Determination of methotrexate in biological samples by solid phase extraction and high performance liquid chromatography

Objective: To establish a method for determining the concentration of methotrexate (MTX) in biological samples. METHODS: Solid phase extraction apparatus and enriched samples were used to determine the MTX content in serum and cerebrospinal fluid by high performance liquid chromatography. Chromatographic conditions: C18 column was used as the analytical column. The mobile phase was methanol-acetonitrile-phosphate buffer (2:7:91), the flow rate was 0.8 mL.min-1, the column temperature was 25 °C, and the detection wavelength was 313 nm. Results: Linearity in the range of 0.05-80 μg.mL-1, the recovery rate was above 90%, the low detection limit of MTXzui in serum was 10 ng.mL-1, and the low detection limit of cui in cerebrospinal fluid was 5 ng.mL-1. . Conclusion: This method is simple, rapid, sensitive and specific. It is suitable for MTX monitoring and pharmacokinetic study in biological samples.
Key words solid phase extraction high performance liquid chromatography methotrexate acute lymphoblastic leukemia

Monitoring the concentration of methotrexate (MTX) in body fluids and guiding its rational use of anti-tetralin (flinic aci d, CF) is a recognized necessary measure for high-dose MTX/CF therapy in children with acute lymphoblastic leukemia. . The methods for determining MTX in biological samples include fluorescence spectrophotometry [1, 2], enzyme inhibition [3], radioimmunoassay [4] and microbial methods [1, 5, 6], but all have defects such as poor specificity, MTX. Metabolites and folic acid analogs interfere with the assay and the accuracy of the results is poor. Most of them are now measured by fluorescence polarization immunoassay, but their specificity is poor, and the kit is imported and expensive, and its monitoring is difficult to synchronize with clinical treatment. High performance liquid chromatography can separate and measure MTX and metabolites, so its specificity and accuracy are better than the above methods. High performance liquid chromatography for the determination of MTX in biological samples (serum and cerebrospinal fluid, etc.) is mainly limited by sample pretreatment methods. The pretreatment methods reported in the literature are ion pair extraction [6,7], treated with Seppak cartridge [8]. After acetonitrile and methanol deproteinization, direct injection [9], etc., but the recovery rate is between 38% and 63%, and extremely unstable; in order to improve the sensitivity, the injection volume is 100 μL, due to impurity removal Incomplete, causing serious contamination of the column limits the use of this method. Through repeated exploration, solid phase extraction purification and enrichment samples, reversed-phase high performance liquid chromatography for the determination of MTX in serum and cerebrospinal fluid, the recovery rate is more than 90% and very stable, the method is simple, rapid, specific, and specific Strong and sensitive, it can fully meet the needs of clinical monitoring and pharmacokinetic research.

1 Instruments and materials

HP1100 series high performance liquid chromatography (HP); DL-1 solid phase extractor, Spe column (Dalian Institute of Chemical Physics, Chinese Academy of Sciences); WH-1 type vortex mixer (Shanghai Huxi Analytical Instrument Factory); MTX reference substance (0138-9702, for quantitative analysis, China National Institute for the Control of Pharmaceutical and Biological Products); methanol and acetonitrile are chromatographically pure, water is heavy distilled water; 0.9% sodium chloride injection (produced in this preparation room), other reagents are Analytical purity.

2 Experimental methods and results

2.1 Chromatographic conditions The column is ODS Hypersil (5 μm, 250 mm × 4.6 mm), and the mobile phase is methanol-acetonitrile-7% sodium dihydrogen phosphate and 1% citric acid buffer (2:7:91,). 0.8 mL.min-1, column temperature: 25 °C, detection wavelength: 313 nm, injection volume: 20 μL. The chromatogram is shown in Figure 1. The endogenous impurities in serum and cerebrospinal fluid did not interfere with MTX detection. MTX and its metabolites reached baseline separation with retention time of 14.3 min and 15.4 min, respectively. Chromatographic analysis was completed within 18 min.

Figure 1. 4.33 μg.mL-1 MTX control solution (A) and ALL children with MTX (3 gm-2 iv) 24 h serum (B) and
Chromatogram of cerebrospinal fluid (C) at 2.5 h
1.MTX 2.MTX metabolites

2.2 Preparation of standard curve accurately weighed 50 mg of MTX reference substance dried to constant weight at 100 °C, placed in a 50 mL volumetric flask, made up to volume with methanol, and made 0.05, 0.1, 0.5, 1.0 with 50% methanol. 2.0, 5.0, 10, 20, 40, 80 μg.mL-1 series standard solution, take 20 μL injection test, and regress the peak area (A) with concentration (C) to obtain the regression equation:
A=44.87C+2.01 r=0.999 9
The results showed that the linear relationship of MTX in the range of 0.05-80 μg.mL-1 was good.

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