CN112345670A

CN112345670A - Method for measuring content of alpha-linolenic acid in semen boitae medicinal material based on HPLC-ELSD

Info

Publication number: CN112345670A
Application number: CN202011378166.9A
Authority: CN
Inventors: 周厚成; 胡昌江; 辜新月; 陈玉梅; 叶志萍; 宋媛; 周维
Original assignee: Sichuan Neo Green Pharmaceutical Technology Development Co ltd
Current assignee: Sichuan Neo Green Pharmaceutical Technology Development Co ltd
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-02-09

Abstract

The invention discloses a method for measuring alpha-linolenic acid content in semen boitae medicinal materials based on HPLC-ELSD, and belongs to the technical field of traditional Chinese medicine analysis, quality identification and standard control. The method has the advantages of simple operation, high analysis speed, accuracy and good repeatability, makes up the defects of blank research on the absolute content of fatty acid components (particularly alpha-linolenic acid) in the platycladi seeds, provides a scientific and reliable analysis method for analyzing the fatty acid components such as the alpha-linolenic acid in the platycladi seeds, and lays a foundation for improving the quality standard of the traditional Chinese medicine platycladi seeds.

Description

Method for measuring content of alpha-linolenic acid in semen boitae medicinal material based on HPLC-ELSD

Technical Field

The invention relates to a method for measuring the content of alpha-linolenic acid in platycladi seed medicinal materials, in particular to a method for measuring the content of alpha-linolenic acid in platycladi seed medicinal materials based on HPLC-ELSD, belonging to the technical field of traditional Chinese medicine analysis, quality identification and standard control.

Background

The semen Platycladi is dry mature seed of Franco of Platylladus orientalis (L.) of Cupressaceae, has sweet and mild taste, has effects of nourishing heart, tranquilizing mind, loosening bowel to relieve constipation, and suppressing sweating, and can be used for treating vexation, insomnia, cardiopalmus, night sweat due to yin deficiency, and constipation due to intestinal dryness. Because the lipid-lowering health-care food is rich in a large amount of fatty oil, the lipid-lowering health-care food can effectively lower blood fat, prevent atherosclerosis, protect cardiac muscle and the like. The main components of the fatty oil are unsaturated fatty acids, including alpha-linolenic acid, linoleic acid, oleic acid, arachidonic acid and the like, wherein the platycladi seed is rich in the unsaturated fatty acids which are necessary for human bodies and belong to the alpha-linolenic acid, and the unsaturated fatty acids are selected as the indexes of the quality control components of the platycladi seed.

At present, the Chinese pharmacopoeia (2020 edition) does not contain a method for measuring the content of alpha-linolenic acid in platycladi seeds, and has almost no technology for measuring the content of alpha-linolenic acid in platycladi seeds in other aspects. In the prior art, GC-MS, pre-column saponification or derivatization HPLC methods are adopted to measure fatty acid in platycladi seeds, such as: in papers "GC-MS comparative analysis studies of fatty acids in arborvitae kernel and arborvitae kernel cream, xunewgang et al, 2009" GC-MS comparative analysis studies of fatty acids in arborvitae leaves and seeds, butyrospermum et al, 2008 ", GC-MS comparative analysis of fatty acids in arborvitae kernel and kernel cream (arborvitae leaves and seeds) is described.

However, the GC and GC-MS methods are easy to generate side reactions such as isomerization, oxidation and decomposition, and the GC-MS method is difficult to popularize in quality control; the pre-column derivatization HPLC method has complicated operation steps and is easy to cause errors, and the methods have certain defects in the aspects of detection sensitivity, reaction completeness, quantitative accuracy and the like.

Therefore, the establishment of an HPLC-ELSD analysis method of alpha-linolenic acid is a technical problem which is urgently needed to be solved at present.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a method for determining the content of alpha-linolenic acid in semen boitae medicinal materials based on HPLC-ELSD. In the technical scheme, the content of alpha-linolenic acid (higher fatty acid) in the platycladi seeds is detected by using a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), the method is simple to operate, high in analysis speed, accurate and good in repeatability, and the defects of blank research on the absolute content of fatty acid components (particularly alpha-linolenic acid) in the platycladi seeds and the like are overcome. The evaporative light scattering detector is a universal quality detector, does not depend on ultraviolet absorption of a detected component, overcomes the difficulties that part of fatty acid structures have no conjugated double bonds and have no absorption and weak absorption in an ultraviolet spectrum, and has the characteristics of good stability, high sensitivity and the like;

the method for measuring the content of alpha-linolenic acid in the platycladi seed medicinal material by HPLC-ELSD provides a scientific and reliable analysis method for the analysis of fatty acid components such as alpha-linolenic acid in the platycladi seed and the like, and lays a foundation for perfecting the quality standard of the traditional Chinese medicine platycladi seed.

In order to achieve the technical purpose, the following technical scheme is proposed:

a method for measuring the content of alpha-linolenic acid in semen boitae medicinal materials based on HPLC-ELSD comprises the following steps:

A. preparation of control solutions: adding methanol into alpha-linolenic acid reference substance to obtain solution containing 0.2mg per 1 mL;

B. preparation of a test solution: sieving the powder with a second sieve, weighing 0.5g, placing in a conical flask with a plug, adding 10mL of methanol, weighing, ultrasonically treating (power 600W, frequency 40kHz) for 30min, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the filtrate;

C. drawing a standard curve: injecting the obtained reference substance solution into HPLC-ELSD for determination, and recording corresponding chromatogram and peak area;

drawing a standard curve by taking the logarithm of the content of the reference substance as a horizontal coordinate X and the logarithm of the peak area as a vertical coordinate Y, and calculating to obtain a regression equation;

D. and (4) calculating a result: injecting the obtained test solution into HPLC-ELSD for determination, and substituting the peak area into the linear regression equation of the obtained standard curve to obtain the content of alpha-linolenic acid in semen Platycladi;

the chromatographic conditions in the high performance liquid chromatograph meet:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m;

column temperature of the chromatographic column: 30 ℃;

mobile phase: methanol is taken as a mobile phase A, and a 1% acetic acid solution is taken as a mobile phase B;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

the measurement conditions in the evaporative light scattering detector satisfy:

temperature of the drift tube: 60 ℃;

air pressure: 350 Kpa;

gain value: 6;

the mobile phase was subjected to gradient elution according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
20	94	6
			30	95	5

。

Preferably, the sampling amount of the control product alpha-linolenic acid is 1.3539-6.7695 mug.

By adopting the technical scheme, the beneficial technical effects brought are as follows:

in the invention, based on an evaporative light scattering detection-high performance liquid chromatography method, specific reference substance types, sample solution extraction and matched chromatographic conditions, a standard curve is drawn to obtain a linear relation between the logarithm of the content of alpha-linolenic acid and the logarithm of the peak area, and then the content of the alpha-linolenic acid in the platycladi seed medicinal material is obtained.

The method has the advantages of simple operation, high analysis speed, good stability, accuracy and good repeatability, makes up the defects of blank research on the absolute content of fatty acid components (especially alpha-linolenic acid) in the platycladi seeds and the like, provides a scientific and reliable analysis method for the analysis of the fatty acid components such as the alpha-linolenic acid in the platycladi seeds, and lays a foundation for improving the quality standard of the traditional Chinese medicine platycladi seeds.

Drawings

FIG. 1 is a chromatogram map of the alpha-linolenic acid content determination specificity of semen Platycladi in example 4;

FIG. 2 is a graph of the standard curve of the control product, alpha-linolenic acid, in example 4.

Detailed Description

In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, reference is made to an apparatus comprising:

high performance liquid chromatograph: shimadzu LC-20AT high performance liquid chromatograph, Agilent 1260 high performance liquid chromatograph, Shimadzu ELSD-LT II evaporation light detector;

an electronic balance: ME204E/02, MS205D Mm, XP26 (Mettler-Tollido instruments, Inc.);

an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);

an ultrasonic cleaner: model KQ600DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

a chromatographic column: phenomenex Luna 5. mu. m C18(2)100A 4.6X 250mm, InertSustain C185. mu.m 4.6X 250mm, Agilent 5 TC-C185. mu.m 4.6X 250 mm;

in the following examples, the reagents involved included:

methanol (chromatographically pure), glacial acetic acid (chromatographically pure), water (ultrapure water), and other reagents are analytically pure;

in the following examples, the test articles referred to include:

the batch number of the arborvitae seed medicinal material is as follows: XLS 2018096, XLS 2018097, XLS201808098, XLS201808099, XLS201808125, XLS201808126, XLS201808154, XLS201808155, XLS201808156, XLS201808422, XLS201808423, XLS201808424, XLS201808425, XLS201808426, XLS 201808427.

In the following examples, reference controls include:

alpha-linolenic acid (China institute for testing and testing food and drug, lot number: 111631-201605, content is 99.8%);

example 1

column temperature of the chromatographic column: 30 ℃;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

temperature of the drift tube: 60 ℃;

air pressure: 350 Kpa;

gain value: 6;

the mobile phase was subjected to gradient elution according to table 1 below, specifically:

TABLE 1 gradient elution

Time of day(min)	Mobile phase A (%)	Mobile phase B (%)
			0	90	10
20	94	6
			30	95	5

。

Example 2

Based on example 1, this example will examine the kind of extraction solvent, extraction method, extraction time, and material ratio in the preparation of the sample solution, and further describe the present technical solution.

First, investigation of extraction solvent

Taking twelve parts of the powder of the product, sieving the powder by a second sieve, weighing 0.5g uniformly, precisely weighing, placing the powder in a conical flask with a plug, adding 10mL of petroleum ether, 10mL of dichloromethane, 10mL of ethyl acetate, 10mL of water, 10mL of methanol and 10mL of ethanol respectively, weighing, ultrasonically extracting (with the power of 600W and the frequency of 40kHz) for 30min, cooling, weighing again, complementing the lost weight with corresponding solvent, shaking up, filtering, taking the subsequent filtrate, and then measuring to obtain the results shown in the following table 2.

TABLE 2 analysis results of different extraction solvents

The results show that: methanol extraction efficiency is the highest, and therefore, methanol is selected as its extraction solvent.

Second, examination of extraction mode

Taking four parts of the powder, sieving by a second sieve, weighing 0.5g, precisely weighing, placing in a conical flask with a plug, respectively adding 10mL of methanol, weighing, respectively heating and refluxing (conventional process conditions) for 30min, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, cooling, weighing again, complementing the weight loss by methanol, shaking uniformly, filtering, taking the subsequent filtrate, and then measuring to obtain the results shown in the following table 3.

TABLE 3 analysis results of different extraction methods

The results show that: when the ultrasonic extraction and reflux extraction are carried out on the test solution, the extraction effect has no great difference, so that the ultrasonic extraction method which is simple and convenient is selected for the test solution extraction method.

Third, investigation of extraction time

Taking six parts of the powder, sieving by a second sieve, weighing 0.5g, placing in a conical flask with a plug, adding 10mL of methanol respectively, weighing, performing ultrasonic extraction (power 600W and frequency 40kHz) for 20min, 30min and 40min respectively, cooling, weighing again, supplementing the weight loss by methanol, shaking uniformly, filtering, taking the subsequent filtrate, and then measuring to obtain the results shown in the following table 4.

TABLE 4 analysis results of different extraction times

The results show that: the ultrasonic extraction is completed within 30min, so the extraction time is selected to be 30 min.

Fourth, survey of material ratio

Taking six parts of the powder, sieving by a second sieve, respectively weighing 0.3g, 0.5g and 0.7g (respectively two parts), placing in a conical flask with a plug, adding 10mL of methanol, weighing, ultrasonically extracting for 30min, cooling, weighing again, complementing the weight loss by methanol, shaking up, filtering, taking the subsequent filtrate, and then measuring to obtain the results shown in the following table 5.

TABLE 5 analysis results of different extraction amounts

The results show that: when the sampling amount of the semen boitae medicinal material is 0.5g, the extraction efficiency of the alpha-linolenic acid content is high, and the peak height is proper, so that the sampling amount of the selected test sample is 0.5 g.

In summary, the following steps: weighing the powder (sieving with a second sieve) about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of methanol, ultrasonically treating (power 600W, frequency 40kHz) for 30 minutes, cooling, weighing, supplementing the lost weight with methanol, shaking, filtering, and collecting the filtrate.

Example 3

In this example, the chromatographic conditions (column temperature, flow rate) in the measurement were examined based on examples 1 to 2 to further describe the present embodiment.

First, column temperature investigation

The column temperatures were set to 25 ℃, 30 ℃ and 35 ℃ respectively, and the chromatographic conditions were the same as in example 1, and the results were as shown in Table 6 below.

TABLE 6 column temperature examination results

Column temperature	Retention time	Number of theoretical plate	Degree of separation	Symmetry factor
						25	10.512	14063	12.92	1.06
30	9.896	14181	12.33	1.04
					35	9.423	13409	11.58	1.04

The results show that: the alpha-linolenic acid can be effectively detected at the column temperature of 25 ℃ and 30 ℃, other measurement conditions are integrated, and the column temperature of 30 ℃ is finally drawn up as the detection column temperature.

Second, investigation of flow velocity

The flow rates were set to 0.8mL/min, 1.0mL/min, and 1.2mL/min, respectively, and the other chromatographic conditions were the same as in example 1, and the results obtained by measuring the symmetry factor of the α -linolenic acid chromatographic peak, the number of theoretical plates, and the like as evaluation indices are shown in Table 7 below.

Table 7 flow rate investigation results

Flow rate of flow	Retention time	Number of theoretical plate	Degree of separation	Symmetry factor
					0.8	12.28	17905	14.30	1.04
1.0	9.896	14181	12.33	1.04
					1.2	8.191	11322	10.50	1.05

The results show that: the three flow rates can effectively separate and measure the alpha-linolenic acid, other measurement conditions are integrated, and the flow rate of 1.0mL/min is finally selected for subsequent investigation.

Example 4

Based on example 1, the following methodology examination was also performed in this example to further illustrate the present technical solution.

First, specificity experiment

1. Preparation of control solutions: dissolving alpha-linolenic acid reference substance in methanol to obtain solution containing 0.2mg per 1 mL;

2. preparation of a test solution: taking semen Platycladi powder, sieving with a second sieve, weighing 0.5g, placing in a conical flask with a plug, adding 10mL of methanol, weighing, ultrasonic treating (power 600W, frequency 40kHz) for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate;

3. preparation of negative control solution: transferring 10mL of methanol into a conical flask with a plug, performing ultrasonic treatment (power 600W and frequency 40kHz) for 30min, filtering, and taking a subsequent filtrate to obtain the final product;

the obtained control solution, test solution and negative control solution were injected into HPLC-ELSD for measurement, and the obtained results are shown in fig. 1, which indicates that: the negative control solution chromatogram has no interference to the measurement of the peak to be measured, which shows that the method has good specificity.

Second, precision test

The control solution was sampled six times continuously, the peak area of α -linolenic acid was recorded, and the RSD value was calculated, and the results are shown in table 8 below.

TABLE 8 results of precision investigation

The results show that: the retention time of each characteristic peak and the RSD value of the peak area meet the requirements, and the instrument has good precision.

Three, linear relationship

Respectively taking 5 muL, 10 muL, 15 muL, 20 muL, 23 muL and 25 muL of alpha-linolenic acid reference substance solution (270.78 mug/mL), injecting HPLC-ELSD, analyzing to obtain peak areas, drawing a standard curve by taking the logarithm of the content (mug) of the reference substance as a horizontal coordinate X and the logarithm of the peak areas as a vertical coordinate Y, and calculating to obtain a regression equation, wherein the obtained results are shown in the following table 9 and figure 2.

TABLE 9 analysis of the alpha-linolenic acid Standard Curve

The results show that: the sample injection range of the alpha-linolenic acid is 1.3539-6.7695 mu g, the linear relation is that y is 1.4627x +5.1068, and R is 0.9998, and the good linear relation is formed.

Fourthly, investigation of stability

Based on the above conditions, the same semen Platycladi sample solution was measured at 0h, 2h, 6h, 12h, 18h, and 24h, respectively, and the results are shown in Table 10 below.

TABLE 10 stability test results

The results show that: under the condition, the RSD value of the alpha-linolenic acid content is 2.00 percent, and the semen boitae medicinal material test solution has good stability within 24 hours.

Fifth, repeatability inspection

The powder was sieved through a second sieve and weighed to 0.5g (six parts), and the same operator prepared a test solution according to example 1, and the α -linolenic acid content in the six test parts was measured and calculated, the results of which are shown in table 11 below.

TABLE 11 results of repeated experiments

The results show that: the RSD value of the alpha-linolenic acid content is 1.85 percent, and the method has good repeatability.

Sixth, investigation of intermediate precision

Selecting different measurement time, different HPLC, different testers (person A and person B), taking 0.5g of semen Platycladi powder (sieved by a second sieve) and six parts to prepare a sample solution, carrying out sample injection measurement under the chromatographic conditions in example 1, and calculating the content of alpha-linolenic acid and RSD value, wherein the results are shown in Table 12 below.

TABLE 12 results of measurement of intermediate precision

The experimental results show that: different testers operate at different time and different chromatographs, the content of alpha-linolenic acid in the platycladi seed medicinal material RSD is less than 2.0%, and the intermediate precision of the analysis method is good.

Seventh, accuracy test

Taking 0.25g of a platycladi seed medicinal material test sample with known alpha-linolenic acid content, adding six parts of alpha-linolenic acid control samples respectively, preparing and measuring a test sample solution according to the method in the example 1, and calculating the recovery rate, wherein the results are shown in the following table 13. The calculation formula is as follows:

recovery (%) - (C-A)/BX 100%

In the formula: a is the measured component content of the test sample;

b is the amount of the added reference substance;

c is an actual measurement value;

TABLE 13 sample recovery test results

The results show that: the average recovery rate is 101.96%, the RSD value is 1.98%, and the method has good accuracy.

Eight, inspection of durability of chromatographic column

Comparing the influence of different chromatographic columns on the measurement of the content of alpha-linolenic acid in the platycladi seed medicinal material, respectively examining the chromatographic columns of Phenomenex Luna 5um C18(2)100A 4.6X 250mm, Inertsustatin C185 mu m 4.6X 250mm and Agilent 5 TC-C185 mu m 4.6X 250mm, taking 0.5g of the platycladi seed medicinal material powder to prepare a test solution, and performing measurement under the same other chromatographic conditions as in example 1 to obtain the results shown in the following table 14.

TABLE 14 durability examination results

The results show that: the analytical chromatographic indexes of different chromatographic columns are good, the RSD values of six measurement results are 1.96%, and the durability of the chromatographic column of the analytical method is good.

Content determination of nine-seed and arborvitae seed medicinal materials

The measurement of the powder of arborvitae seed of different batches was carried out according to the measurement method of example 1, and the results are shown in the following table 15.

TABLE 15 fifteen lots of semen Platycladi test results

Batch number	Alpha-linolenic acid content%
		XLS201808096	0.78
XLS201808097	0.78
		XLS201808098	0.77
XLS201808099	0.96
		XLS201808125	0.85
XLS201808126	0.94
		XLS201808154	1.07
XLS201808155	0.89
		XLS201808156	0.66
XLS201808422	0.94
		XLS201808423	1.40
XLS201808424	1.81
		XLS201808425	1.75
XLS201808426	1.77
		XLS201808427	2.06

In conclusion, we obtain: the content determination method of the semen boitae medicinal material can effectively detect the content of alpha-linolenic acid in the semen boitae medicinal material.

Claims

1. A method for measuring the content of alpha-linolenic acid in semen boitae medicinal materials based on HPLC-ELSD is characterized by comprising the following steps:

B. preparation of a test solution: sieving the powder with a second sieve, weighing 0.5g, placing in a conical flask with a plug, adding 10mL of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate;

column temperature of the chromatographic column: 30 ℃;

flow rate of mobile phase: 1.0 mL/min;

sample introduction amount: 10 mu L of the solution;

temperature of the drift tube: 60 ℃;

air pressure: 350 Kpa;

gain value: 6;

。

2. the HPLC-ELSD-based method for determining the content of alpha-linolenic acid in platycladi seed medicinal material according to claim 1, wherein in the ultrasonic treatment, the power is 600W, and the frequency is 40 kHz.

3. The HPLC-ELSD-based method for determining the content of alpha-linolenic acid in platycladi seed medicinal materials according to claim 1, wherein the sample amount of the control alpha-linolenic acid is 1.3539-6.7695 μ g.