CN115326996B - Method for detecting amino acid in oral cleaning care product containing periplaneta americana extract - Google Patents
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Abstract
The method for detecting amino acid in the oral cleaning care product containing the American cockroach extract comprises the steps of extracting an oral cleaning care product sample containing the American cockroach extract by using an inorganic salt, vortex and ultrasonic auxiliary solvent, centrifugally concentrating, performing pre-column derivatization reaction with phenyl isothiocyanate, measuring an amino acid derivative product in the sample by using a high performance liquid chromatograph, and quantifying by using a retention time qualitative and external standard method. The method reduces the influence of the silica agglomeration effect on the pretreatment efficiency by the auxiliary extraction of inorganic salt; by means of vortex, ultrasonic, centrifugal concentration and the like, the extraction time is shortened, and the extraction rate is improved; under proper alkaline conditions, the derivatization reaction process is accelerated; the damage of PITC to the chromatographic column is reduced by back extraction of a proper organic solvent; by optimizing the gradient elution procedure, interference of the oral cleaning care product matrix is reduced and the separation efficiency is greatly improved. The method is simple and easy to operate, has controllable process and high sensitivity, and is easy to popularize.
Description
Technical Field
The invention belongs to the technical field of detection methods of oral cleaning care products (toothpaste, gel, mouthwash and the like), and particularly relates to a detection method of amino acid content in an oral cleaning care product containing periplaneta americana.
Background
The periplaneta americana is a representative animal medicine, and the ingredients contain more medicinal components such as amino acid, protein, polypeptide and small molecular compounds, wherein most substances are endogenous compounds (existing in vivo), and the other substances are exogenous compounds (released in vivo and in vitro). According to modern pharmacological research, the periplaneta americana extract has the effects of resisting inflammation and bacteria, promoting tissue repair, inhibiting tumor growth, protecting liver, healing wounds and the like. The American cockroach extract is applied to the rehabilitation and refresh liquid, can be externally applied or orally taken, and has the wonderful effects of promoting tissue repair, regulating immune function, repairing bone defect and pulmonary fibrosis, resisting bacteria, diminishing inflammation, removing necrotic tissue and promoting granulation. In recent years, the effects of repairing tissues and healing wounds are explored by researchers in the field of oral care products, so that the effects of repairing oral mucosa, promoting the healing of dental ulcers, reducing gingivitis and the like are achieved. According to the requirements of QB/T2966-2014 'efficacy toothpaste', the efficacy components in the efficacy toothpaste should be qualitatively or quantitatively detected, but no relevant standard is available at present for detecting the content of the efficacy components in the oral cavity cleaning care products, so that a method for detecting the content of the oral cavity cleaning care products, particularly the American cockroach extract in the toothpaste, is established, and has positive significance for guaranteeing the quality of the toothpaste product, guaranteeing the rights and health of consumers and perfecting the toothpaste product inspection technology system. Amino acids are one of various active ingredients contained in the periplaneta americana extract, and the content of the periplaneta americana extract can be indirectly monitored by detecting the content of the amino acids.
The polypeptide is also one of active ingredients contained in the American cockroach extract, and the method is used for detecting the polypeptide by using a Fu Lin Fen method in QBT 5287-2018 American cockroach extract for toothpaste which is an oral cleaning care product, but a Fu Lin Fen reagent can react with phenolic hydroxyl groups under alkaline conditions, and raw materials such as xanthan gum, polyalcohol and the like in toothpaste, gel and mouthwash are easy to react with the Fulin to cause interference, so the method is only suitable for detecting the raw materials of the American cockroach extract and is not suitable for detecting the polypeptide ingredients of the American cockroach extract added into an oral cleaning care product system. Amino acid is one of important active ingredients of the periplaneta americana extract, and the detection method of the amino acid in the periplaneta americana comprises spectrophotometry, chemiluminescence method, liquid chromatography, gas chromatography, fluorescence spectrometry, capillary electrophoresis and the like, and has advantages and disadvantages. The amino acid analyzer is adopted to directly detect the amino acid, but the amino acid analyzer has the defects of strong specificity, expensive instrument, non-common property, high cost and the like. High performance liquid chromatography (High performance liquid chromatography, HPLC) is currently the most widely used method for amino acid analysis and detection, and is also a hot spot method for amino acid research, most amino acids have almost no ultraviolet absorption and no fluorescence emission, so that derivatization of amino acids is required when spectrophotometry, gas chromatography, liquid chromatography and the like are used in combination with ultraviolet or fluorescence detectors, and the derivatization methods can be divided into pre-column derivatization and post-column derivatization. The pre-column derivatization can select reaction conditions relatively freely, more derivatization reagents can be selected, complex instruments are not needed, but by-products after derivatization can cause difficulty in chromatographic separation, and samples are easy to lose or impurities are easy to introduce; the post-column derivatization has few introduced impurities and good reproducibility, the analyte can be separated in the original form, and the existing analysis method is easy to select, but the post-column derivatization has higher requirements on solvent and reaction time, only limited reactions are available at present, and the diffusion problem can exist. As reported in the literature, currently commonly used pre-column derivatization reagents are phthalic aldehyde (OPA), fluorenylmethoxycarbonyl chloride (FMOC-Cl), phenyl Isothiocyanate (PITC), dansyl chloride (Dansyl-Cl), 6-aminoquinoline-N-hydroxysuccinimidyl formate (AQC), and some synthetic novel derivatization reagents. The OPA reaction speed is high, but primary amino acid and secondary amino acid cannot be derived at the same time, and the derived products are unstable; FMOC-Cl is fast and the product is stable, but the reagents themselves and the hydrolysates interfere with the analysis; the Dansyl-Cl product is stable, but has poor reactivity and low speed; although the derivative product of the AQC is stable and simple to operate, the derivative separation degree is not high, and the analysis is needed by using ultra-high performance liquid chromatography; PITC is easy to crystallize when meeting water, has high toxicity and damages to chromatographic columns.
Disclosure of Invention
The invention aims to solve the defects of the prior art, provides a method for establishing the content of free amino acid in the oral cleaning care product containing the American cockroach extract by optimizing pretreatment conditions, derivatization reaction conditions, chromatographic conditions and the like, so as to perfect and standardize a detection system of the oral cleaning care product, ensure the effectiveness and the safety of the oral cleaning care product, maintain the rights and interests of consumers and provide technical support and basis for market supervision of related departments.
The technical scheme adopted by the invention is as follows:
the method for detecting free amino acid in the oral cleaning care product containing the periplaneta americana extract comprises the following steps:
(1) Taking an oral cleaning care product sample containing the periplaneta americana extract and an extractant, wherein the mass ratio of the sample to the extractant is 1:2-1:50, and simultaneously adding inorganic salt accounting for 2-40% of the mass of the sample, and carrying out auxiliary extraction on the sample sequentially by vortex, ultrasonic and centrifugal modes; the method comprises the steps of carrying out a first treatment on the surface of the The extractant is one of methanol-water solution with volume concentration of 25-100%, ethanol-water solution with volume concentration of 25-100%, acetonitrile-water solution with volume concentration of 25-100% and deionized water with volume concentration of 100%; the inorganic salt is one of magnesium sulfate, sodium chloride, potassium chloride and sodium carbonate;
(2) Collecting the supernatant after extraction, concentrating and redissolving at 50-100 ℃;
(3) Adding alkaline acetonitrile solution with the mass of 0.2-1.0% of the concentrated redissolved solution and derivative reagent phenyl isothiocyanate with the mass of 0.2-1.0% of the concentrated redissolved solution into the concentrated redissolved solution for vortex mixing, and reacting for 5-240 min at 20-50 ℃ under alkaline conditions to obtain derivative solution; the alkaline acetonitrile solution is acetonitrile solution containing any one of sodium hydroxide, calcium hydroxide, barium hydroxide and triethanolamine;
(4) Back-extracting the derivatization solution by using an organic solvent to remove excessive derivatization reagent phenyl isothiocyanate; the organic solvent is one of cyclohexane, normal hexane, heptane, hexane and pentane;
(5) Centrifuging the back-extracted solution, removing the upper layer liquid, taking the lower layer liquid, adopting high performance liquid chromatography, sampling under gradient elution condition, detecting peak area at 254nm, and quantifying by retention time qualitative and external standard one-point method.
Further, the chromatographic conditions of the high performance liquid chromatography are as follows:
a. chromatographic column: a C18 chromatographic column or other equivalent chromatographic column special for amino acid, which takes octadecylsilane chemically bonded silica as a filler;
b. mobile phase a: sodium acetate solution at 0.1mol/L and pH 6.5: acetonitrile (v/v) =93.0:7.0;
mobile phase B: acetonitrile: water (v/v) =80.0:20.0;
c. flow rate: 1mL/min;
d. detection wavelength: 254nm;
e. column temperature: 40 ℃;
f. sample injection amount: 5. Mu.L;
the gradient elution sample injection process is as follows:
at 0.01 min, mobile phase A samples 100% and mobile phase B samples 0%;
at 11 min, mobile phase A samples 93.0% and mobile phase B samples 7.0%;
at 13.9 min, mobile phase A was 88.0% injected and mobile phase B was 12.0% injected;
at 14 minutes, mobile phase A samples 85.0% and mobile phase B samples 15.0%;
at 29 minutes, mobile phase A was 66.0% injected and mobile phase B was 34.0% injected;
at 32 minutes, mobile phase A was injected at 30.0% and mobile phase B was injected at 70.0%;
at 35 min, mobile phase A samples 0% and mobile phase B samples 100.0%;
at 42 minutes, mobile phase A samples 0% and mobile phase B samples 100.0%;
at 45 min, mobile phase A samples 100.0% and mobile phase B samples 0%;
at 60 minutes, mobile phase A was 100.0% injected and mobile phase B was 0%.
Further, in the step (1), the vortex speed of the vortex mode is 200-3000 rpm, and the vortex time is 1-20 min; the ultrasonic frequency of the ultrasonic mode is 10-60 Hz, and the ultrasonic time is 1-50 min; the centrifugal speed of the centrifugal mode is 500-100000 rpm, and the centrifugal time is 1-20 min; the auxiliary extraction times are 1-5 times.
Further, the concentration and redissolution are that the supernatant is poured into an evaporation dish for repeated extraction, then evaporated to dryness, cooled to room temperature and then added with methanol for redissolution.
The invention has the following beneficial effects:
the method reduces the influence of the agglomeration effect of the silicon dioxide on the pretreatment efficiency by the auxiliary extraction of the inorganic salt, so that the method is applicable to almost all oral cleaning care products of commercial phosphorus calcium and silicon dioxide matrixes, such as toothpaste, gel, mouthwash and the like. The dispersion of the paste in the extractant is accelerated by vortex, the extraction rate is improved by ultrasonic assistance, the separation of the extraction liquid and the semi-solid phase is accelerated by centrifugation, and then the re-dissolution concentration is carried out, so that the extraction time is shortened, and the extraction rate is improved. The invention adopts derivatization under proper alkaline condition, which accelerates the derivatization reaction process. By performing the stripping with a suitable organic solvent, the damage of PITC to the chromatographic column is reduced. By selecting different inorganic salt buffer solution systems with different pH ranges and different organic solvents with different polarities, and continuously changing the concentration ratio of the mobile phase in the same analysis period to a certain extent, the gradient elution procedure is optimized, the interference of the oral cleaning care product matrix is reduced, and the separation efficiency is greatly improved. The method is simple and easy to operate, has controllable process and high sensitivity, and is easy to popularize. The recovery rate and precision experiment verifies that the method is suitable for detecting the amino acid content in the oral cleaning care product containing the American cockroach extract and controlling the quality.
Drawings
FIG. 1 is a blank (water) derived chromatogram;
FIG. 2 is a five amino acid standard solution derived chromatogram;
fig. 3 is a toothpaste sample solution derived chromatogram.
Detailed Description
The invention is further illustrated below in conjunction with examples.
The method for detecting amino acid in the oral cleaning care product containing the periplaneta americana extract comprises the following steps:
(1) Pretreatment of toothpaste samples: 4.0000g of toothpaste containing the American cockroach extract is precisely weighed, placed in a 50ml centrifuge tube, added with 0.5g of NaCl and 20ml of methanol-water solution with the volume concentration of 50%, vortexed for 1min, the vortexed rotating speed is more than or equal to 2000rpm, then placed in an ultrasonic cleaner for 30min at 35Hz, and then placed in a centrifuge for 6000rmp for 5min. And collecting the supernatant after extraction, repeatedly extracting once, combining the supernatants extracted twice, and concentrating and redissolving. The concentration and redissolution process is as follows: pouring the supernatant into an evaporation dish, evaporating to dryness on a water bath kettle at 80-85 ℃, cooling to room temperature, adding a small amount of methanol to redissolve in a 10mL volumetric flask, fixing the volume to a scale mark by using the methanol, and standing for later use.
(2) Sample derivatization: taking 800 mu L of the solution after pretreatment of the toothpaste sample, placing the solution into an EP tube, adding 400 mu L of each of the prepared PITC-acetonitrile solution and TEA-acetonitrile solution, fully vortex-mixing, standing at 30 ℃ for derivatization for 60min; then 1600 mu L of normal hexane is added, vortex is carried out, the upper layer liquid is taken out by a dropper and discarded, then normal hexane is added for repeated back extraction, the upper layer liquid is discarded, 800 mu L of the lower layer liquid is taken out, a proper amount of ultrapure water is used for dilution, a 0.22 mu m filter head is used for filtration, and the subsequent filtrate is taken for testing.
The invention employs PITC as the derivatizing agent. PITC, although crystallizing when meeting water, is toxic and damaging to the column, is not high in reaction conditions, rapid in reaction, stable in product and can protect the column by back-extracting the derivative reagent.
(3) Setting chromatographic conditions and detecting:
chromatographic column: a Xuexu UItimate AA amino acid special column was used. Mobile phase a was a 0.1mol/L sodium acetate solution (ph=6.5): acetonitrile=93.0:7.0 (v/v). 13.60g of sodium acetate trihydrate is accurately weighed and placed in 1000mL of water, stirred until the sodium acetate trihydrate is fully dissolved, the pH value is adjusted to 6.50 by glacial acetic acid or sodium hydroxide, 930mL of the prepared sodium acetate trihydrate solution and 70mL of acetonitrile are accurately measured, uniformly mixed, and the solution is subjected to suction filtration and then ultrasonic treatment for 30min. Mobile phase B was acetonitrile: water=80.0:20.0 (v/v). Accurately measuring 800mL of acetonitrile and 200mL of water by using a measuring cylinder, carrying out suction filtration by using an organic filter membrane, and carrying out ultrasonic treatment for 30min. Column temperature: 40 ℃; detection wavelength: 254nm; flow rate: 1mL/min; sample injection amount: 5. Mu.L.
The peak area at 254nm was detected by high performance liquid chromatography, and the peak area was determined qualitatively by retention time using the external standard one-point method of the prior art.
TABLE 1 gradient elution program table
| Time (min) | A％ | B％ |
| 0.01 | 100.0 | 0.0 |
| 11 | 93.0 | 7.0 |
| 13.9 | 88.0 | 12.0 |
| 14 | 85.0 | 15.0 |
| 29 | 66.0 | 34.0 |
| 32 | 30.0 | 70.0 |
| 35 | 0.0 | 100.0 |
| 42 | 0.0 | 100.0 |
| 45 | 100.0 | 0.0 |
| 60 | 100.0 | 0.0 |
The detection results are shown in the toothpaste sample solution derivative chromatogram of fig. 3. The retention time/chromatographic peak corresponding to each amino acid (glycine, alanine, proline, valine, leucine) derivative can be seen from the chromatogram, and the content of each amino acid can be calculated according to the peak area.
FIG. 1 is a blank (water) derived chromatogram with HPLC sample introduction of ultrapure water without pretreatment extraction.
(4) And (3) verification:
a. the method is repeated, namely taking the same batch of toothpaste samples containing the American cockroach extract, preparing 6 samples according to the toothpaste pretreatment method in the step (1), carrying out derivatization according to the method in the step (2), and carrying out sample injection measurement to obtain the average values of glycine, alanine, proline, valine and leucine content of 4.40, 16.82, 6.88, 9.62 and 13.75mg/100g, wherein RSD is 4.83%, 4.40%, 5.29%, 4.68% and 4.35% (n=6) respectively, and the result shows that the sample is good in repeated performance.
b. The linear relationship, the detection limit and the quantitative limit are shown in Table 2
TABLE 2 standard curves, linear ranges, correlation coefficients, detection limits and quantification limits for 5 amino acids
FIG. 2 is a chromatogram of five amino acid standard solution derivatives, and as can be seen from FIG. 2, the five amino acid derivatives are fully separated, and the separation degree is greater than 1.5.
c. Stability of the derived product: taking the same sample after derivatization and mixed standard solution, and respectively carrying out sample injection measurement at 0,2,6, 10, 16 and 24 hours, wherein the RSD of peak areas of glycine, alanine, proline, valine and leucine in the sample solution is respectively 0.35%, 2.11%, 2.33%, 0.97% and 1.56% (n=6), and the RSD of peak areas of glycine, alanine, proline, valine and leucine in the mixed standard solution is respectively 0.29%, 1.11%, 0.38%, 0.74% and 2.26% (n=6), so that the sample and standard solution have good stability within 24 hours.
d. Sample labeling recovery rate and precision: see Table 3
Table 3.5 labeled recovery and relative standard deviation of amino acids in toothpaste samples (n=6)
The above embodiment is only one exemplary embodiment of the present invention, and does not limit the scope of the present invention. The specific material ratios and technical parameters in the steps of the method of the present invention are not limited to the above examples. In the method, when the oral cleaning care product sample containing the periplaneta americana extract and the extractant are taken, the mass ratio of the sample to the extractant can be in the range of 1:2-1:50, and the added inorganic salt can be 2-40% of the mass of the sample. The extractant can be any one of methanol-water solution with volume concentration of 25-100%, ethanol-water solution with volume concentration of 25-100%, acetonitrile-water solution with volume concentration of 25-100% and 100% deionized water. The inorganic salt can be any one of magnesium sulfate, sodium chloride, potassium chloride and sodium carbonate. The temperature of the supernatant concentration and re-dissolution can be 50-100 ℃. The addition amount of the alkaline acetonitrile solution and the derivative reagent phenyl isothiocyanate added into the concentrated redissolved solution can be 0.2-1.0% of the mass of the concentrated redissolved solution, and the concentrated redissolved solution can react for 5-240 min at 20-50 ℃ under alkaline conditions, wherein the alkaline acetonitrile solution refers to acetonitrile solution containing any one of sodium hydroxide, calcium hydroxide, barium hydroxide and triethanolamine. The vortex speed of the vortex mode can be 200-3000 rpm, and the vortex time is 1-20 min. The ultrasonic frequency of the ultrasonic mode can be 10-60 Hz, and the ultrasonic time is 1-50 min. The centrifugal speed of the centrifugal mode can be 500-100000 rpm, and the centrifugal time is 1-20 min. The number of auxiliary extractions may be 1 to 5.
Claims (4)
1. The method for detecting free amino acid in the oral cleaning care product containing the periplaneta americana extract is characterized by comprising the following steps:
(1) Taking an oral cleaning care product sample containing the periplaneta americana extract and an extractant, wherein the mass ratio of the sample to the extractant is 1:2-1:50, and simultaneously adding inorganic salt accounting for 2-40% of the mass of the sample, and carrying out auxiliary extraction on the sample sequentially by vortex, ultrasonic and centrifugal modes; the extractant is one of methanol-water solution with volume concentration of 25-100%, ethanol-water solution with volume concentration of 25-100%, acetonitrile-water solution with volume concentration of 25-100% and deionized water with volume concentration of 100%; the inorganic salt is one of magnesium sulfate, sodium chloride, potassium chloride and sodium carbonate;
(2) Collecting the supernatant after extraction, concentrating and redissolving at 50-100 ℃;
(3) Adding alkaline acetonitrile solution with the mass of 0.2-1.0% of the concentrated redissolved solution and derivative reagent phenyl isothiocyanate with the mass of 0.2-1.0% of the concentrated redissolved solution into the concentrated redissolved solution for vortex mixing, and reacting for 5-240 min at 20-50 ℃ under alkaline conditions to obtain derivative solution; the alkaline acetonitrile solution is acetonitrile solution containing any one of sodium hydroxide, calcium hydroxide, barium hydroxide and triethanolamine;
(4) Back-extracting the derivatization solution by using an organic solvent to remove excessive derivatization reagent phenyl isothiocyanate; the organic solvent is one of cyclohexane, normal hexane, heptane, hexane and pentane;
(5) Centrifuging the back-extracted solution, removing the upper layer liquid, taking the lower layer liquid, adopting high performance liquid chromatography, sampling under gradient elution condition, detecting peak area at 254nm, and quantifying by retention time qualitative and external standard one-point method.
2. The method for detecting free amino acids in an oral cleaning care product containing periplaneta americana extract according to claim 1, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
a. chromatographic column: a C18 chromatographic column or other equivalent chromatographic column special for amino acid, which takes octadecylsilane chemically bonded silica as a filler;
b. mobile phase a: sodium acetate solution at 0.1mol/L and pH 6.5: acetonitrile (v/v) =93.0:7.0;
mobile phase B: acetonitrile: water (v/v) =80.0:20.0;
c. flow rate: 1mL/min;
d. detection wavelength: 254nm;
e. column temperature: 40 ℃;
f. sample injection amount: 5. Mu.L;
the gradient elution sample injection process is as follows:
at 0.01 min, mobile phase A samples 100% and mobile phase B samples 0%;
at 11 min, mobile phase A samples 93.0% and mobile phase B samples 7.0%;
at 13.9 min, mobile phase A was 88.0% injected and mobile phase B was 12.0% injected;
at 14 minutes, mobile phase A samples 85.0% and mobile phase B samples 15.0%;
at 29 minutes, mobile phase A was 66.0% injected and mobile phase B was 34.0% injected;
at 32 minutes, mobile phase A was injected at 30.0% and mobile phase B was injected at 70.0%;
at 35 min, mobile phase A samples 0% and mobile phase B samples 100.0%;
at 42 minutes, mobile phase A samples 0% and mobile phase B samples 100.0%;
at 45 min, mobile phase A samples 100.0% and mobile phase B samples 0%;
at 60 minutes, mobile phase A was 100.0% injected and mobile phase B was 0%.
3. The method for detecting free amino acids in an oral cleaning care product containing periplaneta americana extract according to claim 1 or 2, wherein in the step (1), the swirling rotational speed of the swirling means is 200-3000 rpm, and the swirling time is 1-20 min; the ultrasonic frequency of the ultrasonic mode is 10-60 Hz, and the ultrasonic time is 1-50 min; the centrifugal speed of the centrifugal mode is 500-100000 rpm, and the centrifugal time is 1-20 min; the auxiliary extraction times are 1-5 times.
4. The method for detecting free amino acids in an oral cleaning care product containing the periplaneta americana extract according to claim 1 or 2, wherein the concentration and reconstitution are carried out by pouring the supernatant into an evaporation dish for repeated extraction, evaporating to dryness, cooling to room temperature and adding methanol for reconstitution.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957345A (en) * | 2010-03-05 | 2011-01-26 | 浙江京新药业股份有限公司 | Method for detecting active ingredients in American cockroach extract |
| CN102621250A (en) * | 2011-01-30 | 2012-08-01 | 复旦大学 | Liquid chromatogram method for detecting various common amino acids |
| CN107091896A (en) * | 2017-05-26 | 2017-08-25 | 浙江出入境检验检疫局检验检疫技术中心 | The method that SPE liquid chromatography mass/mass spectrography determines nicotinoids drug residue in honey simultaneously |
| AU2020100921A4 (en) * | 2020-06-02 | 2020-07-09 | Dali University | Preparation And Quality Control Method Of Periplaneta Americana Linn. Anti-Tumor Capsule |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957345A (en) * | 2010-03-05 | 2011-01-26 | 浙江京新药业股份有限公司 | Method for detecting active ingredients in American cockroach extract |
| CN102621250A (en) * | 2011-01-30 | 2012-08-01 | 复旦大学 | Liquid chromatogram method for detecting various common amino acids |
| CN107091896A (en) * | 2017-05-26 | 2017-08-25 | 浙江出入境检验检疫局检验检疫技术中心 | The method that SPE liquid chromatography mass/mass spectrography determines nicotinoids drug residue in honey simultaneously |
| AU2020100921A4 (en) * | 2020-06-02 | 2020-07-09 | Dali University | Preparation And Quality Control Method Of Periplaneta Americana Linn. Anti-Tumor Capsule |
Non-Patent Citations (2)
| Title |
|---|
| 柱前衍生化HPLC法测定天麻提取物中18种氨基酸含量;蒋清伟;王威;刘姝畅;张印广;王迷娟;张猛;;中国新药杂志(第15期);全文 * |
| 美洲大蠊乙醇提取物中原儿茶酸超高效液相色谱定量分析方法的建立;郭昕;巩荣艳;朱娟娟;曾成娟;沈咏梅;岳碧松;李静;;四川动物(第02期);全文 * |
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