A UHPLC-QTOF-MS/MS method with a superimposed multiple product ion strategy and esterase inhibitor improved sensitivity for the determination of xylocarpin H in rat plasma

Xylocarpin H is a natural limonoid that has the potential to be a viable therapeutic candidate whether used alone or in combination with other medications. However, the pharmacokinetic properties of xylocarpin H are still not well understood and require further investigation. Sensitive analysis of xylocarpin H using liquid chromatography tandem triple-quadrupole mass spectrometry is challenging due to its structural feature. In the present research, a sensitive UHPLC-QTOF-MS/MS method was developed and validated to determine xylocarpin H in rat plasma. After simple methanol protein precipitation pretreatment with 50 mM esterase inhibitor bis(4-nitrophenyl)-phosphate (BNPP), xylocarpin H and Schisandrin (internal standard) were separated on a Synergi 4 u fusion-RP C-18 column (50 mm x 3.0 mm, 4 mu m) using isocratic elution with a mobile phase consisting of formic acid (0.1%) and acetonitrile, which were detected only within 3 min. The QTOF-MS/MS detection, which is a high-resolution mode, can accurately quantify analytes (ppm<5). The peak area of xylocarpin H was determined by adding the single peak areas of m/z 483.2013, m/z 465.1930 and m/z 345.1333 as the superimposed multiple product ions (SMPI) mode for data processing with optimized the high-resolution extraction widths of fragment ions at +/- 0.05 Da. The S/N of the SMPI mode was found to be approximately two to seven times greater than the independent product ion mode. The linearity, precision, accuracy, and stability were completely validated for the method. Finally, the validated method was employed for the pharmacokinetic study of xylocarpin H in rats. The study indicated that the optimized high-resolution extraction widths of fragment ions and SMPI mode for data processing significantly improve the quantitative sensitivity of QTOF-MS/MS. In addition, esterase inhibitor BNPP was added in the process of sample treatment to inhibit the degradation of xylocarpin H in rat blood, which further improved the sensitivity of the quantitative method.

Automated summary

In this study, a sensitive UHPLC-QTOF-MS/MS method was developed and validated for the determination of xylocarpin H in rat plasma. The optimized high-resolution extraction widths of fragment ions and the superimposed multiple product ions (SMPI) mode significantly improved the quantitative sensitivity of QTOF-MS/MS. The addition of the esterase inhibitor BNPP in the sample treatment process further enhanced the sensitivity of the quantitative method.