WO2021003792A1 - Gas chromatography method for simultaneously detecting and separating multiple fatty acids - Google Patents

Gas chromatography method for simultaneously detecting and separating multiple fatty acids Download PDF

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WO2021003792A1
WO2021003792A1 PCT/CN2019/099735 CN2019099735W WO2021003792A1 WO 2021003792 A1 WO2021003792 A1 WO 2021003792A1 CN 2019099735 W CN2019099735 W CN 2019099735W WO 2021003792 A1 WO2021003792 A1 WO 2021003792A1
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fatty acids
gas chromatography
keep
detector
separation
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PCT/CN2019/099735
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French (fr)
Chinese (zh)
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王强
郭芹
李甜
屈阳
石爱民
刘红芝
刘丽
胡晖
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中国农业科学院农产品加工研究所
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Publication of WO2021003792A1 publication Critical patent/WO2021003792A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

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  • the invention relates to the technical field of food detection. More specifically, the present invention relates to a gas chromatography method for simultaneous detection and separation of multiple fatty acids.
  • Fatty acids are a class of compounds composed of three elements: carbon, hydrogen, and oxygen. They are also one of the main energy sources of the human body; including short-chain fatty acids (C3-C5), medium-chain fatty acids (C6-C12) and long-chain fatty acids (C13- C24).
  • short-chain fatty acids are mainly derived from milk, oligosaccharides, malted barley food, oat bran and corn starch;
  • medium-chain fatty acids are mainly derived from edible oil (especially coconut oil and palm kernel oil), milk and milk
  • the main sources of long-chain fatty acids are meat foods, edible oils and hydrogenated foods.
  • the methods for detecting fatty acids in food mainly include gas chromatography, liquid chromatography, silver ion thin layer chromatography, infrared spectroscopy, mass spectrometry, capillary electrophoresis, etc.
  • gas chromatography is widely used due to its high separation efficiency and low detection limit.
  • detection technology there is an urgent need to establish a high-throughput, accurate identification and rapid detection technology for fatty acids, and the existing high-efficiency separation and detection methods of gas chromatography need to be further improved. Therefore, based on the existing fatty acid gas chromatography analysis method, a new detection method is established to achieve high-throughput, accurate qualitative and quantitative analysis of fatty acids in food, which has good promotion and application value.
  • An object of the present invention is to solve at least the above-mentioned problems and provide at least the advantages described later.
  • Another object of the present invention is to provide a gas chromatography method for simultaneous detection and separation of multiple fatty acids, which can achieve high-efficiency separation and qualitative and quantitative analysis of 72 fatty acids, and has the advantages of high throughput, high sensitivity, and low detection limit. .
  • a gas chromatography method for simultaneous detection and separation of multiple fatty acids including: using a cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column for gas chromatography
  • the specification is 80-200m ⁇ 0.25mm ⁇ 0.20 ⁇ m
  • the chromatographic conditions are:
  • the temperature of the injection port is 200-230°C; the injection volume is 1 ⁇ L; the split ratio is 8-12:1; the nitrogen flow rate is 10-12cm/s; the constant linear velocity mode;
  • the initial temperature of the chromatographic column is 60-80°C, keep it for 4-6min, increase to 150-180°C at a heating rate of 20-30°C/min, keep it for 3-5min, and increase it to 200-225°C at 1-5°C/min , Keep for 40-60min, then increase to 200-230°C at 0.5-2°C/min, keep it for 4-6min;
  • Detector hydrogen flame ionization detector FID; detector temperature 200-230°C; makeup flow rate 2-5mL/min.
  • the chromatographic column specification is 100m ⁇ 0.25mm ⁇ 0.20 ⁇ m.
  • the chromatographic conditions are:
  • Injection port temperature 230°C; injection volume 1 ⁇ L; split ratio 10:1; nitrogen flow rate 10.6cm/s; constant linear velocity mode;
  • the initial temperature of the chromatographic column is 60°C, keep it for 5min, raise it to 160°C at a heating rate of 25°C/min, keep it for 4min, raise it to 225°C at 2°C/min, keep it for 50min, then raise it to 230°C at 1°C/min , Keep for 5min;
  • Detector hydrogen flame ionization detector FID; detector temperature 230°C; makeup flow rate 3mL/min.
  • the various fatty acids include C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and C13-C24 long-chain fatty acids.
  • multiple fatty acids include C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C11: 1-10c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14:1-9c, C15:0, C15:1-10t, C15:1- 10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1-10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19:0, C18:2-9t, 12t, C19:1-7t, C19: 1-10t, C19:1-7c, C19:1-10
  • the detection limit is between 0.000084-0.001276g/100g
  • the quantification limit is between 0.000289-0.004263g/100g.
  • the RSD value of intraday precision is controlled between 0.57-9.81%, and the RSD value of intraday precision is controlled between 0.47-9.87%.
  • the present invention uses the cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column to establish a method that can simultaneously detect 72 C3-C24 series fatty acids with high throughput, including 36 unsaturated fatty acids, 22 There are three types of saturated fatty acids, 12 types of trans fatty acids and 2 types of conjugated fatty acids. Compared with the national standard GB5009.168-2016 "Determination of Fatty Acids in Food" testing method, 35 types of fatty acids are detected, including 18 types of unsaturated fatty acids and 5 types. Saturated fatty acids, 10 trans fatty acids and 2 conjugated fatty acids;
  • the RSD value of the intraday precision of the 72 fatty acid detection methods established by the present invention is controlled within 0.57-9.81%, and the RSD value of the intraday precision is controlled within 0.47-9.87%, which is lower than the national standard GB 5009.168-2016. 10%, far lower than the 15% required in the national standard GB 5009.257-2016, with good stability and meeting the requirements of accurate quantitative analysis;
  • the detection limit of the 72 fatty acid detection methods established in the present invention is between 0.000084-0.001276g/100g, which shows that under the conditions of existing equipment and the parameters used, each fatty acid methyl ester of 0.0013g/100g can be qualitatively
  • the detection is two tenths of the detection limit of the national standard GB 5009.168-2016 (0.0013-0.0066g/100g), and one tenth of the detection limit of the national standard GB 5009.257-2016 (0.012g/100g); the present invention
  • the limit of quantification is between 0.000289-0.004263g/100g, indicating that under the conditions of existing instruments and the parameters used, all fatty acid methyl esters of 0.0043g/100g can be quantitatively analyzed, which is the limit of quantification (0.024) of the national standard GB 5009.257-2016 g/100g), which significantly reduces the detection limit and quantification limit of fatty acids, with good accuracy and accurate identification of fatty acids;
  • the linear relationship of the 72 fatty acid detection methods established by the present invention the instrument response value of each fatty acid is well linearly related to the concentration, and the linear relationship is basically higher than 0.999, which fully meets the requirements of quantitative analysis, and the method has wide adaptability ;
  • the present invention can also realize the high-efficiency separation of 21 trans fatty acids, 3 cis-trans conjugated linoleic acids, and 2 anti-trans conjugated linoleic acids.
  • the national standard GB5009.257-2016 "Trans Fatty Acids in Food Determination ⁇ Detection method, detecting 12 kinds of fatty acids, including 7 kinds of trans fatty acids, 3 kinds of cis-trans conjugated linoleic acid and 2 kinds of anti-trans conjugated linoleic acid.
  • Figure 1 is a diagram of the gas chromatograph temperature rising program of the present invention
  • Figure 2 is one of the gas chromatograms of 72 fatty acid methyl esters of the present invention.
  • Figure 3 is the second gas chromatogram of 72 kinds of fatty acid methyl esters of the present invention.
  • Figure 4 is the third gas chromatogram of 72 fatty acid methyl esters of the present invention.
  • Figure 5 is a gas chromatogram of 26 fatty acid isomers of the present invention.
  • Figure 6 is a gas chromatogram of peanut oil in Example 1 of the present invention.
  • Figure 7 is a gas chromatogram of milk in Example 2 of the present invention.
  • Figure 8 is a gas chromatogram of linseed oil in Example 3 of the present invention.
  • Figure 9 is a gas chromatogram of the milk of Comparative Example 1 of the present invention.
  • Figure 10 is a gas chromatogram of linseed oil in Comparative Example 2 of the present invention.
  • the temperature of the injection port is 200-230°C; the injection volume is 1 ⁇ L; the split ratio is 8-12:1; the nitrogen flow rate is 10-12cm/s; the constant linear velocity mode;
  • the initial temperature of the chromatographic column is 60-80°C, keep it for 4-6min, raise it to 150-180°C at a heating rate of 20-30°C/min, keep it for 3-5min, raise it to 200-225°C at 1-5°C/min , Keep for 40-60min, then increase to 200-230°C at 0.5-2°C/min, keep it for 4-6min;
  • Detector hydrogen flame ionization detector FID; detector temperature 200-230°C; makeup flow rate 2-5mL/min.
  • the specification of the chromatographic column is 100m ⁇ 0.25mm ⁇ 0.20 ⁇ m, and the commonly purchased chromatographic column has this specification.
  • the chromatographic conditions are:
  • Injection port temperature 230°C; injection volume 1 ⁇ L; split ratio 10:1; nitrogen flow rate 10.6cm/s; constant linear velocity mode;
  • the initial temperature of the chromatographic column is 60°C, keep it for 5 minutes, raise it to 160°C at a heating rate of 25°C/min, hold it for 4 minutes, raise it to 225°C at 2°C/min, hold it for 50 minutes, and then increase it at 1°C. /min to 230°C, keep it for 5min;
  • Detector hydrogen flame ionization detector FID; detector temperature 230°C; makeup flow rate 3mL/min.
  • multiple fatty acids include C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and C13-C24 long-chain fatty acids.
  • the reference retention time of 72 kinds of fatty acid methyl esters is shown in Table 1.
  • a variety of fatty acids include C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C11:1-10c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14: 1-9c, C15:0, C15:1-10t, C15:1-10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1- 10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19: 0, C18:2-9t,12t, C19:1
  • the present invention adopts cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column to establish a method that can simultaneously detect 72 C3-C24 series fatty acids with high throughput, including 36 unsaturated fatty acids and 22 saturated fatty acids , 12 kinds of trans fatty acids and 2 kinds of conjugated fatty acids, as shown in Figure 2-4, the peaks are numbered in Arabic numerals, and the numbers in Table 1 correspond to the names of fatty acids.
  • the precision of the instrument is expressed by the relative standard deviation (RSD), which is used to evaluate the reproducibility of the reproducibility of the measurement result of the same sample by the gas chromatography method.
  • RSD relative standard deviation
  • the same gas chromatography analysis method was used to investigate the intra-day (7 determinations in a day) and intra-day (7 consecutive days of determination) precision of 72 fatty acid mixed standard solutions.
  • the intra-day and inter-day precisions of 72 fatty acids were as follows As shown in Table 2, the RSD value of the intraday precision of each fatty acid methyl ester is controlled at 0.57-9.81%, and the daytime precision is controlled at 0.47-9.87%, which is lower than the 10% required in the national standard GB 5009.168-2016, and far lower than the national standard 15% of the requirements in GB5009.257-2016, good stability, and meet the requirements of accurate quantitative analysis.
  • the detection limit and quantification limit of 72 fatty acids are shown in Table 3. It can be seen that the detection limit of the present invention is between 0.000084-0.001276g/100g, indicating that under the conditions of existing equipment and the parameters used, 0.0013g/100g All fatty acid methyl esters can be qualitatively detected, which is two-tenths of the detection limit of the national standard GB 5009.168-2016 (0.0013-0.0066g/100g), and the detection limit of the national standard GB 5009.257-2016 (0.012g/100g)
  • the quantification limit of the present invention is between 0.000289-0.004263g/100g, indicating that under the conditions of existing instruments and the parameters used, each fatty acid methyl ester of 0.0043g/100g can be quantitatively analyzed, which is the national standard GB Two-tenths of the limit of quantification (0.024g/100g) of 5009.257-2016.
  • the invention significantly reduces the detection limit and the determination limit of fatty acids, has good accuracy,
  • the 72 kinds of fatty acid standard curves, correlation coefficients and concentration ranges are shown in Table 4.
  • the 72 kinds of fatty acid methyl ester standard solutions are gradually diluted, and the diluted mixed standard solutions of each concentration are analyzed by gas chromatography according to the established method. Linear fitting was performed within the concentration range, and the linear equation and correlation coefficient of each fatty acid methyl ester were obtained. The linear relationship was basically higher than 0.999, which fully met the requirements of quantitative analysis.
  • the present invention can also realize the high-efficiency separation of 21 trans fatty acids, 3 cis-trans conjugated linoleic acids, and 2 kinds of anti-anti-conjugated linoleic acids.
  • each wave peak is numbered in Arabic numerals, as shown in Table 5.
  • the serial number corresponds to the name of the fatty acid.
  • the unmarked peak between No. 11 and No. 12 is C18:2-9c, 12c (same as No. 41 in Table 1), and the unmarked peak between No. 17 and No. 18 is C18:3- 9c, 12c, 15c (same as No. 49 in Table 1), in order not to affect the display of 26 fatty acid isomers, these two substances are not numbered in Figure 5.
  • Step: Weigh 100mg of peanut oil, place it in a 10mL centrifuge tube, add 2mL of C11:0 internal standard solution, and then add 0.1mL 2mol/L of potassium hydroxide methanol solution, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min.
  • the gas chromatography column is CP Sil 88 (100m ⁇ 0.25mm ⁇ 0.20 ⁇ m), chromatographic condition position: Inlet temperature: 200°C ;Injection volume: 1 ⁇ L; split ratio: 8:1; flow rate: 10cm/s (nitrogen); constant linear velocity mode; oven heating program: 60°C for 5 min, 21°C/min to 150°C, hold for 5 minutes, Raise to 200°C at 1°C/min, hold for 60min, then rise to 200°C at 0.5°C/min, hold for 6min; Detector: hydrogen flame ionization detector (FID); detector temperature: 200°C; makeup flow : 2.0mL/min.
  • FID hydrogen flame ionization detector
  • milk may have 10 fatty acids after detection by this method, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, palmitic acid (C16:0), Stearic acid (C18:0), oleic acid (C18:1-9c) and linoleic acid (C18:2-9c, 12c).
  • Step: Weigh 100mg of linseed oil, place it in a 10mL centrifuge tube, add 2mL of C11:0 internal standard solution, and then add 0.1mL 2mol/L of methanol solution of potassium hydroxide, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min.
  • the gas chromatography column is CP Sil 88 (100m ⁇ 0.25mm ⁇ 0.20 ⁇ m), chromatographic condition position: inlet temperature: 230°C ;Sample volume: 1 ⁇ L; Split ratio: 10:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 60°C for 5min, 25°C/min to 160°C, and hold for 4min , Rise to 225°C at 2°C/min, hold for 50 minutes, then rise to 230°C at 1°C/min, hold for 5 minutes; detector: hydrogen flame ionization detector (FID); detector temperature: 230°C; makeup Flow rate: 3.0mL/min.
  • FID hydrogen flame ionization detector
  • Gas chromatography column is CP Sil 88 (100m ⁇ 0.25mm ⁇ 0.20 ⁇ m), chromatographic condition position: inlet temperature: 270°C ;Sample volume: 1 ⁇ L; Split ratio: 100:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 100°C for 13min, 10°C/min to 180°C, for 6min , Rise to 200°C at 1°C/min, hold for 20min, then rise to 230°C at 4°C/min, hold for 10.5min; detector: hydrogen flame ionization detector (FID); detector temperature: 280°C; tail Blowing flow: 3.0mL/min.
  • FID hydrogen flame ionization detector
  • milk may have 7 fatty acids after detection by this method, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, and palmitic acid (C16:0). It can be seen that compared to Example 2, stearic acid (C18:0), oleic acid (C18:1-9c) and linoleic acid (C18:2-9c, 12c) were not detected.
  • Step: Weigh 100mg of linseed oil, place it in a 10mL centrifuge tube, add 0.1mL of 2mol/L potassium hydroxide methanol solution, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min. Take 20 ⁇ L of supernatant to a 1mL volumetric flask and use Shimadzu GC-2010 gas chromatography for determination.
  • the gas chromatography column is CP Sil 88 (100m ⁇ 0.25mm ⁇ 0.20m), and the chromatographic condition position: inlet temperature: 270°C ;Sample volume: 1 ⁇ L; Split ratio: 100:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 100°C for 13min, 10°C/min to 180°C, for 6min , Rise to 200°C at 1°C/min, hold for 20min, then rise to 230°C at 4°C/min, hold for 10.5min; detector: hydrogen flame ionization detector (FID); detector temperature: 280°C; tail Blowing flow: 3.0mL/min.
  • FID hydrogen flame ionization detector
  • linseed oil may have 7 fatty acids after being tested by this method, including linolenic acid (C18:3-9c, 12c, 15c), oleic acid (C18:1-9c), and linoleic acid (C18: 2-9c, 12c), stearic acid (C18:0) and palmitic acid (C16:0) are higher in content, in addition to C18: 3-6c, 9c, 12c, C20: 1-5c, It can be seen that relative to examples 3C16:1-9c, C17:0, C18:1-11c, C20:0, C18:3-9t, 12t, 15c, C18:3-9c, 12t, 15t, C19:2 -10c, 13c, C18: 3-9t, 12c, 15c, C20: 2-11c, 14c, C22:0, C20: 3-8c, 11c, 14c, C21: 2-12c, 15c and C24:
  • this method is suitable for detecting substances containing C3-C24 fatty acids.
  • peanut oil, linseed oil and milk in the above cases, it can also detect various fatty acid-rich edible oils, meat foods, vegetables, and milk. And dairy products and other fatty acid-rich substances, due to the limited length of the instructions, they will not be detailed one by one.

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Abstract

A gas chromatography method for simultaneously detecting and separating multiple fatty acids, comprising: performing gas chromatographic detection by using a cyanopropyl siloxane strong-polar stationary CP Sil 88 gas chromatographic column with the specification of 80-100 m*0.25 mm*0.20 μm. The chromatographic conditions are that: the sample injection port temperature is 200-230°C, the sample injection quantity is 1 μL, the split ratio is 8-12:1, the nitrogen flow velocity is 10-12 cm/s, a constant linear speed mode is adopted, a detector is a flame ionization detector (FID), the detector temperature is 200-230°C, and the make-up flow is 2-5 mL/min. The method can realize efficient separation and qualitative and quantitative analysis of 72 fatty acids, and has the advantages of high throughput, high sensitivity and low limit of detection.

Description

同时检测分离多种脂肪酸的气相色谱方法Gas chromatography method for simultaneous detection and separation of multiple fatty acids 技术领域Technical field
本发明涉及食品检测技术领域。更具体地说，本发明涉及一种同时检测分离多种脂肪酸的气相色谱方法。The invention relates to the technical field of food detection. More specifically, the present invention relates to a gas chromatography method for simultaneous detection and separation of multiple fatty acids.
背景技术Background technique
脂肪酸是由碳、氢、氧三种元素组成的一类化合物，也是人体主要能量来源之一；包括短链脂肪酸(C3-C5)、中链脂肪酸(C6-C12)和长链脂肪酸(C13-C24)。其中短链脂肪酸的主要来源于牛奶、低聚糖、发芽大麦食品、燕麦麸和玉米淀粉等食品；中链脂肪酸的主要来源于食用油(尤其是椰子油和棕榈仁油)、乳类和奶类等食品；长链脂肪酸的主要来源于肉类食物、食用油和氢化食品等。Fatty acids are a class of compounds composed of three elements: carbon, hydrogen, and oxygen. They are also one of the main energy sources of the human body; including short-chain fatty acids (C3-C5), medium-chain fatty acids (C6-C12) and long-chain fatty acids (C13- C24). Among them, short-chain fatty acids are mainly derived from milk, oligosaccharides, malted barley food, oat bran and corn starch; medium-chain fatty acids are mainly derived from edible oil (especially coconut oil and palm kernel oil), milk and milk The main sources of long-chain fatty acids are meat foods, edible oils and hydrogenated foods.
目前检测食品中脂肪酸的方法主要有气相色谱法、液相色谱法、银离子薄层色谱法、红外光谱法、质谱法、毛细管电泳法等。其中，气相色谱由于分离效率高、检测限低而广为应用。随着检测技术的快速发展，急需建立脂肪酸的高通量、精准识别快检技术，而现有气相色谱高效分离检测方法还有待进一步提高。因此，在现有脂肪酸气相色谱分析方法基础上，建立一种新的检测方法，实现食品中脂肪酸的高通量、精准定性定量分析，具有较好的推广应用价值。At present, the methods for detecting fatty acids in food mainly include gas chromatography, liquid chromatography, silver ion thin layer chromatography, infrared spectroscopy, mass spectrometry, capillary electrophoresis, etc. Among them, gas chromatography is widely used due to its high separation efficiency and low detection limit. With the rapid development of detection technology, there is an urgent need to establish a high-throughput, accurate identification and rapid detection technology for fatty acids, and the existing high-efficiency separation and detection methods of gas chromatography need to be further improved. Therefore, based on the existing fatty acid gas chromatography analysis method, a new detection method is established to achieve high-throughput, accurate qualitative and quantitative analysis of fatty acids in food, which has good promotion and application value.
发明内容Summary of the invention
本发明的一个目的是解决至少上述问题，并提供至少后面将说明的优点。An object of the present invention is to solve at least the above-mentioned problems and provide at least the advantages described later.
本发明还有一个目的是提供一种同时检测分离多种脂肪酸的气相色谱方法，其能够实现了72种脂肪酸的高效分离及定性和定量分析，具有高通量、高灵敏度、低检测限的优点。Another object of the present invention is to provide a gas chromatography method for simultaneous detection and separation of multiple fatty acids, which can achieve high-efficiency separation and qualitative and quantitative analysis of 72 fatty acids, and has the advantages of high throughput, high sensitivity, and low detection limit. .
为了实现根据本发明的这些目的和其它优点，提供了一种同时检测分离多种脂肪酸的气相色谱方法，包括：采用氰丙基硅氧烷强极性固定相CP Sil 88气相色谱柱进行气相色谱检测，规格为80-200m×0.25mm×0.20μm，色谱条件为：In order to achieve these objectives and other advantages according to the present invention, a gas chromatography method for simultaneous detection and separation of multiple fatty acids is provided, including: using a cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column for gas chromatography For detection, the specification is 80-200m×0.25mm×0.20μm, and the chromatographic conditions are:
进样口温度200-230℃；进样量1μL；分流比8-12:1；氮气流速10-12cm/s；恒定线速度模式；The temperature of the injection port is 200-230℃; the injection volume is 1μL; the split ratio is 8-12:1; the nitrogen flow rate is 10-12cm/s; the constant linear velocity mode;
色谱柱初始温度为60-80℃，保持4-6min，以20-30℃/min的加热速率升至150-180℃， 保持3-5min，以1-5℃/min升至200-225℃，保持40-60min，再以0.5-2℃/min升至200-230℃，保持4-6min；The initial temperature of the chromatographic column is 60-80℃, keep it for 4-6min, increase to 150-180℃ at a heating rate of 20-30℃/min, keep it for 3-5min, and increase it to 200-225℃ at 1-5℃/min , Keep for 40-60min, then increase to 200-230℃ at 0.5-2℃/min, keep it for 4-6min;
检测器：氢火焰离子化检测器FID；检测器温度200-230℃；尾吹流量2-5mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 200-230℃; makeup flow rate 2-5mL/min.
优选的是，色谱柱规格为100m×0.25mm×0.20μm。Preferably, the chromatographic column specification is 100m×0.25mm×0.20μm.
优选的是，色谱条件为：Preferably, the chromatographic conditions are:
进样口温度230℃；进样量1μL；分流比10:1；氮气流速10.6cm/s；恒定线速度模式；Injection port temperature 230℃; injection volume 1μL; split ratio 10:1; nitrogen flow rate 10.6cm/s; constant linear velocity mode;
色谱柱初始温度为60℃，保持5min，以25℃/min的加热速率升至160℃，保持4min，以2℃/min升至225℃，保持50min，再以1℃/min升至230℃，保持5min；The initial temperature of the chromatographic column is 60℃, keep it for 5min, raise it to 160℃ at a heating rate of 25℃/min, keep it for 4min, raise it to 225℃ at 2℃/min, keep it for 50min, then raise it to 230℃ at 1℃/min , Keep for 5min;
检测器：氢火焰离子化检测器FID；检测器温度230℃；尾吹流量3mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 230℃; makeup flow rate 3mL/min.
优选的是，多种脂肪酸包括C3-C5短链脂肪酸、C6-C12中链脂肪酸和C13-C24长链脂肪酸。Preferably, the various fatty acids include C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and C13-C24 long-chain fatty acids.
优选的是，多种脂肪酸包括C3:0，C4:0，C5:0，C6:0，C7:0，C8:0，C9:0，C10:0，C11:0，C12:0，C11:1-10c，C13:0，C12:1-11c，C14:0，C13:1-12c，C14:1-9t，C14:1-9c，C15:0，C15:1-10t，C15:1-10c，C16:0，C15:1-14c，C16:1-9t，C16:1-9c，C17:0，C17:1-10t，C17:1-10c，C18:0，C18:1-6t，C18:1-9t，C18:1-11t，C18:1-6c，C18:1-9c，C18:1-11c，C19:0，C18:2-9t,12t，C19:1-7t，C19:1-10t，C19:1-7c，C19:1-10c，C18:2-9c,12c，C20:0，C20:1-11t，C18:3-6c,9c,12c，C20:1-5c，C20:1-8c，C20:1-11c，C19:2-10c,13c，C18:3-9c,12c,15c，C21:0，C18:2-9c,11t，C18:2-10t,12c，C21:1-12c，C20:2-11c,14c，C22:0，C22:1-13t，C20:3-8c,11c,14c，C22:1-13c，C21:2-12c,15c，C20:3-11c,14c,17c，C23:0，C20:4-5c,8c,11c,14c，C23:1-14c，C22:2-13c,16c，C24:0，C20:5-5c,8c,11c,14c,17c，C24:1-15c，C22:3-13c,16c,19c，C22:4-7c,10c,13c,16c，C22:5-4c,7c,10c,13c,16c，C22:5-7c,10c,13c,16c,19c，C22:6-4c,7c,10c,13c,16c,19c。Preferably, multiple fatty acids include C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C11: 1-10c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14:1-9c, C15:0, C15:1-10t, C15:1- 10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1-10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19:0, C18:2-9t, 12t, C19:1-7t, C19: 1-10t, C19:1-7c, C19:1-10c, C18:2-9c,12c, C20:0, C20:1-11t, C18:3-6c,9c,12c, C20:1-5c, C20:1-8c, C20:1-11c, C19:2-10c,13c, C18:3-9c,12c,15c, C21:0, C18:2-9c,11t, C18:2-10t,12c, C21:1-12c, C20:2-11c,14c, C22:0, C22:1-13t, C20:3-8c,11c,14c, C22:1-13c, C21:2-12c,15c, C20: 3-11c,14c,17c,C23:0,C20:4-5c,8c,11c,14c,C23:1-14c,C22:2-13c,16c,C24:0,C20:5-5c,8c, 11c,14c,17c,C24:1-15c,C22:3-13c,16c,19c,C22:4-7c,10c,13c,16c,C22:5-4c,7c,10c,13c,16c,C22: 5-7c, 10c, 13c, 16c, 19c, C22: 6-4c, 7c, 10c, 13c, 16c, 19c.
优选的是，检出限在0.000084-0.001276g/100g之间，定量限在0.000289-0.004263g/100g之间。Preferably, the detection limit is between 0.000084-0.001276g/100g, and the quantification limit is between 0.000289-0.004263g/100g.
优选的是，日内精密度的RSD值控制在0.57-9.81％之间，日间精密度的RSD值控制在0.47-9.87％之间。Preferably, the RSD value of intraday precision is controlled between 0.57-9.81%, and the RSD value of intraday precision is controlled between 0.47-9.87%.
本发明至少包括以下有益效果：The present invention includes at least the following beneficial effects:
第一、本发明采用氰丙基硅氧烷强极性固定相CP Sil 88气相色谱柱，建立能够同时高通量检测72种C3-C24系列脂肪酸的方法，其中包括36种不饱和脂肪酸、22种饱和脂肪酸、12种反式脂肪酸和2种共轭脂肪酸，相比国标GB5009.168-2016《食品中脂肪酸的测定》检测方法，检测脂肪酸种类多35种，包括18种不饱和脂肪酸、5种饱和脂肪酸、10种反式脂肪酸和2种共轭脂肪酸；First, the present invention uses the cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column to establish a method that can simultaneously detect 72 C3-C24 series fatty acids with high throughput, including 36 unsaturated fatty acids, 22 There are three types of saturated fatty acids, 12 types of trans fatty acids and 2 types of conjugated fatty acids. Compared with the national standard GB5009.168-2016 "Determination of Fatty Acids in Food" testing method, 35 types of fatty acids are detected, including 18 types of unsaturated fatty acids and 5 types. Saturated fatty acids, 10 trans fatty acids and 2 conjugated fatty acids;
第二、本发明建立的检测72种脂肪酸方法的日内精密度的RSD值控制在0.57-9.81％，日间精密度的RSD值控制在0.47-9.87％，低于国标GB 5009.168-2016中要求的10％，远低于国标GB 5009.257-2016中要求的15％，稳定性好，满足精准定量分析的要求；Second, the RSD value of the intraday precision of the 72 fatty acid detection methods established by the present invention is controlled within 0.57-9.81%, and the RSD value of the intraday precision is controlled within 0.47-9.87%, which is lower than the national standard GB 5009.168-2016. 10%, far lower than the 15% required in the national standard GB 5009.257-2016, with good stability and meeting the requirements of accurate quantitative analysis;
第三、本发明建立的检测72种脂肪酸方法的检出限在0.000084-0.001276g/100g之间，表明在现有仪器和所用参数的条件下，0.0013g/100g的各脂肪酸甲酯都能够定性检出，是国标GB 5009.168-2016的检出限(0.0013-0.0066g/100g)的十分之二，是国标GB 5009.257-2016的检出限(0.012g/100g)十分之一；本发明的定量限在0.000289-0.004263g/100g之间，表明在现有仪器和所用参数的条件下，0.0043g/100g的各脂肪酸甲酯都能够定量分析，是国标GB 5009.257-2016的定量限(0.024g/100g)的十分之二，显著降低了脂肪酸的检出限和定量限，准确性好，能够实现脂肪酸的精准识别；Third, the detection limit of the 72 fatty acid detection methods established in the present invention is between 0.000084-0.001276g/100g, which shows that under the conditions of existing equipment and the parameters used, each fatty acid methyl ester of 0.0013g/100g can be qualitatively The detection is two tenths of the detection limit of the national standard GB 5009.168-2016 (0.0013-0.0066g/100g), and one tenth of the detection limit of the national standard GB 5009.257-2016 (0.012g/100g); the present invention The limit of quantification is between 0.000289-0.004263g/100g, indicating that under the conditions of existing instruments and the parameters used, all fatty acid methyl esters of 0.0043g/100g can be quantitatively analyzed, which is the limit of quantification (0.024) of the national standard GB 5009.257-2016 g/100g), which significantly reduces the detection limit and quantification limit of fatty acids, with good accuracy and accurate identification of fatty acids;
第四、本发明建立的检测72种脂肪酸方法的线性关系：各脂肪酸的仪器响应值与浓度呈良好的线性相关，线性关系基本都高于0.999，完全满足定量分析的要求，且方法适应性广；Fourth, the linear relationship of the 72 fatty acid detection methods established by the present invention: the instrument response value of each fatty acid is well linearly related to the concentration, and the linear relationship is basically higher than 0.999, which fully meets the requirements of quantitative analysis, and the method has wide adaptability ；
第五、本发明还能实现21种反式脂肪酸、3种顺反共轭亚油酸和2种反反共轭亚油酸的高效分离，相比国标GB5009.257-2016《食品中反式脂肪酸的测定》检测方法，检测脂肪酸种类多12种，包括7种反式脂肪酸、3种顺反共轭亚油酸和2种反反共轭亚油酸。Fifth, the present invention can also realize the high-efficiency separation of 21 trans fatty acids, 3 cis-trans conjugated linoleic acids, and 2 anti-trans conjugated linoleic acids. Compared with the national standard GB5009.257-2016 "Trans Fatty Acids in Food Determination》Detection method, detecting 12 kinds of fatty acids, including 7 kinds of trans fatty acids, 3 kinds of cis-trans conjugated linoleic acid and 2 kinds of anti-trans conjugated linoleic acid.
本发明的其它优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。Other advantages, objectives and features of the present invention will be partially embodied by the following description, and partly will be understood by those skilled in the art through the research and practice of the present invention.
附图说明Description of the drawings
图1为本发明的气相色谱升温程序图；Figure 1 is a diagram of the gas chromatograph temperature rising program of the present invention;
图2为本发明72种脂肪酸甲酯的气相色谱图之一；Figure 2 is one of the gas chromatograms of 72 fatty acid methyl esters of the present invention;
图3为本发明72种脂肪酸甲酯的气相色谱图之二；Figure 3 is the second gas chromatogram of 72 kinds of fatty acid methyl esters of the present invention;
图4为本发明72种脂肪酸甲酯的气相色谱图之三；Figure 4 is the third gas chromatogram of 72 fatty acid methyl esters of the present invention;
图5为本发明26种脂肪酸异构体的气相色谱图；Figure 5 is a gas chromatogram of 26 fatty acid isomers of the present invention;
图6为本发明实施例1花生油的气相色谱图；Figure 6 is a gas chromatogram of peanut oil in Example 1 of the present invention;
图7为本发明实施例2牛奶的气相色谱图；Figure 7 is a gas chromatogram of milk in Example 2 of the present invention;
图8为本发明实施例3亚麻籽油的气相色谱图；Figure 8 is a gas chromatogram of linseed oil in Example 3 of the present invention;
图9为本发明对比例1牛奶的气相色谱图；Figure 9 is a gas chromatogram of the milk of Comparative Example 1 of the present invention;
图10为本发明对比例2亚麻籽油的气相色谱图。Figure 10 is a gas chromatogram of linseed oil in Comparative Example 2 of the present invention.
具体实施方式Detailed ways
下面结合附图对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it with reference to the text of the description.
应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不配出一个或多个其它元件或其组合的存在或添加。It should be understood that terms such as "having", "including" and "including" used herein do not equate the presence or addition of one or more other elements or combinations thereof.
需要说明的是，下述实施方案中所述实验方法，如无特殊说明，均为常规方法，所述试剂和材料，如无特殊说明，均可从商业途径获得。It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.
同时检测分离多种脂肪酸的气相色谱方法，包括：采用氰丙基硅氧烷强极性固定相CP Sil 88气相色谱柱进行气相色谱检测，规格为80-200m×0.25mm×0.20μm，长度可变化，内径、膜厚为定值，购于美国安捷伦科技有限公司，色谱条件为：Gas chromatographic methods for simultaneous detection and separation of multiple fatty acids, including: cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column for gas chromatographic detection, the specification is 80-200m×0.25mm×0.20μm, the length can be Change, inner diameter and film thickness are fixed values, purchased from Agilent Technologies Co., Ltd., the chromatographic conditions are:
进样口温度200-230℃；进样量1μL；分流比8-12:1；氮气流速10-12cm/s；恒定线速度模式；The temperature of the injection port is 200-230℃; the injection volume is 1μL; the split ratio is 8-12:1; the nitrogen flow rate is 10-12cm/s; the constant linear velocity mode;
色谱柱初始温度为60-80℃，保持4-6min，以20-30℃/min的加热速率升至150-180℃，保持3-5min，以1-5℃/min升至200-225℃，保持40-60min，再以0.5-2℃/min升至200-230℃，保持4-6min；The initial temperature of the chromatographic column is 60-80℃, keep it for 4-6min, raise it to 150-180℃ at a heating rate of 20-30℃/min, keep it for 3-5min, raise it to 200-225℃ at 1-5℃/min , Keep for 40-60min, then increase to 200-230℃ at 0.5-2℃/min, keep it for 4-6min;
检测器：氢火焰离子化检测器FID；检测器温度200-230℃；尾吹流量2-5mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 200-230℃; makeup flow rate 2-5mL/min.
在另一个技术方案中，色谱柱规格为100m×0.25mm×0.20μm，通常购买的色谱柱为该规格。In another technical solution, the specification of the chromatographic column is 100m×0.25mm×0.20μm, and the commonly purchased chromatographic column has this specification.
在另一个技术方案中，色谱条件为：In another technical solution, the chromatographic conditions are:
进样口温度230℃；进样量1μL；分流比10:1；氮气流速10.6cm/s；恒定线速度模式；Injection port temperature 230℃; injection volume 1μL; split ratio 10:1; nitrogen flow rate 10.6cm/s; constant linear velocity mode;
如图1所示，色谱柱初始温度为60℃，保持5min，以25℃/min的加热速率升至160℃，保持4min，以2℃/min升至225℃，保持50min，再以1℃/min升至230℃，保持5min；As shown in Figure 1, the initial temperature of the chromatographic column is 60°C, keep it for 5 minutes, raise it to 160°C at a heating rate of 25°C/min, hold it for 4 minutes, raise it to 225°C at 2°C/min, hold it for 50 minutes, and then increase it at 1°C. /min to 230℃, keep it for 5min;
检测器：氢火焰离子化检测器FID；检测器温度230℃；尾吹流量3mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 230℃; makeup flow rate 3mL/min.
在另一个技术方案中，多种脂肪酸包括C3-C5短链脂肪酸、C6-C12中链脂肪酸和C13-C24长链脂肪酸。In another technical solution, multiple fatty acids include C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and C13-C24 long-chain fatty acids.
在另一个技术方案中，72种脂肪酸甲酯的参考保留时间如表1所示，多种脂肪酸包括C3:0，C4:0，C5:0，C6:0，C7:0，C8:0，C9:0，C10:0，C11:0，C12:0，C11:1-10c，C13:0，C12:1-11c，C14:0，C13:1-12c，C14:1-9t，C14:1-9c，C15:0，C15:1-10t，C15:1-10c，C16:0，C15:1-14c，C16:1-9t，C16:1-9c，C17:0，C17:1-10t，C17:1-10c，C18:0，C18:1-6t，C18:1-9t，C18:1-11t，C18:1-6c，C18:1-9c，C18:1-11c，C19:0，C18:2-9t,12t，C19:1-7t，C19:1-10t，C19:1-7c，C19:1-10c，C18:2-9c,12c，C20:0，C20:1-11t，C18:3-6c,9c,12c，C20:1-5c，C20:1-8c，C20:1-11c，C19:2-10c,13c，C18:3-9c,12c,15c，C21:0，C18:2-9c,11t，C18:2-10t,12c，C21:1-12c，C20:2-11c,14c，C22:0，C22:1-13t，C20:3-8c,11c,14c，C22:1-13c，C21:2-12c,15c，C20:3-11c,14c,17c，C23:0，C20:4-5c,8c,11c,14c，C23:1-14c，C22:2-13c,16c，C24:0，C20:5-5c,8c,11c,14c,17c，C24:1-15c，C22:3-13c,16c,19c，C22:4-7c,10c,13c,16c，C22:5-4c,7c,10c,13c,16c，C22:5-7c,10c,13c,16c,19c，C22:6-4c,7c,10c,13c,16c,19c。In another technical solution, the reference retention time of 72 kinds of fatty acid methyl esters is shown in Table 1. A variety of fatty acids include C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C11:1-10c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14: 1-9c, C15:0, C15:1-10t, C15:1-10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1- 10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19: 0, C18:2-9t,12t, C19:1-7t, C19:1-10t, C19:1-7c, C19:1-10c, C18:2-9c,12c, C20:0, C20:1- 11t, C18: 3-6c, 9c, 12c, C20: 1-5c, C20:1-8c, C20:1-11c, C19: 2-10c, 13c, C18: 3-9c, 12c, 15c, C21: 0, C18: 2-9c, 11t, C18: 2-10t, 12c, C21:1-12c, C20: 2-11c, 14c, C22:0, C22:1-13t, C20: 3-8c, 11c, 14c, C22:1-13c, C21: 2-12c, 15c, C20: 3-11c, 14c, 17c, C23:0, C20: 4-5c, 8c, 11c, 14c, C23:1-14c, C22: 2-13c,16c, C24:0, C20: 5-5c, 8c, 11c, 14c, 17c, C24:1-15c, C22: 3-13c, 16c, 19c, C22: 4-7c, 10c, 13c, 16c, C22: 5-4c, 7c, 10c, 13c, 16c, C22: 5-7c, 10c, 13c, 16c, 19c, C22: 6-4c, 7c, 10c, 13c, 16c, 19c.
本发明采用氰丙基硅氧烷强极性固定相CP Sil 88气相色谱柱，建立能够同时高通量检测72种C3-C24系列脂肪酸的方法，其中包括36种不饱和脂肪酸、22种饱和脂肪酸、12种反式脂肪酸和2种共轭脂肪酸，如图2-4所示，各波峰采用阿拉伯数字顺序编号，同表1各序号对应脂肪酸名称。相比国标GB5009.168-2016《食品中脂肪酸的测定》检测方法，检测脂肪酸种类多35种，包括18种不饱和脂肪酸(C11:1-10c、C12:1-11c、C13:1-12c、C15:1-14c、C18:1-6c、C18:1-11c、C19:1-7c、C19:1-10c、C20:1-5c、C20:1-8c、C19:2-10c,13c、C21:1-12c、C21:2-12c,15c、C23:1-14c、C22:3-13c,16c,19c、C22:4-7c,10c,13c,16c、C22:5-4c,7c,10c,13c,16c和C22:5-7c,10c,13c,16c,19c)、5种饱和脂肪酸(C3:0、C5:0、C7:0、C9:0和C19:0)、10种反式脂肪酸(C14:1-9t、C15:1-10t、C16:1-9t、C17:1-10t、C18:1-6t、C18:1-11t、C19:1-7t、C19:1-10t、C20:1-11t和C22:1-13t)和2种共轭脂肪酸(C18:2-9c,11t和C18:2-10t,12c)。The present invention adopts cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatography column to establish a method that can simultaneously detect 72 C3-C24 series fatty acids with high throughput, including 36 unsaturated fatty acids and 22 saturated fatty acids , 12 kinds of trans fatty acids and 2 kinds of conjugated fatty acids, as shown in Figure 2-4, the peaks are numbered in Arabic numerals, and the numbers in Table 1 correspond to the names of fatty acids. Compared with the national standard GB5009.168-2016 "Determination of Fatty Acids in Food" testing method, there are 35 types of fatty acids detected, including 18 types of unsaturated fatty acids (C11:1-10c, C12:1-11c, C13:1-12c, C15:1-14c, C18:1-6c, C18:1-11c, C19:1-7c, C19:1-10c, C20:1-5c, C20:1-8c, C19:2-10c, 13c, C21:1-12c, C21:2-12c,15c, C23:1-14c, C22:3-13c,16c,19c, C22:4-7c,10c,13c,16c, C22:5-4c,7c, 10c, 13c, 16c and C22: 5-7c, 10c, 13c, 16c, 19c), 5 kinds of saturated fatty acids (C3:0, C5:0, C7:0, C9:0 and C19:0), 10 kinds of reaction Fatty acids (C14:1-9t, C15:1-10t, C16:1-9t, C17:1-10t, C18:1-6t, C18:1-11t, C19:1-7t, C19:1-10t , C20:1-11t and C22:1-13t) and 2 kinds of conjugated fatty acids (C18:2-9c,11t and C18:2-10t,12c).
表1Table 1
序号Serial number 脂肪酸名称Fatty acid name 参考保留时间(min)Reference retention time (min)
11 C3:0C3:0 19.08519.085
22 C4:0C4:0 19.95819.958
33 C5:0C5:0 21.23921.239
44 C6:0C6:0 22.45522.455
55 C7:0C7:0 23.60223.602
66 C8:0C8:0 24.73324.733
77 C9:0C9:0 25.87125.871
88 C10:0C10:0 27.10327.103
99 C11:0C11:0 28.48528.485
1010 C12:0C12:0 30.05930.059
1111 C11:1-10cC11:1-10c 30.13930.139
1212 C13:0C13:0 31.85131.851
1313 C12:1-11cC12:1-11c 31.95131.951
1414 C14:0C14:0 33.8733.87
1515 C13:1-12cC13:1-12c 33.99533.995
1616 C14:1-9tC14:1-9t 35.22635.226
1717 C14:1-9cC14:1-9c 35.93235.932
1818 C15:0C15:0 36.09736.097
1919 C15:1-10tC15:1-10t 37.58537.585
2020 C15:1-10cC15:1-10c 38.33538.335
21twenty one C16:0C16:0 38.50238.502
22twenty two C15:1-14cC15:1-14c 38.65838.658
23twenty three C16:1-9tC16:1-9t 39.87339.873
24twenty four C16:1-9cC16:1-9c 40.52240.522
2525 C17:0C17:0 41.03441.034
2626 C17:1-10tC17:1-10t 42.47442.474
2727 C17:1-10cC17:1-10c 43.13243.132
2828 C18:0C18:0 43.6743.67
2929 C18:1-6tC18:1-6t 44.87744.877
3030 C18:1-9tC18:1-9t 44.99544.995
3131 C18:1-11tC18:1-11t 45.13745.137
3232 C18:1-6cC18:1-6c 45.4445.44
3333 C18:1-9cC18:1-9c 45.59345.593
3434 C18:1-11cC18:1-11c 45.83545.835
3535 C19:0C19:0 46.39446.394
3636 C18:2-9t,12tC18: 2-9t, 12t 47.07547.075
3737 C19:1-7tC19:1-7t 47.66347.663
3838 C19:1-10tC19:1-10t 47.80847.808
3939 C19:1-7cC19:1-7c 48.19848.198
4040 C19:1-10cC19:1-10c 48.42948.429
4141 C18:2-9c,12cC18: 2-9c, 12c 48.50148.501
4242 C20:0C20:0 49.27749.277
4343 C20:1-11t/C18:3-6c,9c,12c/C20:1-5cC20:1-11t/C18:3-6c,9c,12c/C20:1-5c 50.80450.804
4444 C20:1-8cC20:1-8c 51.21751.217
4545 C20:1-11cC20:1-11c 51.46151.461
4646 C19:2-10c,13cC19: 2-10c, 13c 51.56551.565
4747 C18:3-9c,12c,15cC18: 3-9c, 12c, 15c 52.11752.117
4848 C21:0C21:0 52.37552.375
4949 C18:2-9c,11tC18: 2-9c, 11t 52.52752.527
5050 C18:2-10t,12cC18: 2-10t, 12c 53.04153.041
5151 C21:1-12cC21:1-12c 54.72354.723
5252 C20:2-11c,14cC20: 2-11c, 14c 54.83454.834
5353 C22:0C22:0 55.74155.741
5454 C22:1-13t/C20:3-8c,11c,14cC22:1-13t/C20:3-8c,11c,14c 57.5857.58
5555 C22:1-13cC22:1-13c 58.34558.345
5656 C21:2-12c,15cC21: 2-12c, 15c 58.47158.471
5757 C20:3-11c,14c,17cC20: 3-11c, 14c, 17c 59.15159.151
5858 C23:0C23:0 59.53459.534
5959 C20:4-5c,8c,11c,14cC20:4-5c,8c,11c,14c 59.91559.915
6060 C23:1-14c/C22:2-13c,16cC23:1-14c/C22:2-13c,16c 62.37462.374
6161 C24:0C24:0 63.67463.674
6262 C20:5-5c,8c,11c,14c,17cC20: 5-5c, 8c, 11c, 14c, 17c 65.19765.197
6363 C24:1-15cC24:1-15c 66.96166.961
6464 C22:3-13c,16c,19cC22: 3-13c, 16c, 19c 67.88467.884
6565 C22:4-7c,10c,13c,16cC22: 4-7c, 10c, 13c, 16c 69.58769.587
6666 C22:5-4c,7c,10c,13c,16cC22: 5-4c, 7c, 10c, 13c, 16c 72.37572.375
6767 C22:5-7c,10c,13c,16c,19cC22: 5-7c, 10c, 13c, 16c, 19c 76.6276.62
6868 C22:6-4c,7c,10c,13c,16c,19cC22: 6-4c, 7c, 10c, 13c, 16c, 19c 80.14780.147
仪器精密度用相对标准偏差(RSD)表示，用于评价气相色谱方法对测定同一个样品结果重复性的再现性。本实验采用同一气相色谱分析方法对72种脂肪酸混标溶液的日内(一天内测定7次)和日间(连续7天测定)精密度进行考察，72种脂肪酸日内精密度和日间精密度如表2所示，各脂肪酸甲酯日内精密度的RSD值控制在0.57-9.81％，日间精密度控制在0.47-9.87％，低于国标GB 5009.168—2016中要求的10％，远低于国标GB5009.257—2016中要求的15％，稳定性好，满足精准定量分析的要求。The precision of the instrument is expressed by the relative standard deviation (RSD), which is used to evaluate the reproducibility of the reproducibility of the measurement result of the same sample by the gas chromatography method. In this experiment, the same gas chromatography analysis method was used to investigate the intra-day (7 determinations in a day) and intra-day (7 consecutive days of determination) precision of 72 fatty acid mixed standard solutions. The intra-day and inter-day precisions of 72 fatty acids were as follows As shown in Table 2, the RSD value of the intraday precision of each fatty acid methyl ester is controlled at 0.57-9.81%, and the daytime precision is controlled at 0.47-9.87%, which is lower than the 10% required in the national standard GB 5009.168-2016, and far lower than the national standard 15% of the requirements in GB5009.257-2016, good stability, and meet the requirements of accurate quantitative analysis.
表2Table 2
Figure PCTCN2019099735-appb-000001
Figure PCTCN2019099735-appb-000001
Figure PCTCN2019099735-appb-000002
Figure PCTCN2019099735-appb-000002
Figure PCTCN2019099735-appb-000003
Figure PCTCN2019099735-appb-000003
Figure PCTCN2019099735-appb-000004
Figure PCTCN2019099735-appb-000004
72种脂肪酸检测限及定量限如表3所示，可以看出，本发明检出限在0.000084-0.001276g/100g之间，表明在现有仪器和所用参数的条件下，0.0013g/100g的各脂肪酸甲酯都能够定性检出，是国标GB 5009.168-2016的检出限(0.0013-0.0066g/100g)的十分之二，是国标GB 5009.257-2016的检出限(0.012g/100g)的十分之一；本发明的定量限在0.000289-0.004263g/100g之间，表明在现有仪器和所用参数的条件下，0.0043g/100g的各脂肪酸甲酯都能够定量分析，是国标GB 5009.257-2016的定量限(0.024g/100g)的十分之二。本发明显著降低了脂肪酸的检出限和测定限，准确性好，能够实现脂肪酸的精准识别。The detection limit and quantification limit of 72 fatty acids are shown in Table 3. It can be seen that the detection limit of the present invention is between 0.000084-0.001276g/100g, indicating that under the conditions of existing equipment and the parameters used, 0.0013g/100g All fatty acid methyl esters can be qualitatively detected, which is two-tenths of the detection limit of the national standard GB 5009.168-2016 (0.0013-0.0066g/100g), and the detection limit of the national standard GB 5009.257-2016 (0.012g/100g) The quantification limit of the present invention is between 0.000289-0.004263g/100g, indicating that under the conditions of existing instruments and the parameters used, each fatty acid methyl ester of 0.0043g/100g can be quantitatively analyzed, which is the national standard GB Two-tenths of the limit of quantification (0.024g/100g) of 5009.257-2016. The invention significantly reduces the detection limit and the determination limit of fatty acids, has good accuracy, and can realize precise identification of fatty acids.
表3table 3
Figure PCTCN2019099735-appb-000005
Figure PCTCN2019099735-appb-000005
Figure PCTCN2019099735-appb-000006
Figure PCTCN2019099735-appb-000006
Figure PCTCN2019099735-appb-000007
Figure PCTCN2019099735-appb-000007
Figure PCTCN2019099735-appb-000008
Figure PCTCN2019099735-appb-000008
72种脂肪酸标准曲线、相关系数和浓度范围如表4所示，72种脂肪酸甲酯标准品溶液进行梯度稀释，将各浓度的稀释混标液按建立的方法进行气相色谱分析，在所考察的浓度范围内进行线性拟合，得出各脂肪酸甲酯的线性方程和相关系数，线性关系均基本高于0.999，完全满足定量分析的要求。The 72 kinds of fatty acid standard curves, correlation coefficients and concentration ranges are shown in Table 4. The 72 kinds of fatty acid methyl ester standard solutions are gradually diluted, and the diluted mixed standard solutions of each concentration are analyzed by gas chromatography according to the established method. Linear fitting was performed within the concentration range, and the linear equation and correlation coefficient of each fatty acid methyl ester were obtained. The linear relationship was basically higher than 0.999, which fully met the requirements of quantitative analysis.
表4Table 4
Figure PCTCN2019099735-appb-000009
Figure PCTCN2019099735-appb-000009
Figure PCTCN2019099735-appb-000010
Figure PCTCN2019099735-appb-000010
Figure PCTCN2019099735-appb-000011
Figure PCTCN2019099735-appb-000011
Figure PCTCN2019099735-appb-000012
Figure PCTCN2019099735-appb-000012
本发明还能实现21种反式脂肪酸、3种顺反共轭亚油酸和2种反反共轭亚油酸的高效分离，如图5所示，各波峰采用阿拉伯数字顺序编号，同表5各序号对应脂肪酸名称，其中11号、12号之间未标记的波峰为C18:2-9c,12c(同表1中41号)，17号、18号之间未标记的波峰为C18:3-9c,12c,15c(同表1中49号)，为了不影响展示26种脂肪酸异构体，图5中未对这两种物质进行编号处理。相比国标GB5009.257-2016《食品中反式脂肪酸的测定》检测方法，检测脂肪酸种类多12种，包括7种反式脂肪酸(C14:1-9t、C15:1-10t、C17:1-10t、C19:1-7t、C19:1-10t、C18:2-9c,12t、C18:2-9t,12c)、3种顺反共轭亚油酸(C18:2-9c,11t、C18:2-10t,12c和C18:2-11c,13t)和2种反反共轭亚油酸(C18:2-9t,11t和C18:2-10t,12t)，26种脂肪酸异构体的参考保留时间如表5所示。The present invention can also realize the high-efficiency separation of 21 trans fatty acids, 3 cis-trans conjugated linoleic acids, and 2 kinds of anti-anti-conjugated linoleic acids. As shown in Figure 5, each wave peak is numbered in Arabic numerals, as shown in Table 5. The serial number corresponds to the name of the fatty acid. The unmarked peak between No. 11 and No. 12 is C18:2-9c, 12c (same as No. 41 in Table 1), and the unmarked peak between No. 17 and No. 18 is C18:3- 9c, 12c, 15c (same as No. 49 in Table 1), in order not to affect the display of 26 fatty acid isomers, these two substances are not numbered in Figure 5. Compared with the national standard GB5009.257-2016 "Determination of Trans Fatty Acids in Food", there are 12 more types of fatty acids detected, including 7 types of trans fatty acids (C14:1-9t, C15:1-10t, C17:1- 10t, C19:1-7t, C19:1-10t, C18: 2-9c, 12t, C18: 2-9t, 12c), 3 kinds of cis-trans conjugated linoleic acid (C18: 2-9c, 11t, C18: 2-10t, 12c and C18: 2-11c, 13t) and 2 kinds of anti-anti-conjugated linoleic acid (C18: 2-9t, 11t and C18: 2-10t, 12t), reference reservation of 26 fatty acid isomers The time is shown in Table 5.
表5table 5
序号Serial number 脂肪酸名称Fatty acid name 参考保留时间(min)Reference retention time (min)
11 C14:1-9tC14:1-9t 35.0935.09
22 C15:1-10tC15:1-10t 37.4537.45
33 C16:1-9tC16:1-9t 39.73639.736
44 C17:1-10tC17:1-10t 42.34142.341
55 C18:1-6tC18:1-6t 44.75144.751
66 C18:1-9tC18:1-9t 44.86744.867
77 C18:1-11tC18:1-11t 45.00945.009
88 C18:2-9t,12tC18: 2-9t, 12t 46.93746.937
99 C19:1-7tC19:1-7t 47.48347.483
1010 C18:2-9c,12t/C19:1-10tC18:2-9c,12t/C19:1-10t 47.68447.684
1111 C18:2-9t,12cC18: 2-9t, 12c 47.86647.866
1212 C18:3-9t,12t,15tC18: 3-9t, 12t, 15t 49.39449.394
1313 C18:3-9t,12t,15c/C18:3-9t,12c,15tC18: 3-9t, 12t, 15c/C18: 3-9t, 12c, 15t 50.24250.242
1414 C18:3-9c,12t,15t/C20:1-11tC18:3-9c,12t,15t/C20:1-11t 50.47150.471
1515 C18:3-9c,12c,15tC18: 3-9c, 12c, 15t 50.67150.671
1616 C18:3-9c,12t,15cC18: 3-9c, 12t, 15c 51.2851.28
1717 C18:3-9t,12c,15cC18: 3-9t, 12c, 15c 51.3851.38
1818 C18:2-9c,11tC18: 2-9c, 11t 52.30252.302
1919 C18:2-11c,13tC18: 2-11c, 13t 52.74252.742
2020 C18:2-10t,12cC18: 2-10t, 12c 52.88452.884
21twenty one C18:2-9t,11t/C18:2-10t,12tC18: 2-9t, 11t/C18: 2-10t, 12t 53.94953.949
22twenty two C22:1-13tC22:1-13t 57.21557.215
<实例1><Example 1>
花生油中脂肪酸的气相色谱定性定量分析Qualitative and quantitative analysis of fatty acids in peanut oil by gas chromatography
步骤：称取100mg花生油，置于10mL离心管中，加入2mL的C11:0内标液，再加入0.1mL 2mol/L的氢氧化钾甲醇溶液，旋涡混匀30s。以4000rpm离心10min。取上清液20μL定容至1mL容量瓶，采用岛津GC-2010气相色谱测定，气相色谱柱为CP Sil 88(100m×0.25mm×0.20μm)，色谱条件位：进样口温度：200℃；进样量：1μL；分流比：8:1；流速：10cm/s(氮气)；恒定线速度模式；柱箱升温程序：60℃保持5min，21℃/min升至150℃，保持5min，以1℃/min升至200℃，保持60min，再以0.5℃/min升至200℃，保持6min；检测器：氢火焰离子化检测器(FID)；检测器温度：200℃；尾吹流量：2.0mL/min。Step: Weigh 100mg of peanut oil, place it in a 10mL centrifuge tube, add 2mL of C11:0 internal standard solution, and then add 0.1mL 2mol/L of potassium hydroxide methanol solution, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min. Take 20μL of the supernatant and dilute to a 1mL volumetric flask, use Shimadzu GC-2010 gas chromatography to determine, the gas chromatography column is CP Sil 88 (100m×0.25mm×0.20μm), chromatographic condition position: Inlet temperature: 200℃ ;Injection volume: 1μL; split ratio: 8:1; flow rate: 10cm/s (nitrogen); constant linear velocity mode; oven heating program: 60°C for 5 min, 21°C/min to 150°C, hold for 5 minutes, Raise to 200°C at 1°C/min, hold for 60min, then rise to 200°C at 0.5°C/min, hold for 6min; Detector: hydrogen flame ionization detector (FID); detector temperature: 200°C; makeup flow : 2.0mL/min.
由图6可知：花生油经本方法检测后共有10种脂肪酸，其中油酸(C18:1-9c)、亚油酸(C18:2-9c,12c)和棕榈酸(C16:0)含量较高，除此之外还有硬脂酸(C18:0)、C18:1-11c、C20:0、C20:1-11c、C18:3-9c,12c,15c、C22:0和C22:1-13c。It can be seen from Figure 6 that there are 10 fatty acids in peanut oil tested by this method, among which oleic acid (C18:1-9c), linoleic acid (C18:2-9c, 12c) and palmitic acid (C16:0) are higher in content , In addition to stearic acid (C18:0), C18:1-11c, C20:0, C20:1-11c, C18:3-9c, 12c, 15c, C22:0 and C22:1- 13c.
<实例2><Example 2>
牛奶中脂肪酸的气相色谱定性定量分析Qualitative and quantitative analysis of fatty acids in milk by gas chromatography
步骤：称取1mL牛奶，置于烧瓶中，加入氨水5mL，混匀。将烧瓶放入70-80℃水浴中，水解20min。每5min振荡一下烧瓶，使黏附在烧瓶壁上的颗粒物混入溶液中。水解完成后，取出烧瓶冷却至室温。水解后的试样，加入10mL95％乙醇，混匀。将烧瓶中的水解液转移到分液漏斗中，用50mL乙醚石油醚混合液冲洗烧瓶和塞子，冲洗液并入分液漏斗中，加盖。振摇5min，静置10min。将醚层提取液收集到250mL烧瓶中。按照 以上步骤重复提取水解液3次，最后用乙醚石油醚混合液冲洗分液漏斗，并收集到250mL烧瓶中。旋转蒸发仪浓缩至干，残留物为脂肪提取物。加入2mL的C11:0内标液，再加入1ml异辛烷和0.1mL 2mol/L的氢氧化钾甲醇溶液，旋涡混匀30s。以4000rpm离心10min。取上清液20μL定容至1mL容量瓶，采用岛津GC-2010气相色谱测定，气相色谱柱为CP Sil 88(100m×0.25mm×0.20μm)，色谱条件位：进样口温度：230℃；进样量：1μL；分流比：11.5:1；流速：11.6cm/s(氮气)；恒定线速度模式；柱箱升温程序：80℃保持4min，29℃/min升至180℃，保持3min，以4.5℃/min升至225℃，保持40min，再以2℃/min升至230℃，保持4min；检测器：氢火焰离子化检测器(FID)；检测器温度：230℃；尾吹流量：3.8mL/min。Step: Weigh 1 mL of milk, place it in a flask, add 5 mL of ammonia, and mix well. Put the flask in a 70-80°C water bath and hydrolyze for 20 min. Shake the flask every 5 minutes to mix the particles adhering to the wall of the flask into the solution. After the hydrolysis was completed, the flask was taken out and cooled to room temperature. Add 10 mL of 95% ethanol to the hydrolyzed sample and mix well. Transfer the hydrolysate in the flask to a separatory funnel, rinse the flask and stopper with 50 mL of ether and petroleum ether mixture, merge the rinse liquid into the separatory funnel, and cover it. Shake for 5 minutes and let stand for 10 minutes. The ether layer extract was collected in a 250 mL flask. Repeat the extraction of the hydrolysate 3 times according to the above steps, and finally rinse the separatory funnel with a mixture of ether and petroleum ether and collect it in a 250 mL flask. The rotary evaporator is concentrated to dryness, and the residue is fat extract. Add 2mL C11:0 internal standard solution, then add 1ml isooctane and 0.1mL 2mol/L potassium hydroxide methanol solution, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min. Take 20μL of the supernatant and dilute to a 1mL volumetric flask, use Shimadzu GC-2010 gas chromatography to determine, the gas chromatography column is CP Sil 88 (100m×0.25mm×0.20μm), chromatographic condition position: inlet temperature: 230℃ ;Sample volume: 1μL; Split ratio: 11.5:1; Flow rate: 11.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 80°C for 4min, 29°C/min to 180°C, hold for 3min , Rise to 225°C at 4.5°C/min, hold for 40 minutes, then rise to 230°C at 2°C/min, hold for 4 minutes; Detector: hydrogen flame ionization detector (FID); detector temperature: 230°C; makeup Flow rate: 3.8mL/min.
由图7可知：牛奶经本方法检测后可能有10种脂肪酸，包括C4:0、C6:0、C8:0、C10:0、C12:0、C14:0、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1-9c)和亚油酸(C18:2-9c,12c)。It can be seen from Figure 7 that milk may have 10 fatty acids after detection by this method, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, palmitic acid (C16:0), Stearic acid (C18:0), oleic acid (C18:1-9c) and linoleic acid (C18:2-9c, 12c).
<实例3><Example 3>
亚麻籽油中脂肪酸的气相色谱定性定量分析Qualitative and quantitative analysis of fatty acids in linseed oil by gas chromatography
步骤：称取100mg亚麻籽油，置于10mL离心管中，加入2mL的C11:0内标液，再加入0.1mL 2mol/L的氢氧化钾甲醇溶液，旋涡混匀30s。以4000rpm离心10min。取上清液20μL定容至1mL容量瓶，采用岛津GC-2010气相色谱测定，气相色谱柱为CP Sil 88(100m×0.25mm×0.20μm)，色谱条件位：进样口温度：230℃；进样量：1μL；分流比：10:1；流速：10.6cm/s(氮气)；恒定线速度模式；柱箱升温程序：60℃保持5min，25℃/min升至160℃，保持4min，以2℃/min升至225℃，保持50min，再以1℃/min升至230℃，保持5min；检测器：氢火焰离子化检测器(FID)；检测器温度：230℃；尾吹流量：3.0mL/min。Step: Weigh 100mg of linseed oil, place it in a 10mL centrifuge tube, add 2mL of C11:0 internal standard solution, and then add 0.1mL 2mol/L of methanol solution of potassium hydroxide, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min. Take 20μL of the supernatant and dilute to a 1mL volumetric flask, use Shimadzu GC-2010 gas chromatography to determine, the gas chromatography column is CP Sil 88 (100m×0.25mm×0.20μm), chromatographic condition position: inlet temperature: 230℃ ;Sample volume: 1μL; Split ratio: 10:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 60°C for 5min, 25°C/min to 160°C, and hold for 4min , Rise to 225°C at 2°C/min, hold for 50 minutes, then rise to 230°C at 1°C/min, hold for 5 minutes; detector: hydrogen flame ionization detector (FID); detector temperature: 230°C; makeup Flow rate: 3.0mL/min.
由图8可知：亚麻籽油经本方法检测后可能有20种脂肪酸，其中亚麻酸(C18:3-9c,12c,15c)、油酸(C18:1-9c)、亚油酸(C18:2-9c,12c)、硬脂酸(C18:0)和棕榈酸(C16:0)含量较高，除此之外还可能有C16:1-9c、C17:0、C18:1-11c、C20:0、C18:3-9t,12t,15c、C18:3-9c,12t,15t、C18:3-6c,9c,12c、C20:1-5c、C19:2-10c,13c、C18:3-9t,12c,15c、C22:0、C20:2-11c,14c、C20:3-8c,11c,14c、C21:2-12c,15c和C24:0。It can be seen from Figure 8 that after linseed oil is tested by this method, there may be 20 fatty acids, including linolenic acid (C18:3-9c, 12c, 15c), oleic acid (C18:1-9c), and linoleic acid (C18: 2-9c, 12c), stearic acid (C18:0) and palmitic acid (C16:0) are higher in content. In addition, there may be C16:1-9c, C17:0, C18:1-11c, C20:0, C18: 3-9t, 12t, 15c, C18: 3-9c, 12t, 15t, C18: 3-6c, 9c, 12c, C20: 1-5c, C19: 2-10c, 13c, C18: 3-9t, 12c, 15c, C22:0, C20: 2-11c, 14c, C20: 3-8c, 11c, 14c, C21: 2-12c, 15c and C24:0.
<对比例1><Comparative Example 1>
牛奶中脂肪酸的气相色谱定性定量分析Qualitative and quantitative analysis of fatty acids in milk by gas chromatography
步骤：称取1mL牛奶，置于烧瓶中，加入氨水5mL，混匀。将烧瓶放入70-80℃水浴中，水解20min。每5min振荡一下烧瓶，使黏附在烧瓶壁上的颗粒物混入溶液中。水解完成后，取出烧瓶冷却至室温。水解后的试样，加入10mL95％乙醇，混匀。将烧瓶中的水解液转移到分液漏斗中，用50mL乙醚石油醚混合液冲洗烧瓶和塞子，冲洗液并入分液漏斗中，加盖。振摇5min，静置10min。将醚层提取液收集到250mL烧瓶中。按照以上步骤重复提取水解液3次，最后用乙醚石油醚混合液冲洗分液漏斗，并收集到250mL烧瓶中。旋转蒸发仪浓缩至干，残留物为脂肪提取物。以4000rpm离心10min。取上清液20μL定容至1mL容量瓶，采用岛津GC-2010气相色谱测定，气相色谱柱为CP Sil 88(100m×0.25mm×0.20μm)，色谱条件位：进样口温度：270℃；进样量：1μL；分流比：100:1；流速：10.6cm/s(氮气)；恒定线速度模式；柱箱升温程序：100℃保持13min，10℃/min升至180℃，保持6min，以1℃/min升至200℃，保持20min，再以4℃/min升至230℃，保持10.5min；检测器：氢火焰离子化检测器(FID)；检测器温度：280℃；尾吹流量：3.0mL/min。Step: Weigh 1 mL of milk, place it in a flask, add 5 mL of ammonia, and mix well. Put the flask in a 70-80°C water bath and hydrolyze for 20 min. Shake the flask every 5 minutes to mix the particles adhering to the wall of the flask into the solution. After the hydrolysis was completed, the flask was taken out and cooled to room temperature. Add 10 mL of 95% ethanol to the hydrolyzed sample and mix well. Transfer the hydrolysate in the flask to a separatory funnel, rinse the flask and stopper with 50 mL of ether and petroleum ether mixture, merge the rinse liquid into the separatory funnel, and cover it. Shake for 5 minutes and let stand for 10 minutes. The ether layer extract was collected in a 250 mL flask. Repeat the extraction of the hydrolysate 3 times according to the above steps, and finally rinse the separatory funnel with a mixture of ether and petroleum ether and collect it in a 250 mL flask. The rotary evaporator is concentrated to dryness, and the residue is fat extract. Centrifuge at 4000 rpm for 10 min. Take 20μL of supernatant and dilute to a 1mL volumetric flask, and use Shimadzu GC-2010 gas chromatography for determination. Gas chromatography column is CP Sil 88 (100m×0.25mm×0.20μm), chromatographic condition position: inlet temperature: 270℃ ;Sample volume: 1μL; Split ratio: 100:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 100°C for 13min, 10°C/min to 180°C, for 6min , Rise to 200°C at 1°C/min, hold for 20min, then rise to 230°C at 4°C/min, hold for 10.5min; detector: hydrogen flame ionization detector (FID); detector temperature: 280°C; tail Blowing flow: 3.0mL/min.
由图9可知：牛奶经本方法检测后可能有7种脂肪酸，包括C4:0、C6:0、C8:0、C10:0、C12:0、C14:0、棕榈酸(C16:0)。可以看出，相对于实例2硬脂酸(C18:0)、油酸(C18:1-9c)和亚油酸(C18:2-9c,12c)未检出。It can be seen from Figure 9 that milk may have 7 fatty acids after detection by this method, including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, and palmitic acid (C16:0). It can be seen that compared to Example 2, stearic acid (C18:0), oleic acid (C18:1-9c) and linoleic acid (C18:2-9c, 12c) were not detected.
<对比例2><Comparative Example 2>
亚麻籽油中脂肪酸的气相色谱定性定量分析Qualitative and quantitative analysis of fatty acids in linseed oil by gas chromatography
步骤：称取100mg亚麻籽油，置于10mL离心管中，再加入0.1mL的2mol/L的氢氧化钾甲醇溶液，旋涡混匀30s。以4000rpm离心10min。取上清液20μL定容至1mL容量瓶，采用岛津GC-2010气相色谱测定，气相色谱柱为CP Sil 88(100m×0.25mm×0.20m)，色谱条件位：进样口温度：270℃；进样量：1μL；分流比：100:1；流速：10.6cm/s(氮气)；恒定线速度模式；柱箱升温程序：100℃保持13min，10℃/min升至180℃，保持6min，以1℃/min升至200℃，保持20min，再以4℃/min升至230℃，保持10.5min；检测器：氢火焰离子化检测器(FID)；检测器温度：280℃；尾吹流量：3.0mL/min。Step: Weigh 100mg of linseed oil, place it in a 10mL centrifuge tube, add 0.1mL of 2mol/L potassium hydroxide methanol solution, vortex and mix for 30s. Centrifuge at 4000 rpm for 10 min. Take 20μL of supernatant to a 1mL volumetric flask and use Shimadzu GC-2010 gas chromatography for determination. The gas chromatography column is CP Sil 88 (100m×0.25mm×0.20m), and the chromatographic condition position: inlet temperature: 270℃ ;Sample volume: 1μL; Split ratio: 100:1; Flow rate: 10.6cm/s (nitrogen); Constant linear velocity mode; Oven heating program: 100°C for 13min, 10°C/min to 180°C, for 6min , Rise to 200°C at 1°C/min, hold for 20min, then rise to 230°C at 4°C/min, hold for 10.5min; detector: hydrogen flame ionization detector (FID); detector temperature: 280°C; tail Blowing flow: 3.0mL/min.
由图10可知：亚麻籽油经本方法检测后可能有7种脂肪酸，其中亚麻酸(C18:3-9c,12c,15c)、油酸(C18:1-9c)、亚油酸(C18:2-9c,12c)、硬脂酸(C18:0)和棕榈酸(C16:0)含量较高，除此之外还可能有C18:3-6c,9c,12c、C20:1-5c，可以看出，相对于 实例3C16:1-9c、C17:0、C18:1-11c、C20:0、C18:3-9t,12t,15c、C18:3-9c,12t,15t、C19:2-10c,13c、C18:3-9t,12c,15c、C20:2-11c,14c、C22:0、C20:3-8c,11c,14c、C21:2-12c,15c和C24:0未检出。It can be seen from Figure 10 that linseed oil may have 7 fatty acids after being tested by this method, including linolenic acid (C18:3-9c, 12c, 15c), oleic acid (C18:1-9c), and linoleic acid (C18: 2-9c, 12c), stearic acid (C18:0) and palmitic acid (C16:0) are higher in content, in addition to C18: 3-6c, 9c, 12c, C20: 1-5c, It can be seen that relative to examples 3C16:1-9c, C17:0, C18:1-11c, C20:0, C18:3-9t, 12t, 15c, C18:3-9c, 12t, 15t, C19:2 -10c, 13c, C18: 3-9t, 12c, 15c, C20: 2-11c, 14c, C22:0, C20: 3-8c, 11c, 14c, C21: 2-12c, 15c and C24:0 unchecked Out.
通过实验验证发现，本方法适用于检测含有C3-C24脂肪酸的物质，除上述案例的花生油、亚麻籽油和牛奶外，还可检测各类富含脂肪酸的食用油、肉类食品、蔬菜、乳及乳制品及其他富含脂肪酸的物质，由于说明书篇幅有限，不作一一进行详述。Through experimental verification, it is found that this method is suitable for detecting substances containing C3-C24 fatty acids. In addition to peanut oil, linseed oil and milk in the above cases, it can also detect various fatty acid-rich edible oils, meat foods, vegetables, and milk. And dairy products and other fatty acid-rich substances, due to the limited length of the instructions, they will not be detailed one by one.
这里说明的设备数量和处理规模是用来简化本发明的说明的。对本发明的应用、修改和变化对本领域的技术人员来说是显而易见的。The number of equipment and processing scale described here are used to simplify the description of the present invention. The applications, modifications and changes to the present invention are obvious to those skilled in the art.
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. For those familiar with the field, it can be easily Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims (7)

  1. 同时检测分离多种脂肪酸的气相色谱方法，其特征在于，包括：采用氰丙基硅氧烷强极性固定相CP Sil 88气相色谱柱进行气相色谱检测，规格为80-200m×0.25mm×0.20μm，色谱条件为：The gas chromatographic method for simultaneous detection and separation of multiple fatty acids is characterized by the use of cyanopropyl siloxane strong polar stationary phase CP Sil 88 gas chromatographic column for gas chromatographic detection, the specification is 80-200m×0.25mm×0.20 μm, the chromatographic conditions are:
    进样口温度200-230℃；进样量1μL；分流比8-12:1；氮气流速10-12cm/s；恒定线速度模式；The temperature of the injection port is 200-230℃; the injection volume is 1μL; the split ratio is 8-12:1; the nitrogen flow rate is 10-12cm/s; the constant linear velocity mode;
    色谱柱初始温度为60-80℃，保持4-6min，以20-30℃/min的加热速率升至150-180℃，保持3-5min，以1-5℃/min升至200-225℃，保持40-60min，再以0.5-2℃/min升至200-230℃，保持4-6min；The initial temperature of the chromatographic column is 60-80℃, keep it for 4-6min, raise it to 150-180℃ at a heating rate of 20-30℃/min, keep it for 3-5min, raise it to 200-225℃ at 1-5℃/min , Keep for 40-60min, then increase to 200-230℃ at 0.5-2℃/min, keep it for 4-6min;
    检测器：氢火焰离子化检测器FID；检测器温度200-230℃；尾吹流量2-5mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 200-230℃; makeup flow rate 2-5mL/min.
  2. 如权利要求1所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，色谱柱规格为100m×0.25mm×0.20μm。The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to claim 1, wherein the chromatographic column specification is 100 m×0.25 mm×0.20 μm.
  3. 如权利要求1所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，色谱条件为：The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to claim 1, wherein the chromatographic conditions are:
    进样口温度230℃；进样量1μL；分流比10:1；氮气流速10.6cm/s；恒定线速度模式；Injection port temperature 230℃; injection volume 1μL; split ratio 10:1; nitrogen flow rate 10.6cm/s; constant linear velocity mode;
    色谱柱初始温度为60℃，保持5min，以25℃/min的加热速率升至160℃，保持4min，以2℃/min升至225℃，保持50min，再以1℃/min升至230℃，保持5min；The initial temperature of the chromatographic column is 60℃, keep it for 5min, raise it to 160℃ at a heating rate of 25℃/min, keep it for 4min, raise it to 225℃ at 2℃/min, keep it for 50min, then raise it to 230℃ at 1℃/min , Keep for 5min;
    检测器：氢火焰离子化检测器FID；检测器温度230℃；尾吹流量3mL/min。Detector: hydrogen flame ionization detector FID; detector temperature 230℃; makeup flow rate 3mL/min.
  4. 如权利要求1-3任一项所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，多种脂肪酸包括C3-C5短链脂肪酸、C6-C12中链脂肪酸和C13-C24长链脂肪酸。The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to any one of claims 1 to 3, wherein the multiple fatty acids include C3-C5 short-chain fatty acids, C6-C12 medium-chain fatty acids, and C13-C24 long-chain fatty acids. fatty acid.
  5. 如权利要求4所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，多种脂肪酸包括C3:0，C4:0，C5:0，C6:0，C7:0，C8:0，C9:0，C10:0，C11:0，C12:0，C11:1-10c，C13:0，C12:1-11c，C14:0，C13:1-12c，C14:1-9t，C14:1-9c，C15:0，C15:1-10t，C15:1-10c，C16:0，C15:1-14c，C16:1-9t，C16:1-9c，C17:0，C17:1-10t，C17:1-10c，C18:0，C18:1-6t，C18:1-9t，C18:1-11t，C18:1-6c，C18:1-9c，C18:1-11c，C19:0，C18:2-9t,12t，C19:1-7t，C19:1-10t，C19:1-7c，C19:1-10c，C18:2-9c,12c，C20:0，C20:1-11t，C18:3-6c,9c,12c，C20:1-5c，C20:1-8c，C20:1-11c，C19:2-10c,13c，C18:3-9c,12c,15c，C21:0，C18:2-9c,11t，C18:2-10t,12c，C21:1-12c，C20:2-11c,14c，C22:0，C22:1-13t，C20:3-8c,11c,14c，C22:1-13c，C21:2-12c,15c， C20:3-11c,14c,17c，C23:0，C20:4-5c,8c,11c,14c，C23:1-14c，C22:2-13c,16c，C24:0，C20:5-5c,8c,11c,14c,17c，C24:1-15c，C22:3-13c,16c,19c，C22:4-7c,10c,13c,16c，C22:5-4c,7c,10c,13c,16c，C22:5-7c,10c,13c,16c,19c，C22:6-4c,7c,10c,13c,16c,19c。The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to claim 4, wherein the multiple fatty acids include C3:0, C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, C11:0, C12:0, C11:1-10c, C13:0, C12:1-11c, C14:0, C13:1-12c, C14:1-9t, C14: 1-9c, C15:0, C15:1-10t, C15:1-10c, C16:0, C15:1-14c, C16:1-9t, C16:1-9c, C17:0, C17:1- 10t, C17:1-10c, C18:0, C18:1-6t, C18:1-9t, C18:1-11t, C18:1-6c, C18:1-9c, C18:1-11c, C19: 0, C18:2-9t,12t, C19:1-7t, C19:1-10t, C19:1-7c, C19:1-10c, C18:2-9c,12c, C20:0, C20:1- 11t, C18: 3-6c, 9c, 12c, C20: 1-5c, C20:1-8c, C20:1-11c, C19: 2-10c, 13c, C18: 3-9c, 12c, 15c, C21: 0, C18: 2-9c, 11t, C18: 2-10t, 12c, C21:1-12c, C20: 2-11c, 14c, C22:0, C22:1-13t, C20: 3-8c, 11c, 14c, C22:1-13c, C21: 2-12c, 15c, C20: 3-11c, 14c, 17c, C23:0, C20: 4-5c, 8c, 11c, 14c, C23:1-14c, C22: 2-13c,16c, C24:0, C20: 5-5c, 8c, 11c, 14c, 17c, C24:1-15c, C22: 3-13c, 16c, 19c, C22: 4-7c, 10c, 13c, 16c, C22: 5-4c, 7c, 10c, 13c, 16c, C22: 5-7c, 10c, 13c, 16c, 19c, C22: 6-4c, 7c, 10c, 13c, 16c, 19c.
  6. 如权利要求5所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，检出限在0.000084-0.001276g/100g之间，定量限在0.000289-0.004263g/100g之间。The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to claim 5, characterized in that the detection limit is between 0.000084-0.001276g/100g, and the quantification limit is between 0.000289-0.004263g/100g.
  7. 如权利要求5所述的同时检测分离多种脂肪酸的气相色谱方法，其特征在于，日内精密度的RSD值控制在0.57-9.81％之间，日间精密度的RSD值控制在0.47-9.87％之间。The gas chromatography method for simultaneous detection and separation of multiple fatty acids according to claim 5, characterized in that the RSD value of intraday precision is controlled between 0.57-9.81%, and the RSD value of intraday precision is controlled between 0.47-9.87% between.
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