1)69-73(2000) 4.Schofield, R.C., Ramanathan, L.V., Murata, K., et al.Development of an assay for methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum by LC-MS/MSMethods in Molecular Biology213-222(2016) 5.Klapkova, E., Kukacka, J., Kotaska, K., et al.The influence of 7-OH methotrexate metabolite on clinical relevance of methotrexate determinationClin. Lab.57(7-8)599-606(2011)
| Cas No. | 5939-37-7 | SDF | |
| 别名 | 7-羟基甲氨蝶呤 | ||
| Canonical SMILES | NC1=C2C(NC(C(CN(C3=CC=C(C=C3)C(N[C@H](C(O)=O)CCC(O)=O)=O)C)=N2)=O)=NC(N)=N1 | ||
| 分子式 | C20H22N8O6 | 分子量 | 470.4 |
| 溶解度 | DMSO: slightly soluble,Ethanol: slightly soluble | 储存条件 | -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.1259 mL | 10.6293 mL | 21.2585 mL |
| 5 mM | 0.4252 mL | 2.1259 mL | 4.2517 mL |
| 10 mM | 0.2126 mL | 1.0629 mL | 2.1259 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Simultaneous Determination of Urine Methotrexate, 7-hydroxy Methotrexate, Deoxyaminopteroic Acid, and 7-Hydroxy Deoxyaminopteroic Acid by UHPLC-MS/MS in Patients Receiving High-dose Methotrexate Therapy
Anal Sci 2020 Dec 10;36(12):1479-1486.PMID:32801284DOI:10.2116/analsci.19P481.
Nephrotoxicity, the most important toxicity in high-dose methotrexate (MTX) therapy, is partly caused by the formation of crystal deposits in the kidney due to poor water solubility of MTX and its metabolites 7-hydroxy Methotrexate (7-OH MTX), deoxyaminopteroic acid (DAMPA) and 7-hydroxy deoxyaminopteroic acid (7-OH DAMPA). Plasma MTX level-guided urine alkalinization, leucovorin rescue and glucarpidase detoxification are common strategies to overcome MTX-related nephrotoxicity. However, overestimation is a problem for MTX analysis by immunoassays due to the cross-reactivity of MTX metabolites (7-OH MTX and DAMPA). An UHPLC-MS/MS method for the simultaneous determination of MTX, 7-OH MTX, DAMPA and 7-OH DAMPA in human urine was developed, validated and applied in clinical practice. Samples were treated by one-step protein precipitation and analyzed within 3 min. The calibration range was 0.02 to 4 μmol/L for MTX and DAMPA, and 0.1 to 20 μmol/L for 7-OH MTX and 7-OH DAMPA. For all analytes, the intra-day and inter-day bias and imprecision were -8.0 to 7.6 and <9.0%, the internal standard normalized recovery and matrix factor were 92.34 to 109.49 and <20.68%. The plasma MTX and 7-OH MTX levels increased with the urine drug levels, age, serum creatinine and alanine transaminase, but urine could not replace blood for MTX monitoring due to their poor correlation (R2, 0.16 to 0.51). Dose-normalized urine and plasma MTX and 7-OH MTX levels were similar between different patient groups (urine pH <7 or ≥7). Due to the large inter-individual variance of the analytes levels in both plasma and urine, these findings should be treated with caution.
Simultaneous determination of plasma methotrexate and 7-hydroxy Methotrexate by UHPLC-MS/MS in patients receiving high-dose methotrexate therapy
J Pharm Biomed Anal 2018 Sep 5;158:300-306.PMID:29909319DOI:10.1016/j.jpba.2018.06.011.
The plasma concentrations of methotrexate (MTX) and its major metabolite 7-hydroxy Methotrexate (7-OH-MTX) are highly correlated with the toxicities in patients with high-dose MTX therapy. Routine monitoring of MTX and 7-OH-MTX plasma levels is useful for dose adjustment of rescue drugs and toxicity prevention. A UHPLC-MS/MS method for simultaneous determination of plasma MTX and 7-OH-MTX was developed, validated, and applied in 181 plasma samples. The ion transition was m/z 455.2 → 308.2 for MTX and m/z 471.2 → 324.1 for 7-OH-MTX. The flow rate was 0.4 mL/min with a run time of 2.6 min. The calibration range was 0.002-2 μM for MTX, and 0.01-10 μM for 7-OH-MTX. The intra-day and inter-day inaccuracy and imprecision were -5.50% to 10.93% and less than 9.20% for both analytes. The internal standard (MTX-D3) normalized recovery and matrix factor were consistent at four quality control levels. 14 h, 38 h, and 62 h after dosing, MTX and 7-OH-MTX plasma levels were significantly higher in patients with impaired renal function compared to those with normal renal function. 7-OH-MTX plasma levels were significantly higher in patients with impaired liver function compared to those with normal liver function.
Effects and interaction of 7-hydroxy Methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay
Cancer Chemother Pharmacol 2005 Sep;56(3):322-7.PMID:15868145DOI:10.1007/s00280-005-1032-1.
In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 microM for MTX alone versus 0.088 microM for the 1:1 mixture with 7-OH MTX, respectively (p< or =0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.
Development of an Assay for Methotrexate and Its Metabolites 7-hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS
Methods Mol Biol 2016;1383:213-22.PMID:26660190DOI:10.1007/978-1-4939-3252-8_23.
Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to 1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.
Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy Methotrexate and DAMPA in serum
J Chromatogr B Analyt Technol Biomed Life Sci 2015 Oct 1;1002:169-75.PMID:26322588DOI:10.1016/j.jchromb.2015.08.025.
A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy Methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.