CN113203811A - Method for rapidly screening illegal addition of bisacodyl in Chinese patent medicines and health-care foods - Google Patents
Method for rapidly screening illegal addition of bisacodyl in Chinese patent medicines and health-care foods Download PDFInfo
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- CN113203811A CN113203811A CN202110487638.2A CN202110487638A CN113203811A CN 113203811 A CN113203811 A CN 113203811A CN 202110487638 A CN202110487638 A CN 202110487638A CN 113203811 A CN113203811 A CN 113203811A
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- KHOITXIGCFIULA-UHFFFAOYSA-N Alophen Chemical compound C1=CC(OC(=O)C)=CC=C1C(C=1N=CC=CC=1)C1=CC=C(OC(C)=O)C=C1 KHOITXIGCFIULA-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 229960000503 bisacodyl Drugs 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 238000012216 screening Methods 0.000 title claims abstract description 32
- 229940079593 drug Drugs 0.000 title claims abstract description 30
- 235000013305 food Nutrition 0.000 title claims abstract description 16
- 239000012488 sample solution Substances 0.000 claims abstract description 49
- 239000000243 solution Substances 0.000 claims abstract description 47
- 239000007788 liquid Substances 0.000 claims abstract description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000012085 test solution Substances 0.000 claims abstract description 16
- 230000014759 maintenance of location Effects 0.000 claims abstract description 12
- 238000001228 spectrum Methods 0.000 claims abstract description 8
- 239000000741 silica gel Substances 0.000 claims abstract description 6
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 6
- 239000012088 reference solution Substances 0.000 claims abstract description 5
- 238000007689 inspection Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 54
- 239000000523 sample Substances 0.000 claims description 42
- 238000012360 testing method Methods 0.000 claims description 38
- 238000001819 mass spectrum Methods 0.000 claims description 34
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- 238000002360 preparation method Methods 0.000 claims description 21
- 239000013558 reference substance Substances 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 17
- 238000007865 diluting Methods 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 235000013402 health food Nutrition 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000004809 thin layer chromatography Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- KXBSWEZJRRWDPK-UHFFFAOYSA-N 4-(2,3-dimethylphenyl)butan-2-one Chemical compound CC(=O)CCC1=CC=CC(C)=C1C KXBSWEZJRRWDPK-UHFFFAOYSA-N 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 239000013074 reference sample Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000012790 confirmation Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
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- 239000000835 fiber Substances 0.000 description 2
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- 238000012795 verification Methods 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010036774 Proctitis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
Abstract
The invention provides a method for rapidly screening bisacodyl illegally added in Chinese patent medicines and health-care foods, which comprises the following steps: (1) dropping the first test solution and the first control solution on a silica gel thin-layer plate, placing under an ultraviolet lamp for inspection, detecting corresponding main spots, and screening by a liquid chromatograph; (2) injecting the second test solution and the second reference solution into a liquid chromatograph, recording a chromatogram and a DAD spectrogram, and if a chromatographic peak consistent with the chromatographic retention time of the reference and the DAD spectrum is detected, confirming by using a liquid chromatogram-mass spectrometer; (3) and injecting the second test solution, the second control solution and the second negative sample solution into a liquid chromatography-mass spectrometer, detecting corresponding chromatographic peaks, and determining illegal addition of bisacodyl. The method has the advantages of high sensitivity, good selectivity, strong corroborability and simple and quick operation, and can be used for qualitative screening and accurate confirmation of illegal addition of bisacodyl in Chinese patent medicines and health-care foods.
Description
Technical Field
The invention relates to the technical field of drug detection, in particular to a method for rapidly screening bisacodyl illegally added in Chinese patent medicines and health-care foods.
Background
At present, in the market, the bisacodyl chemical is illegally added into weight-losing Chinese patent medicines and weight-losing health-care foods and weight-losing products, and is mainly used for treating constipation, cleaning intestinal tracts before and after operations and the like, so that the bisacodyl chemical has the weight-losing effect, but abdominal colic, excessive diarrhea, proctitis and the like can be caused by non-standard use, and the health of people is damaged. At present, the substance lacks a rapid and accurate detection method, main detection means still remain in a non-aqueous titration method, a liquid chromatography method and the like, the detection speed is low, the accuracy is low, and the situations of false positive and false negative are easy to occur.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a method for rapidly screening bisacodyl illegally added into Chinese patent medicines and health-care foods, which can rapidly, sensitively and finally confirm bisacodyl in products and solve the problems of false positive and false negative. The technical scheme adopted by the invention is as follows:
a method for rapidly screening bisacodyl illegally added in Chinese patent medicines and health-care foods comprises the following steps:
(1) dissolving a bisacodyl reference substance in acetone to obtain a first reference substance solution, dissolving a test sample and a negative sample in acetone, diluting to obtain a first test sample solution and a first negative sample solution respectively, taking the first test sample solution and the first reference substance solution, respectively dropping the first test sample solution and the first reference substance solution on the same silica gel thin-layer plate, developing by using dimethylbenzene-butanone as a developing agent, drying in the air, and placing under an ultraviolet lamp for inspection, wherein the first test sample solution thin-layer chromatography cannot detect a main spot with the same position and color as those of the main spot displayed by the first reference substance solution thin-layer chromatography, and if the corresponding main spot is detected, further screening by using a liquid chromatograph;
(2) dissolving a bisacodyl reference substance, a test sample and a negative sample in methanol respectively, dissolving and diluting to obtain a second reference substance solution, a second test sample solution and a second negative sample solution, injecting the second test sample solution and the second reference substance solution into a liquid chromatograph, recording a chromatogram and a DAD (digital-to-analogue) spectrogram, if chromatographic peaks consistent with the reference substance chromatogram retention time and the DAD spectrum are detected, determining that the second test sample solution is positive, and then confirming by using a liquid chromatogram-mass spectrometer;
(3) and (3) taking the second test sample solution with positive detection in the step (2), sequentially injecting a second control sample solution and a second negative sample solution into a liquid chromatogram-mass spectrometer, recording a liquid chromatogram, a primary mass spectrum and a secondary mass spectrum, wherein in the chromatogram of the second test sample solution, a chromatographic peak which is consistent with the chromatographic retention time and the primary mass spectrum of the second control sample solution cannot be detected, and if a corresponding chromatographic peak is detected and the primary mass spectrum and the secondary mass spectrum are consistent, determining that bisacodyl is illegally added.
Preferably, the method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods comprises the following detection conditions of a liquid chromatograph:
liquid chromatography column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
a detector: a diode array detector;
detection wavelength: 265 nm;
scanning wavelength: 210-400 nm;
column temperature: 35 ℃;
flow rate: 1.0 ml/min-1；
Mobile phase: acetonitrile-water, the volume ratio of acetonitrile to water being 45: 55.
Preferably, the method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods comprises the following steps of:
a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
detection wavelength: 265 nm;
mobile phase: acetonitrile-0.01 mol. L-1Ammonium acetate solution (0.1% formic acid), acetonitrile and ammonium acetate solution in a volume ratio of 40:60, adding a solvent to the mixture;
flow rate: 0.3 ml. min-1。
Preferably, the method for rapidly screening the illegal addition of bisacodyl in the Chinese patent medicines and the health foods comprises the following steps of:
an electrospray ionization source is arranged, and ESI + scanning is carried out;
temperature of the drying gas: 350 ℃;
capillary voltage: 3.5 KV;
taper hole voltage: 120V;
temperature of the drying gas: 350 ℃;
flow rate of drying gas: 10 L.min-1；
The scanning mode is as follows: primary mass spectrum full scanning and secondary mass spectrum scanning;
scanning range: 100-1000.
Preferably, the method for rapidly screening the illegal addition of bisacodyl in Chinese patent medicines and health-care foods comprises the following specific steps: the bisacodyl control was dissolved and diluted with acetone to make a first control solution containing 1mg per 1 ml.
Preferably, the method for rapidly screening the illegal addition of bisacodyl in Chinese patent medicines and health-care foods comprises the following specific steps: the bisacodyl control was dissolved and diluted with methanol to make a second control solution containing 1mg per 1 ml.
Preferably, the method for rapidly screening the illegal addition of bisacodyl in Chinese patent medicines and health-care foods comprises the following specific steps: taking the first sample as a solid preparation, grinding, precisely weighing the first sample, dissolving with acetone, and diluting; if the sample is a liquid preparation, shaking up, precisely measuring the dosage once, dissolving with acetone, and diluting; filtering, and taking the filtrate as a test solution.
Preferably, the method for rapidly screening the illegal addition of bisacodyl in the Chinese patent medicines and the health-care foods comprises the following specific preparation method of the second test solution:
if the sample is a solid preparation, grinding, precisely weighing the sample, placing in a measuring bottle, adding methanol, performing ultrasonic treatment, standing at room temperature, diluting with methanol to scale, shaking, and filtering with microporous membrane; if the sample is a liquid preparation, shaking, precisely measuring the dosage once, placing into a measuring flask, adding methanol, shaking, diluting with methanol to scale, shaking, and filtering with microporous membrane; the filtrate was used as the second sample solution.
The invention has the advantages that: the method for rapidly screening the illegal addition of the bisacodyl in the Chinese patent medicine and the health food establishes a method for supplementing and checking the illegal addition of the bisacodyl chemical substances in weight-losing and slimming Chinese patent medicines, weight-losing health food and weight-losing products, uses the thin-layer chromatography and the high-performance liquid chromatography for qualitative screening and quantification, uses the liquid chromatography and the high-performance liquid chromatography for accurate confirmation, can be suitable for the large-batch screening of a base layer, can avoid false positive results, and has the advantages of high sensitivity, good selectivity, strong confirmation and simple and rapid operation through verification, the method can be used for qualitative screening and accurate confirmation of illegal addition of bisacodyl in weight-losing and weight-losing Chinese patent medicines, weight-losing health-care foods and weight-losing products, provides reliable technical support for the fake-fighting work of the weight-losing Chinese patent medicines and the health-care products by a medicine supervision and management department and a public security system, and ensures the food and drug administration safety of people.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a thin layer chromatogram of an example of the present invention.
FIG. 2 is a chromatogram of a second control solution according to an embodiment of the present invention.
FIG. 3 is a spectral scan of a chromatographic peak of a second control solution in accordance with an embodiment of the present invention.
FIG. 4 is a chromatogram of a second sample solution according to an embodiment of the present invention.
FIG. 5 is a scanning spectrum of a second sample solution according to the embodiment of the present invention.
FIG. 6 is a chromatogram of a second negative test sample solution according to the example of the present invention.
FIG. 7 is a scanned spectrum of a second negative test sample according to the present invention.
FIG. 8 is a chromatogram of a second negative sample solution according to an embodiment of the present invention.
FIG. 9 is a mass spectrum of a second negative sample solution according to the example of the present invention.
FIG. 10 is a chromatogram of a second control solution according to an embodiment of the present invention.
FIG. 11 is a mass spectrum of a second control solution according to the present invention.
FIG. 12 is a first order mass spectrum of a second control solution according to an embodiment of the present invention.
FIG. 13 is a second mass spectrum of a second control solution according to an example of the present invention.
FIG. 14 is a chromatogram of a second sample solution according to an embodiment of the present invention.
FIG. 15 is a mass spectrum of a second sample solution according to the embodiment of the present invention.
FIG. 16 is a first order mass spectrum of a second sample solution according to an embodiment of the present invention.
FIG. 17 is a second mass spectrum of a second sample solution according to an embodiment of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Examples
The following detailed description of specific embodiments of the invention refers to the accompanying drawings
A method for rapidly screening bisacodyl illegally added in Chinese patent medicines and health-care foods mainly comprises a high performance liquid chromatography, and the method comprises the following verification contents: specificity, detection limit; and (3) adopting a UHPLC-Q TOF MS instrument by a liquid-mass combination method, and confirming a high-resolution primary mass spectrum and a secondary mass spectrum.
The method comprises the following steps:
(I) test materials
1. And (3) testing the sample: positive sample: baicao princess magic fiber tabletted candy (upgraded version) gift round pink tablets; negative samples: the fruit and vegetable fiber nutrition tablets are presented as samples of the market supervision and management bureau of the Wuxi market.
2. Comparison products: bisacodyl, batch No.: 100181-.
3. Reagent: see Table 1 below
4. The instrument comprises the following steps: see Table 2 below
(II) test method
Screening system
(1) Thin layer chromatography
1. Deployment system
Silica gel GF254The thin layer plate is developed by using xylene-butanone (1:1) as a developing agent, air-dried and inspected under an ultraviolet lamp (366 nm).
2. Sample preparation method
2.1 preparation of the first control solution
The bisacodyl control was dissolved and diluted with acetone to make a first control solution containing 1mg per 1 ml.
2.2 preparation of the first test solution
Taking 2 samples, grinding, adding 5-10ml of acetone, dissolving and filtering, and taking the subsequent filtrate as the first sample solution. The first negative sample was prepared in the same manner.
3. Specificity
Respectively dispensing 10 μ l of the first sample solution and the first reference solution on the same silica gel GF254The thin layer plate is developed by using xylene-butanone (1:1) as a developing agent, air-dried and inspected under an ultraviolet lamp (366 nm). The thin layer chromatogram is shown in FIG. 1, wherein in FIG. 1, the main spot corresponding to the main spot (and color) of the first control sample is detected in the first sample solution, and the negative sampleThe solution was not detected, and the first sample solution in which the main spot was detected was used as a positive sample solution.
(2) High performance liquid chromatography
1. Chromatographic conditions and sample preparation methods:
1.1 chromatographic conditions
A chromatographic column: octadecylsilane chemically bonded silica Xbridge C18(4.6X 250mm, inner diameter 5 μm, batch No. 01313725014030); mobile phase: acetonitrile-water (45:55), diode array detector, detection wavelength of 265nm, scanning wavelength: 210-400 nm; column temperature: 35 ℃; flow rate: 1.0 ml/min-1(ii) a Sample introduction volume: 10 mu l of the mixture; the theoretical plate number is calculated by bisacodyl and should not be lower than 2000.
1.2 sample preparation method
1.2.1 preparation of the second control solution
The bisacodyl control substance is dissolved in methanol and prepared into a solution containing 0.1mg of bisacodyl per 1ml as a second control substance solution.
1.2.2 preparation of the second test solution
Taking 2 positive samples, grinding, adding 15ml of methanol, carrying out ultrasonic treatment for 10 minutes, shaking all the time, filtering by using a 0.45-micrometer filter membrane, taking a subsequent filtrate as a second sample solution, and preparing a second negative sample by the same method.
2. Specificity
Precisely sucking 10 μ l of the second sample solution and 10 μ l of the second reference solution, respectively, injecting into chromatograph, and recording the chromatogram. The concentration is 0.1 mg/ml-1The sample of the second control solution is 10 mul, and the chromatogram and the spectrogram are shown in figures 2 and 3: the 4.5min chromatogram peak in FIG. 2 is the chromatogram peak of the second control, and FIG. 3 is the spectrum scan of the chromatogram peak of the second control.
The second sample solution prepared by the above preparation method was injected into 10. mu.l sample, and the chromatogram is shown in FIGS. 4 and 5: the peak at 4.5min in fig. 4 is the peak of the bisacodyl in the second test sample, and fig. 5 is the scanning spectrum of bisacodyl in the second test sample.
A sample of 10. mu.l of the second negative sample solution prepared by the above preparation method was injected, and the chromatogram and the spectral scan are shown in FIGS. 6 and 7: the peak at 4.5min in fig. 6 is the peak without bisacodyl.
As a result, a chromatographic peak which is consistent with the retention time of the bisacodyl control substance appears in the second test sample solution, the DAD spectrum of the second test sample solution is consistent, a chromatographic peak which is consistent with the retention time of the control substance does not appear in the negative test sample solution, and the second test sample solution which is consistent with the retention time of the bisacodyl control substance appears is taken as a positive sample.
(II), confirmation system-liquid chromatogram-mass spectrum combined method
1. Chromatographic conditions and System suitability test
Octadecylsilane chemically bonded silica gel as filler (chromatographic column specification of 100mm x 2.1mm,1.8 μm), and acetonitrile-0.01 mol. L-1Ammonium acetate solution (0.1% formic acid) (40:60) as mobile phase; flow rate: 0.3 ml. ml-1(ii) a The detection wavelength was 265 nm.
2. Conditions of Mass Spectrometry
Equipped with electrospray ionization source, ESI + scan, dry gas temperature: 350 ℃, capillary voltage: 3.5KV, taper hole voltage: 120V, dry air flow: 10 L.min-1The scanning mode is as follows: primary mass spectrum full scan and secondary mass spectrum scan, wherein the scanning range is as follows: 100-1000
3. Preparation of the second control solution
Precisely weighing bisacodyl reference substance about 10mg, placing in a 100ml measuring flask, adding an appropriate amount of methanol, performing ultrasonic treatment for 10 minutes to dissolve, cooling to room temperature, adding methanol to scale, shaking up, precisely weighing 1ml, placing in a 10ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the final product.
4. Preparation of the second test solution
Taking the second test solution which is positive in the high performance liquid chromatography test as the test solution for the item test, and diluting the second test solution with methanol to be a solution with the concentration similar to that of the reference substance.
5. Assay method
Injecting the second sample solution and the second control solution into a liquid chromatograph by 1 μ l each, and recording the chromatogram and mass spectrogram.
6. As a result: in the chromatogram of the second sample, a chromatographic peak with the same retention time as that of the chromatogram of the control appears, and the primary mass spectrum and the secondary mass spectrum of the chromatogram are consistent with those of the second control, as shown in FIGS. 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17. FIGS. 8 and 9 are chromatograms and mass spectrograms of the second negative sample solution, with no chromatographic peak appearing at 8.75 min; FIGS. 10 and 11 are the second control solution chromatogram and mass spectrum showing a peak of bisacodyl at 8.75 min; FIG. 12 is a first order mass spectrum of the second control, with an excimer ion peak of 362.1415 (the excimer ion of bisacodyl is the bisacodyl molecule ion); FIG. 13 is a secondary mass spectrum of the second control with a major ion fragment of m/z154.0647,167.0724, 184.0753; FIGS. 14 and 15 are chromatograms and mass spectrograms of the second test sample, showing a peak of the chromatogram with a retention time of 8.77min as bisacodyl; FIG. 16 is a primary mass spectrum of a second test article, with an excimer ion peak of 362.1409; FIG. 17 is a secondary mass spectrum of the second test sample, the major ion fragment m/z154.0646,167.0724, 184.0751.
TABLE 3 bisacodyl HPLC/MS
The unknown substance in the sample is consistent with the primary and secondary mass spectra of the reference substance, and the positive sample is [ M + H ]]+362.1409 bisacodyl (C)22H19NO4) Theory [ M + H]+m/z362.1415 with an error of-0.17 ppm and an error of less than 5ppm, the unknown substance in the positive sample can be considered to be bisacodyl C22H19NO4。
The conclusion is as follows:
1. thin-layer chromatography: the specificity test shows that: the positive samples showed a main spot that was consistent in position and color with the control main spot.
2. Liquid chromatography: the specificity test shows that: the positive sample has chromatographic peak with retention time same as that of the reference, and the DAD spectrogram is consistent with that of the reference.
3. Liquid chromatography-mass spectrometry combination: the chromatogram of the test sample has chromatographic peak with retention time of the chromatogram of the reference sample, and its primary mass spectrum and secondary mass spectrumFirst order [ M + H ] consistent with the control]+362.1415, secondary 154.0647,167.0724,184.0753 Positive sample [ M + H]+ m/z362.1409, bisacodyl (C)22H19NO4) Theory [ M + H]+m/z362.1415 with an error of-0.17 ppm and an error of less than 5ppm, the unknown substance in the positive sample can be considered to be bisacodyl C22H19NO4In conclusion, the experimental results meet the requirements, and the method can be used for specially, sensitively, quickly and accurately preliminarily screening and confirming the illegal addition of bisacodyl in weight-reducing and weight-losing Chinese patent medicines and health-care foods.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (8)
1. A method for rapidly screening bisacodyl illegally added in Chinese patent medicines and health-care foods is characterized by comprising the following steps:
(1) dissolving a bisacodyl reference substance in acetone to obtain a first reference substance solution, dissolving a test sample and a negative sample in acetone, diluting to obtain a first test sample solution and a first negative sample solution respectively, taking the first test sample solution and the first reference substance solution, respectively dropping the first test sample solution and the first reference substance solution on the same silica gel thin-layer plate, developing by using dimethylbenzene-butanone as a developing agent, drying in the air, and placing under an ultraviolet lamp for inspection, wherein the first test sample solution thin-layer chromatography cannot detect a main spot with the same position and color as those of the main spot displayed by the first reference substance solution thin-layer chromatography, and if the corresponding main spot is detected, further screening by using a liquid chromatograph;
(2) dissolving the bisacodyl reference substance, the test sample and the negative sample in the step (1) in methanol respectively, dissolving and diluting to obtain a second reference substance solution, a second test sample solution and a second negative sample solution, injecting the second test sample solution and the second reference sample solution into a liquid chromatograph, recording a chromatogram and a DAD spectrogram, if chromatographic peaks consistent with the chromatographic retention time of the reference substance and the DAD spectrum are detected, determining that the second test sample solution is positive, and then confirming by using a liquid chromatography-mass spectrometer;
(3) and (3) taking the second test solution with positive detection in the step (2), sequentially injecting a second reference solution and a second negative sample solution into a liquid chromatogram-mass spectrometer, recording a liquid chromatogram, a primary mass spectrum and a secondary mass spectrum, wherein in the chromatogram of the second test solution, a chromatographic peak which is consistent with the chromatographic retention time and the primary mass spectrum of the second reference solution cannot be detected, and if a corresponding chromatographic peak is detected, determining that bisacodyl is illegally added.
2. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the detection conditions of the liquid chromatograph are as follows:
liquid chromatography column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
a detector: a diode array detector;
detection wavelength: 265 nm;
scanning wavelength: 210-400 nm;
column temperature: 35 ℃;
flow rate: 1.0 ml.min-1；
Mobile phase: acetonitrile-water, the volume ratio of acetonitrile to water being 45: 55.
3. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the chromatographic detection conditions of the liquid chromatography-mass spectrometer are as follows:
a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
detection wavelength: 265 nm;
mobile phase: acetonitrile-0.01 mol.L-1Ammonium acetate solution (0.1% formic acid), acetonitrile and ammonium acetate solution in a volume ratio of 40:60, adding a solvent to the mixture;
flow rate: 0.3 ml.min-1。
4. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 3, wherein the mass spectrum detection conditions of the liquid chromatography-mass spectrometer are as follows:
an electrospray ionization source is arranged, and ESI + scanning is carried out;
temperature of the drying gas: 350 ℃;
capillary voltage: 3.5 KV;
taper hole voltage: 120V;
temperature of the drying gas: 350 ℃;
flow rate of drying gas: 10 L.min-1；
The scanning mode is as follows: primary mass spectrum full scanning and secondary mass spectrum scanning;
scanning range: 100-1000.
5. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the preparation method of the first control solution specifically comprises the following steps: the bisacodyl control was dissolved and diluted with acetone to make a first control solution containing 1mg per 1 ml.
6. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the preparation method of the second control solution comprises the following specific steps: the bisacodyl control was dissolved and diluted with methanol to make a second control solution containing 1mg per 1 ml.
7. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the preparation method of the first test solution specifically comprises the following steps: taking the first sample as a solid preparation, grinding, precisely weighing the first sample, dissolving with acetone, and diluting; if the sample is a liquid preparation, shaking up, precisely measuring the dosage once, dissolving with acetone, and diluting; filtering, and taking the filtrate as a test solution.
8. The method for rapidly screening bisacodyl illegally added to Chinese patent medicines and health foods according to claim 1, wherein the preparation method of the second test solution specifically comprises the following steps:
if the sample is a solid preparation, grinding, precisely weighing the sample, placing in a measuring bottle, adding methanol, performing ultrasonic treatment, standing at room temperature, diluting with methanol to scale, shaking, and filtering with microporous membrane; if the sample is a liquid preparation, shaking, precisely measuring the dosage once, placing into a measuring flask, adding methanol, shaking, diluting with methanol to scale, shaking, and filtering with microporous membrane; the filtrate was used as the second sample solution.
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