CN105974021A - Method for detecting illegally added propoxyadenafil - Google Patents
Method for detecting illegally added propoxyadenafil Download PDFInfo
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- CN105974021A CN105974021A CN201610313677.XA CN201610313677A CN105974021A CN 105974021 A CN105974021 A CN 105974021A CN 201610313677 A CN201610313677 A CN 201610313677A CN 105974021 A CN105974021 A CN 105974021A
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- acetonitrile
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- 238000000034 method Methods 0.000 title claims abstract description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 99
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
- 238000012790 confirmation Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 73
- 238000012360 testing method Methods 0.000 claims description 56
- 239000013558 reference substance Substances 0.000 claims description 50
- 235000010894 Artemisia argyi Nutrition 0.000 claims description 36
- 244000030166 artemisia Species 0.000 claims description 36
- -1 propoxyl group Chemical group 0.000 claims description 36
- 238000002360 preparation method Methods 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 16
- 239000012895 dilution Substances 0.000 claims description 16
- 239000012071 phase Substances 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 238000007689 inspection Methods 0.000 claims description 8
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 claims description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000012490 blank solution Substances 0.000 claims description 5
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 238000001228 spectrum Methods 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 2
- 238000011003 system suitability test Methods 0.000 claims description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims 2
- YSMRWXYRXBRSND-UHFFFAOYSA-N TOTP Chemical compound CC1=CC=CC=C1OP(=O)(OC=1C(=CC=CC=1)C)OC1=CC=CC=C1C YSMRWXYRXBRSND-UHFFFAOYSA-N 0.000 claims 1
- 239000003595 mist Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 10
- 238000001819 mass spectrum Methods 0.000 abstract description 10
- 235000013402 health food Nutrition 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 230000002929 anti-fatigue Effects 0.000 abstract 2
- 238000005728 strengthening Methods 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 69
- 238000005070 sampling Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Substances NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000005464 sample preparation method Methods 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000208340 Araliaceae Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 229940123333 Phosphodiesterase 5 inhibitor Drugs 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- 229940068682 chewable tablet Drugs 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002590 phosphodiesterase V inhibitor Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a drug detection method, and specifically relates to a method for qualitative, quantitative and confirmation method of a propoxyadenafil chemical product illegally added into a kidney-reinforcing and Yang-strengthening Chinese patent medicine and anti-fatigue health food. The principle of the method is as follows: a sample is extracted and diluted with acetonitrile, separated by a C18 column, qualitatively and quantitatively detected by a high-performance liquid chromatography DAD detector, and confirmed by primary and secondary mass spectra. The experimental results of the method all meet the requirements, indicating that the method can be used for exclusive, sensitive, rapid and accurate preliminary screening and confirmation of the propoxyadenafil illegally added into the kidney-reinforcing and Yang-strengthening Chinese patent medicine and the anti-fatigue health food.
Description
Technical field
The present invention relates to medicine detection method, be particularly used in kidney invigorating and YANG supporting class Chinese patent medicine and resisting fatigue class health food
Illegal add propoxyl group Chinese mugwort ground that non-chemically the qualitative examination method of product and confirmation method.In the method, high performance liquid chromatography is used
In qualitative examination；It is positive sample through qualitative examination result, survey report must be provided after liquid-mass chromatography method is confirmed.
Background technology
Propoxyl group Chinese mugwort ground that non-be that non-analog of a kind of Chinese mugwort ground, its chemical constitution is: 5-[[2-propoxyl group-5-(cis-
2,6-lupetazin-4-sulphonyl) phenyl]]-1-methyl-3-n-pro-pyl-7,6-dihydro-1 hydrogen-pyrazolo [4,3, d] is phonetic
Pyridine-7-ketone (structural formula sees below), molecular formula is C24H34N6O4S, molecular weight is 502.64.It is right that the most Canadian TLC company has
According to product, but unnamed, ID:S-0643.According to its structure and Chinese mugwort that non-parent nucleus of ground consistent (as follows), only will on phenyl ring
Ethyoxyl is instead of the structure of propoxyl group, it is proposed that general entitled: that is non-on propoxyl group Chinese mugwort ground, English entitled: propoxy
aildenafil。
At present, market product is found to have illegal this material of interpolation, but without examination, the quantitative and confirmation of this material
Method, and the harm the unknown of this toxicity of compound, in order to ensure the diet drug safety of the people, detect with reference to PDE5 inhibitor
The supplementary method of inspection 2009030 that project is more, and consider that a survey is commented more, screen multiple analog under the conditions of one as far as possible,
On the basis of front-end process verifying data, set up propoxyl group Chinese mugwort ground method for quickly detecting, for supervision application.
Summary of the invention
It is an object of the invention to provide one illegally to add in kidney invigorating and YANG supporting class Chinese patent medicine and resisting fatigue class health food
Adding that non-supplemental method of inspection of propoxyl group Chinese mugwort ground, the principle of the method is: by test sample acetonitrile extraction and dilution, C18Chromatographic column
Separate, confirm according to high performance liquid chromatography DAD detector qualitative and quantitative detection, LC-MS instrument one-level and second order ms figure.
The present invention adopts the following technical scheme that realization:
A kind of with detecting illegal interpolation propoxyl group Chinese mugwort that non-method, comprises the steps:
(1), high performance liquid chromatography
1.1, chromatographic condition and system suitability test
Liquid-phase chromatographic column with octadecylsilane chemically bonded silica as filler (specification as 250mm × 4.6mm, internal diameter 5 μm)；Two
Pole pipe array (DAD) detector, detection wavelength is 230nm, scanning wavelength: 200-400nm；Column temperature: 35 DEG C；Flow velocity: 1.0mL/
min；With phosphoric acid triethylamine solution (take triethylamine 7mL and be diluted with water to 1000mL, adjust pH to 2.8 with phosphoric acid) methanol acetonitrile
(60:20:20) it is mobile phase A, with phosphoric acid triethylamine solution methanol acetonitrile (8:46:46) as Mobile phase B, according to the form below program
Gradient elution.Theoretical cam curve calculates with that non-peak, propoxyl group Chinese mugwort ground, should be not less than 2000.
1.2, the preparation of reference substance solution
Take propoxyl group Chinese mugwort ground those non-control product about 10mg, put in 25mL measuring bottle, add methanol and dissolve and be diluted to scale, shake up, system
Become every 1mL storing solution containing about 0.4-0.5mg, face the used time, take 1mL methanol dilution and be settled to 10mL, obtain (be placed in 8 DEG C with
Lower Refrigerator store).
1.3, the preparation of need testing solution
If test sample is solid preparation, finely ground, precision weighs a dose, puts in 50mL volumetric flask, adds acetonitrile 40mL, ultrasonic
Process 15 minutes, put to room temperature, by dilution in acetonitrile to scale, shake up, filter with microporous filter membrane (0.45 μm)；If test sample is liquid
Body preparation, shakes up, and precision measures a dose, puts in 50mL volumetric flask, adds acetonitrile to 40mL, shakes 3 minutes, add acetonitrile dilute
Releasing to scale, filter with microporous filter membrane (0.45 μm) after shaking up, filtrate, as need testing solution, to obtain final product.
1.4, algoscopy
Take need testing solution and each 10 μ L of reference substance solution respectively, inject chromatograph of liquid, record chromatogram and DAD spectrogram.
1.5, result judges
In test sample chromatogram, the chromatographic peak consistent with reference substance chromatographic retention and DAD spectrum should be detected.If detection
Corresponding chromatographic peak, then use Liquid Chromatography-Mass Spectrometry confirmation.
(2), tablets by HPLC-MS
Measure according to high performance liquid chromatography (2010 editions two annex IX J of Chinese Pharmacopoeia).
2.1, chromatographic condition and system suitability
Chromatographic column with octadecylsilane chemically bonded silica as filler (specification as 150mm × 2.1mm, internal diameter 3 μm), detect ripple
A length of 230nm；With methanol: acetonitrile: the ammonium acetate solution (30:25:45) containing the 0.02mol/L of 0.1% glacial acetic acid is for flowing phase；
Flow velocity: 0.2mL/min.
2.2, Mass Spectrometry Conditions
Being furnished with electro-spray ionization source (ESI), ESI+ scans, dry temperature: 320 DEG C, and sheath gas velocity 40arb assists gas 10
Arb, spray voltage: 4.5KV, scan mode: first mass spectrometric full scan, second order ms full scan, sweep limits: 50 ~ 600m/z.
2.3, the preparation of reference substance solution
Take propoxyl group Chinese mugwort ground those non-control product about 10mg, put in 25mL measuring bottle, add dilution in acetonitrile and be settled to scale, shake up, make
Every 1mL storing solution containing about 0.4mg, faces the used time, takes the solution that appropriate storing solution 50% acetonitrile dilute one-tenth concentration is 10 μ g/mL, i.e.
Obtain (being placed in less than 8 DEG C Refrigerator stores).
2.4, the preparation of need testing solution
Take need testing solution that high performance liquid chromatography inspection is positive as the need testing solution checked, dilute with 50% acetonitrile
It is interpreted as and the solution of reference substance concentration comparable.Prepare blank solution simultaneously.
2.5, algoscopy
Take each 10 μ L of blank solution, need testing solution and reference substance solution respectively, inject LC-MS instrument, record liquid chromatogram
And I and II mass spectrum.
2.6, result judges
In test sample chromatogram, should detect consistent with reference substance chromatographic retention, first mass spectrometric figure and second order ms figure
Chromatographic peak.If detecting corresponding chromatographic peak, it is determined that is non-illegally to the addition of propoxyl group Chinese mugwort ground.
The present invention establishes the illegal propoxyl group that adds in a kind of kidney invigorating and YANG supporting class Chinese patent medicine and resisting fatigue class health food and ends
That non-method of inspection of ground.Use the high performance liquid chromatograph fast qualitative examination that becomes basically universal of basic unit and quantitatively, LC-MS
Instrument accurately confirms, both can be suitably used for basic unit's high-volume screening, has been avoided that again false positive results.Empirical tests, this method is highly sensitive,
Selectivity is good, authenticity is strong, simple and efficient to handle, can be used in kidney invigorating and YANG supporting class Chinese patent medicine and resisting fatigue class health food illegal
Add propoxyl group Chinese mugwort ground that non-qualitatively screening, detection by quantitative and accurately confirm.
Accompanying drawing explanation
Fig. 1 represents that non-range of linearity of propoxyl group Chinese mugwort ground in embodiment.
Fig. 2 represents that in embodiment, concentration is the reference substance solution sample introduction 10 μ L chromatogram of 214 μ g/mL.
Fig. 3 represents that in embodiment, concentration is the reference substance solution sample introduction 10 μ L spectrogram of 214 μ g/mL.
Fig. 4 represents need testing solution sample introduction 10 μ L chromatogram in embodiment.
Fig. 5 represents need testing solution sample introduction 10 μ L spectrogram in embodiment.
Fig. 6 represents embodiment empty contrast color spectrogram.
Fig. 7 A represents reference substance chromatogram in embodiment.
Fig. 7 B represents reference substance first mass spectrometric figure in embodiment.
Fig. 7 C represents reference substance second order ms figure in embodiment.
Fig. 8 A represents sample chromatogram figure in embodiment.
Fig. 8 B represents sample first mass spectrometric figure in embodiment.
Fig. 8 C represents sample second order ms figure in embodiment.
Fig. 9 represents reference substance solution high-resolution first mass spectrometric figure in embodiment.
Figure 10 represents reference substance solution high-resolution second order ms figure in embodiment.
Figure 11 represents three grades of mass spectruies of reference substance solution high-resolution in embodiment.
Figure 12 represents sample solution high-resolution first mass spectrometric figure in embodiment.
Figure 13 represents sample solution high-resolution second order ms figure in embodiment.
Figure 14 represents three grades of mass spectruies of sample solution high-resolution in embodiment.
Detailed description of the invention
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in detail.
A kind of with detecting illegal interpolation propoxyl group Chinese mugwort that non-method, wherein Method validation content includes: efficiently liquid phase
Chromatography is investigated in terms of the range of linearity, repeatability, detection limit, quantitative limit, sample-adding recovery, specificity etc.；Liquid Chromatography/Mass Spectrometry: from
Sub-hydrazine mass spectrum and high resolution mass spectrum first mass spectrometric, second order ms, three grades of mass spectruies confirm.
Concrete grammar comprises the steps:
(), high performance liquid chromatography
(1) test material:
1, test sample: positive: a kind of health promoting wine in market, lot number :/, 125mL/ bottle.Negative sample: cattle colostrums chewable tablet
(solid sample), lot number: 20140203；Certain health promoting wine (fluid sample), lot number: 20130903J.
2, reference substance: propoxyl group Chinese mugwort ground that non-(Sidenafil Analogue), lot number: 1329-054A2, content
99.0%, specification 10mg, purchased from TLCpharmachem company of Canada, 2 ~ 8 DEG C of storages.
3, reagent: see table 1
4, instrument: see table 2
5, chromatographic column: Symmetry, C18,5 μm, 4.6 × 250mm, column temperature: 35 DEG C.
(2) chromatographic condition and sample preparation methods:
1, flowing phase
With phosphoric acid triethylamine solution (take triethylamine 7mL and be diluted with water to 1000mL, adjust pH to 2.8 with phosphoric acid) methanol acetonitrile
(60:20:20) it is mobile phase A, with phosphoric acid triethylamine solution methanol acetonitrile (8:46:46) as Mobile phase B, according to the form below 3 journey
Sequence gradient elution；Detection wavelength is 230nm.Scanning wavelength: 200-400nm.Theoretical cam curve is in terms of that non-peak, propoxyl group Chinese mugwort ground
Calculate, 2000 should be not less than.Flow velocity: 1.0mL/min, sampling volume: 10 μ L.
Table 3 high performance liquid chromatography eluent gradient table
2, the preparation of reference substance solution
Take propoxyl group Chinese mugwort ground those non-control product about 10mg, add acetonitrile 25mL, make every 1mL storing solution containing about 0.4mg, face use
Time, take 2.5mL dilution in acetonitrile to 10mL, obtain (being placed in less than 8 DEG C Refrigerator stores).
3, the preparation of need testing solution
Solid preparation is finely ground, and precision weighs a dose, puts in 50mL volumetric flask, adds acetonitrile 40mL, supersound process 15 minutes,
It is cooled to room temperature, by dilution in acetonitrile to scale, shakes up, filter with microporous filter membrane (0.45 μm)；Liquid preparation shakes up, and precision measures one
Secondary dose, puts in 50mL volumetric flask, adds acetonitrile to 40mL, shakes 3 minutes, adds dilution in acetonitrile to scale, uses micropore after shaking up
Filter membrane (0.45 μm) filters；Filtrate, as need testing solution, to obtain final product.
(3) range of linearity is investigated
Precision measures in above-mentioned reference substance storing solution (428 μ g/mL) 5mL to 10mL measuring bottle, by dilution in acetonitrile to scale, shakes up,
Making every 1mL containing reference substance is the reference substance solution of 214 μ g, dilutes successively, standard curve concentration is 2.14,8.56,42.8,
85.6, the solution of 214,428 μ g/mL, injects 10 μ L in chromatograph of liquid, measures chromatographic peak area, with concentration (μ g/mL) for horizontal
Coordinate, integral area is vertical coordinate, tries to achieve regression equation Y=33790X+1862.8, R2=0.9998, it is seen that concentration is at 428 μ g/
In the range of mL to 2.14 μ g/mL linear, as shown in Figure 1.
(4) repeated experiment
Liquor sample: take positive test sample 1 batch, prepare 6 parts by test sample preparation method, sample introduction 10 L, standard curve method measures
In sample that non-content of propoxyl group Chinese mugwort ground be respectively 0.129,0.130,0.130,0.130,0.130,0.130mg/mL, averagely
0.130mg/mL(is to drink in terms of 100mL every time, equivalent each serving consumption 13mg/ time), RSD is 0.31%, and replica test meets
Requirement.
Solid sample: because not finding solid positive in the detection, therefore the test sample in recovery testu is considered as
Solid sample carries out repeated experiment, sample introduction 10 L, and standard curve method measures that non-content difference of propoxyl group Chinese mugwort ground in sample
Be 4.021,3.990,3.933,3.962,4.013,3.962mg/g, average 3.980mg/g, RSD are 0.9%, replica test
Meet the requirements.
(5) quantitative limit and detection limit
Liquor sample: by 428 μ g/mL reference substance solution dilution variable concentrations, join in negative sample, by sample preparation behaviour
Make, with signal to noise ratio 3 times for the minimum response value of detection limit, 8.56 μ g/mL reference substance solution 1mL join in sample solution and reach
The minimum response value of detection limit, concentration limit is that 0.17 g/mL(is limited to 0.43 with test sample sampling amount 20mL calculating detection
G/mL).
Operate as above, with signal to noise ratio 10 times for the minimum response value of quantitative limit, quantitative limit concentration be 0.34 g/mL(for
Test product sampling amount 20mL calculates and is quantitatively limited to 0.86 g/mL).
Solid sample: by 428 μ g/mL reference substance solution dilution variable concentrations, join in negative sample, prepare by sample
Operation, with signal to noise ratio 3 times for the minimum response value of detection limit, 8.6 μ g/mL reference substance solution 1mL join in sample solution and reach
To the minimum response value of detection limit, concentration limit is that 0.17 g/mL (calculates detection with test sample sampling amount 1g and is limited to 8.6
g/g)。
Operate as above, with signal to noise ratio 10 times for the minimum response value of quantitative limit, the quantitative limit concentration of solid sample is 0.34
G/mL(calculates with test sample sampling amount 1g and is quantitatively limited to 17.1 g/g).
(6) recovery test
Recovery test uses sample-adding absorption method to measure.
Liquor sample: precision weighs above-mentioned known content positive 9 parts, every part of 10mL, is placed in 25mL volumetric flask, essence
Close measuring reference substance solution (428 μ g/mL) 2.5mL, 5.0mL, 7.5mL, each concentration in 3 parts of samples, adds acetonitrile extremely respectively
20mL, shakes 3 minutes, adds dilution in acetonitrile to scale, filters with microporous filter membrane (0.45 μm), takes 10 μ L sample introductions, calculate after shaking up
The response rate (result see table 4).
Table 4 is loaded recovery experiment result (liquor sample)
Solid sample: precision weighs above-mentioned negative sample 9 parts, every part of about 1g, is placed in 50mL volumetric flask, and precision measures reference substance
Solution (392.8 μ g/mL) 2.0mL, 5.0mL, 8.0mL, each concentration in 3 parts of samples, adds acetonitrile about 40mL, ultrasonic place respectively
Manage 15 minutes, be cooled to room temperature, by dilution in acetonitrile to scale, shake up, filter with microporous filter membrane (0.45 μm), take 10 μ L sample introductions, meter
Calculate the response rate (result see table 5).
Table 5 is loaded recovery experiment result (solid sample)
(7) specificity test
Target peak and reference substance solution chromatograph in sample solution, spectrogram are the most consistent.
1, by the reference substance solution sample introduction 10 μ L that concentration is 214 μ g/mL, chromatogram and spectrogram as shown in Figure 2,3: Fig. 2
Middle 16.6min chromatographic peak is the chromatographic peak of reference substance, and Fig. 3 is the spectral scan figure of reference substance chromatographic peak.
2, the need testing solution sample introduction 10 μ L prepared by the method for being prepared as described above, chromatogram and spectrogram as shown in Figure 4,5:
The chromatographic peak of object during 16.6min chromatographic peak is sample in Fig. 4, Fig. 5 is the spectral scan figure of object chromatographic peak in sample.
(), Liquid Chromatography-Mass Spectrometry
(1) ion hydrazine mass spectroscopy result
1, chromatographic condition and system suitability
With octadecylsilane chemically bonded silica as filler (chromatographic column specification as 150mm × 2.1mm, internal diameter 3 μm), with methanol:
Acetonitrile: the ammonium acetate solution (30:25:45) containing the 0.02mol/L of 0.1% glacial acetic acid is flowing phase；Flow velocity: 0.2mL/min；Inspection
Survey wavelength is 230nm.Theoretical cam curve calculates with that non-peak, propoxyl group Chinese mugwort ground, should be not less than 2000.
2, Mass Spectrometry Conditions
Mass spectrum is furnished with electric spray ion source (ESI), the MS detection parameters: ESI+Scanning, dry temperature: 320 DEG C, sheath gas velocity
40arb, assists gas 10 arb, spray voltage: 4.5KV, scan mode: first mass spectrometric full scan, second order ms full scan, scanning
Scope: 50 ~ 600m/z.
3, the preparation of reference substance solution
Take propoxyl group Chinese mugwort ground those non-control product about 10mg, add acetonitrile 25mL, make every 1mL storing solution containing about 0.4mg, face use
Time, take the solution that appropriate storing solution 50% acetonitrile dilute one-tenth concentration is 10 μ g/mL, obtain (being placed in less than 8 DEG C Refrigerator stores).
4, the preparation of need testing solution
Take need testing solution that high performance liquid chromatography inspection the is positive need testing solution as this item inspection, with 50%
Dilution in acetonitrile is and the solution of reference substance concentration comparable.
5, algoscopy
Take need testing solution and each 10 μ L of reference substance solution, inject chromatograph of liquid, record chromatograph, mass spectrum.
6, there is the chromatographic peak identical with reference substance chromatographic retention, and its one-level matter in result: in test sample chromatogram
Spectrum and second order ms are all consistent with reference substance, as shown in Fig. 6,7,8.Fig. 6 is the chromatogram of blank solution, 14.4min does not occurs
Chromatographic peak.Fig. 7 A is reference substance solution chromatogram, occurs that 14.4min chromatographic peak is that is non-on propoxyl group Chinese mugwort ground；Fig. 7 B is comparison
The first mass spectrometric figure of product, quasi-molecular ion peak is 503；Fig. 7 C is the second order ms figure of reference substance, and leading ion fragment is m/z
486、461、446、391、347、327、299、283.Fig. 8 A is sample solution chromatogram, occurs that 14.4min chromatographic peak is the third oxygen
That is non-on base Chinese mugwort ground；Fig. 8 B is the first mass spectrometric figure of sample, and quasi-molecular ion peak is 503；Fig. 8 C is the second order ms figure of sample, main
Wanting fragment ion is m/z 486,461,446,391,347,327,299,283.
That non-HPLC/MS/MS characteristic ion peak, table 6 propoxyl group Chinese mugwort ground
(2) high resolution mass spectrum measurement result
Instrument is Shimadzu Corporation LCMS-IT/TOF, and flow phase: acetonitrile (lark prestige), flow velocity: 0.8ml/min, direct injected, sweeps
Retouch scope: 1 grade of m/z100-2000,2 grades of m/z 100-2000,3 grades of 50-400.
Reference substance [M+H]+: m/z one-level 503.2438, two grade 391.1405,299.1129,283.1178,327.1729,
446.1859,461.1973,347.0808, three grade 166.0988.As shown in Fig. 9,10,11.
Sample [M+H]+: m/z one-level 503.2429, two grade 391.1418,299.1110,283.1170,327.1732,
446.1859,461.1855,347.0775, three grade 166.0974.As shown in Figure 12,13,14.
Unknown material and reference substance one-level in sample, two grades, three grades of mass spectrums the most consistent, sample [M+H]+M/z is 503.2429,
Propoxyl group Chinese mugwort that non-(C of ground24H34N6O4S) theoretical [M+H]+M/z is 503.2435, and error is-1.19ppm, and error is less than 5ppm,
It is believed that unknown material is propoxyl group Chinese mugwort that non-C of ground in sample24H34N6O4S。
Conclusion is as follows:
Liquid chromatography: the range of linearity is investigated, regression equation Y=33790X+1862.8, R2=0.9998, that is non-on propoxyl group Chinese mugwort ground
Concentration is linear in the range of 2.14 μ g/mL to 428 μ g/mL；Replica test RSD fluid sample be RSD be 0.9%, Gu
Body sample is 0.31%；Liquor sample concentration limit is that 0.17 g/mL(is limited to test sample sampling amount 20mL calculating detection
0.43 g/mL), solid sample concentration limit is that 0.17 g/mL(is limited to 8.6 g/ with test sample sampling amount 1g calculating detection
G)；Liquor sample quantitative limit concentration is that 0.34 g/mL(is quantitatively limited to 0.86 g/mL with test sample sampling amount 20mL calculating), Gu
The quantitative limit concentration of body sample is that 0.34 g/mL(is quantitatively limited to 17.1 g/g with test sample sampling amount 1g calculating)；Sample-adding reclaims
Test shows, high, medium and low 3 concentration average recovery rates are respectively 91.4,95.3,94.8%, RSD is respectively 1.2,0.9,
0.5%；Specificity test shows, occurs the chromatographic peak identical with reference substance retention time in sample, and DAD spectrum spectrogram is all with right
Consistent according to product.
, there is the chromatograph identical with reference substance chromatographic retention in Liquid Chromatography-Mass Spectrometry: in test sample chromatogram
Peak, and its first mass spectrometric and second order ms are all consistent with reference substance, one-level [M+H]+M/z 503, two grades [M+H]+M/z:391,
299、283、327、446、461、347、486；High resolution mass spectrum measure unknown material and reference substance one-level in sample, two grades, three grades
Mass spectrum is all consistent, sample [M+H]+M/z is 503.2429, propoxyl group Chinese mugwort that non-(C of ground24H34N6O4S) theoretical [M+H]+M/z is
503.2435, error is-1.19ppm, less than 5ppm, it is believed that in sample, unknown material is that is non-on propoxyl group Chinese mugwort ground
C24H34N6O4S。
To sum up, experimental result is satisfied by requirement, shows that this law can exclusive, sensitive, primary dcreening operation and confirmation the kidney invigorating quickly and accurately
That is non-illegally to add propoxyl group Chinese mugwort ground in tonifying YANG class Chinese patent medicine and resisting fatigue class health food.
It should be noted last that, above example is only in order to illustrate technical scheme and unrestricted, although ginseng
It is described in detail according to the embodiment of the present invention, it will be understood by those within the art that, to technical scheme
Modifying or equivalent, without departure from the spirit and scope of technical scheme, it all should contain the present invention's
In claims.
Claims (1)
1. illegal with adding propoxyl group Chinese mugwort that non-method of detection, it is characterised in that: comprise the steps:
(1), high performance liquid chromatography
1.1, chromatographic condition and system suitability test
Liquid-phase chromatographic column with octadecylsilane chemically bonded silica as filler；Diode array detector, detection wavelength is
230nm, scanning wavelength: 200-400nm；Column temperature: 35 DEG C；Flow velocity: 1.0mL/min；Tricresyl phosphate with volume ratio as 60:20:20
Ethylamine solution methanol acetonitrile is mobile phase A, and the phosphoric acid triethylamine solution methanol acetonitrile with volume ratio as 8:46:46 is
Mobile phase B, by following program gradient elution:
0-6min: mobile phase A 100%；
6-30min: mobile phase A 100%-0%；
30-35min: mobile phase A 0%；
35-36min: mobile phase A 0%-100%；
36-50min: mobile phase A 100%；
1.2, the preparation of reference substance solution
Take those non-control product of propoxyl group Chinese mugwort ground, put in measuring bottle, add methanol and dissolve and be diluted to scale, shake up, make every 1mL and contain
The storing solution of 0.4-0.5mg；
1.3, the preparation of need testing solution
If test sample is solid preparation, finely ground, precision weighs a dose, puts in volumetric flask, adds acetonitrile, after supersound process,
Put to room temperature, by dilution in acetonitrile to scale, shake up, filter with microporous filter membrane；If test sample is liquid preparation, shake up, accurate amount
Take a dose, put in volumetric flask, add acetonitrile extremely, shaking, add dilution in acetonitrile to scale, filter with microporous filter membrane after shaking up；
Filtrate is as need testing solution；
1.4, algoscopy
Take need testing solution and reference substance solution respectively, inject chromatograph of liquid, record chromatogram and DAD spectrogram；
1.5, result judges
In test sample chromatogram, the chromatographic peak consistent with reference substance chromatographic retention and DAD spectrum should be detected, if detection
Corresponding chromatographic peak, then use Liquid Chromatography-Mass Spectrometry confirmation；
(2), tablets by HPLC-MS
2.1, chromatographic condition and system suitability
Chromatographic column with octadecylsilane chemically bonded silica as filler, detection wavelength is 230nm；With volume ratio as 30:25:45
Methanol: acetonitrile: the ammonium acetate solution of the 0.02mol/L containing 0.1% glacial acetic acid is flowing phase；Flow velocity: 0.2mL/min；
2.2, Mass Spectrometry Conditions
Being furnished with electro-spray ionization source, ESI+ scans, dry temperature: 320 DEG C, and sheath gas velocity 40arb assists gas 10arb, spray
Mist voltage: 4.5KV, scan mode: first mass spectrometric full scan, second order ms full scan, sweep limits: 50 ~ 600m/z；
2.3, the preparation of reference substance solution
Take those non-control product of propoxyl group Chinese mugwort ground, put in measuring bottle, add dilution in acetonitrile and be settled to scale, shake up, make every 1mL containing 0.4-
The storing solution of 0.5mg；
2.4, the preparation of need testing solution
Take need testing solution that high performance liquid chromatography inspection is positive as the need testing solution checked, dilute with 50% acetonitrile
It is interpreted as and the solution of reference substance concentration comparable；Prepare blank solution simultaneously；
2.5, algoscopy
Take blank solution, need testing solution and reference substance solution respectively, inject LC-MS instrument, record liquid chromatogram and one,
Second order ms figure；
2.6, result judges
In test sample chromatogram, should detect consistent with reference substance chromatographic retention, first mass spectrometric figure and second order ms figure
Chromatographic peak；If detecting corresponding chromatographic peak, it is determined that is non-illegally to the addition of propoxyl group Chinese mugwort ground.
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| CN109187798A (en) * | 2018-10-09 | 2019-01-11 | 山西省食品药品检验所(山西省药品包装材料监测中心) | Detect that non-, propoxyl group Chinese mugwort that non-, N- ethyl Tadalafei of ground of demethyl card rolling land and hydroxypropyl demethyl Tadalafei method |
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| CN109187798A (en) * | 2018-10-09 | 2019-01-11 | 山西省食品药品检验所(山西省药品包装材料监测中心) | Detect that non-, propoxyl group Chinese mugwort that non-, N- ethyl Tadalafei of ground of demethyl card rolling land and hydroxypropyl demethyl Tadalafei method |
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