Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design / 生物工程学报

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

Simultaneous enantioseparation and simulation studies of atenolol,metoprolol and propranolol on Chiralpak(R) IG column using supercritical fluid chromatography / 药物分析学报

Enantioseparation of three β-blockers,i.e.,atenolol,metoprolol and propranolol,was studied on amylose tris(3-chloro-5-methylphenylcarbamate) immobilized chiral stationary phase using supercritical fluid chromatography (SFC).The effect of organic modifiers (methanol,isopropanol and their mixture),col-umn temperature and back pressure on chiral separation of β-blockers was evaluated.Optimum chro-matographic separation with respect to resolution,retention,and analysis time was achieved using a mixture of CO2 and 0.1％ isopropyl amine in isopropanol:methanol (50:50,V/V),in 75:25 (V/V) ratio.Under the optimized conditions,the resolution factors (Rs) and separation factors (α) were greater than 3.0 and 1.5,respectively.Further,with increase in temperature (25-45 ℃) and pressure (100-150 bars)there was corresponding decrease in retention factors (k),α and Rs.However,a reverse trend (α and Rs)was observed for atenolol with increase in temperature.The thermodynamic data from van't Hoff plots revealed that the enantioseparation was enthalpy driven for metoprolol and propranolol while entropy driven for atenolol.To understand the mechanism of chiral recognition and the elution behavior of the enantiomers,molecular docking studies were performed.The binding energies obtained from simulation studies were in good agreement with the elution order found experimentally and also with the free energy values.The method was validated in the concentration range of 0.5-10 μg/mL for all the enan-tiomers.The limit of detection and limit of quantitation ranged from 0.126 to 0.137 μg/mL and 0.376-0.414 μg/mL,respectively.The method was used successfully to analyze these drugs in pharmaceutical preparations.

Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry / 药物分析学报

Tea is a widely consumed beverage and has many important physiological properties and potential health benefits. In this study, a novel method based on supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) was developed to simultaneously determine 11 amino acids in different types of tea (green teas, Oolong tea, black tea and Pu-erh tea). The separation conditions for the analysis of the selected amino acids including the column type, temperature and backpressure as well as the type of additive, were carefully optimized. The best separation of the 11 amino acids was obtained by adding water (5％, v/v) and trifluoroacetic acid (0.4％, v/v) to the organic modifier (methanol). Finally, the developed SFC-MS method was fully validated and successfully applied to the determination of these amino acids in six different tea samples. Good linearity (r ! 0.993), precision (RSDs 2.99％), accuracy (91.95％e107.09％) as well as good sample stability were observed. The limits of detection ranged from 1.42 to 14.69 ng/mL, while the limits of quantification were between 4.53 and 47.0 ng/mL. The results indicate that the contents of the 11 amino acids in the six different tea samples are greatly influenced by the degree of fermentation. The proposed SFC-MS method shows a great potential for further investi-gation of tea varieties.

Advances of supercritical fluid chromatography in lipid profiling / 药物分析学报

Supercritical fluid chromatography (SFC) meets with great favor due to its high efficiency, low organic solvent consumption, and the specialty for the identification of the isomeric species. This review de-scribes the advances of SFC in targeted and untargeted lipid profiling. The advancement of the SFC in-struments and the stationary phases are summarized. Typical applications of SFC to the targeted and untargeted lipid profiling are discussed in detail. Moreover, the perspectives of SFC in the lipid profiling are also proposed. As a useful and promising tool for investigating lipids in vitro and in vivo, SFC will predictably obtain further development.

Separation and Purification of Costunolide and Dehydrocostus Lactone in Aucklandiae Radix by Supercritical Fluid Chromatography / 中国实验方剂学杂志

Objective:To establish a supercritical fluid chromatography(SFC) method for separating and purifying costunolide and dehydrocostus lactone in Aucklandiae Radix. Method:With supercritical carbon dioxide as the mobile phase,the effect of six factors, such as type of chromatographic columns,modifiers and modifiers ratio, flow rate of mobile phase,pressure and temperature, on the separation process of supercritical fluid chromatography were explored. The target components were separated and prepared by semi-preparative supercritical fluid chromatography. High performance liquid chromatography and nuclear magnetic resonance were used to analyze the components and study the thermodynamic regularity of the chromatographic process. Result:C18 column (10 mm×250 mm,5 μm) was adopted, with supercritical fluid dioxide as the mobile phase,the ratio of methanol was 0.13%,the flow rate was 12 mL·min-1,column pressure was 13 MPa,column temperature was 318℃, and detection wavelength was 225 nm. The sample was injected for 20 times,crude extract was 4 mg,and each target component was collected according to the chromatogram. Its purity was determined to be more than 99%by HPLC,and its structure was determined as costunolide and dehydrocostus lactone by NMR. Under this condition,the SFC separation process was normal-phase chromatography. Conclusion:The method can be used to prepare effective components of Aucklandiae Radix with a high purity and low solvent residue.

Determination of related substances in cinnarizine by supercritical fluid chromatography / 中国药学杂志

OBJECTIVE: To develop a gradient supercritical fluid chromatography method for the separation of cinnarizine and its four related substances. METHODS: Cinnarizine and its four related substances were separated on a Torus DIOL column (3.0 mm×100 mm,1.7 μm) maintained at 40 ℃ with the mobile phase consisting of CO2 and methanol with 0.1% TFA-0.1% TEA at 1.5 mL•min-1, the detection wavelength was set at 230 nm and the back pressure was set at 1.38×107 Pa. RESULTS: Cinnarizine and its four related substances were separated in 4.5 min with satisfying resolutions. Good linear relationships were established between the peak response and the concentration in the range of 2-20 μg•mL-1 for each related substance (r>0.999 9) and the detection limits were 0.7-1.3 ng(S/N≥3). Good linear relationships were established between the peak response and the concentration in the range of 0.05-1.0 μg•mL-1 for cinnarizine (r=1.000 0). The spiked recovery of four related substances of cinnarizine was 98.0%-106.7%, and the RSD was 2.57%-4.44%(n=9). CONCLUSION: The established method can be applied in the simultaneous determination of the related substances of cinnarizine and provide reference for the quality control.

Chiral Separation of Atorvastatin Calcium and Its Enantiomeric Impurity by Supercritical Fluid Chromatography / 中国药学杂志

OBJECTIVE: To develop a supercritical fluid chromatography method for the separation of atorvastatin calcium and its enantiomer, meanwhile assaying the enantiomer. METHODS: Atorvastatin calcium and its enantiomer were separated on a ACQUITY UPC2 Trefoil CEL2 column(3.0 mm×150 mm, 2.5 μm) maintained at 45 ℃ with the mobile phase containing a mixture of CO2 and methanol with 0.1% TFA(78∶22, V/V) at 1.5 mL·min-1, and the detection wavelength was set at 244 nm. The back pressure was set at 13.8 MPa. RESULTS: The enantiomer and atorvastatin calcium were separated successfully in 5 min with a resolution factor of 4.1. Good linear relationship was established between the peak response and the concentration in the range of 2.5-50 μg·mL-1 for enantiomer(r2=0.999 9, n=6), the quantitative limit(S/N=10) was 2.5 μg·mL-1, and the detection limit(S/N=3) was 1.0 μg·mL-1. The spiked recovery of the enantiomer was 100.40%(n=9). CONCLUSION: The proposed method shows high accuracy, repeatability and stability. It can be employed for the quality control and stability research of the enantiomer of atorvastatin calcium.

Chiral Separation of Naproxen Enantiomers by Supercritical Fluid Chromatography / 中国药房

OBJECTIVE:To develop a method for the separation of naproxen enantiomers. METHODS:The supercritical fluid chromatography(SFC)method was adopted. The chiral column,type and proportion of polar additive in mobile phase,back pres-sure and column temperature were optimized using the separation time,capacity factor,separation factor and separation degree as index. RESULTS:The best condition was on the chiral column CHIRALPAK?AD-H,with polar additive of isopropanol (20%), back pressure of 170 bar,column temperature of 20℃and the detection wavelength of 283 nm. Under this condition,baseline sep-aration of naproxen enantiomers can be achieved;separation degree was 4.31 and separation factor was 1.90;RSD of precision,sta-bility and reproducibility tests were no more than 2.65%(n=5). CONCLUSIONS:Established method is simple,reproducible and well-separated,and can be used for chiral separation of naproxen enantiomers.

Techniques and strategies for characterization and isolation of chemical components of traditional Chinese medicines / 国际药学研究杂志

The chemic al composition of traditional Chinese medicine (TCM) is the material base for its prevention and treatment of disease. Characterization, analysis and isolation of chemical constituents, important fields of modern research on TCM, however, are still a great challenge due to the complexity. In recent years, considerable progress has been made in these fields with the application of new chromatographic packing materials and methods, analysis technologies represented by LC-MS and the associated databases and softwares. Here, the advancement of methods and strategies used in the research on the chemical constituents of TCM is briefly reviewed.

Techniques and strategies for characterization and isolation 01 chemical components of traditional chinese medicines / 国际药学研究杂志

The chemical composition of traditional Chinese medicine (TCM) is the material base for its prevention and treatment of disease. Characterization, analysis and isolation of chemical constituents, important fields of modern research on TCM, however, are still a great challenge due to the complexity. In recent years, considerable progress has been made in these fields with the application of new chromatographic packing materials and methods, analysis technologies represented by LC-MS and the associated databases and softwares. Here, the advancement of methods and strategies used in the research on the chemical constituents of TCM is briefly reviewed.

Chiral separation of ezetimibe and R-enantiomer by supercritical fluid chromatography / 中国药学杂志

OBJECTIVE: To develop an isocratic supercritical fluid chromatography method for the separation of ezetimibe and its R-enantiomer. METHODS: Ezetimibe and its R-enantiomer were separated on a Chiralcel OD column (4.6 mm × 250 mm, 10 μm) maitained at 35℃ with the mobile phase containing a mixture of CO2 and methanol with 0.1% TFA-0.1% TEA (90:10, V/V) at 3.0 mL · min-1, and the detection wavelength was set at 235 nm. The back pressure of SF-CO2 was set at 15 MPa. RESULTS: The R-enantiomer and ezetimibe were separated successfully in 15 min with a resolution factor of 1.6. Good linear relationships were established between the peak response and the concentration in the range of 0.010-0.100 mg · mL-1 for each analyte(r =0.999 9 and 0.999 9, respectively, n =7), the quantitation limits (S/N = 10) were 34 and 32 ng, and the detection limits (S/N =3) were 10 and 9 ng, respectively. The spiked recovery of R-enantiomer of ezetimibe was 98.4% (n =9). CONCLUSION: The proposed method shows high accuracy, repeatability and stability. It could be employed for the quality control and stability research of R-enantiomer of ezetimibe and its tablet formulation.

Steroidal saponins from the seeds of Trigonella foenum-graecum L.: Research advances / 国际药学研究杂志

Seeds of Trigonella foenum-graecum L., as a raditional Chinese medicine, are rich n steroidal saponins. The systematic research on steroidal saponins of Trigonella foenum-graecum, including the study on furostanol saponins and spirostanol saponins, the rapid characterization and dentification of steroidal saponins, and he analytical and preparative separation of C-25 R/S-spirostanol saponin diastereomers using supercritical fluid chromatography, was performed. The results of research are helpful to the development and utilization of Trigonella foenum-graecum seeds, and the methods could provide reference for the chemical research on other traditional Chinese medicines.