Marrari P, Pianezzola E, Strolin Benedetti M
Pharmacia, Department of Pharmacokinetics and Metabolism, Milan, Italy.
J Chromatogr B Biomed Appl. 1996 Feb 23;677(1):133-9. doi: 10.1016/0378-4347(95)00437-8.
A sensitive and reproducible HPLC method for the determination of tallimustine (I) in human plasma has been developed and validated. Compound I was extracted from plasma by solid-phase extraction using a C18 cartridge from which the test compound was eluted with a methanol-formic acid mixture. The methanol solution was evaporated to dryness and the residue dissolved in a 0.2 M formic acid in methanol-water (1:1, v/v) mixture, then injected onto the HPLC column. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS, using a 50 mM KH2PO4-acetonitrile mixture as the mobile phase. The flow-rate was 1 ml/min. The eluate was monitored at 314 nm. No peak interfering with that of I was observed when blank human plasma was assayed. Linearity was established in the concentration range 0.5-85.5 nanograms of I per millilitre of plasma. Four calibration curves in plasma, prepared and run on four different days, showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V. = 4.5%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V. ranged from 0.9 to 14.4%. The inter-day precision evaluated at the same concentrations was better than 10.2%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of I, ranged from 86.2 to 108.5%. The limit of quantitation was 0.5 ng/ml plasma. The HPLC method described here was successfully employed for the determination of I in some plasma samples obtained during a phase I clinical trial with the test compound.
已开发并验证了一种灵敏且可重现的高效液相色谱法，用于测定人血浆中的他利莫司（I）。化合物I通过使用C18柱的固相萃取从血浆中提取，测试化合物用甲醇 - 甲酸混合物从柱上洗脱。将甲醇溶液蒸发至干，残余物溶解于甲醇 - 水（1:1，v/v）的0.2 M甲酸混合物中，然后注入高效液相色谱柱。色谱分离通过填充有ODS的反相柱等度进行，使用50 mM KH2PO4 - 乙腈混合物作为流动相。流速为1 ml/min。在314 nm处监测洗脱液。测定空白人血浆时未观察到干扰I峰的峰。在每毫升血浆中I的浓度范围为0.5 - 85.5纳克时建立了线性关系。在四个不同日期制备并运行的血浆中的四条校准曲线显示相关系数高于0.99，斜率具有良好的重现性（变异系数 = 4.5%）。日内精密度在三个浓度（标准曲线的低、中、高范围）下评估，以变异系数表示，范围为0.9%至14.4%。在相同浓度下评估的日间精密度优于10.2%。在相同样品中评估的日间准确度以I的测定值/添加量的比率表示，范围为86.2%至108.5%。定量限为0.5 ng/ml血浆。本文所述的高效液相色谱法已成功用于在一项使用测试化合物的I期临床试验期间获得的一些血浆样品中I的测定。
