CN113125595A - HPLC detection method of sophora flavescens component - Google Patents
HPLC detection method of sophora flavescens component Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 21
- 241000246044 Sophora flavescens Species 0.000 title claims abstract description 17
- ZSBXGIUJOOQZMP-BHPKHCPMSA-N sophoridine Chemical compound C1CC[C@@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-BHPKHCPMSA-N 0.000 claims abstract description 48
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- 229930015582 oxymatrine Natural products 0.000 claims abstract description 25
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- ZSBXGIUJOOQZMP-JLNYLFASSA-N Matrine Chemical compound C1CC[C@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-JLNYLFASSA-N 0.000 claims abstract description 23
- 229930014456 matrine Natural products 0.000 claims abstract description 23
- QMGGMESMCJCABO-JARXUMMXSA-N oxysophocarpine Chemical compound C([C@@H]1CCC[N@+]2(CCC[C@H]3[C@@H]21)[O-])N1[C@@H]3CC=CC1=O QMGGMESMCJCABO-JARXUMMXSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
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- 239000000243 solution Substances 0.000 claims description 37
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- 239000013558 reference substance Substances 0.000 claims description 24
- 238000005303 weighing Methods 0.000 claims description 19
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an HPLC (high performance liquid chromatography) detection method of a traditional Chinese medicine radix sophorae flavescentis component, which comprises the steps of (1) chromatographic condition and system applicability test (2) solution preparation (3) determination and the like. The HPLC detection method for the sophora flavescens component has the advantages of simple operation, high detection efficiency, small error, good reproducibility, strong specificity, more stable detection method, less influenced factors and wide applicability; the method has the advantages that the specificity, the stability, the precision, the accuracy and the like meet the requirement of methodology verification, and the method has strong practicability, can be used for detecting the components of the sophora flavescens (traditional Chinese medicine decoction pieces) produced by various enterprises, can quickly and accurately detect the contents of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, and can be used for inspection analysis and quality judgment of sophora flavescens traditional Chinese medicinal materials and decoction pieces thereof.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an HPLC (high performance liquid chromatography) detection method for a traditional Chinese medicine radix sophorae flavescentis component.
Background
Radix Sophorae Flavescentis is a common Chinese medicinal material, is the dried root of Sophora flavescens Ait of Leguminosae, and has effects of clearing heat, eliminating dampness, killing parasite, and promoting urination. The externally applied medicament is clinically used for treating dysentery with fever, hematochezia, jaundice, anuresis, leucorrhea with red and white discharge, vulvar swelling and pruritus vulvae, eczema, skin pruritus and mange leprosy, and is externally used for treating trichomonas vaginitis. Because of its remarkable therapeutic effect, it is widely used in clinic at present, and its main effective components include oxymatrine, oxysophocarpine, sophoridine, matrine, sophocarpine and other alkaloids.
Oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine are main effective components of radix sophorae flavescentis, and in the current standard of radix sophorae flavescentis, the content of oxymatrine, sophoridine and sophocarpine is not controlled in the part of 2020 edition of Chinese pharmacopoeia, and only oxymatrine and matrine with small content are taken as quality control indexes, which is unreasonable.
The invention aims to provide an HPLC detection method of sophora flavescens constituents, aiming at the defects in the prior art, the method is adopted to detect various alkaloid constituents in sophora flavescens, and has the advantages of simple operation, high detection efficiency, small error, good reproducibility, strong specificity, more stable detection method, wide applicability and capability of effectively controlling the quality of sophora flavescens.
Disclosure of Invention
In order to achieve the purpose, the invention adopts the following technical scheme:
an HPLC detection method of radix Sophorae Flavescentis comprises the following steps:
(1) chromatographic conditions and system applicability test:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column comprises the following components: waters
T3C 18 column, 4.6X 250mm, 5 μm or equivalent column, mobile phase: acetonitrile (a) -0.1% phosphoric acid solution (triethylamine to pH 8.0) (B) and gradient elution (procedure see table 1). Flow rate: 1.0ml/min, detection wavelength: 208nm, column temperature: 25 ℃, sample introduction: 20 mul, the number of theoretical plates is not less than 5000 calculated according to the sophoridine peak.
TABLE 1 gradient elution procedure
(2) Solution preparation:
preparing a test solution: weighing about 0.3g of the product powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, adding 0.5ml of concentrated ammonia test solution for wetting, precisely adding 25ml of trichloromethane, sealing the plug, weighing, carrying out ultrasonic treatment (power 320W and frequency 40kHz) for 45 minutes, cooling, weighing again, supplementing the lost weight with trichloromethane, shaking uniformly, filtering, precisely weighing 10ml of subsequent filtrate, recovering the solvent to dryness, adding an appropriate amount of acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) into the residue to dissolve, transferring to a 10ml measuring flask, adding acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) to scale, shaking, filtering, taking the subsequent filtrate, centrifuging for 5min at 5000 r/min, and filtering with a 0.45 mu m filter membrane to obtain the product solution.
Preparation of control solutions: precisely weighing appropriate amount of oxymatrine reference substance, oxysophocarpine reference substance, sophoridine reference substance, matrine reference substance and sophocarpine reference substance, and quantitatively diluting with acetonitrile-0.1% phosphoric acid solution (pH value is adjusted to 8.0 by triethylamine) (11: 89) to obtain mixed solution containing oxymatrine 150 μ g, oxysophocarpine 100 μ g, sophoridine 75 μ g, matrine 10 μ g and sophocarpine 10 μ g per 1 ml.
(3) And (3) determination:
precisely measuring 20 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram. The content of each component in the test solution is calculated by a standard method except for a reference solution.
In the HPLC detection method of the sophora flavescens component, the detector is an ultraviolet-visible light spectrum detector.
The HPLC detection method of radix Sophorae Flavescentis component comprises detecting radix Sophorae Flavescentis component by HPLC, separating oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, and determining the contents of the five components separately.
The following is a methodological validation of the HPLC detection method of the Sophora flavescens component of the present invention:
1. specificity test
Precisely sucking 20 μ l of each of the control solution, the sample solution and the solvent, measuring by the above detection method, and recording chromatogram (see FIG. 1-1, FIG. 1-2, FIG. 2-1, FIG. 2-2, FIG. 3-1 and FIG. 3-2). As can be seen from the figure, in the chromatogram of the test solution, oxymatrine, oxysophocarpine, sophoridine, matrine, sophocarpine and adjacent peaks can all achieve baseline separation, and the blank solvent has no interference, which indicates that the method has good specificity.
2. Investigation of linear relationships
Accurately weighing 15.28mg of oxymatrine reference substance, 12.16mg of oxysophocarpine reference substance, 12.49mg of sophoridine reference substance, 10.50mg of matrine reference substance and 10.77mg of sophocarpine reference substance, respectively placing in 10ml volumetric flasks, adding absolute ethyl alcohol for dissolving, quantitatively diluting to a scale, shaking up to obtain a reference substance stock solution, accurately measuring a proper amount of the reference substance stock solution, quantitatively diluting with acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) to obtain standard curve solutions with different concentrations of 1-7, taking the prepared standard curve solution, carrying out sample injection measurement, carrying out linear regression on peak area y by concentration x (mg/ml), and obtaining a result: oxymatrine has good linear relation in the range of 0.02839mg/ml to 1.4195mg/ml, the linear equation is y 39320x-43.578, the correlation coefficient r is 1.0000 (see fig. 4), oxymatrine has good linear relation in the range of 0.01946mg/ml to 0.9728mg/ml, the linear equation is y 39964x-0.7437, the correlation coefficient r is 1.0000 (see fig. 5), sophoridine has good linear relation in the range of 0.01543mg/ml to 0.6170mg/ml, the linear equation is y 32570x-6.1755, the correlation coefficient r is 1.0000 (see fig. 6), matrine has good linear relation in the range of 0.002591mg/ml to 0.1036mg/ml, the linear equation is y 44143x-6.2503, the correlation coefficient r is 0.9999 (see fig. 7), sophocarpine has good linear relation in the range of 0.006311mg/ml to 0.4039mg/ml, and the linear equation is y-4138, the correlation coefficient r is 0.9999 (see FIG. 8)
3. Investigation of solution stability
Taking a batch of samples (batch No. 180901) of Guangxi Guisheng Chinese medicinal pharmacy Co., Ltd to prepare a test solution, respectively placing for 0, 8, 16, 20, 24, 40 and 60 hours, then carrying out sample injection measurement, measuring the peak areas of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, wherein the result shows that the test solution is stable within 60 hours, the RSD of oxymatrine peak area is 0.59%, the RSD of oxysophocarpine peak area is 0.47%, the RSD of sophocarpine peak area is 1.55%, the RSD of matrine peak area is 0.92%, the RSD of sophocarpine peak area is 1.14%, and the measurement result is shown in the following table 2.
TABLE 2 solution stability test
4. Sample introduction precision test
Taking a batch of samples (batch No. 180901) of Guangxi Guisheng Chinese medicinal pharmacy Co., Ltd to prepare 1 part of test solution according to the preparation method of the test solution in the detection method, continuously injecting 6 needles, measuring the peak areas of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, and observing the precision of the instrument, wherein the RSD of the peak area of oxymatrine is 0.39%, the RSD of the peak area of oxysophocarpine is 0.50%, the RSD of the peak area of sophocarpine is 0.84%, the RSD of the peak area of matrine is 0.83%, and the RSD of the peak area of sophocarpine is 0.65%, thus indicating that the precision of the instrument is good. The results of the measurements are shown in Table 3 below.
TABLE 3 sample introduction precision experiment
5. Repeatability test
Taking a batch of samples (batch No. 180901) of Guangxi Osmanthus Fragrans Henry Chinese medicinal pharmacy Co., Ltd.), preparing 6 parts of test solution according to the preparation method of the test solution in the detection method, carrying out sample injection measurement, and calculating the content and RSD of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, wherein the results are shown in Table 4.
TABLE 4 repeatability tests
6. Sample application recovery test
Precisely measuring a reference substance solution (solvent: absolute ethyl alcohol): 1700 mul of oxymatrine (1.0695 mg/ml), 1000 mul of oxysophocarpine (1.2160 mg/ml), 700 mul of sophoridine (1.2340 mg/ml), 2000 mul of matrine (0.05250 mg/ml) and 800 mul of sophocarpine (0.1077 mg/ml) are put into the same flat-bottomed flask, 0.15g of a sample (lot 180901) with known content is precisely weighed and placed in the flat-bottomed flask, a 100% recovery rate solution is prepared according to the preparation method of the test sample solution in the detection method, 6 recovery rate solutions are prepared, and the results are determined by sample injection and are shown in Table 5 below. According to the result of the determination, the HPLC detection method for the sophora flavescens component is simple to operate and wide in applicability.
TABLE 5 sample recovery test
The invention has the advantages that
The HPLC detection method for the sophora flavescens component has the advantages of simple operation, high detection efficiency, small error, good reproducibility, strong specificity, more stable detection method, less influenced factors and wide applicability; the specificity, stability, precision, accuracy and the like of the method all meet the requirement of methodology verification, and the method has strong practicability.
Drawings
FIG. 1-1 liquid chromatogram of solvent (Agilent)
FIG. 2-1 liquid chromatogram of sample solution (Agilent)
FIG. 3-1 liquid chromatogram of control solution (Agilent)
FIG. 1-2 liquid chromatogram of solvent (waters)
FIG. 2-2 liquid chromatogram of test solution (waters)
FIG. 3-2 liquid chromatogram of control solution (waters)
FIG. 4 is a linear relationship graph of the concentration x (mg/ml) of oxymatrine in a standard curve solution prepared by the present invention and the peak area y
FIG. 5 is a linear plot of the concentration x (mg/ml) of oxysophocarpine in a standard curve solution prepared according to the present invention versus the peak area y
FIG. 6 is a linear relationship graph of the concentration x (mg/ml) of sophoridine in a standard curve solution prepared according to the present invention and the peak area y
FIG. 7 is a linear relationship graph of matrine concentration x (mg/ml) and peak area y in a standard curve solution prepared by the present invention
FIG. 8 is a linear relationship graph of concentration x (mg/ml) of sophocarpine in a standard curve solution prepared according to the present invention and peak area y
Detailed Description
Example 1
An HPLC detection method of radix Sophorae Flavescentis comprises the following steps:
(1) chromatographic conditions and system applicability test:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column comprises the following components: waters
T3C 18 column, 4.6X 250mm, 5 μm or equivalent column, mobile phase: acetonitrile (a) -0.1% phosphoric acid solution (triethylamine adjusted to pH 8.0) (B) and gradient elution (procedure same as table 1). Flow rate: 1.0ml/min, detection wavelength: 208nm, column temperature: 25 ℃, sample introduction: 20 mul, the number of theoretical plates is not less than 5000 calculated according to the sophoridine peak.
(2) Solution preparation:
preparing a test solution: weighing about 0.3g of the product powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, adding 0.5ml of concentrated ammonia test solution for wetting, precisely adding 25ml of trichloromethane, sealing the plug, weighing, carrying out ultrasonic treatment (power 320W and frequency 40kHz) for 45 minutes, cooling, weighing again, supplementing the lost weight with trichloromethane, shaking uniformly, filtering, precisely weighing 10ml of subsequent filtrate, recovering the solvent to dryness, adding an appropriate amount of acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) into the residue to dissolve, transferring to a 10ml measuring flask, adding acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) to scale, shaking, filtering, taking the subsequent filtrate, centrifuging for 5min at 5000 r/min, and filtering with a 0.45 mu m filter membrane to obtain the product solution.
Preparation of control solutions: precisely weighing appropriate amount of oxymatrine reference substance, oxysophocarpine reference substance, sophoridine reference substance, matrine reference substance and sophocarpine reference substance, and quantitatively diluting with acetonitrile-0.1% phosphoric acid solution (pH value is adjusted to 8.0 by triethylamine) (11: 89) to obtain mixed solution containing oxymatrine 150 μ g, oxysophocarpine 100 μ g, sophoridine 75 μ g, matrine 10 μ g and sophocarpine 10 μ g per 1 ml.
(3) And (3) determination:
precisely measuring 20 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram. The content of each component in the test solution is calculated by a standard method except for a reference solution.
Application effects
The content of each component in 5 samples of the enterprise was analyzed (see Table 6 for results)
TABLE 65 content of Components in samples of the family Enterprise
Therefore, the HPLC detection method for the sophora flavescens component has the advantages of simple operation, high detection efficiency, small error, good reproducibility and wide applicability; the method can be used for detecting multiple batches of radix Sophorae Flavescentis components, can rapidly and accurately detect the contents of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine, and can be used for inspection analysis and quality judgment of radix Sophorae Flavescentis traditional Chinese medicinal materials and decoction pieces thereof.
Claims (3)
1. The HPLC detection method of the sophora flavescens component is characterized by comprising the following steps of:
(1) chromatographic conditions and system applicability test:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column comprises the following components: waters
T3C 18 column, 4.6X 250mm, 5 μm or equivalent column, mobile phase: acetonitrile (a) -0.1% phosphoric acid solution (triethylamine to pH 8.0) (B) and gradient elution (procedure see table 1). Flow rate: 1.0ml/min, detection wavelength: 208nm, column temperature: 25 ℃, sample introduction: 20 mul, the number of theoretical plates is not less than 5000 calculated according to the sophoridine peak.
TABLE 1 gradient elution procedure
(2) Solution preparation:
preparation of a test solution: weighing about 0.3g of the product powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, adding 0.5ml of concentrated ammonia test solution for wetting, precisely adding 25ml of trichloromethane, sealing the plug, weighing, carrying out ultrasonic treatment (power 320W and frequency 40kHz) for 45 minutes, cooling, weighing again, supplementing the lost weight with trichloromethane, shaking uniformly, filtering, precisely weighing 10ml of subsequent filtrate, recovering the solvent to dryness, adding an appropriate amount of acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) into the residue to dissolve, transferring to a 10ml measuring flask, adding acetonitrile-0.1% phosphoric acid solution (pH is adjusted to 8.0 by triethylamine) (11: 89) to scale, shaking, filtering, taking the subsequent filtrate, centrifuging for 5min at 5000 r/min, and filtering with a 0.45 mu m filter membrane to obtain the product solution.
Preparing a reference substance solution: precisely weighing appropriate amount of oxymatrine reference substance, oxysophocarpine reference substance, sophoridine reference substance, matrine reference substance and sophocarpine reference substance, and quantitatively diluting with acetonitrile-0.1% phosphoric acid solution (pH value is adjusted to 8.0 by triethylamine) (11: 89) to obtain mixed solution containing oxymatrine 150 μ g, oxysophocarpine 100 μ g, sophoridine 75 μ g, matrine 10 μ g and sophocarpine 10 μ g per 1 ml.
(3) And (3) determination:
precisely measuring 20 μ l of each of the sample solution and the reference solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram. The content of each component in the test solution is calculated by a standard method except for a reference solution.
2. The HPLC detecting method for Sophora flavescens ait component as claimed in claim 1, wherein the detector is an ultraviolet-visible light spectrum detector.
3. The HPLC method for detecting Sophora flavescens Aiton composition as claimed in claim 1, wherein the content of oxymatrine, oxysophocarpine, sophoridine, matrine and sophocarpine can be determined separately by HPLC.
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