To identify the structures of the related substances in penciclovir cream by two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC-Q TOF/MS) technique.

The first-dimension separation of the related substances in penciclovir cream and its stressed samples according to ICH were carried out on an ODS (250 mm×4.6 mm, 5 μm) column with gradient elution by 0.15% formic acid 10 mmol·L^-1 ammonium formate buffer solution and acetonitrile as mobile phases, and each related substance was enriched separately. The second-dimension gradient elution was performed on a Phenomenex Luna SCX (250 mm×4.6 mm, 5 μm) column with 0.1% formic acid 20 mmol·L^-1 ammonium formate buffer solution and acetonitrile as mobile phases for each of the related substances to achieve good separation with the matrix of penciclovir cream. The accurate mass and elemental composition of the parent ions and their product ions of the related substances were determined by positive electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry, and the structures of all the related substances were elucidated.

Under the established 2D-LC-Q TOF/MS analytical conditions, penciclovir and its related substances were adequately separated, and 21 major related substances were detected and identified in the penciclovir cream and its stressed samples. According to their chromatographic retention behavior, spectral characteristics, mass spectrometry characteristics and their differences from other known related substances of nucleoside drugs and combing with synthesis and formulation process route, their structures can be identified all of which were identified for the first time.

The results can provide reference for the quality control of penciclovir cream.

To establish an HPLC-MS/MS method for the determination of the genotoxic impurities (S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene in rivaroxaban. The factors affecting impurities separation and the response of mass spectrometry were systematically investigated.

A ZORBAX SB-C₁₈ column(150 mm×2.1 mm, 3.5 μm) was used with 10 mmol·L^-1 ammonium formate solution-methanol as mobile phase at flow rate of 0.4 mL·min^-1. And MRM detection by electrospray positive ionization was used.

(S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene had good linear relationships in the range of 0.008-3.4 ng·mL^-1 and 0.2-3.4 ng·mL^-1, respectively. The average recoveries(n=9) were 97.9% and 98.9%, RSD were 0.94% and 0.68% respectively. The limits of quantitation were 0.008 ng·mL^-1 and 0.2 ng·mL^-1 respectively. The results of genotoxic impurity residue determination in 15 batches of rivaroxaban samples were far below the limit value.

The method is sensitive and specific, and can effectively detect trace genotoxic impurities in rivaroxaban.

To establish a method for the detection of genetic impurity chloropropanol in hydroxypropyl distarch phosphate (HPDSP).

The method was performed with Agilent VF-WAX capillary column and temperature programmed by GC-MS. The initial temperature was 80 ℃ for 8 min, then increased to 220 ℃ at the rate of 35 ℃·min^-1 and maintained for 5 min with the flow rate of 2 mL·min^-1. The temperature of inlet was 200 ℃, and the injection volume was 1 μL without split. The detector was electron impact (EI) with selected ion monitoring (SIM) mode and ion source temperature at 230 ℃. The m/z of qualitative ions of 1-chloro-2-propanol were 45, 43, 79, 81, and the m/z of quantitative ion was 79. The m/z of qualitative ions of 2-chloro-1-propanol were m/z 58, 62, 63, 64, 65, and m/z of the quantitative ion was 62.

Good linear was achieved in the range of 20.04 ng·mL^-1 to 1 002 ng·mL^-1 (r=0.999 8, n=7) and the recovery rate ranged from 93.7% to 117.1% for 1-chloro-2-propanol. The contents of chloropropanol in 9 batches were within the range of 0.000 006%-0.000 08%.

The method shows the advantages of high sensitivity, good accuracy, linearity and can be used for the detection of genetic impurities in HPDSP.

To establish a suitable method to determine the structure and source of impurities of colistimethate sodium (CMS) for drug quality control studies.

Frist-dimensional system: using Acquity UPLC^® Peptide CSH C₁₈(150 mm×2.1 mm, 1.7 μm) column, the mobile phase A was phosphate buffer (7.8 g·L^-1 sodium dihydrogen phosphate, adjusted to pH 6.4 with 1 mol·L^-1 sodium hydroxide)- acetonitrile (19∶1), the mobile phase B was phosphate buffer-acetonitrile (1∶1). Gradient elution was performed at a flow rate of 0.3 mL·min^-1. The column temperature was 30 ℃. Second-dimensional system: the Acquity BEH C₁₈ column (50 mm×2.1 mm, 1.7 μm) column was used with ammonium formate(A)-acetonitrile mixture as mobile phase with gradient elution. The flow rate was 0.2 mL·min^-1. The column temperature was 40 ℃. The detection wave length was 210 nm. The ESI source was used in negative ion mode.

The 2D-LC-Q TOF MS method was used to infer the structure of the 55 impurities in CMS, and the main sources were polymyxin E1-I, polymyxin E1-7MOA, polymyxin E3 and polymyxin E6.

The structure and source of impurities in CMS are determined by 2D-LC-Q TOF MS, and the changes in the content of impurities such as manufacturers and production processes are evaluated, which is conducive to improving the production process and controlling drug quality at the source.

To establish an HPLC method for the determination of the related substances in telmisartan.

Waters Symmetry Shield RP8 (150 mm×4.6 mm, 3.5 μm) column was adopted. The mobile phase A was 0.1% phosphoric acid solution (pH was adjusted to 3.0 with triethylamine), and the mobile phase B was acetonitrile. The gradient elution was performed. The flow rate, column temperature and wavelength were 0.8 mL·min^-1, 25 ℃ and 230 nm, respectively.

The resolutions between the peaks of telmisartan and nine known impurities were greater than 1.5. The limits of quantitation of all impurities were less than 40 ng·mL^-1, and the limits of detection of all impurities were less than 10 ng·mL^-1. The standard curves of telmisartan and its impurities were linear within the range 30-600 ng·mL^-1, and the correlation coefficients were all greater than 0.999 5. The average recovery rates of impurities Ⅰ-Ⅸ were 93.8%, 99.8%, 97.5%, 99.2%, 99.8%, 98.3%, 98.4%, 99.2% and 99.7%, respectively, and the RSDs of 9 results were all less than 2.0%. Changing the flow rate, column temperature, wavelength, mobile phase ratio and pH did not affect the detection results of the related substances. The results of maximum single impurity were 0.02% in three batches of telmisartan, and the results of total impurities were less than 0.10%.

The method is highly specific, sensitive, precise and accurate, and can be used for the determination of related substances of telmisartan.

To study and analyze rapid evaporative ionization mass spectrometry (REIMS) fingerprints of samples of Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus in different forms for authenticity discrimination with machine learning.

Aerosol formations from the samples by high temperature of dry burning method were ionized and determined by REIMS with m/z 50-1 200 as scanning range in sensitive mode and positive ion mode. The scanning time was 0.2 s and data was recorded as continuous mode. Then the basic situation of REIMS data distribution was studied and analyzed through the methods of cluster analysis, correlation analysis, similarity analysis and principal component analysis. And then logistic regression model with ridge regression (l2) as penalty parameter and quasi-Newton method (lbfgs) as optimization algorithm was established.

The REIMS fingerprints of the samples showed the characteristics of variety differences. Both cross validation and test set validation had an accuracy of 1.0, and the logistic regression model could accurately predict and distinguish the varieties of the samples.

The application prospect of REIMS technique combined with machine learning in the field of traditional Chinese medicine is very broad.

To investigate the accumulation pattern of metabolites in Glycyrrhiza uralensis by qualitative and quantitative analyses of metabolic constituents in Glycyrrhiza uralensis with different cultivation years, and to search for its differential metabolites.

The separation was performed on an Agilent SB-C₁₈ (100 mm×2.1 mm, 1.8 μm) column with 0.1% formic acid aqueous solution as the mobile phase A and 0.1% formic acid acetonitrile solution as the mobile phase B. The gradient elution was carried out at a flow rate of 0.35 mL·min^-1, and the column temperature was 40 ℃ with an injection volume of 4 μL. The mass spectrometry was performed with positive and negative ions scanning in multiple reaction monitoring mode. The mass spectrometry was performed in multi-response monitoring mode with positive and negative ion scanning. The qualitative and quantitative analyses of the metabolites in Glycyrrhiza uralensis were carried out on the basis of the self-constructed secondary mass spectrometry database, and the multivariate statistical analyses of the metabolites of Glycyrrhiza uralensis with different cultivation were combined with principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and cluster heat map analyses.

(1) A total of 1 038 metabolites were detected from the samples of Glycyrrhiza uralensis with different cultivation years, among which 201 differential metabolites existed between annual and biennial Glycyrrhiza uralensis, 125 up-regulated and 76 down-regulated; 223 differential metabolites existed between biennial and three years old Glycyrrhiza uralensis, 64 up-regulated and 159 down-regulated; 185 differential metabolites existed between annual and three years old Glycyrrhiza uralensis, 59 up-regulated and 126 down-regulated. Four metabolites specific to annual Glycyrrhiza uralensis, six to biennial and one to three-year old were found. (2) K-mean cluster analysis was performed on the differential metabolites, and the differential metabolites were classified according to the different accumulation trends, and it was found that most of the metabolites such as flavonoids, phenolic acids, terpenes, lignans, and coumarins peaked in biennial Glycyrrhiza uralensis, and most of the metabolites such as alkaloids, amino acids and their derivatives peaked in annual Glycyrrhiza uralensis, and a part of the flavonoids, phenolic acids and other metabolites reached peaks in three years old Glycyrrhiza uralensis, suggesting that the metabolism of Glycyrrhiza uralensis in the body reached the peaks. peak value, suggesting that there was a certain pattern of metabolite content changes in Glycyrrhiza uralensis. (3) 160 differential metabolites annotated in Kyoto Encyclopedia of Genes and Genomes (KEGG) datebase and flavonoids, amino acids and their derivatives, and organic acids were the differential metabolites that accounted for a relatively large number of them. A total of 79 differential metabolic pathways were enriched among different comparison groups, among which 6 differential metabolic pathways were highly significantly enriched (P<0.01) and 23 significantly enriched (P<0.05), and the distributions of compounds involved in the above pathways were basically the same as before enrichment in comparison of different cultivation year.

The present study elucidate the differences between the metabolic components of Glycyrrhiza uralensis with different cultivation years, and further analyse the metabolic pathways that might cause the differences through the differential metabolites, which can provide a certain reference basis for the determination of the harvesting year of Glycyrrhiza uralensis and the study of the quality formation mechanism.

To explore the prepararation methods and rules of hair quality control samples which contained ketamine, norketamine or fluoroketamine, and the effects of different preparation and storage conditions on the performance of hair quality control samples.

Different preparation conditions were compared, such as different types of soaking solutions, solutions with different target contents, different amount of blank hair etc. The performances of hair quality control samples which were prepared in different conditions were systematically analyzed, and eighteen kinds of hair quality control samples with different contents of targets and different forms were prepared according to the rules, ACN-DMSO (1∶1, v/v, added 0.02 mol·L^-1 hydrochloric acid) was used to soak blank hair in different time periods to prepare hair quality control samples with a wide coverage. HPLC-MS/MS instruments and BEH C₁₈(100 mm×2.1 mm, 1.7 μm) column were used to test the content of targets in hair. Column temperature was 40 ℃, the mobile phases were aqueous solution with 0.1%(v/v) formic acid (A)-acetonitrile aqueous solution with 0.1%(v/v) formic acid (B), gradient elution (0-9 min, 5% B→100% B; 9-11 min, 100% B; 11-11.1 min, 100% B→5% B; 11.1-13 min, 5% B, a flow rats of 0.4 mL·min^-1, inject volume of 1 μL. The homogeneity, short term and long term stability in different storage conditions (temperature, humidity, light conditions, etc.) were also investigated.

The influences of different preparation conditions on the hair quality control samples were determined, soaking solutions had strong influences on the hair quality control samples, however the amount of blank hair had nearly no effects. All the samples prepared were uniform, F of each sample was lower than F_0.05(table) (3.02) according to the F-test method, and all the samples were stable, during storage at room temperature, refrigerated or frozen conditions for 6 months, the contents of the target substances in each sample were monitored, all of RSDs were <12%, and the t values of each sample were < t_{(0.05)(table)} (2.131 8) according to the T-test method.

This study provides preparation rules of hair quality control samples and also provides detailed reference data for subsequent preparation and storage of related hair quality control samples.

To develop the first batch of traditional Chinese medicine chemical reference substance 2-acetate regaloside A used in standard of Baihe (Juandan) Peifang Keli.

The structure was elucidated by nuclear magnetic resonance spectrometry, mass spectrometry, ultraviolet spectroscopy and infrared spectroscopy. Purity analysis, determination of related substance and investigation of other physical and chemical parameters were carried out by using analytical different techniques such as high performance liquid chromatography, gas chromatography, coulometry and ignition residue method. On the above basis, the mass balance was used for value determination, and the ¹H quantitative nuclear magnetic resonance method was applied for further verification.

The structure of the traditional Chinese medicine chemical reference substance was determined as 2-acetate regaloside A, and it was identified as a new compound by SciFinder search. The calibration value by mass balance method was 99.31%, and result of ¹H quantitative nuclear magnetic resonance method was 100.06% (n=3, RSD=0.47%).

The accurate development and calibration of the first batch of 2-acetate regaloside A fully ensures the implementation of the standard for Baihe (Juandan) Peifang Keli.

To establish an immunochromatographic method suitable for the rapid detection of residues of triazine herbicide prometryn in Chinese medicine, based on self-made prometryn antigen and monoclonal antibody.

An antibody conjugate amount of 7 μg and dilution configuration of 0.1 mol·L^-1 PBS with 1% OVA, 0.1% Tween-80 were determined, and dry test strips with a T-line scribing concentration of 0.05 mg·mL^-1 and a C-line scribing concentration of 0.5 mg·mL^-1 were used. The suitable method was selected to improve the sensitivity of colloidal gold immunochromatography test strip, reduce the matrix effect of traditional Chinese medicine in different parts, and detect a large number of traditional Chinese medicine.

The detection sensitivity of the test strip could reach 1 ng·mL^-1, and the detection limit of the actual sample could reach 0.1 mg·kg^-1. The established method was suitable for the detection of prometryn residues in 40 kinds of Chinese medicinal materials such as Citri Reticulatae Pericarpium, Taraxaci Herba, Notoginseng Radix et Rhizoma andLonicerae Japonicae Flos.

This method has the advantages of rapid and accurately detection, simple pretreatment, simple operation and strong generalization, and can be used as an effective method for rapid screening of prometryn residues in the field, so as to ensure the quality and safety of traditional Chinese medicine. At the same time, this study has a certain reference significance for the development of rapid detection of agricultural residues in many kinds of traditional Chinese medicine.