CN105158400A - Quality control method of cordate houttuynia eye drop - Google Patents
Quality control method of cordate houttuynia eye drop Download PDFInfo
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Abstract
The invention discloses a quality control method of a cordate houttuynia eye drop. The method comprises: thin-layer identification of methyl-nonyl-ketone, measurement of content of methyl-nonyl-ketone, and characteristic spectrum. In accordance with "Quality Standard of Traditional Chinese Medicine Guiding Principles for Validation of Analytical Methods" of Chinese Pharmacopoeia edited in 2010, validation of methods of each detection project of an original standard is performed, and projects with problems are improved. An injection of cordate houttuynia is taken as a research basis; identification of other component is added; and identification of terpineol and bornyl acetate by adopting a CC method is added. According to the requirement, and through research, a preparation method of a solution of a to-be-tested product in thin-layer identification of methyl-nonyl-ketone is changed. By referring to a research content of the injection of cordate houttuynia, identification of components of terpineol, bornyl acetate, terpineol, and the like by adopting a CC method is added, and check of the characteristic spectrum of the components is established to ensure uniform stable quality of the cordate houttuynia eye drop.
Description
Technical field
The present invention relates to a kind of method of quality control, be specifically related to a kind of method of quality control of cordate houttuynia eye drops.
Background technology
Cordate houttuynia is the whole herb with root of Saururaceae per nnial herb heartleaf houttuynia HouttuyniacordataThunb., famous root of picking up the ears, purple Ji, hog snout hole, Rhizoma Rohdeae Japonicae etc., pungent bad smell is had after its Fresh Plants fragmentation, be distributed widely in Central China, the southeast and southwestern each provinces and regions, especially economize in the majority with Hunan, Hubei, Sichuan, Jiangsu etc.Cordate houttuynia has effect of clearing heat and detoxicating, the carbuncle that disappears apocenosis, inducing diuresis for treating strangurtia, clinically to cough for lung carbuncle pyemesis, phlegm heat panting, hot dysentery, the disease such as heat pouring, carbuncle sore tumefacting virus.Research shows, containing compositions such as volatile ingredient, flavonoids, organic acid, sterols, alkaloidss in cordate houttuynia.Cordate houttuynia mainly eats with young stem and leaf and subterranean stem work vegetables, and complete stool can using fresh herb or dry be used as medicine, and is formally defined as one of resource of the great exploitation potential for its of " being medicine, is again food " by health ministry, day by day receives the concern of people.
Cordate houttuynia eye drops is through steam distillation preparation and obtains, and principal ingredient is its volatile ingredient.Prior art has the TLC distinguish of shape, pH value, methylnonanone and the assay of methylnonanone about the project of cordate houttuynia eye drops quality control.But the principal ingredient due to cordate houttuynia eye drops is Herba Houttuyniae extract, and the effective constituent complexity in extract, only differentiate and assay methylnonanone, detection means is too simple, requires larger gap relative to current Drug's control.Therefore existing method of quality control effectively can not control the quality of cordate houttuynia eye drops, thus affect the production of product and ensure the quality of products.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, a kind of method of quality control of cordate houttuynia eye drops is provided, the method is easy and simple to handle, precision and highly sensitive, good stability, ensure that the safe, homogeneous, stable, effective, controlled of the quality of cordate houttuynia eye drops.
Object of the present invention is achieved through the following technical solutions: a kind of method of quality control of cordate houttuynia eye drops, and it comprises the following steps:
S1. the TLC distinguish of methylnonanone
Get methylnonanone reference substance, add absolute ethyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast; Test according to thin-layered chromatography, draw reference substance solution 10 μ l and need testing solution 20 μ l, put on same silica gel g thin-layer plate respectively, put into streak, take cyclohexane-ethyl acetate as developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
S2. the assay of methylnonanone
Chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute; Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak;
The preparation of reference substance solution: get methylnonanone reference substance appropriate, accurately weighed, add absolute ethyl alcohol and make the solution of every 1ml containing 25 μ g, to obtain final product;
The preparation of need testing solution: precision measures testing sample 25ml, by the C that pre-service is good
8solid phase extraction column, with the mixed solution wash-out of ethyl acetate-ethanol, collect eluent and be about 1.8ml, put in 2ml measuring bottle, the mixed solution adding ethyl acetate-ethanol is diluted to scale, shakes up, and to obtain final product;
Determination method: accurate absorption reference substance solution and each 2 μ l of need testing solution, respectively inject gas chromatograph, measure, to obtain final product;
The every 1ml of this product in methylnonanone, must not be less than 4.0 μ g containing flavour amino acid;
S3. characteristic spectrum
A. the preparation of object of reference solution: get alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance in right amount, accurately weighed, add absolute ethyl alcohol respectively and make the solution of every 1ml containing 25 μ g, to obtain final product;
B. the preparation of need testing solution: precision measures testing sample 25ml, by the C that pre-service is good
8solid phase extraction column, with the mixed solution wash-out of ethyl acetate-ethanol, collect eluent and be about 1.8ml, put in 2ml measuring bottle, the mixed solution adding ethyl acetate-ethanol is diluted to scale, shakes up, and to obtain final product;
C. measure: accurate absorption object of reference solution and each 2 μ l of need testing solution respectively, inject gas chromatograph, measures;
Should present 6 characteristic peaks in test sample characteristic spectrum, wherein 4 peaks should be consistent with alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance peak retention time respectively; The peak corresponding to methylnonanone reference substance is S peak, calculates the relative retention time at each characteristic peak and S peak, should setting ± 5% within; Setting is: 0.782 (peak 1), 0.820 (peak 2), 0.878 (peak 3), 0.909 (peak 4), 1.000 (peak S), 1.052 (peaks 5).
Further, the volume ratio of described cyclohexane-ethyl acetate cyclohexane and ethyl acetate is 9:1.
Further, in the mixed solution of described ethyl acetate-ethanol, the volume ratio of ethyl acetate and ethanol is 7:3.
Further, the chromatographic condition of gas chromatograph described in step S3 is: chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute.Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak.
The present invention has the following advantages: the present invention carries out method validation by Chinese Pharmacopoeia 2010 version " quality standards in Chinese drugs analytical approach verification guide principle " to each test item of primary standard, carries out perfect to in-problem project; Utilize the Research foundation of houttuynia cordata injection, increase the discriminating (composition such as terpilenol) of other compositions; Increase and adopt GC method to the discriminating of terpinol, Bronyl acetate etc.As requested, by research, have changed the preparation method of need testing solution in methylnonanone TLC distinguish; With reference to the research contents of houttuynia cordata injection, add the discriminating of GC method to compositions such as terpinol, Bronyl acetate, terpilenols, and set up the characteristic spectrum inspection of this kind, with the stable homogeneous of ensuring the quality of products.Therefore, the invention provides a kind of method of quality control of cordate houttuynia eye drops, the method be easy and simple to handle, precision and highly sensitive, good stability, ensure that the safe, homogeneous, stable, effective, controlled of the quality of cordate houttuynia eye drops.
Accompanying drawing illustrates:
Fig. 1 is the TLC discriminating figure of methylnonanone;
Fig. 2 is the TLC discriminating figure (Tianjin aluminum foil plate) of methylnonanone;
Fig. 3 is the TLC discriminating figure (Qingdao Haiyang prefabricated board) of methylnonanone;
Fig. 4 is the TLC discriminating figure (Merk plate) of methylnonanone;
Fig. 5 is the TLC discriminating figure (hand bed board) of methylnonanone;
Fig. 6 is the TLC discriminating figure of methylnonanone, temperature 30 DEG C, humidity 55%;
Fig. 7 is the TLC discriminating figure of methylnonanone, temperature 18 DEG C, humidity 75%;
Fig. 8 is contrast characteristic spectrum, in figure, and peak 3:4-terpilenol; Peak 4: alpha-terpineol; Peak S: methylnonanone; Peak 5: Bronyl acetate;
Fig. 9 is methylnonanone canonical plotting.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, and protection scope of the present invention is not limited to the following stated:
Embodiment 1:
The method for making of cordate houttuynia eye drops is: get flavour amino acid 2000g and carry out steam distillation, collects just distillate 2000ml, then carries out redistillation, collect re-distilled liquid 1000ml, add equivalent water for injection, then carry out redistillation, collect rectifying liquid 900ml, add sodium chloride 7g, polyoxyethylene sorbitan monoleate 5g and ethyl hydroxy benzoate 0.3g, mixing, injects and makes into 1000ml with water, filters, embedding, to obtain final product.
Embodiment 2:
The method of quality control of the cordate houttuynia eye drops of above-described embodiment 1, it comprises the following steps:
S1. the TLC distinguish of methylnonanone
Get methylnonanone reference substance, add absolute ethyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast; According to thin-layered chromatography test, draw reference substance solution 10 μ l and need testing solution 20 μ l, put respectively on same silica gel g thin-layer plate, put into streak, take cyclohexane-ethyl acetate as developping agent, the volume ratio of cyclohexane-ethyl acetate cyclohexane and ethyl acetate is 9:1, launches, take out, dry, spray with dinitrophenylhydrazine test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
S2. the assay of methylnonanone
Chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute; Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak;
The preparation of reference substance solution: get methylnonanone reference substance appropriate, accurately weighed, add absolute ethyl alcohol and make the solution of every 1ml containing 25 μ g, to obtain final product;
The preparation of need testing solution: precision measures testing sample 25ml, by the C that pre-service is good
8solid phase extraction column, with the mixed solution wash-out of ethyl acetate-ethanol, collect eluent and be about 1.8ml, put in 2ml measuring bottle, the mixed solution adding ethyl acetate-ethanol is diluted to scale, and the volume ratio of ethyl acetate and ethanol is 7:3, shakes up, to obtain final product;
Determination method: accurate absorption reference substance solution and each 2 μ l of need testing solution, respectively inject gas chromatograph, measure, to obtain final product;
The every 1ml of this product in methylnonanone, must not be less than 4.0 μ g containing flavour amino acid;
S3. characteristic spectrum
A. the preparation of object of reference solution: get alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance in right amount, accurately weighed, add absolute ethyl alcohol respectively and make the solution of every 1ml containing 25 μ g, to obtain final product;
B. the preparation of need testing solution: precision measures this product 25ml, by the C that pre-service is good
8solid phase extraction column (100mg:1ml), with the mixed solution wash-out of ethyl acetate-ethanol (7:3), collects eluent and is about 1.8ml, put in 2ml measuring bottle, add above-mentioned mixed solution and be diluted to scale, shake up, to obtain final product.
C. measure: accurate absorption object of reference solution and each 2 μ l of need testing solution respectively, inject gas chromatograph, measures;
Chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute; Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak;
Should present 6 characteristic peaks in test sample characteristic spectrum, wherein 4 peaks should be consistent with alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance peak retention time respectively; The peak corresponding to methylnonanone reference substance is S peak, calculates the relative retention time at each characteristic peak and S peak, should setting ± 5% within; Setting is: 0.782 (peak 1), 0.820 (peak 2), 0.878 (peak 3), 0.909 (peak 4), 1.000 (peak S), 1.052 (peaks 5).
Embodiment 3: the TLC distinguish of methylnonanone
The need testing solution preparation method of primary standard extracts with methenyl choloride after acidifying, the wherein sampling amount of sample comparatively large (at every turn getting 100ml), Extraction solvent used (i.e. methenyl choloride) toxicity is large, now to be revised as under getting [assay] item solution as need testing solution, spot is obvious, easily inspect, and saved sample and solvent, method is easier to save time, environmental protection.In addition cyclohexane lower for the normal hexane toxicity in developping agent is replaced.
Under [assay] item, the preparation method of solution is: precision measures this product 25ml, by the C that pre-service is good
8solid phase extraction column (100mg:1ml), with the mixed solution wash-out of ethyl acetate-ethanol (7:3), collects eluent and is about 1.8ml, put in 2ml measuring bottle, add above-mentioned mixed solution and be diluted to scale, shake up, to obtain final product.
1. technique study and determining
Prepared by need testing solution: get the need testing solution under [assay] item, to obtain final product.
Prepared by reference substance solution: get methylnonanone reference substance (Nat'l Pharmaceutical & Biological Products Control Institute 110834-200502, hereinafter referred to as middle inspection institute), adds absolute ethyl alcohol and makes the solution of every 1ml containing 0.5mg, product solution in contrast.
Prepared by negative control solution: the negative sample getting scarce cordate houttuynia, by the preparation method of need testing solution, is made in the same way of negative control solution.
Draw above-mentioned reference substance solution 10 μ l and need testing solution 20 μ l, put respectively on same silica gel g thin-layer plate, put into streak, with 1. cyclohexane-ethyl acetate (9:1), 2. toluene-ethyl acetate (9:1), 3. cyclohexane-ethyl acetate (17:3) for developping agent, launch, take out, drying, spraying with dinitrophenylhydrazine test solution (facing with newly joining).Result: in test sample chromatogram, all can detect the spot of methylnonanone.During with cyclohexane-ethyl acetate (9:1) for developping agent, spot and R f value is moderate; During with dinitrophenylhydrazine test solution for developer, in sample, methylnonanone spot is obvious, and other spot is more weak, is difficult to inspect, therefore only the discriminating revenue standard text of methylnonanone.
With reference to the TLC distinguish of houttuynia cordata injection publicity standard, with sulfuric acid ethanol (1 → 10) solution of 5% vanillic aldehyde for developer, it is clear that hot blast blows to spot development.Result: in reference substance, the spot of terpinol, Bronyl acetate, terpilenol is obvious, and the spot of methylnonanone is weak compared with other spot under this developer; Have a spot to be difficult to inspect in sample, the interference of other spot is large, therefore this developer non-selected.See Fig. 1, adsorbent in figure: silica gel g thin-layer plate (Tianjin aluminum foil plate), developping agent: cyclohexane-ethyl acetate (9:1), developer: sulfuric acid ethanol (1 → 10) solution of 5% vanillic aldehyde, temperature: 26 DEG C, relative humidity: 60%; 1 is sample 1202118, and 2 is sample 1205102, and 3 is methylnonanone reference substance, and 4 is mixing reference substance (terpinol, Bronyl acetate, terpilenol and methylnonanone), and 5 is sample 1112101.
Final defining method is: get methylnonanone reference substance, adds absolute ethyl alcohol and makes the solution of every 1ml containing 0.5mg, product solution in contrast.Test according to thin-layered chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw the need testing solution 20 μ l under above-mentioned reference substance solution 10 μ l and [assay] item, put respectively on same silica gel g thin-layer plate, put into streak, with cyclohexane-ethyl acetate (9:1) for developping agent, launch, take out, drying, spraying with dinitrophenylhydrazine test solution (facing with newly joining).In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
2. serviceability test:
Adopt dissimilar silica G plate and different humiture, the method that the present invention finally determines is investigated.See Fig. 2 ~ Fig. 7, the temperature of Fig. 2, Fig. 3, Fig. 4 and Fig. 5 is 25 DEG C, relative humidity: 60%, and 1 is negative sample, and 2 is sample 1202118, and 3 is sample 1205102, and 4 is sample 1112101, and 5 is methylnonanone reference substance; Fig. 9 is Qingdao Haiyang prefabricated board, and in figure, 1 is sample 1202118, and 2 is sample 1205102, and 3 is sample 11121014, and 4 is methylnonanone reference substance, and 5 is negative sample.
Embodiment 4: characteristic spectrum
1. method is determined
First investigate the need testing solution used containing surveying under item and chromatographic column thereof, and measure with reference to the chromatographic condition under houttuynia cordata injection [finger-print] item.
Chromatographic condition and system suitability: HP-50+ capillary column (30m × 0.32mm, 0.5 μm); Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute.Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1.
The preparation of object of reference solution: get alpha-terpineol reference substance (middle inspection institute 111859-201102), 4-terpilenol reference substance (ACROS, content 97%), methylnonanone reference substance (middle inspection institute 110834-200502), Bronyl acetate reference substance (middle inspection institute 110759-200303) be appropriate, accurately weighed, add absolute ethyl alcohol respectively and make the solution of every 1ml containing 25 μ g, to obtain final product.
The preparation of need testing solution: method 1: get the need testing solution under [assay] item, to obtain final product; Method 2: with reference to need testing solution preparation method under houttuynia cordata injection publicity standard " finger-print " item.Precision measures this product 120ml, puts in round-bottomed flask, according to determination of volatile oil method (Chinese Pharmacopoeia version in 2010 annex XD), add normal hexane 1.5ml, be slowly heated to boil, and keep micro-and boil 40 minutes, let cool, divide and get normal hexane liquid, add the jolting of 1g anhydrous sodium sulfate, normal hexane liquid is transferred in 5ml measuring bottle, with a small amount of n-hexane anhydrous sodium sulfate, washing lotion is incorporated in measuring bottle, is diluted to scale with normal hexane, shake up, to obtain final product.
Determination method is accurate respectively draws object of reference solution and each 2 μ l of need testing solution, and according to the method test under [assay] item, inject gas chromatograph, measures, to obtain final product.
Result: in the chromatogram of two kinds of need testing solution preparation method gained samples, the number of chromatographic peak is identical, all can detect 6 total characteristic peaks, its relative retention time is all in specialized range.In table 1.
Table 1 two kinds of test sample preparation method comparative feature peak relative retention times
Consider the easy of operation and improve the efficiency checked, finally selecting: the need testing solution got under [assay] item carries out the mensuration of characteristic spectrum.
According to method for selecting, characteristic spectrum investigation is carried out to this product 14 batch sample, result all presents 6 total characteristic peaks, negative noiseless, wherein 4 peaks are consistent with alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance peak retention time respectively; Calculate the relative retention time at each characteristic peak and S peak, should setting ± 5% within.Obtain 14 batches of mean values to be respectively: 0.782 (peak 1), 0.820 (peak 2), 0.878 (peak 3), 0.909 (peak 4), 1.000 (peak S), 1.052 (peaks 5); Use chromatographic fingerprints of Chinese materia medica similarity evaluation software to synthesize 14 batch sample characteristic spectrums, obtain contrast characteristic spectrum.See Fig. 8 and table 2.
Table 214 batch sample characteristic peak relative retention time
Ultimate criterion specifies: should present 6 characteristic peaks in test sample characteristic spectrum, and wherein 4 peaks should be consistent with alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance peak retention time respectively; The peak corresponding to methylnonanone reference substance is S peak, calculates the relative retention time at each characteristic peak and S peak, should setting ± 5% within.Setting is: 0.782 (peak 1), 0.820 (peak 2), 0.878 (peak 3), 0.909 (peak 4), 1.000 (peak S), 1.052 (peaks 5).
2. repeatability is investigated
Precision measures this product (lot number 1202118) 25ml, totally 6 parts, prepares and measures, the results are shown in Table 3 by the method for drafting.
Table 3 sample characteristic peak relative retention time repeatability
3, need testing solution study on the stability
Get this product need testing solution, place different time respectively, measure by drafting method, record retention time, calculates relative retention time RSD, the results are shown in Table 4.
Table 4 sample characteristic peak relative retention time stability
4, durability is investigated
The investigation of 4.1 solid phase extraction column brands
Investigate the C of the Shanghai moon rising sun, Féraud door and Agilent three brands altogether
8solid phase extraction column, has all detected 6 characteristic peaks in results sample.In table 5
Table 5 different brands pillar relative retention time investigates result
4.2 capillary gas chromatographic columns are investigated
Use same chromatographic condition, investigate 1. HP-50+ (30m*0.32mm, 0.5 μm) (standard plan pillar), 2. DB-17 (30m*0.25mm, 0.25 μm) and 3. DB-1 (30m*0.32mm, 1.0 μm) three root chromatogram columns, result: 2. 1. post all can detect 6 characteristic peaks with post, and both retention times are variant, but relative retention time is in specialized range; Post is 3. under same column temperature, 6 characteristic peaks can not completely present, show that polarity and the internal diameter thickness thereof of chromatographic column are large on retention time impact, consider that this product characteristic peak will come qualitative by relative retention time, in order to make method, there is better reappearance and stability, therefore secure type and the specification of chromatographic column in text, namely should use HP-50+ capillary column (30m × 0.32mm, 0.5 μm).
Embodiment 5: assay
Method validation and durability are investigated.
1. instrument and reagent
Instrument: Agilent7890A gas chromatograph-FID
Chromatographic column: 1. HP-50+ (30m*0.32mm, 0.5 μm) (methodological study);
2. DB-17 (30m*0.25mm, 0.25 μm) (durability investigation);
Reagent: be analysis pure.
2. reference substance and sample
Methylnonanone reference substance (middle inspection institute 110834-200502 surveys for containing).
Cordate houttuynia eye drops (Sichuan Sunnyhope Pharmaceutical Co., Ltd., lot number 1202118)
3. assay method
Chromatographic condition and system suitability HP-50+ capillary column (30m × 0.32mm, 0.5 μm); Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute.Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1
Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak.
The preparation of reference substance solution gets methylnonanone reference substance in right amount, accurately weighed, adds absolute ethyl alcohol and makes the solution of every 1ml containing 25 μ g, to obtain final product.
The preparation precision of need testing solution measures this product 25ml, by the C that pre-service is good
8solid phase extraction column (100mg:1ml), with the mixed solution wash-out of ethyl acetate-ethanol (7:3), collects eluent and is about 1.8ml, put in 2ml measuring bottle, add above-mentioned mixed solution and be diluted to scale, shake up, to obtain final product.
Determination method precision draws reference substance solution and each 2 μ l of need testing solution, respectively inject gas chromatograph, measures, to obtain final product.
The every 1ml of this product must not be less than 4.0 μ g containing methylnonanone (C11H22O).
(note: C
8pillar uses the methyl alcohol activation of front 2ml, and rinse loading again well with 10ml water after activation, after upper complete sample, the water in pillar adds eluant, eluent after will extracting again.)
4. methodological study
The investigation of 4.1 linear relationships
Precision measures methylnonanone reference substance (0.09284mg/ml) 0.2 μ l, 1 μ l, 2 μ l and methylnonanone reference substance (0.4642mg/ml) 1 μ l, 2 μ l, 5 μ l, inject gas chromatograph, measure by above-mentioned chromatographic condition, the results are shown in Table 6.
Table 6 methylnonanone linear relationship investigates result
Take peak area as ordinate (Y), sample size (ng) is horizontal ordinate (X) drawing standard curve, sees Fig. 9.Result shows, methylnonanone is good in 18.57 ~ 2321ng scope internal linear relation.
4.2 specificities investigate the sample getting scarce cordate houttuynia, and prepare according to need testing solution preparation method and measure, result is noiseless at peak to be measured place.
4.3 replica test
Get this product (lot number 1202118) 25ml, totally 6 parts, prepare by the method for drafting and measure, the results are shown in Table 7.
Table 7 replica test result
Result shows, selected method favorable reproducibility.
4.3 application of sample recovery tests
Precision measures this product (methylnonanone content is 12.74 μ g/ml) 10ml, totally 6 parts, and precision adds methylnonanone reference substance solution (501.8 μ g/ml) 300 μ l respectively, mixing, all the other, according to drafting method preparation and measuring, calculate, the results are shown in Table 8.
Table 8 methylnonanone average recovery test findings
Result shows: selected method accuracy is good.
5 serviceability tests
5.1 sample introduction precision tests get methylnonanone reference substance solution (0.5018mg/ml) 1 μ l, and continuous sample introduction 5 times, tests by above-mentioned chromatographic condition, and record methylnonanone peak area, calculates, the results are shown in Table 9.
Table 9 Precision test result
Result shows: the sample introduction precision of methylnonanone is good.
Need testing solution 2 μ l and methylnonanone reference substance solution (0.5018mg/ml) 1 μ l is drawn in 5.2 stability tests respectively, the chromatographic condition drafted by text, places different time sample introduction respectively, and record peak area A, calculates RSD, the results are shown in Table 10.
Table 10 solution stability testing result
Result shows: reference substance and need testing solution good at 28 hours internal stabilities.
5.3C
8the solid phase extraction column of different brands is selected in the investigation of solid phase extraction column, to be prepared and to measure, the results are shown in Table 11 by the method drafted to this product.
Table 11 pillar investigates result
5.4 gas chromatographs and chromatographic column are investigated and are selected different chromatographic column, measure, the results are shown in Table 12 by the method drafted.
Table 12 chromatographic column investigates result
Result shows: method for selecting good tolerance.
6. sample determination
By method of the present invention, the sample that collected Various Seasonal is produced is measured, the results are shown in Table 13.
Table 13 sample determination result
| Lot number | 1202103 | 1202118 | 1112101S | 1112102S | 1112103S | 1112104S |
| Content μ g/ml | 11.5 | 12.7 | 6.1 | 6.4 | 5.9 | 7.2 |
| Lot number | 1112105S | 1204107 | 1205102 | 1112101 | 1112102 | 1112103 |
| Content μ g/ml | 6.8 | 9.8 | 9.1 | 9.5 | 9.3 | 9.6 |
| Lot number | 1112104 | 1112105 | ||||
| Content μ g/ml | 8.7 | 8.9 |
7. limit is formulated
Primary standard limit is: every 1ml is containing methylnonanone (C
11h
22o) 2.0 μ g must not be less than.Practical measurement result per sample, consider that otherness and the methylnonanone of Various Seasonal Herba Houttuyniae content slightly adsorb and content decline in the storage of this product, therefore tentative this product limit is: every 1ml contains cordate houttuynia with methylnonanone (C simultaneously
11h
22o) count, 4.0 μ g must not be less than.
Claims (4)
1. a method of quality control for cordate houttuynia eye drops, is characterized in that: it comprises the following steps:
S1. the TLC distinguish of methylnonanone
Get methylnonanone reference substance, add absolute ethyl alcohol and make the solution of every 1ml containing 0.5mg, product solution in contrast; Test according to thin-layered chromatography, draw reference substance solution 10 μ l and need testing solution 20 μ l, put on same silica gel g thin-layer plate respectively, put into streak, take cyclohexane-ethyl acetate as developping agent, launch, take out, dry, spray is with dinitrophenylhydrazine test solution, in test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color;
S2. the assay of methylnonanone
Chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute; Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak;
The preparation of reference substance solution: get methylnonanone reference substance appropriate, accurately weighed, add absolute ethyl alcohol and make the solution of every 1ml containing 25 μ g, to obtain final product;
The preparation of need testing solution: precision measures testing sample 25ml, by the C that pre-service is good
8solid phase extraction column, with the mixed solution wash-out of ethyl acetate-ethanol, collect eluent and be about 1.8ml, put in 2ml measuring bottle, the mixed solution adding ethyl acetate-ethanol is diluted to scale, shakes up, and to obtain final product;
Determination method: accurate absorption reference substance solution and each 2 μ l of need testing solution, respectively inject gas chromatograph, measure, to obtain final product;
The every 1ml of this product in methylnonanone, must not be less than 4.0 μ g containing flavour amino acid;
S3. characteristic spectrum
A. the preparation of object of reference solution: get alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance in right amount, accurately weighed, add absolute ethyl alcohol respectively and make the solution of every 1ml containing 25 μ g, to obtain final product;
B. the preparation of need testing solution: precision measures testing sample 25ml, by the C that pre-service is good
8solid phase extraction column, with the mixed solution wash-out of ethyl acetate-ethanol, collect eluent and be about 1.8ml, put in 2ml measuring bottle, the mixed solution adding ethyl acetate-ethanol is diluted to scale, shakes up, and to obtain final product;
C. measure: accurate absorption object of reference solution and each 2 μ l of need testing solution respectively, inject gas chromatograph, measures;
Should present 6 characteristic peaks in test sample characteristic spectrum, wherein 4 peaks should be consistent with alpha-terpineol reference substance, 4-terpilenol reference substance, methylnonanone reference substance, Bronyl acetate reference substance peak retention time respectively; The peak corresponding to methylnonanone reference substance is S peak, calculates the relative retention time at each characteristic peak and S peak, should setting ± 5% within; Setting is: 0.782(peak 1), 0.820(peak 2), 0.878(peak 3), 0.909(peak 4), 1.000(peak S), 1.052(peak 5).
2. the method for quality control of a kind of cordate houttuynia eye drops according to claim 1, is characterized in that: the volume ratio of described cyclohexane-ethyl acetate cyclohexane and ethyl acetate is 9:1.
3. the method for quality control of a kind of cordate houttuynia eye drops according to claim 1, is characterized in that: in the mixed solution of described ethyl acetate-ethanol, the volume ratio of ethyl acetate and ethanol is 7:3.
4. the method for quality control of a kind of cordate houttuynia eye drops according to claim 1, is characterized in that, the chromatographic condition of gas chromatograph described in step S3 is: chromatographic condition and system suitability: HP-50+ capillary column; Temperature programme: initial temperature is 70 DEG C, keeps 5 minutes; Rise to 140 DEG C with the speed of 5 DEG C per minute, keep 5 minutes, then rise to 250 DEG C with the speed of 20 DEG C per minute; Injector temperature: 230 DEG C; Detecting device: FID; Detector temperature: 280 DEG C; Carrier gas: N
2; Flow rate of carrier gas: 1.0ml/min; Split ratio: 5:1; Number of theoretical plate calculates should be not less than 8000 by methylnonanone peak.
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Cited By (2)
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| CN110470779A (en) * | 2019-09-27 | 2019-11-19 | 江西珍视明药业有限公司 | The method for building up and its finger-print of four taste treasure's layer ice boron eye drops finger-print |
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