CN105203673A - High performance liquid chromatography detection method of compound nightshade inflammation-diminishing tablets - Google Patents
High performance liquid chromatography detection method of compound nightshade inflammation-diminishing tablets Download PDFInfo
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Abstract
The invention discloses a high performance liquid chromatography detection method of compound nightshade inflammation-diminishing tablets. The compound nightshade inflammation-diminishing tablets are mainly prepared from nightshades, hydrocotyle sibthorpioides and pithecellobium clypearia; the detection method is used for mainly detecting the content of gallic acid, quercetin, kaempferol, solasonine and solamargine in the compound nightshade inflammation-diminishing tablets. Phenolic acids substances including the gallic acid, the quercetin, the kaempferol and the like, and alkaloids substances including the solasonine and the solamargine in the inflammation-diminishing tablets can be detected by steps; the detection method has high detection accuracy and rapid detection speed, and can be used for determining the content of the inflammation-diminishing tablets; the method guarantees the accuracy and advancement of quality detection standards of the inflammation-diminishing tablets are guaranteed, and can be used as important indexes of quality control of the inflammation-diminishing tablets.
Description
Technical field
The present invention relates to drug tests.More particularly, the present invention relates to a kind of high-efficiency liquid chromatography method for detecting of Compound Solanum Nigrum sulfathiazole.
Background technology
Herbal mixture is the principal mode of tcm clinical practice medication, is traditional Chinese medicine and pharmacy characteristic and marrow.Chinese medicine be one by multicomponent, the multifactor complex system formed, its chemical composition diversity and complicacy are its curative effect material bases, set up the modern mass hierarchy of control meeting traditional Chinese medicine feature, capture quality analysis of traditional Chinese medicine and evaluate a difficult problem, improving the existing method of quality control of Chinese medicine has become people's active research problem.
Setting up must based on characteristics of Traditional Chinese Medicine to the quality control system of Chinese medicine.Herbal mixture mass action feature determines Chinese medicine and is different from Western medicine.Traditional Chinese medicine quality control method is tackled multiple effective constituent and is controlled.Only in this way, the quality control system set up really could reach and control traditional Chinese medicine quality, guarantee Chinese medicine object safe and effective for medication.It is means that traditional Chinese medicine quality controls to turn to from simple single component content mensuration with advanced technology, multicomponent, multi objective assay.
Compound Solanum Nigrum sulfathiazole is made primarily of black nightshade, pennywort, pithecellobium clypearia, its medicinal active ingredient has gallic acid, Quercetin, Kaempferide, solasonine, solamargine etc., there is clearing heat and detoxicating, cough-relieving apophlegmatic, dispersing swelling and dissipating binds analgesic efficacy, strengthen antibacterial, antiviral, antiphlogistic effects, improve immunity, diuresis antidiarrheal, cures mainly infection and the inflammation such as body surface, respiratory system, digestive system, urinary system.
Compound Solanum Nigrum sulfathiazole, as herbal mixture, only angularly carries out quality control to Compound Solanum Nigrum sulfathiazole from appearance character, simple discriminating and single component assay, obviously its inherent quality can not be accurately described comprehensively, can not ensure its curative effect.Control its effect, according to Chinese medicine polycomponent, Mutiple Targets, multipath action character, its material entirety must be controlled, effectively characterize the quality of Chinese medicine on the whole, set up safe, effective, stable quality standard system.
Compound Solanum Nigrum sulfathiazole is the medicine of new research and development process, and the raw material packet of Compound Solanum Nigrum sulfathiazole contains 3 taste Chinese medicines and there is complicated interaction between traditional Chinese medicine ingredients, cause setting up and reflect that the effective substance method of Compound Solanum Nigrum sulfathiazole inherent quality and curative effect exists larger difficulty fully and effectively, therefore, there is the demand to the detection method that can characterize this medicine of Compound Solanum Nigrum sulfathiazole comprehensively in current this area.
Summary of the invention
In order to solve the problem, the object of this invention is to provide a kind of detection method of Compound Solanum Nigrum sulfathiazole, the method compared with the content of the medicine activity component of complete detection Compound Solanum Nigrum sulfathiazole, thus can characterize and control its inherent quality.
To achieve these goals, the invention provides a kind of high-efficiency liquid chromatography method for detecting of Compound Solanum Nigrum sulfathiazole, described Compound Solanum Nigrum sulfathiazole is made primarily of black nightshade, pennywort, pithecellobium clypearia, and described detection method comprises and adopts high performance liquid chromatography to detect gallic acid, Quercetin, Kaempferide, solasonine, solamargine in described Compound Solanum Nigrum sulfathiazole.
Preferably, the content of the gallic acid in the detection method detection Compound Solanum Nigrum sulfathiazole described in employing, Quercetin, Kaempferide comprises the following steps:
1) chromatographic condition:
Chromatographic column: employing octadecylsilane chemically bonded silica is filling agent;
Mobile phase: the mixed liquor of methyl alcohol and phosphoric acid solution, the volume fraction of phosphoric acid solution is 0.4%, and the volume ratio of methyl alcohol and phosphoric acid water is 55:45;
Flow velocity: 1mL/min;
Column temperature: 25 DEG C;
Gradient wavelength detecting: 0 ~ 15min, determined wavelength is 270nm; 15 ~ 30min, determined wavelength is 362nm;
2) preparation mixing contrast solution: precision takes gallic acid reference substance, Quercetin reference substance, Kaempferol reference substance in right amount, adds methyl alcohol and makes containing gallic acid 0.5mg/mL, the reference substance mixed solution of Quercetin 1mg/mL, Kaempferol 0.5mg/mL;
3) prepare need testing solution: get this product powder and be about 2g, accurately weighed, precision adds methyl alcohol 100ml, weighed weight, refluxing extraction 1h, lets cool, and supplies less loss weight, filter, precision measures filtrate 50mL, adds hydrochloric acid 15mL, water-bath reflux heating and hydrolysis process 2h, filter, filtrate is put in 100mL measuring bottle, use a little methanol wash, washing lotion is incorporated in same measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed Quercetin peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of gallic acid, Quercetin, Kaempferide.
Preferably, the solasonine in the detection method detection Compound Solanum Nigrum sulfathiazole described in employing, solamargine content comprise the following steps:
1) chromatographic condition:
Chromatographic column: chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent;
Mobile phase: acetonitrile and Na
2hPO
4the mixed liquor of solution, Na
2hPO
4for 2mol/L, acetonitrile and Na
2hPO
4the volume ratio of solution is 39:61;
Flow velocity: 1mL/min;
Determined wavelength: 205nm;
Column temperature: 25 DEG C;
2) preparation mixing contrast solution: precision takes solasonine reference substance, solamargine reference substance is appropriate, adds methyl alcohol and makes reference substance mixed solution containing solasonine 1mg/mL, solamargine 1mg/mL;
3) prepare need testing solution: accurately weighed Compound Solanum Nigrum sulfathiazole meal 2g, precision adds 100mL methyl alcohol, accurately weighed, refluxing extraction 1h, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 50mL, evaporate to dryness, adds 1% hydrochloric acid solution 50mL and dissolves, evaporate to dryness, use a little methanol wash, washing lotion is incorporated in 10mL measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance mixed solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed solasonine peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of solasonine, solamargine.
Preferably, detection method described in employing detects the step 1 of content of gallic acid in Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide) described in constant gradient wavelength detecting equiwavelength can also be adopted to detect, wavelength is 254nm.
Preferably, detection method described in employing detects the step 1 of content of gallic acid in Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide) described in mobile phase can also be: mobile phase A is methyl alcohol, Mobile phase B is 0.4% phosphoric acid solution, carry out gradient elution, described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 13min, mobile phase A is: 2%, and Mobile phase B is: 88%;
13 ~ 14min, mobile phase A is: 2% ~ 52%, and Mobile phase B is: 48% ~ 88%;
14 ~ 30min, mobile phase A is: 52%, and Mobile phase B is: 48%;
More than 30min, mobile phase A is: 55%, and Mobile phase B is: 45%.
Preferably, the detection method described in employing detects the step 1 of solasonine in Compound Solanum Nigrum sulfathiazole, solamargine content simultaneously) described phosphoric acid solution can select any one acidic materials in glacial acetic acid, formic acid, trichloroacetic acid to replace.
Preferably, detection method described in employing detects the step 1 of solasonine in Compound Solanum Nigrum sulfathiazole, solamargine content simultaneously) described in mobile phase can also be: mobile phase A is acetonitrile, Mobile phase B is 1% volume fraction phosphoric acid solution, carry out gradient elution, described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 5min, mobile phase A is: 22%, and Mobile phase B is: 78%;
5 ~ 20min, mobile phase A is: 22% ~ 25%, and Mobile phase B is: 78% ~ 75%;
20 ~ 25min, mobile phase A is: 25%, and Mobile phase B is: 75%;
More than 25min, mobile phase A is: 20%, and Mobile phase B is: 80%.
Preferably, the detection method described in employing detects the step 1 of solasonine in Compound Solanum Nigrum sulfathiazole, solamargine content simultaneously) described in Na
2hPO
4ammoniacal liquor, diethylamine, triethylamine, NaH can be selected
2pO
4and Na
3pO
4in any one material replace.
The present invention carries out substep according to the phenolic acids such as gallic acid, Quercetin, Kaempferide in described sulfathiazole and this two class of the alkaloid such as solamargine, solasonine and detects, this detection method testing result degree of accuracy is high, but also can improve detection rates.And use it for the assay of described sulfathiazole, the method ensures accuracy and the advance of described sulfathiazole quality inspection standard, can be used as the important indicator of described sulfathiazole quality control.When detecting the phenolic acid such as gallic acid, Quercetin, Kaempferide in Compound Solanum Nigrum sulfathiazole, the mixed liquor of methyl alcohol and phosphoric acid solution is adopted to carry out wash-out.Because the eluting power of methyl alcohol is stronger, after adding the phosphoric acid solution of 0.4% volume fraction, when detecting the gallic acid in Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide, mobile phase polarity can be adjusted preferably, improve the quality that chromatographic peak goes out peak, such as: reduce hangover, peak type narrows, absorption peak increases.Meanwhile, methanol-eluted fractions ability can also be made to be improved, thus the content of gallic acid in Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide can be detected exactly.But the phosphoric acid solution added will bearing in atmosphere in chromatographic column, and ratio can not be too high, otherwise there is the phenomenon that salts out, be advisable when the volume ratio being therefore the phosphoric acid solution of 0.4% by methyl alcohol in mixed liquor and volume fraction is deployed into 55:45.According to the characteristic of gallic acid, Quercetin, Kaempferide, detect these medicines in the mode of gradient wavelength exactly in different time, different fluctuations.Carry out the de-ability of washing that gradient elution can increase mobile phase when detecting gallic acid, Quercetin, the Kaempferide in Compound Solanum Nigrum antiphlogistic, improve to take off and wash efficiency.
When detecting the alkaloid such as solamargine, solasonine, adopt acetonitrile and Na
2hPO
4the mixed liquor of solution carries out wash-out.Because the eluting power of acetonitrile is very strong, add the Na of 2mol/L
2hPO
4after solution, when detecting the alkaloid such as solamargine, solasonine, mobile phase polarity can be adjusted preferably, improving the quality that chromatographic peak goes out peak, as: hangover reduces, peak type narrows, absorption peak increases.But the Na added
2hPO
4solution will bearing in atmosphere in chromatographic column, and ratio can not be too high, otherwise occur the phenomenon that salts out, therefore by acetonitrile in mixed liquor and 2mol/L
Na
2hPO
4be advisable when the volume ratio of solution is deployed into 39:61.Carry out the de-ability of washing that gradient elution can increase mobile phase when detecting solasonine, the solamargine in Compound Solanum Nigrum antiphlogistic, improve to take off and wash efficiency.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, can implement according to this with reference to instructions word to make those skilled in the art.
Embodiment 1
Gallic acid, Quercetin, Kaempferide, solasonine, solamargine content in isocratic elution detection Compound Solanum Nigrum sulfathiazole are carried out in employing high performance liquid chromatography (HPLC).
1, the content of the gallic acid in high effective liquid chromatography for detecting detection Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide is adopted to comprise the following steps:
1) chromatographic condition:
Chromatographic column: employing octadecylsilane chemically bonded silica is filling agent;
Mobile phase: the mixed liquor of methyl alcohol and phosphoric acid solution, the volume fraction of phosphoric acid solution is 0.4%, and the volume ratio of methyl alcohol and phosphoric acid water is 55:45;
Flow velocity: 1mL/min;
Column temperature: 25 DEG C;
Gradient wavelength detecting: 0 ~ 15min, determined wavelength is 270nm; 15 ~ 30min, determined wavelength is 362nm;
2) preparation mixing contrast solution: precision takes gallic acid reference substance, Quercetin reference substance, Kaempferol reference substance in right amount, adds methyl alcohol and makes containing gallic acid 0.5mg/mL, the reference substance mixed solution of Quercetin 1mg/mL, Kaempferol 0.5mg/mL;
3) prepare need testing solution: get this product powder and be about 2g, accurately weighed, precision adds methyl alcohol 100ml, weighed weight, refluxing extraction 1h, lets cool, and supplies less loss weight, filter, precision measures filtrate 50mL, adds hydrochloric acid 15mL, water-bath reflux heating and hydrolysis process 2h, filter, filtrate is put in 100mL measuring bottle, use a little methanol wash, washing lotion is incorporated in same measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance solution and each 20 μ L of need testing solution respectively, inject high performance liquid chromatograph and carry out rear mensuration:
5) calculate: theoretical cam curve is pressed Quercetin peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of gallic acid, Quercetin, Kaempferide, as shown in table 1.
2, the solasonine in employing high effective liquid chromatography for detecting detection Compound Solanum Nigrum sulfathiazole, solamargine content comprise the following steps:
1) chromatographic condition:
Chromatographic column: chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent;
Mobile phase: acetonitrile and Na
2hPO
4the mixed liquor of solution, Na
2hPO
4for 2mol/L, acetonitrile and Na
2hPO
4the volume ratio of solution is 39:61;
Flow velocity: 1mL/min;
Determined wavelength: 205nm;
Column temperature: 25 DEG C;
1) preparation mixing contrast solution: precision takes solasonine reference substance, solamargine reference substance is appropriate, adds methyl alcohol and makes reference substance mixed solution containing solasonine 1mg/mL, solamargine 1mg/mL;
2) prepare need testing solution: accurately weighed Compound Solanum Nigrum sulfathiazole meal 2g, precision adds 100mL methyl alcohol, accurately weighed, refluxing extraction 1h, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 50mL, evaporate to dryness, adds 1% hydrochloric acid solution 50mL and dissolves, evaporate to dryness, use a little methanol wash, washing lotion is incorporated in 10mL measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
3) measure: accurate absorption reference substance mixed solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
4) calculate: theoretical cam curve is pressed solasonine peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of solasonine, solamargine, as shown in table 1.
Embodiment 2
Gallic acid, Quercetin, Kaempferide, solasonine, solamargine content in gradient degree wash-out detection Compound Solanum Nigrum sulfathiazole are carried out in employing high performance liquid chromatography (HPLC).
1, the content of the gallic acid in high effective liquid chromatography for detecting detection Compound Solanum Nigrum sulfathiazole, Quercetin, Kaempferide is adopted to comprise the following steps:
1) chromatographic condition:
Chromatographic column: employing octadecylsilane chemically bonded silica is filling agent;
Mobile phase: mobile phase A is methyl alcohol, Mobile phase B is 0.4% phosphoric acid solution, carries out gradient elution, and described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 13min, mobile phase A is: 2%, and Mobile phase B is: 88%;
13 ~ 14min, mobile phase A is: 2% ~ 52%, and Mobile phase B is: 48% ~ 88%;
14 ~ 30min, mobile phase A is: 52%, and Mobile phase B is: 48%;
More than 30min, mobile phase A is: 55%, and Mobile phase B is: 45%;
Flow velocity: 1mL/min;
Column temperature: 25 DEG C;
Gradient wavelength detecting: 0 ~ 15min, determined wavelength is 270nm; 15 ~ 30min, determined wavelength is 362nm;
2) preparation mixing contrast solution: precision takes gallic acid reference substance, Quercetin reference substance, Kaempferol reference substance in right amount, adds methyl alcohol and makes containing gallic acid 0.5mg/mL, the reference substance mixed solution of Quercetin 1mg/mL, Kaempferol 0.5mg/mL;
3) prepare need testing solution: get this product powder and be about 2g, accurately weighed, precision adds methyl alcohol 100ml, weighed weight, refluxing extraction 1h, lets cool, and supplies less loss weight, filter, precision measures filtrate 50mL, adds hydrochloric acid 15mL, water-bath reflux heating and hydrolysis process 2h, filter, filtrate is put in 100mL measuring bottle, use a little methanol wash, washing lotion is incorporated in same measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed Quercetin peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of gallic acid, Quercetin, Kaempferide, as shown in table 1.
2, the solasonine in employing high effective liquid chromatography for detecting detection Compound Solanum Nigrum sulfathiazole, solamargine content comprise the following steps:
1) chromatographic condition:
Chromatographic column: chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent;
Mobile phase: mobile phase A is acetonitrile, Mobile phase B is 1% volume fraction phosphoric acid solution, carries out gradient elution, and described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 5min, mobile phase A is: 22%, and Mobile phase B is: 78%;
5 ~ 20min, mobile phase A is: 22% ~ 25%, and Mobile phase B is: 78% ~ 75%;
20 ~ 25min, mobile phase A is: 25%, and Mobile phase B is: 75%;
More than 25min, mobile phase A is: 20%, and Mobile phase B is: 80%.
Flow velocity: 1mL/min;
Determined wavelength: 205nm;
Column temperature: 25 DEG C;
2) preparation mixing contrast solution: precision takes solasonine reference substance, solamargine reference substance is appropriate, adds methyl alcohol and makes reference substance mixed solution containing solasonine 1mg/mL, solamargine 1mg/mL;
3) prepare need testing solution: accurately weighed Compound Solanum Nigrum sulfathiazole meal 2g, precision adds 100mL methyl alcohol, accurately weighed, refluxing extraction 1h, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 50mL, evaporate to dryness, adds 1% hydrochloric acid solution 50mL and dissolves, evaporate to dryness, use a little methanol wash, washing lotion is incorporated in 10mL measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance mixed solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed solasonine peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of solasonine, solamargine, as shown in table 1.
Embodiment 3
Gallic acid, Quercetin, Kaempferide, solasonine, solamargine content in gradient elution mensuration Compound Solanum Nigrum sulfathiazole are carried out in employing ultra-performance liquid chromatography (UPLC).
Ultra-performance liquid chromatography principle in this test with substantially identical with the high performance liquid chromatography in embodiment 2, change place have following some:
(1) granule, high-performance particulate Stationary liquid is used.The chromatographic column of HPLC, as: common 18 alkyl silica gel bonding post, particle diameter is 5um, and UPLC chromatographic column, reach 3.5um, even 1.7um, such aperture is more conducive to separating substances.
(2) use of high pressure pump.Because the chromatographic column particle diameter used reduces, the pressure produced during use also increases exponentially naturally.Therefore the infusion pump of the liquid chromatography also corresponding infusion pump changing over UHV (ultra-high voltage).
(3) sensitive detectors of high-speed sampling speed.
(4) low diffusion, low cross pollution automatic sampler is used.Be equipped with sample introduction probe and pressure in pin and assist sampling technique;
(5) instrument total system optimal design.Chromatographic work station is equipped with multi software platform, realizes the automatic conversion of ultra high efficiency liquid phase analysis method and high efficient liquid phase analysis method.
The result that ultra-performance liquid chromatography detects Compound Solanum Nigrum sulfathiazole is as shown in table 1.
Table 1HPLC method and UPLC method measure Contents of Main Components in Compound Solanum Nigrum sulfathiazole
Same batch sample assay result data is compared see table 1.From table 1 data, can see that high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) and two kinds of elution methods measure Contents of Main Components result in Compound Solanum Nigrum sulfathiazoles basically identical.
According to above 6 batches of assay data analyses, different batches Compound Solanum Nigrum sulfathiazole mass discrepancy is comparatively large, mainly affects by factors such as herbal species, the place of production, processing, preparations and causes.By specifying high-quality Chinese medicine black nightshade, pennywort, pithecellobium clypearia kind and the place of production etc., standardized processing production process, improves or active constituent content in stable Compound Solanum Nigrum sulfathiazole.Solasonine, solamargine have the biologically actives such as anticancer, antibacterial, and content is higher in black nightshade medicinal material, can as the index components evaluating black nightshade quality of medicinal material, simultaneously, gallic acid, Quercetin, Kaempferide content in pennywort and pithecellobium clypearia is higher, can as the index components evaluating pennywort and pithecellobium clypearia quality.Can the every sheet of regulation this product containing gallic acid, Quercetin, Kaempferide, solasonine, solamargine content should be no less than 8mg, 2mg, 0.2mg, 1.5mg, 1.5mg respectively
Although embodiment of the present invention are open as above, but it is not restricted to listed in instructions and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (8)
1. the high-efficiency liquid chromatography method for detecting of a Compound Solanum Nigrum sulfathiazole, it is characterized in that, described Compound Solanum Nigrum sulfathiazole is made primarily of black nightshade, pennywort, pithecellobium clypearia, and described detection method comprises and adopts high performance liquid chromatography to detect gallic acid, Quercetin, Kaempferide, solasonine, solamargine in described Compound Solanum Nigrum sulfathiazole.
2. the high-efficiency liquid chromatography method for detecting of Compound Solanum Nigrum sulfathiazole according to claim 1, is characterized in that, the detection method described in employing detect simultaneously gallic acid in Compound Solanum Nigrum antiphlogistic, Quercetin, Kaempferide content comprise the following steps:
1) chromatographic condition:
Chromatographic column: employing octadecylsilane chemically bonded silica is filling agent;
Mobile phase: the mixed liquor of methyl alcohol and phosphoric acid solution, the volume fraction of phosphoric acid solution is 0.4%, and the volume ratio of methyl alcohol and phosphoric acid water is 55:45;
Flow velocity: 1mL/min;
Column temperature: 25 DEG C;
Gradient wavelength detecting: 0 ~ 15min, determined wavelength is 270nm; 15 ~ 30min, determined wavelength is 362nm;
2) preparation mixing contrast solution: precision takes gallic acid reference substance, Quercetin reference substance, Kaempferol reference substance in right amount, adds methyl alcohol and makes containing gallic acid 0.5mg/mL, the reference substance mixed solution of Quercetin 1mg/mL, Kaempferol 0.5mg/mL;
3) prepare need testing solution: get this product powder and be about 2g, accurately weighed, precision adds methyl alcohol 100ml, weighed weight, refluxing extraction 1h, lets cool, and supplies less loss weight, filter, precision measures filtrate 50mL, adds hydrochloric acid 15mL, water-bath reflux heating and hydrolysis process 2h, filter, filtrate is put in 100mL measuring bottle, use a little methanol wash, washing lotion is incorporated in same measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed Quercetin peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of gallic acid, Quercetin, Kaempferide.
3. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 1, is characterized in that, the detection method described in employing detects solasonine in Compound Solanum Nigrum antiphlogistic simultaneously, solamargine content comprises the following steps:
1) chromatographic condition:
Chromatographic column: chromatographic column adopts octadecylsilane chemically bonded silica to be filling agent;
Mobile phase: acetonitrile and Na
2hPO
4the mixed liquor of solution, Na
2hPO
4for 2mol/L, acetonitrile and Na
2hPO
4the volume ratio of solution is 39:61;
Flow velocity: 1mL/min;
Determined wavelength: 205nm;
Column temperature: 25 DEG C;
2) preparation mixing contrast solution: precision takes solasonine reference substance, solamargine reference substance is appropriate, adds methyl alcohol and makes reference substance mixed solution containing solasonine 1mg/mL, solamargine 1mg/mL;
3) prepare need testing solution: accurately weighed Compound Solanum Nigrum sulfathiazole meal 2g, precision adds 100mL methyl alcohol, accurately weighed, refluxing extraction 1h, lets cool, and supplies the weight of less loss with methyl alcohol, shake up, filter, precision measures subsequent filtrate 50mL, evaporate to dryness, adds 1% hydrochloric acid solution 50mL and dissolves, evaporate to dryness, use a little methanol wash, washing lotion is incorporated in 10mL measuring bottle, adds methyl alcohol to scale, shake up, filter with 0.45nm miillpore filter, get its filtrate;
4) measure: accurate absorption reference substance mixed solution and each 20 μ L of need testing solution respectively, measure after injecting high performance liquid chromatograph;
5) calculate: theoretical cam curve is pressed solasonine peak and calculated, and should be not less than 5000.Record peak area, adopts external standard method to calculate the content of solasonine, solamargine.
4. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 2, is characterized in that, step 1) described in constant gradient wavelength detecting equiwavelength can also be adopted to detect, wavelength is 254nm.
5. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 2, it is characterized in that, step 1) described in mobile phase can also be: mobile phase A is methyl alcohol, Mobile phase B is 0.4% phosphoric acid solution, carry out gradient elution, described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 13min, mobile phase A is: 2%, and Mobile phase B is: 88%;
13 ~ 14min, mobile phase A is: 2% ~ 52%, and Mobile phase B is: 48% ~ 88%;
14 ~ 30min, mobile phase A is: 52%, and Mobile phase B is: 48%;
More than 30min, mobile phase A is: 55%, and Mobile phase B is: 45%.
6. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 2, is characterized in that, step 1) described phosphoric acid solution can select any one material in glacial acetic acid, formic acid, trichloroacetic acid to replace.
7. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 3, it is characterized in that, step 1) described in mobile phase can also be: mobile phase A is acetonitrile, Mobile phase B is 1% volume fraction phosphoric acid solution, carry out gradient elution, described gradient elution program is as follows, and wherein mobile phase ratio is percent by volume:
0 ~ 5min, mobile phase A is: 22%, and Mobile phase B is: 78%;
5 ~ 20min, mobile phase A is: 22% ~ 25%, and Mobile phase B is: 78% ~ 75%;
20 ~ 25min, mobile phase A is: 25%, and Mobile phase B is: 75%;
More than 25min, mobile phase A is: 20%, and Mobile phase B is: 80%.
8. the detection method of Compound Solanum Nigrum sulfathiazole according to claim 3, is characterized in that, step 1) described in Na
2hPO
4ammoniacal liquor, diethylamine, triethylamine, NaH can be selected
2pO
4and Na
3pO
4in any one material replace.
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| CN201510780966.6A CN105203673B (en) | 2015-11-13 | 2015-11-13 | The high-efficiency liquid chromatography method for detecting of Compound Solanum Nigrum sulfathiazole |
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| CN105954390A (en) * | 2016-04-25 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for detecting content of kaempferide in momordica grosvenori tea by using ASE-HPLC |
| CN108205033A (en) * | 2018-01-04 | 2018-06-26 | 吉林师范大学 | A kind of method of solanine and black nightshade polysaccharide in synchronous extracting and developing, measure black nightshade Chinese olive |
| CN109946397A (en) * | 2019-03-26 | 2019-06-28 | 广州莱泰制药有限公司 | A kind of pithecellobium clypearia method of inspection |
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2015
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| 李慧 等: "HPLC测定地锦草中没食子酸、槲皮素及山柰素含量", 《中国实验方剂学杂志》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105954390A (en) * | 2016-04-25 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Method for detecting content of kaempferide in momordica grosvenori tea by using ASE-HPLC |
| CN108205033A (en) * | 2018-01-04 | 2018-06-26 | 吉林师范大学 | A kind of method of solanine and black nightshade polysaccharide in synchronous extracting and developing, measure black nightshade Chinese olive |
| CN109946397A (en) * | 2019-03-26 | 2019-06-28 | 广州莱泰制药有限公司 | A kind of pithecellobium clypearia method of inspection |
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| CN105203673B (en) | 2017-10-31 |
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