CN103675136A - Soft-shelled turtle fatty acid analysis method - Google Patents

Abstract

The invention relates to the fields of aquatic organisms and analytical chemistry, in particular to a method for separating and identifying the composition of fatty acids in soft-shelled turtles through (gas chromatography tandem mass spectrometry) GC-MS combined with retention index. The adipose tissue of Chinese soft-shelled turtle was separated, and the fatty acid was extracted and derivatized to prepare fatty acid methyl ester. The separation and identification method is as follows: dissolve the fatty acid methyl ester of soft-shelled turtle in n-hexane, analyze it with GC-MS to achieve the purpose of separation, calculate the retention index according to the retention time of different fatty acid methyl esters, search the mass spectrum library and combine the retention index Qualitatively, the area normalization method is used to calculate the relative content of each fatty acid methyl ester, so as to achieve the purpose of accurately and quickly analyzing the fatty acid composition of soft-shelled turtle. The characteristics of the present invention: making full use of the method of mass spectrum library search combined with retention index qualitative method, can accurately identify the composition of fatty acids in soft-shelled turtle, especially the operation is simple, and the result is more accurate than the method of mass spectrum library search.

Description

A kind of fatty acid analysis method of soft-shelled turtle

Technical field: This invention clearly belongs to the field of analytical chemistry, and in particular relates to a method for analyzing fatty acids of soft-shelled soft-shelled turtles by gas chromatography mass spectrometry combined with retention index method.

Background technique:

Chinese soft-shelled turtle belongs to Reptiles, Turtles, Turtles, and Soft-shelled Turtles. It is widely distributed in my country except Xinjiang, Tibet, and Qinghai. It is a rare aquatic reptile in my country. Soft-shelled soft-shelled turtle meat is rich in nutrition and high in protein, and is favored by consumers at home and abroad. In addition to being nutritious and delicious, Chinese soft-shelled turtle also has high medicinal value. According to the records in "Bielu of Famous Doctors", soft-shelled turtles are sweet and flat in nature and have the effects of nourishing yin and tonifying kidneys and clearing away deficiency and heat. Turtle shell and turtle blood also have medicinal effects of treating yin deficiency and nourishing blood, and are excellent food therapy products. The fat of Chinese soft-shelled turtle is rich in EPA, DHA and other unsaturated fatty acids, which have the functions of anti-cancer, promoting growth and development, and enhancing memory. It has been used clinically to treat diseases such as hypertension and diabetes. Bai Jing et al. used supercritical CO2 extraction technology and low-temperature concentration process to extract DHA and EPA from Chinese soft-shelled turtle oil, and obtained a content of EPA+DHA in Chinese soft-shelled turtle oil of 26.39%. Li-Hsun Chang et al. used the supercritical CO2 method to extract unsaturated fatty acids from fatty acids in soft-shelled turtles. Chen-Huei Huang et al. found that soft-shelled turtles can synthesize fatty acids with less than 18 carbons, but they cannot synthesize fatty acids with high unsaturation. Unsaturated fatty acids account for more than 70% of the total fatty acids in soft-shelled turtles. By analyzing the composition of fatty acids in soft-shelled soft-shelled turtle fat, especially the composition of unsaturated fatty acids, it provides a basis for the rational utilization of soft-shelled soft-shelled turtle fat.

Gas chromatography-mass spectrometry (GC-MS) combined with mass spectral library search method is to match and compare the mass spectral peaks of the components with the mass spectral database spectra, and use the compounds with high matching degrees as the identification results, which have been widely used in the analysis of animal and plant fatty acids . Due to the large variety of fatty acids, there are mainly three types of fatty acids in terms of structure: straight-chain fatty acids, branched-chain fatty acids, and complex fatty acids. Different chain lengths, double bond positions and numbers, and substituent groups of fatty acids will lead to inaccurate separation. , especially the complex structure of long-chain fatty acids often has isomers, and is affected by factors such as resolution and chromatographic conditions, which will cause deviations in the mass spectrum retrieval results of fatty acids, making it difficult to accurately characterize, and even appear mistake. To improve the accuracy of identification, improved methods using a combination of retention index and mass spectral retrieval can be significantly and increasingly used in qualitative and quantitative analysis. The fatty acids were extracted from the fat of soft-shelled turtle by liquid-liquid extraction and derivatized by methyl esterification, and then the qualitative and quantitative analysis of the fatty acids of soft-shelled turtle was carried out through gas chromatography-mass spectrometry (GC-MS) library search combined with retention index to identify Chinese soft-shelled turtle. Fatty acid composition in soft-shelled turtle fat.

Invention content:

The gas chromatography mass spectrometry combined with the retention index analysis method of the present invention analyzes the Chinese soft-shelled turtle fat, and obtains its accurate fatty acid total ion flow chromatogram, corresponding mass spectrogram and retention index. By comparing the mass spectral library structure of fatty acid methyl esters and the corresponding retention index, the fatty acid of soft-shelled turtle can be qualitatively analyzed.

The method of the present invention comprises the following steps: a method for analyzing Chinese soft-shelled turtle fatty acids comprises the following steps:

(1) After slaughtering soft-shelled turtles, take 10 g of adipose tissue and store at -18°C;

(2) Weigh 1 g of soft-shelled turtle fat, crush it, put it into a 10 mL glass test tube, add 10 mL of extraction solution, mix well, seal and extract for 24 hours; after the extraction is completed, filter with fat-free filter paper, and then add Wash the residue with 5 mL of extract, and save the filtrate twice; the extract is chloroform:methanol with a volume ratio of 2:1;

(3) Take 5 mL of fat extract, add 3 mL of 10% KOH methanol solution, mix well, and place in 80 °C water bath for 1 h; after cooling at room temperature, add 3 mL of 14% BF ₃ methanol solution (v/v) After mixing, react in a water bath at 50 °C for 10 min; after cooling, add 3 mL of n-hexane, shake and vortex, and let it stand at room temperature overnight; take the upper n-hexane layer to the injection vial, freeze at -18 °C, and wait for GC-MS analysis ;

(4) Analyze the fatty acid methyl esters in the sample vial by gas chromatography-mass spectrometry, chromatographic conditions: Agilent DB-5MS quartz capillary column 30.0 m×0.25 mm×0.25 μm; carrier gas is high-purity helium; flow rate 1 mL/min; temperature program: keep at 60°C for 1 min, rise to 160°C at 10°C/min and hold for 4 minutes, rise to 260°C at 5°C/min and hold for 5 minutes, rise to 280°C at 5°C/min and hold Hold for 1 min; split ratio 20:1; injection volume 1 μL; mass spectrometry conditions: EI ion source; electron energy 70 eV; ion source temperature 220 ℃; inlet temperature 240 ℃, mass scanning range: m/z 40~ 600; solvent removal time 2.5 min.

After performing the gas chromatography mass spectrometry analysis, also include the step of calculating the retention index of the analyzed fatty acid methyl esters:

(1) Perform gas chromatography-mass spectrometry analysis on the n-alkane mixed standard (C8-C40) under the conditions of fatty acid methyl ester analysis;

(2) Record the retention time of each n-alkane standard; use the linear temperature rise formula of the retention index: Calculate the RI value of each component, where: t _x , t _n and t _(n+1) are the outflow of the analyzed component and the n-paraffin mixed standard with carbon number between n and n+1 respectively Peak retention time (min);

(3) According to http://webbook.nist.gov/chemistry/ Search literature to obtain the retention index RI of each fatty acid methyl ester in the corresponding chromatographic column, combined with mass spectral library search, and use the area normalization method to calculate the relative percentage of each fatty acid content.

The method of the present invention can not only break the qualitative limitations of conventional gas chromatography mass spectrometry, reduce the errors caused by the incompleteness of the mass spectrum library or the similarity close to the fatty acid analysis of Chinese soft-shelled turtle, but also provide a convenient and accurate qualitative method for the analysis and identification of fatty acids in other organisms , and it can also be used to identify other substances that are difficult to be accurately characterized by gas chromatography mass spectrometry.

Description of drawings

Figure 1 The total ion chromatogram of fatty acid methyl ester of soft-shelled turtle

Detailed ways:

The following provides a specific embodiment of a method for analyzing fatty acids of soft-shelled turtle of the present invention.

Example 1

The method of the present invention is used to analyze the fatty acid composition of the soft-shelled soft-shelled turtle in Poyang Lake. A healthy soft-shelled soft-shelled turtle in Poyang Lake with a weight of about 500g is randomly selected, and the fat tissue around the limbs of the soft-shelled turtle is frozen for future use after slaughter.

Fat extraction: Weigh 1 g of soft-shelled turtle fat, crush it, put it into a 10 mL glass test tube, add 10 mL of extraction solution (chloroform:methanol=2:1, v/v), mix well, seal and extract for 24 h. After the extraction is completed, filter with fat-free filter paper, then add 5 mL of extract to wash the residue, and store the two filtrates together.

Fatty acid methylation: Take 5 mL of the above fat extract, add 3 mL of 10% KOH methanol solution (v/v), mix well, and place in a water bath at 80 °C for 1 h. After cooling at room temperature, add 3 mL of 14% BF3 methanol solution (v/v), mix well, and react in a water bath at 50 °C for 10 min. After cooling, add 3 mL of n-hexane, shake and vortex, and let stand overnight at room temperature. Take the upper n-hexane layer to the injection vial, freeze at -18°C, and analyze by GC-MS.

The gas chromatography mass spectrometry instrument conditions are as follows: GC/MS is QP2010 type, is the product of Shimadzu Corporation

Chromatographic conditions: Agilent DB-5MS quartz capillary column (30.0 m × 0.25 mm × 0.25 μm); carrier gas is high-purity helium; flow rate 1 mL/min; temperature program: 60 ℃ for 1 min, then 10 ℃ /min Rise to 160°C and keep for 4 minutes, rise to 260°C at 5°C/min and keep for 5 minutes, rise to 280°C at 5°C/min and keep for 1 min; split ratio 20:1; injection volume 1 μL.

Mass spectrometry conditions: EI ion source; electron energy 70 eV; ion source temperature 220 °C; inlet temperature 240 °C; mass scanning range: m/z 40-600; solvent removal time 2.5 min.

The linear temperature rise formula using the retention index: Calculate the RI value of each fatty acid methyl ester of soft-shelled turtle, where: t _x , t _n and t _(n+1) [t _n <t _x <t _(n+1) ] are the analyzed components and the number of carbon atoms in the Retention time of the eluting peak of n-paraffin mixed standard between n and n+1 (min)

Perform a mass spectral library search on the mass spectra of each peak in the total ion chromatogram, select the possible substance with the highest similarity, and search the literature to obtain its retention index RI value. Compared with the calculated results, the highest similarity in mass spectrum and retention index The chemical structure is the best identification result, and the relative percentage content of each fatty acid is obtained by using the area normalization method.

Finally, the GC-MS analysis of the fatty acid of soft-shelled soft-shelled turtle in Poyang Lake is shown in Figure 1. Combined with the qualitative analysis of the retention index, it was found that the mass spectrometry analysis results of peak 7 were searched by the Nist library, and the compound with a high matching degree was Hexadecanoic acid, 14-methyl-, methyl ester (similarity 93%) and Heptadecanoic acid, methyl ester (similarity 92%), it is difficult to determine the correct structure of the compound only based on the matching degree of mass spectrum. According to http://webbook.nist.gov/chemistry/ Search for the retention index RI of the literature (DB-5 column), the former retention index RI literature value is 1996, and the latter retention index RI literature value is 2030, by calculating the chromatographic peak The retention index RI value is 2033, so it can be determined that the structure of this chromatographic peak is the latter Heptadecanoic acid, methyl ester. Therefore, the method of the present invention can meet the requirements of fatty acid analysis of soft-shelled turtle.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be considered Within the protection scope 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.