High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma
High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma
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DOI：
10.1016/j.jchromb.2004.11.035
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发表时间：
2005-02-25
影响因子：
3
通讯作者：
Balthasar, JP
中科院分区：
文献类型：
--
作者：
Chen, J;Balthasar, JP
建立了一种快速、灵敏的高效液相色谱(HPLC)分析方法，可在有或无抗拓扑替康(TPT)抗体存在和不存在的情况下，测定小鼠血浆中总的(即结合的和未结合的)和游离(即未结合的)拓扑替康(TPT)。色谱柱为Nova-Pak C18柱(3.9 mm×150 min，4 um)，流动相为10 mM磷酸二氢钾-甲醇-三乙胺(72：26：2(v/v/v)，pH 3.5)。拓扑替康的荧光检测波长为361 nm，发射波长为527 nm。内标保留时间为7.4 m in，吖啶保留时间为9.0 m in。TPT在小鼠血浆和超滤液中的定量下限(LOQ)为0.02 ng，相当于20只小鼠血浆中TPT的浓度为1 ng/ml。该方法在1-500 ng/ml浓度范围内线性关系良好，对小鼠血浆中游离TPT和总TPT的回收率均在理论值的10%以内(分别为1、20和500 ng/ml)。建立了TPT在瑞士Webster小鼠、BALB/c小鼠和高免BALB/c小鼠体内的药代动力学模型。该方法能高灵敏度地测定小鼠血浆中总TPT和游离TPT，并可用于检测抗TPT抗体对TPT处置的影响。(C)2004爱思唯尔B.V保留所有权利。
A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm x 150 min, 4 mum) protected by a Nova-Pak C18 guard column (3.9 nun x 20 mm, 4 mum), where 10 mM KH2PO4-methanol-triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 mul mouse plasma. The assay was shown to be linear over a concentration range of 1-500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT. (C) 2004 Elsevier B.V All rights reserved.