CN115389671A - Method for detecting aloesin content in fresh aloe juice and application thereof - Google Patents
Method for detecting aloesin content in fresh aloe juice and application thereof Download PDFInfo
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- CN115389671A CN115389671A CN202211049910.XA CN202211049910A CN115389671A CN 115389671 A CN115389671 A CN 115389671A CN 202211049910 A CN202211049910 A CN 202211049910A CN 115389671 A CN115389671 A CN 115389671A
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- HKIKAXXIWJHWLY-QEVGBQTESA-N Aloesin Natural products O=C(CC=1Oc2c([C@H]3[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O3)c(O)cc(C)c2C(=O)C=1)C HKIKAXXIWJHWLY-QEVGBQTESA-N 0.000 title claims abstract description 110
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a method for detecting aloesin content in fresh aloe juice and application thereof. The detection method has the advantages that the aloesin peak time is 3-4 min, the aloesin detection quantitative limit can reach 0.001mg/g, the sensitivity is high, the reproducibility is good, the precision is high, the detection standard of the aloe juice is improved, the quality of the aloe medicine is effectively controlled, and the application safety of the aloe medicine is guaranteed.
Description
Technical Field
The invention relates to the field of medicine invention, in particular to a method for detecting aloesin content in fresh aloe juice and application thereof.
Background
The dahurian rose root acne eliminating ointment is a fist product of Guizhou Liangji pharmaceutical industry Limited company, and the prescription of the acne eliminating ointment consists of lightyellow sophora root, fresh aloe juice, rose, perfoliate knotweed herb, borneol, peppermint oil and the like, has the effects of clearing heat, drying dampness, killing parasites and relieving itching, and is mainly used for acne, skin pruritus, eczema and solar dermatitis. The fresh aloe juice in the formula is used as one of main components of the rosa roxburghii acne eliminating cream, is rich in various amino acids, natural whitening and moisturizing factors, monosaccharides, polysaccharides, vitamins, mineral substances, aloe emodin, anthragnonide and other natural substances, has the effects of whitening, moisturizing, resisting aging, diminishing inflammation, sterilizing, preventing mosquito bites, relieving pain, expelling toxins, preventing sun, preventing eczema, repairing damaged skin and the like, and has obvious curative effect on treating the annoying acnes and acnes of teenagers and marks left by the acnes and the acnes. Meanwhile, the literature reports that contact dermatitis (such as rash, erythema, blisters, swelling) occurs after the aloe vera and the preparation thereof are used. The rosa laevigata acne eliminating paste is monitored after being marketed and shows symptoms or allergic reactions such as irritation, burning, desquamation, skin red swelling, pruritus, contact dermatitis (such as erythema, pimple and blister), skin irritation and the like, and the allergic reaction is probably caused by that aloesin in the chemical component of aloe has photosensitivity and can induce a certain allergic reaction of an organism. Because the production and feeding of each batch of the rhaponticum uniflorum acne-eliminating cream can not ensure that the basic source and the production place of the aloe are the same, this also leads to differences in the amount of aloesin in each batch of product, and in order to ensure the quality of the product, the amount of aloesin in the fresh aloe juice of the raw material must be controlled.
Document 1: identifying chemical components of Aloe, analyzing UPLC fingerprint, and selecting Chentong, zhiyangcxia, lifengxia, qinling Ling, jiahongmei, mabaiping, and Zhongmei; the method comprises the steps of collecting aloe chromatographic mass spectrum data by using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), and performing qualitative analysis; the UPLC chromatographic conditions were: a Waters CORTECS T3 (100 mm × 2.1mm,1.6 μm) chromatographic column, with a column temperature of 40 ℃, a detection wavelength of 295nm, acetonitrile (0.1% formic acid) -water (0.1% formic acid) as a mobile phase, a volume flow of 0.3mL/min, gradient elution; the Q-TOF/MS conditions are that an ESI ion source is adopted, an anion scanning mode is adopted, the capillary voltage is 2.3kV, and the ion source temperature is 100 ℃; UPLC characteristic fingerprint graphs of 15 batches of commercially available aloe medicinal materials are established by using software of traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).
The technical problem of document 1 is: (1) Only qualitative analysis is considered, quantitative analysis of aloesin is not mentioned, and the quality of the aloe medicinal material cannot be effectively controlled; (2) The detected quantity of aloesin is high, the aloesin content cannot be effectively controlled, and the potential safety hazard of medication is easily caused; (3) long peak-out time, more impurity peaks and poor resolution; and (4) UPLC-Q-TOF/MS is adopted for detection, so that the detection cost is high.
In order to solve the problems and effectively control the safety of the acne-eliminating aloe cream in China company, the invention team researches a large number of experiments, and researches a method for detecting the aloesin content in fresh aloe juice by using HPLC (high performance liquid chromatography) by carrying out series of researches on an extraction solvent, an extraction mode, extraction time, a mobile phase and a proportion, a wavelength, a column temperature, a flow rate, a chromatographic column and the like of a test solution in a detection method, wherein the method detects that the aloesin peak time is 3-4 min, has high detection rate, has the advantages of high aloesin detection rate limit reaching 0.001mg/g, good reproducibility, good separation effect, high precision and the like, improves the detection standard of the aloe juice, effectively controls the quality of aloe drugs and ensures the use safety of the acne-eliminating aloe cream.
Disclosure of Invention
The invention aims to provide a method for detecting the content of aloesin in fresh aloe juice.
The content detection method comprises the following steps:
(1) Preparation of control solutions: accurately weighing aloesin reference substance 9.0-10.0 mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 15-35 g of fresh aloe juice, adding 50-90% of methanol water 50ml, carrying out ultrasonic treatment for 5-40 min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.05-0.15% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Preferably, the first and second liquid crystal materials are,
the control solution was prepared as follows: precisely weighing 9.2-9.8 mg of aloesin reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking up to obtain the aloesin reference substance.
It is further preferred that the first and second liquid crystal compositions,
the control solution was prepared as follows: precisely weighing aloesin control substance 9.5mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking.
Preferably, the first and second liquid crystal materials are,
the preparation of the test solution comprises the following steps: precisely weighing 25-35 g of fresh aloe juice, adding 50ml of 60-80% methanol water, carrying out ultrasonic treatment for 20-30 min, and filtering to obtain the aloe juice.
It is further preferred that the first and second liquid crystal compositions,
the preparation of the test solution comprises the following steps: precisely weighing 35g fresh Aloe juice, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
Preferably, the first and second liquid crystal materials are,
the chromatographic conditions and the system applicability test are as follows: octadecaneSilane bonded silica gel is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.08-0.12% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
It is further preferred that the first and second liquid crystal compositions,
the chromatographic conditions and the system applicability test are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.09-0.10% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
It is still further preferred that the first and second substrates,
the chromatographic conditions and the system applicability test are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.10% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
The detection limit of the aloesin is 0.001mg/g.
The detection method disclosed by the invention is applied to the aloe medicines.
The invention has the following advantages:
1. according to the invention, through the dissolution condition of the aloe juice and the detection rate of aloesin after being processed by an upper machine, the solvent of the obtained preferable test solution is 80% methanol, the extraction mode is ultrasonic, and the extraction time is 30min.
2. The chromatographic conditions of the invention are screened by mobile phase, chromatographic column, column temperature, flow rate, sample injection volume and the like, and the optimal chromatographic conditions are finally determined by the highest number of aloesin theoretical plates: eighteen in weightAlkyl silane bonded silica gel is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight A,95 to 80% by weight B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate was 1.0ml/min.
3. According to the detection methodology of the invention, the number of theoretical plates of aloesin peak is larger than 2000, continuous sample injection is carried out for 5 needles, RSD of main peak area is 0.1%, and the method accords with the verification standard.
4. The specificity verification result of the invention shows that 10 mul of blank solution, reference solution and test solution are respectively taken and respectively injected into a liquid chromatograph, and chromatogram is recorded, so that the blank solution has no interference at aloesin peak position.
5. The linear relationship verification result of the invention shows that the aloesin solution has good linear relationship in the concentration range of 52.430 mu g/ml-157.290 mu g/ml, and the correlation coefficient r 2 Is 1.
6. The durability verification result shows that when one of the chromatographic conditions of the column temperature and the chromatographic column is changed, the content of the sample solution and the content of the initial condition are changed within the standard range of +/-0.02 mg/g, the system applicability meets the standard specification, and the method has no obvious influence on the analysis method due to the slight change of the chromatographic conditions and has good durability.
7. According to the detection results of different aloe juice sample solutions, the content of aloesin in the aloe juice samples processed in different production places and different processing modes is lower, but the detection rate and the quantitative limit of the method are obviously higher than those of the reference 1, and the quantitative limit of the aloesin can reach 0.001mg/g, so that the detection method is higher in sensitivity, can well detect the content of trace aloesin in the aloe juice, provides a reference basis for aloe evaluation, and ensures the safety of the acne-eliminating paste medicine.
Description of the drawings
FIG. 1 inspection of extraction solvent methanol concentration;
FIG. 2 inspection of extraction patterns;
FIG. 3 Aloesin control solution profile (method 1);
FIG. 4 Aloesin control solution profile (method 2);
FIG. 5 Aloesin control solution profile (method 3);
FIG. 6 shows wavelength selective spectra for aloesin detection;
FIG. 7 is a system suitability map (control solution-1);
FIG. 8 is a system applicability profile (control solution-2);
FIG. 9 System suitability map (control solution-3);
FIG. 10 is a system suitability map (control solution-4);
FIG. 11 System suitability map (control solution-5);
FIG. 12 specificity profile (blank solution);
FIG. 13 specificity profile (aloesin control);
FIG. 14 a specificity profile (test article);
FIG. 15 is a line graph of aloesin;
FIG. 16 Linear relationship investigation plot (Linear-50%);
FIG. 17 Linear relationship investigation map (Linear-80%);
FIG. 18 Linear relationship investigation diagram (Linear-100%);
FIG. 19 Linear relationship investigation profile (Linear-120%);
FIG. 20 Linear relationship investigation profile (Linear-150%).
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
Example 1
(1) Preparation of control solutions: precisely weighing aloesin reference substance 9.5mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 35g fresh Aloe juice, adding 50ml methanol, ultrasonic treating for 30min, and filtering to obtain the final product;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as fillerFilling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 2
(1) Preparation of control solutions: accurately weighing aloesin reference substance 9.2mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 15g fresh Aloe juice, adding 50ml 50% methanol water, ultrasonic treating for 5min, and filtering to obtain the final product;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.05% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight A,95 to 80% by weight B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 3
(1) Preparation of control solutions: precisely weighing aloesin reference substance 9.8mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 35g of fresh aloe juice, adding 50ml of 90% methanol water, performing ultrasonic treatment for 40min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, and 0.15 is used% phosphoric acid solution as mobile phase B, gradient elution: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 4
(1) Preparation of control solutions: accurately weighing aloesin reference substance 9.4mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 20g of fresh aloe juice, adding 50ml of 60% methanol water, performing ultrasonic treatment for 10min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.08 percent phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 5
(1) Preparation of control solutions: precisely weighing aloesin reference substance 9.6mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 30g of fresh aloe juice, adding 50ml of 70% methanol water, performing ultrasonic treatment for 35min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.12% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% by weight of A,80% by weight of B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 6
(1) Preparation of control solutions: precisely weighing aloesin reference substance 9.7mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 25g of fresh aloe juice, adding 50ml of 75% methanol water, carrying out ultrasonic treatment for 35min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.07% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B; 15-25min, 20-A, 80-B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 7
(1) Preparation of control solutions: precisely weighing aloesin reference substance 9.5mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 30g fresh Aloe juice, adding 50ml 60% methanol water, ultrasonic treating for 30min, and filtering to obtain the final product;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is calculated according to aloesin peakNot less than 2000;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Experimental example: in order to prove the scientificity and feasibility of the detection method, the following experimental research of methodology is carried out:
1 test materials, see table 1.
Table 1 test materials table
2 reagents and reagents, see table 2.
TABLE 2 test drugs and reagent tables
3 screening of aloesin content detection method
3.1 screening of test solution preparation method
3.1.1 extraction solvent
The method comprises the following steps: weighing aloe juice about 15g, adding methanol 15ml, shaking, dissolving aloe juice completely, but difficult to filter, and not beneficial to detection.
The second method comprises the following steps: weighing about 35g of aloe juice, adding 35ml of 90% methanol, and shaking to dissolve almost completely, but is difficult to filter and is not suitable for detection.
The third method comprises the following steps: weighing aloe juice 35g, adding 80% methanol 35ml, shaking, dissolving easily, filtering, and detecting trace amount.
The method four comprises the following steps: weighing about 35g of Aloe juice, adding 35ml of 70% methanol, 60% methanol, 50% methanol respectively, shaking to make Aloe juice almost insoluble, and no aloesin detected after processing.
As a result: when the sample solution is prepared from 80% methanol, the aloe juice is easily dissolved, and can be filtered to obtain trace amount. Therefore, 80% methanol is the preferred extraction solvent, and the results are shown in FIG. 1.
3.1.2 examination of extraction methods and extraction times
Weighing aloe juice 35g, adding 80% methanol 50ml, shaking, dissolving aloe juice easily, filtering, detecting trace amount, and performing ultrasonic treatment.
Weighing aloe juice about 35g, adding 80% methanol 50ml, performing ultrasonic treatment for 5min, 10min, 15min, 20 min, 25min, 30min, 35min and 40min to prepare two parts in parallel, filtering, and taking the subsequent filtrate for sample injection analysis, wherein the results are shown in figure 2 and table 3:
TABLE 3 ultrasonic time survey table
As a result: as can be seen from fig. 2 and table 3, the sample solution was prepared, and the sonication time was preferably 30 minutes.
3.2 screening of chromatographic conditions
Aloesin control solution: precisely weighing aloesin control substance 9.5mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the final product with concentration of 186.2000 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30 minutes, and filtering to obtain the aloe juice.
Chromatographic conditions are as follows: see table 4.
TABLE 4 chromatographic conditions (method 1)
TABLE 5 chromatogram Peak Table (method 1)
As a result: see fig. 3, table 5. As can be seen from fig. 3 and table 5, aloesin showed a peak time of 2.3min and a peak time of too early, and the methanol ratio was reduced to 40% for the following examination.
Aloesin control solution: precisely weighing aloesin control substance 9.5mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the final product with concentration of 186.2000 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30 minutes, and filtering to obtain the aloe juice.
Chromatographic conditions are as follows: see table 6.
TABLE 6 chromatographic conditions (method 2)
TABLE 7 chromatogram Peak Table (method 2)
As a result: see fig. 4, table 7. As can be seen from FIG. 4 and Table 7, aloesin was found to have a poor peak shape and a peak appearance time of 2min, and no aloesin was detected in the sample solution. Therefore, this method is not investigated below.
Aloesin control solution: precisely weighing aloesin control substance 9.5mg, placing in 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the final product with concentration of 186.2000 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30 minutes, and filtering to obtain the aloe juice.
Chromatographic conditions are as follows: see table 8.
TABLE 8 chromatographic conditions (method 3)
TABLE 9 chromatogram Peak Table (method 3)
As a result: see fig. 5, table 9. As can be seen from FIG. 5 and Table 9, the number of theoretical plates of aloesin under the chromatographic conditions was high, and the method was used for detecting aloesin in aloe juice.
3.3 determination of the detection wavelength
Precisely weighing 35g of fresh aloe juice, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30min, and filtering to obtain the final product, wherein the absorption spectrum within the range of 190-800 nm is recorded as shown in figure 6.
As a result, at 297nm, aloesin has a maximum absorption, a high peak response and a smooth baseline, and therefore 297nm was chosen as the wavelength for determining the aloesin content in the fresh aloe juice.
4. Methodology investigation
4.1 System applicability
4.1.1 implementation step
Aloesin control solution: precisely weighing 0.010g of aloesin control substance, placing into a 100ml brown measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and making into 98 μ g/ml solution.
Injecting 10 μ l of aloesin reference substance solution into liquid chromatograph, repeatedly injecting sample for 5 times, recording chromatogram, and calculating peak area RSD.
4.1.2 acceptance criteria
In the chromatogram of the control solution, the number of theoretical plates of aloesin peak should not be less than 2000;
RSD of 5-needle repeated sample injection peak area of the reference solution is not more than 2.0%;
4.1.3 the results are shown in Table 10, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11.
TABLE 10 System suitability results
As can be seen from Table 10, the number of theoretical plates of the aloesin peak is greater than 2000, and the RSD of the main peak area is 0.1% after continuous sample injection of 5 needles, which meets the verification standard.
4.2 specificity
4.2.1 implementation step
Blank solution: methanol
Control solution: precisely weighing 0.010g of aloesin control substance, placing into a 100ml brown measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and making into 98 μ g/ml solution.
Test solution: precisely weighing about 35g of aloe juice, placing into a 150ml conical flask, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30 minutes, and filtering to obtain the aloe juice.
Respectively taking 10 μ l of blank solution, reference solution and sample solution, respectively, injecting into a liquid chromatograph, recording chromatogram, and calculating peak area.
Formula for calculation
In the formula:
C to pair : the unit is the concentration of aloesin reference substance solution, and the unit is mug/ml;
A sample (A) : is the peak area of aloesin in the test solution;
A to pair : the peak area average value of the main peak in the 5-needle aloesin reference substance solution is shown;
W sample (A) : weighing the sample solution in unit g;
50: to prepare the diluted volume of the test solution, unit ml.
4.2.2 acceptance criteria
In the blank solution chromatogram, the peak position of aloesin should not interfere.
4.2.3 results, see fig. 12, 13, 14.
As can be seen from fig. 12, 13 and 14, the blank solution did not interfere with aloesin peak positions.
4.3 Linearity and Range
4.3.1 implementation step
Control stock solutions: precisely weighing 0.010g of aloesin control, placing into a 10ml brown measuring flask, dissolving with methanol, diluting to scale, shaking, and making into 980 μ g/ml solution as aloesin control stock solution.
Precisely measuring control stock solutions 0.5ml,0.8ml,1.0ml,1.2ml and 1.5ml respectively, placing in 10ml brown measuring flask, diluting with methanol to scale, shaking to obtain control solutions with control concentrations of 49 μ g/ml,78.4 μ g/ml,98 μ g/ml,117.64 μ g/ml and 147 μ g/ml respectively.
And (3) respectively injecting 10 mu l of the solution into a chromatograph, recording a chromatogram, and performing linear regression on the sample injection concentration by peak area according to a least square method to obtain a standard curve.
4.3.2 acceptance criteria
r 2 More than or equal to 0.995, namely the aloesin solution has good linear relation in the concentration range of 49-147 mu g/ml.
4.3.3 the results are shown in Table 11, FIG. 15, FIG. 16, FIG. 17, FIG. 18, FIG. 19, FIG. 20.
TABLE 11 Linear results
As can be seen from Table 11, the aloesin solution showed a good linear relationship in the concentration range of 52.430. Mu.g/ml to 157.290. Mu.g/ml, and the correlation coefficient r 2 Is 1.
4.4 durability
4.4.1 implementation step
The tolerance of aloe juice with aloesin content should be examined according to the analysis method. Parameters that look at small variations include: column temperature, chromatographic column. Changing certain parameter, keeping other parameters unchanged, preparing reference solution and test solution according to analysis method, and determining aloesin content according to Table 12.
TABLE 12 analysis conditions Change Table
4.4.2 acceptance criteria
Changing the analysis conditions:
in the chromatogram of the control solution, the number of theoretical plates of aloesin peak should not be less than 2000;
RSD of 5-needle repeated sample injection peak area of the reference solution is not more than 2.0%;
the test solution has an aloesin content within 0.02mg/g (absolute) of the initial conditions.
4.4.3 results
The number of theoretical plates of the main peak in the reference solution is larger than 2000 specified in the standard, 5 needles are continuously injected, and the RSD of the main peak area is not larger than 2.0 percent. The results of content durability under each color spectrum condition are shown in tables 13, 14 and 15.
TABLE 13 initial Condition durability test Table
TABLE 14 column temp 35 deg.C durability survey table
Table 15 survey table for changing chromatographic column durability
As can be seen from tables 13, 14 and 15, when one of the chromatographic conditions of the column temperature and the chromatographic column is changed, the content change of the sample solution and the content change of the initial condition are within the standard range of +/-0.02 mg/g, and the system applicability meets the standard specification, so that the method has no obvious influence on the analysis method due to the slight change of the chromatographic conditions, and the method has good durability.
4.5 conclusion, see table 16.
Table 16 analytical methods verification summary table
As can be seen from table 16, the method of analysis of the aloesin content of fresh aloe juice was validated for system suitability, specificity, linearity and range, and durability. The result shows that the method for analyzing the aloesin content can meet the detection requirement of the aloesin content.
4.6 Aloe juice assay comparison
4.6.1 fresh Aloe juice sample solution and cooked Aloe juice sample solution processed by different extraction methods at different places of origin were used to determine aloesin content according to methods of "method 3" and "document 1" under "item 3.2", respectively, and the results are shown in Table 17.
TABLE 17 table for measuring contents of fresh aloe juice and cooked aloe juice
4.6.2 fermented and boiled aloe juice sample solution and the rosa luo acne eliminating cream processed by different extraction modes in different producing areas are taken, and the aloesin content is detected according to a method 3 and a method of a document 1 under the item 3.2 respectively, and the result is shown in a table 18.
TABLE 18 measurement of fermented Aloe juice
As can be seen from the results of the detection of the aloe juice sample solutions in tables 17 and 18, the aloe juice samples treated in different production areas and different treatment modes have lower aloesin content, but the detection rate and the limit of quantitation of the method are obviously higher than those of the comparative reference 1, and the limit of quantitation of the aloesin in the invention can reach 0.001mg/g, so that the detection method has higher sensitivity, can well detect the trace amount of aloesin in the aloe juice, provides a reference for aloe evaluation, and ensures the safety of the acne-eliminating paste medicine.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A method for detecting the aloesin content in fresh aloe juice is characterized in that the content detection method comprises the following steps:
(1) Preparation of control solutions: precisely weighing 9.0-10.0 mg of aloesin reference substance, placing in a 50ml brown measuring flask, adding methanol for dissolving, diluting to scale, and shaking uniformly to obtain;
(2) Preparation of a test solution: precisely weighing 15-35 g of fresh aloe juice, adding 50-90% methanol water 50ml, carrying out ultrasonic treatment for 5-40 min, and filtering to obtain the aloe juice;
(3) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.05-0.15% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B; 15-25min, 20-A, 80-B; the detection wavelength is 297nm,the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak;
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
2. The method for detecting aloesin content in fresh aloe juice according to claim 1, wherein the control solution of step (1) is prepared by: precisely weighing 9.2-9.8 mg of aloesin reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking up to obtain the aloesin reference substance.
3. The method for detecting aloesin content in fresh aloe juice according to claim 2, wherein the control solution of step (1) is prepared by: precisely weighing aloesin control substance 9.5mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking.
4. The method of claim 1, wherein the test solution of step (2) is prepared by: precisely weighing 25-35 g of fresh aloe juice, adding 50ml of 60-80% methanol water, carrying out ultrasonic treatment for 20-30 min, and filtering to obtain the aloe juice.
5. The method of claim 4, wherein the test solution of step (2) is prepared by: precisely weighing 35g fresh Aloe juice, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
6. The method for detecting aloesin content in fresh aloe juice according to claim 1, wherein the chromatographic conditions and system suitability test in step (3) is as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.08-0.12% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight A,95 to 80% by weight B; E15E25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
7. The method for detecting aloesin content in fresh aloe juice according to claim 6, wherein the chromatographic conditions and system applicability test of the step (3) is: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is taken as a mobile phase A, 0.09-0.10% phosphoric acid solution is taken as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight A,95 to 80% by weight B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
8. The method of claim 7, wherein the chromatographic conditions and system suitability test of step (3) is as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Feilomen Titank C 18 250X 4.6mm,5 μm; acetonitrile is used as a mobile phase A, 0.10% phosphoric acid solution is used as a mobile phase B, and gradient elution is as follows: 0 to 15min,5 to 20% by weight, A,95 to 80% by weight, B;15 to 25min,20% A,80% B; the detection wavelength is 297nm, and the column temperature is 30 ℃; the flow rate is 1.0ml/min; the number of theoretical plates is not less than 2000 calculated according to aloesin peak.
9. The method of claim 1, wherein the aloesin detection limit is 0.001mg/g.
10. The method for detecting aloesin content in fresh aloe juice as claimed in claim 1, wherein the detection method is applied to the rosa laevigata acne-eliminating cream drug and the fresh aloe juice.
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| CN115406992A (en) * | 2022-08-30 | 2022-11-29 | 贵州良济药业有限公司 | Method for determining content of aloenin A in aloe medicinal material by using HPLC |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112415116A (en) * | 2020-11-27 | 2021-02-26 | 海南医学院 | LC-MS/MS determination method for aloesin in rat plasma |
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| CN112415116A (en) * | 2020-11-27 | 2021-02-26 | 海南医学院 | LC-MS/MS determination method for aloesin in rat plasma |
Non-Patent Citations (5)
| Title |
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| MICHAEL ZAHN 等: "A Reversed-phase High-performance Liquid Chromatographic Method for the Determination of Aloesin, Aloeresin A and Anthraquinone in Aloe ferox", HYTOCHEMICAL ANALYSIS, vol. 19 * |
| NOBUYUKI OKAMURA 等: "High-performance liquid chromatographic determination of phenolic compounds in Aloe species", JOURNAL OF CHROMATOGRAPHY A, vol. 746, pages 225 - 231, XP004020361, DOI: 10.1016/0021-9673(96)00342-1 * |
| 张慧君, 张新申, 罗仓学, 蒋晓萍, 宋秘钊: "几种树脂对芦荟苦素的吸附性能", 化学研究与应用, no. 02 * |
| 曹元元;郇楠;谢云飞;汪何雅;钟英;姚卫蓉;钱和;: "4种物理条件对库拉索芦荟凝胶中芦荟苷的影响", 食品科技, no. 06 * |
| 李田甜;宋粉云;廖紫琼;谢智勇;: "木立芦荟粉HPLC指纹图谱及化学模式研究", 广东药科大学学报, no. 01 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115406992A (en) * | 2022-08-30 | 2022-11-29 | 贵州良济药业有限公司 | Method for determining content of aloenin A in aloe medicinal material by using HPLC |
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