To analyze and optimize stir-baked with vinegar technology of Genkwa Flos decoction pieces by multi-index weighted scoring method based on orthogonal experiment, and to provide technical index on standardization of stir-baked with vinegar technology of Genkwa Flos decoction pieces.

The orthogonal experiment was employed to analyze and evaluate the four key factors related to stir-baked with vinegar technology including the amount of vinegar, dampening time, temperature and stir-baked time by the six main indicators including the total contents of chlorogenic acid, tiliroside, luteolin, apigenin, hydroxygenkwanin and genkwanin, the content of alcohol extractive and appearance characters.

The amount of vinegar and stir-baked temperature had notable effects, while the dampening time and stir-baked time had no notable effects on the test results. Considering comprehensively the effects of four key factors, the optimized stir-baked with vinegar technology was as follows: add 0.3 kg of vinegar to 1 kg of Genkwa Flos, and fry at 160 ℃ for 10 min after dampening for 15 min.

This study offers guidance and reference for standardizing the stir-baked with vinegar technology of Genkwa Flos decoction pieces.

To establish high-performance liquid chromatography method using cyclodextrin as a mobile phase additive for the determination of ursolic acid and oleanolic acid in Corni Fructus, providing a basis for quality control of Corni Fructus.

Molecular design and response surface method were used to optimize the determination method of ursolic acid and oleanolic acid in Corni Fructus. Agilent Eclipse XDB C₁₈ column (150 mm×4.6 mm, 5 μm) was adopted as the stationary phase, the mobile phase was 3 mmol·L^-1 γ-cyclodextrin solution (contained 0.05% phosphoric acid)-acetonitrile (60∶40), the flow rate was 1.0 mL·min^-1, the column temperature was 25 ℃ and the detecting wavelength was 210 nm.

The resolution of chromatographic peaks of ursolic acid and oleanolic acid was 3.81. Linearities were good in the range of 400-4 000 ng for ursolic acid and in the range of 200-2 000 ng for oleanolic acid. The RSDs for precision, reproducibility, and stability were less than 1.4%, 1.8%, and 1.7%, respectively. The recovery rates were 98.5% (RSD=1.7%) and 99.1% (RSD=2.0%). The contents of ursolic acid in three batches of Corni Fructus were 2.66 mg·g^-1, 2.35 mg·g^-1 and 2.91 mg·g^-1, and those of oleanolic acid were 0.83 mg·g^-1, 0.78 mg·g^-1and 1.12 mg·g^-1, respectively.

This method is simple, accurate, reliable, and environmental friendly, and can be used as a quality control for Corni Fructus.

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).

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.

To establish the UPLC-MS/MS method for simultaneous determination of 9 components (nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) in Desmodium caudatum (Thunb.) DC. and construct a back propagation(BP) neural network model to predict the origin of Desmodium caudatum (Thunb.) DC. from different habitats.

The chromatographic separation was achieved on an Agilent Zorbax SB C₁₈ column (50 mm×3.0 mm，1.8 μm). The mobile phase consisted of methanol -0.1% acctic acid (containing 0.02 mol·L^-1 ammonium acetate) at a flow rate of 0.3 mL·min^-1 with gradient elution, the MS analysis were performed by multiple reaction monitoring (MRM) under ESI⁺ and ESI. A correlation analysis was conducted on the contents of each component, and a BP neural network model was constructed to distinguish Desmodium caudatum (Thunb.) DC. from different habitats.

Under the optimized conditions, 9 components(nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) showed good linear relationships in the ranges of 0.388 8-38.88 ng·mL^-1, 10.07-1 006.6 ng·mL^-1, 34.22-34 221.6 ng·mL^-1, 3.944-394.4 ng·mL^-1, 2.124-212.4 ng·mL^-1, 4.344-434.4 ng·mL^-1, 46.50-4 650.1 ng·mL^-1, 1.649-164.9 ng·mL^-1, 4.880-488.0 ng·mL^-1, respectively (r>0.995 1), whose average recoveries were 96.9%-103.9% (RSDs<1.9%). The contents of the above nine components in 40 batches of Desmodium caudatum (Thunb.) DC. were 1.657-7.407 μg·g^-1, 15.801-64.488 μg·g^-1, 1 068.348-4 270.780 μg·g^-1, 10.608-123.228 μg·g^-1, 3.897-16.802 μg·g^-1, 1.269-97.834 μg·g^-1, 405.285-1 955.796 μg·g^-1, 13.614-36.124 μg·g^-1, 4.417-87.509 μg·g^-1, respectively. According to correlation analysis, four components (swertisin, rutin, spinosin, and luteolin) in Desmodium caudatum (Thunb.) DC. showed a highly linear positive correlation, indicating that these four components had a certain synergistic effect in Desmodium caudatum (Thunb.) DC.. The BP neural network model was constructed to predict Desmodium caudatum (Thunb.) DC. from different habitats, and the accuracy of the test set reached 92.3%.

The method is simple, sensitive and efficient, and can be used for the rapid determination of the components in Desmodium caudatum (Thunb.) DC.. Using the BP neural network model to predict the habitats plays a significant role in tracing the origin of Desmodium caudatum (Thunb.) DC..

To establish a quantitative analysis of multi-components by single marker (QAMS) method for the simultaneous determination of nine effective components including phenolic acids and flavonoids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutoside, isoquercitrin, kaemperol-3-O-rutinoside, 3，4-O-dicaffeoylquinic acid, 3，5-O-dicaffeoylquinic acid, 4，5-O-dicaffeoylquinic acid), in Solidaginis Herba.

Ultra-high performance liquid chromatography was developed on an ACQUITY UPLC^® HSS T3 (100 mm×2.1 mm, 1.8 μm) column with acetonitrile (A)-0.1% phosphoric acid solution (B) as mobile phase in gradient elution. The flow rate was 0.3 mL·min^-1, the detection wavelength was 327 nm, the column temperature was 30 ℃, and the injection volume was 1 μL. Chlorogenic acid and rutoside were chosen as the internal standards for phenolic acids and flavonoids respectively to establish the determine correction factors of neochlorogenic acid, cryptochlorogenic acid，3，4-O-dicaffeoylquinic acid, 3，5-O-dicaffeoylquinic acid, 4，5-O-dicaffeoylquinic acid isoquercitrin and kaemperol-3-O-rutinoside, respectively, by multi-point correction method and their contents were calculated. The results were compared with the results of external standard method to verify their accuracy.

Nine components showed good linear relationships within their own ranges(r≥0.999 5), whose average recoveries were 98.2%-101.7% with the RSDs of 1.0%-1.7%. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutoside, isoquercitrin, kaemperol-3-O-rutinoside, 3，4-O-dicaffeoylquinic acid, 3，5-O-dicaffeoylquinic acid and 4，5-O-dicaffeoylquinic acid, in nine batches of samples were 0.008%-0.014%, 0.055%-0.097%, 0.010%-0.019%, 0.191%-0.412%, 0.018%-0.035%, 0.116%-0.50%, 0.028%-0.048%, 0.111%-0.235% and 0.113%-0.182%. The results obtained by QAMS were consistent with those of external standard method(relative average deviations less than 2.8%).

The simple, accurate and reproducible method can be used for the quality control of Solidaginis Herba.

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-MS/MS method for the simultaneous determination of six characteristic components of Scutellariae Radix including baicalein, baicalin, wogonin, wogonoside, chrysin and oroxylin A in rat plasma, and to compare their pharmacokinetic profiles after oral administration of Scutellariae Radix and Niuhuang Jiedu tablets (NHJDT) to rats.

Rats were given 250 mg·kg^-1 of NHJDT suspension or the prescribed equivalent amount of Scutellariae Radix aqueous extract, and plasma samples were collected at different time intervals. Naringenin was used as internal standard. After precipitated with methanol, the plasma samples were separated on an Inertsil C₈-3 (150 mm×4.6 mm, 5 μm) column by linear gradient elution with 0.1% formic acid solution(A)-0.1% formic acid-methanol(B) as mobile phase. The flow rate was 1.0 mL·min^-1, and the column temperature was 35 ℃. The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source combined with multiple reaction monitoring(MRM) mode in positive ion mode. The pharmacokinetic parameters were calculated and statistically analyzed.

The six characteristic components of Scutellariae Radix were all in good linear relationships in the range of 5-500 ng·mL^-1. The RSDs for accuracy test were in the range of 88.43%-108.6%, and the RSDs for inter-batch and intra-batch precision tests were all below 15%. The matrix effect and plasma stability met the requirements of methodology validation in biological sample analysis. Baicalin and wogonoside were the major components detected in rat plasma after oral gavage of Scutellariae Radix aqueous extract and NHJDT suspension. The AUC_0-t of wogonoside was significantly increased in NHJDT group compared with Scutellariae Radix group. Furthermore, the C_max of wogonoside and baicalin were significantly increased while the T_max was decreased after NHJDT suspension administration.

This method is specific and sensitive for the determination of six characteristic components of Scutellariae Radix, and suitable for pharmacokinetic study of rat plasma. NHJDT with co-existing components enhances the absorption and influences the pharmacokinetic behaviors of active ingredients of Scutellariae Radix.

To investigate the feasibility of applying for bioequivalence exemption based on parallel artificial membrane permeability assay (PAMPA) data by evaluating the solubility and in vitro permeability of levofloxacin, comparing the prescription differences between the generic and reference formulations, assessing the impact of disparate excipients on the permeation behavior of the active pharmaceutical ingredient (API), and predicting the bioequivalence of the two formulations.

The dissolution of the raw material at pH 1.0 to pH 6.8 was determined using high-performance liquid chromatography. The μFlux^TM system was employed to determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media. pH 5.0 fed-state intestinal fluid, pH 6.5 fasted-state intestinal fluid, and pH 7.4 phosphate buffer (16 mL) were precisely added to the donor chamber, while 16 mL of Accepter Sink Buffer was added to the receptor chamber. The rotor speed was set at 200 r·min^-1, and the collection time was 180 min. Determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media, as well as the permeability of the raw material in the reference and generic formulations powders. The impact of newly added excipients and other changed excipients on API was evaluated through a two-tailed t-test. The Macro Flux^TM system was used to measure the dissolution-permeation curves of the reference and generic formulations, intestinal simulation fluids at pH 5.0 and pH 6.5 (1 000 mL) were added to the dissolution cup as dissolution media, with a stirring speed of 75 r·min^-1 using a paddle method, 12 mL of Accepter Sink Buffer was added to the receptor chamber, and the stirring speed of the micro-stirring rod was set at 450 r·min^-1, one tablet of each reference or generic formulation was placed in the dissolution cup, and the dissolution-permeation curves of the formulations were measured. The similarity of dissolution curves was compared, and the permeability rate (J_Flux) and cumulative drug permeation amount (AMT) were calculated to predict the bioequivalence of the two formulations, ensuring that the 90% confidence interval for the geometric mean ratio of J_Flux and AMT of the two formulations fell within the range of 80% to 125%.

Levofloxacin solubility ranged from 16.4 mg·mL^-1 to 62.7 mg·mL^-1 across different mediums, its permeability in pH 5.0 fed-state simulated intestinal fluid, pH 6.5 fasted-state simulated intestinal fluid, and pH 7.4 phosphate-buffered saline was 2.92×10^-6 cm·s^-1, 1.01×10^-5 cm·s^-1, and 1.07×10^-5 cm·s^-1, respectively. The addition of sodium stearyl fumarate showed no significant difference in permeability compared to the original API (P<0.05), and there were no significant differences between the powder API of the reference and generic formulations (P<0.05). The dissolution curves of both formulations were similar, with the 90% confidence interval for JFlux and AMT within the predefined range.

Levofloxacin tablets, classified as BCS Class Ⅰ, demonstrated that the altered excipients in the formulation did not impact on the API’s permeability, confirming bioequivalence between the reference and generic formulations. The bioequivalence exemption study based on PAMPA can be utilized for permeability studies of raw materials, excipient screening and optimization, and prediction of formulation bioequivalence, effectively reducing drug development costs and time. This study provides reference data for pharmaceutical companies applying for bioequivalence exemptions.

To establish a method for identifying Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus.

Chemometrics techniques were used for the analysis of the sample data from determination with Q TOF MS, and characteristic ion pairs were selected as m/z 578.3→164.14 and m/z 578.3→398.31 with triple quadrupole mass spectrometry applied, respectively. Then a specific approach was developed which used Waters Acquisition UPLC CSH (75 mm×2.1 mm, 1.7 μm) as column, and with acetonitrile and 0.1% formic acid solution as the mobile phase, and a flow rate of 0.4 mL·min^-1. Two ion pairs: m/z 578.3→164.14 and m/z 578.3→398.31 were detected by triple quadrupole mass spectrometer with MRM mode.

The validation results through 108 batches of samples indicated that the two characteristic ion pairs, m/z 578.3→164.14 and m/z 578.3→398.31, could effectively distinguish between Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus.

The method had good specificity and sensitivity, and could be used for the detection of adulterated Fritillariae Cirrhosae Bulbus with Fritillariae Ussuriensis Bulbus.