CN108918715A - A method of detection linseed oil middle ring peptide content - Google Patents
A method of detection linseed oil middle ring peptide content Download PDFInfo
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Abstract
The present invention relates to the detection methods of linseed oil middle ring peptide content, preparation, the ultra-performance liquid chromatography measurement of preparation, reference substance solution including test solution.Using content assaying method provided by the invention, sample solution preparation method is simple, reduces the risk of error；When measurement, Chromatogram Baseline is steady, and separating degree is high, and accuracy in detection is high；Method provided by the invention shortens minute simultaneously, improves detection efficiency, reduces the dosage of toxic chemical, more economical environmental protection.
Description
Technical field
The present invention relates to functional food fields, and in particular to a method of detection linseed oil middle ring peptide content.
Background technique
Flax (Linum usitatissimum L.) is also known as flax, is a kind of ancient oil crops, has in China
Extensive distribution and long plantation history.Linseed is the seed of flax, is rich in linseed oil, flax glue, flax protein, wood
Nutrition and the functional components such as phenol element, cyclic peptide, dietary fiber.Linseed oil therein is a kind of natural health-care vegetable oil, is contained
Have flax glue abundant, lignan, dietary fiber, etc. functional components and ω -3 be essential fatty acid alpha-linolenic acid, have and adjust
It saves blood lipid, reduce the functions such as cholesterol, anti-inflammatory, anticancer, enhancing brain and optic nerve function and prevention skin disease.
In recent years, the hydrophobicity annular plant peptide-linseed cyclic peptide with immune function contained in linseed oil by
Extensive concern.Linseed cyclic peptide is made of 8~9 amino acid, and molecular weight has separated mirror about in 1~10kDa at present
25 kinds or more of cyclic peptide is made, wherein the amino acid sequence of cyclic peptide A, C, D, E and molecular formula are determined by researcher, Li Jia
The auspicious equal content (2016) using these four cyclic peptide in high performance liquid chromatography measurement linseed oil, separating degree and repeatability accord with
It closes and requires；But there are this following deficiencies for this method：Firstly, this method pretreatment process is relatively complicated, need ethyl alcohol extraction rich
Collection, the risk that accidental error occurs in operation are larger；Secondly, this method uses component one of of the acetonitrile as mobile phase, and
Accounting can have a certain impact to the health of environment and experimenter 50% or more.
With deepening continuously to linseed cyclic peptide research, for the content of cyclic peptide in better detection linseed oil,
Establish that a kind of easy to operate, stable sample-pretreating method and specificity are strong, separating degree is high, the inspection of the cyclic peptide content of high-efficiency environment friendly
Survey method is imperative.
Summary of the invention
The purpose of the present invention is overcoming defect in the prior art, a kind of assay side of cyclic peptide in linseed oil is provided
Method, sample pre-treatments are easy, stablize, and other compositions do not influence the measurement of cyclic peptide in gained test sample；Measurement is baseline
Steadily, separating degree is good, accuracy is high；Compared with prior art, the retention time at each cyclic peptide peak is shorter, shortens assay
Time, improve the efficiency of detection, and reduce toxic reagent-acetonitrile dosage, more high-efficiency environment friendly.
Specifically, this method includes test sample the present invention provides a kind of method for detecting linseed oil middle ring peptide content
The preparation of solution, the preparation of reference substance solution, ultra-performance liquid chromatography (UPLC) measure cyclic peptide content.
The chromatographic condition of the ultra-performance liquid chromatography is：WatersACQUITYUPLC I-Class ultra high efficiency liquid phase
Chromatographic system and its work station；Chromatographic column is ACQUITYUPLC Peptide CSH C18Column (2.1mm × 100mm, 1.7 μ
m)；Mobile phase is the phosphate aqueous solution (B) of acetonitrile (A) and 0.05% (volume fraction)；Gradient elution；Detection wavelength 200~
400nm；10 μ L of sample volume；35 DEG C of column temperature；Flow velocity 1.0mL/min.
Preferably, the condition of the gradient elution is：0~8min, 30%A；8~20min, 30%A → 50%A；20~
30min, 65%A；
Preferably, the Detection wavelength is 214nm.
The method of reference substance solution preparation is：
Take linseed cyclic peptide A, C, D, E reference substance, after accurately weighed, dissolved and be settled in 10mL volumetric flask with acetonitrile,
The mixed solution that each cyclic peptide concentration is 1mg/mL is obtained, as reference substance solution.
The preparation of the test solution includes the following steps：
(1) extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
In a round bottom flask, acetone, ultrasonic extraction is added in linseed meal；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained Asia after vacuum drying
Flaxseed oil；
(2) preparation of test solution：Linseed oil is taken, with 10~40 times of dilution in acetonitrile, is filtered through 0.45 μm of miillpore filter
Later, as test solution.
Preferably, the process conditions of the ultrasonic extraction are：Solid-liquid ratio 1:6~1:12, it 40~60 DEG C of ultrasonic temperature, extracts
30~60min of time, 180~240W of ultrasonic power, 400~600r/min of mixing speed.
Beneficial effects of the present invention：
The present invention realizes each cyclic peptide using the cyclic peptide content in UPLC method measurement linseed oil, used chromatographic process
Retention time shortens in chromatographic process, improves the efficiency of assay, reduces the usage amount of acetonitrile in mobile phase, more ring
It protects, also reduces testing cost.
The sample-pretreating method that uses of the present invention is simple simultaneously, strong operability, and gained test solution property is stablized,
Separating degree is high.
Method of the present invention, Chromatogram Baseline is steady, and separating degree is good, and accuracy in detection is high.
Specific embodiment
We will the invention will be further described in conjunction with specific embodiments, it is to be noted that, embodiment below is simultaneously
Do not limit protection scope and application range of the present invention.
In following embodiment, unless specifically indicated, reagent of the present invention, instrument, method are the art
Reagent, the instrument, method of routine.
In following embodiment, the instrument being related to includes WatersACQUITYUPLC I-Class ultra performance liquid chromatography system
System and its work station；Chromatographic column ACQUITYUPLC Peptide CSH C18(specification is 2.1mm × 100mm, 1.7 μ to Column
m)；
In following embodiment, the reagent specification being related to is that acetonitrile is chromatographically pure, and phosphoric acid is that analysis is pure, and water is ultrapure water, and
And mentioned reagent can be obtained in conventional commercial channel.
Cyclic peptide A, C, D, E reference substance comes from 98% or more Prairie Tide Chemicals Inc. purity.
The preparation of 1 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：10 are added acetone, open stirring, speed is set as 500r/min, at 50 DEG C
Under with the power ultrasonic extraction 45min of 220W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 20 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The preparation of 2 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：6 are added acetone, open stirring, speed is set as 400r/min, at 40 DEG C
With the power ultrasonic extraction 30min of 180W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 10 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The preparation of 3 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：12 are added acetone, open stirring, speed is set as 600r/min, at 40 DEG C
Under with the power ultrasonic extraction 60min of 240W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 40 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The preparation of 4 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：10 are added acetone, open stirring, speed is set as 600r/min, at 60 DEG C
Under with the power ultrasonic extraction 35min of 240W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 30 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The preparation of 5 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：9 are added acetone, open stirring, speed is set as 500r/min, at 50 DEG C
With the power ultrasonic extraction 45min of 200W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 15 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The preparation of 6 test solution of embodiment
1. the extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；It weighs a certain amount of
Linseed meal in a round bottom flask, by solid-liquid ratio 1：10 are added acetone, open stirring, speed is set as 400r/min, at 50 DEG C
Under with the power ultrasonic extraction 10min of 220W；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed oil after vacuum drying；
2. the preparation of test solution：Linseed oil is taken, with 20 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
The methodological study of 7 ultra-performance liquid chromatography of embodiment detection linseed oil middle ring peptide content
1. chromatographic condition：WatersACQUITYUPLC I-Class ultra performance liquid chromatography system and its work station；Chromatography
Column is ACQUITY UPLC Peptide CSH C18Column (specification be 2.1mm × 100mm, 1.7 μm)；Mobile phase is acetonitrile
(A) with the phosphate aqueous solution (B) of 0.05% (volume fraction)；Gradient elution；Detection wavelength 214nm；10 μ L of sample volume；Column temperature 35
℃；Flow velocity 1.0mL/min.
Condition of gradient elution is：0~8min, 30%A；8~20min, 30%A → 50%A；20~30min, 65%A.
2. the preparation of test solution：Test solution is prepared by the description of embodiment 1.
3. the preparation of reference substance mixed solution：Linseed cyclic peptide A, C, D, E reference substance is taken, it is molten with acetonitrile after accurately weighed
It solves and is settled in 10mL volumetric flask, the mixed solution that each cyclic peptide concentration is 1mg/mL is obtained, as reference substance solution.Sampling knot
Fruit is shown in Table 1.
1 cyclic peptide reference substance sampling record of table
| Sampling amount (g) | Concentration (mg/mL) |
| 9.965 | 0.9965 |
| 10.021 | 1.0021 |
| 10.006 | 1.0006 |
| 9.978 | 0.9978 |
4. linear relationship is investigated
Above-mentioned reference substance mixed solution is taken, respectively the accurate reference substance for drawing 0.1,0.125,0.2,0.4,1.0,2.0mL
Mixed solution is set in 10mL volumetric flask, is added acetonitrile to be settled to scale and is uniformly mixed, and after miillpore filter filters, injects liquid phase
Chromatograph is measured by above-mentioned chromatographic condition, records the retention time and peak area of cyclic peptide respectively, and is vertical sit with peak area
Mark, the concentration of reference substance solution are ordinate, draw standard curve, calculate regression equation and related coefficient, the results are shown in Table 2.
2 linear relationship of table investigates result
The above table 2 the result shows that, cyclic peptide A, C, D, E are linear good within the scope of 0.2~0.01mg/mL of concentration respectively.
5. precision test
Precision draws reference substance mixed solution 1.0mL, sets in 10mL volumetric flask, adds acetonitrile to be settled to scale and mixes equal
It is even, after miillpore filter filters, liquid chromatograph is injected, by above-mentioned chromatographic condition METHOD FOR CONTINUOUS DETERMINATION 6 times, peak area is recorded and calculates
RSD, as a result cyclic peptide C, cyclic peptide E, cyclic peptide D, cyclic peptide A RSD be respectively 0.71%, 0.49%, 0.52%, 0.48%, show instrument
The precision of device is good.
6. stability test
The solution diluted in precision test is taken, 0,2,4,6,8 after preparation hour measurement records peak area
And calculate RSD, as a result cyclic peptide C, cyclic peptide E, cyclic peptide D, cyclic peptide A RSD be respectively 1.2%, 0.80%, 1.2%, 1.5%, show
The precision of instrument is good.
7. repetitive test
6 parts of test solution are taken in parallel, is measured respectively, peak area is recorded, and calculate cyclic peptide content by external standard method, counts simultaneously
Calculate RSD, as a result cyclic peptide C, cyclic peptide E, cyclic peptide D, cyclic peptide A RSD be respectively 1.9%, 1.7%, 1.1%, 1.4%, show the party
The repeatability of method is good.
8. being loaded recovery test
Precision weighs the linseed oil 5.0g prepared in embodiment 1, is divided into tri- groups of L, M, H, it is accurate respectively be added 0.1,
0.2, the reference substance mixed solution of 0.4mL, according to the method in embodiment 1 with 20 times of dilution in acetonitrile, through 0.45 μm of miillpore filter
After filtration, liquid chromatograph is injected, is detected, is measured in parallel twice, calculate sample recovery rate and RSD.It the results are shown in Table 3.
Table 3 is loaded recovery test result table (N=2)
9. sample size measures
It takes linseed to extract linseed oil by embodiment 1, takes three batches of linseed oil as sample, after 20 times of dilution in acetonitrile
As test solution, one point external standard method measures the content of cyclic peptide in sample.It the results are shown in Table 4.
4 three batches of sample cyclic peptide assay results of table
| Cyclic peptide C (μ g/g) | Cyclic peptide E (μ g/g) | Cyclic peptide D (μ g/g) | Cyclic peptide A (μ g/g) | |
| Batch 1 | 191.65 | 175.66 | 30.06 | 499.67 |
| Batch 2 | 188.79 | 176.31 | 32.23 | 502.38 |
| Batch 3 | 192.99 | 176.20 | 31.08 | 500.66 |
It can be seen from the above result that under the conditions of the present invention, the content of cyclic peptide in linseed oil is measured, saves time and molten
Completely, chromatogram baseline is steady, and chromatography peak type is good, is a kind of advanced detection linseed for agent, target component and impurity separation
The method of oily middle ring peptide content.
The assay of cyclic peptide in 8 linseed oil of embodiment
The preparation of test solution, preparation of reference substance solution etc. are same as Example 7 in the present embodiment, and difference exists
In condition of gradient elution is in the chromatographic condition of ultra-performance liquid chromatography：0~6min, 50%A, 0.5mL/min；7~
21min, 65%A, 0.5mL/min；22min, 66%A, 0.5mL/min；23min, 70%A, 0.5mL/min；24~30min,
100%A, 1.0mL/min；31~41min, 50%A, 1.0mL/min.
The parallel sample introduction of every batch of test solution measures 3 times, and results are averaged for content calculating, the results are shown in Table 5.
5 three batches of sample cyclic peptide assay results of table
| Cyclic peptide C (μ g/g) | Cyclic peptide E (μ g/g) | Cyclic peptide D (μ g/g) | Cyclic peptide A (μ g/g) | |
| Batch 1 | 161.52 | 142.69 | 27.51 | 352.01 |
| Batch 2 | 156.32 | 146.28 | 26.73 | 369.25 |
| Batch 3 | 157.29 | 139.97 | 24.12 | 364.29 |
| Average residence time (min) | 12.6 | 15.3 | 19.5 | 22.7 |
The assay of cyclic peptide in 9 linseed oil of embodiment
The preparation of test solution, preparation of reference substance solution etc. are same as Example 7 in the present embodiment, and difference exists
In Detection wavelength is 398nm in the chromatographic condition of ultra-performance liquid chromatography.
The parallel sample introduction of every batch of test solution measures 3 times, and results are averaged for content calculating, the results are shown in Table 6.
6 three batches of sample cyclic peptide assay results of table
| Cyclic peptide C (μ g/g) | Cyclic peptide E (μ g/g) | Cyclic peptide D (μ g/g) | Cyclic peptide A (μ g/g) | |
| Batch 1 | 102.69 | 86.95 | 10.25 | 200.06 |
| Batch 2 | 98.56 | 85.63 | 10.69 | 198.54 |
| Batch 3 | 99.63 | 82.31 | 12.36 | 196.32 |
| Average residence time (min) | 12.9 | 15.6 | 19.9 | 23.4 |
The assay of cyclic peptide in 10 linseed oil of embodiment
The preparation of test solution, preparation of reference substance solution etc. are same as Example 7 in the present embodiment, and difference exists
In Detection wavelength is 256nm in the chromatographic condition of ultra-performance liquid chromatography.
The parallel sample introduction of every batch of test solution measures 3 times, and results are averaged for content calculating, the results are shown in Table 7.
7 three batches of sample cyclic peptide assay results of table
| Cyclic peptide C (μ g/g) | Cyclic peptide E (μ g/g) | Cyclic peptide D (μ g/g) | Cyclic peptide A (μ g/g) | |
| Batch 1 | 126.62 | 98.56 | 15.21 | 249.87 |
| Batch 2 | 122.35 | 95.60 | 13.09 | 245.06 |
| Batch 3 | 124.57 | 94.31 | 15.66 | 246.98 |
| Average residence time (min) | 12.5 | 15.1 | 19.3 | 22.4 |
The assay of cyclic peptide in comparative example linseed oil
Referring to institute in " ethyl alcohol extraction-HPLC method quickly analyzes four kinds of cyclic peptide research in linseed oil " (sharp Jiaxiang, 2016)
The method of offer, wherein
The preparation of test solution：The linseed oil 5g in embodiment 1 is weighed, in 10mL centrifuge tube, and 4mL is added
95% ethyl alcohol, be sufficiently mixed 5min, the high speed centrifugation 5min under revolving speed 6000r/min, after centrifugation, Aspirate supernatant is transferred to
In 50mL round-bottomed flask, be repeated 3 times, the supernatant of collection 40 DEG C at a temperature of rotate, remove solvent, dissolved with chromatography methanol
Cyclic peptide in round-bottomed flask is transferred in 5mL volumetric flask, and with methanol constant volume to scale, is filtered through 0.45 μm of miillpore filter
Afterwards, as test solution.
The preparation of reference substance solution：With the preparation of reference substance solution in embodiment 7.
Chromatographic condition：ODS C18Column (150mm × 4.6mm, 5 μm), mobile phase A are acetonitrile, and Mobile phase B is distilled water, column
Temperature is 35 DEG C, Detection wavelength 214nm, and sample volume is 10 μ L, using gradient elution, quantified by external standard method.Condition of gradient elution is shown in
Table 7, measurement result is shown in Table 8.
7 gradient elution program of table
8 three batches of sample cyclic peptide assay results of table
| Cyclic peptide C (μ g/g) | Cyclic peptide E (μ g/g) | Cyclic peptide D (μ g/g) | Cyclic peptide A (μ g/g) | |
| Batch 1 | 189.82 | 175.26 | 30.02 | 256.37 |
| Batch 2 | 192.35 | 176.98 | 29.86 | 252.78 |
| Batch 3 | 195.19 | 173.09 | 27.45 | 256.77 |
| Average residence time (min) | 15 | 18 | 20 | 26 |
Claims (6)
1. a kind of method for detecting linseed oil middle ring peptide content, preparation, reference substance solution including test sample test solution
Preparation, ultra-performance liquid chromatography (UPLC) measure cyclic peptide content；
The chromatographic condition of the ultra-performance liquid chromatography is：Waters ACQUITY UPLC I-Class ultra high efficiency liquid phase color
Spectra system and its work station；Chromatographic column is ACQUITY UPLC Peptide CSH C18Column (2.1mm × 100mm, 1.7 μ
m)；Mobile phase is the phosphate aqueous solution (B) of acetonitrile (A) and 0.05% (volume fraction)；Gradient elution；Detection wavelength 200~
400nm；10 μ L of sample volume；35 DEG C of column temperature；Flow velocity 1.0mL/min.
2. the method for detection linseed oil middle ring peptide content as described in claim 1, which is characterized in that the gradient elution
Condition is：0~8min, 30%A；8~20min, 30%A → 50%A；20~30min, 65%A.
3. the method for detection linseed oil middle ring peptide content as described in claim 1, which is characterized in that the Detection wavelength is
214nm。
4. the method for detection linseed oil middle ring peptide content as described in claim 1, which is characterized in that the reference substance solution
The method of preparation is：
Take linseed cyclic peptide A, C, D, E reference substance, after accurately weighed, dissolved and be settled in 10mL volumetric flask with acetonitrile, obtained
Each cyclic peptide concentration is the mixed solution of 1mg/mL, as reference substance solution.
5. the method for detection linseed oil middle ring peptide content as described in claim 1, which is characterized in that the test solution
Preparation include the following steps：
(1) extraction of linseed oil：Linseed is selected, cleans, and pulverizer smashes it through 20 meshes；Weigh a certain amount of flax
In a round bottom flask, acetone, ultrasonic extraction is added in seed powder；Extracting solution is filtered, and is concentrated under reduced pressure, is obtained linseed after vacuum drying
Oil；
(2) preparation of test solution：Linseed oil is taken, with 10~40 times of dilution in acetonitrile, after 0.45 μm of miillpore filter filters,
As test solution.
6. the method for detection linseed oil middle ring peptide content as claimed in claim 5, which is characterized in that the linseed oil mentions
It takes in step, the process conditions of ultrasonic extraction are：Solid-liquid ratio 1:6~1:12,40~60 DEG C of ultrasonic temperature, extraction time 30~
60min, 180~240W of ultrasonic power, 400~600r/min of mixing speed.
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| CN113105523A (en) * | 2021-03-19 | 2021-07-13 | 暨南大学 | Preparation method of linseed cyclopeptide mixture with low cyclopeptide A content |
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Application publication date: 20181130 |