A method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry

Abstract

The present invention discloses a method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry, belonging to the technical field of phospholipid detection. This method uses a wet digestion pretreatment method for the sample to be tested. When detecting the phospholipid content by using the mass spectrometry detection method in this method, the use of reagents is reduced, the requirements for the equipment used in digestion are low, the detection time is greatly shortened, the detection efficiency is improved, the detection limit is lower, the reproducibility is better, and the recovery rate is higher. Due to the adoption of mass spectrometry technology, the selectivity for phosphorus ions is enhanced, the interference of impurities on the detection results is eliminated, so the qualitative and quantitative analysis of phospholipids is more accurate, the anti-interference ability is further enhanced, and the accuracy of the detection is ensured. It solves the technical problems such as large deviation in detection results, low efficiency, and weak detection performance caused by the existing spectrophotometry. This method only takes 3 - 3.5 hours to complete, which is not only efficient but also more accurate.

Description

Technical Field

[0001] The present invention belongs to the technical field of phospholipid detection, and particularly relates to a method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry. Background Art

[0002] During the oil refining process, phospholipids in crude oil can cause foaming, color reversion, and oxidation stability of the oil products, affecting the flavor of the oils and fats. Moreover, crude oil foot rich in phospholipids is the main raw material for producing phospholipid health products. In the production process of biodiesel, raw oil with high phospholipid content can cause serious inactivation of lipase. The phospholipid content in liposomes is an important indicator to measure its stability. Therefore, in the production of oil products, the phospholipid content is a very important detection index.

[0003] Currently, the detection methods for phospholipids include colorimetry, turbidimetry, gravimetry, thin-layer chromatography, high-performance liquid chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, etc. Turbidimetry and gravimetry have limited applications due to their low detection accuracy; thin-layer chromatography is greatly affected by manual operation and has poor repeatability; the detection costs of high-performance liquid chromatography, infrared spectroscopy, and nuclear magnetic resonance spectroscopy are all relatively high, and solid professional knowledge is required, which is beyond the reach of ordinary detection personnel.

[0004] The national standard method GB / T 5537-2008 for the determination of phospholipid content in grain and oil inspection uses spectrophotometry and gravimetry to determine the phospholipid content in vegetable oils and fats. However, each of the above detection methods has the following defects: 1. Spectrophotometry requires dry digestion, and high temperature is likely to cause volatilization of phosphorus elements, resulting in low recovery rate. Moreover, molybdenum blue is generated for colorimetric quantification, and the result is easily interfered by other colors, with weak qualitative ability and a large difference from the actual value. 2. A large amount of reagents are required during the processes of dry digestion and quantitative colorimetry, and the detection cycle is relatively long, taking about 12 hours in total, with low efficiency. 3. The detection limits of spectrophotometry and gravimetry are relatively high and are not suitable for the detection of ultra-low concentration phospholipids. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies that a large amount of reagents are required in the prior treatment process of the existing technology, the detection cycle is relatively long, taking about 12 hours in total, with low efficiency, and the recovery rate in the existing method is low, and molybdenum blue is generated for colorimetric quantification, the result is easily interfered by other colors, with weak qualitative ability and a large difference from the actual value, and the detection limit is relatively high and is not suitable for ultra-low concentration phospholipids, etc., and to provide a method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry.

[0006] The technical problem to be solved can be achieved through the following technical solutions:

[0007] A method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry includes the following steps;

[0008] S1. Prepare the sample solution to be measured and the blank control solution:

[0009] Weigh 0.2 - 0.5 g of the homogenized grease sample and place it in a 100 mL glass container. Add 1 - 2 mL of hydrogen peroxide and 6 mL of concentrated nitric acid, cover with a watch glass, pre-digest on a hot plate, and then heat and digest. During the process, add concentrated nitric acid to digest until the solution is clear. Remove the watch glass, drive off the acid until nearly dry, cool, and make up the volume with water to obtain the sample solution to be measured;

[0010] When no grease sample is added, perform the above operations to obtain the blank control solution;

[0011] S2. Prepare the phosphorus standard solution and the scandium internal standard solution;

[0012] S3. Plot the standard curve: Inject the phosphorus standard solution and the scandium internal standard solution into an inductively coupled plasma mass spectrometer to measure the signal response values of the phosphorus element and the internal standard scandium element;

[0013] Use the concentration of the phosphorus element as the abscissa and the ratio of the response values of the phosphorus element to the internal standard scandium element as the ordinate for plotting;

[0014] S4. Determine the phosphorus content in the sample solution to be measured:

[0015] Mix the blank control solution and the sample solution to be measured with the scandium internal standard solution respectively, and inject them into an inductively coupled plasma mass spectrometer to obtain the ratios of the response values of the phosphorus element to the scandium internal standard solution in the blank control solution and the sample solution to be measured respectively;

[0016] S5. Calculate the phospholipid content in the sample to be measured:

[0017] Calculate the phosphorus content in the sample solution to be measured according to the plotted standard curve, and convert it into the phospholipid content.

[0018] Preferably, the grease sample includes any one of lard, beef tallow, fish oil, etc.

[0019] Preferably, the grease sample includes corn oil and / or rice bran oil.

[0020] Preferably, in step S1, the pre-digestion includes preheating on an electric hot plate at 80 - 90 °C for 30 - 35 min.

[0021] Preferably, in step S1, the heating and digestion treatment includes heating the electric hot plate to 150 - 160 °C and treating for 1.5 - 2.5 h.

[0022] Preferably, in step S1, during the heating and digestion treatment, concentrated nitric acid is added; the volume of the added concentrated nitric acid is 4 - 6 mL.

[0023] Preferably, the concentrations of the phosphorus standard solutions are 10, 50, 100, 200, 500, 1000, 2000 μg / L.

[0024] Preferably, the concentration of the scandium internal standard solution is 50 μg / L.

[0025] Preferably, the operating conditions of the inductively coupled plasma mass spectrometer are as follows: radio frequency power (RF): 1500 - 1600 w; helium gas flow rate: 3.5 - 5 mL / min; plasma gas flow rate: 15 L / min; auxiliary gas flow rate: 1.2 mL / min; acquisition mode: peak hopping scan; analysis mode: kinetic energy discrimination mode (KED).

[0026] Preferably, the conversion formula in step S5 is as follows:

[0027]

[0028] Wherein, X -- phospholipid content, unit: milligram per gram (mg / g);

[0029] P 1 -- phosphorus content measured in the test sample solution according to the standard curve, unit: microgram per milliliter (μg / mL);

[0030] P 0 -- phosphorus content measured in the blank control solution according to the standard curve, unit: microgram per milliliter (μg / mL);

[0031] m -- mass of the oil sample, unit: gram (g);

[0032] V 0 -- volume of the test sample solution after constant volume, unit: milliliter (mL);

[0033] D -- dilution factor;

[0034] 26.31 -- milligrams of phospholipid equivalent to per milligram of phosphorus.

[0035] Compared with the prior art, the present invention has at least the following technical effects:

[0036] The present invention provides a method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry. This method uses wet digestion for the oil and fat sample to be measured, in combination with a small amount of hydrogen peroxide and concentrated nitric acid, and only uses an electric hot plate for heating. It can be completed in only 3 - 3.5 hours, reducing the amount of reagent used, greatly shortening the detection time, improving the detection efficiency, and having low requirements for the equipment used. Since the mode of combining mass spectrometry technology with an internal standard detection method is adopted, the selectivity for phosphorus ions is enhanced, further eliminating the interference of impurities on the results, and the detection limit is also lower. Therefore, the qualitative and quantitative determination of phospholipids is more accurate, ensuring the accuracy of the detection, and solving the technical problems such as large deviations in detection results, low efficiency, and weak detection performance caused by the existing spectrophotometry method. Description of the Drawings

[0037] Figure 1 It is a flowchart of the method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry of the present invention. Detailed Embodiments

[0038] The following will describe the implementation scheme of the present invention in detail with specific embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For the specific conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0039] Example 1

[0040] As Figure 1 shown, it specifically includes the following steps:

[0041] 1. Prepare the sample solution to be measured and the blank control solution:

[0042] Accurately weigh 0.2 g of the well-mixed oil and fat sample and place it in a 100 mL high-foot beaker. After adding 1 mL of hydrogen peroxide and 6 mL of concentrated nitric acid, cover it with a watch glass, pre-digest at 80 °C on an electric hot plate for 30 min, then heat to 150 °C for digestion. After 1 h, add 4 mL of nitric acid, digest until the solution is clear, remove the watch glass, drive off the acid until nearly dry, and after cooling, make up the volume to 50 mL with water to obtain the sample solution to be measured; at the same time, make a blank control.

[0043] In the above steps, by adding 1 - 2 mL of hydrogen peroxide, the oil and fat sample can be rapidly oxidized, thereby reducing the amount of nitric acid used and shortening the digestion process; by using pre-digestion and covering with a watch glass, it can effectively prevent the volatilization of hydrogen peroxide and nitric acid due to too high temperature, thus affecting the digestion effect and speed. By adding concentrated nitric acid, the acid concentration can be effectively replenished, the digestion process can be accelerated, and the adverse effects caused by the reduction of acid concentration due to volatilization can be reduced.

[0044] 2. Preparation of phosphorus standard solution and scandium internal standard solution:

[0045] Preparation of 10 mg / L phosphorus standard stock solution: Take 1.00 mL of 1000 mg / L certified phosphorus standard solution and transfer it to a 100 mL volumetric flask, then dilute it to the mark with 5% concentrated nitric acid.

[0046] Preparation of phosphorus standard solutions: Take 0.05 mL, 0.25 mL, 0.50 mL, 1.00 mL, 2.50 mL, 5.00 mL, 10.00 mL of the 10 mg / L phosphorus standard stock solution respectively and transfer them to 50 mL volumetric flasks, then dilute them to the mark with 5% concentrated nitric acid to prepare 10 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L phosphorus standard solutions.

[0047] Preparation of scandium internal standard solution: Take 0.05 mL of 1000 mg / L certified scandium standard solution and transfer it to a 1000 mL volumetric flask, then dilute it to the mark with 5% concentrated nitric acid to obtain a scandium internal standard solution with a concentration of 50 μg / L.

[0048] 3. Plotting the standard curve:

[0049] Set the working conditions of the inductively coupled plasma mass spectrometer: RF power (RF): 1600 W; plasma flow rate: 15 L / min, nebulizer flow rate: 1.0 mL / min; acquisition mode: peak jumping scan; kinetic energy discrimination mode (KED). Then inject the phosphorus standard solutions and the scandium internal standard solution into the inductively coupled plasma mass spectrometer to measure the signal response values of phosphorus element and internal standard scandium element.

[0050] Plot with the concentration of phosphorus element as the abscissa and the ratio of the response values of phosphorus element to internal standard scandium element as the ordinate.

[0051] 4. Determination of phosphorus content in the sample solution to be measured:

[0052] Under the same working conditions of the inductively coupled plasma mass spectrometer as in the above steps, mix the blank control solution and the sample solution to be measured with the scandium internal standard solution respectively, and then inject them into the inductively coupled plasma mass spectrometer to obtain the ratios of the response values of phosphorus element to the scandium internal standard solution in the blank control solution and the sample solution to be measured respectively.

[0053] 5. Calculation of phospholipid content in the sample to be measured

[0054] Calculate the phosphorus content in the sample solution to be measured according to the standard curve plotted above, and convert it into phospholipid content according to the following formula:

[0055]

[0056] Wherein, X is the phospholipid content, with the unit of milligram per gram (mg / g);

[0057] P 1 -- The phosphorus content in the sample solution measured according to the standard curve, with the unit of microgram per milliliter (μg / mL);

[0058] P 0 -- The phosphorus content in the blank control solution measured according to the standard curve, with the unit of microgram per milliliter (μg / mL);

[0059] m is the mass of the sample, with the unit of gram (g);

[0060] V 0 -- The fixed volume of the sample solution, with the unit of milliliter (mL);

[0061] D is the dilution factor;

[0062] 26.31 is the number of milligrams of phospholipid equivalent to each milligram of phosphorus.

[0063] Method verification:

[0064] 1. Standard curve, detection limit, and quantification limit

[0065] Standard curve: With the concentration of phosphorus element from 10 - 2000 μg / L as the abscissa and the ratio of the response value of phosphorus element to the internal standard scandium element as the ordinate, a standard curve is plotted, and the linear relationship equation is obtained as Y = 0.0023x + 0.0306, with the correlation coefficient R 2 being 0.9998, indicating a good linear relationship

[0066] Detection limit and quantification limit: According to the requirements of Appendix A of GB 5009.1 - 2003, 20 blank samples are injected for analysis to calculate the detection limit and quantification limit. The detection limit is obtained as 5 mg / kg, and the quantification limit is 15 mg / kg. Compared with the detection limit (20 mg / kg) of the first spectrophotometry method in GB / T 5537 - 2008, it is lower, the detection is more sensitive, and it is more suitable for the detection of low - concentration phospholipids.

[0067] 2. Recovery rate

[0068] Accurately weigh samples of corn oil, rice oil, lard, beef tallow, and fish oil respectively, add different amounts of phosphorus standard stock solution to them (see the theoretical spiked value for details), and determine and quantitatively calculate according to the method of the present invention. The recovery rate results are shown in Table 1.

[0069] Table 1. Experimental results of sample recovery rate

[0070] Specimen sample Corn oil Rice bran oil Lard Beef tallow Fish oil Background measurement value (μg) 0.40 0.52 12.48 3.06 0.20 Theoretical spike value (μg) 25.00 50.00 10.00 25.00 50.00 Measured value after spiking (μg) 25.51 51.20 22.91 27.77 52.36 Recovery rate (%) 100.4 101.4 104.3 98.8 104.3

[0071] The recovery rate is between 98.8% and 104.3%, meeting the requirements for the spike recovery rate in GB / T 27404-2008 "Laboratory Quality Control Specification for Physical and Chemical Testing of Foods", indicating that this method has a high recovery rate and accurate quantification.

[0072] 3. Precision

[0073] Six precise samples of corn oil, rice bran oil, lard, beef tallow, and fish oil were taken respectively, determined according to the method of the present invention, and quantitatively calculated. The results are shown in Table 2.

[0074] Table 2. Results of the precision experiment of the samples:

[0075]

[0076] As can be seen from Table 2, the relative deviations of the detection precision of the six oil samples are all less than 5%, meeting the requirements of measurement precision, indicating that the detection results of this method of the present invention have small deviations and are more accurate.

[0077] Example 2

[0078] Precise samples of corn oil, rice bran oil, lard, beef tallow, and fish oil were weighed respectively, determined according to the method of the present invention respectively, and the phospholipid content was quantitatively calculated. The results are shown in Table 3.

[0079] Comparative Example 1

[0080] Precise samples of the above-mentioned corn oil, rice bran oil, lard, beef tallow, and fish oil were weighed respectively, and the phospholipid content was determined by the first spectrophotometric method of GB / T5537-2008. The results are shown in Table 3.

[0081] Table 3. Determination results of phospholipid content in corn oil, rice bran oil, lard, beef tallow and fish oil

[0082]

[0083] The current national recommended method for the determination of phospholipid content in grain and oil is GB / T 5537-2008 "Inspection of Grain and Oil - Determination of Phospholipid Content" (i.e., the national standard method), which has many disadvantages, but this national standard method is currently the standard method for the detection of phospholipid content in grain and oil. As can be seen from Table 3, the deviation between the determination results of this method of the present invention and the national standard method is less than 8%, indicating that after overcoming the defects of the national standard method, this method of the present invention can accurately determine the phospholipid content in oil samples.

[0084] It can also be seen from Table 3 that the detection results of corn oil, rice bran oil, lard, and beef tallow by this method of the present invention are slightly greater than those of the national standard method, indicating that this method of the present invention has a better recovery rate and higher accuracy in detection.

[0085] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining the phospholipid content in oils and fats by inductively coupled plasma mass spectrometry, characterized in that, it comprises the following steps; S1. Prepare the sample solution to be measured and the blank control solution: Weigh 0.2 - 0.5 g of the homogenized oil and fat sample and place it in a 100 mL glass container. After adding 1 - 2 mL of hydrogen peroxide and 6 mL of concentrated nitric acid, cover it with a watch glass, pre-digest it on a hot plate, and then carry out heating digestion. During the process, add concentrated nitric acid for digestion until the solution becomes clear. Remove the watch glass, evaporate the acid until nearly dry, cool it, and make the volume constant with water to obtain the sample solution to be measured; the oil and fat sample includes any one of lard, beef tallow, and fish oil; the oil and fat sample includes corn oil and / or rice bran oil; When no oil and fat sample is added, perform the above operations to obtain the blank control solution; The pre-digestion includes preheating on an electric hot plate at 80 - 90 °C for 30 - 35 min; The heating digestion treatment includes heating the electric hot plate to 150 - 160 °C and treating for 1.5 - 2.5 h; During the heating digestion treatment, add concentrated nitric acid; the volume of the added concentrated nitric acid is 4 - 6 mL; S2. Prepare the phosphorus standard solution and the scandium internal standard solution; the concentration of the scandium internal standard solution is 50 μg / L; S3. Draw the standard curve: Inject the phosphorus standard solution and the scandium internal standard solution into the inductively coupled plasma mass spectrometer, and measure the signal response values of the phosphorus element and the internal standard scandium element; The concentrations of the phosphorus standard solution are 10, 50, 100, 200, 500, 1000, 2000 μg / L; Draw with the concentration of the phosphorus element as the abscissa and the ratio of the response values of the phosphorus element and the internal standard scandium element as the ordinate; S4. Determine the phosphorus content in the sample solution to be measured: Mix the blank control solution and the sample solution to be measured with the scandium internal standard solution respectively, and inject them into the inductively coupled plasma mass spectrometer to obtain the ratios of the response values of the phosphorus element and the scandium internal standard solution in the blank control solution and the sample solution to be measured respectively; The operating conditions of the inductively coupled plasma mass spectrometer: Radio frequency power (RF): 1500 - 1600 w; Helium flow rate: 3.5 - 5 mL / min; Plasma flow rate: 15 L / min; Auxiliary gas flow rate: 1.2 mL / min; Acquisition mode: Peak jumping scan; Analysis mode: Kinetic energy discrimination mode (KED); S5. Calculate the phospholipid content in the sample to be measured: Calculate the phosphorus content in the sample solution to be measured according to the drawn standard curve, and convert it into the phospholipid content; The conversion formula is: where X - phospholipid content, unit is milligram per gram (mg / g); P 1 --Phosphorus content in the sample solution to be measured as determined according to the standard curve, in micrograms per milliliter (μg / mL); P 0 --Phosphorus content in the blank control solution as determined according to the standard curve, in micrograms per milliliter (μg / mL); m--mass of the oil sample, in grams (g); V 0 -- Volume of the sample solution to be measured after dilution, unit: milliliter (mL); D - dilution factor; 26.31 - milligrams of phospholipid equivalent to per milligram of phosphorus; The detection limit is 5 mg / kg, and the quantification limit is 15 mg / kg.

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