Fenghan Ganmao granules, originated from the classic famous prescription Mahuang Decoction, consists of ephedra, Pueraria, perilla leaves, Fangfeng, cinnamon twigs, Angelica dahurica, tangerine peel, bitter almond, Platycodon grandiflorum, liquorice, dried ginger11It is made of traditional Chinese medicine and has the effects of relieving sweating on the surface and dispersing wind and cold. It is used to treat cold, body heat, headache, cough, etc. The current quality control standard of the drug is mainly the first volume of the drug standard of the Ministry of Health (the original name of the standard is "fenghan Ganmao Granule"). The overall quality control of the standard is weak, and only the properties and general rules of the preparation are checked^[1]There is a lack of quality control methods such as thin-layer identification and content determination. There are many manufacturers of fenghan Ganmao granules, and there is no literature report on the overall quality and consistency evaluation between different manufacturers and batches. 1Or2Component content determination^[2-4]With few detection indicators, it is difficult to effectively control the overall quality of fenghan Ganmao granules. D-Amygdalin^[5], puerarin^[6-8], cimicifugin glycoside^[9], hesperidin^[10-11], rosmarinic acid^[12], ammonium glycyrrhizinate^[13]etc.14The characteristic chemical components of the main medicinal flavors were used as indicators to evaluate the overall quality of fenghan Ganmao granules. Selected14The chemical constituents include bitter almond, Pueraria, Saposhnikovia divaricata, tangerine peel, perilla leaf, Angelica dahurica and liquorice7The content of these chemical components can reflect the overall quality of fenghan Ganmao granules to a certain extent. Due to the selected14The maximum absorption wavelengths of the chemical components to be measured differ greatly, and cannot be measured at the same wavelength at the same time. In order to take into account the maximum absorption wavelengths of each chemical component, improve sensitivity and reduce interference, this experiment adoptsUHPLCWavelength switching method has a high market share for5Of manufacturing enterprises68The content of the batch samples was determined, and the chemometric analysis was carried out, in order to provide some references for the overall quality evaluation and control of fenghan Ganmao granules and the revision of the standard.

1  Instrument and drug test

1.1  instrument

Thermo 3000Ultra performance liquid chromatograph equipped with diode array detector, Thermo Fisher Scientific, USA;Thomovanquish UHPLCseriesQ ExactiveplusQuadrupole-;XPE26Ppm electronic balance, SwitzerlandMettler ToledoCompany;AE-2401 / 100000 electronic balance, Germany sedolis company;SK250HUltrasonic instrument,50kHz, Shanghai Kedao Ultrasonic Instrument Co., Ltd;10～100 μLPipette gun, GermanyEppendorfCompany;Milli-QUltrapure water preparation system, USAMilliporeCompany.

1.2  Trial drug

Reference puerarin (batch No110752-201816, mass fraction95.4%）, daidzein (batch No111738-201603, mass fraction93.3%）111522-201712, mass fraction96.2%）、5-O-Methyl visamitol glycoside (batch No111523-201811, mass fraction97.4%）, rosmarinic acid (batch No111871-201706, mass fraction90.5%）Ammonium glycyrrhizinate (batch No110731-201720, mass fraction97.7%）, hesperidin (batch No110721-201818, mass fraction96.2%）, daidzein (batch No111502-200402）Provided by National Institutes for food and drug control (nifdc); Reference substanceD-Amygdalin (batch No18032611, mass fraction98.0%）Provided by Chengdu pfeder Biotechnology Co., Ltd; Reference substance3′-Hydroxypuerarin (batch NoMUST-19041601, mass fraction99.59%）、3′-Methoxypuerarin (batch NoMUST-19041602, mass fraction98.47%）, puerarin-6″-O-Xyloside (batch NoMUST-19052801, mass fraction99.45%）, apigenin (batch NoMUST-19022203, mass fraction99.41%）MUST-19022202, mass fraction99.95%）MUST-19022201, mass fraction98.04%）, nobiletin (batch NoMUST-18042205, mass fraction98.86%）Provided by Chengdu Manster Biotechnology Co., Ltd.

Chromatographic grade methanol, chromatographic grade acetonitrile, mass spectrometry grade formic acid, USA.The company has a large number of products in the world, including methanol, acetonitrile, formic acid, formic acid, formic acid, formic acid, formic acid, formic acid, formic acid, formiFisherCompany; Chromatographic grade phosphoric acid, Tianjin kemio Chemical Reagent Co., Ltd; Analytical pure methanol, ethanol and other reagents were purchased from Guangdong Guanghua Technology Co., Ltd; .

1.3  sample

68Batch of fenghan Ganmao granule samples were from5Manufacturing enterprises, see table for information1。

2  Methods and results

2.1  chromatogram、Mass spectrometric conditions

2.1.1  Chromatographic conditions The chromatographic column isAgilentPoroshell 120 EC-C₁₈（150 mm×2.1mm，2.7 μm）; With acetonitrile（A）- 0.1%Phosphoric acid aqueous solution（B）Was the mobile phase, and the gradient elution procedure was0～32 min，2%～14% A；32～57 min，14%～18% A；57～80 min，18%～50% A; volume flow is0.3mL/min; column temperature is25 ℃; Detection wavelength:0～20 minby254 nm；20～22 minby210 nm；22～45 minby254 nm；45～58 minby210 nm；58～61 minby330 nm；61～80 minby254 nm; injection volume is5 μL。UHPLC-Q-Orbitrap HRMSThe analysis process is based on0.1%Formic acid aqueous solution was used as the mobile phaseBThe other conditions are the same as above.

2.1.2  Mass spectrometric conditions The ion source isHESI-II; Ion transfer tube temperature320 ℃, auxiliary gas temperature350 ℃, auxiliary gas pressure10 arb, sheath gas pressure35 arb, spray voltage3.50 kV. scanning mode: positive ionFull MS/dd-MS²Mode,Full MSThe resolution is70 000 FWHM，dd-MS²The resolution is17 500 FWHMThe mass to charge ratio window width ism/z 1, the collision energy gradient is20、40、60 eV, scan range ism/z100～1 500。

2.2  Preparation of solution

2.2.1  Preparation of mixed reference solution  takeD-Amygdalin3′-Hydroxypuerarin, puerarin3′-Methoxypuerarin, puerarin-6″- O-Xyloside, apigenin, daidzein, cimicifugin5-O-A proper amount of methyl visamitol glycoside, hesperidin, rosmarinic acid, hydrated oxidized imperatorin, Ammonium Glycyrrhizinate, oxidized imperatorin reference substance, accurately weighed, plus50%Methanol is made to containD-Amygdalin429.1 μg/mL、3′-Hydroxypuerarin235.8 μg/mL, puerarin341.4 μg/mL、3′-Methoxypuerarin154.6μg/mL, puerarin-6″-O-Xyloside51.5 μg/mL, apigenin glycoside218.3 μg/mL, daidzein233.3 μg/mL, cimicifugin glycoside46.9 μg/mL、5-O-Methyl visamitol glycoside97.2 μg/mL, hesperidin313.2 μg/mL, rosmarinic acid28.5 μg/mL, hydrated oxidized imperatorin44.9 μg/mL, ammonium glycyrrhizinate293.1 μg/mL, oxidized imperatorin11.7 μg/mLOf the mixed reference solution.

2.2.2  Preparation of test solution  Take fenghan Ganmao granule powder4.0 g, accurately weigh, place in a conical flask, and accurately add methanol50 mL, weight and ultrasonic extraction30min, cool, weigh the mass again, make up the lost mass with methanol, shake well, filter, and accurately suck the filtrate5 mL, volatilized dry, used for residue50%Methanol was dissolved and transferred to5 mLDilute to the mark in a measuring flask, shake well, and use0.22 μmAfter filtration by microporous membrane, take the continuous filtrate.

2.3  Methodological review

2.3.1  Linear range inspection  Precision suction“2.2.1”A series of mixed reference solutions with different mass concentrations were prepared by diluting the mixed reference solutions under. According to“2.1.1”The chromatographic conditions were injected in turn, and the chromatographic peak area was recorded. Take the peak area as the ordinate and the compound mass concentration as the abscissa, draw the standard curve and perform linear regression. The results are shown in table2.

2.3.2  Precision test  Take“2.2.1”Mix the reference solution according to“2.1.1”Continuous injection under chromatographic conditions6Times, record the peak area, calculateRSD. the results showed that,14Peak areas of chemical components to be testedRSDby1.09%～2.28%, indicating that the precision of the instrument is good.

2.3.3  stability test   Take the same test solution（S1）, respectively, in0、4、8、12、16、24 hSample injection determination, record the peak area and calculateRSD,14Peak areas of chemical components to be testedRSDby0.30%～2.81%, indicating that the test solution after preparation24hBasically stable.

2.3.4  Repeatability test  Take the same batch of samples（S1）, press“2.2.2”Method under, parallel preparation6Test solution, according to“2.1”According to the chromatographic conditions under, calculate the content of each component. The results showed that,14Mass fraction of chemical components to be testedRSDby0.24%～2.93%, indicating that the assay method has good reproducibility.

2.3.5  Sample recovery test  Accurately weigh the sample powder with known content（S1）9Copies, and the sampling volume of each sample is the same as that of the sample volume during the preparation of the test solution50%, with the current sampling content of0.5、1.0、1.5Times, respectively, precisely add the mixed reference solution, press“2.2.2”The test solution was prepared according to this method, determined according to the content determination method, and the sample recovery was calculated. The results showed that,14The average recovery of the chemical components to be tested was95.24%～100.00%；RSDby1.18%～2.92%It shows that the method has good accuracy and is feasible.

2.4  Sample content determination

Press“2.2.2”Prepare the test solution according to the method below“2.1.1”Injection under chromatographic conditions5 μLAnalyze and calculate the content of each component. Typical chromatograms are shown in Fig1The content determination results are shown in table3。

In order to more intuitively reflect the content distribution of chemical components in the samples of various manufacturers, the14Radar chart of average content distribution of chemical components. Due to the large content difference of different chemical components, the5Among the samples of manufacturers14The average content of each chemical component is added separately, and then the average content of each chemical component of each production enterprise is compared with the corresponding sum to obtain the ratio. The radar map is drawn with the ratio, and the results are shown in Fig2The results showed that the drop points of the content ratios of the chemical components almost did not coincide, and the drop points were widely distributed, indicating that the same chemical component was quite different in different enterprise samples. enterpriseAThe overall outline of the enterprise is relatively large, indicating that the enterprise sample14SpeciationThe content of chemical components is high, suggesting that the quality may be better; While enterprisesEThe overall profile of the enterprise is significantly smaller than that of other enterprises, indicating that in the enterprise sample14The contents of all kinds of chemical components are low, suggesting that the quality of samples from the enterprise may be poor from the perspective of the chemical content of multi-component. withD-Amygdalin, for example,D-Amygdalin is the main active ingredient in bitter almond for relieving asthma and cough^[5], enterpriseAThe average content in the sample is the enterpriseEThe average content of28.5Times（3.591 mg/g vs 0.126 mg/g）, and5Among samples from different manufacturersD-The content of amygdalin varies greatly, which is worthy of attention.
2.5  Statistical analysis of Chemometrics

2.5.1  Similarity evaluation of fingerprint and construction of characteristic fingerprint  Fingerprint can more comprehensively reflect the overall distribution of various chemical components in Chinese patent medicine, especially when the vast majority of effective components are not clear at this stage^[14-15]. will68Batches of fenghan Ganmao granules from different manufacturersUHPLC-UVThe fingerprint was imported into the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine (TCM) issued by the National Pharmacopoeia Commission（2012Version), which is consistent with the reference spectrum (with higher overall chromatographic abundanceA、B、C、D 4Samples from manufacturersUHPLC-UVMap generation) to compare the similarity, and the similarity analysis results are shown in table4. the results showed that,A、B、C、D 4The similarity of the samples of fenghan Ganmao granules from manufacturing enterprises was greater than0.940, indicating that the overall difference between these batches of samples is small; While enterprisesESample similarity in0.856～0.983, indicating that compared with other enterprises, enterprisesEThe overall quality of the samples varied greatly. However, the content determination results showed that the samples from different manufacturers14There are certain differences in the contents of chemical components, which are reported by consulting the literature^[16-17], it may be that there are no missing and increasing chromatographic peaks in the fingerprints of different samples, but only peaksThe area is different, while the peak area of different chemical components is different, up to hundreds of times, which is caused by the large peak masking the small peak information.

The content determination results showed that the chemical components of fenghan Ganmao granules from different manufacturers were quite different, and the overall quality assessment and control were difficult. Therefore, on the basis of multi-component content determination, the characteristic fingerprint of fenghan Ganmao granules was further established, in order to provide reference for the overall quality evaluation and control of fenghan Ganmao granules. 46Batch (excludingEManufacturer22Batch) of fenghan Ganmao granulesUHPLC- UVThe spectral data were imported into the similarity evaluation system of chromatographic fingerprints of traditional Chinese Medicine issued by the National Pharmacopoeia Commission（2012Version) toS1Is the reference spectrum (at46The kurtosis in the chromatogram of the batch of samples is relatively high). After multi-point correction, the chromatographic peaks are matched to generate a characteristic fingerprint. The comparison of reference substance andUHPLC-Q-Exactive- Orbitrap HRMSTechnology VS25Common peaks were identified, of which16The chromatographic peaks were confirmed by comparison with the reference substances,6The chromatographic peaks were identified by high-resolution mass spectrometry, and the characteristic fingerprints of typical fenghan Ganmao granules constructed are shown in Fig3The information of ion fragments of chromatographic peaks analyzed by HRMS but not compared with the reference substance is shown in table5。

2.5.2  Heatmap clustering analysis  In order to more intuitively show the multi-component chemical differences of fenghan Ganmao granules produced by different enterprises, the measured68In batch samples14The contents of chemical components are used as the evaluation indexBid, usingHemI-1.0The software performs heat map clustering analysis by Euclidean distance and far neighbor method, and the results are shown in Fig4Among them, the horizontal clustering samples, the vertical clustering component content, and the change of heat map color bands reflect the content of each component. The heat map analysis results show that among different production enterprises14The contents of these chemical components are quite different. Including manufacturersAThe color of each component strip is warm, indicating that the content of each component is high, and the product quality is good; EThe color of each component strip is cold, indicating that the content of each component contained in it is low, and the product quality is poor, which is consistent with the radar chart analysis result. The cluster analysis results of different production enterprises show that68The overall clustering of batch samples is obvious, and the samples of each enterprise are clustered into one category, indicating that the quality consistency of samples from the same manufacturer is good, while the quality consistency between samples from different manufacturers is poor. C3、C10Grouped together,C1Individually grouped together, and other components grouped together, indicating thatC1（D-Amygdalin)C3(puerarin)C10(hesperidin) was the main component to distinguish the samples.

2.5.3  Principal component analysis（principal component analysis，PCA） PCAIt can quickly extract and reduce the dimension analysis of multivariable in complex information. By reducing the dimension, it can eliminate the overlapping information in many chemical information and generate a new comprehensive variable, namely the principal component. After projection processing, the sample finally falls on the position of the principal component composition plane, which can characterize the overall information of different samples^[23-25]. this study is based on68In the batch of fenghan Ganmao granules14The contents of chemical components were used as variables, and multivariate statistical software was used SIMCA 14.1carry outPCAThe analysis results showed that the largest contribution rate3Principal components, and the cumulative variance contribution rate exceeded90%, indicating that the former3The three principal components can fully reflect the basic characteristics and main information of fenghan Ganmao granules. modelR²_X＝0.963，Q²＝0.911, indicating that the model fits well, and the results are shown in Fig5、6。

From Fig5It can be seen that the samples of various manufacturers are obviously classified,PCAThe overall discrimination of the results is consistent with the Heatmap clustering analysis; enterpriseBThe sample distribution is relatively dispersed, suggesting that there are certain differences in the quality between different batches. chart6Principal component loading diagram, indicating the contribution of each chemical component to the comprehensive effect of principal components, and each1Points represent1Chemical components. The further away from the origin of the load map, the greater the weight of the variable, and the greater the effect of the content of the compound on sample discrimination. （C10）、D-Amygdalin（C1）The larger absolute value in the coordinate system indicates that this2The three components are important indicators to distinguish fenghan Ganmao granules from different manufacturers, which are consistent with the results of Heatmap clustering analysis. Hesperidin is an index component of orange peel, which has pharmacological activities such as enhancing capillary toughness and shortening bleeding time^[10-11].D-Amygdalin is the main active ingredient of bitter almond for relieving cough and asthma, which is easily isomerized intoL-Amygdalin is easily hydrolyzed to glucose, hydrocyanic acid and benzaldehyde under certain temperature and humidity conditions, resulting inD-The content of amygdalin varies greatly, suggesting that manufacturers should pay attention to the quality of raw materials and extraction process of amygdalinD-.

3  discuss

The research group visited the production enterprises in the early stage and found that there were great differences in the source of raw medicinal materials used for feeding, the process parameters in the large-scale production process (such as water addition, volatile oil extraction method and extraction time, total production), and production equipment. The experimental results show that the overall quality consistency (chemical consistency) of different batches of samples from the same enterprise is relatively good, but the overall quality consistency of samples between different enterprises is quite different, which deserves the attention of regulatory authorities and enterprises.

. Through literature research, this study established the prescription of fenghan Ganmao granules7Flavored medicine14Objective to evaluate the overall quality of fenghan Ganmao granules by simultaneous quantitative analysis of chemical components. The research group has tried to simultaneously determine ephedrine hydrochloride and pseudoephedrine hydrochloride, the index components of ephedra, but due to the large difference in chemical properties, poor separation and peak type, it can not meet the determination conditions, so the water vapor distillation method was used to determine them separately. The measurement results showed that5The average law of the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride in fenghan Ganmao granules from different manufacturers wasC（0.545 mg/g）＞A（0.519 mg/g）＞B（0.436 mg/g）＞D（0.211 mg/g）＞E（0.100 mg/g）, manufacturerEThe total amount of the two is only the production enterprisesCOf18.34%。

This experiment established a model based onUHPLCIn fenghan Ganmao granules by wavelength switching method14Methods for simultaneous quantitative analysis of chemical components, and5Of manufacturing enterprises68. Through radar map, fingerprint similarity evaluation, Heatmap clustering analysis andPCAAnd other chemometrics comprehensively evaluated the overall quality of samples from different manufacturers, providing objective data and reference methods for the quality evaluation and control of fenghan Ganmao granules.

References (omitted)  

Come   Source: Xu   Li, Huang Xiaojing, Gao   Peng, zhaoxiaoqin, Wen Yongsheng, Tan   Peng. Study on overall quality evaluation of fenghan Ganmao granules based on multi-component content determination and chemometrics [j]. Chinese herbal medicine, 2020, 51 (14): 3670-3679