CN110596267A - Method for determining phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography - Google Patents

Method for determining phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography Download PDF

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CN110596267A
CN110596267A CN201910872196.6A CN201910872196A CN110596267A CN 110596267 A CN110596267 A CN 110596267A CN 201910872196 A CN201910872196 A CN 201910872196A CN 110596267 A CN110596267 A CN 110596267A
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supernatant
phytic acid
dihydrogen phosphate
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扈战强
胡贤巧
方长云
曹赵云
朱智伟
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China National Rice Research Institute
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China National Rice Research Institute
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Abstract

The invention discloses a method for determining phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography, which comprises the following steps: 1) sample preparation: weighing 0.5-10g of sample powder, adding hydrochloric acid extract to obtain sample solution, performing ultrasonic extraction and centrifugation to obtain supernatant, and filtering, evaporating to dryness, performing ultrasonic dissolution and centrifuging to obtain sample supernatant; 2) solid phase extraction: passing the prepared sample supernatant through a column, and absorbing and loading the eluent after passing the sodium dihydrogen phosphate solution through the column; 3) chromatographic conditions and detection on a computer: the water-resistant chromatographic column is water-resistant C18A column, wherein the mobile phase is sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes; 4) and (3) calculating the result: obtaining the concentration of the organic liquid on the sample according to a standard curve equationThe degree c and the phytic acid content are calculated according to the formula: the phytic acid content is c 5V/(3 m). The method has the advantages of accurate measurement data and low measurement cost in the measurement of the phytic acid content in the grain crops.

Description

Method for determining phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography
Technical Field
The invention relates to the technical field of phytic acid detection, in particular to a method for determining phytic acid content in grain crops by using solid-phase extraction high performance liquid chromatography.
Background
Inositol hexaphosphate, also known as phytic acid, is the main storage form of phosphorus in various plant tissues (especially rice bran and seeds), but human and non-ruminant animals are not able to digest phytic acid and therefore it is neither a source of inositol nor a source of phosphoric acid for the diet. Due to the special molecular structure of the phytic acid, the phytic acid can be combined with metal salt ions and proteins to form a stable chelate, so that the absorption of mineral elements is reduced, the digestion and utilization of the proteins are reduced, the activity of digestive enzyme is reduced, and the environment is adversely affected by high-phosphorus excrement. Phytic acid is therefore also known as an antinutritional factor. Secondly, it is also an anti-cancer new soldier, and scientists have demonstrated that this nutrient has a surprising effect on the help of cancer patients.
The existing detection methods of the phytic acid comprise a ferric trichloride-sulfosalicylic acid method, a quinomolybdic citranone precipitation method, an HPLC-MS method and the like. The principle of the ferric trichloride-sulfosalicylic acid method is that a sample is extracted by an acid solution, the sample is adsorbed and desorbed by anion exchange resin, phytic acid in eluent and ferric trichloride-sulfosalicylic acid mixed solution generate a fading reaction, a spectrophotometer is used for measuring absorbance at a wavelength of 500nm, and the phytic acid content in the sample is calculated. The method is applied to the detection of the phytic acid in edible oil, processed fruits, meat products, fresh shrimps, candies, fruit and vegetable beverages by the national safety standard GB 5009.153-2016, and cannot be applied to grain crops; in addition, the iron ions in the color reagent can also react with other lower inositol phosphates to increase the phytic acid content. The principle of the quinomolybdenyl citraconic precipitation method is that phosphorus ions are decomposed from phosphorus-containing compounds by using concentrated nitric acid, the quinomolybdenyl citraconic reagent is precipitated with the phosphorus ions, then the phosphorus content is determined by a gravimetric method, and the phytic acid content is converted. The principle of the HPLC-MS method is that the mass spectrum of phytic acid and other phosphoinositide compounds and other phosphorus-containing compounds is obtained by liquid chromatography-mass spectrometry, and accurate qualitative and quantitative analysis can be performed.
Disclosure of Invention
The invention aims to solve the problem of detection of phytic acid content in grain crops, and provides a method for determining the phytic acid content in the grain crops by solid-phase extraction-high performance liquid chromatography, which has accurate measurement data and low measurement cost.
In order to achieve the purpose, the invention adopts the following technical scheme that the method for measuring the phytic acid content in the grain crops by using the solid-phase extraction high performance liquid chromatography comprises the following steps:
1) sample preparation: grinding grains, sieving with a 60-mesh sieve, weighing 0.5-10g of sample powder, placing the sample powder into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution into the centrifuge tube to obtain a sample solution, performing ultrasonic extraction on the obtained sample solution at room temperature for 1h, performing centrifugation at 10000r/min for 15min to obtain a supernatant, filtering the obtained supernatant with a 0.45-micron filter membrane, sucking 3mL of the supernatant, placing the supernatant into a 25mL beaker, placing the beaker and the supernatant in a ventilation cabinet, evaporating to dryness in a 40 ℃ water bath, adding 5mL of pure water into the beaker, performing ultrasonic dissolution, transferring into the centrifuge tube, and performing centrifugation at 10000r/min for 15min to obtain a sample supernatant for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: the water-resistant chromatographic column is ZORBAX SB-AqC18Column or Hypersil BDS C18Pillars or other water-resistant C18A column; the mobile phase is 0.025mol/L sodium dihydrogen phosphate solution, and the flow rate is 0.5mL/min, column temperature of 30 ℃, detection wavelength of 254nm, sample amount of 10 mu L, needle washing liquid of 0.025mol/L sodium dihydrogen phosphate solution, and analysis and detection time of 6 minutes; the standard curve adopts an external standard method, and the concentrations of the standard solutions are respectively 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL and 4.0 mg/mL;
4) and (3) calculating the result: the result is marked by mg/g, a standard curve and a standard curve equation are obtained by plotting according to the peak area and the concentration of the standard sample, the concentration c of the organic liquid on the sample is obtained according to the standard curve equation, and the calculation formula of the phytic acid content is as follows: the phytic acid content is c × 5 × V/(3 × m), where V is the volume of the extract, 5 is the volume of the eluate eluted, 3 is the volume of the supernatant withdrawn, and m is the mass of the weighed sample powder.
Preferably, the grain crops comprise indica rice, japonica rice, wheat, corn, sorghum, millet, mung bean, soybean, potato and sweet potato.
The Solid Phase Extraction (SPE) in the scheme is a sample pretreatment technology developed from the middle of eighties, is developed by combining liquid-Solid Extraction and liquid chromatography, is mainly used for separating, purifying and enriching samples, and mainly aims at reducing sample matrix interference and improving detection sensitivity. High Performance Liquid Chromatography (High Performance Liquid Chromatography, HPLC), also known as "High pressure Liquid Chromatography", "High Performance Liquid Chromatography", "High resolution Liquid Chromatography", "modern column Chromatography", etc., is an important branch of Chromatography, wherein Liquid is used as a mobile phase, a High pressure infusion system is adopted to pump mobile phases such as single solvents with different polarities or mixed solvents, buffers, etc. with different proportions into a chromatographic column filled with a stationary phase, and after components in the column are separated, the mobile phases enter a detector for detection, thereby realizing analysis of a sample. The method adopts a measurement method combining solid phase extraction and high performance liquid chromatography to measure the phytic acid content in the grain crops.
Therefore, the invention has the following beneficial effects: in the measurement of the phytic acid content in the grain crops, the measurement data is accurate, and the measurement cost is low.
Drawings
FIG. 1 is a liquid chromatogram of a phytic acid standard sample of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The method for determining the phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography comprises the following steps:
sample preparation: grinding grains, sieving with a 60-mesh sieve, weighing 0.5-10g of sample powder, placing the sample powder into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution into the centrifuge tube to obtain a sample solution, performing ultrasonic extraction on the obtained sample solution at room temperature for 1h, performing centrifugation at 10000r/min for 15min to obtain a supernatant, filtering the obtained supernatant with a 0.45-micron filter membrane, sucking 3mL of the supernatant, placing the supernatant into a 25mL beaker, placing the beaker and the supernatant in a ventilation cabinet, evaporating to dryness in a 40 ℃ water bath, adding 5mL of pure water into the beaker, performing ultrasonic dissolution, transferring into the centrifuge tube, and performing centrifugation at 10000r/min for 15min to obtain a sample supernatant for later use;
solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
chromatographic conditions and detection on a computer: the water-resistant chromatographic column is ZORBAX SB-AqC18Column or Hypersil BDS C18Pillars or other water-resistant C18A column; the mobile phase is 0.025mol/L sodium dihydrogen phosphate solution, the flow rate is 0.5mL/min, the column temperature is 30 ℃, the detection wavelength is 254nm, the sample injection amount is 10 mu L, the needle washing liquid is 0.025mol/L sodium dihydrogen phosphate solution, and the analysis detection time is 6 minutes; the standard curve adopts an external standard method, the concentrations of standard solutions are respectively 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL and 4.0mg/mL, a standard curve equation is obtained by utilizing the peak area and the standard solution concentration, and then the phytic acid in the sample extracting solution is determinedContent (c);
and (3) calculating the result: the result is marked by mg/g, as shown in figure 1, a standard curve and a standard curve equation are obtained by drawing according to the peak area and the concentration of the standard product, the concentration c of the organic liquid on the sample is obtained according to the standard curve equation, and the calculation formula of the phytic acid content is as follows: the phytic acid content is c × 5 × V/(3 × m), where V is the volume of the extract, 5 is the volume of the eluate eluted, 3 is the volume of the supernatant withdrawn, and m is the mass of the weighed sample powder.
The specific use process is, in example 1, the determination of the phytic acid content in indica rice:
1) firstly, husking indica rice, grinding the indica rice, sieving the milled indica rice by a sieve of 60 meshes, weighing about 2.00g of sample powder in a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging the obtained product at 10000r/min for 15min, filtering the obtained supernatant by a 0.45 mu m filter membrane, sucking 3mL of the obtained product in a 25mL beaker, evaporating the obtained product in a 40 ℃ water bath in a ventilation cabinet, adding 5mL of pure water, performing ultrasonic dissolution, transferring the obtained product to the centrifuge tube, centrifuging the obtained product at 10000r/min for 15min, and keeping the obtained supernatant for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. Obtaining a standard curve equation by using the peak area and the concentration of the standard solutionAnd further determining the content of phytic acid in the sample extracting solution.
Example 2 determination of phytic acid content in japonica rice:
1) firstly, husking japonica rice, grinding the japonica rice into powder, sieving the powder by a 60-mesh sieve, weighing about 2.00g of sample powder into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction at room temperature for 1h, centrifuging the powder at 10000r/min for 15min, filtering the supernatant by a 0.45-micron filter membrane, sucking 3mL of the supernatant into a 25mL beaker, evaporating the supernatant in a 40-DEG C water bath in a fume hood to dryness, adding 5mL of pure water, performing ultrasonic dissolution, transferring the solution into the centrifuge tube, centrifuging the powder at 10000r/min for 15min, and reserving the supernatant;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 3 determination of phytic acid content in wheat flour:
1) firstly, grinding wheat to obtain wheat flour, sieving with a 60-mesh sieve, then weighing about 2.00g of sample powder into a 30mL centrifuge tube, then adding 20mL of 0.5mol/L hydrochloric acid extracting solution, ultrasonically extracting for 1h at room temperature, centrifuging for 15min at 10000r/min, filtering supernatant with a 0.45-micron filter membrane, sucking 3mL into a 25mL beaker, evaporating to dryness in a 40 ℃ water bath in a ventilation cabinet, then adding 5mL of pure water, ultrasonically dissolving, then transferring into the centrifuge tube, centrifuging for 15min at 10000r/min, and reserving supernatant;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 4 determination of phytic acid content in corn:
1) firstly, grinding corns, sieving with a 60-mesh sieve, then weighing about 2.00g of sample powder into a 30mL centrifuge tube, then adding 20mL0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging for 15min at 10000r/min, filtering supernate with a 0.45-micron filter membrane, sucking 3mL into a 25mL beaker, evaporating to dryness in a 40 ℃ water bath in a ventilation cabinet, then adding 5mL of pure water, performing ultrasonic dissolution, then transferring into the centrifuge tube, centrifuging for 15min at 10000r/min, and reserving supernate for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 5 determination of phytic acid content in sorghum:
1) firstly, husking and grinding sorghum, sieving with a 60-mesh sieve, then weighing about 2.00g of sample powder into a 30mL centrifuge tube, then adding 20mL0.5mol/L hydrochloric acid extract, ultrasonically extracting for 1h at room temperature, centrifuging for 15min at 10000r/min, filtering supernatant with a 0.45-micron filter membrane, sucking 3mL into a 25mL beaker, evaporating to dryness in a 40 ℃ water bath in a ventilation cabinet, then adding 5mL of pure water, ultrasonically dissolving, then transferring into the centrifuge tube, centrifuging for 15min at 10000r/min, and reserving supernatant;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 6, determination of phytic acid content in millet:
1) firstly, husking and grinding millet, sieving by a 60-mesh sieve, then weighing about 2.00g of sample powder in a 30mL centrifuge tube, then adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging for 15min at 10000r/min, filtering supernate by a 0.45-micron filter membrane, sucking 3mL of the supernate into a 25mL beaker, drying the supernate in a 40 ℃ water bath in a ventilation cabinet by distillation, then adding 5mL of pure water, performing ultrasonic dissolution, then transferring the supernate into the centrifuge tube, centrifuging for 15min at 10000r/min, and reserving the supernate for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. Using peak area and standard solution concentrationAnd obtaining a standard curve equation, and further determining the content of the phytic acid in the sample extracting solution.
Example 7 determination of phytic acid content in mung beans:
1) grinding mung beans, sieving with a 60-mesh sieve, weighing about 0.5g of sample powder into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging at 10000r/min for 15min, filtering the supernatant with a 0.45-micron filter membrane, sucking 3mL of the supernatant into a 25mL beaker, evaporating in a 40-DEG C water bath in a fume hood, adding 5mL of pure water, performing ultrasonic dissolution, transferring into the centrifuge tube, centrifuging at 10000r/min for 15min, and reserving the supernatant;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 8 determination of phytic acid content in soybeans:
1) grinding soybeans, sieving with a 60-mesh sieve, weighing about 0.5g of sample powder into a 30mL centrifuge tube, adding 20mL0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging at 10000r/min for 15min, filtering the supernatant with a 0.45-micron filter membrane, sucking 3mL into a 25mL beaker, evaporating in a 40 ℃ water bath in a fume hood, adding 5mL of pure water, performing ultrasonic dissolution, transferring into the centrifuge tube, centrifuging at 10000r/min for 15min, and reserving the supernatant;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 9 determination of phytic acid content in potatoes:
1) peeling and homogenizing potatoes, weighing about 5.00g of homogenate liquid into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging at 10000r/min for 15min, filtering supernate with a 0.45 mu m filter membrane, sucking 3mL of the supernate into a 25mL beaker, evaporating in a 40 ℃ water bath in a fume hood, adding 5mL of pure water, performing ultrasonic dissolution, transferring into the centrifuge tube, centrifuging at 10000r/min for 15min, and reserving the supernate for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0.025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
Example 10 determination of the phytic acid content in sweet potatoes:
1) peeling sweet potatoes, and then 1: 1, adding water for homogenizing, weighing about 10.0g of homogenate liquid into a 30mL centrifuge tube, then adding 20mL of 0.5mol/L hydrochloric acid extracting solution, performing ultrasonic extraction for 1h at room temperature, centrifuging for 15min at 10000r/min, filtering supernate with a 0.45-micron filter membrane, absorbing 3mL of the supernate into a 25mL beaker, evaporating to dryness in a 40 ℃ water bath in a fume hood, then adding 5mL of pure water, performing ultrasonic dissolution, transferring into the centrifuge tube, centrifuging for 15min at 10000r/min, and reserving the supernate for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: water-tolerant phase chromatographic column: ZORBAX SB-AqC18Column (4.6X 250mm, 5 μm) or Hypersil BDS C18Column (4.6X 250mm, 5 μm) or other water-resistant C18Column, mobile phase: 0025mol/L sodium dihydrogen phosphate (chromatographically pure) solution, flow rate: 0.5mL/min, column temperature: 30 ℃, detection wavelength: 254nm, sample size: 10 mu L, and the needle washing liquid is: 0.025mol/L sodium dihydrogen phosphate solution, and the analysis and detection time is 6 minutes. The standard curve adopts an external standard method, and the concentrations of standard solutions are respectively as follows: 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 4.0 mg/mL. And obtaining a standard curve equation by utilizing the peak area and the concentration of the standard solution, and further determining the content of the phytic acid in the sample extracting solution.
The table for comparing the scheme with the traditional quinomolybdenyl citraconic ketone precipitation method for determining the phytic acid content (mg/g) in the grain crops is as follows:
new method Precipitation method of quinmolybdic citraconic ketone
Indica rice 17.7 18.5
Japonica rice 18.8 19.2
Wheat (Triticum aestivum L.) 2.4 3.2
Corn (corn) 1.6 2.0
Sorghum grain 3.7 3.8
Millet 14.2 15.0
Mung bean 94.3 96.5
Soybean 149.5 156.2
Potato 0.5 0.6
Sweet potato 0.6 0.8
Finally, it should be pointed out that the invention is not limited to the products related to the above embodiments, and all modifications directly derivable or suggested to a person skilled in the art from the disclosure of the present invention are considered to be within the scope of the present invention.

Claims (2)

1. A method for determining the phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography is characterized by comprising the following steps:
1) sample preparation: grinding grains, sieving with a 60-mesh sieve, weighing 0.5-10g of sample powder, placing the sample powder into a 30mL centrifuge tube, adding 20mL of 0.5mol/L hydrochloric acid extracting solution into the centrifuge tube to obtain a sample solution, performing ultrasonic extraction on the obtained sample solution at room temperature for 1h, performing centrifugation at 10000r/min for 15min to obtain a supernatant, filtering the obtained supernatant with a 0.45-micron filter membrane, sucking 3mL of the supernatant, placing the supernatant into a 25mL beaker, placing the beaker and the supernatant in a ventilation cabinet, evaporating to dryness in a 40 ℃ water bath, adding 5mL of pure water into the beaker, performing ultrasonic dissolution, transferring into the centrifuge tube, and performing centrifugation at 10000r/min for 15min to obtain a sample supernatant for later use;
2) solid phase extraction: firstly, 15mL of pure water and 5mL of 0.025mol/L sodium dihydrogen phosphate solution are used for passing through a strong anion solid phase extraction small column for activation, the flow rate is 1mL/min, the washing is continued by 15mL of pure water, then the prepared sample supernatant is passed through the column, the flow rate is 1mL/min, 5mL of eluent 0.0025mol/L sodium dihydrogen phosphate solution is used for passing through the column, the flow rate is 1mL/min, finally, 5mL of eluent 0.025mol/L sodium dihydrogen phosphate solution is used for passing through the column, and the eluent is absorbed and loaded on the machine;
3) chromatographic conditions and detection on a computer: the water-resistant chromatographic column is ZORBAX SB-AqC18Column or Hypersil BDS C18Pillars or other water-resistant C18A column; the mobile phase is 0.025mol/L sodium dihydrogen phosphate solution, the flow rate is 0.5mL/min, the column temperature is 30 ℃, the detection wavelength is 254nm, the sample injection amount is 10 mu L, the needle washing liquid is 0.025mol/L sodium dihydrogen phosphate solution, and the analysis detection time is 6 minutes; the standard curve adopts an external standard method, and the concentrations of the standard solutions are respectively 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1.0mg/mL, 2.0mg/mL and 4.0 mg/mL;
4) and (3) calculating the result: the result is marked by mg/g, a standard curve and a standard curve equation are obtained by plotting according to the peak area and the concentration of the standard sample, the concentration c of the organic liquid on the sample is obtained according to the standard curve equation, and the calculation formula of the phytic acid content is as follows: the phytic acid content is c × 5 × V/(3 × m), where V is the volume of the extract, 5 is the volume of the eluate eluted, 3 is the volume of the supernatant withdrawn, and m is the mass of the weighed sample powder.
2. The method for determining the phytic acid content in the grain crops by the solid-phase extraction high performance liquid chromatography as claimed in claim 1, wherein the grain crops comprise indica rice, japonica rice, wheat, corn, sorghum, millet, mung bean, soybean, potato and sweet potato.
CN201910872196.6A 2019-09-16 2019-09-16 Method for determining phytic acid content in grain crops by solid-phase extraction high performance liquid chromatography Pending CN110596267A (en)

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