A method for the determination of caffeine in urea was developed based on bubble-in-drop single drop microextraction (BID-SDME) followed by gas chromatography/mass spectrometry (GC-MS).Under the optimum conditions including chloroform as extraction solvent, an exposure volume of 1 μL, a bubble volume of 1.6 μL, stirring for 5 min at 300 r/min, 15% (m/V) NaCl, and a distance of 1 cm between bubble and stirring bar, the detection limit of this method was as low as 0.003 mg/L and the linear range was from 0.005 mg/L to 10 mg/L with correlation coefficient of 0.982.The recoveries of caffeine were from 89.2% to 107.5% at different spiked levels in human urine and the relative standard deviation (RSD, n=6) was less than 8%.

Purpose To explore the association of NET-1 expression with the angiogenesis of cervical squamous cell carcinoma and the related molecular mechanism.Methods Immunohistochemistry MaxVision was used to detect the expression of NET-1,NF-κB p65 and VEGF in 80 cases of cervical squamous cancer tissue and 10 cases of chronic cervicitis tissue.The microvascular density (MVD) was assessed by labeling endothelia with CD34-antibody and counted according to Weinner's standard.The eukaryotic expression plasmid pCMV6-NET-1 was transfected into cervical squamous cancer cells by cationic liposome in order to obtain over expression of NET-1 gene.In order to inhibit the activation of NF-kappa B,cervical squamous carcinoma cells were treated with NF-kappa B specific inhibitor PDTC.The protein expression in SiHa cells was assessed by Western blotting.Results The expression level of NET-1 in the cervical squamous carcinoma tissue was significantly higher than that in the tissue of control group.NET-1 expression was positively correlated to the MVD,NF-κB p65 and VEGF expression,respectively.Transient transfection of NET-1 gene significantly increased the nuclear expression of NF-κB p65 and cytoplasm expression of VEGF in the SiHa cells.However,pretreatment of SiHa cells with NF-inhibitor PDTC significantly inhibited NET-1 induced upregulation of VEGF expression.Conclusion NET-1 promotes the angiogenesis of cervical squamous carcinoma through the activation of the NF-κB signaling which upregulate the expression of VEGF in the cancer cells.