Simultaneous Determination of Formic Acid and Trifluoroacetic Acid Residues in Tubulysin Derivatives by HPLC

OBJECTIVE To simultaneously determine the residues of formic acid and trifluoroacetic acid in Tubulysin derivatives using high-performance liquid chromatography(HPLC).

METHODS 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, 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 <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.