CN103675136A - Soft-shelled turtle fatty acid analysis method - Google Patents
Soft-shelled turtle fatty acid analysis method Download PDFInfo
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- CN103675136A CN103675136A CN201310668383.5A CN201310668383A CN103675136A CN 103675136 A CN103675136 A CN 103675136A CN 201310668383 A CN201310668383 A CN 201310668383A CN 103675136 A CN103675136 A CN 103675136A
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Abstract
The invention relates to the field of aquatic organisms and analysis chemistry, in particular to a method for separating and authenticating the components of fatty acid in the body of a soft-shelled turtle by combining GC-MS (gas chromatography-mass spectrometry) with a retention index. Fatty acid methyl esters are prepared by separating the adipose tissue of the soft-shelled turtle, and extracting and deriving the fatty acid from the separated adipose tissue. The method for separating and authenticating the fatty acid comprises the following steps: dissolving fatty acid methyl esters of the soft-shelled turtle in hexane and analyzing by using GC-MS to fulfill the aim of separating; calculating the retention index of fatty acid methyl esters according to the retention time of different fatty acid methyl esters; searching a mass spectrometry library, and meanwhile, carrying out nature determination by combining the retention index, and calculating the relative content of each fatty acid methyl ester by an area normalization method so as to fulfill the aim of analyzing the components of fatty acid of the soft-shell turtle accurately and quickly. The method has the advantages that the components of fatty acid in the soft-shell turtle can be authenticated accurately by fully utilizing the method of searching the mass spectrometry library and nature determination by combining the retention index; particularly, the operation is simple and convenient, and the method has the advantage that the result is accurate compared with the method of searching the mass spectrometry library.
Description
Technical field: this belongs to analytical chemistry field clearly, particularly adopts gaschromatographic mass spectrometry in conjunction with retention index method, to carry out the method for Shelled Turtle Trionyx Sinensis fatty acid analysis.
Background technology:
Shelled Turtle Trionyx Sinensis is subordinate to Reptilia, Chelonia, Trionychidae, soft-shelled turtle genus, is distributed widely in China except Xinjiang, Tibet, and other provinces beyond Qinghai, are the famous and precious aquatic Reptilia of China.Shelled Turtle Trionyx Sinensis fine and tender taste, nutritious, high protein, enjoy domestic and international consumer's favor.Shelled Turtle Trionyx Sinensis, except nutrition delicious food, also has higher medical value.According to < < Mingyi Bielu > >, record, soft-shelled turtle nature and flavor are sweet, flat, there are enriching yin and nourishing kidney, the effect of clearing away deficiency-heat, the medicinal efficacy that turtle shell, soft-shelled turtle blood have more the treatment deficiency of Yin and enriches blood, is dietotherapy good merchantable brand.In Shelled Turtle Trionyx Sinensis fat, be rich in the unsaturated fatty acids such as EPA, DHA, there is the effects such as anticancer, enhancing development, enhancing memory, be used to clinically treat the diseases such as hypertension, diabetes.Bai Jing etc. adopt CO_2 supercritical technology, utilize low-temperature concentration process from Chinese turtle oil, to extract DHA and EPA, and the content that obtains EPA+DHA in Chinese turtle oil is 26.39%.The use supercritical CO 2 methods such as Li-Hsun Chang are extracted unsaturated fatty acid from Shelled Turtle Trionyx Sinensis fatty acid, the discovery Shelled Turtle Trionyx Sinensis self such as Chen-Huei Huang can synthesize 18 fatty acid below carbon, but the fatty acid that can not synthesize high degree of unsaturation finds that in Shelled Turtle Trionyx Sinensis body, unsaturated fatty acid accounts for the more than 70% of total fatty acids simultaneously.Composition by analyzing fatty acid in Shelled Turtle Trionyx Sinensis fat is the composition of unsaturated fatty acid especially, for the reasonable utilization of Shelled Turtle Trionyx Sinensis fat provides foundation.
Gaschromatographic mass spectrometry (GC-MS) is that the mass spectra peak of component and mass spectrometric data storehouse collection of illustrative plates are carried out to matching ratio pair in conjunction with mass spectrum library searching method, and the high compound of the matching degree of usining, as qualification result, has been widely used in the analysis of animal, plant fatty acid.Because fatty acid kind is more; from structure, classify and mainly contain the fatty acid three types of straight chain fatty acid, branched chain fatty acid and Complex Situation; the chain length of fatty acid, position of double bond and quantity and substituted radical difference all can cause separated inaccurate; especially long-chain fatty acid complicated structure often has isomers existence; and the impact that is subject to the factors such as degree of separation and chromatographic condition variation; can make the mass spectrogram result for retrieval of fatty acid occur deviation; make to be difficult to accurately qualitative, even occur mistake.For improving the accuracy of identifying, the modification method that adopts retention index and mass spectrum retrieval to combine can be applied in qualitative and quantitative analysis significantly more and more.Adopt liquid-liquid extraction method from Shelled Turtle Trionyx Sinensis fat, to extract fatty acid and carry out esterification and derive, then pass through gaschromatographic mass spectrometry (GC-MS) library searching in conjunction with retention index, the fatty acid of Shelled Turtle Trionyx Sinensis is carried out to qualitative and quantitative analysis, to differentiate that the fatty acid in Shelled Turtle Trionyx Sinensis fat forms.
Summary of the invention:
Gaschromatographic mass spectrometry of the present invention, in conjunction with retention index analytical Shelled Turtle Trionyx Sinensis fat, has obtained its accurately fatty acid total ions chromatogram, corresponding mass spectrogram and retention index.By comparing mass spectrum spectrum library structure and the corresponding retention index of fatty acid methyl ester, can carry out qualitative analysis to Shelled Turtle Trionyx Sinensis fatty acid.
Method of the present invention comprises the following steps: a kind of Shelled Turtle Trionyx Sinensis fatty acid analysis method, comprises the following steps:
(1) Shelled Turtle Trionyx Sinensis is got adipose tissue 10 g after slaughtering ,-18 ℃ of preservations;
(2) take soft-shelled turtle fat 1 g, after pulverizing, put into 10 mL glass test tubees, add 10 mL extracts, mix rear sealing lixiviate 24 h; After extraction completes, with not fatty Filter paper filtering, then add 5 mL extracts to rinse residue, 2 filtrate merging preserve; Described extract is that volume ratio is the chloroform of 2:1: methyl alcohol;
(3) get fat-extraction liquid 5 mL, add 3 mL 10% KOH methanol solutions, after mixing, be placed in 80 ℃ of water-bath 1 h; After room temperature is cooling, add 3 mL 14% BF
3after methanol solution (v/v) mixes, in 50 ℃ of water-bath 10 min; After cooling, add 3 mL normal hexanes, concussion vortex, room temperature standing over night; Get upper strata normal hexane layer to sample introduction bottle ,-18 ℃ freezing, treats that GC-MS analyzes;
(4) fatty acid methyl ester in sample injection bottle is carried out to gaschromatographic mass spectrometric analysis, chromatographic condition: chromatographic column is Agilent DB-5MS quartz capillary column 30.0 mm * 0.25, m * 0.25 μ m; Carrier gas is high-purity helium; Flow velocity 1 mL/min; Temperature programme: 60 ℃ keep 1 min, rises to 160 ℃ and keep 4 min with 10 ℃/min, with 5 ℃/min, rises to 260 ℃ and keep 5min, with 5 ℃/min, rises to 280 ℃ and keep 1 min; Split ratio 20:1; Sample size 1 μ L; Mass spectrum condition: EI ion gun; Electron energy 70 eV; 220 ℃ of ion source temperatures; 240 ℃ of injector temperatures, mass scanning scope: m/z 40 ~ 600; Solvent mute times 2.5 min.
After carrying out gaschromatographic mass spectrometric analysis, also comprise that the fatty acid methyl ester to analyzing calculates the step that retention index is calculated:
(1) n-alkane hybrid standard product (C8-C40) are carried out to gaschromatographic mass spectrometric analysis with the condition of fatty acid methyl ester analysis;
(2) record the retention time of each n-alkane standard items; Adopt the linear temperature increase formula of retention index:
calculate the RI value of each component, wherein: t
x, t
nand t
(n+1)be respectively the eluting peak retention time (min) of the mixed mark of analyzed component and the carbon number n-alkane between n and n+1 standard items;
(3) according to http://webbook.nist.gov/chemistry/ search document, obtain the retention index RI of corresponding each fatty acid methyl ester of chromatographic column, connexus library searching uses area normalization method to calculate the relative percentage composition of each fatty acid simultaneously.
Method of the present invention not only can be limited to by off the beaten track gaschromatographic mass spectrometry qualitatively, reduce because mass spectral database is incomplete or similarity approaches the error that Shelled Turtle Trionyx Sinensis fatty acid analysis is brought, for the fatty acid analysis of other biological, identify simultaneously provide convenience, quilitative method accurately, but also can apply the method, identify that other gaschromatographic mass spectrometries are difficult to accurately material qualitatively.
Accompanying drawing explanation
Fig. 1 Shelled Turtle Trionyx Sinensis fatty acid methyl ester total ion current figure
Embodiment:
The embodiment of a kind of Shelled Turtle Trionyx Sinensis fatty acid analysis of the present invention method is below provided.
Embodiment 1
Adopt the inventive method to analyze the composition of Poyang Lake Shelled Turtle Trionyx Sinensis fatty acid, randomly draw one of healthy Poyang Lake Shelled Turtle Trionyx Sinensis, the about 500g of body weight, around adipose tissue is freezing standby after slaughtering, to get its four limbs.
Fat-extraction: take Shelled Turtle Trionyx Sinensis fat 1 g, put into 10 mL glass test tubees after pulverizing, add 10 mL extracts (chloroform: methyl alcohol=2:1, v/v), mix rear sealing lixiviate 24 h.After extraction completes, with not fatty Filter paper filtering, then add 5 mL extracts to rinse residue, 2 filtrate merging preserve.
Methyl esterification of fatty acid: get above-mentioned fat-extraction liquid 5 mL, add 3 mL 10% KOH methanol solutions (v/v), after mixing, be placed in 80 ℃ of water-bath 1 h.After room temperature is cooling, add 3 mL 14% BF3 methanol solutions (v/v), after mixing, in 50 ℃ of water-bath 10 min.After cooling, add 3 mL normal hexanes, concussion vortex, room temperature standing over night.Get upper strata normal hexane layer to sample introduction bottle ,-18 ℃ freezing, and GC-MS analyzes.
Gas chromatography mass spectrometer device condition is as follows: GC/MS is QP2010 type, is the product of Shimadzu company
Chromatographic condition: chromatographic column is Agilent DB-5MS quartz capillary column (30.0 mm * 0.25, m * 0.25 μ m); Carrier gas is high-purity helium; Flow velocity 1 mL/min; Temperature programme: 60 ℃ keep 1 min, rises to 160 ℃ and keep 4 min with 10 ℃/min, with 5 ℃/min, rises to 260 ℃ and keep 5min, with 5 ℃/min, rises to 280 ℃ and keep 1 min; Split ratio 20:1; Sample size 1 μ L.
Mass spectrum condition: EI ion gun; Electron energy 70 eV; 220 ℃ of ion source temperatures; 240 ℃ of injector temperatures, mass scanning scope: m/z 40 ~ 600; Solvent mute times 2.5 min.
Adopt the linear temperature increase formula of retention index:
calculate the RI value of each fatty acid methyl ester of Shelled Turtle Trionyx Sinensis, wherein: t
x, t
nand t
(n+1)[t
n<t
x<t
(n+1)] be respectively the eluting peak retention time (min) of the mixed mark of analyzed component and the n-alkane of carbon number between n and n+1 standard items
Mass spectrogram to each peak in total ion current figure carries out mass spectral database retrieval, choose the possible material that similarity is the highest, searching document obtains its retention index RI value, compare with result of calculation, the highest chemical constitution of mass spectrum similarity and the retention index degree of approach of take is best qualification result, uses area normalization method to obtain the relative percentage composition of each fatty acid simultaneously.
Finally Poyang Lake Shelled Turtle Trionyx Sinensis fatty acid is carried out to GC-MS analysis and see Fig. 1, mass spectrometry results in conjunction with the qualitative discovery of retention index peak 7 shows through Nist library searching, the compound that matching degree is higher is Hexadecanoic acid, 14-methyl-, methyl ester(similarity 93%) and Heptadecanoic acid, methyl ester(similarity 92%), only according to mass spectrum matching degree, be difficult to the correct structure of deterministic compound.According to the retention index RI of http://webbook.nist.gov/chemistry/ search document (DB-5 post), the former retention index RI literature value is 1996, the latter's retention index RI literature value is 2030, by calculating the retention index RI value of this chromatographic peak, be 2033, therefore can determine that the structure of this chromatographic peak is the latter Heptadecanoic acid, methyl ester.So the inventive method can meet the requirement of Shelled Turtle Trionyx Sinensis fatty acid analysis.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be considered within the scope of protection of the present invention.
Claims (2)
1. a Shelled Turtle Trionyx Sinensis fatty acid analysis method, comprises the following steps:
(1) Shelled Turtle Trionyx Sinensis is got adipose tissue 10 g after slaughtering ,-18 ℃ of preservations;
(2) take soft-shelled turtle fat 1 g, after pulverizing, put into 10 mL glass test tubees, add 10 mL extracts, mix rear sealing lixiviate 24 h;
After extraction completes, with not fatty Filter paper filtering, then add 5 mL extracts to rinse residue, 2 filtrate merging preserve; Described extract is that volume ratio is the chloroform of 2:1: methyl alcohol;
(3) get fat-extraction liquid 5 mL, add 3 mL 10% KOH methanol solutions, after mixing, be placed in 80 ℃ of water-bath 1 h; After room temperature is cooling, add 3 mL 14% BF
3after methanol solution (v/v) mixes, in 50 ℃ of water-bath 10 min; After cooling, add 3 mL normal hexanes, concussion vortex, room temperature standing over night; Get upper strata normal hexane layer to sample introduction bottle ,-18 ℃ freezing, treats that GC-MS analyzes;
(4) fatty acid methyl ester in sample injection bottle is carried out to gaschromatographic mass spectrometric analysis, chromatographic condition: chromatographic column is Agilent DB-5MS quartz capillary column 30.0 mm * 0.25, m * 0.25 μ m; Carrier gas is high-purity helium; Flow velocity 1 mL/min; Temperature programme: 60 ℃ keep 1 min, rises to 160 ℃ and keep 4 min with 10 ℃/min, with 5 ℃/min, rises to 260 ℃ and keep 5min, with 5 ℃/min, rises to 280 ℃ and keep 1 min; Split ratio 20:1; Sample size 1 μ L; Mass spectrum condition: EI ion gun; Electron energy 70 eV; 220 ℃ of ion source temperatures; 240 ℃ of injector temperatures, mass scanning scope: m/z 40 ~ 600; Solvent mute times 2.5 min.
2. the method for claim 1, is characterized in that: after carrying out gaschromatographic mass spectrometric analysis, also comprise that the fatty acid methyl ester to analyzing calculates the step that retention index is calculated:
(1) n-alkane hybrid standard product C8-C40 is carried out to gaschromatographic mass spectrometric analysis with the condition of fatty acid methyl ester analysis;
(2) record the retention time of each n-alkane standard items; Adopt the linear temperature increase formula of retention index:
calculate the RI value of each component, wherein: t
x, t
nand t
(n+1)be respectively the eluting peak retention time (min) of the mixed mark of analyzed component and the carbon number n-alkane between n and n+1 standard items;
(3) according to http://webbook.nist.gov/chemistry/ search document, obtain the retention index RI of corresponding each fatty acid methyl ester of chromatographic column, connexus library searching uses area normalization method to calculate the relative percentage composition of each fatty acid simultaneously.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104034837A (en) * | 2014-06-16 | 2014-09-10 | 南宁学院 | Method for determining fatty-acid components of tissues of fish body |
| CN104165955A (en) * | 2014-06-27 | 2014-11-26 | 浙江大学 | An establishing method of a breast milk fatty acid spectrum database |
| CN104807936A (en) * | 2015-05-05 | 2015-07-29 | 西北农林科技大学 | Method for fast measuring goat mammary epithelial cell fatty acid ingredients |
| CN105572286A (en) * | 2016-01-22 | 2016-05-11 | 浙江大学 | Determination method for turtle fishy smell matter |
| CN106390134A (en) * | 2016-10-24 | 2017-02-15 | 中国水产科学研究院长江水产研究所 | Preparation method and application of biological medicinal bait for trionyx sinensis |
| CN107449853A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of to differentiate tai-chu race and the method for east Guangdong large yellow croaker |
| CN109828047A (en) * | 2019-02-25 | 2019-05-31 | 中国农业科学院油料作物研究所 | The kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease |
| CN110879263A (en) * | 2019-12-06 | 2020-03-13 | 中国水产科学研究院淡水渔业研究中心 | Analysis method for identifying different geographical groups of eriocheir sinensis |
| CN115453030A (en) * | 2022-09-28 | 2022-12-09 | 博莱克科技（武汉）有限公司 | Method for improving gas chromatography detection efficiency |
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2013
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| 彭德姣等: "斑点叉尾鮰鱼内脏油脂成分的GC-MS检测", 《安全与检测》 * |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104034837A (en) * | 2014-06-16 | 2014-09-10 | 南宁学院 | Method for determining fatty-acid components of tissues of fish body |
| CN104165955A (en) * | 2014-06-27 | 2014-11-26 | 浙江大学 | An establishing method of a breast milk fatty acid spectrum database |
| CN104165955B (en) * | 2014-06-27 | 2016-03-09 | 浙江大学 | The method for building up in breast milk fatty acids modal data storehouse |
| CN104807936A (en) * | 2015-05-05 | 2015-07-29 | 西北农林科技大学 | Method for fast measuring goat mammary epithelial cell fatty acid ingredients |
| CN105572286A (en) * | 2016-01-22 | 2016-05-11 | 浙江大学 | Determination method for turtle fishy smell matter |
| CN106390134A (en) * | 2016-10-24 | 2017-02-15 | 中国水产科学研究院长江水产研究所 | Preparation method and application of biological medicinal bait for trionyx sinensis |
| CN107449853A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of to differentiate tai-chu race and the method for east Guangdong large yellow croaker |
| CN109828047A (en) * | 2019-02-25 | 2019-05-31 | 中国农业科学院油料作物研究所 | The kit of the accurate Structural Identification of unsaturated fatty acid in a kind of grease |
| CN109828047B (en) * | 2019-02-25 | 2021-08-31 | 中国农业科学院油料作物研究所 | Kit for identifying accurate structure of unsaturated fatty acid in grease |
| CN110879263A (en) * | 2019-12-06 | 2020-03-13 | 中国水产科学研究院淡水渔业研究中心 | Analysis method for identifying different geographical groups of eriocheir sinensis |
| CN115453030A (en) * | 2022-09-28 | 2022-12-09 | 博莱克科技（武汉）有限公司 | Method for improving gas chromatography detection efficiency |
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Application publication date: 20140326 |