OBJECTIVE To establish a ultra-high-performance liquid chromatography-mass spectrum/mass spectrum(UPLC-MS/MS) method for the determination of anlotinib in human plasma and assessment of clinical application. METHODS Zanubrutinib was used as internal standard and the extraction process was performed through protein precipitation method using acetonitrile, followed by separation on an Ultimate XB-C18(100 mm×2.1 mm, 3.0 μm) column using acetonitrile and 10 mmol·L−1 ammonium acetate-0.1% formic acid step-elution gradient. The flow rate was 0.6 mL·min−1 and injection volume was 5 μL. The mass analysis was performed by positive ion electrospray ionization in multiple-reaction monitoring mode, and the mass spectrometer was set at m/z 408.1→339.1 for anlotinib and m/z 472.2→290.1 for internal standard, respectively. The specificity, standard curve and lower limit of quantification, precision and recovery, matrix effect and stability of the method and clinical application were investigated. RESULTS The method was validated over the concentration range of 1.0−100.0 ng·mL−1, with R2=0.998 4. The precision RSD was<9%, the recovery and matrix effect were 104.81%−107.32% and 102.54%−105.26%, respectively, and this method had good stability and was not affected by matrix effect. The method had been used for determined 52 advanced non-small cell lung cancer patients treated with anlotinib. The trough plasma concentration (Ctrough) was measured on day 43 after initiation of anlotinib treatment. Anlotinib Ctrough were higher than lower limit of quantitation (1.0 ng·mL−1) from 52 patients. The plasma concentration of anlotinib Ctrough was (11.38±4.29)ng·mL−1 with 37.66% coefficients of variation, which were shown large inter-patient variability. CONCLUSION This method is high sensitivity, specificity and accurate, and suitable for determination of anlotinib in human plasma.

Through PCR amplification, 1.3 kb of 5'-proximal promoter (TA, 1.3 kb) of the beta-actin gene of white cloud mountain minnow Tanichthys albonubes was obtained. Using Genome Walker, a 1.7 kb 5'-upstream sequence from the proximal promoter of the beta-actin gene was isolated, and a further promoter (3.0 kb in size) was amplified according to the isolated 5'-proximal and upstream sequences (TLA, 3.0 kb). Both the 1.3 kb and 3.0 kb promoters contain elements that were critical to the transcription activity of other species, including the CCAAT Box (-89 approximately -85), CArG Box (-59 approximately -49), TATA Box (-26 approximately -20). Results of putative transcription binding sites analysis of the promoters by software TRANSFAC 6.0 revealed the presence of E-box, several transcript binding sites NF-Y, SP1 (Stimulating Protein 1), AP1 (Activator Protein 1), and some more transcription binding sites existing in the further promoter. The two promoter sequences were inserted into the expression vector to construct the recombinant expression vector, pTA-DsRed and pTLA-DsRed, respectively. The vectors were microinjected into the fertilized eggs of Tanichthys albonubes and higher positive rate was obtained and stronger red fluorescence was observed in pTLA-DsRed transgenic fish. RT-PCR analysis showed that RFP (Red fluorescent protein) mRNA level in pTLA-DsRed transgenic fish was 35.7% higher than that of the pTA-DsRed transgenic fish of 15-days-post-hatched. The present study showed that both the proximal and further promoter sequences have effective transcription activities and the 3.0 kb promoter possesses higher potent activity than that of the 1.3 kb promoter.
5' Untranslated Regions ; genetics ; Actins ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Cyprinidae ; genetics ; Genetic Vectors ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism