23 results about "Reversed-Phase Liquid Chromatography" patented technology

The invention discloses a method for improving ion pair reversed-phase liquid chromatography separation by using inert gas atmosphere protection and a chromatographic instrument mobile phase management system, and aims to prevent oxidation denaturation of an ion pair reagent in a mobile phase by using inert gas and prolong the service life of the mobile phase so as to improve the stability and reproducibility of the method. The method comprises the following steps: carrying out headspace replacement on a container filled with a mobile phase by using inert gas, pre-filling the inert gas protection box by using the inert gas, placing the container filled with the mobile phase in the inert gas protection box, carrying out sealed storage, and carrying out small-amount timed supplement on the inert gas protection box by using the inert gas. The method disclosed by the invention can be applied to reversed-phase liquid chromatography separation of a mobile phase added with an ion pair reagent and qualitative and quantitative analysis of combination of the reversed-phase liquid chromatography separation and an optical detector or a mass spectrum; after the method provided by the invention is used, the reproducibility and the stability of each analysis result are remarkably improved.

This invention belongs to the field of synthetic biology and microbial engineering, and discloses a chemical-biological method for preparing the isopentenyl bibenzyl compound 2-isopentenyl-3,3',4',5-tetrahydroxybibenzyl (NPB-1575) which has anti-ischemic stroke activity. The specific disclosed method includes: (1) chemically synthesizing the substrate dihydropiperidine; (2) constructing an engineered Escherichia coli strain based on the isopentenyl utilization pathway, co-expressing hydroxyethylthiazol kinase, isopentenyl phosphokinase, isopentenyl pyrophosphate isomerase and specific isopentenyl transferase, and efficiently synthesizing the isopentenyl donor; (3) using this strain to catalyze dihydropiperidine in whole cells, the yield in a 200L fermenter can reach 2.54g / L, which is nearly 40 times higher than the existing method; (4) obtaining NPB-1575 with a purity >98.5% by macroporous adsorption resin and normal / reversed phase chromatography and recrystallization. This method uses readily available raw materials, involves simple steps, is environmentally friendly, and yields high purity, laying the foundation for the development of NPB-1575 as a new drug for ischemic stroke and showing significant application potential.

The invention relates to the technical field of chromatography, in particular to a preparation method and application of primary hepatic stellate cell membrane biological chromatography for screening anti-hepatic fibrosis active ingredients. The method has the advantages that the primary hepatic stellate cell membrane is used for packing the chromatographic column; the characteristic that primary hepatic stellate cells are closer to in-vivo cells is utilized, the advantage that more real drug response can be provided can be provided, and the method is applied to screening and identification of active ingredients of anti-hepatic fibrosis traditional Chinese medicines. The chromatographic column disclosed by the invention integrates the advantages of primary hepatic stellate cells and a comprehensive two-dimensional cell membrane chromatographic analysis system. Establishment of a comprehensive two-dimensional primary hepatic stellate cell membrane chromatography-reversed phase chromatography system is realized for the first time, and the method can be applied to screening of anti-hepatic fibrosis active components.

The present invention discloses a method for preparing high-purity small molecule peptide-like compounds by chromatography. Specifically, a purification method for small molecule peptide-like compounds is disclosed, which comprises the following steps: using ion exchange chromatography or reverse phase chromatography to elute the crude product of the small molecule peptide-like compound. The purification method provided by the present invention is applicable to the high-efficiency commercial production of high-purity small molecule peptide-like compounds with a chromatographic purity of 98.5% and low impurities.

The present disclosure discusses a method of separating and / or purifying polynucleotides. The method includes injecting a sample into a chromatographic column that is packed with a porous sorbent having a pore size that substantially excludes the polynucleotides from the sorbent. This restricted access to the sorbent allows separation of large polynucleotides from each other and from smaller molecular weight impurities.

The present invention refers to a method for the purification of a collagen-like protein mixture, comprising or consisting of the following steps: a) providing the collagen-like protein mixture in a solvent to form a collagen-like protein solution, followed by heating of the collagen-like protein solution followed by allowing to cool the collagen-like protein solution and b) purifying the collagen-like protein solution of a) by a reversed-phase chromatography which includes a mobile phase in form of at least one mobile phase solvent and a stationary phase in form of at least one column. Furthermore, the present invention refers to the collagen-like protein obtained by the method according to the invention. In addition, the present invention refers to the use of the collagen-like protein obtained by the method according to the invention for medical applications.

The invention provides a method for determining related substances in drops containing vitamin D3, and belongs to the technical field of medicine detection. The method comprises the following steps: mixing contents of drops, acetone and methanol, extracting, performing phase separation, and collecting an upper methanol extract; removing matrix components and a solvent, and redissolving with methanol to prepare a redissolved solution; carrying out saponification reaction on the mixed reconstitution fluid and a sodium methoxide reagent to prepare a test solution; detecting the test solution by adopting a reverse phase chromatography, and collecting an effluent component with the retention time of 11.5-15.5 minutes; detecting an effluent component by adopting normal phase chromatography; the related substances include (5E, 7E)-9, 10-open-loop cholest-5, 7, 10 (19)-triene-3 beta-alcohol, (6E)-9, 10-open-loop cholest-5 (10), 6, 8-triene-3 beta-alcohol, and / or 9, 10-open-loop cholest-6, 8, 10 (5)-triene-3 beta-alcohol. The method is accurate in detection and high in sensitivity.

The present disclosure discusses a method of separating and / or purifying acidic peptides by the use of a mobile phase having a pH greater than or about equal to the isoelectric point of one or more of the metal oxides in the flow path.

The invention discloses a preparation method of p-hydroxymethylpeptide and application of the p-hydroxymethylpeptide in agriculture, and relates to the technical field of biochemistry and agriculture, and the p-hydroxymethylpeptide is prepared by carrying out reflux reaction on L-serine and ethylene glycol in anhydrous DMF (Dimethyl Formamide) at 130 DEG C for 10 hours, cooling, filtering, alternately washing with ethanol and water, and carrying out vacuum drying; the p-hydroxymethylpeptide can also be prepared by the following steps: carrying out fermentation culture on the trichonema, centrifuging, washing, freeze-drying, carrying out methanol ultrasonic extraction to obtain a crude extract, and carrying out macroporous resin and reversed-phase chromatography separation and purification; the p-hydroxymethylpeptide provided by the invention is simple in synthesis process, mild in reaction condition, high in product purity and suitable for large-scale preparation; the p-hydroxymethylpeptide-based potassium composition for promoting crop absorption has specificity on crop potassium synergism, shows good stability on crops such as wheat and the like, has a good effect and has a good crop popularization and application prospect.

The invention relates to the technical field of medicine quality control, in particular to a detection method of an antibody N-glycoform. The detection method comprises the following steps: carrying out qualitative detection on the N-glycoform of an antibody to be detected by adopting mass spectrometry; and carrying out quantitative detection on each N-glycotype which is qualitatively detected by adopting a reverse phase chromatography method. Compared with a traditional detection method, the method can be suitable for detection of drugs with approximate N glycoforms, and an accurate detection result is provided.

The invention provides a preparation method of hydrophobic impurities in a semeglutide injection or bulk drug. The preparation method comprises the following steps: carrying out illumination destruction on the semeglutide injection or bulk drug under the illumination conditions of visible light with the illumination intensity of 4500lx + / -500lx and near ultraviolet light with the energy not lower than 200W. Hr / m < 2 >; carrying out primary purification on the destroyed sample obtained in the step 1) by adopting a reverse phase chromatography; and refining and purifying the solution obtained in the step 2) by adopting a reverse phase chromatography to obtain the high-purity hydrophobic impurities. According to the method disclosed by the invention, the semeglutide injection or the raw material medicine is damaged through illumination, so that the content of hydrophobic impurities is remarkably increased, and the preparation efficiency is greatly improved; according to the method disclosed by the invention, the high-purity semeglutide hydrophobic impurity mixture can be quickly obtained through one-time purification by virtue of reversed-phase purification and high resolution capability, the steps are few, the speed is high, and the purity is high (greater than or equal to 98%).

The invention relates to a method for detecting related substances in an atavalopag starting material, which adopts a high performance liquid reversed phase chromatography, takes water and acetonitrile as mobile phases, and can quickly, effectively and accurately monitor the related substances in the atavalopag starting material.

The invention discloses a method for separating and detecting enantiomers in fluralana and intermediates thereof, S-fluralana, R-fluralana, S-intermediates and R-intermediates can be simultaneously detected by adopting a reverse phase chromatography, a test solution, a reference solution and a system applicability solution are prepared by dissolving with a diluent, and the test solution, the reference solution and the system applicability solution are subjected to quantitative analysis. And determining a chromatogram by adopting specific reversed-phase high-performance liquid chromatography conditions, and calculating the content of the enantiomers in the fluralana and the intermediate according to the chromatogram. The separation and detection method can effectively realize complete separation of S-fluralana, R-fluralana, S-intermediate and R-intermediate, has good convenience, specificity, high sensitivity, repeatability and accuracy, can effectively determine the content of enantiomers in fluralana and intermediate, is beneficial to guarantee the quality of fluralana and intermediate, and has a good application prospect. And the method has important significance on splitting and quality control of the fluralana.

The invention relates to the technical field of anthocyanin preparation and purification, in particular to a high-purity anthocyanin standard sample and a preparation method thereof. According to the method, by combining reversed-phase phenyl chromatography and reversed-phase C18 chromatography and optimizing the relationship between the sample loading amount and the purity, the anthocyanin standard sample with the purity higher than 98% is obtained from acidolysis products of grape seed extracts through purification. Due to the fact that the removal effects of the two chromatography methods on different impurities are different, different types of impurities can be removed through combined use of the two chromatography methods, the sample loading capacity is increased, the preparation efficiency is improved, and the recovery rate can be higher than 90%. The method is simple and economical, and the obtained cyanine standard sample is very high in purity, can be used as a reference substance for quality standard research work of cyanine extract containing cyanine, and provides effective assistance for related scientific research work.

This invention discloses a method for determining ceftazidime and its formulation polymers, comprising the following steps: 1) preparing a system suitability solution; 2) preparing a reference solution; 3) preparing a test solution for ceftazidime for injection; 4) determination: injecting the system suitability solution, reference solution, and test solution into a liquid chromatograph, respectively, and performing determination using reversed-phase chromatography, wherein the mobile phase of the reversed-phase chromatography contains mobile phase A and mobile phase B, mobile phase A being an aqueous formic acid solution and mobile phase B being methanol. The chromatogram obtained by the method of this invention has a stable baseline, can detect multiple polymeric impurities of ceftazidime, and exhibits good separation of each impurity; the method of this invention has good specificity and system suitability, and its linearity, sensitivity, repeatability, intermediate precision, and robustness are all good; furthermore, the test solution and reference solution of this invention have high stability.

The invention belongs to the technical field of drug detection and analysis, and provides a correction method for DAR value detection of an antibody-coupled drug based on dual-wavelength detection and application of the correction method, and the method comprises the following steps: (S1) carrying out reduction treatment on the antibody-coupled drug, and then carrying out detection by adopting reversed-phase chromatography or affinity chromatography, the detection wavelength is set to be 280 nm and the special absorption wavelength of the selected linker-load is a nm, and obtaining an atlas according with the separation degree; respectively obtaining response peak areas under the two detection wavelengths through integration; and (S2) correcting the corresponding response peak area under 280nm through each response peak area under a nm, calculating the percentage of the corrected peak area through the corrected peak area, and calculating the average DAR value according to the contribution of the similar components to the DAR by using the corrected peak area percentage. The correction method is wide in application range and can be applied to DAR value calculation correction of various types of antibody conjugates.

The invention belongs to the field of polypeptide analysis, and discloses a preparation method of a semeglutide related substance, the difference between the semeglutide related substance and the semeglutide is that glutamic acid at the No.15 site of the semeglutide is changed from '-NH-CH (CH2CH2COOH)-CO-' to '-NH-CH (COOH) CH2CH2-CO-', and the semeglutide related substance can be used for preparing the semeglutide related substance. The preparation method comprises the following steps: destroying a semeglutide sample at a high temperature of 40-50 DEG C for 3-15 days, and carrying out primary purification and refined purification on the destroyed sample by adopting a reverse phase chromatography to obtain the high-purity semeglutide related substance. The research successfully establishes a method for preparing the semeglutide related substance based on high-temperature forced degradation combined with a preparative liquid chromatography technology, and the method has the characteristics of high product purity, good reproducibility and good tolerance to different salt type buffer systems. The establishment of the preparation method provides reliable technical support for impurity spectrum research and quality standard formulation of the semeglutide and acquisition of related impurity reference substances.

The invention discloses an analysis method for isomers of methyloxyethyl acetoacetate, comprising the following steps: S1, using a reverse phase column, taking acetonitrile: sodium hydrogen phosphate solution = 60:40 as a mobile phase, a flow rate of 0.8-1.2 mL / min, a column temperature of 43-47°C, a running time of 30 min, and an injection volume of 10 μL; S2, weighing 40 mg of methyloxyethyl acetoacetate, adding it into a solution prepared by acetonitrile and sodium hydrogen phosphate solution in S1 and fixing the volume to 10 mL; S3, respectively performing three groups of data, wherein the first group is prepared and measured immediately, the second group is left to stand for 24 h, and the third group is left to stand for 48 h; S4, accurately measuring 10 μL of the test solution, injecting it into a liquid chromatograph, and recording a chromatogram. The invention optimizes chromatographic conditions, adopts reverse phase chromatography, uses a reverse phase HPLC system, completes the analysis of methyloxyethyl acetoacetate isomers, takes time as a reference axis, can clearly identify the tautomers of the product in a liquid phase detection process, takes acetonitrile: disodium hydrogen phosphate solution = 60:40 as a mobile phase, adopts a reverse phase column for separation, has a short analysis time, and has the advantages of simple and rapid operation.

The invention relates to a high-purity subtilucent 37 peptide reference substance and a production method and application thereof based on biosynthesis industrialization, and belongs to the field of biomedicine.The production method comprises the steps that bacillus subtilis serves as a production strain, and fermentation liquor is obtained through primary seed tank fermentation and seed tank fermentation; carrying out ceramic membrane separation, double-effect concentration, salting-out and tubular centrifugation on the fermentation liquid to obtain a precipitate which is a crude product of the tri17 peptide; the preparation method comprises the following steps: redissolving a crude product of the withered grass tri17 peptide with a 20% ethanol aqueous solution, centrifuging, filtering, dewatering, carrying out ion exchange chromatography, carrying out ultrafiltration concentration, freeze-drying, and subpackaging to obtain a reference substance of the withered grass tri17 peptide; wherein the elution step of hydrophobic chromatography is water-5% ethanol water-50% ethanol water elution. Reversed phase chromatography, ion exchange chromatography and size exclusion chromatography detection results show that the purity of the reference substance is greater than 99.9%, the endotoxin is less than 10 EU / mL, the sterility test and the abnormal toxicity meet the high-purity bulk drug standard, and the reference substance can be used as a crude drug of the triticin.

The invention discloses a method for reducing the peroxide value of phospholipid through reverse phase chromatography, which comprises the following steps: (1) carrying out gradient elution by taking a low-carbon alcohol solution of phospholipid as a sample, adopting a reverse phase silica gel chromatographic column, taking the low-carbon alcohol as a mobile phase A and taking water as a mobile phase B, detecting the wavelength of 250nm, and collecting a low-POV component; and (2) removing the solvent from the low-POV component to obtain the phospholipid product with reduced peroxide value. The method is simple and convenient to operate, can effectively reduce the peroxide value in phospholipid, and is suitable for industrial production.

The present disclosure relates to hybrid bonded siliceous materials. The materials of the present disclosure exhibit enhanced base stability and reduced baseline noise. Thus, the materials disclosed herein are suitable for use across a range of chromatography applications, including size exclusion chromatography and reverse phase chromatography.

The invention is directed to a method for determining amino acid sequence mutations in a produced polypeptide, comprising the following steps of a) providing a sample of a produced polypeptide, b) incubating the polypeptide in the sample with a protease, c) performing a two dimensional analysis using reversed phase chromatography coupled with a high resolution mass spectroscopy (FT-ICR / FT-orbitrap) and MS / MS analysis of the amino acid sequence fragments of the peptides, d) data evaluation by comparing the LC-MS data sets obtained for the samples side by side with the data set of a reference sample, by searching for differences in the signal intensities at given retention times and by evaluation of differential signals with respect to amino acid sequence mutations. The reference sample for data evaluation (d) can be either a well characterized standard or one of the samples to be analyzed.

This invention relates to the field of pharmaceutical analysis technology and discloses a method for detecting the content of ezetimibe and rosuvastatin calcium tablets using a reversed-phase chromatography system. The method employs high-performance liquid chromatography (HPLC) with 0.1% trifluoroacetic acid solution as mobile phase A, acetonitrile as mobile phase B, and methanol as mobile phase C. Gradient elution is performed using an octadecylsilane-bonded silica column. The detection wavelength is 242 nm, and the injection volume is 10 μL. The content of ezetimibe and rosuvastatin calcium is calculated using the external standard method. This method can effectively separate the two main components from various degradation impurities, exhibiting good peak shape and excellent resolution. It meets the requirements for pharmaceutical quality control in terms of specificity, limit of quantitation, limit of detection, linear range, precision, accuracy, repeatability, and robustness. The system is stable and provides a reliable technical solution for the quality detection of ezetimibe and rosuvastatin calcium tablets.