To establish an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of daidzein, formononetin, genkwanin, quercetin, rutin, apigenin, kaempferol, luteolin and puerarin in Tongfu Jingyaotong tincture.

The Waters ACQUITY UPLC HSS T3 Column (100 mm×2.1 mm, 1.8 μm) was used with methanol and 0.1% formic acid (gradient elution) as the mobile phase at a flow rate of 0.3 mL·min^-1. Column temperature was 45 ℃, and injection volume was 2.0 μL. Electrospray ion source was adopted with positive and negative ion modes and multi-reaction monitoring and acquisition.

Apigenin, genkwanin and daidzein had good linearity in the range of 0.2-1 000 ng·mL^-1, puerarin, rutin, luteolin, kaempferol and formononetin in the range of 0.2-2 000 ng·mL^-1, and quercetin in the range of 1.0-5 000 ng·mL^-1. The resolution was good, and the RSDs of precision, repeatability, reproducibility and stability test were all below 5% (n=6). The average recoveries ranged from 82.7% to 111.8% with RSDs (n=6) of 2.0%-5.4%. The contents of daidzein, formononetin, genkwanin, quercetin, rutin, apigenin, kaempferol, luteolin and puerarin in 6 batches of Tongfu Jingyaotong tincture were 8.46-17.97 μg·mL^-1, 0.62-1.67 μg·mL^-1, 0.03-0.08 μg·mL^-1, 1.34-2.22 μg·mL^-1, 0.70-1.14 μg·mL^-1, 0.48-0.99 μg·mL^-1, 0.20-0.90 μg·mL^-1, 0.10-0.16 μg·mL^-1 and 167.95-227.75 μg·mL^-1, respectively.

This method has strong specificity, wide linear range, accuracy and efficiency, and can detect the contents of 9 active ingredients in Tongfu Jingyaotong tincture at the same time, which offers an effective quality control method for Tongfu Jingyaotong tincture.

To establish and optimize a rapid direct analysis of real time tandem mass spectrometry (DART-MS/MS) method for the rapid detection of 12 benzodiazepines in blood and urine that can be used in forensic toxicology work.

A DART ion source was used in conjunction with an API4000 Q Trap mass spectrometer. A DART 12Dip-It^TM autosampling module with a module travel speed of 0.6 mm·s^-1, a sample volume of 5 μL, and a gate voltage of 200 V were applied. The mass spectrometry section scans in positive ion mode using multiple reaction monitoring (MRM) mode. After further optimization, the DART-MS/MS method was validated and applied to real case samples.

Ethyl acetate was selected as the extractant for liquid-liquid extraction and the temperature of the carrier gas heater was optimized. The method has good selectivity and does not interfere with delayed effects. The linearity was good, and the limits of detection (LODs) for the targets in blood and urine were in the ranges of 0.5-10 ng·mL^-1 and 0.2-2 ng·mL^-1, respectively. And the limits of quantification (LOQs) were in the ranges of 1-50 ng·mL^-1 and 0.5-5 ng·mL^-1, respectively. The recoveries ranged from 78.8% to 119%, and the matrix effects ranged from -17.5% to 18.5%. The intra- and inter-day precisions were not greater than 14.4% for the high and intermediate concentrations, and not greater than 18.1% at the limits of quantification. This method enables fast and accurate examination of case samples.

The method is fast and convenient, with good sensitivity, and can be applied to the research and work of rapid detection of toxicants to improve the efficiency of detection.

To establish an HPLC-MS/MS method for the determination of FCN-437c in human plasma and its application to the phase I clinical study of FCN-437c.

Following protein precipitation, plasma was injected and measured using HPLC-MS/MS method. The analytes were separated on a YMC Triart PFP column (50 mm×2.1 mm, 5 μm) using 0.5% formic acid (containing 5 mmol·L^-1 of ammonium acetate, A) and acetonitrile (B) as the mobile phase with gradient elution at the flow rate of 0.5 mL·min^-1, the column temperature was set at 35 ℃, the injection amount was 2 μL, and the injector temperature was 10 ℃. MS detection was performed with multiple reaction monitoring (MRM) mode using positive electrospray ionization. The ion transitions were m/z 549.4→449.4 for FCN-437c and m/z 552.3→449.3 for FCN-437-D3, respectively. Other mass spectrometry parameters were TEM, 500 ℃, GS1, 276 kPa, GS2, 207 kPa, DP, 100 V, CXP, 25 V.

The linear range of FCN-437c in human plasma was 5-1 000 ng·mL^-1 (r=0.999 0). The lower limit of quantification was 5 ng·mL^-1. The intra-batch and inter-batch precisions were less than 2.0% and 4.1%, respectively. The average recovery was 104.0% (FCN-437c), 78.6% (FCN-437-D3), and the internal standard normalized matrix factor was 100%-102%. The stock solution of FCN-437c was stable at 4 ℃ for 202 d, the working solution of FCN-437c and internal standard were stable at room temperature for 24 h. FCN-437c in human plasma was investigated to be stable at room temperature for 20 h, four cycles of freeze-thaw, -20 ℃ for 134 d, -80 ℃ for 662 d, as well as for 24 h in the autosampler after treatment. The whole blood samples were stable at room temperature for 4 h. This method was applied to the determination FCN-437c in human plasma, and the deviation between the test results and the initial values of 97.0% ISR samples was within ±20%. The accuracy was 102.0%-108.0% after 10-fold dilution of plasma samples. The cumulative ratio of R_AUC0-24 and R_{C max} was 1.33 times and 1.59 times when FCN-437c was administered continuously compared with single administration.

This method is simple, accurate, robust and specific, which can meet the requirements of quantitative analysis of FCN-437c in human plasma and also can be used to determine FCN-473c in human plasma of the phase I clinical study.

To establish a UPLC-MS/MS method for the simultaneous determination of 47 pesticide residues in Sucrose.

The residues in the sample were extracted by acidic acetonitrile, concentrated by blow dry with nitrogen gas and then analyzed by using LC-MS/MS with multiple reaction monitoring(MRM). Blank matrix standard curve with internal standard method was used to determine the residue contents.

Each substance to be tested had good linearity relationship within certain concentration (r>0.995). The limits of quantification (LOQ) were 0.01-2.00 μg·kg^-1. The recoveries of 47 pesticide residues at three levels of 10, 25 and 50 μg·kg^-1 were from 71.0% to 106.0% with the RSD of 0.90%-8.5% in sucrose. 2 batches in 15 batches of sucrose samples were detected thiamethoxam and thiamethoxam, while the rest were not detected.

The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements of the determination of analysis, and this developed procedure is suitable for the determination of pesticide residues in Sucrose, can be used for quality control of sucrose.

To establish a fast, accurate and sensitive method by UPLC-DAD-Q-TOF-MS/MS for the detection of 20 kinds of illegally added chemicals such as azilsartan, nifedipin, lacidipine、nebivolo in anti-hypertensive health foods.

Waters HSS T3 C₁₈（100 mm×2.1 mm, 1.8 μm ） column was used with mobile phase of 0.01 mol·L^-1 ammonium acetate aqueous solution-methanol and gradient elution（0 min, 5% A; 0-8 min, 5% A→95% A; 8-11 min, 95% A; 11-11.2 min, 95% A→5% A; 11.2-12 min, 5%). The flow rate was 0.3 mL·min^-1. The injection volume was 1 μL. ESI⁺, ESI^- scan and MS/MS mode were applied. Qualitative and quantitative analyses were carried out by UPLC-DAD-Q-TOF-MS/MS.

The method was validated. A good linear relationship was showed in the concentration of 1-50 μg·mL^-1, （RSD<3%）and the recovery was obtained in range of 80.5%-98.7%. Illegally added chemical drugs were found in 15 batches of samples tested by this method. Two or more types of drugs were detected in 14 batches of samples. Torsemide, candesartan cilexetil, lacidipine were detected in 9 samples. Lacidipine and nebivolol were found in one of the sample.

This method is fast, accurate and sensitive with a high degree of separation. It can be used as a powerful tool for the fast detection of illegally added chemicals in anti-hypertensive health foods.

To establish a liquid chromatography mass spectrometry (LC-MS) method for the detection of nepasaikosaponin K in Bupleurum marginatum var. stenophyllum, which could be used to quickly identify whether Bupleurum marginatum var. stenophyllum was substituted for Bupleurum or was mixed with Bupleurum. At the same time, nepasaikosaponin K was detected in 16 batches of Bupleurum marginatum var. stenophyllum and 157 batches of bupleurum decoction pieces.

Ultra-high performance liquid chromatography (UPLC) coupled with triple quadruple mass spectrometry (QqQ MS) was used to detect nepasaikosaponin K in Bupleurum and Bupleurum marginatum var. stenophyllum. Determination was carried out with the application of a Phenomenex Kinetex C₁₈ (2.1 mm×100 mm, 1.7 μm) column at temperature of 25 ℃. The mobile phase was composed of acetonitrile(A) and water(B) with gradient elution(0-5 min 15% A→25% A; 5-0 min, 25% A→30% A; 30-35 min, 30% A→90% A; 35-36 min, 90% A→15% A; 36-40 min, 15% A) at a flow rate of 0.3 mL·min^-1. The electrospray ionization(ESI^-) source was performed in multiple reaction monitoring (MRM) mode of the transitions of m/z 943.5→797.3, m/z 943.5→635.3, and m/z 943.5→781.3.

The contents of nepasaikosaponin K detected in 16 batches of Bupleurum marginatum var. stenophyllum were much higher than minimum requirements. Nepasaikosaponin K was detected in 8 out of 157 batches of Bupleurum.

The established method is verified by methodology to be specific, so as to develop a supplementary test method for the components of Bupleurum and Bupleurum marginatum var. stenophyllum. to improve the quality control standard and authentic identification investigation, and to effectively control the quality of Bupleurum decoction pieces and guarantee its clinical efficacy.

To establish an amino solid-phase extraction-HPLC method for the determination of three related substances in eldecalcitol soft capsules.

The eldecalcitol soft capsules were extracted by an amino solid-phase extraction method, followed by eluting with ethyl acetate: n-hexane and anhydrous ethanol. The residue was then dissolved with acetonitrile and water after vacuum evaporation. An ODS column (250 mm×4.6 mm, 5 μm) was used. Water-acetonitrile with gradient elution was employed. The flow rate was 1.0 mL·min^-1, the injection volume was 50 μL, and the detection wavelength was set at 265 nm.

Interference from blank excipients was eliminated with pretreatment. Baseline separation and good selectivity of eldecalcitol and other impurities was achieved. The stability of the test and reference solution was investigated at 4 ℃. The area change percentage of the impurities in the test solution were 98.6%-102.3% at 24 h. The area change percentage of the impurities in the reference solution were 101.2%, 96.9%, 98.5% and 96.2% at 24 h, respectively. The quantitation limits of eldecalcitol, trans impurity, pre-eldecalcitol and tachy impurity were 0.107 μg·mL^-1, 0.102 μg·mL^-1, 0.128 μg·mL^-1 and 0.063 μg·mL^-1, respectively. The detection limits were 0.054 μg·mL^-1, 0.051 μg·mL^-1, 0.064 μg·mL^-1, 0.031 μg·mL^-1, respectively. The linear ranges of 4 components were 0.1-6.3 μg·mL^-, 0.1-1.2 μg·mL^-, 0.1-5.6 μg·mL^-and 0.1-1.1 μg·mL^-1, respectively. The correlation coefficients（n=5） were 0.999 9, 1.000, 1.000 and 0.999 4, respectively. The average recoveries （n=6）of tachy impurity, pre-eldecalcitol and trans impurity were 106.6%, 88.6% and 102.8% with RSDs of 6.7%, 3.1% and 2.1%, respectively. In the three batches of samples, no impurities were detected except pre-eldecalcitol. The contents of pre-eldecalcitol were 5.0%, 5.0% and 5.1%, respectively.

The method is simple to operate, economical and practical with high safety, strong specificity, good repeatability and high accuracy. The method provides a reliable basis for the study of drug quality such as eldecalcitol soft capsules and drugs with similar oily substrate.

To determine the content of acrylamide in “8 famous medicines from Zhejiang”, and to carry out the preliminary risk assessment based on the determination results.

After optimization of QuEChERS extraction, the content of acrylamide was determined by the method of isotope-labeled internal standard-ultra high performance liquid chromatography-tandem mass spectrometry (isotope-labeled IS-UHPLC-MS/MS). The determination was carried out with a Phenomenex Kinetex C₁₈ column (100 mm×2.1 mm, 1.7 μm) as the stationary phase, gradient elution was carried out with methanol-0.1% formic acid aqueous solution as the mobile phase, ¹³C₃ acrylamide as the internal standard, and multiple reaction monitoring (MRM) mode was used to detect the residual acrylamide.

The content ranges of acrylamide in 247 batches of samples were 0-26 403 μg·kg^-1. These contents exceeded the regulations stipulated by some nations or organizations, and posed certain safety risks in long-term use.

The method established in this study is rapid, simple, sensitive and reproducible, and will lay a scientific basis for the quality control and safety evaluation of acrylamide in “8 famous medicines from Zhejiang” and other genuine medicinal materials.

To study the toxicity of heavy metal cadmium and screen detoxification drugs base on Caenorhabditis elegans.

The microfluidics chip technology was used as the drug screening platform. The 96-well plate exposure pretest was used, and K-medium blank group was set up. The exposure group consisted of nematodes exposed to heavy metal cadmium at the environmental relevant concentration of 0.25-15.0 μg·mL after homogenization. Vitamin C and calcium disodium ethylenediamine tetraacetate were selected as detoxification group. （10±2） nematodes were added into each orifice plate. The number of nematodes was observed and recorded under a microscope, and paid attention to the changes in the structure of the vulva. The experimental data were processed and analyzed by origin 2019b software.

Compared with blank group, the fatality rates of C. elegans in exposed group were respectively 0%, 1.67%, 4.76%, 67.46%, 100% and 100% at 0.25, 1.50, 5.0, 10.0, 12.5, 15.0 μg·mL^-1 concentration, compared with blank group. The structure of the nematode vulva was deformed, from slight protrusion to severe protrusion, and even a tuberous protrusion, and finally ruptured. The mortality of detoxification group was reduced to different degrees compared with exposure group.

Compared with blank control group, with the increase of cadmium exposure concentration and exposure time, cadmium can inhibit the survival of C. elegans, and damage the structure of C. elegans vulva to varying degrees. The results of detoxification group showed that vitamin C had little detoxification effect on cadmium. The complexation of calcium disodium ethylenediamine tetraacetate with cadmium could prolong the survival time of C. elegans to a large extent.

To establish a chromatogram-effect correlated quality evaluation system for the antibacterial activity of Flos Lonicerae extract by data analysis of components.

The best liquid chromatographic conditions were optimized by investigating the conditions of mobile phase, wavelength and flow rate, and the quantitative fingerprint of 50 batches of Flos Lonicerae extract was established. The best antibacterial concentration of the extract was obtained by microdilution method, and the antibacterial rates of 50 batches of samples were determined. Similarity analysis, cluster analysis, principal component analysis, grey correlation analysis and mathematical model establishment by support vector machine were used to analyze the quantitative fingerprints and antibacterial rates of 50 batches of samples.

Eighteen common peaks were selected from the 50 quantitative fingerprints of Flos Lonicerae extract, and the similarities were between 0.608-1. Six chemical components were identified (peak 4: neochlorogenic acid, peak 8: chlorogenic acid, peak 9: cryptochlorogenic acid, peak 16: isochlorogenic acid B, peak 17: isochlorogenic acid A and peak 18: isochlorogenic acid C). The average antibacterial rates of 50 batches of extracts were between 3.93%-70.50%. The results of principal component analysis and cluster analysis were highly consistent, and the results of grey correlation analysis showed that all components were positively correlated with antibacterial effect. The relative deviations between the predicted data of the mathematical model and the experimental data were all below 2%.

The HPLC conditions of Flos Lonicerae extract are stable and reliable, and the quality evaluation system of antibacterial activity of Flos Lonicerae extract established can evaluate its quality based on antibacterial rate.