CN101285802B - Qualitative analysis detection method for high polarity sugar-reducing chemical medicament in traditional Chinese medicine - Google Patents
Qualitative analysis detection method for high polarity sugar-reducing chemical medicament in traditional Chinese medicine Download PDFInfo
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Abstract
The invention discloses a qualitative analysis detection method for illegally mixed high-polar chemical anti-diabetic components in anti-diabetic traditional Chinese medicine products. In the method, a moving phase is a phosphate-acetonitrile moving phase system; the flow rate is between 0.8 and 1.2ml/min; a chromatographic column adopts a Hypersil amino chromatographic column, the column temperature is between 20 and 45 DEG C, metformin hydrochloride, phenformin hydrochloride, acarbose and voglibose can realize the complete separation. When retention time of a chromatographic peak in an anti-diabetic traditional Chinese medicine product is consistent with that of anti-diabetic medicine and the apparent absorption is shown out, which indicate that the anti-diabetic medicine is contained in the sample to be tested. The method has the advantages of quickness, simplicity, convenience, high sensitivity, strong specialization, broad coverage and so on.
Description
Technical field
The invention provides the qualitative analysis detection method of illegally mixing high polarity hypoglycemic chemicals in a kind of blood-sugar lowering tcm drug series products, belong to the drug quality detection architecture.
Background technology
Diabetes have become our times sex hygiene problem, and China's diabetes prevalence also increases year by year, and newly-increased patient is mainly diabetes B, and oral antidiabetic drug occupies an important position in this class patient's treatment.In recent years, clinical hypoglycemic medicine commonly used has biguanides and alpha-glucosidase restrainer etc., some traditional Chinese medicine antidiabetic preparationses that also occur among the people, wherein some Chinese medicine preparation adds western medicine composition without authorization, arbitrarily exaggerative curative effect, the patient who has thinks that these Chinese medicine preparations among the people are pure Chinese medicinal preparations by mistake, spinoff is little, can take the clinical patient who these Chinese medicine antidiabetic drugs poisonings occur taking in a large number.Therefore, be necessary to set up sensitivity, reliable analytical method is identified the antidiabetic drug that illegally mixes in the traditional Chinese medicine antidiabetic preparations.
The insulin that the hypoglycemic chemicals generally can be divided into sulfonylureas, biguanides, alpha-glucosidase inhibitor, thiazolidinediones medicine, non-sulfonylurea promotes several big classes such as secrete pharmaceutical, wherein biguanides, alpha-glucosidase inhibitor class hypoglycemic chemicals have higher water-soluble, polarity is higher, be high polarity hypoglycemic chemicals, notable difference arranged with the polarity of other several big classes.The typical case of biguanides is represented as Metformin hydrochloride, DB2; The typical case of alpha-glucosidase inhibitor class medicine is represented as acarbose, voglibose.
At present, the existing hypoglycemic chemicals that adopts high-efficient liquid phase chromatogram technology to detect and mix in the blood-sugar lowering tcm drug series products, it as application number 200610016419.1 Chinese patent, with the content of high-performance liquid chromatography method methyl propynyl benzylamin hydrochloride, chromatographic condition and system suitability test are: with octadecylsilane chemically bonded silica is filling agent; Methyl alcohol-0.05mol/L potassium dihydrogen phosphate (10% phosphoric acid is transferred pH=3) 15:85 is a moving phase; The detection wavelength is 217nm; 40 ℃ of column temperatures, theoretical cam curve are pressed the methyl propynyl benzylamin hydrochloride cutting edge of a knife or a sword and are calculated, and should be not less than 5000.But this patented method scope of application is narrow, only can detect a kind of hypoglycemic chemicals of methyl propynyl benzylamin hydrochloride.
" the liquid chromatography-mass spectrography analyses of 6 kinds of oral antidiabetic drug " (analytical test journal, the 19th the 6th phase of volume, in November, 2000, Page 5-8) adopt following high-efficient liquid phase chromatogram condition that melbine, DB2 are detected in the literary composition: moving phase is that volume ratio is methyl alcohol-5mmol/L ammonium acetate buffer (pH3.5) of 65: 35, flow velocity 0.5ml/min; Column temperature is a room temperature; Sample size is 20 μ L;
Adopt the RP-HPLC method that biguanides is detected in " RP-HPLC detects the Western medicine sugar-lowering components in the traditional Chinese medicine health care product " (the 40th the 4th phase of volume of Chinese Pharmaceutical Journal, Page 316-317) literary composition; Chromatographic condition is: C18 chromatographic column (Yi Lite 4.6mm * 250mm, 5 μ m; Moving phase: methyl alcohol-ion pair test solution (sodium heptanesulfonate 0.5056g adds triethylamine 1.4mL, adds water to 1000mL, with phosphorus acid for adjusting pH value to 3.5 ± 0.05) (30: 70); Flow velocity: 1.0mLmin
-1Sample size is: 10 μ L, 40 μ gmL
-1, diode array detector and UV detect wavelength 233nm; Column temperature is a room temperature; Number of theoretical plate calculates and should all be not less than 2000 by two reference substance peaks;
" reversed phase liquid chromatography is measured six kinds of antidiabetic drug western medicine compositions simultaneously " (Modern Scientific Instruments 2006,6, Page89-91) adopt reversed phase liquid chromatography to measure six kinds of hypoglycemic western medicine compositions such as Metformin hydrochloride in the hypoglycemic sample, DB2, Glipizide, gliclazide, glibenclamide, Glimepiride simultaneously in the literary composition.Chromatographic column is an Agilent C18 post, and moving phase is acetonitrile-0.02% phosphoric acid solution, gradient elution, wavelength 229nm, flow velocity 1.0mL/min.Can realize the separation fully of six components in 17 minutes, each component has good linear relationship within the specific limits;
" liquid chromatography-series connection quadrupole rod mass spectrometry method is measured the research of illegally mixing insoral and gliclazide in the traditional Chinese medicine antidiabetic preparations " (Pharmaceutical Analysis magazine 200525, Page 1453-1455) provides a kind of insoral that illegally mixes and specificity method of Gliclazide of detecting, the HPLC condition is: chromatographic column: ODS C18 (250mm * 4.6mm, 5 μ m); Moving phase: methyl alcohol-acetate buffer (pH4.2) (50: 50); Flow velocity: 1.0mL min
-130 ℃ of column temperatures; Sample size: 20 μ L; Detect wavelength: 240nm;
" the liquid chromatograph mass spectrography method is measured biguanides chemicals in the Chinese medicine preparation " (the 16th the 17th phase of volume of drug identification, Page 15-16) measures the biguanides chemicals that illegally mixes in the pure traditional Chinese medicine antidiabetic preparations with liquid chromatography-electron spray iontrap mass spectrometry combination usage, chromatographic condition is: chromatographic column: Agelent C18 post (150mm * 4.6mm, 5 μ m); Moving phase: 0.02mol/L ammonium acetate solution (pH transfers to 3.5 with acetic acid)-methyl alcohol (70: 30); Flow velocity 0.3ml/min; 30 ℃ of column temperatures; Sample size: 10 μ L; Detect wavelength: 235nm;
But all do not relate to the detection of alpha-glucosidase inhibitor class medicine acarbose, voglibose in the above-mentioned document; " reversed-phased high performace liquid chromatographic-diode array detector is measured the chemicals in the blood-sugar lowering tcm drug preparation " (Chinese pharmacist, 2005 the 8th the 10th phases of volume, Page 827-829) uses reversed-phased high performace liquid chromatographic-photodiode array detection (RP-HPLC---3 kinds of chemicals-insorals, acarbose, glibenclamide that DAD) may exist in the mensuration traditional Chinese medicine antidiabetic preparations, chromatographic condition is: Agilent ZORB-AX ODS C18 (250mm * 4.6mm, 5 μ m); Moving phase: acetate buffer (10mmol/L ammonium acetate, the 20mmol/L triethylamine is regulated pH5.5 with acetic acid) and acetonitrile, gradient elution, flow velocity: 0.6mL min
-1Detect wavelength: 230nm; But this arts demand gradient elution, slightly complicated, and also disengaging time is longer, is unfavorable for Rapid identification.
In view of this, the present invention just sets about from the polarity of hypoglycemic chemicals, proposed illegally to mix in a kind of blood-sugar lowering tcm drug series products the qualitative analysis detection method of high polarity hypoglycemic chemicals, Metformin hydrochloride, DB2, acarbose, voglibose can be realized separating fully, have quick, easy, highly sensitive, advantages such as specificity strong, broad covered area.
Summary of the invention
The object of the present invention is to provide the qualitative analysis detection method of illegally mixing high polarity hypoglycemic chemicals in the blood-sugar lowering tcm drug series products.
Briefly, illegally mix the qualitative analysis detection method of high polarity hypoglycemic chemicals in the blood-sugar lowering tcm drug series products provided by the invention, comprising:
(1) method: high performance liquid chromatography;
(2) moving phase: phosphate-acetonitrile moving phase system, form by A and B, A is the mixed solution of 0.06% potassium dihydrogen phosphate and 0.0279% sodium hydrogen phosphate, and B is an acetonitrile, and volume ratio A: B is 30: 70, and flow velocity is 0.8-1.2ml/min;
(3) the amino chromatographic column of chromatographic column: Hypersil, column temperature 20-45 ℃;
(4) ultraviolet detection wavelength: the detection wavelength is 195nm;
(5) one or more high polarity hypoglycemic chemicalses are mixed, make reference substance solution, carry out stratographic analysis then, obtain the retention time of various high polarity hypoglycemic chemicalses;
(6) blood-sugar lowering tcm drug series products to be analyzed is made need testing solution, carry out stratographic analysis then;
When the retention time of the retention time of blood-sugar lowering tcm drug series products chromatographic peak and high polarity hypoglycemic chemicals is consistent, and tangible absorption is arranged, illustrate and contain this kind hypoglycemic chemical drug in the testing sample; Otherwise then do not have.
Wherein, described flow rate of mobile phase is preferably 1.0ml/min, and described chromatographic column column temperature is preferably 30 ℃.The amino chromatographic column specification of described Hypersil is preferably particle diameter 5 μ m, 4.6mm * 250mm.
Described high polarity hypoglycemic chemicals is Metformin hydrochloride, DB2, acarbose and voglibose.
Described reference substance solution and need testing solution solvent are methyl alcohol.
In the described reference substance solution, contain the Metformin hydrochloride that concentration is 10 μ g/ml, concentration is the DB2 of 5 μ g/ml, and concentration is the acarbose of 1000 μ g/ml, concentration is one or more in the voglibose of 500 μ g/ml, and solvent is methyl alcohol.
Described need testing solution preparation process is: capsule and tablet are all got 5 or 5, and capsule removes capsule shells, and big honeyed pills is got half; porphyrize or be cut into small pieces; medicine powder or fritter are changed in the 25ml volumetric flask, add 20ml methyl alcohol, sonicated 15 minutes; be chilled to room temperature; add methanol constant volume, shake up, filter; filtrate is crossed 0.45 μ m filter membrane, promptly gets need testing solution.
Sample size is 20 μ l during stratographic analysis.
Of the present invention being described in detail as follows:
In view of the method complexity of illegally mixing the hypoglycemic chemicals in the present detection blood-sugar lowering tcm drug series products, disengaging time is longer, be unfavorable for shortcomings such as fast detecting, the applicant is through long-term practice, just set about from the polarity of hypoglycemic chemicals, the chromatographic column and the condition that flow to equate are improved, the qualitative analysis detection method of illegally mixing high polarity hypoglycemic chemicals in a kind of quick, easy, highly sensitive detection blood-sugar lowering tcm drug series products is provided.
The present invention adopts high performance liquid chromatography as detecting the qualitative analysis detection method of illegally mixing high polarity hypoglycemic chemicals in the blood-sugar lowering tcm drug series products, and moving phase is phosphate-acetonitrile moving phase system; The moving phase system is made up of A and B, and A is the mixed solution of 0.06% potassium dihydrogen phosphate and 0.0279% sodium hydrogen phosphate, and B is an acetonitrile, and volume ratio A: B is 30: 70, and the flow velocity of moving phase is 0.8-1.2ml/min; Preferred 1.0ml/min.The amino chromatographic column of chromatographic column adopting Hypersil, column temperature 20-30 ℃, preferred 30 ℃.Can select the specification of chromatographic column according to actual needs, as particle diameter 5 μ m, 4.6mm * 250mm.The ultraviolet detection wavelength is 195nm.Sample size is 20 μ l.
During operation, at first need various high polarity hypoglycemic chemicalses are mixed,, carry out stratographic analysis then, obtain the retention time of various high polarity hypoglycemic chemicalses to obtain reference substance solution; So-called high polarity hypoglycemic chemicals is meant Metformin hydrochloride, DB2, acarbose and voglibose here.For convenience of disposable all the high polarity hypoglycemic chemicalses that illegally mix in the blood-sugar lowering tcm drug series products that detect, also in order to keep the consistance of experiment condition, avoid the influence of other factors to experimental result, usually all high polarity hypoglycemic chemicalses are mixed, to obtain reference substance solution, the reference substance solution solvent is a methyl alcohol.As Metformin hydrochloride, DB2, acarbose and voglibose are dissolved in the methyl alcohol, Metformin hydrochloride concentration is 10 μ g/ml, DB2 concentration is 5 μ g/ml, acarbose concentration is 1000 μ g/ml, voglibose concentration is 500 μ g/ml, can obtain reference substance solution.Certainly also can select in the high polarity hypoglycemic chemicals one or more according to actual conditions.Reference substance solution is carried out stratographic analysis, obtain chromatogram, obtain the retention time of various high polarity hypoglycemic chemicalses.It is slightly variant, as shown in table 1 that retention time is looked used the different meetings of instrument, but as long as reference substance solution and need testing solution chromatographic condition are in full accord, can't influence the sensitivity of net result.
The retention time that the high polarity biased sample of table 1 is analyzed by different high performance liquid chromatographs
| Tianjin, island 2010A | Tianjin, island 20AT | PE200 | |
| DB2 Metformin hydrochloride voglibose acarbose | 5.355 8.634 14.072 23.043 | 5.048 7.626 14.757 22.921 | 5.230 8.364 13.815 20.543 |
Then blood-sugar lowering tcm drug series products to be analyzed is made need testing solution, for stratographic analysis.The solvent of need testing solution is similarly methyl alcohol, and its preparation process is: capsule and tablet are all got 5 or 5, and capsule removes capsule shells; big honeyed pills is got half, and porphyrize or be cut into small pieces changes medicine powder or fritter in the 25ml volumetric flask over to; add 20ml methyl alcohol; sonicated 15 minutes is chilled to room temperature, adds methanol constant volume; shake up; filter, filtrate is crossed 0.45 μ m filter membrane, promptly gets need testing solution.
The chromatogram of comparison reference substance solution and need testing solution when the retention time of the retention time of blood-sugar lowering tcm drug series products chromatographic peak and high polarity hypoglycemic chemicals is consistent, and has tangible absorption, illustrates and contains this kind hypoglycemic chemical drug in the testing sample; Otherwise then do not have.
The qualitative analysis detection method of hypoglycemic chemicals of the present invention, can detect the high polarity hypoglycemic chemicals that illegally mixes in the blood-sugar lowering tcm drug series products, Metformin hydrochloride, DB2, acarbose, voglibose can be realized separating fully, have quick, easy, highly sensitive, advantages such as specificity strong, broad covered area.
Description of drawings
The high polarity hypoglycemic of Fig. 1 chemical drug reference substance solution is analyzed chromatogram
1, DB2 2, Metformin hydrochloride
3, voglibose 4, acarbose
The typical curve of the high polarity hypoglycemic of Fig. 2 chemicals
Fig. 2-1 DB2 solution typical curve
Fig. 2-2 Metformin hydrochloride solution typical curve
Fig. 2-3 acarbose solution typical curve
Fig. 2-4 voigelibo sugar juice typical curve
Embodiment
Embodiment 1
1, high-efficient liquid phase chromatogram condition
Moving phase is phosphate-acetonitrile moving phase system; The moving phase system is made up of A and B, and A is the 600mg potassium dihydrogen phosphate, and the 279mg sodium hydrogen phosphate is dissolved in 1 liter of pure water, and B is an acetonitrile, and volume ratio A: B is 30: 70, and the flow velocity of moving phase is 1.0ml/min.Chromatographic column is the amino chromatographic column of Hypersil, 30 ℃ of column temperatures, and specification is particle diameter 5 μ m, 4.6mm * 250mm.The detection wavelength is 195nm.Sample size is 20 μ l.
2, the analysis of reference substance solution
High polarity hypoglycemic medicine is mixed, be made into methanol solution, wherein acarbose concentration is 1000 μ g/ml, DB2 concentration is 5 μ g/ml, Metformin hydrochloride concentration is that 10 μ g/ml, voglibose concentration are 500 μ g/ml, and the analysis collection of illustrative plates of analysis of control product solution is seen Fig. 1.
3, blood-sugar lowering tcm drug series products to be analyzed is analyzed
Blood-sugar lowering tcm drug series products to be analyzed is made the test sample methanol solution, and preparation process is: capsule and tablet are all got 5 or 5, and capsule removes capsule shells; big honeyed pills is got half, and porphyrize or be cut into small pieces changes medicine powder or fritter in the 25ml volumetric flask over to; add 20ml methyl alcohol; sonicated 15 minutes is chilled to room temperature, adds methanol constant volume; shake up; filter, filtrate is crossed 0.45 μ m filter membrane, promptly gets need testing solution.Then need testing solution is carried out stratographic analysis.
4, comparison
The chromatogram of comparison need testing solution and reference substance solution when the retention time of the retention time of blood-sugar lowering tcm drug series products chromatographic peak and high polarity hypoglycemic chemicals is consistent, and has tangible absorption, illustrates and contains this kind hypoglycemic chemical drug in the testing sample; Otherwise then do not have.
Flow rate of mobile phase is 0.8ml/min in the high-efficient liquid phase chromatogram condition, 20 ℃ of column temperatures, and all the other are with embodiment 1.
Embodiment 3
Flow rate of mobile phase is 1.2ml/min in the high-efficient liquid phase chromatogram condition, 45 ℃ of column temperatures, and all the other are with embodiment 1.
Embodiment 4
Flow rate of mobile phase is 0.9ml/min in the high-efficient liquid phase chromatogram condition, 25 ℃ of column temperatures, and all the other are with embodiment 1.
Experimental example 1
This experimental example is the investigation to the inventive method sensitivity, the range of linearity, precision, the recovery etc.
1, the preparation of solution
The preparation of reference substance solution: precision takes by weighing reference substance, places the volumetric flask of certain scale, behind dissolve with methanol, to scale, is made into the stock solution of prescribed concentration with methanol constant volume.
Standard serial solution: use microscale sampler, in volumetric flask, add volume required stock solution respectively, be diluted to scale, be made into required standard serial solution with methyl alcohol.
The preparation of need testing solution: capsule and tablet are all got 5 or 5, and capsule removes capsule shells, and big honeyed pills is got half; porphyrize or be cut into small pieces changes medicine powder or fritter in the 25ml volumetric flask over to, adds 20ml methyl alcohol; sonicated 15 minutes; be chilled to room temperature, add methanol constant volume, shake up; filter; after filtrate crossed 0.45 μ m filter membrane, change in the high performance liquid chromatogram sample bottle, analyze by high performance liquid chromatograph.
The preparation of moving phase: take by weighing the quality of required medicine, be dissolved in a certain amount of pure water, regulate pH (when needing) with the acid or the alkali of appointment, constant volume is crossed 0.45 μ m filter membrane suction filtration, ultrasonic degas.
2, integral condition
Chromatogram analysis of spectrum software is Class-VP version 6.1.Integral parameter width is 5, and slope is 200, and drift is 1000.
3, detectability is measured
Use microscale sampler, the reference substance solution that the operation by 1 will accurately prepare is diluted, and is made into the different serial solution of concentration, is measured by the chromatographic determination condition of embodiment 1 respectively by high performance liquid chromatograph, determines the detectability of different samples.The detectability measurement result sees Table 2.
The detectability measurement result of table 2 hypoglycemic medicine
| Nomenclature of drug | Sampling volume (μ l) | Detect concentration (signal to noise ratio 3: 1) (μ g/ml) | Detectability (signal to noise ratio 3: 1) (ng) |
| DB2 Metformin hydrochloride acarbose |
20 20 20 20 | 0.1 0.2 3 3 | 2 4 60 60 |
4, the drafting of typical curve
Operation by 1 accurately prepares reference substance solution and standard serial solution, and the sample solution of preparation is carried out under the chromatographic determination condition by high performance liquid chromatograph, and measurement result is by 2 integrations.With tester concentration is horizontal ordinate, is ordinate drawing standard curve with the peak area.The range of linearity, typical curve equation and the related coefficient (r2) of different hypoglycemic medicines see Table 3.Typical curve is seen Fig. 2.
The typical curve equation of table 3 hypoglycemic chemical drug solution
| Nomenclature of drug | The range of linearity (μ g) | The typical curve equation | Related coefficient (r 2) |
| DB2 | 5～200 | Y＝119398.5597X+203734.6513 | 0.9992 |
| Metformin hydrochloride acarbose voglibose | 5～300 100～5000 300～10000 | Y＝83949.0550X+5321.7566 Y＝9700.9505X+811145.0748 Y＝2835.0297X+179764.6260 | 0.9999 0.9981 0.9999 |
5, precision is measured
Operation by 1 accurately prepares the solution of basic, normal, high three concentration of reference substance, and the reference substance solution of preparation is by carrying out under the High Performance Liquid Chromatography condition determination, and measurement result is by 2 integrations.With the corresponding equation of linear regression of peak area substitution of each sample determination, calculating concentration.
5.1 withinday precision
The difference sample introduction is 5 times in 1 day, calculates withinday precision.Result sees Table 4.
Table 4 withinday precision (n=5)
5.2 day to day precision
Each sample analysis is 1 time in 1 day, and METHOD FOR CONTINUOUS DETERMINATION 5 days is calculated day to day precision.Result sees Table 5.
Table 5 day to day precision (n=5)
6, determination of recovery rates
Hypoglycemic pcm and other Chinese patent drug are handled by the preparation of need testing solution respectively; under the hypoglycemic medicine high-efficient liquid phase chromatogram condition, analyze; selection does not have the Chinese patent drug of interference as the test sample sample to measuring hypoglycemic medicine; test sample capsule and tablet are all got 5 or 5; capsule removes capsule shells; big honeyed pills is got half; test sample is by porphyrize or be cut into small pieces, and medicine powder or fritter are changed in the 25ml volumetric flask, adds a certain amount of methyl alcohol; accurately add hypoglycemic reference substance stock solution or reference substance; be made into final concentration and precision and measure consistent low of concentration; in; the application of sample sample of a Senior Three concentration, sonicated 15 minutes is chilled to room temperature; add methanol constant volume; shake up, filter, filtrate is crossed 0.45 μ m filter membrane after; change in the high performance liquid chromatogram sample bottle, analyze by high performance liquid chromatograph.Every duplicate samples is measured 1 time, and measurement result with the peak area substitution equation of linear regression of being measured, by calculating concentration, is investigated the recovery by 2 integral condition integrations.Measurement result sees Table 6.Except that indivedual chemical drugs average recovery when the low concentration on the low side and relative deviation big, different hypoglycemic chemical drugs average recovery in different samples has all kept a higher level, and the relative deviation of the recovery in different Chinese patent drugs is also less.
Table 6 (one) the DB2 recovery
Table 6 (two) the Metformin hydrochloride recovery
Table 6 (three) the acarbose recovery
Table 6 (four) the voglibose recovery
7, the analysis of actual sample
The hypoglycemic pcm that buys on the market and health products and the non-hypoglycemic pcm of part are made need testing solution by the preparation method of need testing solution, reference substance solution is made mixed solution by the preparation method of reference substance solution, the concentration of high polarity hypoglycemic chemical drug mixed solution is acarbose concentration 1000 μ g/ml, DB2 concentration 5 μ g/ml, Metformin hydrochloride concentration 10 μ g/ml, voglibose concentration 500 μ g/ml, need testing solution is analyzed under high polarity hypoglycemic medicine analysis condition, per minute is analysed the once high polarity reference substance of 3 high polarity need testing solutions analysis mixed solution, after analyzing end, the retention time that compares the chromatographic peak of need testing solution and reference substance solution, chromatographic peak retention time unanimity, and tangible absorption is arranged, just can judge and contain corresponding reference substance chemical additive in this test sample.Analysis result sees Table 7.
Table 7 actual sample analysis result
| Sample | The hypoglycemic chemical drug that detects | Sample | The hypoglycemic chemical drug that detects | Sample | The hypoglycemic chemical drug that detects |
| Sample 1 | Sample 18 | |
|||
| |
Sample 19 | Sample 36 | |||
| Sample 3 | |
Sample 37 | |||
| Sample 4 | Sample 21 | Sample 38 | |||
| Sample 5 | Sample 22 | Sample 39 |
| Sample 6 | Sample 23 | Sample 40 | DB2 | |||
| Sample 7 | Sample 24 | Sample 41 | ||||
| Sample 8 | |
DB2 | Sample 42 | Voglibose | ||
| Sample 9 | Sample 26 | Sample 43 | ||||
| |
Sample 27 | Sample 44 | ||||
| Sample 11 | Sample 28 | Sample 45 | Metformin hydrochloride | |||
| Sample 12 | Sample 29 | Sample 46 | ||||
| Sample 13 | |
Sample 47 | ||||
| Sample 14 | Sample 31 | Sample 48 | ||||
| |
Sample 32 | DB2 | Sample 49 | |||
| Sample 16 | Sample 33 | | Sample | 50 | Acarbose | |
| Sample 17 | Sample 34 |
The compare of analysis of 8 actual samples
8.1 the compare of analysis of different instruments
Use different high performance liquid chromatographs, the treatment conditions by 7 are handled the back and are analyzed actual sample by high polarity analysis condition, and analysis result sees Table 8.
The different instruments of table 8 are to the analysis result of actual sample
| Sample 18 | |||
| Sample 19 | |||
| |
|||
| Sample 21 | |||
| Sample 22 | |||
| Sample 23 | |||
| Sample 24 | |||
| |
DB2 | DB2 | DB2 |
| Sample 26 | |||
| Sample 27 | |||
| Sample 28 | |||
| Sample 29 | |||
| |
| Sample 31 | |||
| Sample 32 | DB2 | DB2 | DB2 |
| Sample 33 | DB2 | DB2 | DB2 |
| Sample 34 | |||
| |
|||
| Sample 36 | |||
| Sample 37 | |||
| Sample 38 | |||
| Sample 39 | |||
| Sample 40 | DB2 | DB2 | DB2 |
| Sample 41 | |||
| Sample 42 | Voglibose | Voglibose | Voglibose |
| Sample 43 | |||
| Sample 44 | |||
| Sample 45 | Metformin hydrochloride | Metformin hydrochloride | Metformin hydrochloride |
| Sample 46 | |||
| Sample 47 | |||
| Sample 48 | |||
| Sample 49 | |||
| |
Acarbose | Acarbose | Acarbose |
8.2 different medicine inspecting institute are to the compare of analysis of actual sample
The sample label is distributed to different medicine inspecting institute with the form of blind sample, and different medicine inspecting institute uses high performance liquid chromatograph, and the treatment conditions by 7 are handled the back and analyzed actual sample by high polarity analysis condition, and analysis result sees Table 9.
The different medicine inspecting institute of table 9 are to the analysis result of actual sample
| C medicine inspecting institute | The X of D medicine inspecting institute | F medicine inspecting institute | H medicine inspecting institute | X medicine inspecting institute | |
| Sample 1 | |||||
| |
|||||
| Sample 3 | |||||
| Sample 4 | |||||
| Sample 5 | |||||
| Sample 6 | |||||
| Sample 7 |
| Sample 8 | |||||
| Sample 9 | |||||
| |
|||||
| Sample 11 | |||||
| Sample 12 | |||||
| Sample 13 | |||||
| Sample 14 | |||||
| |
|||||
| Sample 16 | |||||
| Sample 17 | |||||
| Sample 18 | |||||
| Sample 19 | |||||
| |
|||||
| Sample 21 | |||||
| Sample 22 |
| Sample 23 | |||||
| Sample 24 | |||||
| |
DB2 | DB2 | DB2 | DB2 | DB2 |
| Sample 26 | |||||
| Sample 27 | |||||
| Sample 28 | |||||
| Sample 29 | |||||
| |
|||||
| Sample 31 | |||||
| Sample 32 | DB2 | DB2 | DB2 | DB2 | DB2 |
| Sample 33 | DB2 | DB2 | DB2 | DB2 | DB2 |
| Sample 34 | |||||
| |
|||||
| Sample 36 | |||||
| Sample 37 | |||||
| Sample 38 | |||||
| Sample 39 | |||||
| Sample 40 | DB2 | DB2 | DB2 | DB2 | DB2 |
| Sample 41 | |||||
| Sample 42 | Voglibose | Voglibose | Voglibose | Voglibose | Voglibose |
| Sample 43 | |||||
| Sample 44 | |||||
| Sample 45 | Metformin hydrochloride | Metformin hydrochloride | Metformin hydrochloride | Metformin hydrochloride | Metformin hydrochloride |
| Sample 46 |
| Sample 47 | |||||
| Sample 48 | |||||
| Sample 49 | |||||
| |
Acarbose | Acarbose | Acarbose | Acarbose | Acarbose |
9, the checking of analysis result
Use liquid chromatograph mass spectrography method (HPLC/MS) that analysis result is measured.
9.1 LC-MS condition
Use the chromatographic column of octadecylsilane chemically bonded silica as filling agent, gradient elution, mobile phase A is 0.1% formic acid solution, and ammoniacal liquor is transferred pH to 3.5, and Mobile phase B is an acetonitrile, and the gradient elution table sees Table 10.
Table 10 liquid-matter coupling liquid phase gradient elution table
| Time (branch) | A | B％ | |
| 0 10 18 25 26 36 | 85 40 20 20 85 85 | 15 60 80 80 15 15 |
Detect wavelength: 230nm, flow velocity: 0.2ml/min.The mass spectrum condition: the ESI source, source voltage 4.5KV, 270 ℃ of capillary temperatures, capillary voltage 20V, sheath gas velocity 50arb, positive ion detects, and scan mode adopts full scan one-level mass spectrum, full scan second order ms (MS2), mass number scope 100～1000.
The preparation of reference substance solution: precision takes by weighing reference substance 10mg, places the volumetric flask of 100ml, add an amount of dissolving of methyl alcohol after, be diluted to scale with methyl alcohol, shake up, precision is measured 5ml, places the volumetric flask of 50ml, adds methyl alcohol and is diluted to scale, shakes up, promptly.(containing reference substance 10 μ g among every 1ml).
The preparation of need testing solution: pill and tablet are all got 5 or 5, and capsule removes capsule shells, and big honeyed pills is got half; porphyrize or be cut into small pieces changes medicine powder or fritter in the 100ml volumetric flask over to, and it is an amount of to add methyl alcohol; sonicated 15 minutes is chilled to room temperature, adds methanol constant volume; shake up, filter, precision is measured subsequent filtrate 5ml; put in the volumetric flask of 50ml; add methyl alcohol and be diluted to scale, shake up, promptly.
Determination method: accurate respectively each the 10 μ l of above-mentioned two kinds of solution that draw inject liquid chromatograph-mass spectrometer, carry out the LC-MS analysis, record liquid chromatogram and one-level mass spectrum and second order ms figure, by with the contrast of reference substance liquid chromatogram retention time, one-level mass spectrum and second order ms figure, determine whether contain the reference substance material in the test sample.
Analysis result sees Table 11.
10, efficient liquid phase chromatographic analysis result's judgement
The analysis result and the liquid-matter coupling technique of different instruments, different medicine inspecting institute are compared discovery, no matter use the high performance liquid chromatograph of which kind of model, also no matter analyze by that medicine inspecting institute, every liquid-matter coupling technique detects the sample that mixes chemical Antidiabetic Drug, and other liquid phase analysis result shows this material and significantly absorbs.Because liquid-matter coupling technique is the most convictive technology of generally acknowledging detecting aspect the adulteration, also is the analyzing and testing result of recognition of state, by and liquid-matter coupling technique relatively, can verify the accuracy of the method for being set up.By relatively finding, if in hypoglycemic pcm or the blood sugar reducing health product sample, have and the consistent absorption peak of chemical sugar-lowering reference substance retention time, and absorption peak is obvious, just can determine to contain in this sample the hypoglycemic chemical drug that mixes.
Table 11 liquid-matter coupling method is to the analysis result of actual sample
| Sample | The hypoglycemic chemical drug that detects | Sample | The hypoglycemic chemical drug that detects | Sample | The hypoglycemic chemical drug that detects | |
| Sample 1 | Sample 18 | |
||||
| |
Sample 19 | Sample 36 | ||||
| Sample 3 | |
Sample 37 | ||||
| Sample 4 | Sample 21 | Sample 38 | ||||
| Sample 5 | Sample 22 | Sample 39 | ||||
| Sample 6 | Sample 23 | Sample 40 | DB2 | |||
| Sample 7 | Sample 24 | Sample 41 | ||||
| Sample 8 | |
DB2 | Sample 42 | Voglibose | ||
| Sample 9 | Sample 26 | Sample 43 | ||||
| |
Sample 27 | Sample 44 | ||||
| Sample 11 | Sample 28 | Sample 45 | Metformin hydrochloride | |||
| Sample 12 | Sample 29 | Sample 46 | ||||
| Sample 13 | |
Sample 47 | ||||
| Sample 14 | Sample 31 | Sample 48 | ||||
| |
Sample 32 | DB2 | Sample 49 | |||
| Sample 16 | Sample 33 | | Sample | 50 | Acarbose | |
| Sample 17 | Sample 34 |
11. conclusion
Use the high-efficient liquid phase technique of being set up to can be used for the qualitative analysis of the hypoglycemic chemical drug in the hypoglycemic pcm.Be used for qualitative analysis and can well determine the kind of the hypoglycemic chemical drug that Chinese patent drug mixes.Particularly to the analysis of high polarity hypoglycemic chemical drug, because high polarity hypoglycemic chemical drug is very short in the liquid chromatography retention time, high polarity hypoglycemic chemical drug has no idea to analyze owing to mix with solvent peak.Illegal manufacturer has also utilized this point to mix these chemical drugs just in hypoglycemic pcm and health products, to strengthen its curative effect, actual sample has also detected DB2, acarbose, Metformin hydrochloride and voglibose really in some hypoglycemic pcms and blood sugar reducing health product, and content is very high, uses these medicines will damage the healthy of diabetic greatly.
The method that this project is set up has been investigated method sensitivity, the range of linearity, precision, the recovery of being set up, the result shows that this method has good reappearance, sensitivity, the recovery can satisfy the requirement of analyzing and testing, the high polarity hypoglycemic chemical drug that these factors can make these class methods perform well in studying to mix in hypoglycemic pcm and the blood sugar reducing health product.
Claims (7)
1. illegally mix the qualitative analysis detection method of high polarity hypoglycemic chemicals in the blood-sugar lowering tcm drug series products, it is characterized in that, comprising:
(1) method: high performance liquid chromatography;
(2) moving phase: phosphate-acetonitrile moving phase system, form by A and B, A is the mixed solution of mass percent 0.06% potassium dihydrogen phosphate and 0.0279% sodium hydrogen phosphate, and B is an acetonitrile, and volume ratio A: B is 30: 70, and flow velocity is 0.8-1.2ml/min;
(3) the amino chromatographic column of chromatographic column: Hypersil, column temperature 20-45 ℃;
(4) ultraviolet detection wavelength: the detection wavelength is 195nm;
(5) high polarity hypoglycemic chemicals is mixed, make reference substance solution, carry out stratographic analysis then, obtain the retention time of various high polarity hypoglycemic chemicalses;
(6) blood-sugar lowering tcm drug series products to be analyzed is made need testing solution, carry out stratographic analysis then; Consistent and tangible absorption is arranged when the retention time of the retention time of blood-sugar lowering tcm drug series products chromatographic peak and high polarity hypoglycemic chemicals, illustrate and contain this kind hypoglycemic chemicals in the testing sample; Otherwise then do not have; Described high polarity hypoglycemic chemicals is selected from Metformin hydrochloride, DB2, acarbose and voglibose.
2. qualitative analysis detection method according to claim 1 is characterized in that, described flow rate of mobile phase is 1.0ml/min, and described chromatographic column column temperature is 30 ℃.
3. qualitative analysis detection method according to claim 1 is characterized in that, the amino chromatographic column specification of described Hypersil is particle diameter 5 μ m, 4.6mm * 250mm.
4. qualitative analysis detection method according to claim 1 is characterized in that, described reference substance solution and need testing solution solvent are methyl alcohol.
5. qualitative analysis detection method according to claim 1, it is characterized in that, in the described reference substance solution, contain multiple in the voglibose that Metformin hydrochloride that concentration is 10 μ g/ml, DB2 that concentration is 5 μ g/ml, acarbose that concentration is 1000 μ g/ml and concentration is 500 μ g/ml, solvent is methyl alcohol.
6. qualitative analysis detection method according to claim 1 is characterized in that, described need testing solution preparation process is: capsule is got 5, tablet is got 5, and capsule removes capsule shells, and big honeyed pills is got half, porphyrize or be cut into small pieces changes medicine powder or fritter in the 25ml volumetric flask over to, adds 20ml methyl alcohol, sonicated 15 minutes is chilled to room temperature, adds methanol constant volume, shake up, filter, filtrate is crossed 0.45 μ m filter membrane, promptly gets need testing solution.
7. the described qualitative analysis detection method of claim 1 is characterized in that, sample size is 20 μ l during stratographic analysis.
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| CN104597171A (en) * | 2013-10-31 | 2015-05-06 | 江苏万邦生化医药股份有限公司 | High performance liquid chromatography analysis method of acarbose and its preparation |
| CN105277705A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Preparation method and detection method for biguanide drug residue detection kit |
| CN112858512A (en) * | 2021-01-15 | 2021-05-28 | 北京万辉双鹤药业有限责任公司 | Method for determining impurities of hypoglycemic drug |
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