Using orthogonal array to obtain gradient liquid chromatography conditions of enhanced peak intensity to determine geniposide and genipin with electrospray tandem mass spectrometry

The conditions to determine geniposide and genipin using gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS) via electrospray ionization were obtained using fractional factorial experimental design approaches, guided with Taguchi orthogonal arrays to enhance peak intensity. Geniposide, the major iridoid glycoside component of Gardenia herbs, which has been recognized to have choleretic effects, is transformed to genipin in animals. In this paper, the gradient establishment times, ionization source temperatures, and the concentrations of volatile additive ammonium acetate were investigated under the guidance of experimental designs to obtain LC-MS/MS signals of the highest peak intensity. Using geniposide and genipin standards, the methods are validated at the concentration ranges of 0.5-1000 ng/mL and 10-5000 ng/mL using ammonium adducts. The correlation coefficients of geniposide and genipin standard curves are greater than 0.999. Compared with the sensitivities of previously published LC-MS/MS methods, the methods developed in this work provide 6-fold sensitivity improvement. The lowest concentrations of geniposide and genipin, 0.19 and 2 ng/mL, respectively, to generate detectable LC-MS/MS signal peaks are one order of magnitude lower than the repoered values in previous publications. The measurement accuracy and precision of geniposide are within 23% and 15%, respectively. The accuracy and precision of genipin are within 16% and 12.5%, respectively. When the validated calibration curves of geniposide and genipin are used to determine spiked control samples in rat blood dialysates, the geniposide determination errors are within 15% accuracy and within 5.8% precision, respectively, and the genipin determination errors are within 23% accuracy and within 3.6% precision, respectively.