Method for quantifying sterculia nobilis acid with high sensitivity and high precision

Abstract

The invention provides a liquid chromatography-mass spectrometry analysis method for high-sensitivity and high-precision quantitative analysis of free sterculia nobilis acid and ester-bound sterculia nobilis acid based on a 2-pyridine formylhydrazine derivative reaction, and belongs to the technical field of detection. After derivation of the 2-pyridine carbohydrazide and the sterculia nobilis acid is adopted, the signal response of the sterculia nobilis acid in the mass spectrum is improved; the reaction conditions of the 2-pyridine carbohydrazide and sterculia nobilis acid are determined, so that the derivative reaction has the advantages of simplicity in operation, mild reaction conditions, high reaction efficiency and the like. The invention further provides an optimal extraction mode, an optimal liquid chromatographic column type and optimal mass spectrum parameters of free sterculia nobilis acid and ester combined sterculia nobilis acid, so that the detection limit of sterculia nobilis acid is as low as 5 mu g / kg, the quantification limit is as low as 12.5 mu g / kg, the detection limit is more than 200 times that of a reported detection method, and high-sensitivity detection of sterculia nobilis acid is realized. The method disclosed by the invention provides a method support for quantitative analysis and function evaluation of free and ester-bound sterculia nobilis acid in feed and food.

Description

Technical Field

[0001] The present disclosure relates to the field of detection technologies, and particularly to a method for highly sensitive and high-precision quantitative determination of sterculic acid. Background Art

[0002] Sterculic acid is an anti-nutritional factor in cottonseed meal, and its chemical structure contains an unsaturated three-membered ring. Sterculic acid mainly exists in the seed oils of Malvaceae plants such as cottonseed oil and sterculia oil, etc. It can inhibit the activity of stearoyl-CoA desaturase 1 (SCD1), thereby affecting the formation of unsaturated double bonds at the delta-9 position of the fatty acid carbon chain and changing the degree of unsaturation of fatty acids in animals. Long-term consumption of sterculic acid will lead to disorders of animal lipid metabolism and the occurrence of lipid metabolism diseases. When using feed containing cotton meal to feed laying hens, it will reduce the egg production rate and growth performance, and even produce inferior eggs such as "pink eggs" and "rubber eggs", and the main inducement is sterculic acid in cotton meal. Therefore, accurately evaluating the content of sterculic acid in foods and feeds is a necessary measure to achieve precise nutrition and reduce the negative effects of sterculic acid.

[0003] The cyclopropene structure of sterculic acid can react with carbon disulfide to form a carbon-sulfur double bond, which polymerizes to form a red product. However, this reaction can only determine the presence of the cyclopropene structure and cannot achieve quantitative analysis of sterculic acid in feeds. After saponifying and methyl-esterifying ester-bound sterculic acid, gas chromatography and gas chromatography-mass spectrometry can be used to achieve quantitative analysis of sterculic acid in cottonseed oil. However, due to the similar retention times of sterculic acid and linoleic acid, the accurate integration of the quantitative determination of low-content sterculic acid is affected by high-content linoleic acid, resulting in deviation of the quantitative results of sterculic acid. After reacting 2-bromoacetophenone with sterculic acid and combining with reversed-phase high-performance liquid chromatography technology, the separation of sterculic acid and linoleic acid in cottonseed oil can be achieved, and quantitative analysis can be carried out by combining with the external standard method, improving the quantitative accuracy of sterculic acid. However, the sensitivity of the ultraviolet detector is low, and it can only determine the content of ester-bound sterculic acid in cottonseed oil and cottonseeds, and it is difficult to determine the sterculic acid in defatted cottonseed meal and feeds. Therefore, it is necessary to develop a method for highly sensitive and high-precision determination of the content of free and ester-bound sterculic acid to solve the above technical problems. Summary of the Invention

[0004] In view of the problems such as low sensitivity and low precision in the existing methods for detecting sterculic acid in cottonseed meal and feeds, the object of the present invention is to provide a highly sensitive and high-precision detection method for quantitative determination of sterculic acid, including the following steps:

[0005] 1) Extraction of sterculic acid: Directly extract free sterculic acid using methanol or saponify ester-bound sterculic acid into free sterculic acid using an alkaline methanol solution;

[0006] 2) Sterculic acid derivation: Use 2-pyridinecarboxylic hydrazide to derivatize sterculic acid in the sample to be tested, improving the mass spectrometry signal response. The reaction formula is as follows Figure 1 ;

[0007] 3) Sterculic acid LC-MS / MS detection:

[0008] Liquid phase conditions: The mobile phase is 0.1% formic acid in water and 0.1% formic acid in acetonitrile; the chromatographic column is a C8 chromatographic column (2.1×100 mm, 1.8 μm); the mobile phase gradient is as follows:

[0009] Time (min) Flow rate (mL / min) A（%） B（%） 0 0.4 50 50 1.00 0.4 50 50 6.00 0.4 0 100 7.00 0.4 0 100 7.10 0.4 50 50 9.00 0.4 50 50

[0010] Mass spectrometry conditions: The ionization mode is positive ion electrospray ionization (ESI + ); the mass spectrometry scanning mode is multiple reaction monitoring (MRM); the parent ion is 414, and the daughter ions are 138 and 120.

[0011] One of the objectives of the present invention is to react 2-pyridinecarboxylic hydrazide with sterculic acid to improve the sensitivity of quantitative analysis of sterculic acid.

[0012] Another objective of the present invention is to improve the reaction efficiency of 2-pyridinecarboxylic hydrazide and sterculic acid by optimizing the reaction conditions.

[0013] Another objective of the present invention is to improve the extraction efficiency of two types of sterculic acid by optimizing the extraction methods of free sterculic acid and ester-bound sterculic acid.

[0014] Another objective of the present invention is to establish a quantitative method for high-sensitivity and high-accuracy liquid chromatography-tandem mass spectrometry of sterculic acid by optimizing the liquid chromatography and mass spectrometry conditions.

[0015] In a specific embodiment, free sterculic acid is extracted using an 80%-100% methanol solution, and the ratio of the sample to the methanol solution is 1:3 - 1:5.

[0016] In a specific embodiment, the sample containing ester-bound sterculic acid is first saponified with an alkali solution. The ratio of the sample to the alkali solution is 1:3 - 1:5; the alkali solution composition is 2%-5% NaOH methanol solution; the hydrolysis time is 4 - 14 hours; the hydrolysis temperature is 30 - 80 °C.

[0017] In a specific embodiment, for the extracted sterculic acid, derivatization is carried out using 2-pyridinecarboxylic hydrazide. The derivatization solution is: 250 - 1000 mM EDC, 0 - 60 mM HOAt, 4 - 80 mM 2-pyridinecarboxylic hydrazide, the reaction temperature is 4 - 50 °C, and the reaction time is 15 - 90 min.

[0018] In a specific embodiment, where liquid chromatography-mass spectrometry is used for quantification, ionization is performed using an electrospray ionization source in the positive ion mode, data is acquired in the multiple ion scanning mode, the parent ion is 414, and the daughter ions are 138 and 120; the collision energy for generating daughter ion 138 is 20 - 30 eV, the collision energy for generating daughter ion 120 is 30 - 50 eV, and the declustering voltage is 20 - 50 V.

[0019] To achieve the above objectives, we adopt the following operations to determine the optimal technical solution:

[0020] (1) Since sterculic acid contains a carboxyl group with strong electronegativity, sterculic acid can only be analyzed by mass spectrometry in the negative ion mode, resulting in low detection sensitivity. To solve this technical bottleneck, the present invention selects 2-pyridinecarboxylic hydrazide to react with sterculic acid. Since 2-pyridinecarboxylic hydrazide carries three nitrogen atoms that are easily positively charged, mass spectrometry detection of sterculic acid in the positive ion mode is achieved. Since the hydrazide structure of 2-pyridinecarboxylic hydrazide is connected to the carboxyl group of sterculic acid, the electronegativity of sterculic acid is further eliminated, ultimately achieving an improvement in the detection sensitivity of sterculic acid ( Figure 1 ).

[0021] (2) To determine the derivatization reaction conditions of 2-pyridinecarboxylic hydrazide and sterculic acid, by comparing the concentrations of the carboxyl activator EDC, the catalyst HOAt, and 2-pyridinecarboxylic hydrazide, the derivatization reaction time, and the reaction temperature, it is determined that when using 250 - 1000 mmol / L EDC ( Figure 2 ), 0 - 60 mmol / L HOAt ( Figure 3 ), 4 - 80 mmol / L 2-pyridinecarboxylic hydrazide ( Figure 4 ), the reaction temperature of 4 - 50 °C ( Figure 5 ), and the reaction time of 15 - 90 min ( Figure 6 ), sterculic acid can react efficiently with 2-pyridinecarboxylic hydrazide.

[0022] (3) To improve the extraction efficiency of free sterculic acid and ester-bound sterculic acid, the extraction of free sterculic acid with methanol, acetonitrile, or a mixed solution of methanol and dichloromethane was compared respectively, and it was determined that methanol has the highest efficiency in extracting free sterculic acid ( Figure 7 ). For ester-bound sterculic acid, three extraction methods were compared to optimize the extraction effect of ester-bound sterculic acid. The results confirmed that Method 1, which first uses an extraction system of dichloromethane - methanol - water to extract ester-bound sterculic acid and then hydrolyzes it, has poor results. Method 2, which first hydrolyzes the bound sterculic acid into free sterculic acid and then performs extraction and purification, can improve the extraction effect, but the recovery rate still does not meet the methodological requirements. Method 3, which directly performs derivatization reaction and detection after hydrolyzing ester-bound sterculic acid, can obtain higher extraction efficiency and meet the methodological technical requirements ( Figure 8 )

[0023] (4) To further improve the sensitivity and accuracy of the quantitative analysis of sterculic acid, the peak height and signal-to-noise ratio of sterculic acid in three types of chromatographic columns were compared respectively, and it was confirmed that both the peak height and signal-to-noise ratio of sterculic acid in the C8 chromatographic column were better than those in the C 18 chromatographic column and the T3 chromatographic column. Through daughter ion scanning, the parent ion of sterculic acid was determined to be 414, and the characteristic ions were 138 and 120 respectively. By optimizing the declustering voltage, it was determined that the signal of sterculic acid was stronger when the declustering voltage was 20 - 50 V ( Figure 9 ). By optimizing the collision energy, it was determined that when the collision energy was 20 - 30 eV, the response of daughter ion 138 was stronger; when the collision energy was 30 - 50 eV, the response of daughter ion 120 was stronger ( Figure 10 ).

[0024] Technical effects

[0025] 1. The derivatization reaction of sterculic acid provided by the present invention with 2-pyridinecarboxylic hydrazide is simple to operate and has mild reaction conditions. The derivatization reaction can be completed within 1 hour at room temperature. The reaction product of sterculic acid and 2-pyridinecarboxylic hydrazide has a strong response in the mass spectrometry signal, and the sensitivity of sterculic acid after derivatization is increased by nearly 20 times.

[0026] 2. By comparing different solvents and procedures, the best extraction method for free sterculic acid and ester-bound sterculic acid was determined, which efficiently enables the accurate detection of the contents of free sterculic acid and ester-bound sterculic acid in the sample, and creatively realizes the high-efficiency detection of two types of sterculic acid substances in the same sample.

[0027] 3. By optimizing the liquid chromatography and mass spectrometry conditions, the detection limit of the sample was effectively reduced. The detection limits of free sterculic acid and ester-bound sterculic acid can reach 5 μg / kg, and the quantitative limits can both reach 12.5 μg / kg, which is more than 200 times the sensitivity of the reported sterculic acid detection methods.

[0028] 4. The method of the present invention has high detection stability. The within-batch RSD range of free sterculic acid is 1.01% - 2.21%, and the between-batch RSD range is 1.61% - 2.38%; the within-batch RSD range of ester-bound sterculic acid is 0.79% - 2.01%, and the between-batch RSD range is 3.40% - 6.22%.

[0029] In summary, the quantitative method of sterculic acid provided by the present disclosure has the advantages of flexible use, simple operation, high sensitivity, and good stability, providing a method support for the quantitative analysis and functional evaluation of free and ester-bound sterculic acid in feeds and foods. Description of the drawings

[0030] To more clearly illustrate the technical solutions in the present disclosure or related technologies, the following will briefly introduce the accompanying drawings required for use in the embodiments or related technology descriptions. Obviously, the accompanying drawings in the following descriptions are only embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0031] Figure 1 This is the reaction formula of sterculic acid and 2-pyridinecarboxylic hydrazide involved in this embodiment;

[0032] Figure 2 This is the comparison of mass spectrometry signals before and after the reaction of sterculic acid and 2-pyridinecarboxylic hydrazide involved in this embodiment;

[0033] Figure 3 This is the influence of the EDC concentration in the derivatization reaction solution on the derivatization efficiency involved in this embodiment;

[0034] Figure 4 This is the influence of the HOAt concentration in the derivatization reaction solution on the derivatization efficiency involved in this embodiment;

[0035] Figure 5 This is the influence of the 2-pyridinecarboxylic hydrazide concentration in the derivatization reaction solution on the derivatization efficiency involved in this embodiment;

[0036] Figure 6 This is the influence of the derivatization reaction temperature on the derivatization efficiency involved in this embodiment;

[0037] Figure 7 This is the influence of the derivatization reaction time on the derivatization efficiency involved in this embodiment;

[0038] Figure 8 This is the comparison of the extraction conditions of free sterculic acid involved in this embodiment.

[0039] Figure 9 This is the comparison of the extraction conditions of ester-bound sterculic acid involved in this embodiment.

[0040] Figure 10 This is the influence of the collision voltage on the mass spectrometry signal response of sterculic acid involved in this embodiment. Detailed implementation manners

[0041] The following further describes the present invention in combination with specific embodiments. The advantages and features of the present invention will become clearer with the description. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention can be modified or replaced, but such modifications or replacements all fall within the protection scope of the present invention.

[0042] Example 1: Effect of EDC Concentration on the Derivatization Efficiency of Sterculic Acid

[0043] 1) Using 2 μg / mL of stearic acid as the substrate, in a derivatization solution of 15 mmol / L HOAt and 40 mmol / L 2-pyridinecarboxylic hydrazide, add 250, 500, 750, and 1000 mmol / L of EDC respectively, and react at 20 °C for 60 min.

[0044] 2) After the reaction, the derivatized product was detected by LC-MS / MS. The liquid phase conditions were as follows:

[0045] Mobile phase: 0.1% formic acid in water and 0.1% formic acid in acetonitrile;

[0046] Chromatographic column: C8 chromatographic column (2.1×100 mm, 1.8 μm);

[0047] Mobile phase gradient:

[0048] Time (min) Flow rate (mL / min) A B 0 0.4 50 50 1.00 0.4 50 50 6.00 0.4 0 100 7.00 0.4 0 100 7.10 0.4 50 50 9.00 0.4 50 50

[0049] The mass spectrometry conditions were as follows:

[0050] Ionization was carried out using an electrospray ion source in positive ion mode. Data was collected in multiple ion scanning mode. The declustering voltage was set to 30 V, the collision energy for generating daughter ion 138 was 20 eV, and the collision energy for generating daughter ion 120 was 50 eV;

[0051] 3) The LC-MS / MS detection results showed that EDC at 250 - 1000 mmol / L could promote the derivatization reaction.

[0052] Example 2: Effect of HOAt Concentration on the Derivatization Efficiency of Sterculic Acid

[0053] Using 2 μg / mL of stearic acid as the substrate, in a derivatization solution of 500 mmol / L EDC and 40 mmol / L 2-pyridinecarboxylic hydrazide, add 0, 15, 30, 45, and 60 mmol / L of HOAt respectively, and react at 20 °C for 60 min. After the reaction, the derivatized product was detected using the liquid phase conditions of Example 1. The mass spectrometry conditions were a declustering voltage of 20 V, a collision energy of 20 eV for daughter ion 138, and a collision energy of 50 eV for daughter ion 120. The results showed that HOAt at 15 - 60 mmol / L could promote the derivatization reaction.

[0054] Example 3: Effect of 2-Pyridinecarboxylic Hydrazide Concentration on the Derivatization Efficiency

[0055] Using 2 μg / mL of sterculic acid as the substrate, in a derivatization solution containing 500 mmol / L EDC and 15 mmol / L HOAt, 4, 10, 20, 40, and 80 mmol / L of 2-pyridinecarboxylic hydrazide were added respectively, and the reaction was carried out at 20 °C for 60 min. After the reaction, the derivatized products were detected using the liquid phase conditions of Example 1, and the mass spectrometry conditions were that the declustering voltage was 50 V, the collision energy of the daughter ion 138 was 30 eV, and the collision energy of the daughter ion 120 was 30 eV. The results showed that 2-pyridinecarboxylic hydrazide at 4 - 80 mmol / L could promote the derivatization reaction.

[0056] Example 4: Influence of derivatization temperature on derivatization efficiency

[0057] Using 2 μg / mL of sterculic acid as the substrate, in a derivatization solution containing 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L of 2-pyridinecarboxylic hydrazide, the reaction was carried out at 4, 20, 37, and 50 °C for 60 min respectively. After the reaction, the derivatized products were detected using the liquid phase and mass spectrometry conditions of Example 1. The results showed that 4 - 50 °C could all promote the derivatization reaction.

[0058] Example 5: Influence of derivatization time on derivatization efficiency

[0059] Using 2 μg / mL of sterculic acid as the substrate, in a derivatization solution containing 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L of 2-pyridinecarboxylic hydrazide, the reaction was carried out at 20 °C for 15, 30, 60, and 90 min respectively. After the reaction, the derivatized products were detected using the liquid phase and mass spectrometry conditions of Example 1. The results showed that 15 - 90 min could all promote the derivatization reaction.

[0060] Example 6: Influence of different organic reagents on the extraction efficiency of free sterculic acid

[0061] Weigh 1.0 g of the feed sample containing sterculic acid, add 5 mL of methanol, acetonitrile, or a mixed solution of methanol and dichloromethane respectively, homogenize and then centrifuge at 14000 r / min for 10 min. The supernatant was reacted with a derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L of 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivatized products were detected using the liquid phase and mass spectrometry conditions of Example 1, and the results showed that the extraction efficiency was the highest when using methanol.

[0062] Example 7: Influence of methanol concentration on the extraction efficiency of free sterculic acid

[0063] Weigh 1.0 g of the feed sample containing sterculic acid, add 5 mL of methanol solutions with concentrations of 80%, 90%, and 100% respectively, homogenize, and then centrifuge at 14000 r / min for 10 min. The supernatant reacts with the derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivatized product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that within the range of 80 - 100% methanol, a high extraction effect can be obtained.

[0064] Example 8: Influence of Different Extraction Methods on the Extraction Effect of Ester-Bound Sterculic Acid

[0065] Weigh 1.0 g of the feed sample containing sterculic acid, and extract ester-bound sterculic acid using three methods respectively. For Method 1, add 5 mL of a dichloromethane-methanol-water mixed system (v / v / v = 2:1:1) to the sample for homogenization and extraction, collect the organic phase and dry it. After drying, add 5 mL of 2% NaOH methanol solution to the sample, hydrolyze at 50 °C for 12 h, neutralize the hydrolyzate with 10% hydrochloric acid methanol solution and make up the volume for use. For Methods 2 and 3, add 5 mL of 2% NaOH methanol solution to the sample, hydrolyze at 50 °C for 12 h, and neutralize the hydrolyzate with 10% hydrochloric acid methanol solution. For Method 2, continue to extract with a dichloromethane-methanol-water mixed system (v / v / v = 2:1:1), collect the lower organic phase and make up the volume for use. For Method 3, directly make up the volume of the neutralized hydrolyzate for use. React the three extraction solutions with the derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivatized product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that after hydrolyzing the ester-bound sterculic acid by Method 3 and directly performing the derivatization reaction and detection, a high extraction effect can be obtained.

[0066] Example 9: Influence of the Volume of Different Extraction Solutions on the Extraction Effect of Ester-Bound Sterculic Acid

[0067] Weigh 1.0 g of the feed sample containing sterculic acid, add 3, 4, and 5 mL of 2% NaOH methanol solution respectively, hydrolyze at 50 °C for 12 h, neutralize the hydrolyzate with 10% hydrochloric acid methanol solution and make up the volume, and react with the derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivatized product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that within the range of 3 - 5 mL of the hydrolyzate, a high extraction effect can be obtained.

[0068] Example 10: Influence of the Concentration of Different Extractants on the Extraction Effect of Ester-Bound Sterculic Acid

[0069] Weigh 1.0 g of the feed sample containing sterculic acid, add 5 mL of 2%, 4%, and 5% NaOH methanol solutions respectively, hydrolyze at 50 °C for 12 h, neutralize and make up the volume of the hydrolyzate with 10% hydrochloric acid methanol solution, and react with the derivative solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivative product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that within the range of 2 - 5%, a high extraction effect can be obtained for the hydrolyzate.

[0070] Example 11: Influence of Different Hydrolysis Temperatures on the Extraction Effect of Ester-Bound Sterculic Acid

[0071] Weigh 1.0 g of the feed sample containing sterculic acid, add 5 mL of 2% NaOH methanol solution, and hydrolyze at 30 °C, 50 °C, and 80 °C for 12 h respectively. Neutralize and make up the volume of the hydrolyzate with 10% hydrochloric acid methanol solution, and react with the derivative solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivative product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that within the range of 30 - 80 °C for the hydrolysis temperature, a high extraction effect can be obtained.

[0072] Example 12: Influence of Different Hydrolysis Times on the Extraction Effect of Ester-Bound Sterculic Acid

[0073] Weigh 1.0 g of the feed sample containing sterculic acid, add 5 mL of 2% NaOH methanol solution, and hydrolyze at 50 °C for 4 h, 8 h, 10 h, 12 h, and 14 h respectively. Neutralize and make up the volume of the hydrolyzate with 10% hydrochloric acid methanol solution, and react with the derivative solution composed of 500 mmol / L EDC, 15 mmol / L HOAt, and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivative product is detected using the liquid phase and mass spectrometry conditions of Example 1. The results show that within the range of 4 - 14 h for the hydrolysis time, a high extraction effect can be obtained.

[0074] Example 13: Quantitative Analysis of Free Sterculic Acid in Cottonseed Meal

[0075] Weigh 1.0 g of cottonseed meal as the sample to be tested, add 5 mL of 100% methanol solution, homogenize, centrifuge at 14000 r / min for 10 min, dilute the supernatant, and react with the derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivative product is detected using the liquid phase and mass spectrometry conditions of Example 1, and the free sterculic acid content is detected to be 0.46 mg / kg.

[0076] Example 14: Quantitative analysis of ester-bound sterculic acid in cottonseed meal-containing feed

[0077] Weigh 1.0 g of cottonseed meal-containing feed as the sample to be tested, add 5 mL of 2% NaOH methanol solution, hydrolyze at 50 °C for 12 h, neutralize and make up the volume of the hydrolysis solution with 10% hydrochloric acid methanol solution, and react with the derivatization solution composed of 500 mmol / L EDC, 15 mmol / L HOAt and 40 mmol / L 2-pyridinecarboxylic hydrazide at 20 °C for 60 min. After the reaction, the derivative product is detected using the liquid phase and mass spectrometry conditions of Example 1, and the ester-bound sterculic acid content is detected to be 425.65 mg / kg.

Claims

1. A method for highly sensitive and high-precision quantification of sterculic acid, characterized in that, Comprising: Extracting free sterculic acid and ester-bound sterculic acid in the sample to be tested; performing a derivatization reaction on the extracted sterculic acid with 2-pyridinecarboxylic hydrazide to enhance the mass spectrometry signal response; quantitatively analyzing free sterculic acid and ester-bound sterculic acid in the sample to be tested respectively using liquid chromatography-tandem mass spectrometry.

2. The method according to claim 1, wherein the free sterculic acid is extracted using an 80%-100% methanol solution.

3. The method according to claim 1, wherein the sample containing ester-bound sterculic acid is first saponified with an alkali solution, and the alkali solution consists of a 2%-5% NaOH methanol solution; the hydrolysis time is 4-14 hours; the hydrolysis temperature is 30-80 °C.

4. For the sterculic acid extracted as described in claims 2 and 3, derivatization is performed using 2-pyridinecarboxylic hydrazide, and the derivatization solution is: 250-1000 mmol / L EDC, 0-60 mmol / L HOAt, 4-80 mmol / L 2-pyridinecarboxylic hydrazide, the reaction temperature is 4-50 °C, and the reaction time is 15-90 min.

5. For the derivative product produced as described in claim 4, using liquid chromatography-tandem mass spectrometry, ionization is performed in the positive ion mode and data is collected in the multiple ion scanning mode. The parent ion of the derivative product is 414, and the declustering voltage is 20-50 V; the daughter ions generated by fragmentation of the parent ion are 138 and 120, the collision energy for generating the daughter ion 138 is 20-30 eV, and the collision energy for generating the daughter ion 120 is 30-50 eV.

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