Method for detecting quality of egg butter

Abstract

The invention relates to the technical field of quality standard detection of traditional Chinese medicinal materials, in particular to a quality detection method of egg butter. According to the method, beta-sitosterol is used as a quality detection index of the egg butter, and quality evaluation can be better carried out on the skin repair effect of the egg butter for treating burns and scalds. The invention newly establishes a thin-layer chromatography identification method taking beta-sitosterol as a contrast, and the method has the advantages of uniform color spot shape, good separation and clear color development. In addition, a high performance liquid chromatography method is adopted, silica gel is used as a stationary phase, and a normal-phase chromatographic column (SiO2, 250 * 4.6 mm) is adopted; n-hexane-petroleum ether (30-60 DEG C)-isopropanol (98: 1: 1) is used as a mobile phase; the detection wavelength is 210 nm, the column temperature is 20 DEG C, the flow velocity is 1.0 ml / min, and 10 microliters of sample is injected, so that the determination effect is better, and the repeatability, the stability and the precision are good.

Description

Technical Field

[0001] The present invention relates to the technical field of quality standard detection of Chinese medicinal materials, and specifically to a method for detecting the quality of egg yolk oil. Background Art

[0002] Egg yolk oil is the oil refined from the cooked egg yolks of the domestic chicken (Gallus gallus domesticus Brisson) of the Phasianidae family. Its preparation method is to take fresh eggs, cook them, remove the shells and take the yolks, place them in a pot and heat them over a low fire. After the water evaporates, then use a high fire to boil out the egg yolk oil, and it is obtained. Egg yolk oil is not specifically listed in "Compendium of Materia Medica", but only under the item of egg yolk; Liu Yuxi's "Chuanxin Fang": Five eggs, remove the whites and take the yolks, and add hair as big as an egg. Mix the flavors in an iron pot and boil with charcoal fire. At first, it is very dry, and soon the hair becomes charred, and then liquid comes out. Immediately take it and place it in a porcelain bowl until the liquid is exhausted, and apply it to the hot sores, and then sprinkle it with the powder of Sophora flavescens root. "There is liquid coming out" is the egg yolk oil. "Compendium of Materia Medica" treats smelly sores on the feet: One cooked egg yolk, one qian of yellow wax, fry the oil and apply it. There is a fifth-generation inheritor of the oral and heart-to-heart teaching of the Xiong family of Miao medicine in Guizhou, Xiong Anfu, in Longguang Town, Anlong County, our province. Mr. Xiong used the "Miao Medicine King's All-purpose Ointment" formulated with egg yolk oil to treat many burn and scald patients and achieved good curative effects.

[0003] Since the national drug standards and the 2003 edition and 2019 edition of the quality standards for Chinese medicinal materials and ethnic medicinal materials in Guizhou Province do not include egg yolk oil, and the quality standards for egg yolk oil (prepared slices) in Sichuan Province, Heilongjiang Province, and Hunan Province still have the deficiency that the evaluation of the skin repair for treating burn and scald by egg yolk oil is not accurate enough. In order to further standardize and improve the quality standard of egg yolk oil, a new quality standard for egg yolk oil needs to be established. Summary of the Invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention provides a method for detecting the quality of egg yolk oil, specifically as follows:

[0005] A method for detecting the quality of egg yolk oil uses the content of β-sitosterol as an index for detecting the quality of egg yolk oil.

[0006] This method can use β-sitosterol as a reference substance and adopt thin-layer chromatography for identification, specifically including the following steps:

[0007] (1) Take the powder of the medicinal material to be tested, add chloroform, ultrasonically treat it, filter, evaporate the filtrate to dryness, dissolve the residue in methanol to make a test solution;

[0008] (2) Separately take a reference substance of β-sitosterol and make a reference solution by dissolving it in methanol;

[0009] (3) Test by thin layer chromatography. Pipette the above-mentioned test solution and reference solution respectively onto the same silica gel G thin layer plate to form strip bands. Use petroleum ether - ethyl acetate = 5:1 as the developing solvent. Develop, take out, air dry, spray with 5% ethanolic phosphomolybdic acid solution, and heat at 105°C until the spots are clearly developed;

[0010] (4) In the chromatogram of the test sample, spots of the same color should appear at the positions corresponding to the chromatogram of the reference substance.

[0011] Further, the petroleum ether is petroleum ether with a boiling range specification of 60 - 90°C.

[0012] Further, the specific steps of this method are as follows:

[0013] (1) Take 2 g of the medicinal material powder to be tested, add 30 ml of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution;

[0014] (2) Separately take β-sitosterol reference substance, and make a solution containing 1 mg per 1 ml in methanol as the reference solution;

[0015] (3) Test by thin layer chromatography. Pipette 5 μl of the above-mentioned test solution and 2 μl of the reference solution respectively onto the same silica gel G thin layer plate to form strip bands. Use petroleum ether - ethyl acetate = 5:1 as the developing solvent. Develop, take out, air dry, spray with 5% ethanolic phosphomolybdic acid solution, and heat at 105°C until the spots are clearly developed;

[0016] (4) In the chromatogram of the test sample, spots of the same color should appear at the positions corresponding to the chromatogram of the reference substance.

[0017] This method can also use β-sitosterol as the reference substance and adopt high performance liquid chromatography to determine the content of β-sitosterol in egg yolk oil. The high performance liquid chromatography uses silica gel as the filler; uses n-hexane - petroleum ether - isopropanol = 98:1:1 as the mobile phase; has a detection wavelength of 210 nm; the petroleum ether is petroleum ether with a boiling range specification of 30 - 60°C.

[0018] Further, the high performance liquid chromatography uses a normal phase chromatographic column: SiO2, 250*4.6 mm; the column temperature is 20°C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

[0019] Further, for the high performance liquid chromatography, the reference solution is prepared as follows: Take an appropriate amount of β-sitosterol reference substance, accurately weigh it, and make it in dichloromethane. Specifically, make a solution containing 0.25 mg per 1 ml, and that is it.

[0020] Further, in the high performance liquid chromatography method, the test solution is prepared as follows: Take the sample to be tested, add potassium hydroxide methanol solution, heat under reflux, cool, shake and extract with n-hexane, recover the solvent to dryness, dissolve the residue in dichloromethane, add dichloromethane, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0021] The specific parameters of this method are as follows: Take 0.2 g of the sample to be tested and place it in a 100 ml stoppered conical flask. Add 20 ml of 13% potassium hydroxide methanol solution (take 13 g of potassium hydroxide, dissolve it in 20 ml of water, dilute it to 100 ml with methanol, and shake well), heat under reflux at 65 °C for 90 minutes, cool, shake and extract with n-hexane 4 times, 20 ml each time. Combine the extraction solutions, recover the solvent to dryness, dissolve the residue in dichloromethane, transfer it to a 10 ml volumetric flask, add dichloromethane to the scale, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0022] During the determination, accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph for determination to obtain the result. The final result contains β-sitosterol (C 29 H 50 O) not less than 1.0%.

[0023] The quality of egg yolk oil can also be detected by simultaneously using the above two methods.

[0024] Compared with the prior art, the technical effects of the present invention are as follows:

[0025] (1) β-sitosterol in egg yolk oil has the effects of reducing cholesterol, inhibiting tumors, repairing tissues, and promoting wound healing. In this application, β-sitosterol is used as an index for the quality detection of egg yolk oil, which can better evaluate the quality of egg yolk oil for the skin repair effect of treating burns and scalds.

[0026] (2) Referring to the thin layer chromatography identification method for the quality standard of egg yolk oil in Hunan Province and Sichuan Province, this application examines the effects of sample pretreatment methods, developing agents, and color developing agents on chromatographic spots, and newly establishes a thin layer chromatography identification method with β-sitosterol as the reference. Each chromatographic spot of this method has a uniform shape, good separation, and clear color development, and it is an excellent thin layer chromatography identification method for egg yolk oil with β-sitosterol as the reference.

[0027] (3) In this application, silica gel was used as the stationary phase, and a normal-phase chromatographic column (Elite SiO2, 250 * 4.6 mm, batch number 5 - 11476) was adopted; n-hexane - petroleum ether (30 - 60 °C) - isopropanol (98:1:1) was used as the mobile phase; the detection wavelength was 210 nm, the column temperature was 20 °C, the flow rate was 1.0 ml / min, and 10 μl was injected. At this time, the determination effect was better, with good repeatability, stability, and precision. It is an excellent method for the determination of β-sitosterol in egg yolk oil. Description of the Drawings

[0028] Figure 1 It is the result diagram of 2.1, where 1 is the reference substance, and 2, 3, 4, 5 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: benzene - chloroform - acetone (8∶5∶1); color-developing agent: 20% perchloric acid solution.

[0029] Figure 2 It is the result diagram of 2.2, where 1 is the reference substance, and 2, 3, 4, 5 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: petroleum ether (60 - 90 °C) - ether (7:3); color-developing agent: 30% phosphomolybdic acid ethanol solution.

[0030] Figure 3 It is the result diagram of 3.1, where 1 is the reference substance, and 2, 3, 4 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: benzene - chloroform - acetone (8∶5∶1); color-developing agent: 20% perchloric acid solution.

[0031] Figure 4 It is the result diagram of 3.2, where 1 is the reference substance, and 2, 3, 4 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: petroleum ether (60 - 90 °C) - ether (7:3); color-developing agent: 30% phosphomolybdic acid ethanol solution.

[0032] Figure 5 It is the result diagram of 3.3, where 1 is the reference substance, and 2, 3 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: petroleum ether (60 - 90 °C) - ethyl acetate (3∶1); color-developing agent: 30% phosphomolybdic acid ethanol solution.

[0033] Figure 6 It is the result diagram of 4.1, where 1 is the reference substance, and 2, 3 are the test samples; sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; developing agent: petroleum ether (60 - 90 °C) - ethyl acetate (5∶1); color-developing agent: 10% sulfuric acid ethanol solution.

[0034] Figure 7It is the result diagram of 4.2, where 1 is the reference substance, and 2 and 3 are the test samples; Sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (5:1); Color developer: 30% ethanolic solution of phosphomolybdic acid.

[0035] Figure 8 It is the result diagram of 4.3, where 1 is the reference substance, and 2, 3, and 4 are the test samples; Sample application volume: 2 μl of the reference substance solution, 10 μl of the test sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (5:1); Color developer: 5% ethanolic solution of phosphomolybdic acid.

[0036] Figure 9 It is the result diagram of 5.2(1) silica gel G thin layer plate, where 1 is the reference substance, and 2, 3, and 4 are the samples; Temperature: 28 °C; Humidity: 65%; Support: silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (5:1); Color developer: 5% ethanolic solution of phosphomolybdic acid.

[0037] Figure 10 It is the result diagram of 5.2(2) silica gel H thin layer plate, where 1 is the reference substance, and 2, 3, and 4 are the samples; Temperature: 28 °C; Humidity: 65%; Support: silica gel H thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (5:1); Color developer: 5% ethanolic solution of phosphomolybdic acid.

[0038] Figure 11 It is the result diagram of 5.2(3) silica gel GF 254 thin layer plate, where 1 is the reference substance, and 2, 3, and 4 are the samples; Temperature: 28 °C; Humidity: 65%; Support: silica gel GF 254 thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (5:1); Color developer: 5% ethanolic solution of phosphomolybdic acid.

[0039] Figure 12 It is the result diagram of 5.3 petroleum ether (60 - 90 °C) - ethyl acetate (4.7:1) group, where 1 is the reference substance, and 2, 3, and 4 are the samples; Temperature: 23.5 °C; Humidity: 54%; Support: silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing solvent: petroleum ether (60 - 90 °C) - ethyl acetate (4.7:1); Color developer: 5% ethanolic solution of phosphomolybdic acid.

[0040] Figure 13It is the result diagram of the petroleum ether (60 - 90°C)-ethyl acetate (5:1) group at 5.3. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; temperature: 23.5°C; humidity: 54%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0041] Figure 14 It is the result diagram of the petroleum ether (60 - 90°C)-ethyl acetate (5.3:1) group at 5.3. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; temperature: 23.5°C; humidity: 54%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5.3:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0042] Figure 15 It is the result diagram of the circular dot sample application group at 5.4. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; sample application method: circular dot sample application; temperature: 29°C; humidity: 62%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0043] Figure 16 It is the result diagram of the strip sample application group at 5.4. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; sample application method: strip sample application; temperature: 29°C; humidity: 62%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0044] Figure 17 It is the result diagram at 5.5. Among them, 1 is the reference substance, and 2, 3, and 4 are the sample volumes of 3 μl, 4 μl, and 5 μl respectively; temperature: 28°C; humidity: 65%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 3 μl, 4 μl, and 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0045] Figure 18 It is the result diagram of the thin-layer plate produced by Qingdao Ocean Chemical Co., Ltd. at 5.6. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; carrier: silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd.); temperature: 24°C; humidity: 62%; carrier: silica gel G thin-layer plate; sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; developing agent: petroleum ether (60 - 90°C)-ethyl acetate (5:1); color-developing agent: 5% phosphomolybdic acid ethanol solution.

[0046] Figure 19 It is the result diagram of the thin layer plate produced by Qingdao Dingkang Silicone Co., Ltd. on May 6. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Carrier: Silica gel G thin layer plate (Qingdao Dingkang Silicone Co., Ltd.); Temperature: 24 °C; Humidity: 62%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0047] Figure 20 It is the result diagram of the 4 °C group in 5.7. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Temperature: 4 °C; Humidity: 52%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0048] Figure 21 It is the result diagram of the 25 °C group in 5.7. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Temperature: 25 °C; Humidity: 52%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0049] Figure 22 It is the result diagram of the 40 °C group in 5.7. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Temperature: 40 °C; Humidity: 52%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0050] Figure 23 It is the result diagram of the 32% humidity group in 5.8. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Temperature: 28 °C; Humidity: 32%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0051] Figure 24 It is the result diagram of the 58% humidity group in 5.8. Among them, 1 is the reference substance, and 2, 3, and 4 are samples; Temperature: 28 °C; Humidity: 58%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution and 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0052] Figure 25It is the result diagram of the 88% humidity group in 5.8. Among them, 1 is the reference substance, and 2, 3, and 4 are the samples; Temperature: 28 °C; Humidity: 88%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0053] Figure 26 It is the result diagram of the reproducibility investigation (20230422, 20230504, 20230409, 20230403) in 5.9. Among them, 1 is the reference substance, 2, 3: Sample 20230422; 4, 5: Sample 20230504; 6, 7: Sample 20230409; 8, 9: Sample 20230403; Temperature: 24 °C; Humidity: 56%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0054] Figure 27 It is the result diagram of the reproducibility investigation (20230606, 20230901, 20230903, 20230904) in 5.9. Among them, 1 is the reference substance, 2, 3, 4, 5: Samples 20230606, 20230901, 20230903, 20230904; Temperature: 24 °C; Humidity: 56%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0055] Figure 28 It is the result diagram of the reproducibility investigation (20230905, 20230910, 20230815) in 5.9. Among them, 1 is the reference substance, 2, 3, 4: Samples 20230905, 20230910, 20230815; Temperature: 24 °C; Humidity: 56%; Carrier: Silica gel G thin layer plate; Sample application volume: 2 μl of the reference substance solution, 5 μl of the sample solution; Developing agent: Petroleum ether (60 - 90 °C) - Ethyl acetate (5:1); Color developer: 5% Phosphomolybdic acid ethanol solution.

[0056] Figure 29 It is the DAD scan diagram of the β - sitosterol reference substance solution.

[0057] Figure 30 It is the result diagram of the purity inspection of the main component peak of the test solution.

[0058] Figure 31 It is the detection chromatogram of the reversed - phase column C18, 250*4.6mm.

[0059] Figure 32 It is the detection chromatogram of a normal-phase column SiO2, 250*4.6mm.

[0060] Figure 33 It is the detection chromatogram of an amino column 250*4.6mm.

[0061] Figure 34 It is the detection chromatogram of a silica gel column 250*4.6mm.

[0062] Figure 35 It is the detection chromatogram of n-hexane - petroleum ether (30 - 60°C) - isopropanol (95:2:3).

[0063] Figure 36 It is the detection chromatogram of n-hexane - petroleum ether (30 - 60°C) - isopropanol (96:4:2).

[0064] Figure 37 It is the detection chromatogram of n-hexane - petroleum ether (30 - 60°C) - isopropanol (96:2:2).

[0065] Figure 38 It is the detection chromatogram of n-hexane - petroleum ether (30 - 60°C) - isopropanol (98:1:1).

[0066] Figure 39 It is the detection chromatogram of n-hexane - isopropanol (98:2).

[0067] Figure 40 It is the detection chromatogram of n-hexane - isopropanol (99:1).

[0068] Figure 41 It is the trend chart of the linear investigation result.

[0069] Figure 42 It is the detection chromatogram of a Zhongpu Red SiO2 250*4.6mm column.

[0070] Figure 43 It is the detection chromatogram of an Elite SiO2 250*4.6mm column.

[0071] Figure 44 It is the detection chromatogram of a Thermo Fisher U3000 liquid chromatograph.

[0072] Figure 45 It is the detection chromatogram of a Thermo Fisher Vanquish-core liquid chromatograph.

[0073] Figure 46 It is the chromatogram of a Shimadzu LC-20AT reference solution.

[0074] Figure 47 It is the chromatogram of a Shimadzu LC-20AT test solution. Specific implementation manners

[0075] The technical solution of the present invention will be further limited in combination with specific implementation manners below, but the scope of protection required is not limited only to the description made.

[0076] Example 1

[0077] Identification of egg yolk oil

[0078] β-sitosterol in egg yolk oil has the effects of reducing cholesterol, inhibiting tumors, repairing tissues, and promoting wound healing. Referring to the thin-layer identification method of the quality standards of egg yolk oil in Hunan Province and Sichuan Province, the effects of sample pretreatment methods, developing agents, and color-developing agents on chromatographic spots were investigated, and a thin-layer chromatography identification method with β-sitosterol as a reference was established.

[0079] 1 Instruments and test drugs

[0080] Instruments: CBL9960A ultrasonic cleaner, Sartorius BS124S ten-thousandth electronic balance; Tianjin Test 202-2AB electrothermal constant temperature drying oven; Tianjin Test DK-99-II electrothermal constant temperature water bath.

[0081] Reagents and test drugs: β-sitosterol reference substance (National Institutes for Food and Drug Control, batch number 110851-201909); chloroform, ethanol, methanol, petroleum ether (60-90 °C), ethyl acetate, sulfuric acid are all of analytical grade, and water is purified water; silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd., batch number 20210408; Qingdao Dingkang Silica Gel Co., Ltd., batch number 20190813); silica gel GF 254 Thin-layer plate (Qingdao Ocean Chemical Co., Ltd., batch number 20210728); silica gel H thin-layer plate (Qingdao Ocean Chemical Factory Branch, batch number 20120218); egg yolk oil samples (batch numbers 20230403, 230903 from Yikang Natural Spice Oil Refinery in Jishui County, Jiangxi Province, batch numbers 20230409, 20230815 from Jiangxi Yisenyuan Plant Spice Co., Ltd., batch numbers 20230422, 20230910 from Jishui County Junda Natural Spice Oil Factory, batch numbers 20230504, 230905 from Guangzhou Jingjing Biotechnology Co., Ltd., batch number 20230606 from Jiangxi Global Natural Spice Co., Ltd., batch number 230901 from Hongmao Vegetable Oil Refinery in Qingyuan District, Ji'an City, batch number 230904 from Jiangxi Yuanshangcao Spice Co., Ltd.).

[0082] 2 Preparation method of test solution Method 1

[0083] Take 2g of this product, add 50ml of petroleum ether (60-90℃) to dissolve, wash with dilute ethanol 3 times, 15ml each time, discard the dilute ethanol solution, evaporate the petroleum ether solution, add 2ml of chloroform to dissolve the residue, add it to the treated neutral alumina column (100-200 mesh, 10g, inner diameter 10-15mm), elute with 20ml of petroleum ether (60-90℃) and 20ml of benzene in turn, discard the eluent, and then elute with 20ml of a mixed solution of benzene-acetone (4:1), collect the eluent, evaporate to dryness, and add 3ml of chloroform to dissolve the residue as the test solution. Take another β-sitosterol reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution.

[0084] 2.1 Thin layer chromatography

[0085] Take 10 μl of the test solution of item "2" and 2 μl of the reference solution, spot them on the same silica gel G thin layer plate, use benzene-chloroform-acetone (8:5:1) as the developing agent, develop, take out, dry, spray with 20% perchloric acid solution, and heat at 105℃ until the spots are clearly colored. Figure 1 .

[0086] The test results show that: in the chromatogram of the test sample, the spots at the positions corresponding to the chromatogram of the reference sample are not clearly colored, so they are not included in the standard.

[0087] 2.2 Thin layer chromatography method 2

[0088] According to the thin layer chromatography method of the Chinese Pharmacopoeia 2020 edition, Part IV General Rules 0502 test, take 10 μl of the test solution of item "2" and 2 μl of the reference solution, respectively, spot them on the same silica gel G thin layer plate, use petroleum ether (60-90°C)-ether (7:3) as the developing agent, develop, take out, dry, spray with 30% phosphomolybdic acid ethanol solution, and heat at 105°C until the spots are clearly colored. Results are shown in Figure 2 .

[0089] The test results show that when using different developing agents and different color developers, the test sample shows spots corresponding to the chromatogram of the reference sample, but the Rf value is low and the separation is not good, so it is not included in the standard.

[0090] 3 Preparation method of test solution 2

[0091] Take 2g of this product, add 50ml of petroleum ether (60-90℃) to dissolve, wash with dilute ethanol 3 times, 15ml each time, discard the dilute ethanol solution, evaporate the petroleum ether solution to dryness, add 2ml of chloroform to dissolve the residue, and use it as the test solution. Take β-sitosterol reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference substance solution.

[0092] 3.1 Thin layer chromatography method 1

[0093] Absorb 10 μl of the test solution of item “3” and 2 μl of the reference solution, respectively spot them on the same silica gel G thin-layer plate, use benzene-chloroform-acetone (8:5:1) as the developing solvent, develop, take out, air dry, spray with 20% perchloric acid solution, heat at 105 °C until the spots are clearly developed. The results are shown in Figure 3 .

[0094] The test results show that: in the chromatogram of the test sample, the spots at the positions corresponding to the chromatogram of the reference substance are not clearly developed, so it is not included in the standard.

[0095] 3.2 Thin-layer chromatography method two

[0096] Absorb 10 μl of the test solution of item “3” and 2 μl of the reference solution, respectively spot them on the same silica gel G thin-layer plate, use petroleum ether (60-90 °C)-ethyl ether (7:3) as the developing solvent, develop, take out, air dry, spray with 30% ethanolic solution of phosphomolybdic acid, heat at 105 °C until the spots are clearly developed. The results are shown in Figure 4 .

[0097] The test results show that: in the chromatogram of the test sample, spots of the same color appear at the positions corresponding to the chromatogram of the reference substance, but the separation is not good, so it is not included in the standard.

[0098] 3.3 Thin-layer chromatography method three

[0099] Absorb 10 μl of the test solution of item “3” and 2 μl of the reference solution, respectively spot them on the same silica gel G thin-layer plate, use petroleum ether (60-90 °C)-ethyl acetate (3:1) as the developing solvent, develop, take out, air dry, spray with 30% ethanolic solution of phosphomolybdic acid, heat at 105 °C until the spots are clearly developed. The results are shown in Figure 5 .

[0100] The test results show that: in the chromatogram of the test sample, spots of the same color appear at the positions corresponding to the chromatogram of the reference substance, but the correspondence is not very neat and there are many impurity spots, which is not conducive to analysis, so it is not included in the standard.

[0101] 4 Preparation method of the test solution four

[0102] Take 2 g of this product, add 30 ml of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness on a water bath, dissolve the residue in 2 ml of methanol to prepare the test solution. Separately take β-sitosterol reference substance, dissolve it in methanol to prepare a solution containing 1 mg per 1 ml as the reference solution.

[0103] 4.1 Thin-layer chromatography method one

[0104] Take 10 μl of the test solution and 2 μl of the reference solution under item "4" and spot them on the same silica gel G thin layer plate, use petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. Figure 6 .

[0105] The test results show that: in the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the reference sample, but the color of the spots is Therefore, it is not included in the standard.

[0106] 4.2 Thin layer chromatography method 2

[0107] Take 10 μl of the test solution and 2 μl of the reference solution under item "4" and spot them on the same silica gel G plate respectively. Use petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing solvent, develop, take out, dry, spray with 30% phosphomolybdic acid ethanol solution, and heat at 105°C until the spots are clearly colored. Figure 7 .

[0108] The test results show that: in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference sample, but the leading spot is too dark in color and needs further adjustment of the developer concentration, so it is not included in the standard.

[0109] 4.3 Thin layer chromatography method 3

[0110] Take 10 μl of the test solution and 2 μl of the reference solution under item "4" and spot them on the same silica gel G plate respectively. Use petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing solvent, develop, take out, dry, spray with 5% phosphomolybdic acid ethanol solution, and heat at 105°C until the spots are clearly colored. Figure 8 .

[0111] The test results show that after the color developer 30% phosphomolybdic acid ethanol solution is changed to 5% phosphomolybdic acid ethanol solution, the chromatographic spots are uniform in shape, well separated and clearly colored, so this method is selected for subsequent methodological verification.

[0112] 5. Methodological Validation

[0113] 5.1 Solution preparation

[0114] Take 2g of sample (batch number 20230422), add 30ml of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate on a water bath, add 2ml of methanol to the residue to dissolve it, and use it as the test solution. Take β-sitosterol reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference substance solution.

[0115] 5.2 Carrier Investigation

[0116] Using (1) silica gel G thin layer plate, (2) silica gel H thin layer plate, and (3) silica gel GF 254 thin layer plates as carriers, according to the thin layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), 2 μl of the reference solution under item "5.1" and 5 μl of the test solution were taken and spotted on the same thin layer plate in a strip shape respectively. Petroleum ether (60 - 90 °C) - ethyl acetate (5:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 5% ethanolic phosphomolybdic acid solution, heated at 105 °C until the spots developed clearly, and examined under daylight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference substance. The results are shown in Figures 9 - 11 .

[0117] From the above Figures 9 - 11 it can be seen that among the three thin layer plates, the chromatographic spots using silica gel G thin layer plate as the carrier had the best separation, clear color development, and moderate Rf value. Therefore, silica gel G thin layer plate was selected as the carrier.

[0118] 5.3 Investigation of the proportion of the developing solvent

[0119] Using petroleum ether (60 - 90 °C) - ethyl acetate (4.7:1), (5:1), and (5.3:1) as the developing solvents respectively, according to the thin layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), 2 μl of the reference solution under item "5.1" and 5 μl of the test solution were taken and spotted on the same silica gel G thin layer plate in a strip shape respectively. After development, the plate was taken out, dried, sprayed with 5% ethanolic phosphomolybdic acid solution, heated at 105 °C until the spots developed clearly, and examined under daylight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference substance. The results are shown in Figures 12 - 14 .

[0120] From the above Figures 12 - 14 it can be seen that there were no significant differences in the resolution and color development clarity of the chromatographic spots detected with different proportions of petroleum ether (60 - 90 °C) - ethyl acetate (4.7:1), (5:1), and (5.3:1), indicating good durability of the developing solvent petroleum ether (60 - 90 °C) - ethyl acetate (5:1).

[0121] 5.4 Investigation of the spotting method

[0122] The dot sampling and strip sampling were investigated separately. According to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition), 2 μl of the reference substance solution and 5 μl of the test solution under item "5.1" were respectively taken and spotted on the same silica gel G thin-layer plate to form dots and strips. Petroleum ether (60 - 90 °C) - ethyl acetate (5:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 5% ethanolic phosphomolybdic acid solution, heated at 105 °C until the spots developed clearly, and inspected under daylight. In the chromatogram of the test solution, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference substance. The results are shown in Figures 15 - 16 。

[0123] As can be seen from the above Figures 15 - 16 : When strip sampling was used, the spots in the chromatograms of the reference substance and the test solution developed clearly, and the resolution was good. Therefore, strip sampling was selected.

[0124] Investigation of the sample application volume

[0125] The sample application volumes of 3 μl, 4 μl, and 5 μl were investigated separately. According to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition), 2 μl of the reference substance solution and 3 μl, 4 μl, and 5 μl of the test solution under item "5.1" were respectively taken and spotted on the same silica gel G thin-layer plate to form strips. Petroleum ether (60 - 90 °C) - ethyl acetate (5:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 5% ethanolic phosphomolybdic acid solution, and heated at 105 °C until the spots developed clearly. In the chromatogram of the test solution, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference substance. The results are shown in Figure 17 。

[0126] From Figure 17 it can be seen that when the sample application volume was 3 - 5 μl, the chromatographic spots developed gradually darker, and the resolution was good for all, with no significant difference. Considering the differences in the β-sitosterol content in different batches of samples, the sample application volume of 5 μl with clearer color development and the reference substance of 2 μl were selected as the sample application volumes for this method.

[0127] Investigation of thin-layer plates from different manufacturers

[0128] The thin-layer plates produced by Qingdao Ocean Chemical Co., Ltd. and Qingdao Dingkang Silica Gel Co., Ltd. were investigated separately. 2 μl of the reference substance solution and 5 μl of the test solution under item ":5.1" were taken. According to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition), they were respectively spotted on the same silica gel G thin-layer plate to form strips. Petroleum ether (60 - 90 °C) - ethyl acetate (5:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 5% ethanolic phosphomolybdic acid solution, and heated at 105 °C until the spots developed clearly. In the chromatogram of the test solution, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference substance. The results are shown separately in Figures 18 - 19 。

[0129] From the above Figures 18 - 19 It can be seen that the sample chromatographic spot separation is good when the TLC plates of Qingdao Ocean Chemical Co., Ltd. and Qingdao Dingkang Silica Gel Co., Ltd. are used for development. f The value is moderate and the color is clear, indicating that thin layer plates from different manufacturers have no significant effect on this method.

[0130] 5.7 Temperature investigation

[0131] 4℃, 25℃, and 40℃ were examined respectively. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2μl of the reference solution and 5μl of the test solution under "5.1" were taken and spotted on the same silica gel G thin layer plate to form strips, and developed with petroleum ether (60-90℃)-ethyl acetate (5:1) as the developing agent. Take it out, dry it, spray it with 5% phosphomolybdic acid ethanol solution, and heat it at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference sample. The results are shown in Figures 20 - 22 .

[0132] From the above Figures 20 - 22 It can be seen that the chromatographic separation is good when the sample is developed in the temperature range of 4℃~40℃, R f The value is moderate, and temperature has no significant effect on the separation of thin layer chromatography.

[0133] 5.8 Humidity investigation

[0134] The humidity was investigated at 32%, 58%, and 88% respectively. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 2μl of the reference solution and 5μl of the test solution under "5.1" were taken and spotted on the same silica gel G thin layer plate to form strips. Petroleum ether (60-90°C)-ethyl acetate (5:1) was used as the developing agent, and developed at relative humidity of 32%, 58%, and 88%, respectively. Take it out, dry it, spray it with 5% phosphomolybdic acid ethanol solution, heat it at 105°C until the spots are clearly colored, and inspect it under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference sample. The results are shown in the figure. Figures 23 - 25 .

[0135] From the above Figures 23 - 25 It can be seen that the sample is developed in the humidity range of 32% to 88%, and the chromatographic separation is good. f The value is moderate, indicating that humidity has no significant effect on the separation of TLC.

[0136] 5.9 Repeatability Study

[0137] Take 2 g each of 11 batches of egg yolk oil (20230422, 20230504, 20230409, 20230403, 20230606, 20230901, 20230903, 20230904, 20230905, 20230910, 20230815), add 30 ml of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness on a water bath, dissolve the residue in 2 ml of methanol to prepare the test solution. Separately, take a β-sitosterol reference substance, add methanol to make a solution containing 1 mg per 1 ml as the reference solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, absorb 2 μl of the reference solution and 5 μl of the test solution, respectively spot them on the same silica gel G thin-layer plate to form strip bands, use petroleum ether (60 - 90 °C)-ethyl acetate (5:1) as the developing agent, develop, take out, air dry, spray with 5% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed. In the test solution chromatogram, at the position corresponding to the reference solution chromatogram, spots of the same color are shown, respectively seen Figures 26 - 28 。

[0138] The above test results show that: in the test solution chromatogram, at the position corresponding to the reference solution chromatogram, spots of the same color are shown, and the repeatability test of samples from different batches is good.

[0139] Based on the comprehensive results of the above various methodological validations, it shows that this method has good repeatability, is stable and reliable, and can be included in the quality standard.

[0140] Example 2

[0141] Determination of β-sitosterol content in egg yolk oil

[0142] 1 Instruments and reagents

[0143] Thermo Fisher U3000 high performance liquid chromatograph; Thermo Fisher Vanquish-core high performance liquid chromatograph; Shimadzu LC-20AT high performance liquid chromatograph; Sartorius BS124S one-ten-thousandth electronic balance; Sartorius CPA225D one-hundred-thousandth electronic balance; Tianjin Test DK-98-IIA type electrothermal constant temperature water bath.

[0144] β - Sitosterol reference substance (National Institutes for Food and Drug Control, batch number 110851 - 201909, content 92.7%); Dichloromethane (analytical grade); n - Hexane (chromatographic grade, analytical grade); Isopropanol (chromatographic grade); Petroleum ether (30 - 60 °C) (chromatographic grade); Potassium hydroxide (analytical grade); Methanol (analytical grade); Purified water; Ultra - pure water; Egg yolk oil samples (batch numbers 20230403, 230903 from Yikang Natural Spice Oil Refinery in Jishui County, Jiangxi Province, batch numbers 20230409, 20230815 from Jiangxi Yisenyuan Plant Spice Co., Ltd., batch numbers 20230422, 20230910 from Jishui County Junda Natural Spice Oil Factory, batch numbers 20230504, 230905 from Guangzhou Jingjing Biotechnology Co., Ltd., batch number 20230606 from Jiangxi Global Natural Spice Co., Ltd., batch number 230901 from Hongmao Vegetable Oil Refinery in Qingyuan District, Ji'an City, batch number 230904 from Jiangxi Yuanshangcao Spice Co., Ltd.).

[0145] 2 Methods and Results

[0146] 2.1 Selection of Chromatographic Conditions

[0147] Using silica gel as the stationary phase, a normal - phase chromatographic column (Elite SiO 2 , 250 * 4.6 mm, batch number 5 - 11476) was used; The mobile phase was n - hexane - petroleum ether (30 - 60 °C) - isopropanol (98:1:1); The detection wavelength was 210 nm, the column temperature was 20 °C, the flow rate was 1.0 ml / min, and the injection volume was 10 μl, and the determination effect was better.

[0148] 2.1.1 Wavelength Selection

[0149] The instrument was Shimadzu LC - 20AT, the detector was SPD - M20A, a normal - phase chromatographic column (SiO 2 , 250 * 4.6 mm) was used, the column temperature was 20 °C; The flow rate was 1.0 ml / min; The detection wavelength range was 190 - 400 nm; Using n - hexane - petroleum ether (30 - 60 °C) - isopropanol (98:1:1) as the mobile phase, the areas of the target peaks were measured at 209 nm, 210 nm, and 211 nm respectively. The results showed that the peak areas showed a decreasing trend. The shorter the wavelength, the greater the influence of the solvent. The detection wavelength in the literature was mostly selected as 210 nm. At this wavelength, no impurities were detected in the reference substance peak and the target peak of the test sample. Therefore, 210 nm was selected as the detection wavelength. The results are shown in Figures 29 - 30 .

[0150] 2.1.2 Selection between Reverse - Phase and Normal - Phase Chromatography

[0151] A comparative test between the reverse phase method and the normal phase chromatography method was carried out. The results showed that more impurity peaks were detected by the reverse phase chromatography method, the main component peak and the impurity peaks could not be separated, and there was little room to adjust the resolution using a single-component solvent (methanol) as the mobile phase. While fewer impurity peaks of the test sample were detected by the normal phase chromatography method, the main component peak and the impurity peaks were completely separated, the peak shape was symmetrical, and the number of theoretical plates was relatively high. Therefore, the normal phase chromatography method was selected. The results are shown in Figures 31 - 32 。

[0152] 2.1.3 Selection of different types of normal phase columns

[0153] A comparison was made between a normal phase amino column and a silica gel column. The results showed that the resolution of the main component peak measured by the amino column did not meet the requirements, and the resolution of the silica gel column was 2.0, which was better than that of the amino column. Therefore, a SiO 2 250*4.6 mm chromatographic column was selected. The results are shown in Figures 33 - 34 。

[0154] 2.1.4 Selection of mobile phase

[0155] Mobile phases with different compositions and ratios were investigated. The following were used as mobile phases for separate determinations: ① n-hexane - isopropanol (98:2), ② n-hexane - isopropanol (99:1) 【3】 , ③ n-hexane - petroleum ether (60 - 90 °C) - isopropanol (98:1:1), ④ n-hexane - petroleum ether (30 - 60 °C) - isopropanol (96:2:2), ⑤ n-hexane - petroleum ether (30 - 60 °C) - isopropanol (95:3:2), ⑥ n-hexane - petroleum ether (30 - 60 °C) - isopropanol (94:4:2), ⑦ n-hexane - petroleum ether (30 - 60 °C) - isopropanol (94:4:2). The test results showed that the resolution of the main component peak of the test sample detected by mobile phase ⑦ n-hexane - petroleum ether (30 - 60 °C) - isopropanol (98:1:1) was greater than 2.0, the peak symmetry and the number of theoretical plates were good, and the main component eluted within 30 minutes. Therefore, n-hexane - petroleum ether (30 - 60 °C) - isopropanol (98:1:1) was selected as the mobile phase. The results are shown in Figures 35 - 40 。

[0156] 2.2 Solution preparation

[0157] Preparation of reference solution: Weigh an appropriate amount of β-sitosterol reference substance accurately and dissolve it in dichloromethane to prepare a solution containing 0.25 mg per 1 ml, which is the reference solution.

[0158] Preparation of test solution 【9】:Take 0.2 g of this product, place it in a 100-ml stoppered conical flask, add 20 ml of 13% potassium hydroxide methanol solution (take 13 g of potassium hydroxide, dissolve it in 20 ml of water, dilute it to 100 ml with methanol, and shake well), heat under reflux at 65 °C for 90 minutes, cool, extract 4 times with n-hexane by shaking, 20 ml each time, combine the extraction solutions, recover the solvent to dryness, dissolve the residue in dichloromethane, transfer it to a 10-ml volumetric flask, add dichloromethane to the scale, shake well, filter, and take the subsequent filtrate, that is obtained.

[0159] Determination: Accurately pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine.

[0160] 2.2.1 Investigation of dissolution solvents

[0161] Use methanol, petroleum ether (30 - 60 °C), n-hexane, dichloromethane, and methanol-dichloromethane (1:1) mixture as the dissolution solvents for the residue respectively. The results show that methanol and petroleum ether (30 - 60 °C) cannot completely dissolve the residue, n-hexane can completely dissolve it, but it is not easy to filter, the methanol-dichloromethane (1:1) mixture can completely dissolve the residue, but methanol has an impact on the baseline, dichloromethane can not only dissolve the residue but also is easy to filter and has no impact on the detection baseline. Therefore, dichloromethane is selected as the dissolution solvent.

[0162] 2.2.2 Investigation of saponification time

[0163] Set the heating reflux time of the sample to be 30, 60, 90, and 120 minutes respectively. Weigh 4 portions of this product, 0.2 g each, with the heating reflux time being 30, 60, 90, and 120 minutes respectively. Prepare the test solution according to the method in item "2.2", and determine. The results are shown in Table 1.

[0164] Table 1 Results of investigation of saponification time

[0165]

[0166] The test results show that: the content is the highest when heating under reflux for 90 minutes, and it decreases after 120 minutes. Therefore, it is more suitable to choose the heating reflux time of 90 minutes.

[0167] 2.2.3 Investigation of saponification temperature

[0168] Set the heating reflux temperature of the sample to be 60, 65, and 70 °C. Weigh 3 portions of this product, 0.2 g each. Prepare the test solution according to the method in item "2.2", and determine. The results are shown in Table 2.

[0169] Table 2 Results of investigation of saponification temperature

[0170]

[0171] The test shows that the content is the lowest when the water bath temperature is 60°C, the highest when the temperature is 65°C, slightly lower at 70°C than at 65°C, and the average relative deviation between the two is 1.3%. Therefore, the saponification temperature of water bath heating can be selected as 65°C.

[0172] 2.2.4 Investigation of extraction times

[0173] Set the extraction times of the sample to 3, 4, and 5 times respectively. Weigh 0.2 g of this product, prepare the test solution according to the method in item "2.2", and determine. The results are shown in Table 3.

[0174] Table 3 Results of investigation of extraction times

[0175]

[0176]

[0177] The test results show that after 4 extractions, the content no longer increases, indicating that the extraction is complete after 4 extractions. Therefore, 4 shaking extractions are selected.

[0178] 3 Investigation of linear relationship

[0179] The concentration of the β-sitosterol reference substance is set to 0.25 mg / ml. Therefore, the linear concentrations are set to 1.0, 0.5, 0.25, 0.125, and 0.0625 mg / ml for linear investigation. Accurately weigh 10.8933 mg of the β-sitosterol reference substance and place it in a 10 ml volumetric flask to obtain a concentration of 1.009812 mg / ml. Take an appropriate amount and dilute it successively to solutions of 0.504909 mg / ml, 0.252453 mg / ml, 0.1262265 mg / ml, and 0.06311325 mg / ml. The test results are shown in Table 4. Figure 41 .

[0180] Table 4 Results of linear investigation

[0181]

[0182] It can be seen from the test results that within the concentration range of 0.06311325 mg / ml to 1.009812 mg / ml of the reference substance, the measured correlation coefficient r is 0.99995, indicating that the linear relationship of this method is good.

[0183] 4 Precision test

[0184] Take the β-sitosterol reference substance solution with a concentration of 0.252453 mg / ml and inject it continuously for 6 times for determination. The results are shown in Table 5.

[0185] Table 5 Results of precision investigation

[0186]

[0187] It can be seen from the above test results that the RSD of the peak areas obtained from 6 injections of the reference solution was 0.99%, indicating that the precision test was good.

[0188] 5 Repeatability test

[0189] Six portions of egg yolk oil (20230422), each weighing 0.2 g, were accurately weighed, and the test solution was prepared according to the method described in item "2.2", and determined. The results are shown in Table 6.

[0190] Table 6 Results of repeatability investigation

[0191]

[0192] The results of the repeatability test showed that the average value of the repeated tests was 1.0112% and the RSD was 0.34%, indicating that the repeatability of this method was good.

[0193] 6 Determination of quantitative limit and detection limit

[0194] The β-sitosterol reference solution for content determination (concentration: 0.252453 mg / ml) was successively diluted and determined. The concentration at a signal-to-noise ratio of 10 was taken as the quantitative limit, and the concentration at a signal-to-noise ratio of 3 was taken as the detection limit. The results are shown in Table 7.

[0195] Table 7 Results of investigation on quantitative limit and detection limit

[0196]

[0197] The test results showed that the quantitative limit of this method was 5.04906 μg / ml and the detection limit was 1.009812 μg / ml.

[0198] 7 Recovery test

[0199] 21.73 mg of β-sitosterol reference substance was weighed and placed in a 200-ml volumetric flask. Methanol was added to dissolve it to obtain a stock solution containing 0.10071855 mg per 1 ml. Nine portions of the sample (20230422), each weighing 0.1 g, were accurately weighed and divided into three groups. 8 ml of the stock solution was accurately added to each portion in the first group, 10 ml of the stock solution was accurately added to each portion in the second group, and 12 ml of the stock solution was accurately added to each portion in the third group. The test solution was prepared according to the method described in item "2.2", and determined, and the recovery rate was calculated. The results are shown in Table 8.

[0200] Table 8 Results of recovery test

[0201]

[0202]

[0203] The results of the recovery test showed that: the average recovery rate at the 80% level concentration was 101.62%, and the RSD was 1.20%; the average recovery rate at the 100% level concentration was 103.79%, and the RSD was 0.63%; the average recovery rate at the 120% level concentration was 100.96%, and the RSD was 0.77%. The RSD of each level concentration met the requirements, indicating that the recovery test of this method was good.

[0204] 8 Content determination

[0205] The test solution was prepared according to the method described in item "2.2", and the contents of 11 batches of egg yolk oil were detected. The results are shown in Table 9.

[0206] Table 9 Results of content determination

[0207]

[0208] The test results showed that: the average content of 11 batches of samples was 1.31%. The limit range was set at 80% of the average content, that is, 1.31% * 80% = 1.0%. Therefore, the content limit of this product was tentatively set as not less than 1.0% of β-sitosterol.

[0209] 9 Durability investigation

[0210] 9.1 Investigation of chromatographic columns from different manufacturers

[0211] The separation degree, theoretical plate number, and peak area of the main component peaks detected by using Zhongpu Red and Elite SiO 2 250*4.6mm chromatographic column had no obvious difference, and the retention time differed by about 1 time, but it did not affect the quantitative detection. Therefore, SiO 2 250*4.6mm chromatographic column was suitable for this method. The results are shown in Figures 42 - 43 .

[0212] 9.2 Investigation of different instruments

[0213] The detection was carried out on Thermo Fisher U3000, Thermo Fisher Vanquish-dore, and Shimadzu LC-20AT liquid chromatographs respectively. The results showed that the separation degree of the target peaks met the requirements, and the peak theoretical plate number and symmetry were good. The results are shown in Figures 44 - 47 .

[0214] 9.3 Investigation of column temperature

[0215] The column temperature was set at 15, 20, 25, and 30 °C respectively. The reference solution and the test solution were injected respectively for determination. The results showed that with the increase of the column temperature, the peak retention time, separation degree, and theoretical plate number also increased, and the separation degree met the requirements. The results are shown in Table 10. (Zhongpu Red sio 2 250*4.6mm column)

[0216] Table 10 Column temperature investigation results

[0217]

[0218] 9.4 Flow rate investigation

[0219] The column flow rate was set at 0.8, 1.0, and 1.2 ml / min, and the reference solution and the test solution were injected respectively. The results showed that the separation degree of the main component peak in the test solution met the requirements under different column flow rates, and the number of theoretical plates did not change significantly. The results are shown in Table 11. 2 250*4.6mm column)

[0220] Table 11 Flow rate investigation results

[0221]

[0222] The above test results show that this content determination method has good repeatability and stability and is included in the quality standards.

[0223] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for detecting the quality of egg butter, characterized in that, the content of β-sitosterol is used as an index for detecting the quality of egg butter.

2. The method for detecting the quality of egg butter according to claim 1, characterized in that, specifically, β-sitosterol is used as a reference substance, and thin-layer chromatography is used for identification.

3. The method for detecting the quality of egg butter according to claim 2, characterized in that, using β-sitosterol as a reference substance and performing identification by thin-layer chromatography specifically includes the following steps: (1) Take the powder of the medicinal material to be tested, add chloroform, ultrasonically treat, filter, evaporate the filtrate to dryness, dissolve the residue in methanol to obtain a test solution; (2) Take another β-sitosterol reference substance, and make a reference solution by adding methanol; (3) According to the thin-layer chromatography test, suck the above-mentioned test solution and reference solution, and spot them on the same silica gel G thin-layer plate to form strip bands respectively. Use petroleum ether-ethyl acetate = 5:1 as the developing agent, develop, take out, dry in the air, spray with 5% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed; (4) In the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, spots of the same color should appear.

4. The method for detecting the quality of egg butter according to claim 3, characterized in that, the petroleum ether is petroleum ether with a boiling range specification of 60-90 °C.

5. The method for detecting the quality of egg butter according to claim 3, characterized in that, the specific steps are as follows: (1) Take 2 g of the powder of the medicinal material to be tested, add 30 ml of chloroform, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of methanol to obtain a test solution; (2) Take another β-sitosterol reference substance, and make a solution containing 1 mg per 1 ml in methanol as a reference solution; (3) According to the thin-layer chromatography test, suck 5 μl of the above-mentioned test solution and 2 μl of the reference solution, and spot them on the same silica gel G thin-layer plate to form strip bands respectively. Use petroleum ether-ethyl acetate = 5:1 as the developing agent, develop, take out, dry in the air, spray with 5% ethanolic phosphomolybdic acid solution, and heat at 105 °C until the spots are clearly developed; (4) In the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference substance, spots of the same color should appear.

6. The method for detecting the quality of egg butter according to claim 1, characterized in that, specifically, β-sitosterol is used as a reference substance, and high performance liquid chromatography is used to determine the content of β-sitosterol in egg butter.

7. The method for detecting the quality of egg butter according to claim 6, characterized in that, for the high performance liquid chromatography method, silica gel is used as the filler; n-hexane - petroleum ether - isopropanol = 98:1:1 is used as the mobile phase; the detection wavelength is 210 nm; the petroleum ether is petroleum ether with a boiling range specification of 30-60 °C.

8. The method for detecting the quality of egg butter according to claim 6, characterized in that, The high performance liquid chromatography method uses a normal phase chromatographic column: SiO 2 , 250 * 4.6 mm; the column temperature is 20 °C, the flow rate is 1.0 ml / min, and the injection volume is 10 μl.

9. The method for detecting the quality of egg butter according to claim 6, characterized in that, for the high performance liquid chromatography method, the reference solution is prepared by the following method: Take an appropriate amount of β-sitosterol reference substance, accurately weigh it, and make it with dichloromethane.

10. The method for detecting the quality of egg butter according to claim 6, characterized in that, in the high performance liquid chromatography method, the test solution is prepared by the following method: taking the sample to be tested, adding potassium hydroxide methanol solution, heating under reflux, cooling, shaking and extracting with n-hexane, recovering the solvent to dryness, dissolving the residue with dichloromethane, adding dichloromethane, shaking well, filtering, and taking the subsequent filtrate to obtain the test solution.

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