Etienne M C, Oster W, Milano G
Centre Antoine Lacassagne, Oncopharmacology unit, Nice, France.
Cancer Chemother Pharmacol. 1996;38(4):343-8. doi: 10.1007/s002800050493.
The purpose of the present study was to develop and validate a stereo-specific high-performance liquid chromatography (HPLC) assay for rogletimide (Rog) and rogletimide-N-oxide (Nox) isomers in plasma. The assay was performed with a chiral cellulose-[4-methylbenzoate]ester column (Chiracel OJ). Optimal separation was achieved isocratically with a mobile phase consisting of n-hexane/anhydrous ethanol (65/35, v/v) at a flow rate of 0.9 ml/min, with the column being thermostated at +35 degrees C (UV detection at 257 nm). Under these conditions, retention times were approximately 17, 28, 31 and 76 min for R-Rog, S-Rog, R-Nox and S-Nox, respectively. S-aminoglutethimide (S-Ag) served as the internal standard (retention time 70 min). An extraction procedure from plasma samples was developed on Bond Elut RP8 500-mg cartridges; conditioning was performed with 5 ml methanol and 5 ml water, after which 1 ml plasma that had previously been spiked with 5 microM S-Ag was applied. Washing was done with 6 ml water and elution, with 4 ml methanol. After evaporation to dryness, residues were dissolved in 400 microliters anhydrous ethanol and 12-48 microliters was injected onto the HPLC system. Blank plasma from healthy donors showed the random presence of a small interference eluting at the retention time of R-Rog, precluding the accurate quantification of R-Rog concentrations below 2.5 microM. Reproducibility assays demonstrated the need to use an internal standard. Taking into account the internal standard, at 2.5 microM the intra- and inter-assay coefficients of variation were 10.5% and 21.0% for R-Rog 5.5% and 8.7% for S-Rog, 7.6% and 20.8% for R-Nox and 11.7% and 6.4% for S-Nox, respectively. The detection limit was 2.5 microM for R-Rog, 0.5 microM for S-Rog, 0.25 microM for R-Nox and 0.5 microM for S-Nox. Linearity was satisfactory at concentrations ranging from 2.5 to 10 microM for R-Rog, from 0.5 to 10 microM for S-Rog, from 0.25 to 2.5 microM for R-Nox and from 0.50 to 2.5 microM for S-Nox. This assay was applied to plasma obtained from rog-letimide-treated breast cancer patients receiving conventional oral doses and demonstrated its feasibility with regard to sensitivity. The preliminary pharmacokinetic results reported herein suggest for the first time that both the R-Rog and S-Rog isomers are metabolized into rogletimide-N-oxide.
本研究的目的是开发并验证一种用于测定血浆中罗格列胺（Rog）和罗格列胺 - N - 氧化物（Nox）异构体的立体特异性高效液相色谱（HPLC）分析方法。该分析使用手性纤维素 - [4 - 甲基苯甲酸酯]酯柱（Chiralcel OJ）进行。以正己烷/无水乙醇（65/35，v/v）为流动相，流速为0.9 ml/min，在柱温为 +35℃（257 nm 处紫外检测）的条件下等度洗脱，实现了最佳分离。在此条件下，R - Rog、S - Rog、R - Nox和S - Nox的保留时间分别约为17、28、31和76分钟。S - 氨基谷氨酰胺（S - Ag）用作内标（保留时间70分钟）。在Bond Elut RP8 500 - mg柱上开发了从血浆样品中提取的方法；先用5 ml甲醇和5 ml水进行柱平衡，然后加入1 ml预先加入5 μM S - Ag的血浆。用6 ml水洗涤，并用4 ml甲醇洗脱。蒸发至干后，残渣溶于400 μl无水乙醇中，取12 - 48 μl注入HPLC系统。健康供体的空白血浆在R - Rog保留时间处有少量随机干扰峰出现，这使得低于2.5 μM的R - Rog浓度无法准确定量。重现性分析表明需要使用内标。考虑到内标，在2.5 μM时，R - Rog的批内和批间变异系数分别为10.5%和21.0%，S - Rog为5.5%和8.7%，R - Nox为7.6%和20.8%，S - Nox为11.7%和6.4%。R - Rog的检测限为2.5 μM，S - Rog为0.5 μM，R - Nox为0.25 μM，S - Nox为0.5 μM。R - Rog在2.5至10 μM浓度范围内、S - Rog在0.5至10 μM浓度范围内、R - Nox在0.25至2.5 μM浓度范围内以及S - Nox在0.50至2.5 μM浓度范围内线性良好。该分析方法应用于接受常规口服剂量罗格列胺治疗的乳腺癌患者的血浆，证明了其在灵敏度方面的可行性。本文报道的初步药代动力学结果首次表明，R - Rog和S - Rog异构体均代谢为罗格列胺 - N - 氧化物。
