Method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels in fresh eating period

Abstract

The invention belongs to the technical field of analysis and testing of fresh corn, and particularly relates to a method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels in a fresh period. The method comprises the following steps: preparing sweet and waxy corn kernels in a fresh eating period into a to-be-detected solution; the method comprises the following steps: respectively and quantitatively taking standard substances of B vitamins, dissolving the standard substances with acetonitrile, acetic acid and formic acid water, fixing the volume, diluting into mixed standard substance solutions with a series of concentrations, and making a standard curve of each standard substance; the acetonitrile-acetic acid-formic acid aqueous solution is obtained by adding acetonitrile, glacial acetic acid and formic acid into water; and detecting the solution to be detected, substituting into the standard curve, and calculating to obtain the content of each group B vitamin. The method disclosed by the invention can be used for simultaneously detecting eight B vitamins in sweet and waxy corn kernels in the fresh eating period, so that the detection steps are saved, and the detection limit of the B vitamins in the corn is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of fresh corn analysis and testing, and specifically relates to a method for simultaneously detecting eight kinds of B vitamins in sweet and glutinous corn kernels at the fresh stage (i.e., milk-ripening stage). Background Art

[0002] B vitamins are water-soluble vitamins that play an important role in biological metabolism. B vitamins include vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine), vitamin B7 (biotin), vitamin B9 (folic acid) and vitamin B 12 (Cobalamin). A lack of B vitamins in the human body can lead to beriberi, neurodegenerative diseases, pellagra, amnesia, vascular diseases, diabetes and many other diseases. However, since the human body lacks the key enzymes to synthesize B vitamins from scratch, it can only obtain the B vitamins needed by the human body from the diet.

[0003] Cereal crops are one of the important sources of B vitamins. Through the analysis of the content of B vitamins in cereal crops such as wheat, rice, triticale, millet, sorghum, and corn, it was found that the content of B vitamins in different cereals varies greatly, and more than half of the vitamin B6 in corn appears in the form of pyridoxine. Sweet corn and glutinous corn are types of corn that are eaten fresh at the milky stage. They are rich in starch, protein, amino acids, vitamins, minerals and other nutrients. Understanding the distribution of B vitamins in fresh corn and deeply exploring the functional nutritional value of fresh corn are of great significance to enriching my country's food nutrition database and breeding resources.

[0004] Previous methods for detecting B vitamins mostly used milk powder and health foods with stable properties as research sample matrices. However, in the detection of sweet and glutinous corn kernel samples during the milk maturity period, the B vitamins in fresh corn kernel samples during this period are unstable. At present, the main analytical methods for the content of B vitamins include spectrophotometry, liquid chromatography, high performance liquid chromatography-mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, etc. Among them, the national standard detection methods for water-soluble vitamins are mostly for the purpose of detecting a single vitamin. The research methods for the simultaneous detection of multiple water-soluble vitamins are mainly liquid chromatography-tandem mass spectrometry and liquid chromatography. High performance liquid chromatography mainly uses ultraviolet detectors for detection, which has a high detection limit for B vitamins, severe interference, and cannot simultaneously separate multiple B vitamin components. Ultra-high performance liquid chromatography has greatly improved the separation effect and sensitivity compared with high performance liquid chromatography, but these existing methods still detect fewer types of B vitamin components.

[0005] In summary, it is necessary to develop a method for the simultaneous analysis of multiple B vitamin components in sweet and waxy corn kernels during the fresh consumption period. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for simultaneously detecting 8 B vitamins in sweet and glutinous corn kernels during the fresh eating period, which can simultaneously detect multiple B vitamins in sweet and glutinous corn kernels during the fresh eating period with a low detection limit.

[0007] The object of the present invention is to provide a method for simultaneously detecting eight kinds of B vitamins in sweet and waxy corn kernels during fresh consumption, comprising:

[0008] Prepare a test solution using fresh corn kernels, wherein the fresh corn kernels are fresh sweet corn kernels or fresh glutinous corn kernels;

[0009] Quantitatively take standard products of 8 kinds of B vitamins respectively, dissolve and fix the volume with acetonitrile, acetic acid and formic acid water, dilute to a mixed standard solution with a series of concentrations, and prepare a standard curve of each standard product; the acetonitrile, acetic acid and formic acid water is obtained by adding acetonitrile, glacial acetic acid and formic acid to deionized water, and the volume fractions of acetonitrile, glacial acetic acid and formic acid are 5% to 5.5%, 1% and 0.3% to 0.5% respectively, and the solvent is deionized water;

[0010] The solution to be tested was detected by ultra-high performance liquid chromatography-tandem mass spectrometry, and the content of each B vitamin was calculated by substituting it into the standard curve;

[0011] The eight B vitamins are vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9 and vitamin B 12 .

[0012] Acetonitrile, acetic acid and formic acid water are the reagents for dissolving standard substances developed by the present invention, in which 8 kinds of B vitamins can be well separated and dispersed, so as to be easily identified and detected by instruments, thereby being able to detect more kinds of B vitamins than the prior art.

[0013] Preferably, the quantitative analysis of the test solution using ultra-high performance liquid chromatography-tandem mass spectrometry can greatly improve the sensitivity, and has the characteristics of high separation efficiency, fast analysis speed, accurate results, etc., and is more suitable for the separation and determination of multiple vitamins.

[0014] Preferably, in the above method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption, the vitamin B6 includes at least one of pyridoxine, pyridoxal, and pyridoxamine; and the vitamin B3 includes at least one of nicotinic acid and nicotinamide. The method of the present invention also has a good effect on the separation and detection of these B vitamins in different forms.

[0015] Preferably, the method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption, and the method for preparing the test solution is as follows:

[0016] The sweet and glutinous corn kernels in the fresh-eating stage are quickly frozen with liquid nitrogen, and then ground below 0°C to obtain a sample to be tested; and the B vitamins in the sample to be tested are extracted with an extracting solution to obtain a test solution.

[0017] The above-mentioned method for obtaining corn kernels and method for preparing the test solution are used to reduce the loss of B vitamins during sample processing.

[0018] Preferably, in the above method for simultaneously detecting 8 kinds of B vitamins in sweet and glutinous corn kernels during fresh consumption, the extract is a methanol-formic acid extract with a volume fraction of 80% to 82%, wherein the volume fraction of methanol in the methanol-formic acid extract with a volume fraction of 80% to 82% is 80% to 82%, the volume of formic acid is 1%, and deionized water is supplemented to 100%. The above extract is fully extracted, and many kinds of B vitamins are extracted, thereby reducing the residue and loss of B vitamins in corn kernels, and the recovery rate of the spiked experiment is high and the standard deviation is low.

[0019] Preferably, the method for simultaneously detecting eight kinds of B vitamins in sweet and waxy corn kernels during fresh consumption, and the method for extracting the B vitamins in the sample to be tested with the extracting solution is as follows:

[0020] Adding the extract and calcium sulfate to the sample to be tested, extracting, centrifuging, collecting the supernatant, filtering the supernatant with an aqueous filter membrane to remove impurities, and obtaining a solution to be tested;

[0021] For every gram of sample to be tested, 5 mL to 15 mL of extract and 0.1 g to 0.2 g of calcium sulfate are added.

[0022] Calcium sulfate is insoluble in formic acid and methanol and will not enter into the methanol-formic acid extract with a volume fraction of 80% to 82%. Calcium sulfate is an inorganic salt that promotes the dissolution of B vitamins, and a wide variety of B vitamins are extracted.

[0023] Preferably, in the above method for simultaneously detecting eight kinds of B vitamins in sweet and glutinous corn kernels during the fresh-eating period, the precipitate after centrifugation repeats the following steps 2 to 3 times: adding extracting solution, extraction, centrifugation, collecting new supernatant and new precipitate; collecting and mixing all supernatants, filtering with an aqueous filter membrane to remove impurities, and obtaining a test solution. That is, calcium sulfate is added only during the first extraction, and no calcium sulfate is added during subsequent repeated extractions. The types and quality of B vitamins extracted in the first extraction are the largest, and the addition of calcium sulfate promotes the dissolution of B vitamins. Since calcium sulfate has fully played its role in the first extraction, no additional calcium sulfate needs to be added during subsequent extractions, thereby achieving the purpose of saving reagents.

[0024] Preferably, in the above method for simultaneously detecting eight B vitamins in sweet and glutinous corn kernels during the fresh-eating period, the extraction refers to oscillating the extraction with a vortex oscillator for 15 minutes to 20 minutes.

[0025] Preferably, in the above method for simultaneously detecting eight B vitamins in sweet and glutinous corn kernels during the fresh consumption period, the centrifugation conditions are: 4° C., 12,000 r / min, centrifugation for 10 min to 15 min.

[0026] Preferably, in the above method for simultaneously detecting eight kinds of B vitamins in sweet and glutinous corn kernels during the fresh eating period, the corn kernels are kernels in the middle of the cobs of sweet and glutinous corn collected during the fresh eating period, which are quickly frozen in liquid nitrogen and then placed on dry ice.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The use of formic acid in the acetonitrile, acetic acid, formic acid and water of the present invention can improve the detection stability and reduce the detection limit of 8 types of B vitamins to 0.01 μg / mL, thereby allowing more types of B vitamins to be detected.

[0029] In the present invention, the test solution is prepared by extracting with a methanol-formic acid extract having a volume fraction of 80% to 82%, and then testing is performed by using calcium sulfate, extraction, centrifugation, and aqueous phase membrane filtration. Calcium sulfate is insoluble in formic acid and methanol and will not enter the 80% to 82% methanol-formic acid extract. Calcium sulfate is an inorganic salt and promotes the dissolution of B vitamins. Many types of B vitamins are extracted. Eight types of B vitamins can be detected at one time, reducing the number of tests and saving energy and protecting the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the ion chromatogram of thiamine standard solution.

[0031] Figure 2 This is the ion chromatogram of riboflavin standard solution.

[0032] Figure 3 This is the ion chromatogram of niacinamide standard solution.

[0033] Figure 4 This is the ion chromatogram of a standard solution of pantothenic acid.

[0034] Figure 5 This is the ion chromatogram of a standard solution of biotin.

[0035] Figure 6 This is the ion chromatogram of a standard solution of folic acid.

[0036] Figure 7 This is the ion chromatogram of the standard solution of cobalamin. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is clearly and completely described below in conjunction with specific embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0038] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0039] The present invention provides a method for simultaneously detecting eight kinds of B vitamins in sweet and waxy corn kernels during fresh consumption, wherein the eight kinds of B vitamins refer to vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9 and vitamin B 12 , it detects a wide variety of B vitamins with high accuracy and low detection limit.

[0040] The method for simultaneously detecting eight kinds of B vitamins in sweet and waxy corn kernels during fresh consumption is described in detail below.

[0041] 1. Reagents

[0042] Acetonitrile: chromatographic grade.

[0043] Methanol: chromatographic grade.

[0044] Formic acid: chromatographic grade.

[0045] Acetonitrile, acetic acid, formic acid and water: Pipette 5 mL of acetonitrile, 1 mL of glacial acetic acid and 0.3 mL of glacial acetic acid, dilute to 100 mL with deionized water and mix well.

[0046] 80% methanol-formic acid extract by volume: dilute 80 mL of methanol to 100 mL with deionized water, add 1 mL of formic acid, and mix well.

[0047] 82% methanol-formic acid extract by volume: dilute 82 mL of methanol to 100 mL with deionized water, add 1 mL of formic acid, and mix well.

[0048] 0.1% by volume formic acid methanol solution: dilute 1 mL of formic acid to 1000 mL with methanol and mix well.

[0049] 0.1% by volume formic acid aqueous solution: dilute 1 mL of formic acid to 1000 mL with deionized water and mix well.

[0050] 2. Standard products

[0051] The purity of thiamine, riboflavin, niacin, niacinamide, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, folic acid and cobalamin is ≥98%.

[0052] Except as otherwise noted above, all reagents used in this experiment were of analytical grade and the water used was deionized water.

[0053] 3. Equipment

[0054] Ultra-high performance liquid chromatography-tandem mass spectrometry (Waters H-Class UPLC and SCIEX TripleQuad4500, Z-17).

[0055] Vortex mixer.

[0056] Ultrasonic cleaning machine.

[0057] Analytical balance: sensitivity 0.0001g.

[0058] 4. Experimental corn materials

[0059] Table 1 Test corn materials

[0060] Sample No. Variety name Variety Type 1 Shenke Nuo 602 Glutinous corn 2 Suhuanuo No.2 Glutinous corn 3 Fresh Jade Glutinous Rice No. 4 Glutinous corn 4 Colorful sweet sticky 6 Glutinous corn 5 Huyu Nuo No.2 Glutinous corn 6 Jingnuo No.6 Glutinous corn 7 Huyunuo No.3 Glutinous corn 8 Wan Nuo 2000 Glutinous corn 9 Jingke Nuo 928 Glutinous corn 10 Crisp King Sweet Corn 11 Sweet Jade No.4 Sweet Corn 12 Shenketian 811 Sweet Corn 13 Sweet No. 90 Sweet Corn 14 Migo Sweet Corn

[0061] Experimental Example 1

[0062] The method for determining the content of B vitamins in a mixed standard solution comprises the following steps:

[0063] (1) Preparation of mixed standard solution

[0064] Take 0.0500g of each standard of B vitamins (thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, biotin, folic acid, and cobalamin), dissolve them in acetonitrile, acetic acid, and formic acid water, and dilute to 50mL in a brown volumetric flask. When using, accurately pipette an appropriate amount of mixed standard working solution and dilute it with water so that the concentration of each standard becomes 0.01μg / mL, 0.1μg / mL, 1.0μg / mL, 10μg / mL, 100μg / mL, 500μg / mL, and 1000μg / mL to obtain a mixed standard solution. The preparation method of acetonitrile, acetic acid, and formic acid water is as follows: take 5mL of acetonitrile, 1mL of glacial acetic acid, and 0.5mL of formic acid, dilute to 100mL with deionized water, and mix well. The mixed standard solution is used to evaluate the detection limit of 8 B vitamins.

[0065] In addition, since vitamin B6 includes pyridoxine, pyridoxal, and pyridoxamine, and vitamin B3 includes nicotinic acid and nicotinamide, and structural analogs exist, and the peak times are difficult to distinguish, the present invention also separately configures a series of concentration standard solutions of pyridoxal, pyridoxamine, and nicotinic acid, such as 0.01 μg / mL, 0.1 μg / mL, 1.0 μg / mL, 10 μg / mL, 100 μg / mL, 500 μg / mL, and 1000 μg / mL.

[0066] (2) Ultra-high performance liquid chromatography-tandem mass spectrometry detection.

[0067] (2.1) The chromatographic conditions are as follows:

[0068] Chromatographic column: Waters HSS T3 column, 2.1 mm × 100 mm × 1.8 μm.

[0069] Column temperature: 45℃.

[0070] Injection volume: 3 μL.

[0071] Flow rate: 0.38mL / min.

[0072] Mobile phase: A is 0.1% by volume formic acid methanol solution; B is 0.1% by volume formic acid aqueous solution; gradient elution conditions, see Table 2.

[0073] Table 2 Mobile phase gradient elution conditions

[0074]

[0075]

[0076] (2.2) Mass spectrometry conditions are as follows:

[0077] (a) Ion source: electrospray ionization (ESI).

[0078] (b) Curtain gas (CUR): 30 psi; Collision gas (CAD): 7 psi; Ionization voltage (IS): 5500 V; Spray gas (GS1): 50 psi; Auxiliary heating gas (GS2): 50 psi; Temperature (TEM): 550°C.

[0079] (c) Scanning mode: positive ion scanning.

[0080] (d) Detection method: multiple reaction monitoring (MRM).

[0081] The standard curve was prepared with the concentration of each standard as the horizontal axis and the response peak area as the vertical axis. The results are shown in Table 3.

[0082] Table 3 Standard curve of standard products

[0083]

[0084] The results of Experimental Example 1 show that the detection limit of the solvent used to dissolve the standard in Experimental Example 1 is as low as 0.01 μg / L, and the correlation coefficients are all greater than 0.99.

[0085] In addition, in order to assist in determining the peak time, the present invention tested the single substance standard of each B vitamin. Figure 1 This is the ion chromatogram of thiamine standard solution. Figure 2 This is the ion chromatogram of riboflavin standard solution. Figure 3 This is the ion chromatogram of niacinamide standard solution. Figure 4 This is the ion chromatogram of a standard solution of pantothenic acid. Figure 5 This is the ion chromatogram of a standard solution of biotin. Figure 6 This is the ion chromatogram of a standard solution of folic acid. Figure 7 The ion chromatogram of the standard solution of cobalamin. The parameters of the ultra-high performance liquid chromatography-tandem mass spectrometer for the detection of single substance standards of each B vitamin are shown in Table 4.

[0086] Table 4 Parameters for testing single substance standards of each B vitamin

[0087]

[0088] Example 1

[0089] A method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption, comprising the following steps:

[0090] (1) Sample preparation

[0091] Fresh corn Shenketian 811 ears were collected during the fresh-eating period, and after quick freezing in liquid nitrogen, the kernels in the middle of the ears were peeled off on dry ice, quick frozen in liquid nitrogen, and then ground with a grinder at 0°C to obtain samples to be tested.

[0092] Accurately weigh 0.1000 g of the sample to be tested into a 2 mL centrifuge tube.

[0093] Add 0.5 mL of 80% methanol formic acid extract and 0.01 g of calcium sulfate, vortex oscillator for 15 min, centrifuge at 4°C and 12000 r / min for 10 min, and obtain the first supernatant and the first residual precipitate. The first supernatant is transferred to another centrifuge tube. Repeat the above steps of "add 0.5 mL of 80% methanol formic acid extract, vortex oscillator for 15 min, centrifuge at 4°C and 12000 r / min for 10 min" for the first residual precipitate twice, that is, add 0.5 mL of 80% methanol formic acid extract to the first residual precipitate, vortex oscillator for 15 min, centrifuge at 4°C and 12000 r / min for 10 min, and collect the supernatant for the second time; add 0.5 mL of 80% methanol formic acid extract to the second residual precipitate, vortex oscillator for 15 min, centrifuge at 4°C and 12000 r / min for 10 min, and collect the supernatant for the third time.

[0094] The first supernatant, the second supernatant and the third supernatant were combined, mixed evenly, and filtered with a 0.45 μm aqueous filter membrane to obtain a test solution.

[0095] (2) Qualitative testing

[0096] The retention time of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, biotin, folic acid, and cobalamin on the total ion current chromatogram of the sample and the characteristic ions corresponding to each chromatographic peak were compared with the corresponding retention time and characteristic ions corresponding to each chromatographic peak of the standard for qualitative analysis.

[0097] The relative deviation of the retention time between the test solution and the standard is no more than 2.5%.

[0098] The allowable differences between the base peak percentages of the three characteristic ions of each B vitamin (thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, biotin, folic acid, and cobalamin) and the standard are: when the base peak percentage>50%, the allowable difference is ±20%; when 20%≤base peak percentage≤50%, the allowable difference is ±25%; when 10%<base peak percentage<20%, ​​the allowable difference is ±30%; when the base peak percentage≤10%, the allowable difference is ±50%.

[0099] The qualitative test results showed that the retention times of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, biotin, folic acid, and cobalamin and the characteristic ions corresponding to each chromatographic peak were all within the allowable error range.

[0100] (3) Quantitative detection

[0101] The solution to be tested was tested according to the method of "(2) ultra-high performance liquid chromatography-tandem mass spectrometry" in Example 1, and then substituted into the standard curve of Example 1 to calculate the content of each B vitamin. Note that the response values ​​of the components to be tested in the solution to be tested should be within the linear range of the instrument. If the response value of the solution to be tested exceeds the linear range of the standard curve, it should be diluted with water before analysis.

[0102] The content of the B vitamin component in the test solution is measured in mass fraction, in micrograms per kilogram (μg / kg), and is calculated according to the following formula:

[0103]

[0104] Where: X is the content of B vitamins in the solution to be tested, in micrograms per kilogram (μg / kg); C i is the concentration of B vitamins obtained from the standard curve, in micrograms per liter (μg / L); V is the volume of the solution to be tested, in milliliters (mL); m is the mass of the sample to be tested, in grams (g);

[0105] The calculation result shall be rounded to two significant figures.

[0106] The method of Example 2 was repeated 10 times to examine the stability. The results are shown in Table 5.

[0107] Table 5 Stability test results of Example 1 (Shen Ketian 811)

[0108] B vitamins ingredients Average μg / kg Standard Deviation Thiamine 114.11 2.32％ Riboflavin 135.15 2.53％ Niacinamide 849.85 2.16％ Pantothenic acid 325.06 2.60％ Pyridoxine 346.84 2.70％ Biotin 168.74 2.11％ Folic acid 250.10 1.89％ Cobalamin - -

[0109] Note: In label 5, “-” means not detected.

[0110] Compared with the prior art study "Comparative analysis of B vitamin content in sweet corn and glutinous corn inbred lines", the present invention tests more types of B vitamins and reduces the standard deviation of the test results.

[0111] In addition, the other experimental corn materials in Table 1 were tested, and the seven B vitamins in Table 5 except cobalamin were detected, and after 10 repetitions, the standard deviation was between 1.89% and 2.70%, and the detection limit was 0.01 μg / mL. On the one hand, it shows that the method of the present invention has good reproducibility and low detection limit, and on the other hand, it also shows that cobalamin was not detected in corn.

[0112] Comparative Example 1

[0113] A method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption is basically the same as the operation of Example 1, the only difference being that only the extract is added to the sample to be tested, and no calcium sulfate is added.

[0114] The method of the control example 1 was repeated 10 times to examine the stability, and the results are shown in Table 6. Compared with the stability test results of Example 1 in Table 5, the standard deviation is larger, and only 5 B vitamins are detected.

[0115] Table 6 Comparative Example 1 Stability Test Results (Shen Ketian 811)

[0116]

[0117]

[0118] Example 2

[0119] A method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption, comprising the following steps:

[0120] (1) Sample preparation

[0121] During the fresh-eating period, the ears of fresh corn Shenke Nuo 602 were collected, quick-frozen in liquid nitrogen, and the kernels in the middle of the ears were peeled off on dry ice, quick-frozen in liquid nitrogen, and then ground at 0°C using a grinder to obtain the samples to be tested.

[0122] Accurately weigh 0.1000 g of the sample to be tested into a 5 mL centrifuge tube.

[0123] Add 1.5 mL of 82% methanol-formic acid extract and 0.02 g of calcium sulfate, vortex and extract for 20 min, centrifuge at 4°C and 12000 r / min for 15 min to obtain the first supernatant and the first residual precipitate, and transfer the first supernatant to another centrifuge tube. The first residual precipitate was extracted three times by repeating the above steps of "adding 1.5 mL of 82% methanol and formic acid extract, vortexing and extracting for 20 min, and centrifuging at 4°C and 12000 r / min for 15 min", that is, 1.5 mL of 82% methanol and formic acid extract was added to the first residual precipitate, vortexed and extracted for 20 min, centrifuged at 4°C and 12000 r / min for 15 min, and the supernatant was collected for the second time; 1.5 mL of 82% methanol and formic acid extract was added to the second residual precipitate, vortexed and extracted for 15 min, centrifuged at 4°C and 12000 r / min for 15 min, and the supernatant and the third residual precipitate were collected for the third time; 1.5 mL of 82% methanol and formic acid extract was added to the third residual precipitate, vortexed and extracted for 20 min, centrifuged at 4°C and 12000 r / min for 15 min, and the supernatant was collected for the fourth time.

[0124] The first supernatant, the second supernatant, the third supernatant and the fourth supernatant were combined, mixed evenly, and filtered through a 0.45 μm aqueous filter membrane to obtain a test solution.

[0125] (3) Quantitative detection

[0126] The solution to be tested was tested according to the method of "(3) Quantitative detection" in Experimental Example 1.

[0127] The method of Example 2 was repeated 10 times to examine the stability. The results are shown in Table 7.

[0128] Table 7 Stability test results of Example 2

[0129] B vitamins ingredients Average μg / kg Standard Deviation Thiamine 165.842 2.88％ Riboflavin 631.87 2.64％ Niacinamide 368.22 2.16％ Pantothenic acid 234.81 2.71％ Pyridoxine 236.42 2.60％ Biotin 155.07 2.11％ Folic acid 423.85 2.08％ Cobalamin - -

[0130] Note: “-” in Table 7 means not detected.

[0131] Compared with the prior art study "Comparative analysis of B vitamin content in sweet corn and glutinous corn inbred lines", the present invention tests more types of B vitamins and reduces the standard deviation of the test results.

[0132] In addition, the other test corn materials in Table 1 were tested, and the seven B vitamins in Table 7 except cobalamin were detected. After repeating 10 times, the standard deviation was between 2.08% and 2.88%, and the detection limit was 0.01 μg / mL.

[0133] Experimental Example 3

[0134] Spike recovery experiment

[0135] Thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxine, biotin, folic acid and cobalamin were added to the test solution of Shenketian 811 obtained in Example 1 to test the content. The results are shown in Tables 8 and 9.

[0136] Table 8 Recovery rate of low concentration addition

[0137]

[0138]

[0139] Table 9 Recovery rate of high concentration addition

[0140] B vitamins ingredients Added concentration (μg / kg) Recovery rate (%) Standard deviation (%) Thiamine 300 100.18 2.44 Riboflavin 300 106.77 1.26 Niacinamide 300 99.48 2.52 Pantothenic acid 300 98.28 2.11 Pyridoxine 300 98.41 2.08 Biotin 300 99.40 1.25 Folic acid 300 101.05 2.07 Cobalamin 300 101.11 2.00

[0141] The results in Tables 8 and 9 show that, regardless of whether the addition amount is low or high, the standard deviation is less than 3% and the recovery rate is higher than 98%.

[0142] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0143] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. A method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption, characterized in that: include: Prepare a test solution using fresh corn kernels, wherein the fresh corn kernels are fresh sweet corn kernels or fresh glutinous corn kernels; Quantitatively take each standard of 8 kinds of B vitamins, dissolve and fix the volume with acetonitrile, acetic acid and formic acid water, dilute to a mixed standard solution with a series of concentrations, and prepare a standard curve for each standard; the volume fractions of acetonitrile, glacial acetic acid and formic acid in the acetonitrile, acetic acid and formic acid water are 5% to 5.5%, 1% and 0.3% to 0.5% respectively; The solution to be tested is tested and substituted into the standard curve to calculate the content of each B vitamin; The eight B vitamins are vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9 and vitamin B 12 .

2. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 1, characterized in that: The vitamin B6 includes at least one of pyridoxine, pyridoxal and pyridoxamine.

3. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 1, characterized in that: Vitamin B3 includes at least one of niacin and niacinamide.

4. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 1, characterized in that: The method for preparing the test solution is as follows: The fresh sweet corn or glutinous corn kernels are quickly frozen and then ground below 0°C to obtain a sample to be tested; and the B vitamins in the sample to be tested are extracted with an extracting solution to obtain a test solution.

5. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 4, characterized in that: The extract is a methanol-formic acid extract with a volume fraction of 80% to 82%. In the methanol-formic acid extract with a volume fraction of 80% to 82%, the volume fraction of methanol is 80% to 82%, the volume of formic acid is 1%, and deionized water is supplemented to 100%.

6. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 5, characterized in that: The method of extracting B vitamins from the sample to be tested using the extracting solution is as follows: Adding the extract and calcium sulfate to the sample to be tested, extracting, centrifuging, collecting the supernatant, filtering the supernatant with an aqueous filter membrane to remove impurities, and obtaining a solution to be tested; For every gram of sample to be tested, 5 mL to 15 mL of extract and 0.1 g to 0.2 g of calcium sulfate are added.

7. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 6, characterized in that: Repeat the following steps 2 to 3 times for the precipitate after centrifugation: add extraction solution, extract, centrifuge, collect new supernatant and new precipitate; Collect and mix all the supernatants, and filter them through an aqueous filter membrane to remove impurities to obtain a test solution.

8. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 6, characterized in that: Extraction refers to oscillating extraction for 15 minutes to 20 minutes.

9. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 6, characterized in that: The centrifugal conditions are: 4°C, 12000r / min for 10min to 15min.

10. The method for simultaneously detecting eight B vitamins in sweet and waxy corn kernels during fresh consumption according to claim 1, characterized in that: The corn kernels are kernels in the middle of fresh cobs of sweet corn or glutinous corn collected during the fresh-eating period, which are quickly frozen and then placed on dry ice and peeled off.

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