Method for identifying grease auxiliary materials by using sterol spectrogram

Abstract

The invention relates to a method for identifying grease auxiliary materials by using a sterol spectrogram, in particular to identification and adulteration judgment of grease auxiliary materials such as soybean oil and olive oil. According to the technical scheme, the method comprises the following steps: extracting unsaponifiable matters from the grease pharmaceutical excipients, separating and collecting sterol components by using HPLC (High Performance Liquid Chromatography), derivatizing a sterol sample, and detecting the composition and content of sterol by adopting a GC method, so that the method is suitable for determining the sterol components in the grease pharmaceutical excipients, and is good in applicability.

Description

Technical Field

[0001] The invention relates to a method for identifying oil auxiliary materials by using sterol spectra, in particular to the identification and adulteration judgment of oil auxiliary materials such as soybean oil and olive oil. Background Art

[0002] As an important source of nutrients for humans, oils and fats can not only meet the fatty acids and other nutrients required by the human body, but also provide energy for basic human activities. They are indispensable substances for the human body to maintain normal life activities. As pharmaceutical excipients, oils and fats can be used in injections, ointments, emulsions and other dosage forms. For example, water-in-oil fat emulsions made from vegetable oils as raw materials, supplemented with phospholipids as emulsifiers, and adding appropriate amounts of isotonic regulators, antioxidants, etc., are a high-energy parenteral nutrition agent that can provide energy and necessary fatty acids for patients who cannot eat and are severely malnourished, and play an irreplaceable role in clinical practice. The vegetable oils currently used as raw materials for fat emulsions mainly include soybean oil, peanut oil, coconut oil, corn oil, olive oil, etc.

[0003] Sterols are chemical substances with cyclopentane polyhydrophenanthrene as the main structure and containing alcoholic hydroxyl groups. Plant-derived oils and fats are rich in plant sterols. According to reports, up to 250 types of plant sterols have been identified from plants. Plant sterols can lower blood lipids and cholesterol, and also have the effects of preventing cardiovascular diseases, anti-inflammatory, anti-tumor and immunomodulation. The composition and content of sterols in different types of vegetable oils vary greatly and have certain regularities. The type of oil can be preliminarily judged based on the composition and content of sterols. Therefore, it is of great significance to establish an efficient and fast analytical method for sterol composition.

[0004] Sterols can be determined by different methods. In the early days, visible light colorimetry, digitonin precipitation method, enzyme method, etc. were commonly used. The disadvantages of these methods are that they are not specific and can only determine the total amount of sterols, but cannot analyze the components of sterols. TLC method is also used for determination, which mainly judges the proportion of sterols based on the color and size of the color spots. Although this method can determine the components of sterols, it has low sensitivity and poor accuracy. Currently, the more advanced method is capillary gas chromatography. Most sterol components are well separated on the DB-5 capillary column. Sterols that have not been derivatized can be detected by FID or MS. Sterols that have been derivatized by silanization can obtain better peak shape, higher separation and sensitivity, and the sample has higher stability. The most important thing is that this method can quantitatively detect the content of sterol components.

[0005] The sterol composition of vegetable oils from different plant sources varies greatly. Therefore, the sterol composition can be used as a fingerprint of plant-derived oils. The oils from different plant sources can be clearly distinguished according to the differences in sterol composition and content, and further used for the identification of oil varieties and the identification of adulteration. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an analytical method with better peak shape, higher separation and sensitivity, and capable of quantitatively detecting the content of sterol components, so as to identify oil and fat excipients based on the obtained sterol spectrum.

[0007] The technical solution of the present invention is:

[0008] The present invention uses a method in which unsaponifiable matter is extracted from oils and fats, separated by HPLC, the collected sterol components are subjected to a derivatization reaction, and finally separated and detected by GC. The present application analyzes the sterol composition of two common oil excipients, soybean oil and olive oil, identifies the differences in sterol composition and content in different oils and fats, and evaluates the process consistency of the oils and fats.

[0009] The present invention relates to a method for identifying oil and fat auxiliary materials by using a sterol spectrum, comprising extracting unsaponifiable matter from the oil and fat auxiliary materials, separating and collecting sterol components from the unsaponifiable matter by using an HPLC method, performing a derivatization treatment on the collected fractions, performing separation and analysis by using a GC method, and identifying the oil and fat auxiliary materials according to the sterol spectrum.

[0010] In the method of the present invention, the method for extracting unsaponifiable matter from oil and fat auxiliary materials is to add potassium hydroxide ethanol solution to the oil and fat auxiliary materials, heat them in a water bath for reflux saponification, and then extract them with ether.

[0011] In the method of the present invention, the chromatographic column used in the HPLC method is a Kromasil 60-5SIL silica gel column with a specification of 250×4.6 mm and 5 μm; a pre-column with the same filler with a specification of 5×4.6 mm and 5 μm; the mobile phase is 99:1 n-hexane-isopropanol, the detection wavelength is 210 nm, and the flow rate is 1 ml / min.

[0012] In the method of the present invention, the GC method adopts a capillary chromatographic column with a stationary liquid of 5% phenyl-95% methyl polysiloxane, the temperature of the injection port is 290°C, the temperature of the detector is 290°C, the heating program is: the initial column temperature is 270°C, maintained for 50 minutes, and the temperature is increased to 290°C at a rate of 5°C / min and maintained for 25 minutes; the carrier gas is helium.

[0013] In the method of the present invention, the derivatization method is to take a sample, add 0.2 ml of anhydrous pyridine and 0.2 ml of a 99:1 mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS), seal, mix evenly, heat at 80°C for 20 minutes, take out, cool, and take the liquid layer as the test sample derivatization solution.

[0014] In the method of the present invention, the oil auxiliary materials are soybean oil and olive oil.

[0015] In the method of the present invention, the sterol spectrum includes chromatographic peaks of cholesterol, rapeseed sterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol and β-sitosterol.

[0016] Beneficial effects of the present invention:

[0017] 1. The method adopted by the present invention first extracts unsaponifiable matter from oil-based pharmaceutical excipients, then separates and collects sterol components by HPLC, and after the sterol samples are derivatized, the composition and content of sterols are detected by GC. The method is suitable for the determination of sterol components in oil-based pharmaceutical excipients, and the method has good applicability.

[0018] 2. The detection and analysis method provided by the present invention has simple operation steps and requires a short time.

[0019] 3. The method adopted in the present invention has better peak shape, higher separation and sensitivity, and can quantitatively detect the content of sterol components, and is suitable for identifying oil and fat excipients based on sterol spectra. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a typical chromatogram for the determination of olive oil sterols in Example 1, wherein A: blank; B: reference substance derivatization solution; C: test substance derivatization solution.

[0021] Figure 2 This is a typical chromatogram for the determination of soybean sterols in Example 1, wherein A: reference substance derivatization solution; B: test substance derivatization solution. DETAILED DESCRIPTION

[0022] The present invention is further described below by way of examples. The following description is only for the purpose of explaining the present invention and is not intended to limit its contents.

[0023] Example 1

[0024] 1. Unsaponifiable matter extraction

[0025] Weigh 5g of soybean oil and olive oil accurately, place in a 250ml reflux bottle, add 50ml of potassium hydroxide ethanol solution, heat in a water bath and reflux for 1h to saponify, transfer to a separatory funnel after cooling, wash with water, add the washing liquid to the separatory funnel, extract with ether 3 times, 100ml each time, combine the ether extract, wash with water, discard the water layer, wash the ether layer with 3% potassium hydroxide solution and water 3 times, 40ml each time, and finally wash the ether layer repeatedly with 40ml of water until the last washing liquid does not show red after adding 2 drops of phenolphthalein indicator solution. Transfer the ether extract to a constant weight evaporating dish, evaporate to dryness in a 50℃ water bath, dissolve the residue with 6ml of acetone, and evaporate the acetone under air flow. Dry at 105℃ to constant weight, calculate the content of unsaponifiable matter, and the results are shown in Table 1.

[0026] Table 1 Unsaponifiable matter content results

[0027]

[0028] 2. Separation of sterols

[0029] Take the solution that meets the requirements after titration with ethanol sodium hydroxide titration solution (0.1 mol / L) to the endpoint under the unsaponifiable matter, evaporate to dryness in a water bath, dissolve the residue in 6 ml of acetone, evaporate to dryness at room temperature, and dry the residue at 105°C for about 15 minutes as the test sample. Take another sunflower seed oil, prepare the unsaponifiable matter in the same way and treat it in the same way as a control.

[0030] Take the test and control substances, dissolve them with ether three times, 4 ml each time, transfer them to a test tube, evaporate to dryness under a nitrogen flow, add mobile phase to dissolve, make a solution containing about 40 mg of residue per 1 ml, filter, and take the filtrate. Take cholesterol and β-sitosterol reference substances, dissolve and dilute them with mobile phase to make a solution containing 40 mg per 1 ml, as the solution for cholesterol and β-sitosterol positioning. Use Kromasil 60-5SIL silica gel column (250×4.6mm, 5μm) and pre-column (5×4.6mm, 5μm) with the same filler, with n-hexane-isopropanol (99:1) as the mobile phase, the detection wavelength is 210nm, and the flow rate is 1ml·min -1 , injection. The control solution showed two main chromatographic peaks at 23-32 minutes. The eluates of the control solution, the test solution, cholesterol and β-sitosterol positioning solution during this period were collected and placed in test tubes respectively. Each test tube collected the eluate obtained by two injections and evaporated to dryness under nitrogen flow.

[0031] 3. Sample Derivatization

[0032] Take the above residue, add 0.2ml anhydrous pyridine and 0.2ml N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS) (99:1) mixture, seal, mix evenly, heat at 80℃ for 20 minutes, take out, cool, and take the liquid layer as the test sample derivatization solution. Take the control solution, cholesterol and β-sitosterol positioning solution and treat them in the same way to obtain the corresponding derivatization solution.

[0033] 4. Determination of sterols

[0034] The content of the samples was measured by gas chromatography, using DB-5MS UI (30m×0.32mm, 1.0μm) and DB-5 (30m×0.25mm, 0.25μm) columns respectively, with injection port temperature at 290°C, detector temperature at 290°C, programmed temperature increase: initial column temperature at 270°C, maintained for 50 min, then increased to 290°C at a rate of 5°C per minute, maintained for 25 min; the carrier gas was helium.

[0035] Take the reference derivatization solution, cholesterol derivatization solution, β-sitosterol derivatization solution and test sample derivatization solution and inject them separately. The reference derivatization solution should show 4 main chromatographic peaks. Locate each sterol peak according to the retention time relative to the β-sitosterol peak in Table 2. Typical chromatograms of reference and test samples are shown in Figure 1 and Figure 2 .

[0036] Table 2 Retention time of each sterol peak relative to β-sitosterol peak

[0037]

[0038]

[0039] 5. Test results

[0040] In the gas chromatogram of the test solution, the total peak area of ​​15 sterol peaks from cholesterol to △7-oatsterol was calculated, and the content of each sterol in the test sample was calculated by area normalization method. The sterol composition of 8 batches of olive oil and 8 batches of soybean oil calculated according to the above method is shown in Table 3 and Table 4.

[0041] The results show that the sterol composition of vegetable oils from different plant sources varies greatly. Therefore, the sterol composition can be used as a fingerprint of plant-derived oils. According to the difference in sterol composition and content, oils from different plant sources can be clearly distinguished, which can be further used for the identification of oil varieties and the identification of adulteration.

[0042] Table 3 Olive oil sterol composition results

[0043]

[0044]

[0045] Table 4 soybean oil sterol composition results

[0046]

Claims

1. A method for identifying oil and fat auxiliary materials using sterol spectra, characterized in that: Unsaponifiable matter is extracted from oil and fat auxiliary materials, and then sterol components are separated and collected from the unsaponifiable matter by HPLC. The collected fractions are derivatized and separated and analyzed by GC. The oil and fat auxiliary materials are identified based on the sterol spectrum.

2. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The method for extracting unsaponifiable matter from oil and fat auxiliary materials comprises the following steps: adding potassium hydroxide ethanol solution to the oil and fat auxiliary materials, heating in a water bath for reflux saponification, and then extracting with ether.

3. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The HPLC method uses a Kromasil 60-5SIL silica gel column with a size of 250×4.6 mm and 5 μm; a pre-column with the same filler with a size of 5×4.6 mm and 5 μm; a mobile phase of 99:1 n-hexane-isopropanol, a detection wavelength of 210 nm, and a flow rate of 1 ml / min.

4. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The GC method uses a capillary column with a stationary liquid of 5% phenyl-95% methylpolysiloxane, an injection port temperature of 290°C, a detector temperature of 290°C, a heating program: an initial column temperature of 270°C, maintained for 50 minutes, heated to 290°C at a rate of 5°C / min, and maintained for 25 minutes; the carrier gas is helium.

5. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The derivatization method is to take a sample, add 0.2 ml of anhydrous pyridine and 0.2 ml of a 99:1 mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS), seal, mix evenly, heat at 80°C for 20 minutes, take out, cool, and take the liquid layer as the test sample derivatization solution.

6. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The oil auxiliary materials are soybean oil and olive oil.

7. The method for identifying oil and fat auxiliary materials according to claim 1, characterized in that: The sterol spectrum includes chromatographic peaks of cholesterol, rapeseed sterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol and beta-sitosterol.

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