CN117741010A - Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract - Google Patents
Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract Download PDFInfo
- Publication number
- CN117741010A CN117741010A CN202311835197.6A CN202311835197A CN117741010A CN 117741010 A CN117741010 A CN 117741010A CN 202311835197 A CN202311835197 A CN 202311835197A CN 117741010 A CN117741010 A CN 117741010A
- Authority
- CN
- China
- Prior art keywords
- belladonna extract
- belladonna
- scopoletin
- solution
- methanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001106067 Atropa Species 0.000 title claims abstract description 100
- RODXRVNMMDRFIK-UHFFFAOYSA-N scopoletin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2 RODXRVNMMDRFIK-UHFFFAOYSA-N 0.000 title claims abstract description 99
- XEHFSYYAGCUKEN-UHFFFAOYSA-N Dihydroscopoletin Natural products C1CC(=O)OC2=C1C=C(OC)C(O)=C2 XEHFSYYAGCUKEN-UHFFFAOYSA-N 0.000 title claims abstract description 53
- FWYIBGHGBOVPNL-UHFFFAOYSA-N scopoletin Natural products COC=1C=C2C=CC(OC2=C(C1)O)=O FWYIBGHGBOVPNL-UHFFFAOYSA-N 0.000 title claims abstract description 53
- OTHYPAMNTUGKDK-UHFFFAOYSA-N (3-acetylphenyl) acetate Chemical compound CC(=O)OC1=CC=CC(C(C)=O)=C1 OTHYPAMNTUGKDK-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 229960001550 hyoscyamine sulfate Drugs 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 126
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims abstract description 13
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 10
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000010828 elution Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 44
- 239000012488 sample solution Substances 0.000 claims description 31
- 239000000047 product Substances 0.000 claims description 30
- 238000001514 detection method Methods 0.000 claims description 20
- 239000012224 working solution Substances 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 10
- YDBYJHTYSHBBAU-YFKPBYRVSA-N S-methyl-L-methioninate Chemical group C[S+](C)CC[C@H](N)C([O-])=O YDBYJHTYSHBBAU-YFKPBYRVSA-N 0.000 claims description 9
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 9
- 229910052782 aluminium Inorganic materials 0.000 claims description 9
- 239000002775 capsule Substances 0.000 claims description 9
- -1 scopoletin lactone Chemical class 0.000 claims description 9
- 239000013558 reference substance Substances 0.000 claims description 8
- 238000004811 liquid chromatography Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- YGXVNFMRNNRJJE-UHFFFAOYSA-N [Na].CCCCCCC Chemical group [Na].CCCCCCC YGXVNFMRNNRJJE-UHFFFAOYSA-N 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- 239000004411 aluminium Substances 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 17
- 241000208278 Hyoscyamus Species 0.000 abstract description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 244000025254 Cannabis sativa Species 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 30
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 12
- 210000000692 cap cell Anatomy 0.000 description 12
- 239000003814 drug Substances 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 9
- 239000012982 microporous membrane Substances 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- STECJAGHUSJQJN-UHFFFAOYSA-N N-Methyl-scopolamin Natural products C1C(C2C3O2)N(C)C3CC1OC(=O)C(CO)C1=CC=CC=C1 STECJAGHUSJQJN-UHFFFAOYSA-N 0.000 description 3
- HOBWAPHTEJGALG-JKCMADFCSA-N [(1r,5s)-8-methyl-8-azoniabicyclo[3.2.1]octan-3-yl] 3-hydroxy-2-phenylpropanoate;sulfate Chemical compound [O-]S([O-])(=O)=O.C([C@H]1CC[C@@H](C2)[NH+]1C)C2OC(=O)C(CO)C1=CC=CC=C1.C([C@H]1CC[C@@H](C2)[NH+]1C)C2OC(=O)C(CO)C1=CC=CC=C1 HOBWAPHTEJGALG-JKCMADFCSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229960002028 atropine sulfate Drugs 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical compound CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- STECJAGHUSJQJN-FWXGHANASA-N scopolamine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-FWXGHANASA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- RKUNBYITZUJHSG-FXUDXRNXSA-N (S)-atropine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@H]3CC[C@@H](C2)N3C)=CC=CC=C1 RKUNBYITZUJHSG-FXUDXRNXSA-N 0.000 description 1
- 229930000680 A04AD01 - Scopolamine Natural products 0.000 description 1
- 241001465356 Atropa belladonna Species 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- STECJAGHUSJQJN-GAUPFVANSA-N Hyoscine Natural products C1([C@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-GAUPFVANSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 229960003210 hyoscyamine Drugs 0.000 description 1
- 229930005342 hyoscyamine Natural products 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- MOYZEMOPQDTDHA-YQYZCKNZSA-N norhyoscine Chemical compound C1([C@H](C(=O)OC2C[C@@H]3N[C@@H]([C@H]4O[C@H]43)C2)CO)=CC=CC=C1 MOYZEMOPQDTDHA-YQYZCKNZSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 229960002646 scopolamine Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for simultaneously detecting henbane sulfate and scopoletin in a belladonna extract or a belladonna extract-containing product. Detecting belladonna extract and product containing belladonna extract by high performance liquid chromatography; wherein the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, mobile phase A is methanol, mobile phase B is 0.01mol/L sodium heptanesulfonate solution, and gradient elution method is adopted to measure hyoscyamine sulfate and scopoletin under different wavelengths. The method can effectively detect the contents of the hyoscyamine sulfate and scopoletin in the belladonna extract and the belladonna extract-containing products, judge the quality of belladonna grass, and ensure the safety and effectiveness of the raw materials and the finished products.
Description
Technical Field
The invention belongs to the technical field of medicine analysis and detection, and particularly relates to a method for simultaneously detecting hyoscyamine sulfate and scopoletin aiming at raw material belladonna extract or a product containing the belladonna extract.
Background
In the prior art, the belladonna extract is prepared by processing dried whole herb of belladonna Atropa belladonna L. The literature reports that scopolamine present in belladonna grass is mainly (-) scopolamine (i.e. hyoscyamine), containing only a trace of (+) scopolamine (atropine sulfate). The atropine sulfate and scopoletin in the first belladonna extract of the 2020 edition of the current Chinese pharmacopoeia are respectively measured by adopting different treatment methods and chromatographic conditions. And the difference of each component in different parts of the whole herb is large, and the integrity of the belladonna herb medicine cannot be reflected by taking a single component as an index when evaluating the quality integrity of the belladonna extract.
The product containing belladonna extract, such as vitamin U belladonna aluminum capsule II, is prepared by extracting atropine sulfate with chloroform for 6 times, evaporating, and transferring the residue with 5mL methanol to constant volume. The method is complex in operation and has more influence factors.
Disclosure of Invention
In order to solve the problems in the prior art, the invention establishes a method for simultaneously detecting the hyoscyamine sulfate and scopoletin in the belladonna extract or the product containing the belladonna extract. The method has simple pretreatment operation, can effectively detect the contents of the hyoscyamine sulfate and the scopoletin in the belladonna extract or the product containing the belladonna extract under the same chromatographic condition, can judge the quality of belladonna grass used and the quality of the product containing the belladonna extract, and ensures the safety of the bulk drug and the finished drug.
In order to achieve the above purpose, the invention adopts the following technical scheme: a method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or belladonna extract-containing product comprises the following steps:
1) Pretreating belladonna extract or belladonna extract-containing product to obtain sample solution.
2) Measuring the sample solution by adopting a liquid chromatography method, and measuring the hyoscyamine sulfate and scopoletin in the sample solution; wherein the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, mobile phase A is methanol, mobile phase B is heptane sodium sulfonate solution, and gradient elution method is adopted for detection.
Further, in the step 1), for the belladonna extract, 0.5mL of the belladonna extract is taken, dissolved by 50% methanol and fixed to 50mL, after filtration by a microporous filter membrane, 1mL of a subsequent filtrate is precisely measured, and the solution of the belladonna extract for sample is formed by shaking uniformly with 50% methanol to 10 mL.
Further, in the step 1), for the product containing the belladonna extract, a product which is equivalent to 50mg of the product containing the belladonna extract is mixed with a proper amount of 50% methanol, the mixture is subjected to ultrasonic treatment for 15min, the volume is fixed to 50mL by 50% methanol, the mixture is centrifuged, the supernatant is taken, and a sample solution for the product containing the belladonna extract is formed after filtration by a microporous filter membrane.
Further, the concentration of the sodium heptanesulfonate solution is 0.009-0.011mol/L.
Still further, the concentration of the sodium heptanesulfonate solution was 0.01mol/L.
Further, the method comprises the following steps: preparing standard series working solutions by using a standard reference substance of hyoscyamine sulfate and scopoletin; and (3) measuring the standard series working solution by adopting a liquid chromatography method, and drawing a standard curve.
Further, in the standard series working solution, the concentration of the hyoscyamine sulfate is 4.098-40.98 mug/mL; the scopoletin lactone has a concentration of 0.2097-2.097 μg/mL.
Further, the liquid chromatography test conditions were: the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is methanol, the mobile phase B is sodium heptanesulfonate solution, the flow rate is 1.0mL/min, the sample injection amount is 100 mu L, and the column temperature is 35 ℃;
the gradient elution procedure was as follows:
| time min | Mobile phase a | Mobile phase B |
| 0 | 35 | 65 |
| 30 | 100 | 0 |
| 31 | 35 | 65 |
| 45 | 35 | 65 |
Further, hyoscyamine sulfate was measured at a wavelength of 210nm, and scopoletin was measured at a wavelength of 344 nm.
Further, the belladonna extract-containing product is vitamin U belladonna aluminum capsule II.
The beneficial effects of the invention are as follows:
1. according to the invention, by adopting a specific chromatographic column and a mobile phase, the actual contents of the belladonna extract and the product containing the belladonna extract, namely the hyoscyamine sulfate and the scopoletin can be effectively utilized, so that whether the contents of the belladonna extract and the product containing the belladonna extract, namely the hyoscyamine sulfate and the scopoletin meet the regulations can be effectively judged, and the safety of the raw material medicine and the finished medicine can be ensured.
2. The belladonna extract can be used alone or in combination with other drugs to form a compound medicine, such as vitamin U belladonna aluminum capsule II, which contains other medicinal components and is likely to influence the determination of hyoscyamine sulfate and scopoletin. The invention provides a detection method of henbane sulfate and scopoletin in a belladonna extract product, which detects the belladonna extract product by using high performance liquid chromatography; the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, and the mobile phase is a mixed solution of methanol and sodium heptanesulfonate solution, so that the detection can be accurately carried out.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1a is a graph showing the detection result of a CAPCELL PAK C chromatographic column on a hyoscyamine sulfate control.
FIG. 1b is a graph of the detection results of scopoletin control on CAPCELL PAK C column.
Fig. 2a is a graph showing the detection result of the belladonna extract on the henbane sulfate by CAPCELL PAK C chromatographic column.
FIG. 2b is a graph showing the detection result of scopoletin in belladonna extract by CAPCELL PAK C column chromatography.
Fig. 3a is a graph showing the results of detection of hyoscyamine sulfate in belladonna extract with methanol-0.01% phosphoric acid solution as mobile phase.
FIG. 3b is a graph showing the results of detection of scopoletin in belladonna extract with methanol-0.01% phosphoric acid solution as mobile phase.
FIG. 4a is a high performance liquid chromatogram at a wavelength of 210nm for a negative solution.
FIG. 4b is a high performance liquid chromatogram at 344nm wavelength of the negative solution.
Fig. 5a is a graph showing the results of high performance liquid phase detection of hyoscyamine sulfate in vitamin U belladonna aluminum capsule II (lot number 191005) from the pharmaceutical company, inc.
Fig. 5b is a graph showing the results of high performance liquid phase detection of scopoletin in vitamin U belladonna aluminum capsule II (lot number 191005) from the modified pharmaceutical company, inc.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
High performance liquid chromatograph: waters, inc. of America.
Preparation of standard solution: the hyoscyamine sulfate and scopoletin standard reference substances are taken and dissolved in 50% methanol to prepare mixed standard solution stock solution. And (3) carrying out gradient dilution on the mixed standard solution stock solution to prepare a standard series of working solutions. In the standard series working solution, the concentration of the hyoscyamine sulfate is 4.098-40.98 mug/mL; the scopoletin lactone concentration is 0.2097-2.097 μg/mL.
Selection of chromatographic column
The method comprises the following steps:
1) 1mL of mixed standard solution with the concentration of the henbane amine sulfate of 10 mug/mL and the concentration of the scopoletin of 0.5 mug/mL is taken for high performance liquid chromatography test.
2) Taking 0.5mL of belladonna extract solution (modified pharmaceutical industry group Co., ltd., T220601 batch number), dissolving with 50% methanol, fixing the volume to 50mL, shaking uniformly, filtering with microporous membrane, precisely measuring 1mL of the subsequent filtrate, fixing the volume to 10mL with 50% methanol, shaking uniformly, forming a sample solution of belladonna extract, and taking 1mL of sample solution for high performance liquid chromatography.
The high performance liquid chromatography test conditions are as follows:
chromatographic column: octadecylsilane chemically bonded silica column (CAPCELL PAK C column) and octyl silane chemically bonded silica column, respectively.
Mobile phase a: a methanol solution; mobile phase B:0.01mol/L sodium heptanesulfonate solution; flow rate: 1.0mL/min, the sample injection amount is 100 mu L, and the column temperature is 35 ℃; the gradient elution procedure is as in table 1.
TABLE 1
| Time min | Mobile phase a | Mobile phase B |
| 0 | 35 | 65 |
| 30 | 100 | 0 |
| 31 | 35 | 65 |
| 45 | 35 | 65 |
FIG. 1a is a graph showing the detection result of a CAPCELL PAK C chromatographic column on a hyoscyamine sulfate control. FIG. 1b is a graph of the detection results of scopoletin control on CAPCELL PAK C column. Fig. 2a is a graph showing the detection result of the belladonna extract on the henbane sulfate by CAPCELL PAK C chromatographic column. FIG. 2b is a graph showing the detection result of scopoletin in belladonna extract by CAPCELL PAK C column chromatography. As can be seen from fig. 1a, 1b, 2a and 2b, the separation effect of the hyoscyamine sulfate and scopoletin in the reference substance and belladonna extract is good by adopting CAPCELL PAK C chromatographic column, the retention time is reasonable, and the analysis efficiency is high. And the hyoscyamine sulfate and scopoletin in the belladonna extract can not be effectively separated from other components by adopting an octyl silane bonded silica gel chromatographic column. Thus, octadecylsilane chemically bonded silica columns are preferred in the present invention.
(II) selection of mobile phase System
The method comprises the following steps: taking 0.5mL of belladonna extract solution (modified pharmaceutical industry group Co., ltd., T220601 batch number), dissolving with 50% methanol, fixing the volume to 50mL, shaking, filtering with microporous membrane, precisely measuring 1mL of the subsequent filtrate, fixing the volume to 10mL with 50% methanol, shaking, forming belladonna extract sample solution, taking 1mL of the sample solution, and performing high performance liquid chromatography test.
The high performance liquid chromatography test conditions are as follows:
chromatographic column: CAPCELL PAK C18 chromatography column.
Mobile phase system 1: mobile phase a: methanol; mobile phase B:0.01mol/L sodium heptanesulfonate solution.
Mobile phase system 2: mobile phase a: methanol; mobile phase B:0.01% phosphoric acid solution.
Flow rate: 1.0mL/min, the sample injection amount is 100 mu L, and the column temperature is 35 ℃; the gradient elution procedure is as in table 1.
Fig. 3a is a graph showing the detection result of hyoscyamine sulfate in belladonna extract with methanol-0.01% phosphoric acid solution as mobile phase, and fig. 3b is a graph showing the detection result of scopoletin in belladonna extract with methanol-0.01% phosphoric acid solution as mobile phase. It can be seen from fig. 2a and fig. 2b that the mobile phase system eluted with a gradient of 0.01mol/L sodium heptanesulfonate solution is adopted, the hyoscyamine sulfate and scopoletin in the belladonna extract can be effectively separated from other components, and from fig. 3a and fig. 3b, the mobile phase system eluted with a gradient of 0.01% phosphoric acid solution is adopted, the retention time of the hyoscyamine sulfate is short, the effective separation of other components can not be realized, and the measurement result is inaccurate. Thus, in the present invention, mobile phase a is preferably methanol; mobile phase B was a 0.01mol/L sodium heptanesulfonate solution.
Example 2
A method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or belladonna extract-containing product comprises the following steps:
step 1: drawing a standard curve
The hyoscyamine sulfate and scopoletin standard reference substances are taken and dissolved in 50% methanol to prepare mixed standard solution stock solution.
And (3) carrying out gradient dilution on the mixed standard solution stock solution to prepare a standard series of working solutions. In the standard series working solution, the concentration of the hyoscyamine sulfate is 4.098-40.98 mug/mL; the scopoletin lactone concentration is 0.2097-2.097 mug/mL, 1mL of mixed standard solution is respectively taken, the standard series working solution is measured by adopting high performance liquid chromatography, and a standard curve is drawn.
Step 2: preparation of sample solution:
for the belladonna extract, 0.5mL of the belladonna extract is taken, dissolved by 50% methanol and subjected to constant volume to 50mL, shaking is carried out, after microporous membrane filtration, 1mL of continuous filtrate is precisely measured, 50% methanol is used for constant volume to 10mL, shaking is carried out, and a sample solution for the belladonna extract is formed.
For the product containing the belladonna extract, mixing the product which is equivalent to 50mg of the product containing the belladonna extract with a proper amount of 50% methanol, carrying out ultrasonic treatment for 15min, fixing the volume to 50mL by using 50% methanol, shaking uniformly, centrifuging, taking supernatant, and filtering by a microporous filter membrane to form a sample solution containing the belladonna extract product.
Step 3: high performance liquid chromatography assay
Taking 1mL of standard series working solution or sample solution to be tested in a chromatographic bottle, and performing high performance liquid chromatography test.
The high performance liquid chromatography test conditions are as follows:
chromatographic column: octadecylsilane chemically bonded silica column (CAPCELL PAK C18 column);
mobile phase system: mobile phase a: a methanol solution; mobile phase B:0.01mol/L sodium heptanesulfonate solution;
flow rate: 1.0mL/min, the sample injection amount is 100 mu L, and the column temperature is 35 ℃;
the gradient elution procedure is as in table 1, with each sample equilibrated for 10min.
Hyoscyamine sulfate was measured at a wavelength of 210nm and scopoletin was measured at a wavelength of 344 nm.
(one) Effect of negative solution
Preparation of a negative solution: the preparation is formulated according to the formula of vitamin U belladonna aluminum capsule II, and belladonna extract is not added to obtain a negative sample. Mixing a negative sample corresponding to 50mg of belladonna extract with a proper amount of 50% methanol, performing ultrasonic treatment for 15min, fixing the volume to 50mL with 50% methanol, centrifuging, collecting supernatant, filtering with microporous membrane to obtain a negative solution, and performing high performance liquid chromatography test. Measured at a wavelength of 210nm and measured at a wavelength of 344 nm.
FIG. 4a is a high performance liquid chromatogram of a negative solution at a wavelength of 210 nm. FIG. 4b is a high performance liquid chromatogram of the negative solution at a wavelength of 344 nm. As can be seen from fig. 4a and 4b, the negative solution showed no corresponding chromatographic peaks at the corresponding retention times of hyoscyamine sulfate (210 nm) and scopoletin (344 nm) with 50% methanol as solvent, indicating that other components in the vitamin U belladonna aluminum capsule ii do not interfere with the measurement of hyoscyamine sulfate (210 nm) and scopoletin (344 nm), therefore 50% methanol is preferred as solvent in the present invention.
(II) quantitative analysis
And (3) performing high performance liquid chromatography test analysis on the prepared standard series working solution, and drawing a standard curve. Obtaining the standard curve, linear equation, correlation coefficient and linear range of the corresponding hyoscyamine sulfate and scopoletin lactone.
The linear equation of the hyoscyamine sulfate is y= 114993x-8131, and the correlation coefficient is 1.000, which shows that the hyoscyamine sulfate has good linear relation between peak area and mass concentration in the range of 4.098-40.98 mug/mL.
The scopoletin lactone linear equation is y= 355477x-218.68, and the correlation coefficient is 1.000, which shows that scopoletin lactone is in the range of 0.2097-2.097 mug/mL, and the peak area and mass concentration are in good linear relation.
As above, the method of the invention has high sensitivity and good reproducibility, and is suitable for qualitative and quantitative analysis of the hyoscyamine sulfate and scopoletin in the belladonna extract or the belladonna extract-containing products.
(III) repeatability and recovery
1. Repeatability of
Mixing 0.5mL of belladonna extract solution (modified pharmaceutical Co., ltd., batch T220601) with appropriate amount of 50% methanol solution, filtering with microporous membrane, and fixing volume with 50% methanol to 50mL to obtain sample solution of belladonna extract. Taking 1mL of sample solution to be tested in a chromatographic bottle, and measuring the sample solution to be tested by adopting a high performance liquid chromatography for 6 times in parallel to obtain RSD of the hyoscyamine sulfate and scopoletin respectively.
The results showed that 6 parts of sample solutions were prepared and tested together, the average contents of hyoscyamine sulfate and scopoletin were 99.6% and 102.2%, respectively, and the RSD% was 0.6% and 1.1%, respectively, with good reproducibility.
2. Recovery rate
Another group of belladonna extract (0.25 mL) was precisely measured, and six parts were added, respectively, with 0.25mL of the mixed standard solution (concentration of hyoscyamine sulfate 10. Mu.g/mL, concentration of scopoletin 0.5. Mu.g/mL). Dissolving with 50% methanol, fixing volume to 50mL, shaking, filtering with microporous membrane, precisely measuring 1mL, and fixing volume to 10mL with 50% methanol solution to obtain sample solution of belladonna extract. Taking 1mL of sample solution to be tested in a chromatographic bottle, performing high performance liquid chromatography test, and calculating the recovery rate of the hyoscyamine sulfate and scopoletin.
The results showed that the average recovery of hyoscyamine sulfate and scopoletin was 100.3% and 100.7%, respectively, and RSD% (n=6) was 1.9% and 2.8%, respectively. The method has good accuracy.
3. Sample solutions were taken and assayed at 0, 2, 4, 8, 12, and 24 hours, and the results showed that the sample solutions were stable within 24 hours.
(IV) measurement of actual sample
1. Preparation of standard reference substance solution:
taking a proper amount of each of the hyoscyamine sulfate reference substance and the scopoletin reference substance, adding 50% methanol solution for dissolution, and quantitatively diluting to prepare a solution containing 10 mug of the hyoscyamine sulfate and 0.5 mug of the scopoletin in each 1mL of the mixed standard solution.
2. Determination of raw material drug belladonna extract
Taking 0.5mL of belladonna extract, dissolving with 50% methanol, fixing volume to 50mL, filtering with microporous membrane, precisely measuring 1mL, and fixing volume to 10mL with 50% methanol solution to obtain sample solution of belladonna extract. 1mL of the sample solution was taken in a chromatographic bottle, and the sample solution was measured by high performance liquid chromatography, and the results are shown in Table 2.
3. Determination of finished drug containing belladonna extract
Mixing the above medicinal materials with 50mg of belladonna extract, ultrasonic treating for 15min, adding 50% methanol to 50mL, shaking, centrifuging, collecting supernatant, and filtering with microporous membrane to obtain sample solution containing belladonna extract. 1mL of the sample solution was taken in a chromatographic bottle, and the sample solution was measured by high performance liquid chromatography, and the results are shown in Table 2.
TABLE 2
Although this example only provides an example of a belladonna extract containing product being a vitamin U belladonna aluminum capsule II, it will be appreciated that the method of the invention is equally applicable to the detection of other belladonna extract containing samples.
Claims (10)
1. The method for simultaneously detecting the hyoscyamine sulfate and scopoletin in the belladonna extract or the belladonna extract-containing product is characterized by comprising the following steps of:
1) Pretreating belladonna extract or belladonna extract-containing product to obtain sample solution;
2) Measuring the sample solution by adopting a liquid chromatography method, and measuring the hyoscyamine sulfate and scopoletin in the sample solution; wherein the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, mobile phase A is methanol, mobile phase B is heptane sodium sulfonate solution, and gradient elution method is adopted for detection.
2. The method according to claim 1, wherein in step 1), 0.5mL of the belladonna extract is taken, dissolved with 50% methanol and fixed to a volume of 50mL, and after filtration with a microporous filter membrane, 1mL of the subsequent filtrate is precisely measured, and the solution is shaken to a volume of 10mL with 50% methanol to form a sample solution of the belladonna extract.
3. A method according to claim 1, wherein in step 1), 50mg of the product containing the belladonna extract is mixed with a proper amount of 50% methanol, sonicated for 15min, and the volume is set to 50mL with 50% methanol, centrifuged, and the supernatant is collected and filtered through a microporous filter membrane to form a sample solution containing the belladonna extract product.
4. The method of claim 1, wherein the concentration of the sodium heptanesulfonate solution is 0.009-0.011mol/L.
5. The method according to claim 4, wherein the concentration of the sodium heptanesulfonate solution is 0.01mol/L.
6. The method according to claim 1, characterized in that the method further comprises the step of: preparing standard series working solutions by using a standard reference substance of hyoscyamine sulfate and scopoletin; and (3) measuring the standard series working solution by adopting a liquid chromatography method, and drawing a standard curve.
7. The method of claim 6, wherein the concentration of the hyoscyamine sulfate in the standard series of working solutions is 4.098-40.98 μg/mL; the scopoletin lactone has a concentration of 0.2097-2.097 μg/mL.
8. The method of any one of claims 1-7, wherein the liquid chromatography test conditions are: the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, the mobile phase A is methanol, the mobile phase B is sodium heptanesulfonate solution, the flow rate is 1.0mL/min, the sample injection amount is 100 mu L, and the column temperature is 35 ℃;
the gradient elution procedure was:
。
9. The method of claim 1, wherein the hyoscyamine sulfate is measured at a wavelength of 210nm and the scopoletin is measured at a wavelength of 344 nm.
10. A method according to any one of claims 1 to 7, wherein the belladonna-containing extract product is vitamin U belladonna aluminium capsule ii.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311835197.6A CN117741010A (en) | 2023-12-28 | 2023-12-28 | Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311835197.6A CN117741010A (en) | 2023-12-28 | 2023-12-28 | Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117741010A true CN117741010A (en) | 2024-03-22 |
Family
ID=90283169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311835197.6A Pending CN117741010A (en) | 2023-12-28 | 2023-12-28 | Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117741010A (en) |
-
2023
- 2023-12-28 CN CN202311835197.6A patent/CN117741010A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101884706B (en) | Method for detecting phlegm eliminating and cough relieving granules | |
| CN113466355A (en) | Construction method of high performance liquid phase characteristic spectrum of caulis sinomenii | |
| CN107315058A (en) | A kind of method of total ginkgoic acid in detection ginkgo biloba succi | |
| CN108627581B (en) | Method for determining content of rhynchophylline and isorhynchophylline in children's Qixing tea granules | |
| CN107991408B (en) | Method for measuring content of index component in four-flavor Zhenzhi eye drops | |
| CN117741010A (en) | Method for simultaneously detecting hyoscyamine sulfate and scopoletin in belladonna extract or product containing belladonna extract | |
| CN108445115A (en) | A kind of method that high performance liquid chromatography detects neoline and/or songorine and/or Fuziline | |
| CN115524424A (en) | Capsella bursa-pastoris sample quality control method | |
| CN112578035B (en) | High performance liquid chromatography detection method for patulin in pig excreta | |
| HAN et al. | Simultaneous determination of cephaeline and emetine in ipecac and its preparations using RP-HPLC | |
| CN110687224B (en) | Method for measuring triptolide A in tripterygium wilfordii medicinal material and tripterygium wilfordii multi-glycoside tablet prepared from tripterygium wilfordii medicinal material | |
| CN110031577B (en) | Quality detection method and identification application of traditional Chinese medicine or traditional Chinese medicine composition preparation | |
| CN111208214B (en) | Method for detecting tween content in pharmaceutical preparation by liquid phase method | |
| CN112649525A (en) | Method for simultaneously measuring geniposide and amygdalin in children heat-clearing lung-ventilating plaster | |
| CN111337599A (en) | Pretreatment method, morphine detection method and application | |
| CN110907548B (en) | Method for detecting biapenem and/or related substances | |
| CN113063883B (en) | Method for simultaneously identifying 7 effective components in three-flavor bistort rhizome oral liquid | |
| CN115097040B (en) | UPLC characteristic spectrum construction method and application of semen momordicae | |
| CN117347532B (en) | HPLC characteristic spectrum detection method for Guizhi sugar-cellulitis granule preparation | |
| CN112326806B (en) | Albizzia julibrissin formula particle HPLC characteristic spectrum and construction method and application thereof | |
| CN112816609B (en) | Method for detecting creatine phosphate sodium residue in preparation production process | |
| CN101352558A (en) | Quality control method of granular formulation for treating gastricism without aversion to cold | |
| CN116068098A (en) | Method for detecting 5-hydroxytryptamine in calf serum | |
| CN118050451A (en) | Liquid chromatography qualitative and quantitative detection method for centipede formula particles | |
| CN112394110A (en) | Method for detecting content of hydroxychloroquine sulfate ester impurities in hydroxychloroquine sulfate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |