To perform a comprehensive analysis of extractables in rubber by pseudotargeted metabolomics.

The quality control(QC) solutions and test solutions were prepared by extracting the six kinds of rubbers with water, pH 2.6 phosphate buffer, pH 9.18 phosphate buffer, and 15% ethanol solution, respectively. The QC solutions were detected by LC-30A ultra performance liquid chromatography-Q TOF-9030 mass spectrometer in ESI⁺/ESI^- mode. The mobile phases were 0.1% formic acid-water and 0.1% formic acid-acetonitrile/0.05% ammonium-water and 0.05% ammonium -5% water-methanol. The separation chromatographic column was Waters Acquity BEH C8/HSS T3. TOF MS and MS/MS DDA scanning modes were used to detect the QC solutions. The scan results of QC solution were processed with MSConvert and MRM-Ion_Pair_Finder software to obtain MRM parameters. The extracts were detected by ExionLC UPLC-Qtrap6500+ mass spectrometer in ESI⁺/ESI^- mode with the same mobile phase and column as before, and Scheduled-MRM scan mode was applied to detect the extracts.

The 1 399 unknown compounds and 116 known compounds were analyzed. By extraction solution, it was found that pH 9.18 phosphate buffer and 15% ethanol solution could extract more compounds, and 27 compounds with large differences in different extraction solutions were identified. By rubbers, the affinities of different rubbers were determined by 25 differences.

In this study, the results of the extraction of six rubbers under four extracts are analyzed. There are differences between extracts under different extraction conditions. Analytical methods can be specifically developed for these key variables for quality control follow-up studies to ensure the stability and consistency of drug quality. The variables in different rubbers are also analyzed, which can be used to make a general class determination of unknown rubber samples.

To study and establish a method based on gas chromatography and chemometrics techniques for distinguishing Artemisiae Argyi Folium and its adulterants Artemisiae Mongolica Folium.

Gas chromatography method was established with Agilent HP-5 19091J (30 m×0.32 mm, 0.25 μm) as chromatographic column, and hydrogen flame ion detector (FID) as detector. After the chemical composition of 21 chromatographic peaks in the chromatogram were identified, and the peak area data of the 21 chromatographic peaks in 29 batches of samples were determined. Similarity analysis, correlation analysis, cluster analysis, principal component analysis and orthogonal partial least squares-discriminant analysis were applied to analyze the chromatographic data.

The results of chemometric analysis indicated that tpeak 20 (chamazulene), peak 3 (1, 8-cineole) and peak 19((1S, 8aα)-decahydro-1, 4aβ-dimethyl-7β-isopropenyl-1-naphthol) were the differential characteristic chromatographic peaks between Artemisiae Argyi Folium and its adulterants Artemisiae Mongolica Folium. The ratios of the peak areas of peak 3 to peak 20 were in the ranges of 54.50-348.39 and 0.16-0.87 respectively, and the ratios of the peak areas of peak 19 to peak 20 were in the ranges of 18.55-128.46 and 0.01-0.14 respectively. These significant differences could be used for the identification of Artemisiae Argyi Folium and its adulterant Artemisiae Mongolica Folium.

The research findings can be used for the identification of Artemisiae Argyi Folium and its adulterant Artemisiae Mongolica Folium, and these have certain reference significance for the research and analysis of Artemisiae Argyi Folium and related drugs.

To establish the first national standard for oncolytic activity assay of herpes simplex virus type 1(HSV-1).

According to the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2020 edition), the liquid and freeze-dried standard for oncolytic activity of HSV-1 were prepared and tested, of which the stability were evaluated by thermal acceleration test. The oncolytic activity of the standard was calibrated collaboratively by U-2 OS cells/CCK-8 method in 3 laboratories. The liquid standard was compared with the freeze-dried standard, and the more suitable one was selected as the national standard.

The prepared standard substance was all qualified, among which the moisture content of the freeze-dried standard was 1.09% and the dispensing accuracy was 0.15%. The results of stability test were calculated by Arrhenius formula. It was preliminarily predicted that it would take 7.7 years for the oncolytic activity of liquid standard to decrease by 10% at -70 ℃, and it would take 6.1×10⁵ years for the oncolytic activity of lyophilized standard to decrease by 10% at -70 ℃. Compared with liquid standard, the stability of lyophilized standard was greatly improved. Twenty-one times of collaborative calibration tests by 3 laboratories showed that the oncolytic activity liquid standard was 7.08×10⁴ U·mL^-1 and the oncolytic activity liquid standard was 1.82×10⁴ U·vial^-1. After lyophilized, the oncolytic activity of the bulk of HSV-1 standard decreased from 7.08×10⁴ U·mL^-1 to 3.03×10⁴ U·mL^-1. However, the good S-shaped dose-response curve still appeared after 100 times of pre-dilution, which did not affect the requirements of its use as a standard. When the liquid standard was used as the sample and the freeze-dried standard was used as the standard for calibration, the geometric coefficient of variation (GCV) of the results of the 3 laboratories decreased from 64.4% to 29.2%, and the precision of the experiment was greatly improved.

The batch of freeze-dried HSV-1 standards for oncolytic activity assay meets the relavant requirements, and is more suitable for use as a national standard than liquid standards. Its oncolytic activity is assigned a value of 1.82×10⁴ U·vial^-1.

To determine seven impurities in oxytocin for injection and investigate the limit values.

HPLC and principal component self-control with correction factor were adopted. The determination was performed on a Waters Xbridge C₁₈ column(150 mm×4.6 mm, 5 μm). The mobile phase consisted of 0.1 mol·L^-1 dihydrogen phosphate solution (adjusted to pH 5.4)-acetonitrile (90∶10, phase A), and acetonitrile (phase B) with gradient elution at a flow rate of 1.5 mL·min^-1. The column temperature was maintained at 32 ℃, and the detection wavelength was set at 220 nm. The injection volume was 100 μL. The linear equations of oxytocin, impurities Ac-Oxy, Oxy[Glu⁴], Oxy[+Gly¹⁰], Oxy[-NH₂], Oxy[trisulfide], Oxy[cis-dimer] and Oxy[trans-dimer] were drawn. The correction factors of each impurity related to oxytocin were calculated by slope. The contents of impurities in 3 batches of oxytocin for injection were determined and compared with the results of impurity reference method.

The limits of quantification for seven impurities were 2.75-5.66 ng, while the detection limits were 1.38-2.83 ng. The linear ranges of seven impurities were 0.03-3.40 μg·mL^-1 with good linearity(r>0.999). The correction factors of Ac-Oxy, Oxy[Glu⁴] and Oxy[-NH₂] were 1.1, while the correction factors of Oxy[+Gly¹⁰] and Oxy[trisulfide] were 1.2 and 0.9, respectively. The correction factors of Oxy[cis-dimer] and Oxy[trans-dimer] were both 1.3. The seven impurities were determined in 3 batches of samples by principal component self-control with correction factor. The contents of impurity Ac-Oxy were 0.96%, 0.93% and 1.01%, respectively. The contents of impurity Oxy[Glu⁴] were 0.07%, 0.06% and 0.08%, respectively. The contents of impurity Oxy[+Gly¹⁰] were 0.07%, 0.04% and 0.04%, respectively. The contents of impurity Oxy[-NH₂] were 0.09%, 0.05% and 0.07%, respectively. The contents of impurity Oxy[trans-dimer] were 0.27%, 0.18% and 0.22%, respectively. The maximum single impurity contents were 0.18%-0.19%, while the total impurity contents were 1.88%-2.06%. Compared the results measured by principal component self-control with correct factor method and the impurity reference method, there was no significant difference between two methods (p>0.05).

The method is proved to be simple, repeatable and accurate for the content determination of related substances in oxytocin for injection.

To explore the correlations between opalescence and molecular size, purity or aggregation tendency in the antibody formulation.

Studied on the opalescence phenomena of various antibodies, including the influence of different concentrations, pH/buffer, excipients, as well as the influence of environmental factors such as temperature, light, freeze-thaw and agitation, and the correlations had been analyzed between opalescence and molecular size, purity (aggregation), K_D or T_agg.

The degree of antibody opalescence was positively correlated with the molecular size, the deepening of the opalescence indicated that the molecular size was increased and the more tendency of molecular aggregation. For example, the opalescence of C molecule obviously enhanced under high temperature, and the result of turbidity changed from 3.3 NTU to 13.6 NTU, the molecular size changed from 13.1 nm to 40.6 nm, and the K_D changed from positive to negative. The T_agg changed from 59.6 ℃ to below 50 ℃.

The opalescence can be affected by buffer system, pH, excipients and ambient temperature, the degree of opalescence increased, indicating the stability become worse and the molecules prone to aggregate. This study provides important reference for the analysis and evaluation of opalescence in the antibody formulation development.

To establish a method for quality evaluation of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] dispensing granules by combining characteristic chromatogram, quantitative analysis of multi-components by single marker (QAMS) and chemical pattern recognition analysis.

The high performance liquid chromatography (HPLC) characteristic chromatogram was established by 15 batches of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] standard decoctions and 3 batches of dispensing granules. The contents of 6 components were determined by QAMS. The chromatographic separation was achieved on a Acclaim^TM RSLC 120 C₁₈ column (100 mm×2.1 mm, 2.2 μm), with the mobile phase comprising of acetonitrile -0.05% phosphoric acid flowing at 0.4 mL·min^-1 in a gradient elution manner. And the detection wavelength was set at 327 nm. The similarity evaluation system of fingerprint of traditional Chinese medicine was used to determine the common peak for similarity evaluation. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were applied for chemical pattern recognition. The transfer rates of the 6 components from decoction pieces to standard decoctions and dispensing granules were calculated.

The similarities of characteristic chromatograms of 15 batches of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] standard decoctions and 3 batches of dispensing granules were all above 0.85. And 8 common characteristic peaks were identified. The results of HCA and PCA indicated the similarity of ingredients in formula granules to those in standard decoctions. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C in standard decoctions were 1.87-5.23, 7.44-15.26, 2.85-8.18, 3.05-6.14, 0.99-3.93 and 3.23-10.38 mg·g^-1 and the transfer rates of these components from decoction pieces to standard decoction were 23.85%-37.28%, 19.57%-31.93%, 28.15%-45.88%, 22.34%-36.59%, 16.64%-28.36% and 21.81%-39.19%, respectively. The contents and transfer rates of these 6 compounds in dispensing granules were close to that of standard decoctions.

The characteristic chromatogram and QAMS method established can be used for quality control and process research of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] dispensing granules.

To develop an HPLC method for the determination of related substances and assay of levamisole hydrochloride tablets.

The chromatographic separation of related substances was performed on a Thermo Hypsil C₁₈ column(100 mm×4.6 mm, 3 μm). A gradient elution was applied with a mobile phase composed of 0.75% monobasic ammonium phosphate solution(adjusted at pH 6.5 with diiopropylamine) and acetonitrile. The assay analytical column was packed with IntertSustain C₁₈ column(150 mm×4.6 mm, 5 μm). The mobile phase was 70 volumes of 0.75% monobasic ammonium phosphate solution(adjusted at pH 7.0 with diiopropylamine) with 30 volumes of acetonitrile. The wavelength detection was set at 215 nm, the injection volumn was 10 μL, and the flow rate was 1.0 mL·min^-1.

Levamisole and its impurities were separated well by related substances HPLC method above. Impurities A, B, C, D, E showed the good linearities in the concentration ranges of 10.87-25.37 μg·mL^-1, 11.62-27.10 μg·mL^-1, 12.38-28.90 μg·mL^-1, 30.89-72.07 μg·mL^-1, 12.41-28.95 μg·mL^-1 (r>0.999). The average correction factors of impurities A, B, C, D, E determined by three columns and liquid chromatographies were 1.6, 1.4, 2.6, 1.2, 2.4, respectively, the recovery rates(n=9) were 98.1%, 99.0%, 98.6%, 100.1%, 99.9% and the RSDs were 0.63%, 0.79%, 0.92%, 0.96%, 0.33%. The separation of levamisole and impurity C was good by assay HPLC method. Levamisole showed the good linearity in the concentration range of 0.10 14-0.304 3 mg·mL^-1 (r=0.999 9). The average recovery rate(n=9) of levamisole was 100.2%, RSD=0.52%. Ten batches levamisole hydrochloride tablets from five domestic pharmaceutical enterprises were determined by the above related substances and assay HPLC method, the total impurities mass were 0.05%-0.62%, and the assays were 99.5%-104.3%.

The established method is high selection and accurate. It is suitable applied for determination of related substances and assay of levamiole hydrochloride tablets.

To determine the contents of Cd and As in Isatidis Radix and to explore the application of target-organ toxicity dose(TTD) modification of hazard index (HI) method in assessing the risk of combined exposure to heavy metals and harmful elements in traditional Chinese medicines (TCMs).

According to the monitoring data of Cd and As in Isatidis Radix, the exposure doses were calculated. The health risk caused by combined exposure to Cd and As was preliminary screened by HI method. Moreover, a more accurate TTD method was used.

The qualified rates of Cd in 29 batches of Isatidis Radix were 100%. However, 5 batches of As contents exceeded the limit standard. The HQ values of As in 5 batches of Isatidis Radix were more than 1. The results of TTD method showed that for the end points of cardiovascular system, blood, nervous system, the HI values of 5 batches of Isatidis Radix were more than 1, and the health risk was not acceptable.

The cumulative risk assessment of heavy metals in TCMs is proposed in this study, which offers novel ideas for the development of risk assessment methods of exogenous harmful residues in TCMs, and provides technical support for formulating scientific limit standards.

To develop an HPLC method for the separation of perindopril tert-butylamine and its epimer [(±)-1”-epi-perindopril tert-butylamine] and the determination of the epimer.

Perindopril and (±)-1”-epi-perindopril were separated on an Agilent Poroshell CS-C₁₈ column (100 mm×3.0 mm, 2.7 μm) maintained at 50 ℃ with the mobile phase containing a mixture of 0.15% sodium heptanesulfonate solution (adjusted to pH 2.0 with phosphoric acid) and acetonitrile-pentanol(217∶3)(82∶18, V/V) at 0.8 mL·min^-1, and the detection wavelength was set at 215 nm. The injection volume was 2 μL.

(±)-1”-epi-perindopril and perindopril were separated successfully in 25 min with peak to valley ratio more than 3.0 or a resolution factor of 1.7. Good linear relationships were established between the peak response and the concentration in the range of 2-2 000 μg·mL^-1 for the epimer and perindopril tert-butylamine(r>0.999). The quantitative limits(S/N= 10) were both about 1.0 μg·mL^-1, and the detection limits(S/N=3) were both 0.3 μg·mL^-1. The spiked recovery of the epimerer was 97.2% (RSD=1.8%, n=9). The content of (±)-1”-epi-perindopril tert-butylamine in 10 batches of samples ranged from 0.025% to 0.078%.

The proposed method enhances the resolution efficiency, shows high accuracy, repeatability and stability. It can be effectively employed for the quality control of perindopril tert-butylamine．

To establish a method for the rapid identification of the authenticity of Gastrodiae Rhizoma herbs based on enzymatic recombinase amplification (ERA) technique.

Following the principle of ERA primer design, Oligo 7.0 software was applied to screen and optimize the ERA-specific primers of Tianma based on the ITS2 genome sequences of Gastrodia elata and its common artifacts. Primer Premier 5.0 software was applied to design the specific PCR primers for the identification of Gastrodia elata, and through the optimization of ERA and PCR reaction system, the optimal reaction time for ERA was finally determined to be 17 min, the optimal reaction temperature was 40 ℃, the optimal annealing temperature for PCR was 57 ℃, and the cycle was 32 times, and the established method was verified for sensitivity and specificity, and the samples of asparagus available in the traditional Chinese medicine The sensitivity and specificity of the established method were verified, and the commercially available asparagus samples in the market were selected for testing.

The designed primers for the specific identification of ERA in Gastrodia elata did not cross-react with its common forgeries, and the specificity was good, a repeatability results showed that the three repeatability tests were consistent, with no false positives or false negatives；the sensitivity of this method for Gastrodia elata genomic DNA was 1 pg·μL^-1, which was higher than that of conventional PCR；the ERA technique can be used for the rapid identification of commercially available samples of Gastrodia elata and the results of the assay are the same as those of the PCR method.

The established detection method is simple, rapid, with high specificity and sensitivity, and provides a new means for the authentication of the Gastrodiae Rhizoma.