Target-specific affinity separation of the bioactive compounds from herbal extract using the spin column packed with the immobilized protein microspheres prior to LC-MS analysis.

Excellent pretreatments before instrumental analysis are critical for separation and determination of target compounds for discovery of new drugs from herb medicines. We developed a rapid and highly-selective method to separate the bioactive compounds from herbal extract using protein affinity-selection spin column, which was packed with the new sorbent materials from integrating the recombinant ß2-adrenoceptor (ß2-AR) directly out of cell lysates onto the surface of microspheres. Protein affinity-selection spin column was placed in a centrifugal tube, where after the non-specific binders were released to the filtrate under the operational centrifugation, the specific binders on the spin column were cleaned with a washing solvent for LC-MS analysis. The known agonists of ß2-AR were retained/released on protein affinity-selection spin column but not on control column, demonstrating the method with good recovery (79.4∼95.7 %) and high repeatability (RSD < 3.5 %). The adsorption features of three ligands on the spin column were described best by Prism saturation binding model, and the high-affinity binding and the large binding capacity of the spin column make it feasible to trap the trace analytes effectively. It was applied in separating bioactive compounds from Alstoniae Scholaris extract, two of which were identified as picrinine and oleanolic acid in combination with LC-MS and verified as the potential agonists towards ß2-AR though molecular docking and cell experiments. Our study demonstrated that, the spin column with the immobilized protein sorbents in the centrifugal filter device represents a promising tool, enabling rapid and target-specific affinity separation of the bioactive compounds from herbal extract.

The screened compounds from Ligustri Lucidi Fructus using the immobilized calcium sensing receptor column exhibit osteogenic activity in vitro.

Calcium sensing receptor (CaSR) has become the novel target of treating osteoporosis with herbal medicine Ligustri Lucidi Fructus (LLF), however, the bioactive compounds responsible for anti-osteoporosis are hard to clarify due to the complexity and diversity of chemical constituents in it. Herein, the immobilized CaSR column was packed with stationary phase materials, which were derived from integrating CLIP-tagged CaSR directly out of crude cell lysates onto the surface of silica gels (5.83 mg/g) in a site-specific covalent manner. The column had a great specificity of recognizing agonists and kept a good stability for at least 3 weeks. The two compounds from LLF extract were screened and identified as olenuezhenoside and ligustroflavone using the immobilized CaSR column in conjunction with mass spectrometry. Molecular docking predicted that both compounds were bound in venus flytrap (VFT) domain of CaSR by the formation of hydrogen bonds. Cellular results showed that both compounds exhibited the distinct osteogenic activity by enhancing the proliferation, differentiation and mineralization of osteoblastic cells. Our study demonstrated that, the immobilized protein column enables to screen the bioactive compounds rapidly from herbal extract, and the newly discovered natural product ligands towards CaSR, including olenuezhenoside and ligustroflavone, will be the candidates for the treatment of osteoporosis.

[Determination of T-2 toxin in cereal grains by high performance liquid chromatography with fluorescence detection after immunoaffinity column clean-up and precolumn derivatization].

A method has been developed for the determination of T-2 toxin in cereal grains by high performance liquid chromatography with fluorescence detection after immunoaffinity column clean-up and precolumn derivatization. The derivatization reaction was used to develop a sensitive, reproducible and accurate method for the determination of T-2 toxin in wheat, corn, barley and rice. T-2 toxin was extracted with methanol-water (80: 20, v/v), purified by immunoaffinity columns containing antibodies specific for T-2 toxin, and quantified by reversed-phase high performance liquid chromatography with fluorescence detection (excitation wavelength, 381 nm; emission wavelength, 470 nm) after derivatization with 1-anthroylnitrile (1-AN) and 4-dimethylaminopyridine (DMAP). ZORBAX Eclipse XDB-C18 column and mobile phase of acetonitrile-water (80: 20, v/v) were used for the analysis. Recoveries from the different cereals spiked with T-2 toxin at levels ranging from 0.01 to 1.5 microg/g were from 79.7% to 94.5%, the relative standard deviations were lower than 7% and the limit of detection was 0.01 microg/g based on a signal-to-noise ratio of 3: 1.

[Simultaneous determination of twelve sulfonyl urea herbicide residues in rice by high performance liquid chromatography with solid-phase extraction].

A high performance liquid chromatographic (HPLC) method with solid-phase extraction was developed for screening of 12 sulfonyl urea herbicides in rice. The sample was cleaned up and extracted with ENVI-18 (C18) and ENVI-Carb (GCB) columns. The separation was performed on a Symmetryshield RP8 column (4.6 mm i.d. x 150 mm)with a linear gradient elution (acetonitrile and 5 mmol/L acetic acid as mobile phase), and the wavelength of an ultraviolet detector was set to 240 nm for the detection. The linear range was 0.1 - 10 mg/L, and the correlation coefficients were 0.9983-0.9999. Recoveries from rice samples spiked with 12 sulfonyl urea herbicides at spiking levels ranging from 0.01-0.50 microg/g were from 72.2% to 106.5%, with relative standard deviations from 0.6% to 6.4%. The lowest detection limits (S/N = 3) were 0.01-0.02 microg/g. The results indicate that the method is easier, faster, sensitive and has better purification effect. It also demonstrates that this multiresidue analytical method can meet the requirements for simultaneous determination of sulfonylurea herbicides in rice.

[Simultaneous determination of triazine herbicide residues in maize by gas chromatography].

A gas chromatographic method was developed for the determination of simeton, simazine, atrazine, propazine, terbumeton, terbuthylazine, cyprazine, simetryn, prometryn, terbutryn, methoprotryne, hexazinone residues in maize. The triazine herbicide residues were extracted with acetonitrile by blender and then cleaned up on a strong cation-exchange (SCX) solid-phase extraction cartridge. The SCX cartridge was conditioned with acetone, methylene chloride, washed with methylene chloride, acetone and eluted with methanol-water (9: 1, v/v) solution saturated potassium chloride. As a result, most interfering impurities were removed in the SPE cleanup procedure. The gas chromatographic analysis was performed on a capillary column (DB-5, 30 m x 0.25 mm i. d. x 0.25 microm) and determined by nitrogen phosphorus detector (NPD). Twelve triazine herbicides were effectively separated on this column. Diazinon was used as the internal standard for the determination. Linear correlation coefficients of the 12 herbicides were higher than 0.998 in the range of 0.01 to 2.0 mg/L. The limits of quantitation of the method were 0.01 mg/kg for these compounds. Average recoveries of these herbicides from spiked samples ranged from 84.0% to 106.8% at fortification levels of 0.01 - 0.5 mg/kg and the relative standard deviations (RSDs) were between 0.9% and 4.7%. The method is suitable for the simultaneous determination of a wide range of triazine herbicides in maize in commodities inspection.