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.