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To establish a hollow fiber centrifugal ultrafiltration (HFCF-UF) coupled with ultra-high performance liquid chromatography (UPLC) method for the analysis of free methotrexate (MTX) concentration in human plasma and apply to clinical therapeutic drug monitoring.
Plasma samples 500 μL were prepared with HFCF-UF. A Waters UPLC BEH C18 column (50.0 mm×2.1 mm, 1.7 μm) was used. The mobile phase consisted of methanol and 0.05 mol·L-1 phosphate buffer (pH 6.2) (17∶83, v/v) at a flow rate of 0.2 mL·min-1. The column temperature was maintained at 30 ℃. The detected wavelength was 302 nm. The specificity, linear relationship, lower limit of quantification (LLOQ), precision, recovery rate and stability of this method were investigated, and it was applied to the determination of clinical plasma samples.
The good linear relationship were obtained between concentration of free MTX in human plasma from 0.05-10.00 μmol·L-1, y=0.297x+0.001 (r2=0.999). The LLOQ of MTX free plasma concentration analysis was 0.05 μmol·L-1. The method recovery rates of free MTX were 95.77%-100.52%. The absolute recovery rates of free MTX was 84.09%-92.93%. The intra-day and inter-day relative standard deviation (RSD) were all less than 7.0 %. It was successfully used to analyze free MTX concentration in 52 plasma samples from patients with MTX chemotherapy. The average free plasma concentration of MTX in these 52 patients was 0.68 μmol·L-1, with the maximum value of 9.32 μmol·L-1 and the minimum value of 0.056 μmol·L-1.
The developed method is simple, accurate and sensitive, which is suitable for the analysis of free MTX on clinical therapeutic drug monitoring.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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