Simultaneous determination of corynoline and acetylcorynoline in human urine by LC–MS/MS and its application to a urinary excretion study

• An LC–MS/MS method for determination of CRL and ACR in urine was developed.
• The method was highly sensitive with the LLOQ of 3.0 pg/mL for both analytes.
• The method was applied to a urinary excretion study in healthy human volunteers.
• The urinary excretion profiles of CRL and ACR in human were first reported.

Corynoline and acetycorynoline, the major active components derived from Corydalis bungeana Herba, showed multiple pharmacological activities. However, quantification of the two compounds in human urine has not been reported. A simple liquid chromatography with tandem mass spectrometry method for the simultaneous determination of corynoline and acetycorynoline in human urine has been developed and fully validated. The analytes were extracted from urine samples by simple liquid-liquid extraction. Chromatographic separation was achieved on a Hedera ODS-2C18 column with the mobile phase of water (containing 0.5% formic acid) and acetonitrile (28:72, v/v) at a flow rate of 0.4 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring via an electrospray ionization source in positive mode. The monitored ion transitions were m/z 368.1 → 289.1 for corynoline, m/z 410.2 → 289.2 for acetycorynoline and m/z 380.2 → 243.2 for donepezil (internal standard), respectively. The calibration curves were linear (correlation coefficients > 0.9970) over the concentration ranges of 3.0–3000 pg/mL for corynoline and 3.0–1000 pg/mL for acetycorynoline. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 3.0 pg/mL for both analytes. The intra- and inter-day precision was lower than 10% in terms of relative standard deviation for the low, medium, and high quality control samples, and lower than 16% for the LLOQ samples of the analytes. The accuracy was within ±10% in terms of relative error for both analytes. The method was successfully applied to a urinary excretion study after oral administration of the Chinese medicine formula Shuanghua Baihe tablets in healthy volunteers. The urinary excretion profiles of corynoline and acetycorynoline in human were first reported. The results of this study suggest that renal excretion was not the main excretion pathway of corynoline and acetycorynoline in humans.