CN108627599A - A kind of Cortex Eucommiae is the content assaying method that raw material is prepared into drug containing Cortex Eucommiae - Google Patents
A kind of Cortex Eucommiae is the content assaying method that raw material is prepared into drug containing Cortex Eucommiae Download PDFInfo
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Abstract
It is the content assaying method that raw material is prepared into drug the present invention provides a kind of Cortex Eucommiae or containing Cortex Eucommiae, it is to refer to spectrogram spectrum detection using HPLC, and operating procedure is as follows：A, the preparation of test solution：Take measuring samples, water extraction be added, lets cool, then weighed weight, then weighed weight, the weight of less loss is supplied with water, is shaken up, filters, take subsequent filtrate to get；B, the preparation of reference solution：Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, be dissolved in water to get；C, accurate reference solution of drawing injects liquid chromatograph with test solution respectively, is eluted as mobile phase using acetonitrile, water.Content assaying method of the present invention is more simple and fast than official method, time saving and energy saving, cost-effective, adopts water as Extraction solvent, eliminates organic solvent extraction (removing methanol after such as methanol extraction) step, had not only saved the time but also cost-effective；And reference substance preparation solvent for use is consistent with test sample solvent.Detection method is suitable for Cortex Eucommiae standard decoction, Cortex Eucommiae granule, the inspection of stir-baked CORTEX EUCOMMIAE with salt solution standard decoction, stir-baked CORTEX EUCOMMIAE with salt solution granule content.It can be used as Cortex Eucommiae granule, stir-baked CORTEX EUCOMMIAE with salt solution granule quality standard.
Description
Technical field
It is the content assaying method that raw material is prepared into drug the present invention relates to a kind of Cortex Eucommiae or containing Cortex Eucommiae.
Background technology
Eucommia ulmoides are the dry bark of Eucommiaceae plant Cortex Eucommiae (EucommiaulmoidesOliv.), and tcm clinical practice is normal
Lumbago due to the kidney deficiency is treated with it, muscles and bones is powerless, blood leaking in gestation, fetal irritability, the diseases such as hypertension.The main chemical composition of Cortex Eucommiae has pine
Lipidol diglucoside (pinoresinoldiglucoside, PDG), chlorogenic acid, aucubin, gutta-percha etc., generally with
Pinoresinol diglucoside is leading indicator ingredient.Versions in 2000 and version Chinese Pharmacopoeia in 2005 are all made of Soxhlet extraction legal system
Standby sample, but the method is cumbersome time-consuming, and extraction time is up to 12 hours, cannot handle more batches of samples simultaneously；And in pharmacopeia color
Under spectral condition, chromatography peak type is bad, and separating degree is undesirable.
Jiang Xinyi, etc., in Cortex Eucommiae Pinoresinol diglucoside assay sample treatment optimization, research and hand over
Stream, the 1st phase page 30 of volume 33 in 2009；Use 60% methanol for Extraction solvent；Reference substance adds mobile phase to be formulated.
Invention content
It is the content assaying method that raw material is prepared into drug the present invention provides a kind of Cortex Eucommiae or containing Cortex Eucommiae.
Assay method of the present invention is to refer to spectrogram spectrum detection using HPLC, and operating procedure is as follows：
A, the preparation of test solution：
Measuring samples are taken, water extraction is added, lets cool, then weighed weight, then weighed weight, the weight of less loss is supplied with water,
Shake up, filter, take subsequent filtrate to get；
B, the preparation of reference solution：
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, be dissolved in water to get；
C, accurate reference solution of drawing injects liquid chromatograph with test solution respectively, using acetonitrile, water as mobile phase
It is eluted, chromatographic condition is：
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Acetonitrile-water (15: 85) is mobile phase；Detection wavelength is 227nm；Column temperature is 25-35 DEG C, flow velocity 0.8-1.2ml/min；It is theoretical
Plate number is calculated by Pinoresinol diglucoside peak should be not less than 3000.
Wherein, the drug includes Eucommia ulmoides, Cortex Eucommiae standard decoction, Cortex Eucommiae granule, stir-baked CORTEX EUCOMMIAE with salt solution standard decoction,
Stir-baked CORTEX EUCOMMIAE with salt solution granule.
Wherein, a steps extracting method is ultrasound or refluxing extraction.
Wherein, a steps extracting method is ultrasonic extraction, and extracting parameter is：Power 600W, frequency 40kHz；Extraction time
10-30min。
Wherein, the extraction time is 10min.
Wherein, it is 250mm that the octadecylsilane chemically bonded silica described in step c, which is the column length of filler, and internal diameter is
4.6mm, granularity are 5 μm；Column temperature is 30 DEG C；Flow velocity is 1.0ml/min.
Content assaying method of the present invention is more simple and fast than official method, time saving and energy saving, cost-effective, and it is molten to adopt water as extraction
Agent eliminates organic solvent extraction (removing methanol after such as methanol extraction) step, had not only saved the time but also cost-effective；And reference substance
It is consistent with test sample solvent to prepare solvent for use.Detection method is suitable for Cortex Eucommiae standard decoction, Cortex Eucommiae granule, salt
The inspection of Cortex Eucommiae standard decoction, stir-baked CORTEX EUCOMMIAE with salt solution granule containing survey.It can be used as Cortex Eucommiae granule, stir-baked CORTEX EUCOMMIAE with salt solution granule quality mark
It is accurate.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 Pinoresinol diglucoside uv absorption spectras
Fig. 2 column temperatures are investigated
Fig. 3 flow velocity investigation tables
Fig. 4 Cortex Eucommiae standard decoctions sample, Pinoresinol diglucoside reference substance
Fig. 5 Cortex Eucommiae granules sample, Pinoresinol diglucoside reference substance, blank solvent
Fig. 6 Pinoresinol diglucoside uv absorption spectras
Fig. 7 stir-baked CORTEX EUCOMMIAE with salt solution standard decoctions sample, Pinoresinol diglucoside reference substance
Fig. 8 stir-baked CORTEX EUCOMMIAE with salt solution granules sample, Pinoresinol diglucoside reference substance, blank
Specific implementation mode
1 Cortex Eucommiae standard decoction assay of the present invention of test example
Chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Acetonitrile-water (15: 85) is mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Number of theoretical plate presses Pinoresinol diglucoside
Peak, which calculates, should be not less than 3000.
It is prepared by reference substance solution
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, add water that solution of every 1ml containing 0.3mg is made, i.e.,
.
It is prepared by test solution
This product about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water 50ml, close plug is added in precision, and weighed weight surpasses
Sonication (power 600W, frequency 40kHz) 10 minutes, lets cool, then weighed weight, the weight of less loss is supplied with water, is shaken up, filter
Cross, take subsequent filtrate to get.
Measuring method
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
Chromatography condition is investigated with system suitability
Mobile phase selects
With acetonitrile-water (15: 85) be mobile phase when, investigate mobile phase fluctuation Pinoresinol diglucoside chromatographic peak is divided
From effect, it is shown in Table 1.
Pinoresinol diglucoside chromatographic peak performance indicator under the different mobile phases of table 1
The result shows that water does not influence the separation of Pinoresinol diglucoside and detection significantly in mobile phase, equal energy
Good separation and detection are carried out to it；Therefore determine that acetonitrile-water (15: 85) is the flowing of Cortex Eucommiae standard decoction content assaying method
Phase.
Detection wavelength determines
On the basis of the experiment condition drafted above, Pinoresinol diglucoside is carried out using diode array detector
All band scans, and sees Fig. 1.
The result shows that Pinoresinol diglucoside ultraviolet maximum absorption wavelength is near 227nm, therefore Cortex Eucommiae standard decoction contains
Measurement determines Detection wavelength and is determined as 227nm.
Column temperature is investigated
On the basis of the experiment condition drafted above, investigated when being respectively 25 DEG C, 30 DEG C, 35 DEG C to column temperature.With pine
Lipidol diglucoside separating degree, theoretical cam curve etc. are used as evaluation index.It is shown in Table 2, Fig. 2.
The analysis result of the different column temperatures of table 2
The result shows that when column temperature is 25 DEG C, 30 DEG C, 35 DEG C, number of theoretical plate, the separating effect of chromatographic peak conform to
It asks.Consider separating degree size and environment influence, therefore select 30 DEG C as column temperature.
Flow velocity is investigated
It is respectively 0.8ml/min, 1.0ml/min, 1.2ml/min to flow velocity on the basis of the experiment condition drafted above
Shi Jinhang is investigated, using Pinoresinol diglucoside separating degree, number of theoretical plate etc. as evaluation index.It is shown in Table 3, Fig. 3.
3 analysis result different in flow rate of table
The result shows that three kinds of flow velocitys can be detached and be detected to Pinoresinol diglucoside, analysis time is considered, most
Determine that flow velocity is 1.0ml/min eventually.
In conclusion finally determining that chromatographic condition is：Using octadecylsilane chemically bonded silica as filler, (column length is
250mm, internal diameter 4.6mm, granularity are 5 μm)；With acetonitrile-water (15: 85) for mobile phase；Detection wavelength is 227nm；Column temperature is
30℃；Number of theoretical plate is calculated by Pinoresinol diglucoside peak should be not less than 3000.
Test solution preparation method is investigated
Extraction solvent is investigated
6 parts of this product fine powder each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, is to test sample Extraction solvent respectively
It is investigated when methanol, 60% methanol, water 50ml, close plug, weighed weight, 15 points of supersound process (power 600W, frequency 40kHz)
Clock is let cool, then weighed weight, and the weight of less loss is supplied with Extraction solvent, is shaken up, filtration, take subsequent filtrate to get.Respectively take 10 μ l
Injecting chromatograph, analysis calculate Pinoresinol diglucoside content under different extracting modes, the results are shown in Table 4.
The analysis result of 4 different solvents of table
The result shows that the extraction efficiency highest of water, using water as its Extraction solvent.
Extracting mode is investigated
4 parts of this product each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water 50ml is added in precision, and close plug is weighed heavy
Amount is investigated when being respectively reflux, ultrasound to test sample extracting method, 15 minutes extraction times, is let cool, then weighed weight,
The weight that less loss is supplied with water, shakes up, filtration, take subsequent filtrate to get.Calculate separately different extracting mode rosin spirit glucosulfones
Glycosides content.It the results are shown in Table 5.
The different extracting mode analysis results of table 5
The result shows that Pinoresinol diglucoside content obtained by refluxing extraction is compared with ultrasonic extraction height, but without significant difference, with
Ultrasonic extraction simple to operation is extracting mode.
Extraction time is investigated
6 parts of this product (lot number DZBT180201) each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water is added in precision
50ml, close plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz), be respectively 10 minutes to test sample extraction time,
It is investigated, is let cool at 15 minutes, 30 minutes, then weighed weight, the weight of less loss is supplied with water, is shaken up, filtered, take continuous filter
Liquid to get.Analysis calculates Pinoresinol diglucoside content under different extraction times, the results are shown in Table 6.
The analysis result of the different extraction times of table 6
The result shows that when extraction time is 10 minutes, you can fully extraction.Therefore test sample extraction time is determined as 10 points
Clock.
In conclusion this product about 0.5g is taken, and it is accurately weighed, it sets in conical flask with cover, water 50ml is added in precision, and close plug claims
Determine weight, be ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, lets cool, then weighed weight, the weight of less loss is supplied with water,
Shake up, filter, take subsequent filtrate to get.
2 Cortex Eucommiae standard decoction content assaying method of embodiment
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler (column length 250mm,
Internal diameter is 4.6mm, and granularity is 5 μm)；With acetonitrile-water (15: 85) for mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Stream
Speed is 1ml per minute；Number of theoretical plate is calculated by Pinoresinol diglucoside peak should be not less than 3000.
The preparation of reference solution takes Pinoresinol diglucoside reference substance appropriate, accurately weighed, adds water that every 1ml is made
Solution containing 0.3mg to get.
Test solution preparation takes this product about 0.5g, accurately weighed, sets in conical flask with cover, and water 50ml is added in precision, close
Plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, let cool, then weighed weight, less loss is supplied with water
Weight shakes up, filtration, take subsequent filtrate to get.
Measuring method precision draw 10 μ l of test solution, inject liquid chromatograph, measure to get.
Methodological study
Specificity is tested
Test solution and reference substance solution (0.3mg/ml) are prepared by the method for drafting, is detected.As a result see Fig. 4.
Bright by chart, sample chromatogram figure is corresponding with reference substance chromatographic peak on corresponding position, shows this method specificity
It is good.
Precision is investigated
It takes reference substance solution (0.3mg/ml) continuous sample introduction 6 times, records the peak area of Pinoresinol diglucoside, calculate
RSD values, the results are shown in Table.
7 precision of table investigates result
The result shows that precision investigate in Pinoresinol diglucoside peak area RSD values be 1.88%, this method into
Sample precision is good
Linear relationship
Take various concentration Pinoresinol diglucoside reference substance solution, concentration be respectively 0.0174mg/mL,
0.0348mg/mL, 0.0696mg/mL, 0.1392mg/mL, 0.2783mg/mL, 0.5566mg/mL, sample size are 10 μ l.Injection
Liquid chromatograph, analysis obtain peak area, with Pinoresinol diglucoside reference substance concentration (X, mg/mL) for abscissa, peak face
Product (Y) is that ordinate draws response curve, the results are shown in Table 7.
7 Pinoresinol diglucoside standard curve analysis result of table
The result shows that：Pinoresinol diglucoside concentration range is in 0.0174~0.5566mg/mL, linear relationship y
=15491.5349x+17.1509, R2=0.9999, it is in good linear relationship.
Repeatability
6 parts of same test sample is taken, respectively accurately weighed 0.5g, confession is made according to the method drafted by same operating personnel
Test sample solution calculates the Pinoresinol diglucoside content of 6 parts of test samples, the results are shown in Table 8.
8 repeated experiment result of table
The result shows that the RSD values of Pinoresinol diglucoside content are 0.41%, this method repeatability is good.
Intermediate precision
(1) different instruments are investigated
On the basis of the experiment condition drafted above, precision weighs Cortex Eucommiae standard decoction freeze-dried powder (lot number：
DZBT180201) two parts, test solution is prepared, respectively in 1290 type high performance liquid chromatograph of Agilent, 1260 type of Agilent
Investigated that (chromatographic column is Phenomenex Luna on high performance liquid chromatograph, Shimadzu LC-AD type high performance liquid chromatographs
5μm C18(2)100A 250×4.60mm 5micron).Pinoresinol diglucoside content is calculated, acquired results are shown in Table 9.
The different instrument experiment results of table 9
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.71%, this method Intermediate precision
Well.
(2) different personnel and time are investigated
On the basis of the experiment condition drafted above, by different personnel (A, B), in different time (T1, T2), precision claims respectively
Each portion of Cortex Eucommiae standard decoction freeze-dried powder is taken, test sample is prepared, is measured.Calculate gained Pinoresinol diglucoside content knot
Fruit is shown in Table 10.
The different personnel of table 10 investigate result with the time
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.45%, this method Intermediate precision
Well.
Sample recovery rate
Test sample (content 2.29% of Pinoresinol diglucoside) about 0.25g of known content is taken, totally 6 parts, precision claims
It is fixed, it is accurate respectively that a certain amount of Pinoresinol diglucoside reference substance (purity is added：91.7%) it, is supplied by the method drafted
The preparation of test sample solution and measurement calculate the rate of recovery, the results are shown in Table.Calculation formula is as follows：
Table 11 is loaded recovery experiment result
The result shows that rate of recovery mean value is 99.63%, RSD values as a result are 3.47%, and this method accuracy is good.
Durability is investigated
(1) chromatographic column durability is investigated
It is respectively Agilent 5TC-C to chromatographic column on the basis of the experiment condition drafted above18(2)250×4.6mm、
PhenomenexLuna 5μm C18(2)100A 250×4.60mm 5micron、Diamonsil C18 5μm,4.6×250mm
Shi Jinhang is investigated.It is shown in Table 12.
12 durability of table investigates result table
The result shows that the analysis chromatography index of different chromatographic columns is good, the RSD values of 6 measurement results are 2.72%, we
Method chromatographic column good tolerance.
(2) stability experiment
Same test solution is taken, respectively 0,3,6,9,12, measures Pinoresinol diglucoside peak area for 24 hours, as a result
It is shown in Table 13.
13 Pinoresinol diglucoside stability experiment result of table
The result shows that under this experiment condition, the RSD values of Pinoresinol diglucoside content are 0.61%, and test sample is molten
Liquid is good in 24 hours internal stabilities.
Sample size measures verification
It using Cortex Eucommiae standard decoction freeze-dried powder as test sample, prepares test solution by the method drafted and measures, record peak
Area calculates the content of Pinoresinol diglucoside, the results are shown in Table 14.
14 batches of Cortex Eucommiae standard decoction Pinoresinol diglucoside contents of table
As seen from table, the content of Cortex Eucommiae standard decoction Pinoresinol diglucoside is in 1.27%~2.21%, average value
1.56%, SD 0.23
3 Cortex Eucommiae granule quality testing of the present invention of embodiment
Material, reagent and instrument
Laboratory apparatus and material
High performance liquid chromatograph：1290 type high performance liquid chromatograph of Agilent, 1260 type high performance liquid chromatograph of Agilent,
Shimadzu LC-AD type high performance liquid chromatographs；
Electronic balance：ME204E/02, MS205DU, XP26 (plum Teller-support benefit Instrument Ltd.)；
Ultrapure water machine：Cellular type 1810A (Shanghai Moller scientific instrument Co., Ltd)；
Ultrasonic cleaner：KQ5200DB types (600W, 40KHz；Kunshan Ultrasonic Instruments Co., Ltd.)；
Chromatographic column：Agilent 5TC-C18(2)250×4.6mm、PhenomenexLuna 5μm C18(2)100A 250
×4.60mm 5micron、Dismonsil C18 5μm,4.6×250mm。
Reagent and reagent
Acetonitrile, phosphoric acid are chromatographically pure, and water is ultra-pure water, remaining reagent is that analysis is pure.
Pinoresinol diglucoside (National Institute for Food and Drugs Control, lot number：111537-201706, content with
91.7% meter)
(prepared by Sichuan new green medicine company development in science and technology Co., Ltd, lot number for Cortex Eucommiae granule：SY1804001、
SY1804002、SY1804003)。
Chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Acetonitrile-water (15: 85) is mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Number of theoretical plate presses Pinoresinol diglucoside
Peak, which calculates, should be not less than 3000.
The preparation of solution
It is prepared by reference substance solution
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, add water that solution of every 1ml containing 0.3mg is made, i.e.,
.
It is prepared by test solution
This product under content uniformity item is taken, it is finely ground, about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water is added in precision
50ml, close plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, let cool, then weighed weight, mended with water
The weight of sufficient less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
Methodological study
Specificity is tested
Test solution and reference substance solution (0.3mg/ml) are prepared by the method for drafting, is detected.As a result see Fig. 5.
Bright by chart, sample chromatogram figure is corresponding with reference substance chromatographic peak on corresponding position, shows this method specificity
It is good.
Precision is investigated
It takes reference substance solution (0.3mg/ml) continuous sample introduction 6 times, records the peak area of Pinoresinol diglucoside, calculate
RSD values, the results are shown in Table 15.
15 precision of table investigates result
The result shows that precision investigate in Pinoresinol diglucoside peak area RSD values be 0.49%, this method into
Sample precision is good
Linear relationship
Take various concentration Pinoresinol diglucoside reference substance solution, concentration be respectively 0.0174mg/mL,
0.0348mg/mL, 0.0696mg/mL, 0.1392mg/mL, 0.2783mg/mL, 0.5566mg/mL, sample size are 10 μ l.Injection
Liquid chromatograph, analysis obtain peak area, with Pinoresinol diglucoside reference substance concentration (X, mg/mL) for abscissa, peak face
Product (Y) is that ordinate draws response curve, the results are shown in Table 16.
16 Pinoresinol diglucoside standard curve analysis result of table
The result shows that：Pinoresinol diglucoside concentration range is in 0.0174~0.5566mg/mL, linear relationship y
=15491.5349x+17.1509, R2=0.9999, it is in good linear relationship.
Repeatability
6 parts of same test sample is taken, respectively accurately weighed 0.5g, confession is made according to the method drafted by same operating personnel
Test sample solution calculates the Pinoresinol diglucoside content of 6 parts of test samples, the results are shown in Table 17.
17 repeated experiment result of table
The result shows that the RSD values of Pinoresinol diglucoside content are 1.49%, this method repeatability is good
Intermediate precision
(1) different instruments are investigated
On the basis of the experiment condition drafted above, precision weighs Cortex Eucommiae standard decoction freeze-dried powder (lot number:SY1804001)
Two parts, test solution is prepared, respectively in 1290 type high performance liquid chromatograph of Agilent, 1260 type high performance liquid chromatography of Agilent
Investigated that (chromatographic column is 5 μm of C of Phenomenex Luna on instrument, Shimadzu LC-AD type high performance liquid chromatographs18(2)
100A 250×4.60mm 5micron).Pinoresinol diglucoside content is calculated, acquired results are shown in Table 18.
The different instrument experiment results of table 18
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.67%, this method Intermediate precision
Well.
(2) different personnel and time are investigated
On the basis of the experiment condition drafted above, by different personnel (A, B), in different time (T1, T2), precision claims respectively
Each portion of Cortex Eucommiae standard decoction freeze-dried powder is taken, test sample is prepared, is measured.Calculate gained Pinoresinol diglucoside content knot
Fruit is shown in Table 19.
The different personnel of table 19 investigate result with the time
The result shows that the assay result RSD values of Pinoresinol diglucoside are 0.78%, this method Intermediate precision
Well.
Sample recovery rate
Test sample (content 0.71% of Pinoresinol diglucoside) about 0.25g of known content is taken, totally 6 parts, precision claims
It is fixed, it is accurate respectively that a certain amount of Pinoresinol diglucoside reference substance (purity is added：91.7%) it, is supplied by the method drafted
The preparation of test sample solution and measurement calculate the rate of recovery, the results are shown in Table 20.Calculation formula is as follows：
Table 20 is loaded recovery experiment result
The result shows that rate of recovery mean value is 94.96%, RSD values as a result are 1.25%, and this method accuracy is good.
Durability is investigated
(1) chromatographic column durability is investigated
It is respectively Agilent 5TC-C to chromatographic column on the basis of the experiment condition drafted above18(2)250×4.6mm、
PhenomenexLuna 5μm C18(2)100A 250×4.60mm 5micron、Diamonsil C18 5μm,4.6×250mm
Shi Jinhang is investigated.It is shown in Table 21.
21 durability of table investigates result table
The result shows that the analysis chromatography index of different chromatographic columns is good, the RSD values of 6 measurement results are 1.98%, we
Method chromatographic column good tolerance.
(2) stability experiment
Same test solution is taken, respectively 0,2,4,8,12, measures Pinoresinol diglucoside peak area for 24 hours, as a result
It is shown in Table 22.
22 Pinoresinol diglucoside stability experiment result of table
The result shows that under this experiment condition, the RSD values of Pinoresinol diglucoside content are 0.97%, and test sample is molten
Liquid is good in 24 hours internal stabilities.
Sample size measures verification
It using 3 batches of Cortex Eucommiae granules as test sample, prepares test solution by the method drafted and measures, record peak face
Product, calculates the content of Pinoresinol diglucoside, the results are shown in Table 23.
23 batches of Cortex Eucommiae granule assay results of table
Conclusion：The content assaying method of Cortex Eucommiae standard decoction can effectively detect Cortex Eucommiae granule.
4 stir-baked CORTEX EUCOMMIAE with salt solution standard decoction quality determining method of the present invention of test example
Instrument
High performance liquid chromatograph：1290 type high performance liquid chromatograph of Agilent, 1260 type high performance liquid chromatograph of Agilent,
Shimadzu LC-AD type high performance liquid chromatographs；
Electronic balance：ME204E/02, MS205DU, XP26 (plum Teller-support benefit Instrument Ltd.)；
Ultrapure water machine：Cellular type 1810A (Shanghai Moller scientific instrument Co., Ltd)；
Ultrasonic cleaner：KQ5200DB types (600W, 40KHz；Kunshan Ultrasonic Instruments Co., Ltd.)；
Chromatographic column：Agilent 5TC-C18(2)250×4.6mm、PhenomenexLuna 5μm C18(2)100A 250
×4.60mm 5micron、Dismonsil C18 5μm,4.6×250mm。。
Reagent and reagent
Pinoresinol diglucoside (National Institute for Food and Drugs Control, lot number：111537-201706, content with
91.7% meter).
Acetonitrile, phosphoric acid are chromatographically pure, and water is ultra-pure water, remaining reagent is that analysis is pure.
(prepared by Sichuan new green medicine company development in science and technology Co., Ltd, lot number for stir-baked CORTEX EUCOMMIAE with salt solution standard decoction freeze-dried powder：
YDZBT180322、YDZBT180323、YDZBT180324、YDZBT180325、YDZBT180326、YDZBT180327、
YDZBT180328、YDZBT180329、YDZBT180330、YDZBT180331、YDZBT180332、YDZBT180333、
YDZBT180334、YDZBT180335、YDZBT180201。)
Chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Acetonitrile-water (15: 85) is mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Number of theoretical plate presses Pinoresinol diglucoside
Peak, which calculates, should be not less than 1000.
It is prepared by reference substance solution
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, add water that solution of every 1ml containing 0.3mg is made, i.e.,
.
It is prepared by test solution
This product about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water 50ml, close plug is added in precision, and weighed weight surpasses
Sonication (power 600W, frequency 40kHz) 10 minutes, lets cool, then weighed weight, the weight of less loss is supplied with water, is shaken up, filter
Cross, take subsequent filtrate to get.
Measuring method
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
Chromatography condition is investigated with system suitability
Mobile phase selects
With acetonitrile-water (15: 85) be mobile phase when, investigate mobile phase fluctuation Pinoresinol diglucoside chromatographic peak is divided
From effect, it is shown in Table 24.
Pinoresinol diglucoside chromatographic peak performance indicator under the different mobile phases of table 24
The result shows that phosphoric acid water has an impact the separation and detection of Pinoresinol diglucoside in mobile phase, separating degree is not
If mobile phase is water；Therefore determine that acetonitrile-water (15: 85) is the mobile phase of Cortex Eucommiae standard decoction content assaying method.
Detection wavelength determines
On the basis of the experiment condition drafted above, Pinoresinol diglucoside is carried out using diode array detector
All band scans, and sees Fig. 6.
The result shows that Pinoresinol diglucoside ultraviolet maximum absorption wavelength is near 227nm, therefore Cortex Eucommiae standard decoction contains
Measurement determines Detection wavelength and is determined as 227nm.
Column temperature is investigated
On the basis of the experiment condition drafted above, investigated when being respectively 25 DEG C, 30 DEG C, 35 DEG C to column temperature.With pine
Lipidol diglucoside separating degree, theoretical cam curve etc. are used as evaluation index.It is shown in Table 25.
The analysis result of the different column temperatures of table 25
The result shows that when column temperature is 25 DEG C, 30 DEG C, 35 DEG C, number of theoretical plate, the separating effect of chromatographic peak conform to
It asks.Consider separating degree size and environment influence, therefore select 30 DEG C as column temperature.
Flow velocity is investigated
It is respectively 0.8ml/min, 1.0ml/min, 1.2ml/min to flow velocity on the basis of the experiment condition drafted above
Shi Jinhang is investigated, using Pinoresinol diglucoside separating degree, number of theoretical plate etc. as evaluation index.It is shown in Table 26.
26 analysis result different in flow rate of table
The result shows that three kinds of flow velocitys can be detached and be detected to Pinoresinol diglucoside, consider analysis time and
Experimental Habits, it is final to determine that flow velocity is 1.0ml/min.
In conclusion finally determining that chromatographic condition is：Using octadecylsilane chemically bonded silica as filler, (column length is
250mm, internal diameter 4.6mm, granularity are 5 μm)；With acetonitrile-water (15: 85) for mobile phase；Detection wavelength is 227nm；Column temperature is
30℃；Number of theoretical plate is calculated by Pinoresinol diglucoside peak should be not less than 3000.
Test solution preparation method is investigated
Extraction solvent is investigated
6 parts of this product fine powder each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, is to test sample Extraction solvent respectively
It is investigated when methanol, 60% methanol, water 50ml, close plug, weighed weight, 15 points of supersound process (power 600W, frequency 40kHz)
Clock is let cool, then weighed weight, and the weight of less loss is supplied with Extraction solvent, is shaken up, filtration, take subsequent filtrate to get.Respectively take 10 μ l
Injecting chromatograph, analysis calculate Pinoresinol diglucoside content under different extracting modes, the results are shown in Table 27.
The analysis result of 27 different solvents of table
The result shows that the extraction efficiency highest of water, using water as its Extraction solvent.
Extracting mode is investigated
4 parts of this product each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water 50ml is added in precision, and close plug is weighed heavy
Amount is investigated when being respectively reflux, ultrasound to test sample extracting method, 15 minutes extraction times, is let cool, then weighed weight,
The weight that less loss is supplied with water, shakes up, filtration, take subsequent filtrate to get.Calculate separately different extracting mode rosin spirit glucosulfones
Glycosides content.It the results are shown in Table 28.
The different extracting mode analysis results of table 28
The result shows that Pinoresinol diglucoside content obtained by refluxing extraction is compared with ultrasonic extraction height, but without significant difference, with
Ultrasonic extraction simple to operation is extracting mode.
Extraction time is investigated
6 parts of this product each about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water 50ml is added in precision, and close plug is weighed heavy
Amount is ultrasonically treated (power 600W, frequency 40kHz), when being respectively 10 minutes, 15 minutes, 30 minutes to test sample extraction time
Investigated, let cool, then weighed weight, the weight of less loss is supplied with water, is shaken up, filter, take subsequent filtrate to get.Analysis calculates
Pinoresinol diglucoside content, the results are shown in Table 29 under different extraction times.
The analysis result of the different extraction times of table 29
The result shows that when extraction time is 10 minutes, you can fully extraction.Therefore test sample extraction time is determined as 10 points
Clock.
In conclusion this product about 0.5g is taken, and it is accurately weighed, it sets in conical flask with cover, water 50ml is added in precision, and close plug claims
Determine weight, be ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, lets cool, then weighed weight, the weight of less loss is supplied with water,
Shake up, filter, take subsequent filtrate to get.
Stir-baked CORTEX EUCOMMIAE with salt solution standard decoction content assaying method
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler (column length 250mm,
Internal diameter is 4.6mm, and granularity is 5 μm)；With acetonitrile-water (15: 85) for mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Stream
Speed is 1ml per minute；Number of theoretical plate is calculated by Pinoresinol diglucoside peak should be not less than 3000.
The preparation of reference solution takes Pinoresinol diglucoside reference substance appropriate, accurately weighed, adds water that every 1ml is made
Solution containing 0.3mg to get.
Test solution preparation takes this product about 0.5g, accurately weighed, sets in conical flask with cover, and water 50ml is added in precision, close
Plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, let cool, then weighed weight, less loss is supplied with water
Weight shakes up, filtration, take subsequent filtrate to get.
Measuring method precision draw 10 μ l of test solution, inject liquid chromatograph, measure to get.
Methodological study
Specificity is tested
Test solution and reference substance solution (0.3mg/ml) are prepared by the method for drafting, is detected.As a result see Fig. 7.
Bright by chart, sample chromatogram figure is corresponding with reference substance chromatographic peak on corresponding position, shows this method specificity
It is good.
Precision is investigated
It takes reference substance solution (0.3mg/ml) continuous sample introduction 6 times, records the peak area of Pinoresinol diglucoside, calculate
RSD values, the results are shown in Table 30.
30 precision of table investigates result
The result shows that precision investigate in Pinoresinol diglucoside peak area RSD values be 1.55%, this method into
Sample precision is good
Linear relationship
Take various concentration Pinoresinol diglucoside reference substance solution, concentration be respectively 0.0174mg/mL,
0.0348mg/mL, 0.0696mg/mL, 0.1392mg/mL, 0.2783mg/mL, 0.5566mg/mL, sample size are 10 μ l.Injection
Liquid chromatograph, analysis obtain peak area, with Pinoresinol diglucoside reference substance concentration (X, mg/mL) for abscissa, peak face
Product (Y) is that ordinate draws response curve, the results are shown in Table 31.
31 Pinoresinol diglucoside standard curve analysis result of table
The result shows that：Pinoresinol diglucoside concentration range is in 0.0174~0.5566mg/mL, linear relationship y
=15491.5349x+17.1509, R2=0.9999, it is in good linear relationship.
Repeatability
6 parts of same test sample is taken, respectively accurately weighed 0.5g, confession is made according to the method drafted by same operating personnel
Test sample solution calculates the Pinoresinol diglucoside content of 6 parts of test samples, the results are shown in Table 32.
32 repeated experiment result of table
The result shows that the RSD values of Pinoresinol diglucoside content are 1.52%, this method repeatability is good
Intermediate precision
(1) different instruments are investigated
On the basis of the experiment condition drafted above, precision weighs Cortex Eucommiae standard decoction freeze-dried powder (lot number：
DZBT180201) two parts, test solution is prepared, respectively in 1290 type high performance liquid chromatograph of Agilent, 1260 type of Agilent
Investigated that (chromatographic column is Phenomenex Luna on high performance liquid chromatograph, Shimadzu LC-AD type high performance liquid chromatographs
5μm C18(2)100A 250×4.60mm 5micron).Pinoresinol diglucoside content is calculated, acquired results are shown in Table 33.
The different instrument experiment results of table 33
The result shows that the assay result RSD values of Pinoresinol diglucoside are 2.00%, this method Intermediate precision
Well.
(2) different personnel and time are investigated
On the basis of the experiment condition drafted above, by different personnel (A, B), in different time (T1, T2), precision claims respectively
Each portion of Cortex Eucommiae standard decoction freeze-dried powder is taken, test sample is prepared, is measured.Calculate gained Pinoresinol diglucoside content knot
Fruit is shown in Table 34.
The different personnel of table 34 investigate result with the time
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.29%, this method Intermediate precision
Well.
Sample recovery rate
Test sample (content 1.22% of Pinoresinol diglucoside) about 0.5g of known content is taken, totally 6 parts, precision claims
It is fixed, it is accurate respectively that a certain amount of Pinoresinol diglucoside reference substance (purity is added：91.7%) it, is supplied by the method drafted
The preparation of test sample solution and measurement calculate the rate of recovery, the results are shown in Table 35.Calculation formula is as follows：
Table 35 is loaded recovery experiment result
The result shows that rate of recovery mean value is 96.07%, RSD values as a result are 3.41%, and this method accuracy is good.
Durability is investigated
(1) chromatographic column durability is investigated
It is respectively 5 TC-C of Agilent to chromatographic column on the basis of the experiment condition drafted above18(2)250×
4.6mm、PhenomenexLuna 5μm C18(2)100A 250×4.60mm 5micron、Dismonsil C18 5μm,4.6
It is investigated when × 250mm.It is shown in Table 36.
36 durability of table investigates result table
The result shows that the analysis chromatography index of different chromatographic columns is good, the RSD values of 6 measurement results are 2.60%, we
Method chromatographic column good tolerance.
(2) stability experiment
Same test solution is taken, respectively 0,2,4,8,12, measures Pinoresinol diglucoside peak area for 24 hours, as a result
It is shown in Table 37.
37 Pinoresinol diglucoside stability experiment result of table
The result shows that under this experiment condition, the RSD values of Pinoresinol diglucoside content are 1.02%, and test sample is molten
Liquid is good in 24 hours internal stabilities.
Sample size measures verification
It using stir-baked CORTEX EUCOMMIAE with salt solution standard decoction freeze-dried powder as test sample, prepares test solution by the method drafted and measures, record
Peak area calculates the content of Pinoresinol diglucoside, the results are shown in Table 38.
38 batches of stir-baked CORTEX EUCOMMIAE with salt solution standard decoction Pinoresinol diglucoside contents of table
By chart it is found that the content average value of Pinoresinol diglucoside is 1.38%, SD 0.24.
1 stir-baked CORTEX EUCOMMIAE with salt solution granule quality determining method of the present invention of test example
Laboratory apparatus and material
High performance liquid chromatograph：1290 type high performance liquid chromatograph of Agilent, 1260 type high performance liquid chromatograph of Agilent,
Shimadzu LC-AD type high performance liquid chromatographs；
Electronic balance：ME204E/02, MS205DU, XP26 (plum Teller-support benefit Instrument Ltd.)；
Ultrapure water machine：Cellular type 1810A (Shanghai Moller scientific instrument Co., Ltd)；
Ultrasonic cleaner：KQ5200DB types (600W, 40KHz；Kunshan Ultrasonic Instruments Co., Ltd.)；
Chromatographic column：Agilent 5TC-C18(2)250×4.6mm、PhenomenexLuna 5μm C18(2)100A 250
×4.60mm 5micron、Dismonsil C18 5μm,4.6×250mm。
Reagent and reagent
Acetonitrile, phosphoric acid are chromatographically pure, and water is ultra-pure water, remaining reagent is that analysis is pure.
Pinoresinol diglucoside (National Institute for Food and Drugs Control, lot number：111537-201706, content with
91.7% meter)
(prepared by Sichuan new green medicine company development in science and technology Co., Ltd, lot number for stir-baked CORTEX EUCOMMIAE with salt solution granule：SY1804004、
SY1804005、SY1804006)。
Chromatographic condition and system suitability
Using octadecylsilane chemically bonded silica as filler (column length 250mm, internal diameter 4.6mm, granularity are 5 μm)；With
Acetonitrile-water (15: 85) is mobile phase；Detection wavelength is 227nm；Column temperature is 30 DEG C；Number of theoretical plate presses Pinoresinol diglucoside
Peak, which calculates, should be not less than 3000.
The preparation of solution
It is prepared by reference substance solution
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, add water that solution of every 1ml containing 0.3mg is made, i.e.,
.
It is prepared by test solution
This product under content uniformity item is taken, it is finely ground, about 0.5g is taken, it is accurately weighed, it sets in conical flask with cover, water is added in precision
50ml, close plug, weighed weight are ultrasonically treated (power 600W, frequency 40kHz) 10 minutes, let cool, then weighed weight, mended with water
The weight of sufficient less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
Methodological study
Specificity is tested
Test solution and reference substance solution (0.3mg/ml) are prepared by the method for drafting, is detected.As a result see Fig. 8.
Bright by chart, sample chromatogram figure is corresponding with reference substance chromatographic peak on corresponding position, shows this method specificity
It is good.
Precision is investigated
It takes reference substance solution (0.3mg/ml) continuous sample introduction 6 times, records the peak area of Pinoresinol diglucoside, calculate
RSD values, the results are shown in Table 39.
39 precision of table investigates result
The result shows that precision investigate in Pinoresinol diglucoside peak area RSD values be 0.49%, this method into
Sample precision is good
Linear relationship
Take various concentration Pinoresinol diglucoside reference substance solution, concentration be respectively 0.0174mg/mL,
0.0348mg/mL, 0.0696mg/mL, 0.1392mg/mL, 0.2783mg/mL, 0.5566mg/mL, sample size are 10 μ l.Injection
Liquid chromatograph, analysis obtain peak area, with Pinoresinol diglucoside reference substance concentration (X, mg/mL) for abscissa, peak face
Product (Y) is that ordinate draws response curve, the results are shown in Table 40.
40 Pinoresinol diglucoside standard curve analysis result of table
The result shows that：Pinoresinol diglucoside concentration range is in 0.0174~0.5566mg/mL, linear relationship y
=15491.5349x+17.1509, R2=0.9999, it is in good linear relationship.
Repeatability
Take same test sample (lot number：Lot number SY1804004) 6 parts, respectively accurately weighed 0.5g, by same operating personnel
Test solution is made according to the method drafted, calculates the Pinoresinol diglucoside content of 6 parts of test samples, the results are shown in Table 41.
41 repeated experiment result of table
The result shows that the RSD values of Pinoresinol diglucoside content are 0.78%, this method repeatability is good
Intermediate precision
(1) different instruments are investigated
On the basis of the experiment condition drafted above, precision weighs stir-baked CORTEX EUCOMMIAE with salt solution granule (lot number:SY1804004) two
Part, test solution is prepared, respectively in 1290 type high performance liquid chromatograph of Agilent, 1260 type high performance liquid chromatography of Agilent
Investigated that (chromatographic column is 5 μm of C of Phenomenex Luna on instrument, Shimadzu LC-AD type high performance liquid chromatographs18(2)
100A 250×4.60mm 5micron).Pinoresinol diglucoside content is calculated, acquired results are shown in Table 42.
The different instrument experiment results of table 42
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.68%, this method Intermediate precision
Well.
(2) different personnel and time are investigated
On the basis of the experiment condition drafted above, by different personnel (A, B), in different time (T1, T2), precision claims respectively
Each portion of stir-baked CORTEX EUCOMMIAE with salt solution granule is taken, test sample is prepared, is measured.Gained Pinoresinol diglucoside content results are calculated,
It is shown in Table 43.
The different personnel of table 43 investigate result with the time
The result shows that the assay result RSD values of Pinoresinol diglucoside are 1.57%, this method Intermediate precision
Well.
Sample recovery rate
Test sample (content 0.71% of Pinoresinol diglucoside) about 0.25g of known content is taken, totally 6 parts, precision claims
It is fixed, it is accurate respectively that a certain amount of Pinoresinol diglucoside reference substance (purity is added：91.7%) it, is supplied by the method drafted
The preparation of test sample solution and measurement calculate the rate of recovery, the results are shown in Table.Calculation formula is as follows：
Table 44 is loaded recovery experiment result
The result shows that rate of recovery mean value is 94.96%, RSD values as a result are 1.58%, and this method accuracy is good.
Durability is investigated
(1) chromatographic column durability is investigated
It is respectively 5 TC-C of Agilent to chromatographic column on the basis of the experiment condition drafted above18(2)250×
4.6mm、PhenomenexLuna 5μm C18(2)100A 250×4.60mm 5micron、Diamonsil C18 5μm,4.6
It is investigated when × 250mm.It is shown in Table 45.
45 durability of table investigates result table
The result shows that the analysis chromatography index of different chromatographic columns is good, the RSD values of 6 measurement results are 2.33%, we
Method chromatographic column good tolerance.
(2) stability experiment
Same test solution is taken, respectively 0,2,4,8,12, measures Pinoresinol diglucoside peak area for 24 hours, as a result
It is shown in Table 46.
46 Pinoresinol diglucoside stability experiment result of table
The result shows that under this experiment condition, the RSD values of Pinoresinol diglucoside content are 1.81%, and test sample is molten
Liquid is good in 24 hours internal stabilities.
Sample size measures verification
It using 3 batches of stir-baked CORTEX EUCOMMIAE with salt solution granules as test sample, prepares test solution by the method drafted and measures, record peak face
Product, calculates the content of Pinoresinol diglucoside, the results are shown in Table 47.
47 batches of stir-baked CORTEX EUCOMMIAE with salt solution granule assay results of table
Conclusion：The content assaying method of Cortex Eucommiae standard decoction can effectively detect Cortex Eucommiae granule.
Claims (6)
1. a kind of Cortex Eucommiae is the content assaying method that raw material is prepared into drug containing Cortex Eucommiae, it is characterised in that：It is to use HPLC
Refer to spectrogram spectrum detection, operating procedure is as follows：
A, the preparation of test solution：
Measuring samples are taken, water extraction is added, lets cool, then weighed weight, then weighed weight, the weight of less loss is supplied with water, is shaken up,
Filtration, take subsequent filtrate to get；
B, the preparation of reference solution：
Take Pinoresinol diglucoside reference substance appropriate, it is accurately weighed, be dissolved in water to get；
C, accurate reference solution of drawing injects liquid chromatograph with test solution respectively, is carried out as mobile phase using acetonitrile, water
Elution, chromatographic condition are：
Using octadecylsilane chemically bonded silica as filler；With acetonitrile-water (15: 85) for mobile phase；Detection wavelength is 227nm；
Column temperature is 25-35 DEG C, flow velocity 0.8-1.2ml/min；Number of theoretical plate should be not less than by the calculating of Pinoresinol diglucoside peak
3000。
2. assay method according to claim 1, it is characterised in that：The drug includes Eucommia ulmoides, Cortex Eucommiae standard
Decoction, Cortex Eucommiae granule, stir-baked CORTEX EUCOMMIAE with salt solution standard decoction, stir-baked CORTEX EUCOMMIAE with salt solution granule.
3. assay method according to claim 1, it is characterised in that：A step extracting methods are ultrasound or refluxing extraction.
4. assay method according to claim 3, it is characterised in that：A step extracting methods are ultrasonic extraction, extracting parameter
For：Power 600W, frequency 40kHz；Extraction time 10-30min.
5. assay method according to claim 4, it is characterised in that：The extraction time is 10min.
6. assay method according to claim 1, it is characterised in that：Octadecylsilane chemically bonded silica described in step c
Column length for filler is 250mm, and internal diameter 4.6mm, granularity is 5 μm；Column temperature is 30 DEG C；Flow velocity is 1.0ml/min.
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| CN109596763A (en) * | 2018-12-21 | 2019-04-09 | 广东方制药有限公司 | The construction method and discrimination method of the characteristic spectrum of a kind of Cortex Eucommiae and stir-baked CORTEX EUCOMMIAE with salt solution |
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Application publication date: 20181009 |
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