A method for determining the content of medium-chain triglycerides by HPLC-ELSD

Abstract

This invention proposes an HPLC-ELSD method for determining the content of medium-chain triglycerides. Using tricaprylic acid glyceride and tricaprylic acid glyceride as controls, the method employs HPLC-ELSD based on a C18 column for separation, captures signals using an evaporative light scattering detector, and performs linear fitting using the logarithmic response values ​​and concentration values ​​of tricaprylic acid glyceride and tricaprylic acid glyceride. The logarithmic peak area of ​​the main peak of the test sample is substituted into the linear equation for calculation. The content of tricaprylic acid glyceride and dicaprylic acid-capric acid glyceride in the test sample is calculated using the regression equation of tricaprylic acid glyceride, and the content of tricaprylic acid glyceride and dicaprylic acid-capric acid glyceride in the test sample is calculated using the regression equation of tricaprylic acid glyceride. The total content of the four main triglycerides is calculated as the triglyceride content. This method overcomes the drawback of the difficulty in obtaining dicaprylic acid-capric acid glyceride and dicaprylic acid-capric acid glyceride controls, and offers high sensitivity and accuracy. It is simple, rapid, and suitable for industrial production and quality control.

Description

Technical Field

[0001] This invention relates to the field of instrumental analysis and detection technology, specifically to a method for determining the content of medium-chain triglycerides using HPLC-ELSD. Background Technology

[0002] Glycerides can be classified into short-chain triglycerides (SCTs), medium-chain triglycerides (MCTs), and long-chain triglycerides (LCTs) based on their fatty acid chain length. Medium-chain triglycerides are relatively rare in nature, primarily found in breast milk, cow's milk and its products, palm kernel oil, and coconut oil. They are characterized by high water solubility and a high hydrolysis rate, exhibiting unique characteristics in digestion, absorption, hydrolysis, and metabolism. Ingested MCTs are broken down into glycerol and medium-chain fatty acids (MCFAs) by lipases in the stomach and duodenum. In the small intestine, they can be absorbed directly as glycerol and MCFAs without bile emulsification, passing directly through the small intestinal capillaries into the portal vein and then rapidly transported to the liver. The rate of MCFA oxidation and metabolism in the liver is as fast as that of glucose, and its mitochondrial oxidation across the membrane does not rely on carnitine as a carrier. This unique absorption and metabolic pathway of MCTs is of great significance for nutritional therapy in some clinical diseases.

[0003] Medium-chain triglycerides (MCTs) can be used as active pharmaceutical ingredients (APIs) in nutritional fat emulsion injections, primarily in medium / long-chain fat emulsion injections, such as Fresenius Kabi Huare Pharmaceutical Co., Ltd.'s medium / long-chain fat emulsion injection (C6~C24), marketed under the name "Lineng". They can also be used as solvents in soft capsules and ointments. For drugs like idecalciferol, which are highly lipid-soluble and almost insoluble in water, MCTs are key excipients.

[0004] In the field of pharmaceutical formulation, particularly in delivery systems involving poorly soluble drugs (such as self-microemulsions, fat emulsion injections, and soft capsules), medium-chain triglycerides (MCTs) are crucial as high-performance oil-soluble solvents and carriers. The success of original formulations depends not only on their active ingredients but also heavily on the specific composition and properties of their complex excipient systems. For generic drug development, one of the core challenges in achieving pharmaceutical consistency with the original product lies in the precise quality control of key excipients such as MCTs.

[0005] Currently, commercially available medium-chain triglycerides (MCTs) are mainly differentiated based on the ratio of carbon atoms in the fatty acid chains bound to their triglyceride molecules (C8 caprylic acid and C10 decanoic acid). Examples include Captex® 8000 (C8:C10 approximately 100:0), Captex® 810 (C8:C10 approximately 70:30), and Captex® 812 (C8:C10 approximately 60:40) produced by the IOI Group. Although these products all meet the general definition of "medium-chain triglycerides" in the Chinese Pharmacopoeia: this product is derived from coconut... Cocos nucifera L. The hard, dried part of the endosperm or oil palm Elaeis guineensis Caprylic acid (C8H) isolated from the fatty oil extracted from the dried part of Jacq endosperm. 16 O2), decanoic acid (C 10 H 20 A mixture of triglycerides obtained by esterification of saturated fatty acids such as O2 with glycerol. Contains caprylic acid (C8H2O). 16 O2) and decanoic acid (C 10 H 20 The total amount of O2 must not be less than 95.0%. However, the specific caprylic / capric acid ratio (i.e., glyceride composition) varies, which directly and significantly affects the key quality attributes of the formulation. 1. Drug solubility: Triglycerides of different chain lengths have different saturated solubilities for specific drugs, and deviations in the ratio may cause the drug to precipitate during storage; 2. Self-emulsification efficiency and droplet size: In self-microemulsification systems, the composition of the oil phase is the thermodynamic driving force and size determinant for the formation of nanodroplets. Changes in the proportion will alter the HLB value of the system, affecting its self-emulsification performance and the size of the formed droplets, thereby influencing the rate and extent of drug absorption in vivo; 3. Metabolic rate and safety: C8 and C10 have different metabolic pathways and rates in the body, and changes in their ratio may affect the safety and metabolic kinetics of parenteral nutrition preparations that use MCT as an energy source.

[0006] Current MCT quality standards (such as those in various pharmacopoeias) typically only control conventional items such as acid value, iodine value, peroxide value, and fatty acid composition. Fatty acid composition analysis (usually determined by GC after methylation) can only reflect the distribution of total fatty acids, but cannot directly and accurately reveal the specific molecular composition of its original glycerides (such as CCC, CCL, CLL, LLL, etc., where C represents caprylic acid and L represents decanoic acid). Therefore, MCT products from two suppliers may have similar total caprylic acid / decanoic acid ratios, but their glyceride molecular distributions may differ significantly. This difference at the molecular level is a potential source of inconsistencies in formulation behavior.

[0007] There are various existing methods for detecting triglyceride content. Patent document CN111077103A discloses a near-infrared quantitative analysis model stored in a cloud database. This model precisely detects and controls the amount of triacetylglycerol added to cigarette filters, thereby stabilizing filter hardness and improving cigarette smoking quality. The method uses the industry standard YC / T 331-2010, "Determination of Triacetylglycerol in Cellulose Acetate Filters by Gas Chromatography," to obtain the accurate triacetylglycerol content in the sample filter. Combining the near-infrared spectrum of the filter sample with the accurate triacetylglycerol content, a near-infrared quantitative analysis model for detecting tributyric acid glycerol is established using partial least squares (PLS). This method utilizes a near-infrared spectrometer, but the instrument is not widely available, and the algorithm is complex, requiring combinations of smoothing, reciprocal processing, standard normal transformation, and multivariate scattering correction.

[0008] Patent document CN103926353A discloses a gas chromatography method for determining triacetylglycerol, using area normalization for quantification. The article "Improvement of the Determination Method for Triethylglycerol Content in Pharmaceutical Excipients" by Liu Yanming et al. also established a gas chromatography-internal standard method for determining triacetylglycerol. However, both methods are limited by the limitations of gas chromatography and are not suitable for determining the content of medium-chain or medium-long-chain fatty acid triglycerides with high boiling points. Furthermore, the patent's quantification method, area normalization, requires all components to elute with consistent responses. In reality, diglycerides, monoglycerides, fatty acids, and free glycerol can all elute, but these substances have large polarity differences and lack consistent responses. Additionally, impurities such as water do not respond in the gas phase. Therefore, this patent detects relative content and cannot assess absolute content.

[0009] Patent document CN117517508A discloses a method for determining glyceryl monostearate and glyceryl distearate in benvomod cream using high performance liquid chromatography-electrospray ionization (HPLC-ESI) detector. The method uses a C18 column as the stationary phase, water and acetonitrile as the mobile phase, gradient elution, and ESI detector to determine glyceryl monostearate and glyceryl distearate, quantifying by external standard method. However, this method requires an expensive and not widely available ESI detector, and the mobile phase of water and acetonitrile has limited solubility in oils. The analysis time is up to one hour, making it uneconomical and impractical.

[0010] Patent document CN116893233A discloses a method for simultaneously determining the content of soybean oil and medium-chain triglycerides in medium- and long-chain fat emulsion injections. Specifically, it uses silica gel as the packing material, n-hexane-isopropanol-glacial acetic acid (98.9-1.0-0.1%) as the mobile phase, evaporative light scattering detector, and commercial batch material as a reference. The method involves logarithmic linear regression fitting of the logarithmic concentration of the reference solution with the corresponding peak area, and then inputting the sample solution into the calculation to determine the content of soybean oil and medium-chain triglycerides. This method is based on a normal-phase separation mechanism and uses commercial soybean oil and medium-chain triglycerides as controls. It cannot specifically calculate the content of each triglyceride component, and the calculation of commercial batch soybean oil and medium-chain triglycerides is recorded as 100%, which does not represent the true content, resulting in poor accuracy.

[0011] Patent document CN110632193A discloses a method for determining the content of 1,3-dioleoyl-2-palmitoylglycerol triglyceride in infant formula milk powder using high performance liquid chromatography (HPLC) with external standard method. Specifically, it uses dichloromethane and acetonitrile as the mobile phase, a C18 column, gradient elution, and linear fitting with a series of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) standard working solutions to calculate the OPO content in the test sample. This method, using dichloromethane as the mobile phase, is not optimal in terms of safety and column tolerance, and is also cumbersome and costly.

[0012] Zhou Xiaohong et al.'s paper, "Determination of Trioleic Acid Glycerides in Ganoderma Lucidum Spore Oil by HPLC-ELSD," describes a method for determining trioleic acid glycerides in Ganoderma lucidum spore oil using acetonitrile-isopropanol (51:49) isocratic elution on a C18 column. This method is simple, accurate, and reproducible. However, spore oil mainly contains oleic acid, linoleic acid, palmitic acid, and stearic acid, which may result in at least 20 components. This paper only detects trioleic acid glycerides and does not consider all glycerides.

[0013] Fang Xinxin et al. proposed a method for the simultaneous determination of mono-, di-, and triglycerides and free glycerol in glycerol behenate by gel permeation chromatography. The method used two styrene-divinylbenzene columns (5 μm, 100A, 300 mm × 7.8 mm) in series, with tetrahydrofuran as the mobile phase, a differential refractive index detector, and peak area normalization. However, the method was not very sensitive or accurate.

[0014] In summary, while existing methods for detecting glycerides each have their advantages, they generally suffer from drawbacks such as complex operation, high cost, lack of versatility, inability to evaluate the content of individual components, and a lack of methods for detecting and evaluating components with different contents of medium-chain triglycerides. Summary of the Invention

[0015] This invention proposes an HPLC-ELSD method for determining the content of medium-chain triglycerides, which can simultaneously determine the content of different triglycerides to meet the actual requirements for quality control of raw materials or products.

[0016] The technical solution of this invention is implemented as follows: A method for determining the content of medium-chain triglycerides by HPLC-ELSD, comprising the following steps: S1. Take tricaprylic acid glyceride and tricaprylic acid glyceride standards, prepare standard solutions of different concentrations respectively, perform HPLC-ELSD detection, and plot standard curves respectively; S2. The medium-chain triglyceride test solution was subjected to HPLC-ELSD detection to obtain the peak areas of tricaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, and tricaprylic acid glyceride; the contents of tricaprylic acid glyceride and dicaprylic acid monocaprylic acid glyceride in the test sample were calculated from the standard curve of tricaprylic acid glyceride; the total content of tricaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, and tricaprylic acid glyceride was taken as the total content of triglycerides. The HPLC-ELSD conditions are as follows: octadecyl bonded silica gel is used as the packing material or a column with equivalent performance; the mobile phase is a mixture of methanol and isopropanol with a volume percentage of (70-90):(10-30); and the ELSD drift tube temperature is 40-80℃.

[0017] Furthermore, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0018] Furthermore, the volume ratio of methanol to isopropanol in the mobile phase is 10:90.

[0019] Furthermore, the tricaprylic acid glyceride and tricaprylic acid glyceride standards are prepared into standard solutions of different concentrations using methanol and / or isopropanol as solvents.

[0020] Furthermore, any of the standard solutions of different concentrations are selected from any 3-5 concentrations in the range of 0.01-5.0 mg / mL; for example, 0.02 mg, 0.1 mg, 0.2 mg, 0.5 mg, and 1.0 mg / mL.

[0021] Furthermore, the concentration of triglycerides in the medium-chain triglyceride test sample is 0.5-5.0 mg / mL. The test sample can be any sample containing tricaprylic acid glyceride, dicaprylic acid-capric acid glyceride, dicaprylic acid-capric acid glyceride, and tricaprylic acid glyceride, such as industrial products or grain and oil products.

[0022] Furthermore, the column temperature of the chromatographic column is 25-45℃.

[0023] Furthermore, the carrier gas flow rate of the evaporative light scattering drift tube is 1.5-2.5 L / min.

[0024] Furthermore, the injection volume for the HPLC-ELSD detection process is 5-20 μL.

[0025] Furthermore, the high-performance liquid chromatography method can generally employ liquid chromatography, and the brand can be selected from Agilent, Shimadzu, Thermo Fisher Scientific, and Waters, etc. The evaporative light scattering detector used can be from Agilent, Alltech, and Waters, etc.

[0026] Furthermore, in the chromatographic column, the particle size of the packing material can be conventional in the art, preferably 3-5 μm. The column length can be conventional in the art, preferably 5-25 cm. The inner diameter of the column can be conventional in the art, preferably 4.6 mm. The chromatographic column can be purchased from Agilent Technologies or Phylogen Technologies.

[0027] Furthermore, the peak area of ​​the tricaprylic acid glyceride and tricaprylic acid glyceride can be confirmed by the characteristic peak of the standard and then the content can be calculated; the characteristic peak and mass-to-charge ratio (m / z) of the dicaprylic acid monocaprylic acid glyceride and dicaprylic acid monocaprylic acid glyceride are obtained by liquid chromatography-mass spectrometry to identify the substance components at the corresponding residence time, and the content is calculated based on the peak area of ​​the component.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: The detection method of this invention directly uses tricaprylic acid glyceride and tricaprylic acid glyceride as controls, and separates them using HPLC-ELSD based on a C18 column, capturing the signal with an evaporative light scattering detector. Linear fitting is performed using the logarithmic response values ​​and concentration values ​​of tricaprylic acid glyceride and tricaprylic acid glyceride. The logarithm of the peak area of ​​the main peak of the test sample is substituted into the linear equation for calculation. The content of tricaprylic acid glyceride and dicaprylic acid glyceride in the test sample is calculated using the regression equation of tricaprylic acid glyceride, and the content of tricaprylic acid glyceride and dicaprylic acid glyceride in the test sample is calculated using the regression equation of tricaprylic acid glyceride. The total content of the four main glycerides is taken as the triglyceride content. This method overcomes the deficiency of the difficulty in obtaining dicaprylic acid glyceride and dicaprylic acid glyceride reference standards, and has high sensitivity and accuracy. It is simple and rapid, and suitable for industrial production and quality control. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a standard curve of the tricaprylic acid glyceride reference standard described in the embodiments of the present invention.

[0031] Figure 2 This is a standard curve of tridecanoic acid glyceride reference standard described in the embodiments of the present invention.

[0032] Figure 3 This is a reference chromatogram for liquid chromatography detection of the test sample in this embodiment of the invention. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] In some embodiments, we collected data from five different MCT manufacturers and two related formulations, all of which meet the relevant standards for MCT in the Chinese Pharmacopoeia, but the glyceride composition varies significantly among different manufacturers. See Table 1 for details.

[0035] Table 1:

[0036] To directly and intuitively present the differences in core molecular composition among different manufacturers and models of MCT, and to facilitate in-depth consistency comparison with original MCT excipients in formulation development, formulation research, and the establishment of higher-level excipient quality control standards, it is necessary to distinguish and quantify these differences. Therefore, it is essential to propose a highly efficient analytical method specifically for determining the triglyceride content (i.e., the relative composition of different triglyceride molecules) in MCT.

[0037] This invention proposes a method for determining four major triglycerides in medium-chain triglycerides using HPLC-ELSD, comprising the following steps: (1) Prepare a methanol-isopropanol mobile phase; (2) Prepare a series of standard solutions of tricaprylic acid glyceride and tricaprylic acid glyceride; (3) Take appropriate amounts of tricaprylic acid glyceride and tricaprylic acid glyceride reference standards, dissolve and dilute them with isopropanol to prepare a mixed solution containing 0.02~1.0 mg of the above reference standards per 1 ml for detection, and obtain the peak areas of tricaprylic acid glyceride and tricaprylic acid glyceride in each standard working solution; calculate the regression equation of tricaprylic acid glyceride by the logarithm of the concentration of tricaprylic acid glyceride in the reference solution and the corresponding logarithm of the peak area, and calculate the regression equation of tricaprylic acid glyceride by the logarithm of the concentration of tricaprylic acid glyceride in the reference solution and the corresponding logarithm of the peak area. (4) Prepare and test the medium-chain triglyceride test solution.

[0038] The chromatographic conditions are as follows: The chromatographic column used was octadecyl bonded silica gel (4.6 mm × 25 cm, 5 μm or equivalent performance). The mobile phase was a mixture of methanol and isopropanol, with isopropanol content of 10-30% and a flow rate of 0.8-1.2 ml per minute. The detector was an evaporative light scattering detector (drift tube temperature of 60-80 °C and carrier gas flow rate of 1.5-2.5 L per minute).

[0039] The selection of experimental conditions for the above methods is explained below: A. Selection of Column Length Experimental tests revealed that the content of four major triglycerides in medium-chain triglycerides could be detected with column lengths of 150 mm, 200 mm, and 250 mm, without affecting the detection results of each triglyceride component.

[0040] B. Selection of Sample Solvent

[0041] Medium-chain triglycerides are lipid-soluble and readily soluble in petroleum ether, soybean oil, methanol, etc. Clear solutions can be obtained using both methanol and isopropanol. This method can also be extended to the detection of medium-chain triglycerides in fat emulsion injections. Vegetable oils (soybean oil) are highly soluble in isopropanol, and this method is also applicable to the detection of such samples; therefore, isopropanol is preferred as the solvent.

[0042] C. Selection of Calculation Method

[0043] Tricaprylic acid glyceride and tricaprylic acid glyceride are triglycerides formed from single fatty acids. The degree of reaction and the purity of the fatty acids are easily controlled, and standards are readily available. The other two components in medium-chain triglycerides (dicaprylic acid-capric acid glyceride and dicaprylic acid-capric acid glyceride) require different synthesis processes. Besides controlling the degree of reaction and the ratio of caprylic to capric acid, they also need to be separable from randomly bound fatty acid glycerides. Furthermore, dicaprylic acid-capric acid glyceride exhibits positional isomerism, making pure products difficult to obtain. To approximate the actual content of each glyceride in the sample as closely as possible, this invention selects the following calculation method: ①Calculate the four triglycerides using trioctylglycerol as the linear arithmetic ester; ②Calculate the four triglycerides using tridecanoic acid glyceride as the linear arithmetic component; ③Calculate tricaprylate and dicaprylate-capric acid glyceride linearly using tricaprylate, and calculate tricaprylate and dicaprylate-capric acid glyceride linearly using tricaprylate; ④ Any of the above, but corrected by the molar mass of each component.

[0044] In the above embodiments, the peak area of ​​the tricaprylic acid glyceride and tricaprylic acid glyceride can be confirmed by the characteristic peak of the standard and then the content can be calculated; the characteristic peak and mass-to-charge ratio (m / z) of the dicaprylic acid monocaprylic acid glyceride and dicaprylic acid monocaprylic acid glyceride are obtained by liquid chromatography-mass spectrometry to identify the substance components at the corresponding residence time, and the content is calculated based on the peak area of ​​the component.

[0045] In one embodiment, for the same test sample, the content of each component and the total content were calculated using the above four different calculation methods, and the results are shown in Table 2.

[0046] Table 2:

[0047] As shown above, method 1 results in a lower total glyceride content, while method 2, using linear calculations based on tricaprylate, results in a higher total glyceride content. Method 4, however, requires knowing the molecular weights of the components and performing molecular weight correction, making it more complex, but it approximates the true values ​​most closely. Method 3, using linear calculations based on tricaprylate to determine both tricaprylate and dicaprylate, is relatively simple, and the data is close to that of method 4.

[0048] Example 1

[0049] Tricaprylic acid glyceride standard: Source: Maclean's; Batch No.: C12635118, Purity 99.16%; Tridecanoic acid glyceride standard: Source: Sigma-Aldrich, Lot No.: SLCM1123; Medium-chain triglyceride sample, source: Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd., batch number Y003C211101 Chromatographic conditions: Octadecyl bonded silica gel was used as the packing material (4.6 mm × 25 cm, 5 μm or equivalent column), and the column temperature was 25 °C; methanol-isopropanol (9:1) was used as the mobile phase, and the flow rate was 1.0 mL per minute; the detector was an evaporative light scattering detector (reference conditions: drift tube temperature 60 °C, carrier gas flow rate 1.5 L per minute).

[0050] Chromatographic-mass spectrometry conditions: Octadecyl bonded silica gel as the stationary phase (4.6 mm × 25 cm, 5 μm or equivalent column), column temperature 25 °C; methanol-isopropanol (9:1) as the mobile phase, flow rate 1.0 ml / min; Shimadzu LCMS2050, ion source: ESI; SCAN mode, mass-to-charge ratio range: 300~800; Test solution: Take an appropriate amount of sample and dilute it with isopropanol to a solution containing approximately 2 mg per 1 ml.

[0051] Reference solution: Take appropriate amounts of tricaprylic acid glyceride and tricaprylic acid glyceride reference standards, dissolve and dilute them with isopropanol to prepare a mixed solution containing 0.02 mg, 0.1 mg, 0.2 mg, 0.5 mg and 1.0 mg of the above reference standards per 1 ml.

[0052] The operation steps are as follows: 1) Accurately measure 5 μl of both the reference solution and the test solution, and inject them into the liquid chromatograph. Calculate the regression equation for tricaprylic acid glycerol using the logarithm of the tricaprylic acid glycerol concentration in the reference solution and the corresponding logarithm of the peak area. Calculate the regression equation for tricaprylic acid glycerol using the logarithm of the tricaprylic acid glycerol concentration in the reference solution and the corresponding logarithm of the peak area. The regression equation curve for tricaprylic acid glycerol is shown below. Figure 1 As shown, the regression equation curve for tridecanoic acid glyceride is as follows: Figure 2 As shown.

[0053] 2) Inject the test solution into the liquid chromatograph, obtain the chromatogram, and confirm the substance corresponding to the retention time (RT) in the chromatogram by obtaining the mass spectrum under the same chromatographic conditions. The results are shown in Table 3.

[0054] Table 3:

[0055] like Figure 3 As shown in the figure, the characteristic peaks identified correspond to the following: peak 1 is tricaprylic acid glyceride; peak 2 is 1,2-dicaprylic-3-decanoic acid glyceride (1,3-dicaprylic-2-decanoic acid glyceride); peak 3 is 1,3-dicoctanoic-2-octanoic acid glyceride (1,2-dicoctanoic-3-octanoic acid glyceride); and peak 4 is tricaprylic acid glyceride.

[0056] 3) Content Calculation

[0057] Based on the peak areas corresponding to each component in the chromatogram, the contents of tricaprylic acid glyceride and dicaprylic acid monocaprylic acid glyceride in the test sample were calculated using the regression equation of tricaprylic acid glyceride. Table 4 shows the detection results of six different samples.

[0058] Table 4:

[0059] Example 2 Recovery rate experiment

[0060] Blank solution: Isopropanol

[0061] Test solutions and linear solutions: see the repeatability section; 80% accuracy solution: Weigh 0.2g of the test sample accurately into a 20ml volumetric flask, dissolve and dilute to the mark with isopropanol; take 1ml of this solution into a 10ml volumetric flask, accurately add 0.8ml each of the stock solutions of tricaprylic acid glyceride and tridecanoic acid glyceride, dissolve and dilute to the mark with isopropanol; prepare 3 portions in the same manner.

[0062] 100% accuracy solution: Accurately weigh 0.2 g of the test sample into a 20 ml volumetric flask, dissolve and dilute to the mark with isopropanol; take 1 ml of this solution into a 10 ml volumetric flask, accurately add 1 ml each of tricaprylic acid glyceride and tricaprylic acid glyceride stock solutions, dissolve and dilute to the mark with isopropanol. Prepare 3 aliquots using the same method.

[0063] 120% accuracy solution: Accurately weigh 0.2 g of the test sample into a 20 ml volumetric flask, dissolve and dilute to the mark with isopropanol; take 1 ml of this solution into a 10 ml volumetric flask, accurately add 1.2 ml each of the stock solutions of tricaprylic acid glyceride and tridecanoic acid glyceride, dissolve and dilute to the mark with isopropanol. Prepare 3 aliquots using the same method.

[0064] Take the reference solution, test solution, and each accuracy solution separately, inject them for analysis, and record the chromatograms.

[0065] The results of the accuracy test for medium-chain triglyceride content are as follows:

[0066] The method achieved recoveries of 99.4% and 102.6% for tricaprylic acid glyceride and tricaprylic acid glyceride, respectively, with RSD values ​​of 1.8% and 1.2%, respectively. The method exhibits high recovery rates, meeting the requirements.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of medium-chain triglycerides using HPLC-ELSD, characterized in that the steps include... include: S1. Take tricaprylic acid glyceride and tricaprylic acid glyceride standards, prepare standard solutions of different concentrations respectively, perform HPLC-ELSD detection, and plot standard curves respectively; S2. The medium-chain triglyceride test solution was subjected to HPLC-ELSD detection to obtain the peak areas of tricaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, and tricaprylic acid glyceride; the contents of tricaprylic acid glyceride and dicaprylic acid monocaprylic acid glyceride in the test sample were calculated from the standard curve of tricaprylic acid glyceride; the total content of tricaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, dicaprylic acid monocaprylic acid glyceride, and tricaprylic acid glyceride was taken as the total content of triglycerides. The HPLC-ELSD conditions are as follows: octadecyl bonded silica gel is used as the packing material or a column with equivalent performance; the mobile phase is a mixture of methanol and isopropanol with a volume percentage of (70-90):(10-30); and the ELSD drift tube temperature is 40-80℃.

2. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8-1.2 mL / min.

3. The method according to claim 1, characterized in that, The volume ratio of methanol to isopropanol in the mobile phase is 10:

90.

4. The method according to claim 1, characterized in that, The trioctanoic acid glyceride and tridecanoic acid glyceride standards were prepared into standard solutions of different concentrations using methanol and / or isopropanol as solvents.

5. The method according to claim 1 or 3, characterized in that, The standard solutions of any different concentrations are selected from any 3 to 5 concentrations ranging from 0.01 to 5.0 mg / mL.

6. The method according to claim 1, characterized in that, The concentration of triglycerides in the medium-chain triglyceride test sample is 0.5-5.0 mg / mL.

7. The method according to claim 1, characterized in that, The column temperature of the chromatographic column is 25-45℃.

8. The method according to claim 1, characterized in that, The carrier gas flow rate of the evaporative light scattering drift tube is 1.5-2.5 L / min.

9. The method according to claim 1, characterized in that, The injection volume for the HPLC-ELSD detection process is 5-20 μL.

10. The method according to claim 1, characterized in that, The characteristic peaks of dioctanoic acid monocaprylic acid glyceride and dioctanoic acid monocaprylic acid glyceride were obtained by liquid chromatography-mass spectrometry (LC-MS).

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