CN115406992A - Method for determining content of aloenin A in aloe medicinal material by using HPLC - Google Patents
Method for determining content of aloenin A in aloe medicinal material by using HPLC Download PDFInfo
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- CN115406992A CN115406992A CN202211058913.XA CN202211058913A CN115406992A CN 115406992 A CN115406992 A CN 115406992A CN 202211058913 A CN202211058913 A CN 202211058913A CN 115406992 A CN115406992 A CN 115406992A
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Abstract
The invention relates to a method for determining aloenin A content in an aloe medicinal material by using HPLC, the detection method has the advantages that the aloenin A peak output time is 5.09min and 6.43min respectively, the number of theoretical plates is 3793 and 5579 respectively, the separation degree is 4.0, the detection limit of the aloenin A can reach 0.012mg/g, the aloenin A content detection method can be used for micro detection of the aloenin A in the aloe medicinal material, the precision is high, the reproducibility is good, the separation effect is good, and the safety of the aloe medicinal material can be effectively controlled.
Description
Technical Field
The invention relates to the field of medicine invention, in particular to a method for determining aloenin A content in an aloe medicinal material by using HPLC.
Background
Aloe is a traditional common traditional Chinese medicine, and has been fully applied in the fields of food, medicine, beauty and health care, etc. With the progress of science and technology, people pay more and more attention to beauty and health care. The aloe is rich in natural vitamins, amino acids and other nutrient substances beneficial to human body, and aloe is rich in aloe polysaccharide, phenolic compounds and other effective components for beautifying and protecting skin. The active ingredients of the aloe make the beauty function of the aloe more prominent, and people pay more and more attention to the beauty and health care aspect, which results in that various aloe beauty and health care products are produced endlessly. However, as people research aloe more and more deeply, adverse reaction events caused by aloe in beauty and health care application often react with symptoms such as skin redness and swelling, pruritus, contact dermatitis (such as erythema, pimple and blister), skin irritation and the like or allergic reaction after the aloe is used, and the allergic reaction is probably caused by that the chemical component aloenin A in the aloe has photosensitivity and can induce a certain allergic reaction of organisms.
Document 1: chemical component identification and UPLC fingerprint analysis of aloe medicinal material, chen Tongtong, nafii, li Fengxia, qin Lingling, gu Gongmei, ma Baiping, zou Zhongmei; discloses the identification of main chemical components in aloe, the establishment of UPLC characteristic fingerprint, and the evaluation of the quality of aloe medicinal materials of 15 batches sold in the market. The method adopts ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) to collect aloe chromatographic mass spectrometry data and perform 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 fingerprints of 15 batches of commercially available aloe medicinal materials are established by using software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and the common peaks of aloin A, aloin B, aloenin B, aloinoside A and aloinoside B in the fingerprints can be used for distinguishing plant-based atoms of the aloe medicinal materials.
The technical problem of document 1 is: (1) Only the qualitative analysis for identifying the main chemical components in the aloe is examined, and the safety of the aloe medicinal material cannot be accurately and effectively controlled; (2) When the UPLC characteristic fingerprint spectrum method is used for measuring the content of the aloenin A, the detection rate is extremely low, the detection limit is high, the actual content of the aloenin A with lower content cannot be reflected, and the potential safety hazard of the aloe drug is easily caused; (3) poor repeatability, multiple impurity peaks and poor resolution; and (4) UPLC-Q-TOF/MS is adopted for detection, so that the detection cost is high.
Aloe is also one of the main raw materials of a fist product, namely the acne eliminating cream from the false Chinese medicine industry Limited company in Guizhou, and the quality safety of the product is more important, but at present, no quantitative detection method for aloenin A in aloe medicinal materials exists, so that the safety of the acne eliminating cream from the false Chinese angelica cannot be better controlled.
Aiming at the technical problems, the content of aloenin A in the aloe medicinal material needs to be effectively controlled, a large amount of experimental researches are carried out by an invention team, a method for detecting the content of aloenin A in the aloe medicinal material by using HPLC (high performance liquid chromatography) is researched by carrying out series researches on an extraction solvent, an extraction mode, extraction time, a mobile phase, a ratio, a wavelength, a chromatographic column and the like of a sample solution to be detected, the peak time of the aloenin A in the method is respectively 5.09min and 6.43min, the theoretical plate number is 3793 and 5579, and the separation degree is 4.0, when the chromatographic condition is used for detecting the aloenin A in the aloe medicinal material, the detection limit of the aloenin A in the aloe medicinal material can reach 0.012mg/g, the accuracy is high, the reproducibility is good, the separation effect is good, the content of the aloenin A in the aloe medicinal material can be effectively detected, and the safety of the acne eliminating paste with the rhapontes is ensured.
Disclosure of Invention
The invention aims to provide a method for determining the content of aloenin A in an aloe medicinal material by using HPLC, and the determination method can be applied to determination of the rosa multiflora acne eliminating cream medicament and fresh aloe juice.
The determination method comprises the following steps:
(1) Preparation of control solutions: accurately weighing 8-12 mg of the aloenin A reference substance into a 50ml brown measuring flask, adding methanol to dissolve and dilute the aloenin A reference substance to a scale, and shaking up to obtain the aloenin A reference substance;
(2) Preparation of a test solution: precisely weighing 30-40 g of fresh aloe juice, putting the fresh aloe juice into a 150ml conical flask, adding 50ml of 60-90% methanol water, carrying out ultrasonic treatment for 20-40 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking methanol-0.5-1.5% acetic acid solution with the proportion of 40-60; the detection wavelength is 296nm, and the column temperature is 28-32 ℃; the flow rate is 0.8 to 1.2ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: precisely sucking 5-10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.
Preferably, the first and second liquid crystal materials are,
the control solution was prepared as follows: precisely weighing 9-11 mg of aloenin A reference substance, placing the aloenin A reference substance into a 50ml brown measuring flask, adding methanol to dissolve and dilute the aloenin A reference substance to a scale, and shaking up the aloenin A reference substance to obtain the aloenin A reference substance.
It is further preferred that the first and second liquid crystal compositions,
the control solution was prepared as follows: accurately weighing 10.12mg of aloenin A reference substance, placing into 50ml brown measuring flask, adding methanol to dissolve, diluting 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 32-38 g of fresh aloe juice, adding 50ml of 65-85% methanol water, carrying out ultrasonic treatment for 25-35 min, and filtering to obtain the aloe juice.
It is further preferred that the first and second liquid compositions are,
the preparation of the test solution comprises the following steps: precisely weighing 35.15g 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: to be provided withOctadecylsilane chemically bonded silica is used as a filler; the chromatographic column is Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking methanol-0.9-1.1% acetic acid solution with the proportion of 45-55; the detection wavelength is 296nm, and the column temperature is 29-31 ℃; the flow rate is 0.9-1.1 ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-1.0% acetic acid solution with the proportion of 50; the detection wavelength is 296nm, 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 aloenin A peak.
The chromatographic column Shijin-Pack scanner C of the invention 18 250X 4.6mm,5 μm, and may be Philomo Titank C 18 250×4.6mm，5μm。
The aloenin A of the invention has the peak time of 5.09min and 6.43min respectively, the theoretical plate numbers of 3793 and 5579 respectively, and the separation degree of 4.0.
Advantageous effects
1. According to the invention, through the investigation of chromatographic conditions, the result shows that the aloenin A peak time is 5.09min and 6.43min respectively, the theoretical plate number is 3793 and 5579 respectively, and the separation degree is 4.0, so that the chromatographic conditions can be effectively used for detecting the content of aloenin A in the aloe medicinal material, the reproducibility is good, the separation effect is good, the accuracy and the precision are high, the effective content of aloenin A in the aloe medicinal material can be effectively controlled, and the safe use of the aloe medicinal material is easy to control.
2. According to the invention, through the investigation of the preparation method of the test solution, the result shows that the preparation method takes 80% methanol as the solvent, adopts ultrasonic extraction mode and takes 30min of extraction time, the aloe dissolution condition is good and the detection rate of aloenin A is high.
3. The detection methodology of the invention verifies that the theoretical plate number of the aloenin A is more than 2000, 5 needles are continuously injected, and the RSD of the main peak area is 0.1 percent and meets 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 the peak position of aloenin A.
5. The linear relationship verification result of the invention shows that the aloenin A solution has good linear relationship in the concentration range of 49-147, and the correlation coefficient r 2 Is 0.9998.
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 aloenin A in aloe juice samples treated in different production places and different treatment modes is lower, but the detection rate and the quantitative limit of the method are obviously higher than those in reference 1, and the quantitative limit of aloesin in the invention can reach 0.012mg/g, so that the detection method has higher sensitivity, can well detect the trace content of aloenin A in the aloe juice, provides a reference basis for aloe evaluation, and ensures the safety of the acne-eliminating paste medicine.
Drawings
FIG. 1 inspection of extraction solvent methanol concentration;
FIG. 2 inspection of the manner of extraction;
FIG. 3 Aloining A control solution profile (method 1);
FIG. 4 Aloining A control solution profile (method 2);
FIG. 5 Aloining A control solution profile (method 3);
FIG. 6 Aloining A control solution profile (method 4);
FIG. 7 Aloining A control solution profile (method 5);
FIG. 8 is a wavelength selective spectrum of aloenin A detection;
FIG. 9 System suitability map (control solution-1);
FIG. 10 is a system suitability map (control solution-2);
FIG. 11 System suitability map (control solution-3);
FIG. 12 System suitability map (control solution-4);
FIG. 13 System suitability map (control solution-5);
FIG. 14 specificity profile (blank solution);
FIG. 15 is a specific spectrum (aloenin A control);
FIG. 16 is a specific map (test article);
FIG. 17 is a line graph of aloenin A;
FIG. 18 Linear relationship investigation plot (Linear-50%);
FIG. 19 Linear relationship investigation profile (Linear-80%);
FIG. 20 Linear relationship investigation diagram (Linear-100%);
FIG. 21 Linear relationship investigation plot (Linear-120%);
FIG. 22 Linear relationship investigation map (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: accurately weighing 10.12mg of aloenin A reference substance, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-1.0% acetic acid solution with the proportion of 50; the detection wavelength is 296nm, 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 aloenin A 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 8mg of aloenin A reference substance, placing into a 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, placing the fresh aloe juice into a 150ml conical flask, adding 50ml of 60% methanol water, carrying out ultrasonic treatment for 20 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-0.5% acetic acid solution with the proportion of 40; the detection wavelength is 296nm, and the column temperature is 28 ℃; the flow rate is 0.8ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: precisely sucking 5 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: accurately weighing 12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 40g of fresh aloe juice, placing the fresh aloe juice into a 150ml conical flask, adding 50ml of 90% methanol water, carrying out ultrasonic treatment for 40 minutes, 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 Shim-Pack Scepter C 18 250X 4.6mm,5 μm; taking a methanol-1.5% acetic acid solution with the proportion of 60; the detection wavelength is 296nm, and the column temperature is 32 ℃; the flow rate is 1.2ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A 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: precisely weighing 9mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 32g of fresh aloe juice, placing the fresh aloe juice into a 150ml conical flask, adding 50ml of 65% methanol water, carrying out ultrasonic treatment for 25 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-0.9% acetic acid solution with the proportion of 45; the detection wavelength is 296nm, and the column temperature is 29 ℃; the flow rate is 0.9ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: precisely sucking 6 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 11mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparing a test solution: precisely weighing 38g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 85% methanol water, performing ultrasonic treatment for 35 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-1.1% acetic acid solution with the ratio of 55; the detection wavelength is 296nm, and the column temperature is 31 ℃; the flow rate is 1.1ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: precisely sucking 8 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 9mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparing a test solution: precisely weighing 36g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 75% methanol water, performing ultrasonic treatment for 32 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-0.7% acetic acid solution with the proportion of 48; the detection wavelength is 296nm, 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 aloenin A 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: accurately weighing aloenin A reference substance 10.12mg, placing into 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% 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; taking a methanol-1.0% acetic acid solution with the proportion of 50; the detection wavelength is 296nm, 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 aloenin A 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 8
(1) Preparation of control solutions: accurately weighing 8mg of aloenin A reference substance, placing into 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, placing the fresh aloe juice into a 150ml conical flask, adding 50ml of 60% methanol water, carrying out ultrasonic treatment for 20 minutes, 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; 60 of methanol-0.5% acetic acid solution as mobile phase; the detection wavelength is 296nm, and the column temperature is 28 ℃; the flow rate is 0.8ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 9
(1) Preparation of control solutions: accurately weighing 12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking;
(2) Preparation of a test solution: precisely weighing 40g of fresh aloe juice, placing into a 150ml conical flask, adding 50ml of 90% methanol water, performing ultrasonic treatment for 40 minutes, 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; taking a methanol-1.5% acetic acid solution with the proportion of 60; the detection wavelength is 296nm, and the column temperature is 32 ℃; the flow rate is 1.2ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A 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.
Experimental example: in order to prove the scientificity and the concordance of the detection method, the following methodology experimental research 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 investigation of method for detecting content of aloenin A
3.1 examination of preparation method of test solution
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 aloe juice easily, filtering, and detecting trace amount.
The method 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 hardly dissolve, and making Aloe extract A not 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 about 35g of aloe juice, adding 50ml of 80% methanol, performing ultrasonic treatment for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes and 40 minutes to prepare two parts in parallel, filtering, and taking the 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 examination of chromatographic conditions
1) Aloenin a control solution: accurately weighing 10.12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the product with concentration of 198.3520 μ g/ml.
2) Fresh aloe juice sample solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
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, aloenin a has two peaks, peak emergence times are 2.69min and 2.82min, and the peak shapes are all shoulder peaks, and the adjustment of the mobile phase gradient is considered.
Aloenin a control solution: accurately weighing 10.12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the product with concentration of 198.3520 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g of fresh aloe juice, placing in 150ml conical flask, adding 50ml of 80% methanol water, performing ultrasonic treatment for 30min, and filtering to obtain the final product.
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, after the mobile phase gradient is adjusted, the aloenin a peak time and peak shape are unchanged, and the next step is to change the chromatographic column.
Aloenin a control solution: accurately weighing 10.12mg of the aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve, diluting to scale, and shaking to obtain the final product with concentration of 198.3520 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
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 aloenin a peak appearance time was 5.01min and 6.37min, respectively, the theoretical plate number was less than 2000 (1540), and the theoretical plate number was not changed much by replacing 0.03% phosphoric acid solution with 0.1% phosphoric acid solution. Because the phosphoric acid solution has high viscosity, the next step is to consider that 1% acetic acid solution is used to replace 0.1% phosphoric acid solution, and to see whether the number of theoretical plates can be increased.
Aloenin a control solution: accurately weighing 10.12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the product with concentration of 198.3520 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
Chromatographic conditions are as follows: see table 10.
TABLE 10 chromatographic conditions (method 4)
TABLE 11 chromatogram Peak Table (method 4)
As a result: see fig. 6, table 11. As can be seen from fig. 6 and table 11, the aloenin a peak time is 5.09min and 6.43min, the theoretical plate number is 3793 and 5579, the separation degree is 4.0, and the chromatographic condition can be used for detecting the content of aloenin a in the aloe juice.
Aloenin a control solution: accurately weighing 10.12mg of aloenin A reference substance, placing into a 50ml brown measuring flask, adding methanol to dissolve and dilute to scale, and shaking to obtain the product with concentration of 198.3520 μ g/ml.
Fresh aloe juice sample solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
Chromatographic conditions are as follows: see table 12.
TABLE 12 chromatographic conditions (method 5)
TABLE 13 chromatogram Peak Table (method 5)
As a result: see fig. 7, table 13. As is clear from FIG. 7 and Table 13, titank C was used as a Feilorman 18 Chromatographic column, aloenin A peak time of 6.09min and 7.64min, theoretical plate number of 3577 and 5421, separation degree of 3.8, shim-Pack scanner C for comprehensive evaluation 18 Is superior to the Philomen chromatographic column.
3.3 determination of the detection wavelength
Precisely weighing 35g of fresh aloe juice, adding 50ml of 80% methanol water, carrying out ultrasonic treatment for 30min, and filtering to obtain the aloe juice, wherein the absorption spectrum in the range of 190-800 nm is recorded as shown in figure 8.
As a result, the aloenin A has maximum absorption at 296nm, the peak response is high, and the base line is stable, so that 296nm is selected as the detection wavelength of the aloenin A content.
4 methodological investigation
4.1 analytical methods
4.1.1 preparation of the solution
Mobile phase: precisely measuring 10ml of glacial acetic acid, adding water to dilute into 1000ml, mixing uniformly to obtain 1% acetic acid solution, filtering with 0.45 μm filter membrane, degassing, and scaling up or down according to requirement.
80% aqueous methanol solution: weighing 400ml of methanol and 100ml of water, and mixing uniformly to obtain the methanol-water composite material.
Aloenin a control stock solution: precisely weighing 0.010g of aloenin A control, placing into a 10ml brown measuring flask, adding methanol to dissolve and dilute to scale, shaking up, and making into solution with concentration of about 980 μ g/ml.
Aloenin a control solution: precisely measuring 1.0ml of the aloenin A reference substance stock solution, placing into a 10ml brown measuring flask, adding methanol to dilute to scale, shaking, and making into solution with concentration of about 98 μ g/ml.
Test solution: precisely weighing 35.15g fresh Aloe juice, placing in 150ml conical flask, adding 50ml 80% methanol water, ultrasonic treating for 30min, filtering with 0.45 μm filter membrane, precisely weighing 1.0ml of Aloe A reference stock solution, placing in 10ml brown flask, and diluting with the filtrate to desired volume.
4.1.2 chromatographic conditions, see Table 14.
TABLE 14 chromatographic conditions
4.1.3 calculation formula
In the formula:
c, pair: the concentration of the aloenin A reference substance solution is unit of mug/ml;
c, storage: the concentration of the aloenin A reference stock solution is unit of mug/ml;
a sample A: the peak area of aloenin A in the test solution is shown;
a pair: the peak area average value of the main peak in the 5-needle aloenin A reference substance solution is shown;
and (2) W sample: weighing sample amount of the test solution in unit g;
50: to prepare a diluted volume of test solution, unit ml.
4.2 System applicability
4.2.1 procedures
Continuously feeding sample of 4.1.1 Aloe extract A reference solution for 5 times, and recording chromatogram. RSD values for the main peak areas were calculated and reported.
4.2.2 acceptance criteria
In the chromatogram of the control solution, the number of theoretical plates of aloenin A peak is not less than 2000;
RSD of 5-needle repeated sample injection peak area of the reference solution is not more than 2.0%;
4.2.3 the results are shown in Table 15, FIG. 9, FIG. 10, FIG. 11, FIG. 12, FIG. 13.
TABLE 15 System suitability results
As can be seen from Table 15, the number of theoretical plates of the aloenin A peak is greater than 2000, the RSD of the main peak area is 0.1% after continuous sample injection of 5 needles, and the verification standard is met.
4.3 specificity
4.3.1 procedures
And (3) respectively injecting 10 mu l of blank solution and 10 mu l of reference solution into a chromatograph, and determining whether the blank solvent interferes with the detection of the main peak. The chromatogram is recorded and processed.
4.3.2 acceptance criteria
In the blank solution chromatogram, the aloenin A peak position should not interfere.
4.3.3 results, see fig. 14, 15, 16.
As can be seen from FIGS. 14, 15 and 16, the blank solution did not interfere with aloenin A peak position. The specificity of the method was proven to meet the acceptance criteria.
4.4 Linear and Range
4.4.1 procedure
Control stock solutions: precisely weighing about 0.010g of aloenin A control, placing into a 10ml brown measuring flask, dissolving with methanol, diluting to scale, shaking, and making into 980 μ g/ml solution as aloenin A 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,117.6 μ g/ml and 147 μ g/ml respectively.
And respectively injecting 10 mu l of the solution into a chromatograph, recording a chromatogram, and performing linear regression on the injection concentration by peak area according to a least square method to obtain a standard curve.
4.4.2 acceptance criteria
r 2 More than or equal to 0.995, namely the aloenin A solution has good linear relation in the concentration range of 49-147 mu g/ml.
4.4.3 the results are shown in Table 16, FIG. 17, FIG. 18, FIG. 19, FIG. 20, FIG. 21, FIG. 22.
TABLE 16 Linear results
As can be seen from Table 16, the aloenin A solution has a good linear relationship in the concentration range of 49.490 μ g/ml-148.470 μ g/ml, and the correlation coefficient r 2 Is 0.9998.
4.5 durability
4.5.1 implementation step
The tolerance of aloe juice aloenin A content should be examined. 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 aloenin A content according to analysis condition change table. The durability conditions and sample injection are shown in Table 17.
TABLE 17 analysis conditions Change Table
4.5.2 acceptance criteria
Changing the analysis conditions:
in the chromatogram of the control solution, the number of theoretical plates of aloenin A 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 aloenin A content within 0.02mg/g (absolute) compared to the initial condition.
4.5.3 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 Table 18.
Table 18 content durability results
As can be seen from Table 18, 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 both changed within the standard range of 0.02mg/g, and the system applicability meets the standard specification, which indicates that the minor change of the chromatographic condition has no obvious influence on the analysis method, and indicates that the method has good durability.
4.6 conclusion, see table 19.
Table 19 analytical methods verification summary table
As can be seen from table 19, the method of analyzing the aloenin a 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 content of the aloenin A can meet the detection requirement of the content of the aloenin A.
4.7 aloenin A content determination and comparison
4.7.1 the aloenin A content was measured in the fresh aloe juice sample solution and the cooked aloe juice sample solution which were treated in different extraction manners at different places of origin by the methods of "method 4" and "document 1" under "item 3.2", respectively, and the results are shown in Table 20.
TABLE 20 table for measuring contents of fresh aloe juice and cooked aloe juice
4.7.2 fermented and boiled aloe juice sample solutions and the rosa luo acne eliminating cream processed by different extraction modes in different producing areas are taken, the content of aloenin A is detected according to a method 4 under the item 3.2 and a method of a document 1 respectively, and the result is shown in a table 21.
TABLE 21 measurement table of content of fermented aloe juice
As can be seen from the results of the detection of the aloe juice sample solutions in tables 20 and 21, the aloe juice samples treated in different production places and different treatment modes have lower aloenin A content, but the detection rate and the limit of quantitation of the method are obviously higher than those of the reference 'document 1', and the limit of quantitation of aloesin in the invention can reach 0.012mg/g, so that the detection method has higher sensitivity, can well detect the trace amount of aloenin A in the aloe juice, provides reference basis for aloe evaluation, and ensures the safety of the acne-eliminating cream medicine.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto 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 determining aloenin A content in an aloe medicinal material by using HPLC is characterized in that the determination method comprises the following steps:
(1) Preparation of control solutions: accurately weighing 8-12 mg of aloenin A reference substance, placing the aloenin A reference substance into a 50ml brown measuring flask, adding methanol to dissolve and dilute the aloenin A reference substance to a scale, and shaking up the aloenin A reference substance to obtain the aloenin A reference substance;
(2) Preparation of a test solution: precisely weighing 30-40 g of fresh aloe juice, putting the fresh aloe juice into a 150ml conical flask, adding 50ml of 60-90% methanol water, carrying out ultrasonic treatment for 20-40 minutes, 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 Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking methanol-0.5-1.5% acetic acid solution with the proportion of 40-60; the detection wavelength is 296nm, and the column temperature is 28-32 ℃; the flow rate is 0.8 to 1.2ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak;
(4) The determination method comprises the following steps: respectively and precisely sucking 5-10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, and measuring to obtain the final product.
2. The HPLC method for determining aloenin A content in aloe medicinal material according to claim 1, wherein the control solution prepared in step (1) is prepared by: precisely weighing 9-11 mg of aloenin A reference substance, placing the aloenin A reference substance into a 50ml brown measuring flask, adding methanol to dissolve and dilute the aloenin A reference substance to a scale, and shaking up the aloenin A reference substance to obtain the aloenin A reference substance.
3. The HPLC method for determining aloenin A content in aloe medicinal material according to claim 2, wherein the control solution prepared in step (1) is prepared by: accurately weighing 10.12mg of aloenin A reference substance, placing into 50ml brown measuring flask, adding methanol to dissolve, diluting to scale, and shaking.
4. The HPLC method for determining aloenin A content in aloe vera as claimed in claim 1, wherein the sample solution prepared in step (2) is prepared by: precisely weighing 32-38 g of fresh aloe juice, adding 50ml of 65-85% methanol water, carrying out ultrasonic treatment for 25-35 min, and filtering to obtain the aloe juice.
5. The HPLC method for determining aloenin A content in aloe medicinal material according to claim 4, wherein the sample solution of step (2) is prepared by: precisely weighing 35.15g fresh Aloe juice, adding 50ml 80% methanol water, ultrasonic treating for 30min, and filtering.
6. The HPLC method for determining aloenin A content in aloe medicinal material according to claim 1, wherein the chromatographic conditions and system applicability test in step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking methanol-0.9-1.1% acetic acid solution with the proportion of 45-55; the detection wavelength is 296nm, and the column temperature is 29-31 ℃; the flow rate is 0.9-1.1 ml/min; the number of theoretical plates is not less than 2000 calculated according to aloenin A peak.
7. The HPLC method for determining aloenin A content in aloe medicinal material according to claim 6, wherein the chromatographic conditions and system applicability test in step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the chromatographic column is Shim-Pack scanner C 18 250X 4.6mm,5 μm; taking a methanol-1.0% acetic acid solution with the proportion of 50; the detection wavelength is 296nm, 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 aloenin A peak.
8. The HPLC method for determining aloenin A content in aloe vera as claimed in claim 1, wherein the determination method in step (4): precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
9. The HPLC method for determining aloenin A content in aloe vera as claimed in claim 1, wherein said chromatographic column Shim-Pack scanner C 18 250X 4.6mm,5 μm, and may be Philomo Titank C 18 250×4.6mm，5μm。
10. The HPLC method for determining the aloenin A content in an aloe medicinal material according to any one of claims 1-9, wherein the aloenin A peak time is 5.09min and 6.43min, the theoretical plate number is 3793 and 5579, and the separation degree is 4.0.
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