CN109655536B - Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride - Google Patents
Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride Download PDFInfo
- Publication number
- CN109655536B CN109655536B CN201811390546.7A CN201811390546A CN109655536B CN 109655536 B CN109655536 B CN 109655536B CN 201811390546 A CN201811390546 A CN 201811390546A CN 109655536 B CN109655536 B CN 109655536B
- Authority
- CN
- China
- Prior art keywords
- acid diglyceride
- capric acid
- caprylic acid
- content
- mobile phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention relates to a method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride, which comprises the following steps: (1) preparation of a test solution: dissolving medium chain triglyceride in methanol, diluting, and shaking to obtain sample solution; (2) preparation of control solutions: adding methanol into caprylic acid diglyceride and capric acid diglyceride reference substances to dissolve and dilute, so as to obtain a mixed reference substance solution; (3) content determination: precisely measuring the test solution and the mixed reference solution, injecting into a liquid chromatograph, and recording the chromatogram; and respectively calculating the content of caprylic acid diglyceride and capric acid diglyceride in the test sample by peak areas according to an external standard method. The determination method of the invention has strong practicability, simple and convenient operation, accurate result and good reproducibility, provides data support and reference for the quality standard formulation and preparation process screening of the medium chain triglyceride and ensures the scientificity and advancement of the determination of the diglyceride.
Description
Technical Field
The invention relates to a method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride, belonging to the field of detection of medicinal raw materials and food.
Background
Medium-chain triglycerides (MCT), also known as Medium-chain oils, are triglyceride mixtures of saturated fatty acids esterified with glycerol, and fatty acid-caprylic acid mixed acids extracted from the hard dried endosperm fraction of coconut milk l. Typical MCT is considered to be saturated caprylic triglyceride or saturated capric triglyceride or a mixed saturated caprylic-capric triglyceride, which is a modified product of natural oil esters. The application for years at home and abroad proves that the product is a safe and reliable product, can be widely used as an emulsifier, a lubricant and a stabilizer, and can be used as a raw and auxiliary material for the production of medicines, foods, health-care products, cosmetics and the like. The medium chain triglycerides currently used in pharmaceutical production are used as raw materials for the production of medium/long chain fat emulsion injection.
The medical medium chain triglyceride is prepared by using Medium Chain Fatty Acid (MCFA) and glycerol as raw materials through a chemical or enzyme catalysis process, wherein diglyceride is one of main byproducts. Diacylglycerol (DG) is structural lipid in which one fatty acid in Triacylglycerol (TG) is replaced by alkyl, is a trace component in natural vegetable fat, mainly contains two isomers of 1,3-DG and 1,2-DG, the content of the isomer is usually less than 5%, the isomer is a well-known safe food component, and the diester is also a multifunctional additive, so that the diester is wide in application prospect in the industries of food, medicine, chemical engineering and the like. In order to obtain a medium-chain structure ester raw material meeting the requirements of medical injection fat emulsion, the control of the by-product diglyceride is needed.
At present, no report about a method for measuring the absolute amount of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride is available in domestic and foreign patents and documents. Currently, the most commonly used methods for analyzing diglycerides include Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), and Gas Chromatography (GC). The TLC method has the disadvantages of low accuracy, the GC method has the disadvantages of high temperature column requirement, large fluctuation of response value, poor reproducibility and the like, and the HPLC method is most suitable for analyzing the diglyceride content. The HPLC method is generally characterized by various methods such as HPLC-UV, HPLC-ELSD and the like according to the types of detectors. The obvious disadvantage of HPLC-UV is that the ultraviolet detector is very sensitive to the number of double bonds of fatty acid, so that substances with different saturation degrees can generate response values with great difference, and the feasibility in quantitative analysis of grease is poor. The HPLC-ELSD method belongs to a quality type detection method, is the most suitable and universal ester analysis method, and has the advantages of high resolution, high speed, high sensitivity, repeated use of chromatographic columns and the like.
The total content of diglyceride in the medium chain triglyceride is not more than 2 percent, because the components are complex and low in content, the measurement by using a high performance liquid phase can be influenced by the interference of the main component peak of the triglyceride, and the diglyceride also has isomers, so the absolute quantity measurement of the caprylic diglyceride and the capric diglyceride after the chromatographic peaks are completely separated is very difficult.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium chain triglyceride.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride comprises the following steps:
(1) preparation of a test solution: dissolving medium chain triglyceride in methanol, diluting, and shaking to obtain sample solution;
(2) preparation of control solutions: adding methanol into caprylic acid diglyceride and capric acid diglyceride reference substances for dissolving and diluting to obtain a mixed reference substance solution of caprylic acid diglyceride and capric acid diglyceride;
(3) content determination: precisely measuring the test solution and the mixed reference solution, injecting into a liquid chromatograph, and recording the chromatogram; and respectively calculating the content of caprylic acid diglyceride and capric acid diglyceride in the test sample by peak areas according to an external standard method.
As a preferred embodiment of the detection method of the present invention, in the step (1), the mass-to-volume ratio of the medium chain triglyceride to the methanol is 200 mg: 10 mL.
As a preferred embodiment of the detection method of the present invention, in the step (2), caprylic acid diglyceride and capric acid diglyceride are precisely weighed, and dissolved and diluted by adding methanol to prepare a mixed control solution containing caprylic acid diglyceride 70 μ g, 150 μ g, 210 μ g, 300 μ g and 450 μ g and capric acid diglyceride 30 μ g, 60 μ g, 90 μ g, 120 μ g and 180 μ g respectively per 1mL of methanol.
In a preferred embodiment of the detection method of the present invention, in the step (3), the liquid chromatography conditions are:
a chromatographic column: a C18 chromatography column;
a detector: an evaporative light scattering detector;
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein A (76%) in the mobile phase and B (24%) in the mobile phase are obtained; 10-30 min, wherein A (76% → 30%) in the mobile phase and B (24% → 70%) in the mobile phase; 30-40 min, wherein A (30%) in the mobile phase and B (70%) in the mobile phase are obtained; 40-50 min, mobile phase A (30% → 76%), mobile phase B (70% → 24%); 50-60 min, wherein the content of A in the mobile phase is 76%, and the content of B in the mobile phase is 24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
respectively and precisely measuring 20 μ L of the test solution and the mixed reference solution, injecting into high performance liquid chromatograph, and measuring.
As a preferred embodiment of the detection method of the present invention, in the step (3), the external standard method specifically comprises: precisely measuring 20 mu L of mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a liquid chromatograph, measuring peak areas of the mixed reference substance solutions with different concentrations, taking the logarithm value of the peak area of the mixed reference substance solution as a vertical coordinate, taking the logarithm of the concentration of the corresponding mixed reference substance solution as a horizontal coordinate as a standard curve, and respectively calculating to obtain a linear regression equation; and precisely measuring 20 mu L of sample solution, injecting into a liquid chromatograph, measuring peak areas of caprylic acid diglyceride and capric acid diglyceride in the sample, and calculating the contents of the caprylic acid diglyceride and the capric acid diglyceride by using regression equations respectively.
The caprylic acid diglyceride has two isomers of 1, 3-caprylic acid diglyceride and 1, 2-caprylic acid diglyceride, so when the content of the 1, 3-caprylic acid diglyceride and the 1, 2-caprylic acid diglyceride is calculated, the caprylic acid diglyceride is used as a reference, and the content of the caprylic acid diglyceride is the sum of the two contents. Similarly, the capric acid diglyceride has two isomers of 1, 3-capric acid diglyceride and 1, 2-capric acid diglyceride, so when the content of the 1, 3-capric acid diglyceride and the 1, 2-capric acid diglyceride is calculated, the capric acid diglyceride is used as a reference, and the content of the capric acid diglyceride is the sum of the two.
In a preferred embodiment of the detection method of the present invention, the specification of the C18 chromatographic column is 250mm × 4.6mm, 5 μm.
As a preferred embodiment of the detection method of the present invention, the drift tube temperature of the detector is 72 ℃, the carrier gas flow rate is 2.0mL/min, the gain value is 4, and the mode is the split mode.
As a preferred embodiment of the detection method of the present invention, the content of the caprylic acid diglyceride is the sum of the contents of 1, 3-caprylic acid diglyceride and 1, 2-caprylic acid diglyceride, and the caprylic acid diglyceride in the mixed control solution is used as a control; the content of the capric acid diglyceride is the sum of the contents of the 1, 3-capric acid diglyceride and the 1, 2-capric acid diglyceride, and the capric acid diglyceride in the mixed reference solution is used as a reference.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention establishes a simple, accurate and reliable determination method, adopts a liquid chromatography separation technology and an evaporative light scattering detector for detection, utilizes a liquid phase external standard method to calculate the content of the diglyceride component in medium chain triglyceride, directly dissolves a sample with methanol, does not need steps of hydrolysis, oxidase, derivatization, esterification and the like, and can utilize a liquid phase to determine and calculate the content of the caprylic acid diglyceride component and the capric acid diglyceride component;
(2) the liquid phase chromatographic system has good separation effect, the chromatographic peak shape of the diglyceride component of the test sample is good, the separation degree between each chromatographic peak is excellent, and the separation degrees reach baseline separation, so the detection method can accurately and effectively determine the content of the diglyceride component in the medium chain triglyceride sample;
(3) the determination method of the invention has strong practicability, simple and convenient operation, accurate result and good reproducibility, provides data support and reference for the quality standard formulation and preparation process screening of the medium chain triglyceride and ensures the scientificity and advancement of the determination of the diglyceride.
Drawings
FIG. 1 is an HPLC chromatogram of a caprylic acid diglyceride control solution of the present invention.
FIG. 2 is an HPLC chromatogram of a capric acid diglyceride control solution of the invention.
FIG. 3 is an HPLC chromatogram of the separated fraction 1, 3-caprylic acid diglyceride of the medium chain triglyceride of the present invention.
FIG. 4 is an HPLC chromatogram of the separated fraction 1, 2-caprylic acid diglyceride of the medium chain triglyceride of the present invention.
FIG. 5 is an HPLC chromatogram of the separated 1, 3-decanoic acid diglyceride of the medium chain triglyceride of the present invention.
FIG. 6 is an HPLC chromatogram of the separated fraction 1, 2-decanoic acid diglyceride of the medium chain triglyceride of the present invention.
FIG. 7 is an HPLC chromatogram of a sample of a medium chain triglyceride of the present invention.
FIG. 8 is an HPLC chromatogram (0-20 min enlarged view) of a sample of a medium-chain triglyceride of the present invention.
FIG. 9 is an HPLC chromatogram (0-20 min enlarged view) of a mixed control solution for repeated measurement of chain triglycerides in the present invention.
FIG. 10 is an HPLC chromatogram (0-20 min enlarged view) of a test sample for repeated determination of chain triglycerides in the present invention.
FIG. 11 is an HPLC chromatogram (0-20 min enlarged view) of a test sample for 0-hour stability measurement of a chain triglyceride in the present invention.
FIG. 12 is an HPLC chromatogram (0-20 min enlarged view) of a test sample for 24-hour stability measurement of a chain triglyceride in the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
Specificity test of the detection method of the invention
One embodiment of the method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in the chain triglyceride in the invention comprises the following steps:
(1) preparation of a test solution: taking 250mg of medium chain triglyceride, adding 10mL of methanol for dissolving and diluting, and shaking up to obtain a medium chain triglyceride sample solution; respectively taking 10mg of 1, 3-caprylic acid diglyceride (MTG-DG-1), 1, 2-caprylic acid diglyceride (MTG-DG-2), 1, 3-capric acid diglyceride (MTG-DG-5) and 1, 2-capric acid diglyceride (MTG-DG-6) which are separated by confirming the structure of medium-chain triglyceride, adding 10mL of methanol to dissolve and dilute, and shaking up to obtain a diglyceride test solution;
(2) preparation of control solutions: taking caprylic acid diglyceride and capric acid diglyceride, respectively 10mg and 10mg, and dissolving in 1mL of methanol to obtain caprylic acid diglyceride reference solution and capric acid diglyceride reference solution;
(3) and (3) determination:
the liquid chromatography conditions were:
a chromatographic column: c18 column (250 mm. times.4.6 mm, 5 μm);
a detector: an evaporative light scattering detector (drift tube temperature 72 ℃, carrier gas flow rate 2.0mL/min, gain 4, mode split);
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein A (76%) in the mobile phase and B (24%) in the mobile phase are obtained; 10-30 min, wherein A (76% → 30%) in the mobile phase and B (24% → 70%) in the mobile phase; 30-40 min, wherein A (30%) in the mobile phase and B (70%) in the mobile phase are obtained; 40-50 min, mobile phase A (30% → 76%), mobile phase B (70% → 24%); 50-60 min, wherein the content of A in the mobile phase is 76%, and the content of B in the mobile phase is 24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
precisely measuring 20 mu L of caprylic acid diglyceride reference solution and capric acid diglyceride reference solution with different concentrations, injecting into a liquid chromatograph, and recording chromatograms.
The results of measuring caprylic acid diglyceride control solution and capric acid diglyceride control solution, and the medium chain triglyceride structure confirmed that 1, 3-caprylic acid diglyceride (MTG-DG-1), 1, 2-caprylic acid diglyceride (MTG-DG-2), 1, 3-capric acid diglyceride (MTG-DG-5), and 1, 2-capric acid diglyceride (MTG-DG-6) were separated, and 1, 3-caprylic acid diglyceride, 1, 2-caprylic acid diglyceride, 1, 3-capric acid diglyceride, and 1, 2-capric acid diglyceride in the test solutions are shown in Table 1. An HPLC (high performance liquid chromatography) spectrum of a caprylic acid diglyceride reference substance solution, an HPLC spectrum of a capric acid diglyceride reference substance solution, an HPLC spectrum of a separated component 1, 3-caprylic acid diglyceride of medium chain triglyceride, an HPLC spectrum of a separated component 1, 2-caprylic acid diglyceride of medium chain triglyceride, an HPLC spectrum of a separated component 1, 3-capric acid diglyceride of medium chain triglyceride, an HPLC spectrum of a separated component 1, 2-capric acid diglyceride of medium chain triglyceride and an HPLC spectrum of a medium chain triglyceride test sample are respectively shown in figures 1-7, and an enlarged view of the medium chain triglyceride test sample at 0-20 min is shown in figure 8.
TABLE 1 isolation of medium chain triglycerides
As can be seen from table 1, the peak discharge time of caprylic acid diglyceride and capric acid diglyceride components (including 1, 3-caprylic acid diglyceride, 1, 2-caprylic acid diglyceride, 1, 3-capric acid diglyceride, and 1, 2-capric acid diglyceride) separated from medium-chain triglyceride measured under the chromatographic conditions of the present invention corresponds to the peak discharge time of caprylic acid diglyceride and capric acid diglyceride components in the medium-chain triglyceride test sample. 1, 3-caprylic acid diglyceride and 1, 2-caprylic acid diglyceride are isomers, so when the content of the 1, 3-caprylic acid diglyceride and the 1, 2-caprylic acid diglyceride is calculated, the caprylic acid diglyceride is used as a reference substance, and the content of the caprylic acid diglyceride is the sum of the two; similarly, 1, 3-capric acid diglyceride and 1, 2-capric acid diglyceride are isomers, so when the content of the 1, 3-capric acid diglyceride and 1, 2-capric acid diglyceride is calculated, the capric acid diglyceride is used as a reference substance, and the content of the capric acid diglyceride is the sum of the two. According to the results, the peak separation degree and the theoretical plate number of each component meet the requirements, which indicates that the method has good specificity, so that the content of caprylic acid diglyceride and capric acid diglyceride can be calculated by adopting an external standard method.
Example 2
Linearity test of the detection method of the invention
One embodiment of the method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in the chain triglyceride in the invention comprises the following steps:
(1) preparation of a test solution: taking 200mg of medium chain triglyceride, adding 10mL of methanol for dissolving and diluting, and shaking up to obtain a test solution;
(2) preparation of control solutions: precisely weighing caprylic acid diglyceride and capric acid diglyceride reference substances, adding methanol for dissolving and diluting, and preparing mixed reference substance solutions containing caprylic acid diglyceride 70 mug, 150 mug, 210 mug, 300 mug and 450 mug and capric acid diglyceride 30 mug, 60 mug, 90 mug, 120 mug and 180 mug respectively per 1mL of methanol;
(3) and (3) determination:
the liquid chromatography conditions were:
a chromatographic column: c18 column (250 mm. times.4.6 mm, 5 μm);
a detector: an evaporative light scattering detector (drift tube temperature 72 ℃, carrier gas flow rate 2.0mL/min, gain 4, mode split);
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein A (76%) in the mobile phase and B (24%) in the mobile phase are obtained; 10-30 min, wherein A (76% → 30%) in the mobile phase and B (24% → 70%) in the mobile phase; 30-40 min, wherein A (30%) in the mobile phase and B (70%) in the mobile phase are obtained; 40-50 min, mobile phase A (30% → 76%), mobile phase B (70% → 24%); 50-60 min, wherein the content of A in the mobile phase is 76%, and the content of B in the mobile phase is 24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
precisely measuring 20 mu L of mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a liquid chromatograph, recording a chromatogram, measuring peak areas of the mixed reference substance solutions with different concentrations, taking a logarithm value of the peak area of the mixed reference substance solution as a vertical coordinate, taking a logarithm of the concentration of the corresponding mixed reference substance solution as a horizontal coordinate as a standard curve, and respectively calculating to obtain a linear regression equation; the results are shown in Table 2, from which the standard curve equation for caprylic acid diglyceride is shown: 2.0467x +5.7424, coefficient of correlation r20.9984, indicating good linearity of the method in the concentration range of 0.07285mg/ml to 0.4371 mg/ml; taking the logarithm of the peak area of the capric acid diglyceride as an ordinate and taking the logarithm of the concentration of the corresponding standard solution as an abscissa to make a standard curve, and the result is shown in table 2, and the standard curve equation of the capric acid diglyceride can be known from the result: 1.3592x +5.5447, coefficient of correlation r2＝0.9990The method is shown to be linear in the concentration range of 0.03025 mg/ml-0.1815 mg/ml.
TABLE 2 Linear relationship test results
Example 3
Repeatability test of the detection method of the invention
One embodiment of the method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in the chain triglyceride in the invention comprises the following steps:
(1) preparation of a test solution: taking 200mg of 6 parts of medium-chain triglyceride in the same batch, adding 10mL of methanol for dissolving and diluting, and shaking up to obtain a test solution;
(2) preparation of control solutions: precisely weighing caprylic acid diglyceride and capric acid diglyceride reference substances, adding methanol for dissolving and diluting, and preparing mixed reference substance solutions containing caprylic acid diglyceride 70 mug, 150 mug, 210 mug, 300 mug and 450 mug and capric acid diglyceride 30 mug, 60 mug, 90 mug, 120 mug and 180 mug respectively per 1mL of methanol;
(3) and (3) determination:
the liquid chromatography conditions were:
a chromatographic column: c18 column (250 mm. times.4.6 mm, 5 μm);
a detector: an evaporative light scattering detector (drift tube temperature 72 ℃, carrier gas flow rate 2.0mL/min, gain 4, mode split);
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein A (76%) in the mobile phase and B (24%) in the mobile phase are obtained; 10-30 min, wherein A (76% → 30%) in the mobile phase and B (24% → 70%) in the mobile phase; 30-40 min, wherein A (30%) in the mobile phase and B (70%) in the mobile phase are obtained; 40-50 min, mobile phase A (30% → 76%), mobile phase B (70% → 24%); 50-60 min, wherein the content of A in the mobile phase is 76%, and the content of B in the mobile phase is 24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
precisely measuring 20 mu L of mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a liquid chromatograph, recording a chromatogram, measuring peak areas of the mixed reference substance solutions with different concentrations, taking a logarithm value of the peak area of the mixed reference substance solution as a vertical coordinate, taking a logarithm of the concentration of the corresponding mixed reference substance solution as a horizontal coordinate as a standard curve, and respectively calculating to obtain a linear regression equation; in addition, precisely measuring 20 mu L of sample solution, injecting the sample solution into a liquid chromatograph, measuring peak areas of caprylic acid diglyceride and capric acid diglyceride in the sample, and respectively calculating the contents of 6 batches of caprylic acid diglyceride and capric acid diglyceride by using a regression equation.
The 0-20 min enlarged view of the HPLC chromatogram of the mixed control solution for the repeatability determination of the medium chain triglyceride is shown in FIG. 9, and the 0-20 min enlarged view of the HPLC chromatogram of the test sample is shown in FIG. 10.
The results of the content reproducibility of caprylic acid diglyceride are shown in table 3, and the results of the content reproducibility of capric acid diglyceride are shown in table 4. As can be seen from tables 3 and 4, the method has good repeatability in determining the content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride.
TABLE 3 content repeatability results for caprylic acid diglyceride
TABLE 4 content repeatability results for capric acid diglycerides
Example 4
Stability test of the detection method of the present invention
One embodiment of the method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in the chain triglyceride in the invention comprises the following steps:
(1) preparation of a test solution: taking 1 part of medium chain triglyceride (200 mg), adding 10mL of methanol for dissolving and diluting, and shaking up to obtain a test solution;
(2) and (3) determination:
the liquid chromatography conditions were:
a chromatographic column: c18 column (250 mm. times.4.6 mm, 5 μm);
a detector: an evaporative light scattering detector (drift tube temperature 72 ℃, carrier gas flow rate 2.0mL/min, gain 4, mode split);
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein A (76%) in the mobile phase and B (24%) in the mobile phase are obtained; 10-30 min, wherein A (76% → 30%) in the mobile phase and B (24% → 70%) in the mobile phase; 30-40 min, wherein A (30%) in the mobile phase and B (70%) in the mobile phase are obtained; 40-50 min, mobile phase A (30% → 76%), mobile phase B (70% → 24%); 50-60 min, wherein the content of A in the mobile phase is 76%, and the content of B in the mobile phase is 24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
precisely measuring the same test solution, precisely sucking 20 mu L of the test solution respectively at 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, injecting the sample solution into a liquid chromatograph, recording a chromatogram, measuring peak areas of the components, and respectively calculating RSD of caprylic acid diglyceride peak and capric acid diglyceride area to be 2.5% and 2.9% within 24 hours, wherein the results are shown in Table 5, which indicates that the sample solution is relatively stable within 24 hours. FIG. 11 is an enlarged view of 0-20 min of the HPLC chromatogram of the test sample for measuring the stability of the medium chain triglyceride at 0 hour, and FIG. 12 is an enlarged view of 0-20 min of the HPLC chromatogram of the test sample for measuring the stability of the medium chain triglyceride at 24 hours.
TABLE 5 content repeatability results for capric acid diglycerides
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
Claims (6)
1. A method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride is characterized in that four compounds, namely 1, 3-caprylic acid diglyceride, 1, 2-caprylic acid diglyceride, 1, 3-capric acid diglyceride and 1, 2-capric acid diglyceride, can be separated simultaneously, and comprises the following steps:
(1) preparation of a test solution: dissolving medium chain triglyceride in methanol, diluting, and shaking to obtain sample solution;
(2) preparation of control solutions: adding methanol into caprylic acid diglyceride and capric acid diglyceride reference substances for dissolving and diluting to obtain a mixed reference substance solution of caprylic acid diglyceride and capric acid diglyceride;
(3) content determination: precisely measuring the test solution and the mixed reference solution, injecting into a liquid chromatograph, and recording the chromatogram; respectively calculating the content of caprylic acid diglyceride and capric acid diglyceride in the test sample by peak areas according to an external standard method;
wherein, in the step (3), the liquid chromatography conditions are as follows:
a chromatographic column: a C18 chromatography column;
a detector: an evaporative light scattering detector; the temperature of a drift tube of the detector is 72 ℃, the flow rate of carrier gas is 2.0mL/min, the gain value is 4, and the mode is a shunting mode;
mobile phase: gradient elution is carried out by taking an acetonitrile-water mixed solvent with a volume ratio of 8:2 as a mobile phase A and taking an acetonitrile-isopropanol mixed solvent with a volume ratio of 1:1 as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-10 min, wherein the content of the active phase is A76% and the content of the active phase is B24%; 10-30 min, wherein A in the mobile phase is 76% → 30%, and B in the mobile phase is 24% → 70%; 30-40 min, wherein the content of the mobile phase A is 30% and the content of the mobile phase B is 70%; 40-50 min, wherein A in the mobile phase is 30% → 76%, and B in the mobile phase is 70% → 24%; 50-60 min, wherein the content of the active phase is A76% and the content of the active phase is B24%;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
respectively and precisely measuring 20 μ L of the test solution and the mixed reference solution, injecting into high performance liquid chromatograph, and measuring.
2. The assay of claim 1, wherein in step (1), the mass to volume ratio of medium chain triglycerides to methanol is 200 mg: 10 mL.
3. The detection method according to claim 1, characterized in that in the step (2), caprylic acid diglyceride and capric acid diglyceride control substances are precisely weighed, methanol is added for dissolution and dilution, and mixed control substance solutions with caprylic acid diglyceride respectively being 70 mug, 150 mug, 210 mug, 300 mug and 450 mug and capric acid diglyceride respectively being 30 mug, 60 mug, 90 mug, 120 mug and 180 mug are prepared for each 1mL of methanol.
4. The detection method according to claim 1, wherein in the step (3), the external standard method specifically comprises the following operations: precisely measuring 20 mu L of mixed reference substance solutions with different concentrations, injecting the mixed reference substance solutions into a liquid chromatograph, measuring peak areas of the mixed reference substance solutions with different concentrations, taking the logarithm value of the peak area of the mixed reference substance solution as a vertical coordinate, taking the logarithm of the concentration of the corresponding mixed reference substance solution as a horizontal coordinate as a standard curve, and respectively calculating to obtain a linear regression equation; and precisely measuring 20 mu L of sample solution, injecting into a liquid chromatograph, measuring peak areas of caprylic acid diglyceride and capric acid diglyceride in the sample, and calculating the contents of the caprylic acid diglyceride and the capric acid diglyceride by using regression equations respectively.
5. The detection method of claim 4, wherein the C18 chromatographic column has a specification of 250mm x 4.6mm, 5 μm.
6. The detection method according to claim 5, wherein the content of caprylic acid diglyceride is the sum of the contents of 1, 3-caprylic acid diglyceride and 1, 2-caprylic acid diglyceride, and caprylic acid diglyceride in the mixed control solution is used as a control; the content of the capric acid diglyceride is the sum of the contents of the 1, 3-capric acid diglyceride and the 1, 2-capric acid diglyceride, and the capric acid diglyceride in the mixed reference solution is used as a reference.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811390546.7A CN109655536B (en) | 2018-11-21 | 2018-11-21 | Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811390546.7A CN109655536B (en) | 2018-11-21 | 2018-11-21 | Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109655536A CN109655536A (en) | 2019-04-19 |
| CN109655536B true CN109655536B (en) | 2021-11-12 |
Family
ID=66112250
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811390546.7A Active CN109655536B (en) | 2018-11-21 | 2018-11-21 | Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109655536B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018944A1 (en) * | 1998-09-25 | 2000-04-06 | Conlinco, Inc. | Triacylglycerols of enriched cla content |
| CN101708156A (en) * | 2009-12-10 | 2010-05-19 | 中国人民解放军第二军医大学 | Camptothecin medicament injection solution and injection and preparation method thereof |
| CN102539586A (en) * | 2010-12-14 | 2012-07-04 | 上海良友（集团）有限公司 | Method for isolating and detecting oxidized triglyceride (ox-TG) of edible vegetable oil and application of the method |
| CN106018611A (en) * | 2016-06-21 | 2016-10-12 | 广州白云山汉方现代药业有限公司 | Testing method for detecting content of medium chain triglyceride with gas chromatography internal standard method |
| CN107334063A (en) * | 2017-07-11 | 2017-11-10 | 桂林医学院 | A kind of oatmeal containing medium chain fatty acid ester and preparation method thereof |
-
2018
- 2018-11-21 CN CN201811390546.7A patent/CN109655536B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018944A1 (en) * | 1998-09-25 | 2000-04-06 | Conlinco, Inc. | Triacylglycerols of enriched cla content |
| CN101708156A (en) * | 2009-12-10 | 2010-05-19 | 中国人民解放军第二军医大学 | Camptothecin medicament injection solution and injection and preparation method thereof |
| CN102539586A (en) * | 2010-12-14 | 2012-07-04 | 上海良友（集团）有限公司 | Method for isolating and detecting oxidized triglyceride (ox-TG) of edible vegetable oil and application of the method |
| CN106018611A (en) * | 2016-06-21 | 2016-10-12 | 广州白云山汉方现代药业有限公司 | Testing method for detecting content of medium chain triglyceride with gas chromatography internal standard method |
| CN107334063A (en) * | 2017-07-11 | 2017-11-10 | 桂林医学院 | A kind of oatmeal containing medium chain fatty acid ester and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Qualitative and quantitative analysis of lipid classes in fish oils by thin-layer chromatography with an Iatroscan flame ionization detector (TLC-FID) and liquid chromatography with an evaporative light scattering detector (LC-ELSD);Indrasena, WM 等;《Journal of Liquid Chromatography & Related Technologies》;20051231;第28卷(第16期);第2581-2595页 * |
| 甘油二酯异构体高效液相色谱分析法的建立;樊巧英 等;《中国油脂》;20170831;第42卷(第8期);第131-135页 * |
| 甘油二酯的制备及其高效液相色谱的检测技术;赵欣欣 等;《食品工业》;20161130;第37卷(第11期);第228-231页 * |
| 食用植物油中甘油三酯色谱分析方法研究进展;籍淑贤 等;《分析测试学报》;20140131;第33卷(第1期);第112-118页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109655536A (en) | 2019-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bravi et al. | Fatty acids by high-performance liquid chromatography and evaporative light-scattering detector | |
| CN106018611B (en) | A kind of assay method of gas chromatogram fixative detection medium chain triglyceride content | |
| Hewawasam et al. | A validated method for analyzing polyunsaturated free fatty acids from dried blood spots using LC–MS/MS | |
| CN103018363B (en) | Method for detecting free fatty acid content in compound traditional Chinese medicine | |
| Guo et al. | Determination of underivatized long chain fatty acids using HPLC with an evaporative light-scattering detector | |
| CN102156174A (en) | Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography | |
| Delmonte | Evaluation of poly (90% biscyanopropyl/10% cyanopropylphenyl siloxane) capillary columns for the gas chromatographic quantification of trans fatty acids in non-hydrogenated vegetable oils | |
| Fontell et al. | Some new methods for separation and analysis of fatty acids and other lipids | |
| Nguyen et al. | Chiral high-performance liquid chromatographic separation of evodiamine enantiomers and rutaecarpine, isolated from Evodiae fructus | |
| Baškirovs et al. | Analytical scale supercritical fluid chromatography for the analysis of nine tocochromanols in 24 different cold-pressed plant oils: Method development, validation, and isolation of tocotrienols and plastochromanol-8 | |
| CN109387587B (en) | Detection method of L-2-amino-5-guanidino valeric acid enantiomer | |
| CN109655536B (en) | Method for detecting content of caprylic acid diglyceride and capric acid diglyceride in medium-chain triglyceride | |
| Liu et al. | Quantitative polyunsaturated fatty acid analysis of chia seed oil by high-performance liquid chromatography | |
| CN108181395A (en) | A kind of method of triglycerides ingredient in on-line checking fish oil using high performance liquid chromatography | |
| CN109142585B (en) | Method for detecting isomer of sodium pantothenate | |
| Porwal et al. | Assessment by HPLC of the degradation behavior of acitretin under hydrolytic, oxidative, photolytic and thermal stress conditions | |
| CN115326909A (en) | Method for detecting free fatty acid in human serum | |
| Saravanan et al. | Development And Validation of Stability Indicating RP-HPLC method for the simultaneous estimation of Metoprolol Succinate and Cilnidipine in bulk and pharmaceutical dosage form | |
| CN107621509A (en) | The method for quantitatively determining of the ester of 1,3 two oleic acid, 2 palmitic acid three in baby formula milk powder | |
| CN111044640B (en) | Method for determining content of gamma-aminobutyric acid in feed additive by GC (gas chromatography) method | |
| Sevim et al. | Validation of high performance liquid chromatographic and spectrophotometric methods for the determination of the antiparkinson agent pramipexole dihydrochloride monohydrate in pharmaceutical products | |
| King et al. | A HPLC method to monitor the occurrence of lipid peroxidation in intravenous lipid emulsions used in parenteral nutrition using in-line UV and charged aerosol detection | |
| CN101464455A (en) | Detection method for octopamine and its precursor compound tyramine content in fish sauce | |
| CN114216976B (en) | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography | |
| Yue et al. | Free fatty acids profile analysis of alcohol extract of Aconitum taipeicum Hand.-Mazz. with gas chromatography-mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |