Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry.

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC-MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00-1000 ng/mL for forsythin, 2.50-2500 ng/mL for KD-2-Glc, and 5.00-5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC-MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.

Analysis of codon use features of stearoyl-acyl carrier protein desaturase gene in Camellia sinensis.

The stearoyl-acyl carrier protein desaturase (SAD) gene widely exists in all kinds of plants. In this paper, the Camellia sinensis SAD gene (CsSAD) sequence was firstly analyzed by Codon W, CHIPS, and CUSP programs online, and then compared with genomes of the tea plant, other species and SAD genes from 11 plant species. The results show that the CsSAD gene and the selected 73 of C. sinensis genes have similar codon usage bias. The CsSAD gene has a bias toward the synonymous codons with A and T at the third codon position, the same as the 73 of C. sinensis genes. Compared with monocotyledons such as Triticum aestivum and Zea mays, the differences in codon usage frequency between the CsSAD gene and dicotyledons such as Arabidopsis thaliana and Nicotiana tobacum are less. Therefore, A. thaliana and N. tobacum expression systems may be more suitable for the expression of the CsSAD gene. The analysis result of SAD genes from 12 plant species also shows that most of the SAD genes are biased toward the synonymous codons with G and C at the third codon position. We believe that the codon usage bias analysis presented in this study will be essential for providing a theoretical basis for discussing the structure and function of the CsSAD gene.