Latest ArticlesTo establish the quantitative analysis of multi-components by single marker（QAMS） method to simultaneously determine the contents of rutin，hyperoside，isoquercetin and quercetin in Zanthoxyli Pericarpium and Pericarpium Zanthoxyli Armati，and compare the differences in flavonoids contents.
Methanol extract of Zanthoxyli Pericarpium was analyzed on an Agilent Eclipse Plus C18 (250 mm×4.6 mm，5 μm) chromatographic column. The mobile phase was acetonitrile(A)-0.1% formic acid(B) in a gradient elution. The flow rate was 1.0 mL·min-1. The column temperature was 35 ℃，and the detection wavelength was 360 nm. The injection volume was 10 μL. Using hyperoside as the internal standard substance，the relative correction factors for the other 3 components were calculate and the flavonoids contents in Zanthoxyli Pericarpium and Pericarpium Zanthoxyli Armati were determined.
The 4 components had good linear relationship within their respective ranges (r≥0.999 9). The average recovery rates of Zanthoxyli Pericarpium were 99.3%-105.1% (n=6)，while thoes of Pericarpium Zanthoxyli Armati were 91.9%-99.8% (n=6). The relative average deviations of content of Zanthoxyli Pericarpium were 0.05%-3.37%，and the relative average deviations of content of Pericarpium Zanthoxyli Armati were 0.02%-3.28%. The results obtained by QAMS method were close to those obtained by the external standard method(ESM).
This method of QAMS method is accurate and reliable. The total amount of flavonoids in Pericarpium Zanthoxyli Armati is significantly higher than that in Zanthoxyli Pericarpium. Which provides reference for the comprehensive evaluation of the quality research of Zanthoxyli Pericarpium and Pericarpium Zanthoxyli Armati.
To establish an HPLC method for the determination of related substances in olaparib raw materials.
By using YMC-Pack pro C18 AS(150 mm×4.6 mm，3 μm) column，0.05 mol·L-1 potassium dihydrogen phosphate (pH 3.0 adjusted with dilute phosphoric acid)-methanol-acetonitrile (90:5:5) was as mobile phase A and methanol-acetonitrile-water (45:45:10) was as mobile phase B，in gradient elution，with flow rate of 0.8 mL·min-1. The column temperature was 30 ℃，the detection wavelength was 210 nm，and the injection volume was 10 μL.
The solvent blank did not interfere with the determination of relevant substances in the test solution，the known impurities Ⅰ-Ⅶ and unknown impurities in the system suitability solution were well separated in the chromatographic system. Olaparib had a good linear relationship with its peak area in the concentration range of 0.083-333.133 μg·mL-1. The concentrations of impurities Ⅰ-Ⅶ were 0.05-3.0 μg·mL-1，there were good linear relationships with their peak area，and their r were 0.999 7 or above. The average recoveries of impurities Ⅰ-Ⅶ were all above 92.0%，and RSDs were less than 4.0%.
The HPLC method for the detection of the related substances of olaparib raw materials has strong specificity，high sensitivity，high accuracy and good precision，which can meet the requirements for the detection of related substances of olaparib raw materials.
To establish a method for determination genotoxic impurity furfural in furosemide and its tablets. To analyze and evaluate the measurement results to provide a reference for national drug supervision and ensure drug safety.
The chromatographic column was filled with octadecylsilane bonded silica gel，and the gradient elution was performed with phosphoric acid solution and acetonitrile as the mobile phase. The detection wavelength was 276 nm. The method was validated and used to determine the furfural content in 6 batches of furosemide and 148 batches of furosemide tablets.
The detection limit of furfural was 4 ng·mL-1 and the quantitation limit was 12 ng·mL-1. The linearity between 0.012 0-0.602 3 μg·mL-1 (r=1.000) was good. The average recoveries of furosemide and its tablets were 99.4% (RSD=0.99%) and 101.4% (RSD=1.3%). Furfural was detected in all batches of raw materials and tablet samples. The furfural content in the tablet products，which used the raw materials from company I，was significantly higher than that of the products obtained from company H.
This method has strong specificity，high sensitivity and good accuracy. It can be used for the determination of genotoxic impurity furfural in furosemide and its tablets. By analyzing the measurement results and discussing the sources and control strategies of furfural，quality control of furosemide tablets can be achieved.
To analyze the peptide components of bran-fried Bombyx batryticatus decoction and virtually screen anti-epileptic peptides with GAT-1 binding activity.
The peptide compounds in bran-fried Bombyx batryticatus decoction were extensively identified by micro-flow liquid chromatography-tandem mass spectrometry（μLC-MS/MS） technology. Potential anti-epileptic peptides were screened through bioinformatics and in silico simulations，including bioactivity prediction，blood-brain barrier permeability prediction，toxicity prediction，and molecular docking. Finally，molecular dynamics simulations were employed to analyze the interactions between peptides and the target protein.
A total of 384 peptides were identified from bran-fried Bombyx batryticatus decoction，and 19 peptides with higher binding affinities to GAT-1 than that of the control drug were screened as potential anti-epileptic peptides. The FDHFDFDAF exhibited the highest binding affinity，followed by the EHYAWGIK. Two peptides primarily interacted with GAT-1 through hydrogen bonds，hydrophobic，and electrostatic interactions. Molecular dynamics simulations confirmed the binding stability of FDHFDFDAF and EHYAWGIK to GAT-1.
Peptidomics and in silico virtual screening techniques facilitate the rapid discovery of active peptide components in animal-derived traditional Chinese medicine. This study has significant reference value for researching anti-epileptic peptides in bran-fried Bombyx batryticatus decoction，and provides new insights for the study of peptide components in animal-derived traditional Chinese medicine.
To analyze the difference of chemical constituents in rhizomes and leaves of Polygonatum sibiricum from Shaanxi Province.
Ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry(UPLC-Q-Orbitrap HRMS) was developed to determine the chemical constituents in rhizomes and leaves of Polygonatum sibiricum from Shaanxi Province. The data were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The structures of chemical markers in rhizomes and leaves of Polygonatum sibiricum were identified based on accurate primary mass spectrometry and secondary mass spectrometry fragment ion，combined with the reference map，software database searching and related literature.
A total of 45 compounds were identified，there were 26 chemical ingredients with significant differences distinguished by the method of OPLS-DA，including 10 amino acids，6 flavonoids，3 organic acids，2 saccharides，1coumarin and 4 alkaloids.
The chemical markers of amino acids，organic acids and alkaloids are mainly distributed in rhizomes，and the chemical markers of flavonoids are mainly concentrated in leaves part of the plant. It is suggested that the potential multiple utilization of rhizomes and leaves of Polygonatum sibiricum from Shaanxi Province.
To establish a new HPLC method for the determination of the related substances in tranexamic acid injection by screening new type of HPLC columns，which can avoid the use of ion pair reagents in current methods and reduce salt concentration.
The method was carried out using a Thermo Mixed-mode WCX column (150 mm×4.6 mm，5 μm)，the mobile phase consisted of 10 mmol·L-1 sodium dihydrogen phosphate solution (adjust the pH value to 5.2±0.05 with sodium hydroxide solution)-water-acetonitrile （50:5:45），the flow rate was 1.0 mL·min-1，the column temperature was 25 ℃，the detection wavelength was 210 nm and the injection volume was 20 μL.
The chromatographic peaks of tranexamic acid and its four impurities B，C，D and E were all separated. Good linear relationship was shown between the concentration of all the five compounds and their corresponding peak areas (r≥0.999). The LODs were 0.34，0.50，0.005 6，0.002 1，0.12 μg·mL-1. The average recoveries (n=9) were 97.4%，100.5%，98.4% and 96.6% with RSDs of 3.9%，0.24%，0.52% and 1.4%，respectively. The test solution and standard solution were all stable within 22 h. The detection results of 5 batches of tranexamic acid injection showed that the number of unspecified impurities and the total impurities content by using the new method were better than the current ChP method.
The established method is specified and sensitive. Good separation can be achieved with low concentration of phosphate. Its applicable to the determination of related substances in tranexamic acid injection.
To establish an innovative analytical method based on high resolution sampling two-dimensional chromatography (HiRes 2D-LC) for determination of the content of vitamin D3 in vitamin D drops.
Two-dimensional liquid chromatography was used. Thermo HYPERSIL Gold Silica (100 mm×2.1 mm, 1.9 μm) column was used in the first dimension with n-hexane-n-amyl alcohol (996∶4) as mobile phase. The flow rate was 0.2 mL·min-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. In the second dimension liquid chromatography, ShimPack Velox Hilic (50 mm×2.1 mm, 2.7 μm) was used as the column with n-hexane-n-pentanol-isopropanol (98∶1∶1) as the mobile phase. The flow rate was 0.5 mL·min-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. A six-position 14-way valve and was equipped with 2 multi-center cutting valves was equipped to make multiple consecutive cuts of the pre-vitamin D3 peak and vitamin D3 peak.
The calibration curves showed a good linearity at the range of 1.018 4-5.092 mg·mL-1 (r≥0.999 8). The precision test showed the RSDs of the peak area of pre-vitamin D3 and vitamin D3 were 0.95% and 0.40%, respectively. The repeatability test showed the RSD of vitamin D3 content was 0.41%. The average recovery rate (n=9) was 101.4%. The test solution was stable at 4 ℃ and 10 ℃ for 12 h, and the RSDs were 0.58% and 0.66%, respectively. The contents of vitamin D3 in the samples of vitamin D drops measured by this method were 100.4%, 101.6%, 100.9%, 101.6%, 102.7% and 101.6%, which was basically consistent with the results measured by the fourth method in General Chapter 0722 of ChP 2020 Vol Ⅳ.
This method has a good specificity and high sensitivity to accurately determine the content of vitamin D3 in vitamin D drops.
Therapeutic oligonucleotides (OGNs) drugs are artificially synthesized single or double stranded short nucleic acids, typically 15 to 30 base pairs in length. OGNs have been rapidly developed as new therapeutic drugs with increasing attention in the discovery and development of drugs concerning various disease fields. Compared with Europe and America, there are currently no other OGNs drugs listed in China, except for Spinraza, which has been approved for marketing as an orphan drug. The development of OGNs in China started relatively late and is still in its early stages of development. However, the OGNs drug market in China is anticipated to grow quickly due to the country’s large population, high patient demand, ongoing support for the development of oligonucleotide drugs in the future, and the steady maturation of related technologies by domestic businesses. Because of their special physicochemical characteristics, OGNs drugs are challenging to design biological analysis techniques. Currently, there are few reports on quantitative analysis methods for oligonucleotide drugs in China. Therefore, the development of sensitive and reliable bioanalysis methods for oligonucleotides is the key to investigate oligonucleotides’ pharmacokinetic and pharmacodynamic properties. Liquid chromatography-mass spectrometry (LC-MS) can quantify OGNs and their metabolites concurrently, compared with traditional ELISA approaches. Numerous benefits come with using LC-MS, in particular, the extensive use of high-resolution mass spectrometry allows for the identification of metabolites, which provides details on base composition and sequence structure, in addition to quantitative information about target oligonucleotides. It has now emerged as the go-to technique for OGN quantitative analysis. The application of LC-MS in the identification of therapeutic oligonucleotide medicines is the primary focus of this paper, which also discusses its benefits and drawbacks. Lastly, it looks at the LC-MS development trend for oligonucleotide detection, which includes a lower detection level and potential general methods.
To establish an UPLC quantitative analysis method for the simultaneous determination of 13 components in Yankening tablets, including phellodendrine, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion.
Agilent Eclipse Plus C18(100 mm×2.1 mm, 1.8 μm) column was used with acetonitrile -0.1% phosphoric acid as mobile phase, and gradient elution at a flow rate of 0.3 mL·min-1. The detection wavelengths were 210 nm and 254 nm. The column temperature was 40 ℃.
Phellodendrine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride, baicalin, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion showed good linear relationships within their concentration range of 0.97-48.63 μg·mL-1, 0.95-47.52 μg·mL-1, 0.86-43.15 μg·mL-1, 0.86-43.19 μg·mL-1, 0.89-44.37 μg·mL-1, 1.00-49.84 μg·mL-1, 1.02-51.01 μg·mL-1, 0.97-48.31 μg·mL-1, 0.99-49.50 μg·mL-1, 1.04-51.80 μg·mL-1, 1.00-50.04 μg·mL-1, 1.00-49.80 μg·mL-1, 1.01-50.64 μg·mL-1. The average recoveries(n=6) were 95.2%, 96.7%, 95.9%, 98.3%, 94.1%，97.6%, 99.2%, 96.6%, 95.5%, 97.2%, 97.0%, 97.8%, 98.7%, RSD values were all less than 2.0%. The contents of the 13 chemical components in 3 batches of samples were 1.367-1.488 mg·g-1(calculated as phellodendrine hydrochloride), 0.378-0.412 mg·g-1(calculated as coptisine hydrochloride), 4.611-5.505 mg·g-1, 0.324-0.407 mg·g-1(calculated as palmatine hydrochloride), 3.665-3.878 mg·g-1(calculated as berberine hydrochloride), 1.107-1.682 mg·g-1, 0.392-0.941 mg·g-1, 0.076-0.105 mg·g-1, 0.097-0.116 mg·g-1, 1.059-1.213 mg·g-1, 0.149-0.167 mg·g-1, 0.213-0.239 mg·g-1, 0.047-0.059 mg·g-1.
The method is accurate, high analysis efficiency, good repeatability, it can be used to control the quality of Yankening tablets.
To establish an HPLC method of the content and related substances of compound amino acid injection(3AA).
RP-HPLC was adopted to determine compound amino acid injection(3AA), combining with the use of two-dimensional column switching-LC/MSn method was applied to separate and identify the impurities. The determination was performed on Capcell PAK AQ C18(250 mm×4.6 mm, 3 μm) column with 0.2 mol·L-1 sodium dihydrogen phosphate solution (adjusted pH to 2.8 with phosphoric acid) -acetonitrile (98∶2) as mobile phase at the flow rate of 1.0 mL·min-1. The column temperature was 40 ℃, and the detection wavelength was 210 nm. And the injection volume was 20 μL. The LC/MSn method was performed on a Thermo Accucore AQ C18 (100 mm×4.6 mm, 2.6 μm) column with 0.1% formic acid solution as mobile phase A, 0.1% formic acid solution-acetonitrile as mobile phase B, at a flow rate of 0.4 mL·min-1, and at a column temperature of 40 ℃. The mass spectrometry conditions were performed using an ESI ionisation source in the positive-ion scanning mode with a scan range of m/z 100-1 000, and the secondary mass spectrum was carried out by data-dependent scanning.
The related substances were completely separated from the main constituents in RP-HPLC. The standard curve of valine was linear over the range of 1.263-5.050 mg·mL-1, with the average recovery of 99.0% (n=9). The standard curve of isoleucine was linear over the range of 1.350-5.402 mg·mL-1, with the average recovery of 99.4%(n=9). The standard curve of leucine was linear over the range of 1.647-6.588 mg·mL-1, with the average recovery of 99.5%(n=9). The main impurities in the three batches of samples were all process impurities introduced from the raw materials, with methionine content of 4.344 μg·mL-1, 3.751 μg·mL-1, 4.503 μg·mL-1, respectively, phenylalanine content of 4.636 μg·mL-1, 4.889 μg·mL-1, 4.753 μg·mL-1, respectively. The maximum single impurity contents were 0.01%, 0.02% and 0.01%, respectively.
The method is proved by the methodology validation that it can be used for the quality control of compound amino acid injection(3AA).
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