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 determine the in vitro release degree of musk sustained-release mini-tablets by reciprocating cylinder method.

Gas chromatography was used to establish the in vitro analysis of muscone, which is the main active ingredient of sustained-release mini-tablets. Comparing its equilibrium solubility in different concentrations of Tween-80, hexadecyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), to determine the type of release medium. The effects of residence time, drip time, reciprocation rate, and screen mesh sizes on the release behaviours of muscone were investigated. Results were compared with that of the paddle method. Meanwhile, a preliminary investigation into the mechanism of its release was also carried out.

The linearity of the constructed analytical method was better, the RSD values of precision, repeatability and stability were less than 3%, and the spiked sample recovery was qualified. The release medium for the reciprocating cartridge method was 200 mL of 0.05% Tween-80, with a dwell and drip time of 20 s, a reciprocation rate of 15 dip·min^-1, and upper and lower screen mesh sizes of 20 and 40 respectively. Compared with the paddle method, Muscone was completely released under different pH conditions in the reciprocating cylinder method, and the release curve increased smoothly.

The study can provide some references for the reciprocating cylinder method in the in vitro evaluation of sustained-release prescriptions of traditional Chinese medicine.

To study different batches of Xiaoer Resuqing granules by combining the method of fingerprint, multi-index component content determination and chemical pattern recognition analysis technology, and to provide the basis for its quality evaluation.

The chromatographic SuperLu C₁₈(250 mm×4.6 mm, 5 μm) column was adopted with mobile phase composed of methanol (A)-0.1% phosphoric acid (B) with gradient elution at a flow rate of 1.0 mL·min^-1. The column temperature was 30 ℃, and the detection wavelength was 280 nm. Eleven characteristic components were identified, and the contents of seven active components, including puerarin, forsythiaside A, phillyrin, baicalin, wogonoside, baicalein and wogonin were determined. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used to classify 15 batches of samples, and orthogonal least partial squares discriminant analysis (OPLS-DA) was performed. To screen out the difference components between batches of Xiaoer Resuqing granules.

The HPLC fingerprint of Xiaoer Resuqing granules and the content determination method of 7 components were established, the results of the two methods met the requirements. Twenty-eight common peaks were identified, 11 chromatographic peaks were identified, and the similarities of 15 batches of samples ranged from 0.995 to 0.999. HCA and PCA results showed that 15 batches were clustered into two categories, and OPLS-DA screened out 13 markers that caused the differences among the 15 batches of samples. There was little difference in the contents of ingredients.

The established HPLC fingerprint of Xiaoer Resuqing granules and the determination method of 7 components are specific, accurate and reliable. Combined with chemical pattern recognition, it can be effectively used for the quality control of Xiaoer Resuqing granules.

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 establish an HPLC characteristic chromatogram for Hyssopus cuspidatu Boriss. and control quality and identity authentication for the herb.

The HPLC method was used, and the mobile phases were acetonitrile (A) and 0.1% aqueous phosphoric acid (B) in gradient elution(0-15 min, 10% A; 15-30 min, 10% A→15% A; 30-40 min, 15% A→18% A; 40-60 min, 18% A→23% A; 60-70 min, 23% A→55% A; 70-85 min, 55% A→60% A; 85-85.1 min, 60% A→10% A; 85.1-90 min, 10% A). The column temperature was 30 ℃, the flow rate was 1.0 mL·min^-1, the detection wavelength was 330 nm and the injection volume was 20 μL. Fifteen batches of Hyssopus cuspidatu Boriss. and its adulterants, notably Cynanchum officinale, were analyzed to construct characteristic chromatogram. The authenticity and quality of Hyssopus cuspidatu Boriss. were assessed through principal component analysis(PCA), cluster analysis, and similarity analysis.

Analysis of the characteristic chromatogram of ten Hyssopus cuspidatu Boriss. batches revealed fifteen distinct peaks, with six compounds identified as isodendrobioside, molluscoside, rosmarinic acid, salvianolic acid, geranylgeranyl, and trichothecenes. Cluster analysis segregated the Hyssopus cuspidatu Boriss. samples into three distinct groups, primarily influenced by geographical (latitude and longitude) and environmental (climate) factors. PCA results indicated that four principal components accounted for 92.914% of the variance. The similarities for the ten batches of Hyssopus cuspidatu Boriss. ranged from 0.896 to 0.997, with no significant difference observed between the wild and cultivated varieties in terms of overall HPLC profile and relative peak content. The overall profiles of the HPLC characteristic chromatogram and the contents of the main components of the Hyssopus cuspidatu Boriss. and the Nepeta bracteata Benth. were significantly different.

The HPLC characteristic chromatogram method developed in this study is reproducible, stable, and practical for the quality control and authentication of Hyssopus cuspidatu Boriss. Moreover, it offers a reliable analytical technique for the quality evaluation and authentication of this herb, providing substantial data support.

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 develop a method for characterizing in vitro release of cyclosporine ophthalmic emulsion.

The Franz diffusion cell and the polyvinylidene fluoride membrane were adopted with buffer-ethanol (40∶60) as receiving media. The sampling time was set at 60, 125, 190, 255, 320, 385 min, respectively.

The in vitro release method showed that the inertia of membrane, specificity, sensitivity and selectivity met the requirements. The validation of HPLC showed that the quantitative limit of the method was 0.07 μg·mL^-1 and a good linear relationship between the concentration range of 0.07-44.62 μg·mL^-1. The average recovery was 98.9%. Compared with the original preparation by FDA guideline, the in vitro release of the self-developed preparation was the same as the reference preparation.

This method is suitable for the in vitro release evaluation of cyclosporine eye drops.

To establish a stable and reliable gas chromatography-tandem mass spectrometry method for the determination of N-nitrosodimethylamine (NDMA) in metformin hydrochloride sustained-release tablets, and to optimize extraction conditions by investigating the effects of isopropyl alcohol and thioglycerin on the stability of the tested solution.

Separation was achieved on AB-InoWax capillary column (30 m×0.25 mm×0.25 μm) with polyethylene glycol as the stationary phase. Electron ion(EI) source and multiple reaction monitoring (MRM) mode were used. Quantitative determination was performed by both external standard and internal standard.

The addition of thioglycerin could significantly improve the stability of the test solution. NDMA showed good linearity within the concentration range of 0.25-50 ng·mL^-1(r>0.999). The detection limit of the method was 0.1 ng·g^-1 and the quantification limit of the method was 0.2 ng·g^-1. The average recoveries (n=9) were 97.3% and 94.9% while using external standard and internal standard, respectively. Precision, repeatability and stability were good with RSD less than 8%. Ninety batches of metformin hydrochloride sustained release tablets were tested. NDMA content in all detected samples were all less than 30% of the acceptable limit set by the National Medical Products Administration and FDA.

This method shows satisfactory sensibility, specificity, accuracy, stability and durability, which is suitable for quantitative analysis of NDMA in metformin hydrochloride sustained-release tablets, providing technical support for the quality and safety of related products.

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 a method for determination of potential anti-vascular disease active components of Semen raphani based on cell membrane chromatographic (CMC) model of human umbilical vein cell (HUVEC).

The HUVEC cell membrane chromatography coupled with HPLC-ESI IT TOF MS system were used to screen the active components of Semen raphani. The selected active components were further applied to ox-LDL induced HUVEC to verify their protective effects.

Two retained components were selected from Semen raphani by this method. One component was identified as erucinic acid by comparing with the reference material. Compared with the model group, the cell survival rates of the erucinic acid pretreatment groups increased significantly. The amount of ICAM-1 and VCAM-1 decreased in a dose-dependent manner. Bcl-2 protein levels decreased and Bax protein levels increased, with statistical significance (P<0.05).

The method can rapidly obtain active ingredients from complex traditional Chinese medicines. It provides a reference for the application of cell membrane chromatography and the development of Semen raphani.