HPLC-VWD法同时测定一种Tubulysin衍生物中甲酸及三氟乙酸残留

Simultaneous Determination of Formic Acid and Trifluoroacetic Acid in a Tubulysin Derivative by HPLC-VWD

关键词:  Tubulysin  甲酸  三氟乙酸  验证

Key words:  Tubulysin  formic acid  trifluoroacetic acid  validation

摘要:

目的 采用高效液相色谱 - 紫外法（HPLC-VWD）同时测定一种Tubulysin衍生物中甲酸及三氟乙酸残留。方法 采用 Welch LP- C18 色谱柱（250 mm×4.6 mm，5 μm），流速1.0 mL·min-1，柱温40℃，紫外检测器波长210 nm。流动相A：水（内含0.7%磷酸和0.5%氨水）；流动相B：50%乙腈，梯度洗脱。结果 甲酸和三氟乙酸线性曲线分别为y=1.1408x+0.0459、y=0.4233x-0.0413，相关系数r均为1.00；重复性RSD（n=6）分别为0.4%、0.2%，中间精密度RSD（n=12）分别为0.5%、1.1%；准确度回收率平均值分别为100.5%、101.2%，RSD（n=12）分别为0.6%、2.0%。两者的检测范围分别为2.40~287.29 μg·mL-1，2.62~62.76 μg·mL-1。检测限分别为0.19 μg·mL-1、0.51 μg·mL-1。不同放置时间（0~24 h）、不同流速（1.0 ±0.2 mL·min-1）、不同柱温（40±2℃）、不同检测波长（210±2 nm）、不同批号色谱柱的条件下，结果的RSD均＜5.0%。结论 所建立的方法重复性、耐用性好，可用于同时测定一种Tubulysin衍生物中甲酸及三氟乙酸含量。

Abstract:

ABSTRACT: OBJECTIVE The study aims to simultaneously determine the residues of formic acid and trifluoroacetic acid in a Tubulysin derivative using high-performance liquid chromatography with ultraviolet detection (HPLC-VWD). METHOD The analysis was performed on a Welch LP-C18 chromatographic column (250 mm×4.6 mm, 5 μm) with a flow rate of 1.0 mL·min-1, a column temperature maintained at 40°C, and a detection wavelength set at 210 nm. Mobile phase A consisted of water containing 0.7% phosphoric acid and 0.5% ammonia, while mobile phase B was 50% acetonitrile with gradient elution. RESULTS The linear calibration curves were y=1.1408x+0.0459 for formic acid and y=0.4233x-0.0413 for trifluoroacetic acid, with correlation coefficients (r) of 1.00 for both cases. The repeatability RSD (n=6) was 0.4% and 0.2%, and the intermediate precision RSD (n=12) was 0.5% and 1.1%, respectively. Mean recovery rates were 100.5% and 101.2% with RSD (n=12) being 0.6% and 2.0% respectively. The detection ranges were 2.40~287.29 μg·mL-1 for formic acid and 2.62~62.76 μg·mL-1 for trifluoroacetic acid, with the detection limits of 0.19 μg·mL-1 and 0.51 μg·mL-1 respectively. Under varied conditions, such as different standing times (0~24 h), flow rate variations (1.0±0.2 mL·min-1), column temperature fluctuations (40±2°C), detection wavelength changes (210±2 nm), and batches of column, the RSD values were invariably below 5.0%. CONCLUSION The established method demonstrates good repeatability and robustness, which is suitable for simultaneous determination of formic acid and trifluoroacetic acid content in Tubulysin derivatives.