CN105004830B - Measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously - Google Patents
Measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously Download PDFInfo
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Abstract
The invention discloses and a kind of measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously, do are described 5 kinds of saponin constituents Ophiopojaponin? C, deacetylate Ophiopojaponin? A, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D '. Above-mentioned 5 kinds of saponin constituents can effectively be detected and separate by the inventive method, accurately and reliably, easy quickly. Utilize the detection method of the present invention, both 5 ophiopogonin component contents quality of medicinal material standard Radix Ophiopogonis as index can have been set up out in Radix Ophiopogonis, can also be the quality standard setting up out genuine medicinal materials Radix Ophiopogonis of different sources, thus providing effective guarantee for overall monitor quality Radix Ophiopogonis.
Description
Technical field
The present invention relates to Control of drug quality method and technology field, be specifically related to measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously.
Background technology
Derive from the dried root of Liliaceae Ophiopogon plant Ophiopogonjaponicus (L.f) Ker-Gawl. Radix Ophiopogonis, be one of parts of generic medicinal plants, be mainly used in treatment dryness of the lung dry cough, deficiency of YIN consumptive disease is coughed, sore throat pharyngalgia, Tianjin wound is thirsty, and interior-heat such as is quenched one's thirst at the disease.The ground such as main product Sichuan, Zhejiang, Hubei, Shandong, Anhui, define the genuine kinds such as river Radix Ophiopogonis, RADIX OPHIOPOGONIS from Hangzhou of China.
In Radix Ophiopogonis, steroidal saponin constituents is its representative effective ingredient, and " Chinese Pharmacopoeia " version in 2010 adopts UV-spectrophotometry that the total saponin content of Radix Ophiopogonis has been measured under item one Radix Ophiopogonis, establishes quality standard with total saponin content. But, the total saponin content of official method is relatively big by ectocine, and its repeatability and precision are all relatively low. Meanwhile, what official method measured is total saponin content, saponin content concrete in Radix Ophiopogonis is not monitored, and this quality standard cannot embody the quality of medical material Radix Ophiopogonis comprehensively.
For solving the problems referred to above, have been reported and the specific saponin constituent of some in Radix Ophiopogonis is detected. Ophiopogonin D is that research is more at present, has the saponin constituent of multiple pharmacological effect. Therefore, Yu Jianping et al. is with it for index composition, have detected ophiopogonin D content in different sources, different growth years Radix Ophiopogonis, find that Zhejiang in the same place of production is produced ophiopogonin D content and had bigger difference (ELSD-HPLC method measures the technique study of ophiopogonin D content in Zhejiang Radix Ophiopogonis, river Radix Ophiopogonis, new Chinese medicine and clinical pharmacology, volume the 4th phase July the 13rd in 2002,253-255 page). Therefore, the height of ophiopogonin D content is not suitable as Zhejiang and produces the index of quality Radix Ophiopogonis, is not suitable for setting up Zhejiang according to it and produces the genuine medicinal materials quality standard of Radix Ophiopogonis.
Visible, if only setting up quality standard using a certain given activity component content in Radix Ophiopogonis as quality index, also existing when judging the quality of medical material Radix Ophiopogonis and judging risk greatly by accident, also cannot accurately know the place of production of medical material Radix Ophiopogonis.
Due to ophiopogonin D ' also there is certain pharmacologically active, Jia Cheng et al. reports and measures ophiopogonin D and ophiopogonin D in Radix Ophiopogonis medical material simultaneously ' method of content, find in river Radix Ophiopogonis of different batches, ophiopogonin D and ophiopogonin D ' stable content, (ELSD_HPLC method measures ophiopogonin D, D ' content in medical material Radix Ophiopogonis, R&D of modern TCM and practice, the 26th volume the 3rd phase in 2012,79-81 page). But, the Radix Ophiopogonis in other places of production is not detected by it, only using ophiopogonin D and ophiopogonin D ' content judge that Radix Ophiopogonis, quality yet suffered from bigger risk as quality index, not high using it as quality standard reliability.
Thus, taking into account the active component of Chinese crude drug is various, and the impact of extraneous many factors can be subject to, not high as the index reliability of quality standard using less active component content. It is presently required and selects in Radix Ophiopogonis the content of more active component as index, to set up the standard of quality of medicinal material a kind of Radix Ophiopogonis relatively reliable, reflection more comprehensively. Simultaneously as the genuineness of medical material is had special requirement by Chinese medicine, usage and the drug effect of different sources medical material there are differences, it is also desirable to set up the quality standard of each genuine medicinal materials, effectively distinguish with the Radix Ophiopogonis to different sources.
For foundation and the application thereof of above-mentioned quality standard, need badly and a kind of can accurately detect the method for the content of more active component in Radix Ophiopogonis. But, do not have the report of related detecting method at present.
Summary of the invention
Consider in prior art that the active component of detection is less, although and the detectable composition of finger printing is more, but it is set up and implementation condition is harsh, and the specificity of given activity composition is strong, thus can not the content of Accurate Determining active component.
Therefore, for solving the problems referred to above, the invention provides one and can measure various active composition in Radix Ophiopogonis, and the method simply easily implemented, it is a kind of to measure HPLC-ELSD (HPLC ELSD detector) method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously, described 5 kinds of saponin constituents are OphiopojaponinC (CAS 911819-08-4), deacetylate OphiopojaponinA (CAS 313054-32-9), 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ', comprise the following steps:
(1) foundation of saponin constituent standard curve:
A, reference substance solution preparation:
Take OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ' reference substance, mixing, add methanol and be configured to mixing reference substance solution;
B, reference substance solution mensuration:
The mixing reference substance solution of preparation series concentration, it is injected separately into high performance liquid chromatograph, gradient elution, measure each chromatographic peak peak area, obtain OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ' standard curve;
Chromatographic condition is as follows:
Evaporative light scattering detector;
Chromatographic column: octadecylsilane chemically bonded silica filler;
Mobile phase: with acetonitrile for mobile phase A, water or 0.1% phosphate aqueous solution are Mobile phase B;
Gradient elution: 0��45min, mobile phase A: 35 �� 55%, Mobile phase B: 65 �� 45%;
(2) assay of saponin constituent in testing sample:
C, need testing solution preparation:
Take Radix Ophiopogonis powder to be measured, add methanol supersound extraction, filter, filtering residue adds methanol supersound extraction, filters, and removes solvent, residue is dissolved in water, water-saturated n-butanol repeatedly extracts, united extraction liquid, and ammonia solution washs, remove ammoniacal liquor, n-butyl alcohol liquid is recycled to dry, and residue adds methanol and dissolves, and filters to obtain need testing solution;
D, need testing solution mensuration:
Take need testing solution; inject high performance liquid chromatograph; with identical for step b chromatographic condition detection, obtain OphiopojaponinC in testing sample, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D according to the standard curve of step (1) ' content.
It is further preferred that the drift tube temperature of described evaporative light scattering detector is 100 DEG C, gas flow rate is 3.0L/min.
Further preferably; in step a; in described reference substance solution, every 1mL solution is containing phiopojaponinC8.80 �� g, deacetylate OphiopojaponinA7.80 �� g, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis 16.80 �� g, ophiopogonin D 9.80 �� g and ophiopogonin D ' 10.60 �� g.
It is further preferred that in step c, the particle diameter of described Radix Ophiopogonis powder was No. four sieves. It is further preferred that in step c, the preparation method of described need testing solution is: took the Radix Ophiopogonis powder 3.0g of No. 4 sieves, accurately weighed, adding methanol 50mL, supersound extraction 45min, filter, filtering residue adds methanol 30mL, supersound extraction 30min, sucking filtration, merging filtrate, recycling design is to dry; The residue 10mL that adds water makes dissolving, and water-saturated n-butanol repeatedly extracts, united extraction liquid, and ammonia solution washs, and removes ammoniacal liquor, and n-butyl alcohol liquid is recycled to dry, and residue adds methanol and dissolves, and is settled to 5mL, filters to obtain need testing solution.
It is further preferred that the length of described chromatographic column is 250mm, internal diameter is 4.6mm, and the particle diameter of filler is 5 ��m. It is further preferred that described chromatographic column is Kromasil100-5C18 chromatographic column.
It is further preferred that the column temperature of described chromatographic condition is 35 DEG C.
It is further preferred that the volume flow of described chromatographic condition is 1.0mL/min.
It is further preferred that described Radix Ophiopogonis is river Radix Ophiopogonis.
OphiopojaponinC, deacetylate OphiopojaponinA and 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis are steroidal saponin, it may have certain physiologically active. Inventor selects their content and ophiopogonin D, ophiopogonin D ' content together with detect, using as the index judging quality Radix Ophiopogonis comprehensively.
Finally, the present invention, by screening to extracting method and chromatographic condition, has been successfully established and has measured the HPLC-ELSD method of 5 ophiopogonin component contents in Radix Ophiopogonis simultaneously, the method accurately and reliably, easy quickly. Utilize the detection method of the present invention, both 5 ophiopogonin component contents quality of medicinal material standard Radix Ophiopogonis as index can have been set up out in Radix Ophiopogonis, can also be the quality standard setting up out genuine medicinal materials Radix Ophiopogonis of different sources, thus providing effective guarantee for overall monitor quality Radix Ophiopogonis.
Obviously, the foregoing according to the present invention, according to ordinary technical knowledge and the customary means of this area, without departing under the above-mentioned basic fundamental thought premise of the present invention, it is also possible to make the amendment of other various ways, replacement or change.
The detailed description of the invention of form by the following examples, is described in further detail the foregoing of the present invention again. But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below. All technology realized based on foregoing of the present invention belong to the scope of the present invention.
Accompanying drawing explanation
In following accompanying drawing, 1.OphiopojaponinC; 2. deacetylate OphiopojaponinA; 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis; 4. ophiopogonin D; 5. ophiopogonin D '.
Fig. 1 is the HPLC-ELSD figure of mixing reference substance solution.
Fig. 2 is HPLC-ELSD figure (sample lot number: 201501) of need testing solution.
Fig. 3 is HPLC-ELSD figure during water extraction.
Fig. 4 is HPLC-ELSD figure during methanol extraction.
Fig. 5 is HPLC-ELSD figure during 80% methanol extraction.
Fig. 6 is HPLC-ELSD figure during dehydrated alcohol extraction.
Fig. 7 is HPLC-ELSD figure during 95% ethanol extraction.
Fig. 8 is HPLC-ELSD figure during 80% ethanol extraction.
Fig. 9 is mobile phase is HPLC-ELSD figure during acetonitrile-water.
Figure 10 is mobile phase is HPLC-ELSD figure during acetonitrile-0.1% phosphoric acid water.
Figure 11 is mobile phase is HPLC-ELSD figure during methanol-water.
Figure 12 is the HPLC-ELSD figure under gradient elution program of the present invention.
Figure 13-16 is the HPLC-ELSD figure under other gradient elution program.
Detailed description of the invention
Instrument and material:
Agilent1200 high performance liquid chromatograph (Agilent company of the U.S., including binary pump, automatic sampler, AlltechELSD-2000 evaporative light scattering detector), BP121S 100,000/electronic balance (Sartorius AG), KQ-500DB numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
Acetonitrile is chromatographically pure, and water is ultra-pure water, and all the other reagent are analytical pure; Ophiopogonin D (lot number: MUST-14052901), ophiopogonin D ' (lot number: MUST-14061901), OphiopojaponinC (lot number: MUST-14071203), 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis (lot number: MUST-14070210), deacetylate OphiopojaponinA (lot number: MUST-14072002) reference substance is provided by Man Site bio tech ltd, Chengdu, and mass fraction is all >=98%;Detection sample is that medical material Radix Ophiopogonis is collected in Santai area in 2015,10 totally batches, is accredited as the dried root of Ophiopogonjaponicus (L.f) Ker-Gawl. through Chengdu University of Traditional Chinese Medicine professor Li Min.
Embodiment 1 the inventive method
1 chromatographic condition
Chromatographic column is Kromasil100-5C18 post (4.6mm �� 250mm, 5 ��m); Mobile phase is acetonitrile (A)-0.1% phosphoric acid water (B) solution, gradient elution: 0��45min, 35%��55%A, volume flow 1.0mL/min; Column temperature 35 DEG C, drift tube temperature 100 DEG C, gas flow rate 3.0L/min; Sample size 15 �� L.
Prepared by 2 solution
Prepared by 2.1 mixing reference substance solution
Accurately weighed OphiopojaponinC, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, deacetylate OphiopojaponinA, ophiopogonin D, ophiopogonin D ', 5 kinds of reference substance 44.00,84.00,39.00,99.00,53.00 �� g; add methanol ultrasonic dissolution and be settled to 5mL, making the reference substance test solution of concentration respectively 8.80,16.80,7.80,19.80,10.60 �� g/mL. According to chromatographic condition sample introduction under " 1 " item, result is as shown in Figure 1.
The result of Fig. 1 proves, above-mentioned 5 kinds of saponin constituents can effectively be detected and separate by the inventive method.
Prepared by 2.2 sample solutions
Take Radix Ophiopogonis powder (crossing No. 4 sieves) 3.0g, accurately weighed, add methanol 50mL, supersound extraction 45min, filters, and filtering residue adds methanol 30mL, supersound extraction 30min, sucking filtration, merging filtrate, recycling design is to dry, and the residue 10mL that adds water makes dissolving, extracts 5 times with water-saturated n-butanol jolting, each 15mL, merges n-butyl alcohol liquid, washs 2 times with ammonia solution, each 5mL, discards ammoniacal liquor, and n-butyl alcohol liquid is recycled to dry. Residue methanol dissolves and to be settled to 5mL weighed, and 0.45 ��m of filter membrane filters, and obtains filtrate and need testing solution.
3 linear relationships are investigated
Precision measure mixing reference substance solution 0.1,0.2,0.5,1.0,2.0,5.0,10.0mL, put respectively in 10mL measuring bottle, add methanol constant volume to scale, shake up, respectively according to chromatographic condition sample introduction successively under " 1 " item, record chromatographic peak area. With peak area for vertical coordinate (Y), reference substance mass concentration is abscissa (X), and drawing standard curve carries out linear regression. Regression equation and the range of linearity are in Table 1.
Result investigated by table 1 linear relationship
It is shown that each reference substance is good in respective mass concentration scope internal linear relation, it was demonstrated that the inventive method range of linearity is wide, accuracy is high.
4 precision tests
Accurate absorption mixes reference substance solution; sample introduction 6 times are repeated respectively under " 1 " item chromatographic condition; sampling volume is 15 �� L; be calculated with reference substance peak area, ophiopogonin D, ophiopogonin D ', OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis peak area RSD respectively 0.23%, 0.34%, 0.30%, 0.21%, 0.19%.
Prove that the inventive method precision is high.
5 stability tests
Take Radix Ophiopogonis to be measured (batch 201501) need testing solution; by chromatographic condition operation under " 1 " item; respectively 0,2,4,6,8,12,24h sample introduction; measure ophiopogonin D, ophiopogonin D ', OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis peak area; RSD respectively 1.20%, 1.13%, 1.07%, 1.68%, 1.55% is obtained, it was shown that Radix Ophiopogonis, need testing solution was stable in 24h by calculated by peak area.
Prove that the inventive method stability is high.
6 replica tests
Take same batch of (lot number 201501) Radix Ophiopogonis powder 3.0g; parallel weighed 6 parts; by method operation under " 2.2 " item; preparation need testing solution Radix Ophiopogonis 6 parts; every part of accurate 15 �� L of absorption measure by chromatographic condition under " 1 " item, record ophiopogonin D, ophiopogonin D respectively ', OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis mass fraction RSD respectively 1.35%, 0.92%, 1.88%, 1.04%, 1.96%.
Proof the inventive method is reproducible.
7 average recovery tests
Take 6 parts of medical material Radix Ophiopogonis (lot number 201501) powder measured, every part of 1.5g, accurately weighed, every part adds same amount of ophiopogonin D, ophiopogonin D ', OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis reference substance, by method operation under " 2.2 " item, preparation need testing solution Radix Ophiopogonis, measure by condition under " 1 " item, result average recovery rate respectively 98.77%, 97.58%, 103.09%, 95.75%, 102.47%, RSD respectively 1.28%, 0.82%, 1.45%, 1.33%, 0.88%.
8 sample determinations
Be taken at difference base, each small towns, Santai County, Genuine producing area Radix Ophiopogonis, river collect 10 parts Radix Ophiopogonis medical material sample, weigh powder respectively and be about 3.0g, accurately weighed, by method operation under " 2.2 " item, preparation need testing solution Radix Ophiopogonis, each lot number 3 parts, it is measured by chromatographic condition under " 1 " item, and calculate the mass fraction of 5 kinds of saponin constituents in sample, using average as final tested volume, result is in Table 2.
Radix Ophiopogonis locality 2 batches, garden town, 2 batches, Luxi County town, 2 batches, keffel township, 1 batch, Xin De town, Liu Ying 1 crowd, hold 1 batch greatly, compete for first place 1 batch.
The chromatogram of lot number 201501 sample is as shown in Figure 2.
5 kinds of composition measurement results (n=3) in table 2 medical material Radix Ophiopogonis
Figure it is seen that 5 kinds of saponin constituents in Radix Ophiopogonis effectively detect and separate, and not by the interference of other compositions in Radix Ophiopogonis.
Result proves, the inventive method can effectively determine the content of 5 kinds of saponin constituents in different batches Radix Ophiopogonis. Meanwhile, in different batches Radix Ophiopogonis, above-mentioned 5 kinds of saponin contents are stable, illustrate the content selecting above Multiple components as Radix Ophiopogonis quality index be effective, it is possible to set up out the quality standard of genuine medicinal materials according to it.
The contrast of embodiment 2 present invention process parameter and other technological parameters
The contrast of the present embodiment is that the sample being 201501 with lot number carries out; During single factor test change, other conditions are all consistent with the method in embodiment 1.
1, Extraction solvent
Extraction solvent is as follows: water, methanol, 80% methanol, dehydrated alcohol, 95% ethanol and 80% ethanol, and result is respectively as shown in figures 3-8.
From the results, it was seen that during methanol extraction, separate good between each composition, therefore select methanol as the Extraction solvent of the present invention.
2, mobile phase
Mobile phase is as follows: acetonitrile-water, acetonitrile-0.1% phosphoric acid water and methanol-water, and result is respectively as shown in figs. 9-11.
From the results, it was seen that when acetonitrile-water and acetonitrile-0.1% phosphoric acid water are as mobile phase, it is possible to being efficiently separated by each composition, peak shape is better, therefore selects acetonitrile-water and acetonitrile-0.1% phosphoric acid water as the mobile phase of chromatographic condition of the present invention.
3, elution requirement
Multiple elution programs have been screened by the present invention, and screening scope, from mobile phase A (acetonitrile) 100%, tunes up the ratio of Mobile phase B (water) gradually, and finishing screen have selected suitable elution program, and result is as shown in figs. 12-16.Wherein, Figure 12 is the gradient elution program of the present invention.
It is shown that the gradient elution program of the present invention, obtain separating effect in the short period of time best, the collection of illustrative plates that peak shape is best.
In sum, the present invention has been successfully established and has measured the HPLC-ELSD method of 5 ophiopogonin component contents in Radix Ophiopogonis simultaneously, the method accurately and reliably, easy quickly. Utilize the detection method of the present invention, both 5 ophiopogonin component contents quality of medicinal material standard Radix Ophiopogonis as index can have been set up out in Radix Ophiopogonis, can also be the quality standard setting up out genuine medicinal materials Radix Ophiopogonis of different sources, thus providing effective guarantee for overall monitor quality Radix Ophiopogonis.
Claims (10)
1. measure the HPLC-ELSD method of 5 kinds of saponin constituents in Radix Ophiopogonis simultaneously; it is characterized in that: described 5 kinds of saponin constituents are OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ', comprise the following steps:
(1) foundation of saponin constituent standard curve:
A, reference substance solution preparation:
Take OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ' reference substance, mixing, add methanol and be configured to mixing reference substance solution;
B, reference substance solution mensuration:
The mixing reference substance solution of preparation series concentration, it is injected separately into high performance liquid chromatograph, gradient elution, measure each chromatographic peak peak area, obtain OphiopojaponinC, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D ' standard curve;
Chromatographic condition is as follows:
Detector: evaporative light scattering detector;
Chromatographic column: octadecylsilane chemically bonded silica filler;
Mobile phase: with acetonitrile for mobile phase A, water or 0.1% phosphate aqueous solution are Mobile phase B;
Gradient elution: 0��45min, mobile phase A: 35 �� 55%, Mobile phase B: 65 �� 45%;
(2) assay of saponin constituent in testing sample:
C, need testing solution preparation:
Take Radix Ophiopogonis powder to be measured, add methanol supersound extraction, filter, filtering residue adds methanol supersound extraction, filters, merging filtrate, removing solvent, residue is dissolved in water, and water-saturated n-butanol repeatedly extracts, united extraction liquid, ammonia solution washs, and removes ammoniacal liquor, and n-butyl alcohol liquid is recycled to dry, residue adds methanol and dissolves, and filters to obtain need testing solution;
D, need testing solution mensuration:
Take need testing solution; inject high performance liquid chromatograph; with identical for step b chromatographic condition detection, obtain OphiopojaponinC in testing sample, deacetylate OphiopojaponinA, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis, ophiopogonin D and ophiopogonin D according to the standard curve of step (1) ' content.
2. HPLC-ELSD method according to claim 1, it is characterised in that: the drift tube temperature of described evaporative light scattering detector is 100 DEG C, and gas flow rate is 3.0L/min.
3. HPLC-ELSD method according to claim 1; it is characterized in that: in step a; in described reference substance solution, every 1mL solution is containing OphiopojaponinC8.80 �� g, deacetylate OphiopojaponinA7.80 �� g, 3-O-��-L-rhamnose-(1 �� 2)-��-glucose aglycon Radix Ophiopogonis 16.80 �� g, ophiopogonin D 9.80 �� g and ophiopogonin D ' 10.60 �� g.
4. HPLC-ELSD method according to claim 1, it is characterised in that: in step c, the particle diameter of described Radix Ophiopogonis powder was No. four sieves.
5. HPLC-ELSD method according to claim 4, it is characterised in that: in step c, the preparation method of described need testing solution is:
Took the Radix Ophiopogonis powder 3.0g of No. 4 sieves, accurately weighed, add methanol 50mL, supersound extraction 45min, filter, filtering residue adds methanol 30mL, supersound extraction 30min, sucking filtration, merging filtrate, and recycling design is to dry;
The residue 10mL that adds water makes dissolving, and water-saturated n-butanol repeatedly extracts, united extraction liquid, and ammonia solution washs, and removes ammoniacal liquor, and n-butyl alcohol liquid is recycled to dry, and residue adds methanol and dissolves, and is settled to 5mL, filters to obtain need testing solution.
6. the HPLC-ELSD method according to any one of claim 1-5, it is characterised in that: the length of described chromatographic column is 250mm, and internal diameter is 4.6mm, and the particle diameter of filler is 5 ��m.
7. HPLC-ELSD method according to claim 6, it is characterised in that: described chromatographic column is Kromasil100-5C18 chromatographic column.
8. the HPLC-ELSD method according to any one of claim 1-5, it is characterised in that: the column temperature of described chromatographic condition is 35 DEG C.
9. the HPLC-ELSD method according to any one of claim 1-5, it is characterised in that: the volume flow of described chromatographic condition is 1.0mL/min.
10. the HPLC-ELSD method according to any one of claim 1-5, it is characterised in that: described Radix Ophiopogonis is river Radix Ophiopogonis.
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| CN113552266A (en) * | 2021-09-22 | 2021-10-26 | 山东省食品药品检验研究院 | Detection method of radix ophiopogonis raw material in Naolingsu preparation |
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