CN105116086A - HPLC-ELSD content determination method for paeoniflorin and albiflori in red paeony roots - Google Patents
HPLC-ELSD content determination method for paeoniflorin and albiflori in red paeony roots Download PDFInfo
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- CN105116086A CN105116086A CN201510509089.9A CN201510509089A CN105116086A CN 105116086 A CN105116086 A CN 105116086A CN 201510509089 A CN201510509089 A CN 201510509089A CN 105116086 A CN105116086 A CN 105116086A
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Abstract
The invention discloses a HPLC-ELSD content determination method for paeoniflorin and albiflori in red paeony roots. The method includes the steps that 1, a test product solution is prepared; 2, comparison product solutions are prepared; 3, detection is performed, namely, the paeoniflorin comparison product solution and the albiflori comparison product solution in the step2 are taken to be detected under the detection conditions of C18 columns, isocratic elution is performed with an acetonitrile-0.05% formic acid aqueous solution as a mobile phase, HPLC-ELSD detection is performed through an ELSD detector, and curves are drawn to obtain standard curve equations; HPLC-ELSD detection is performed on the test product solution in the step1 under the conditions through the ELSD detector, obtained peak areas of the paeoniflorin and the albiflori are substituted into the standard curve linear regression equations, and then the contents of the paeoniflorin and the albiflori in the red paeony roots can be obtained. The method is good in specificity, durability, precision and accuracy and suitable for global quality control over the Chinese herb-red paeony roots.
Description
Technical field
The present invention relates to the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe, belong to biomedicine field.
Background technology
The radix paeoniae rubrathe is the dry root of cohosh Chinese herbaceous peony (PaeonialactifloraPall.) or the river radix paeoniae rubrathe (PaeoniaveitchiiLynch), has long medication history.Studied discovery in recent years, the radix paeoniae rubrathe has the pharmacological action widely such as anticoagulation, antithrombotic, antiatherosclerosis, cardioprotection and liver, protection cerebral ischemia induced brain injury, has broad application prospects.
Radix paeoniae rubrathe principle active component is the monoterpene glycosides compounds such as Paeoniflorin, albiflorin, Hydroxy peoniflorin, benzoylpaeoniflorin, and general name total paeony glycoside, wherein Paeoniflorin and albiflorin account for 70% of total glycosides.Paeoniflorin has the effect of hemangiectasis, antalgic and sedative, anti-inflammatory antiulcer, antipyretic spasmolysis, diuresis, and albiflorin has antalgic and sedative, anticonvulsant action, to immune system, to smooth muscle effect, anti-inflammatory, resisting pathogenic microbes, protects effect of liver.Clinical practice many employings compatibility of Chinese medicine, action mode is Mutiple Targets and is that Multiple components in Chinese medicine plays drug effect jointly.Most researchers adopts high performance liquid chromatography (DAD detecting device) to evaluate the quality of the radix paeoniae rubrathe, DAD detecting device requires that material has ultraviolet emission group, the ultraviolet emission group of the Paeoniflorin in the radix paeoniae rubrathe and this kind of methods of glycosides of albiflorin is near ultraviolet band, molar absorptivity is less, adopts DAD quantitatively to have certain restriction to these two kinds of materials.
Summary of the invention
Not enough for prior art, the object of the invention is to provide the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe, the method utilizes HPLC-ELSD method to measure the content of Paeoniflorin in the radix paeoniae rubrathe and albiflorin simultaneously, the total quality of Lactiflora is better controlled, and radix paeoniae rubrathe clinical application is laid the foundation.
The technical solution used in the present invention is:
In the radix paeoniae rubrathe, a HPLC-ELSD content assaying method for Paeoniflorin and albiflorin, comprises the steps:
1) preparation of need testing solution, pulverizes the root of the dry radix paeoniae rubrathe, sieves, take 1g radix paeoniae rubrathe powder, and soak 1h, heating and refluxing extraction 1h with 10mL70% methanol aqueous solution, cooling is weighed, and supplies weightlessness, gets solution and filters, get subsequent filtrate as need testing solution;
2) preparation of reference substance solution, precision takes Paeoniflorin and albiflorin, is dissolved to volumetric flask, constant volume, shakes up with 70% methanol-water, obtains the Paeoniflorin reference substance solution of 10mg/mL and the albiflorin reference substance solution of 3mg/mL respectively;
3) detect, get 2 respectively) in Paeoniflorin reference substance solution, albiflorin reference substance solution detect, testing conditions is C
18post, with acetonitrile-0.05% aqueous formic acid for mobile phase carries out isocratic elution, ELSD detecting device is adopted to carry out HPLC-ELSD detection, calculate the peak area under same retention time respectively, with paeoniflorin content x for horizontal ordinate, peak area y is ordinate drawing standard curve, obtains the typical curve equation of Paeoniflorin; With albiflorin content x for horizontal ordinate, peak area y is ordinate drawing standard curve, obtains the typical curve equation of albiflorin; Get 1) in need testing solution be C at high performance liquid chromatography testing conditions
18post, with acetonitrile-0.05% aqueous formic acid for mobile phase carries out isocratic elution, adopt ELSD detecting device to carry out HPLC-ELSD detection, the peak area of gained Paeoniflorin and albiflorin is brought in typical curve equation of linear regression, the content of Paeoniflorin and albiflorin in the radix paeoniae rubrathe can be drawn.
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) in acetonitrile and 0.05% aqueous formic acid all use 0.45 μm of filtering with microporous membrane, ultrasonic 30min in Ultrasound Instrument.
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) in the volume ratio of acetonitrile and 0.05% formic acid water be (10-20): (90-80).
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) in the volume ratio of acetonitrile and 0.05% formic acid water be 14:86.
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) middle C
18the specification of post is 250mm × 4.6mm × 5 μm, column temperature 30 DEG C.
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) middle C
18post, the flow velocity of acetonitrile and 0.05% formic acid water is 1mL/min, sample size 5 μ L.
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in described a kind of radix paeoniae rubrathe, described step 3) in ELSD drift tube temperature be 110 DEG C, carrier gas is nitrogen, and nebulizer gas pressure is 0.45MPa, and airshed is 3L/min.
The present invention has following beneficial effect:
Method of the present invention can be used for the assay of Paeoniflorin and albiflorin, and specificity, durability, precision, accuracy well, are applicable to the global quality control of Lactiflora.
ELSD is as a kind of common detector, its response does not rely on optical property, it produces response to nearly all material, adopts ELSD those the target component of uv absorption or end absorption can not had to set up science method of quality control accurately for Chinese patent drug, the prepared slices of Chinese crude drugs and granule.The invention provides and a kind ofly utilize HPLC-ELSD method simultaneously to the method for Paeoniflorin in the radix paeoniae rubrathe and albiflorin assay, can be better controlled the quality of Lactiflora, the content of HPLC-ELSD Simultaneously test Paeoniflorin and albiflorin is adopted in the present invention, relative to only quantitative to Paeoniflorin, the present invention can the quality standard of the control radix paeoniae rubrathe of science more comprehensively.
The present invention have selected volatile formic acid and mixes with water, select isocratic elution simply and easily, fast and effeciently Paeoniflorin and albiflorin can be separated, avoid and adopt difficult volatility acids (as phosphoric acid) to carry out gradient elution and cause the time long, mobile phase is wasted, and is not suitable for the problems such as ELSD detecting device.
Different chromatographic columns has different separating effects, present invention uses acetonitrile and 0.05% aqueous formic acid volume ratio is the proportioning of 14:86, adopts DiamonsilC
18post 250mm × 4.6mm × 5 μm, column temperature 30 DEG C, makes Paeoniflorin and albiflorin in the radix paeoniae rubrathe reach a good separating effect.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of Paeoniflorin standard items in the embodiment of the present invention.
Fig. 2 is the high-efficient liquid phase chromatogram of albiflorin standard items in the embodiment of the present invention.
Fig. 3 is the high-efficient liquid phase chromatogram of radix paeoniae rubrathe extract test sample in the embodiment of the present invention.
Fig. 4 is the linear relationship curve of Paeoniflorin standard items in the embodiment of the present invention.
Fig. 5 is the linear relationship curve of albiflorin standard items in the embodiment of the present invention.
Fig. 6 is that in the embodiment of the present invention, mobile phase acetonitrile-0.05% aqueous formic acid volume ratio is the high-efficient liquid phase chromatogram of 20:80.
Wherein 1-albiflorin, 2-Paeoniflorin.
Embodiment
The HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in embodiment 1, a kind of radix paeoniae rubrathe
Specifically comprise the steps:
One, the preparation of need testing solution
Be the root pulverizing of the dry radix paeoniae rubrathe of Xifeng, Liaoning by the place of production, cross 20 mesh sieves, take radix paeoniae rubrathe powder 1g, soak 1h, heating and refluxing extraction 1h with 10mL70% methanol aqueous solution, be cooled to room temperature and weigh, supply weightlessness, get solution and filter, get subsequent filtrate as need testing solution.By above-mentioned operation repetitive three parts.
Two, the preparation of reference substance solution
Precision takes Paeoniflorin and albiflorin, is dissolved to volumetric flask, constant volume, shakes up with 70% methanol-water, obtains the Paeoniflorin reference substance solution of 10mg/mL and the albiflorin reference substance solution of 3mg/mL respectively.
Three, detect
The testing conditions of efficient liquid phase and ELSD
High performance liquid chromatography: Agilent1200 high performance liquid chromatograph, DiamonsilC
18post 250mm × 4.6mm × 5 μm, column temperature 30 DEG C, mobile phase is acetonitrile-0.05% aqueous formic acid, and flow velocity is 1mL/min, sample size 5 μ L.Mobile phase used all uses 0.45 μm of filtering with microporous membrane, ultrasonic 30min in Ultrasound Instrument.
The screening of mobile phase
By methyl alcohol and formic acid proportioning, and acetonitrile and formic acid water proportioning are entered HPLC and are compared, and methanol system not as the baseline stability of acetonitrile system, therefore selects acetonitrile system.Select acetonitrile-0.05% aqueous formic acid volume ratio to be 20:80 respectively when other condition is the same, 14:86, 10:90 tri-kinds compares, when 20:80, albiflorin goes out peak too early as shown in Figure 6, though good with the degree of separation of Paeoniflorin chromatographic peak, undesirable with the separating effect of the chromatographic peak before it but (baseline is just separated just), and Paeoniflorin appearance time is longer when 10:90, as shown in Figure 3 when 14:86 appearance time relative to 10:90 the short and good separating effect at peak, therefore select the acetonitrile of volume ratio 14:86 and 0.05% aqueous formic acid as mobile phase.
ELSD testing conditions: ELSD drift tube temperature is 110 DEG C, carrier gas is nitrogen, and nebulizer gas pressure is 0.45MPa, and airshed is 3L/min, and ram cuts out.
The drafting of typical curve
From the Paeoniflorin prepared and albiflorin reference substance solution, drawing a certain amount of liquid respectively, to be configured to concentration be respectively 1, 1.5, 2, 3, 4, the Paeoniflorin solution of 5mg/mL and 0.05, 0.15, 0.3, 0.9, 1.2, the albiflorin solution of 1.5mg/mL, detect by above-mentioned HPLC and testing conditions respectively, high-efficient liquid phase chromatogram is as shown in Fig. 1 (Paeoniflorin) and 2 (albiflorins), calculate the peak area under same retention time respectively, with content x for horizontal ordinate, peak area y is ordinate drawing standard curve, Paeoniflorin typical curve equation is y=4274227.26x+2271908.47 as shown in Figure 4, R
2=0.9967, paeoniflorin content is good in 1-5mg/mL scope internal linear relation.Albiflorin typical curve equation is y=1837551.31x+124037.06, R as shown in Figure 5
2=0.9976, the content of albiflorin is good in 0.05-1.5mg/ml scope internal linear relation.
Detect the content of Paeoniflorin and albiflorin in the radix paeoniae rubrathe to be measured
Three parts of need testing solutions are detected according to above-mentioned HPLC and ELSD testing conditions, high-efficient liquid phase chromatogram as shown in Figure 3, the peak area of gained Paeoniflorin and albiflorin brings typical curve equation of linear regression into, show that the content of Paeoniflorin in the radix paeoniae rubrathe of Xifeng, three parts of Liaoning is respectively 1.0121mg/mL, 0.9900mg/mL, 1.0475mg/mL, relative standard deviation is 2.33%.The content of albiflorin is respectively 0.3351mg/mL, 0.3314mg/mL, 0.3303mg/mL, and relative standard deviation is 0.62%.
Four, methodological study
1, detectability and quantitative limit
Get each standard items working curve least concentration progressively to dilute, inserting needle detects, respectively using signal and noise ratio (S/N) for 3:1 and 10:1 is as quantitative limit and detectability, Paeoniflorin be quantitatively limited to 0.015mg/mL, detection is limited to 0.007mg/mL, albiflorin be quantitatively limited to 0.045mg/mL, detect be limited to 0.015mg/mL.
2, instrument precision experiment
Get a sample, enter 6 pins continuously, record the peak area of Paeoniflorin and albiflorin in spectrogram respectively, the RSD of Paeoniflorin peak area is 1.1%, the RSD of retention time is 0.4%, and the RSD of albiflorin peak area is 0.6%, and the RSD of retention time is 0.3%, RSD<3%, shows that instrument precision is good.
3, repeatability experiment
Get the radix paeoniae rubrathe 1g that the place of production is Xifeng, soak 1h, heating and refluxing extraction 1h with 10mL70% methanol solution, weightlessness is supplied in cooling, get solution and cross 0.45 μm of filter membrane, do 6 parts, inserting needle by above-mentioned parallel behaviour, record the peak area of Paeoniflorin and albiflorin in spectrogram respectively, the RSD of Paeoniflorin peak area is 0.3%, the RSD of albiflorin peak area is 2.6%, RSD<3%, shows that method has good reappearance.
4, stability test
Get a sample respectively 0,2,4,8,12,24,48h enters HPLC and analyzes, and records the peak area of Paeoniflorin and albiflorin in spectrogram respectively, the RSD of Paeoniflorin peak area is 2.6%, the RSD of albiflorin peak area is 2.7%, RSD<3%, and sample stability is good.
5, withinday precision and day to day precision
Get a sample respectively 0,1,2,3,4,5,6h and the 1st, 2,3,4d enters HPLC and analyzes, record the peak area of Paeoniflorin and albiflorin in spectrogram respectively, the RSD of Paeoniflorin peak area is 1.1%, and the RSD of albiflorin peak area is 0.6%, RSD<3%, shows that its withinday precision is good.Get same sample respectively the 1st, 2,3, within 4 days, enter HPLC to analyze, record the peak area of Paeoniflorin and albiflorin in spectrogram respectively, the RSD of Paeoniflorin peak area is 2.3%, and the RSD of albiflorin peak area is 1.4%, RSD<3%, shows that its day to day precision is good.
6, application of sample recovery experiment
Getting the place of production is that the radix paeoniae rubrathe 1g of Xifeng is placed in tool plug triangular flask, take 9 parts, be divided into 3 groups, add 80% respectively wherein, 100%, Paeoniflorin contained in 120% sample solution and the amount of lactone glycoside, soak 1h, heating and refluxing extraction 1h with 10mL70% methanol aqueous solution, be cooled to room temperature to weigh, supply weightlessness, get solution and filter, get subsequent filtrate as need testing solution.Measure its mean value of average recovery of high, medium and low three kinds of concentration as following table:
Table 1 Paeoniflorin and albiflorin average recovery experimental data table (n=3)
Claims (7)
1. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in the radix paeoniae rubrathe, is characterized in that, comprise the steps:
1) preparation of need testing solution, pulverizes the root of the dry radix paeoniae rubrathe, sieves, take 1g radix paeoniae rubrathe powder, and soak 1h, heating and refluxing extraction 1h with 10mL70% methanol aqueous solution, cooling is weighed, and supplies weightlessness, gets solution and filters, get subsequent filtrate as need testing solution;
2) preparation of reference substance solution, precision takes Paeoniflorin and albiflorin, is dissolved to volumetric flask, constant volume, shakes up with 70% methanol-water, obtains the Paeoniflorin reference substance solution of 10mg/mL and the albiflorin reference substance solution of 3mg/mL respectively;
3) detect, get 2 respectively) in Paeoniflorin reference substance solution, albiflorin reference substance solution detect, testing conditions is C
18post, with acetonitrile-0.05% aqueous formic acid for mobile phase carries out isocratic elution, ELSD detecting device is adopted to carry out HPLC-ELSD detection, calculate the peak area under same retention time respectively, with paeoniflorin content x for horizontal ordinate, peak area y is ordinate drawing standard curve, obtains the typical curve equation of Paeoniflorin; With albiflorin content x for horizontal ordinate, peak area y is ordinate drawing standard curve, obtains the typical curve equation of albiflorin; Get 1) in need testing solution be C at high performance liquid chromatography testing conditions
18post, with acetonitrile-0.05% aqueous formic acid for mobile phase carries out isocratic elution, adopt ELSD detecting device to carry out HPLC-ELSD detection, the peak area of gained Paeoniflorin and albiflorin is brought in typical curve equation of linear regression, the content of Paeoniflorin and albiflorin in the radix paeoniae rubrathe can be drawn.
2. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 1, it is characterized in that, described step 3) in acetonitrile and 0.05% aqueous formic acid all use 0.45 μm of filtering with microporous membrane, ultrasonic 30min in Ultrasound Instrument.
3. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 1, it is characterized in that, described step 3) in the volume ratio of acetonitrile and 0.05% formic acid water be (10-20): (90-80).
4. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 3, is characterized in that, described step 3) in the volume ratio of acetonitrile and 0.05% formic acid water be 14:86.
5. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 1, is characterized in that, described step 3) in C
18the specification of post is 250mm × 4.6mm × 5 μm, column temperature 30 DEG C.
6. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 1, is characterized in that, described step 3) in C
18post, the flow velocity of acetonitrile and 0.05% formic acid water is 1mL/min, sample size 5 μ L.
7. the HPLC-ELSD content assaying method of Paeoniflorin and albiflorin in a kind of radix paeoniae rubrathe as claimed in claim 1, it is characterized in that, described step 3) in ELSD drift tube temperature be 110 DEG C, carrier gas is nitrogen, nebulizer gas pressure is 0.45MPa, and airshed is 3L/min.
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Cited By (1)
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