Ultra performance liquid chromatography tandem mass spectrometry assay for determination of kukoamine B in human blood and urine

• As far as we know, this is the first paper which describes the quantitation method for kukoamine B (KB).
• In this paper, an UPLC–MS/MS method was developed and validated to quantify KB in human blood and urine, and was successfully applied to the first-in-human pharmacokinetic study of KB in Chinese healthy volunteers.
• The run time of this method is only 1.5 min.

In this paper, we report a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method which is capable of quantifying kukoamine B (KB) levels in human blood and urine. Following solid phase extraction and direct dilution process, the analyte and its internal standard (D5-KB) run on an Acquity UPLC® HSS T3 column (2.1 × 50 mm i.d., 1.8 μm) by using a gradient elution method (run time was 1.5 min). The mass spectrometric analysis was performed by using an API-5500 mass spectrometer coupled with an electro-spray ionization source. The MRM transitions of m/z 531.3+ → 222.1+ and 536.3+ → 222.1+ were used to quantify KB and D5-KB respectively. This assay method has been fully validated in terms of selectivity, linearity, lower limit of quantification, precision, accuracy, stability, recovery and matrix effect. The concentration range of this method is 10.0–2000.0 ng mL−1 in blood and 0.5–500.0 ng mL−1 in urine. Linearity (R2) of calibration curves were 0.9964 ± 0.0022 and 0.9935 ± 0.0053 for blood and urine, respectively (regression equation: y = ax + b). The precision (RSD%) of quality control samples is less than 10.3% for blood and less than 10.5% for urine. The accuracy (RE%) is within −4.0–11.3% and −11.7–12.5% for blood and urine respectively. KB was stable after 4 h in ice-water bath, 1 freeze/thaw cycles and 180 days at −80 °C for blood samples; and was stable after 3 h at room temperature, 3 freeze/thaw cycles and 180 days at −80 °C for urine samples. Recoveries of KB were 4.7 ± 0.9% in blood and 96.5 ± 1.3% in urine, respectively. Additionally, the applicability of this method has been proved by analyzing clinical samples from pharmacokinetic study of KB in human.