A type of testosterone-imprinted polymer film grafted from porous silica was prepared by covalently binding azo-initiators and then photo-grafting. Elemental analysis and infrared (IR) spectroscopy attested the polymer formation, The material could be polymerized within 10-60min with reproducible grafting kinetics, controlla-ble film thickness, and obviously specific recognition ability to testosterone with the imprinting factor of i.52. Due to its uniform size, spherical shape, controllable film thickness, and accessible sites near or at the surface, this polymer could serve as a sensing element, solid-phase extraction material, or chromatographic stationary phase to selectively recognize or separate testosterone.

Based on the 2-propanol-salt-H2O system, a new and simple pretreatment method was developed for the analysis of testosterone and epitestosterone in human urine coupled with high-performance liquid chromatography (HPLC). The influence factors on partition behaviors of the steroids were studied including the type and amount of salts, the volume of 2-propanol, the concentration of the analytes, and temperature. When K3PO4, K2HPO4, K2CO3 and KOH were chosen as phase separation salts, the enrichment factors were 12, 10, 12 and 13, respectively, and the extraction efficiencies for testosterone and epitestosterone were all 80-90%. In the optimal system of 2-propanol-K3PO4-H2O, the recoveries of the spiked standards for the analytes were all 92-102% with relative standard deviation of 2.3-7.2%. Combined with a HPLC method, this extraction technique has been successfully applied to the analysis of testosterone and epitestosterone in human urine with the detection limits of 1 ng/ml and the linear ranges of 10-500 ng/ml for both compounds. Compared with conventional liquid-liquid extraction or solid-phase extraction, this novel method is much simpler and more environmentally friendly due to the direct injection of the upper phase into HPLC system, no use of harmful organic solvent, and low consumption of extractant.

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl)/salt aqueous two-phase systems (ATPS) was presented as a simple, rapid and effective sample pretreatment technique coupled with high-performance liquid chromatography (HPLC) for analysis of the major opium alkaloids in Pericarpium papaveris. To find optimal conditions, the partition behaviors of codeine and papaverine in ionic liquid/salt aqueous two-phase systems were investigated. Various factors were considered systematically, and the results indicated that both the pH value and the salting-out ability of salt had great influence on phase separation. The recoveries of codeine and papaverine were 90.0-100.2% and 99.3-102.0%, respectively, from aqueous samples of P. papaveris by the proposed method.

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (kass and kdiss) and equilibrium dissociation constants (KD) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10-5M, which is within the range of 10-4-10-8M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanninng of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.