CN114577943B - Method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder - Google Patents
Method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder Download PDFInfo
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- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 title claims abstract description 58
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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Abstract
The invention discloses a method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder, which comprises the following steps: adding a Streptomyces griseus protease solution into a sample for enzymolysis to extract aspartic acid and glutamic acid, and deriving through AQC; hydrochloric acid is added into the sample to hydrolyze and extract aspartic acid and glutamic acid, and the sample is derivatized by AQC; preparing a standard solution: and (3) measuring by adopting liquid chromatography-mass spectrometry. By combining acid hydrolysis and innovative enzymolysis technology, an indirect determination method of aspartic acid and glutamic acid in UPLC-MS/MS infant formula milk powder and prepared milk powder products is established, and a technical reference is provided for accurate determination of aspartic acid and glutamic acid in milk powder.
Description
Technical Field
The invention belongs to the field of food detection, and particularly relates to an indirect measurement method for aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder.
Background
Infant formula milk powder and prepared milk powder are the most main milk products in China. The milk protein in the infant formula milk powder and the prepared milk powder contains all essential amino acids required by the growth and development of human bodies of corresponding people, and is a high-efficiency absorption complete protein. The protein is a mark nutrient component for evaluating the nutrition value and quality of the milk powder, and the basic constituent unit amino acid component of the protein, so that the quality of the infant formula milk powder and the quality of the prepared milk powder can be indirectly evaluated by the analysis and detection of the amino acid content. Aspartic acid and glutamic acid are important amino acids constituting protein amino acids, but there is no corresponding national standard detection method.
The detection standard of amino acid in domestic food is GB 5009.124-2016 (determination of amino acid in food), and the standard adopts acid hydrolysis to determine the content of 16 amino acids in food. Since the pretreatment of GB 5009.124-2016 adopts acid hydrolysis, asparagine and glutamine are completely converted into aspartic acid and glutamic acid in the hydrolysis process, so that the contents of aspartic acid and glutamic acid cannot be accurately measured. At present, most of amino acid detection reported at home and abroad adopts an acid hydrolysis method, and the accurate measurement of aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder is freshly reported.
Currently, amino acid detection techniques mainly include an amino acid analyzer, a high performance liquid chromatograph, a liquid chromatograph-tandem mass spectrometer, and the like. Among them, the amino acid analyzer has specific use, poor flexibility and long analysis time. Liquid chromatography has wide universality, but has high requirements on chromatographic separation conditions, and infant formula milk powder and prepared milk powder products have complex matrixes, contain a large amount of nutrient components such as vitamins and the like, and can interfere with the determination of target compounds. The liquid chromatography-tandem mass spectrometry can realize the accurate qualitative and quantitative determination of co-elution compounds with different mass-to-charge ratios, can effectively reduce the chromatographic running time, has the advantages of high sensitivity, good selectivity, high analysis speed and the like, and is more suitable for the analysis and detection of target compounds in complex matrixes. Because the amino acid compound has strong polarity and small molecular weight, characteristic ions in a complex matrix are severely interfered by the matrix, and the amino acid compound is not suitable for direct detection of a mass spectrum detector. The chemical derivatization method can change the chromatographic and mass spectrum characteristics of the target object, improve the chromatographic separation effect and improve the mass spectrum signal intensity. Typical derivatizing agents are Phenyl Isothiocyanate (PITC), 2, 4-Dinitrofluorobenzene (DNFB), phthalaldehyde (OPA), 9-fluorenyl ester (FMOC), OPA-FMOC combination, dansyl chloride (Dansyl-Cl), 6-aminoquinolinyl-N-hydroxy-succinimidyl formate (AQC), ninhydrin, and the like. The AQC and primary amine or secondary amine react to generate stable derivative urea, the operation is simple, the derivatization speed is high, and excessive reagent can be naturally hydrolyzed without special treatment or extraction; and is not interfered by sample matrix, and is more suitable for analyzing amino acid in complex matrixes such as infant formula milk powder, prepared milk powder and the like compared with other derivative reagents.
Disclosure of Invention
The research adopts AQC pre-column derivatization, and in view of the problem of inaccurate detection of aspartic acid and glutamic acid and the complex characteristics of infant formula milk powder and a prepared milk powder substrate, an indirect determination method of the aspartic acid and the glutamic acid in UPLC-MS/MS infant formula milk powder and the prepared milk powder is established by adopting the combination of acid hydrolysis and innovative enzymolysis technology, so that technical reference is provided for accurate determination of the aspartic acid and the glutamic acid in the milk powder.
The invention aims to provide a method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder. The method can accurately determine the contents of asparagine and glutamine. The accurate determination of aspartic acid and glutamic acid is realized by acid hydrolysis and enzyme hydrolysis and adopting a differential method.
The invention is realized by the following technical scheme:
a method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder comprises the following steps:
(1) Adding a Streptomyces griseus protease solution into a sample for enzymolysis to extract aspartic acid and glutamic acid, and deriving through AQC;
(2) Hydrochloric acid is added into the sample to hydrolyze and extract aspartic acid and glutamic acid, and the sample is derivatized by AQC;
(3) Preparing a standard solution:
standard stock solution 10.0 mmol/L: accurately weighing a proper amount of standard substances in a 10 mL volumetric flask respectively, and fixing the volume to the scale by using 0.1mol/L hydrochloric acid respectively;
standard working solution: diluting the standard stock solutions to prepare mixed standard solutions, and gradually diluting until the components are serial standard working solutions of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 100.0 and 200.0 mu mol/L before use;
(4) Liquid chromatography-mass spectrometry was used: the aspartic acid content is the concentration of aspartic acid in step (2) minus the aspartic acid content measured in step (1); the glutamic acid content is the concentration of glutamic acid in step (2) minus the glutamic acid content measured in step (1).
Further, the conditions of the liquid chromatography are: chromatographic column: accQ-Tag Ultra C18,2.1 mm ×100 mm,1.7 μm; mobile phase: A. 10 mmol/L ammonium formate buffer solution, B, acetonitrile; sample injection volume is 1 mu L; column temperature 40 ℃; the detection wavelength 260nm and the gradient elution procedure are shown in the table:
further, the mass spectrometry conditions were: ion source: a jet electrospray ion source; ionization mode: positive ion mode (esi+); the temperature of the drying gas is 250 ℃; the dry air flow is 7L/min; atomization air pressure 45 psi; sheath gas temperature is 350 ℃; sheath air flow, 12L/min; capillary voltage 4000V; nozzle voltage 0V; acquisition mode: multiple reaction monitoring.
Specifically, the specific steps of the step (1) are as follows:
extracting: after uniformly mixing the samples, weighing 0.1g into a 50 mL threaded centrifuge tube with a cover, adding 0.5 mL of 10mg/mL Streptomyces griseus protease solution, 0.1mol/L of 3.0 mL Tris buffer solution with pH of 8.5 and 200 mu L of methanol, uniformly mixing by vortex, carrying out ultrasonic treatment for 20 min, oscillating for 18 h-20 h in a 50 ℃ constant temperature water bath oscillator, and then placing the samples at 90 ℃ for 15-20 min; taking out the sample, cooling to room temperature, transferring the sample solution into a 25 mL volumetric flask, and fixing the volume to the scale with water; after shaking fully and evenly, accurately measuring 100 mu L to 5 mL volumetric flask, adding 50 mu L of 12 amino acid mixed standard solution internal standard, then using ultrapure water to fix the volume to 5 mL, and filtering by a 0.22 mu m organic phase microporous filter membrane for later use;
and (3) derivatization: transferring 10 mu L of standard working solution or the sample extracting solution to the bottom of a 6 mm multiplied by 50 mm test tube, adding 70 mu L of AccQ-Fluor borate buffer solution, mixing by vortex, adding 20 mu L of AccQ-Fluor derivative (6-aminoquinolinyl-N-hydroxy-succinimidyl formate), immediately mixing by vortex, standing at room temperature for 1 min, transferring the sample solution into a micro automatic sample injection bottle, sealing by cover, heating on a 55 ℃ heating device for 10 min, cooling to room temperature, and performing machine measurement.
Specifically, the specific steps of the step (2) are as follows: after uniformly mixing the samples, weighing 0.1g of the samples into a hydrolysis tube, adding 10 mL of 6mol/L hydrochloric acid, filling high-purity nitrogen, sealing, putting the sealed hydrolysis tube into an electrothermal blowing incubator at 110 ℃, hydrolyzing 22 h, taking out, and cooling to room temperature; transferring all liquid of the sample solution in the hydrolysis tube into a 25 mL volumetric flask, and fixing the volume to a scale by using water; after shaking fully and evenly, accurately measuring 100 mu L to 5 mL volumetric flask, adding 50 mu L of 12 amino acid mixed standard solution internal standard, then using ultrapure water to fix the volume to 5 mL, and filtering by a 0.22 mu m organic phase microporous filter membrane for later use;
and (3) derivatization: transferring 10 mu L of standard working solution or the sample extracting solution to the bottom of a 6 mm multiplied by 50 mm test tube, adding 70 mu L of AccQ-Fluor borate buffer solution, mixing by vortex, adding 20 mu L of AccQ-Fluor derivative 6-aminoquinolinyl-N-hydroxy-succinimidyl formate, mixing by vortex immediately, standing at room temperature for 1 min, transferring the sample solution to a micro automatic sample injection bottle, sealing by a cover, heating on a 55 ℃ heating device for 10 min, cooling to room temperature and measuring by a machine.
Advantageous effects
In view of the problem of inaccurate detection of aspartic acid and glutamic acid and no problem of related national standard detection methods, the analysis method for indirectly measuring the aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder products by UPLC-MS/MS is developed.
By examining the influence of enzyme hydrolysis conditions such as enzyme concentration, enzyme hydrolysis time and temperature on asparagine and glutamine, an innovative enzyme hydrolysis technology is developed, and the contents of asparagine and glutamine are accurately measured. The accurate determination of aspartic acid and glutamic acid is realized by acid hydrolysis and enzyme hydrolysis and adopting a differential method.
Aiming at the characteristics of matrix complexity and no blank matrix in the infant formula milk powder and the prepared milk powder, an internal standard method is adopted for quantification, so that the accuracy of the method is improved; the matrix effect is evaluated by adopting an internal standard instead of the target object, and the matrix effect investigation is applied to the selection of chromatographic conditions, so that the interference of the co-effluent on the target object is effectively reduced, the matrix effect is reduced, and the accuracy and the stability of the analysis method are improved.
The research fills the blank and technical defect of detection standards of aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder, effectively solves the technical problem of inaccurate detection of aspartic acid and glutamic acid in amino acid detection, and has important significance for production quality control and government supervision of infant formula milk powder and prepared milk powder.
Drawings
FIG. 1 effect of different hydrolysis modes on extraction effect of asparagine and glutamine;
FIG. 2 effects of different enzymatic hydrolysis conditions on asparagine and glutamine extraction;
FIG. 3 effect of different inactivation methods on asparagine and glutamine extraction;
FIG. 4 effect of different chromatographic columns on 4 amino acid matrix effects;
FIG. 5 MRM chromatogram of amino acids and their internal standard solutions.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1
1. Materials and methods
1.1 Materials and reagents
Standard substance: aspartic acid (Asp) (purity. Gtoreq.99.7%), glutamic acid (Glu) (purity. Gtoreq.98.0%), glutamine (Gln) (purity. Gtoreq.99.2%), all purchased from Dr. Ehrenstorfer GmbH company; asparagine (Asn) (purity not less than 98.7%) Shanghai Annotation laboratory technologies, inc.;
amino acid mixed standard solution internal standard (wherein citrulline-D 2 Glutamic acid-D 3 Aspartic acid-D 3 The equal concentration is 500 mu mol/L, glycine- 13 C- 15 N concentration is 2500. Mu. Mol/L) Cambridge Isotope Laboratories Inc.
The infant formula milk powder and the prepared milk powder for experiments are all sold in the market.
Waters AccQ-Tag amino acid assay kit (AccQ-Fluor kit (comprising boric acid buffer 1, derivatization agent powder 2A, dilution 2B)); streptomyces griseus protease (activity. Gtoreq.3.5U/mg) Sigma-Aldrich company; hydrochloric acid (concentration is more than or equal to 36%, high-grade pure) national medicine group chemical reagent company; tris biological engineering (Shanghai) Co., ltd; acetonitrile (chromatographic purity), fisher, USA; ammonium formate (chromatographic purity) Fisher, usa; organic microporous filter membrane (0.22 mu m) Shanghai An Spectrum experiment technology Co., ltd.
1.2 Apparatus and device
An Agilent1290 information ii liquid chromatography system and an Agilent 6470 triple quadrupole liquid chromatography system, equipped with Jet electrospray (Jet ESI) ionization source, were used for data acquisition and analysis (Agilent company, usa) using Agilent Mass Hunter acquisition software (version b.08.00) and Agilent Mass Hunter quantitative analysis software (version b.07.00); AB204-S electronic balance Mettler Toledo, switzerland; SB-800DTD ultrasonic cleaner, china Ningbo New Zhi Biotechnology Co., ltd; constant temperature water bath oscillator, youlibo technology (Beijing); MS3 vortex mixer, IKA company, germany; milli-Q ultra-pure water preparation apparatus, millipore company, U.S.A..
1.3 Method of
1.3.1 Preparation of standard solution
Standard stock (10.0 mmol/L): accurately weighing a proper amount (accurate to 0.1 mg) of standard substances in a 10 mL volumetric flask, and respectively using 0.1mol/L hydrochloric acid to fix the volume to the scale.
Standard working solution: diluting the above stock solutions to obtain mixed standard solution, and gradually diluting until each component is 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 100.0, 200.0 μmol/L (each containing glycine- 13 C- 15 N concentration is 25. Mu. Mol/L, and other labeled amino acids are 5. Mu. Mol/L).
1.3.2 Preparation of AccQ-Fluor derivatization agent
1 mL of AccQ-Fluor diluent in the 2B bottle is sucked, the 2B bottle is placed into a 2A bottle filled with AccQ-Fluor derivative powder, the 2A bottle is sealed by a cover, vortex 10 s is heated on a 55 ℃ heating device until the AccQ-Fluor derivative powder is completely dissolved, and the heating time is not more than 10 min. Sealing, and storing in a drier at room temperature for one week.
1.3.3 Sample pretreatment
1.3.3.1 Measurement of glutamine and asparagine:
extracting: after the samples are uniformly mixed, 0.1g (accurate to 0.0001 and g) of the mixture is weighed into a 50 mL screw-thread centrifuge tube with a cover, 0.5 mL Streptomyces griseus protease solution (10 mg/mL), 3.0 mL Tris buffer (0.1 mol/L, pH 8.5) and 200 mu L methanol are added, the mixture is uniformly mixed by vortex, after ultrasonic treatment for 20 min, 18 h-20 h of oscillation is carried out in a 50 ℃ constant-temperature water bath oscillator, and then the samples are heated at 90 ℃ for 15-20 min. The sample was removed, cooled to room temperature, and the sample solution was transferred to a 25 mL volumetric flask and scaled with water. After shaking up fully, 100. Mu.L to 5 mL volumetric flask is measured accurately, 50. Mu.L of amino acid mixed standard solution internal standard is added, then ultrapure water is used for constant volume to 5 mL, and the mixture is filtered by a 0.22 μm organic phase microporous filter membrane for later use.
And (3) derivatization: transferring 10 μl of standard working solution or the above sample extract to the bottom of 6 mm ×50 mm test tube, adding 70 μl of AccQ-Fluor borate Buffer (Buffer 1), mixing by vortex, adding 20 μl of AccQ-Fluor derivatization agent, immediately mixing by vortex, standing at room temperature for 1 min, transferring the sample solution into a micro-automatic sample bottle, sealing with cover, heating at 55deg.C for 10 min, cooling to room temperature, and measuring by machine.
1.3.3.2 Determination of total amount of aspartic acid (containing a portion converted from asparagine to aspartic acid), total amount of glutamic acid (containing a portion converted from glutamine to glutamic acid):
extracting: after the samples are evenly mixed, 0.1g (accurate to 0.0001 and g) of the mixture is weighed into a hydrolysis tube, 10 mL of 6mol/L hydrochloric acid is added, high-purity nitrogen is filled into the hydrolysis tube, the sealing is carried out, the sealed hydrolysis tube is put into an electrothermal blowing constant temperature box at 110 ℃, after 22 h of hydrolysis, the hydrolysis tube is taken out, and the temperature is cooled to room temperature. The entire sample solution in the hydrolysis tube was transferred to a 25 mL volumetric flask and the volume was set to the scale with water. After shaking up fully, 100. Mu.L to 5 mL volumetric flask is measured accurately, 50. Mu.L of amino acid mixed standard solution internal standard is added, then ultrapure water is used for constant volume to 5 mL, and the mixture is filtered by a 0.22 μm organic phase microporous filter membrane for later use.
And (3) derivatization: step (a) and the derivative operation step in 1.3.3.1.
1.3.3.3 Determination of aspartic acid and glutamic acid:
the molar concentration of aspartic acid measured in step 1.3.3.2 minus the molar concentration of asparagine measured in 1.3.3.1 is the molar concentration of aspartic acid, yielding the content of aspartic acid; the molar concentration of glutamic acid measured in step 1.3.3.2 minus the molar concentration of glutamine measured in step 1.3.3.1 is the molar concentration of glutamic acid, and the glutamic acid content is obtained.
1.3.3.4 Blank test
The other operation steps are the same as those of the sample except that the sample is not added.
1.3.4 Analysis conditions
1.3.4.1 Chromatographic conditions
Chromatographic column: accQ-Tag Ultra C18 (2.1 mm X100 mm,1.7 μm); mobile phase: A. ammonium formate buffer (10 mmol/L), B.acetonitrile; sample injection volume is 1 mu L; column temperature 40 ℃; the detection wavelength is 260nm and the gradient elution procedure is shown in Table 1:
table 1 HPLC gradient elution procedure
Table 1 Gradient elution program
1.3.4.2 Mass spectrometry conditions
Ion source: jet electrospray ion source (Jet ESI); ionization mode: positive ion mode (esi+); the temperature of the drying gas is 250 ℃; the dry air flow is 7L/min; atomization air pressure 45 psi; sheath gas temperature is 350 ℃; sheath gas flow rate, 12L/min, capillary voltage 4000V; nozzle voltage 0V; acquisition mode: multiple Reaction Monitoring (MRM); the mass spectral parameters are shown in table 2.
TABLE 2 Mass Spectrometry parameters for amino acid derivatives
Table 2 Optimized parameters of MS/MS for amino acids derivatives
Note that: * to quantify ions
1.4 Evaluation of matrix Effect
The Matrix effect (Matrix effect) was evaluated by adding a co-level isotope internal standard to the pure solvent and the sample, and measuring the peak area response values of the two. Matrix effect factor (MEF,%) = (a-B)/a×100, where MEF is a matrix effector, a is a peak area response value of an internal standard in a pure solvent, and B is a peak area response value of an internal standard in a sample extract. MEF is 0, which indicates no matrix effect, and the larger the absolute value is, the stronger the matrix effect is, and the matrix effect is not obvious in the range of-15%.
1.5 Data processing
Data acquisition and processing were completed by Agilent Mass Hunter acquisition software and Agilent Mass Hunter quantitative analysis software, which are compatible with the instrument, origin 8.0 was plotted.
2. Results and analysis
2.1 Optimization of pretreatment conditions
2.1.1 Selection of hydrolysis mode
Hydrolysis is critical to the accuracy of amino acid analysis. Since asparagine and glutamine are completely hydrolyzed into aspartic acid and glutamic acid during acid hydrolysis, resulting in inaccurate measurement values of aspartic acid and glutamic acid, the accurate measurement of aspartic acid and glutamic acid is premised on the accurate measurement of asparagine and glutamine first. The experiment uses a milder enzymatic hydrolysis mode to hydrolyze asparagine and glutamine.
The effect of enzyme hydrolysis, weakened acid hydrolysis and combination mode thereof on asparagine and glutamine extraction effect was examined experimentally (fig. 1). The specific investigation conditions were as follows:
A. carrying out enzymolysis on the sample according to sample pretreatment 1.3.3.1, wherein the enzymolysis is carried out on the sample according to 18-h-20 h;
B. dissolving the sample with 0.1mol/L hydrochloric acid, hydrolyzing at 121 ℃ for 30min, and adjusting the pH of the sample solution to pH8.5; then carrying out enzymolysis on the sample according to sample pretreatment 1.3.3.1, wherein the enzymolysis is carried out on the sample by 18-h-20 h;
C. dissolving the sample with Tris buffer (0.1 mol/L, pH 8.5), and hydrolyzing at 121deg.C for 30min; then carrying out enzymolysis on the sample according to sample pretreatment 1.3.3.1, wherein the enzymolysis is carried out on the sample by 18-h-20 h;
D. the sample is subjected to enzymolysis according to sample pretreatment 1.3.3.1 for 18 h-20 h, and then the pH of the sample solution is adjusted to be 1.0, and the sample solution is hydrolyzed for 30min at 121 ℃;
experiments show (figure 1) that the extraction effect of Asn and Gln in the single enzyme hydrolysis mode (A) sample is good and the recovery rate is normal. The enzyme hydrolysis and the weakened acid hydrolysis are combined to form a hydrolysis method (B, D), the extraction of Asn and Gln are reduced to different degrees, and the influence trend of the recovery rate is basically consistent; when buffer hydrolysis is combined with enzymatic hydrolysis (D), gln extraction is normal with recovery, while Asn is reduced to some extent. Therefore, the Asn and Gln hydrolysis modes select the simple enzymatic hydrolysis mode (A).
Since the enzyme hydrolysis method is milder, asp and Glu cannot be hydrolyzed completely, and Asp and Glu obtained by pretreatment 1.3.3.2 acid hydrolysis are the sum of Asp and Asn and Glu is the sum of Glu and Gln, the molar concentration of Asp and Glu is obtained by subtracting the molar concentration of Asn and Glu obtained by enzyme hydrolysis from the molar concentration of Asp and Glu obtained by acid hydrolysis, respectively, so that the content of Asp and Glu is indirectly measured.
2.1.2 Optimization of enzymatic hydrolysis conditions
The selection of enzyme types and experiments examine the influence of Streptomyces griseus protease, pancreatin, lipase and amylase on experimental results. It was found experimentally that pancreatic enzymes, lipases and amylases do not hydrolyze proteins to free aspartic acid, glutamic acid and tryptophan. The reason is probably that pronase is a non-specific proteolytic enzyme mixture, can split peptide bonds of protein, has extremely strong proteolytic action and can digest the protein into single amino acid, so that the experiment adopts pronase for enzymolysis.
The enzymolysis condition is a key factor for accurately measuring the aspartic acid and the glutamic acid, so the experiment respectively examines the influence of enzyme concentration, enzymolysis temperature and enzymolysis time on the extraction effect of asparagine and glutamine (figure 2).
The effect of enzyme concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0. 20.0 mg/mL on the extraction effect of the target in the sample was examined experimentally (FIG. 2). The results showed that as the enzyme concentration increased, the target content gradually increased, and Asn and Gln did not increase any more when the concentration exceeded 10.0 mg/mL. The concentration of the experimentally selected enzyme was therefore 10.0. 10.0 mg/mL.
The effect of different temperatures (40, 45, 50, 55, 60 ℃) on the extraction of Asn and Gln was compared (fig. 2). Experiments show that the extraction effect of the target substance gradually increases along with the increase of the temperature, and the target substance has obvious descending trend after the temperature exceeds 50 ℃. The enzymolysis temperature in this experiment was determined to be 50 ℃.
The effect on the extraction effect of 2 amino acids when the enzymolysis time of the sample is 14, 16, 18, 20 and 22 h respectively was examined experimentally (fig. 2). The research shows that with the increase of the enzymolysis time, the enzymolysis efficiency is increased, the extraction efficiency is improved, the extraction efficiency is stable after 18 h, and Asn and Gln have different degrees of descending trend after exceeding 20 h. Therefore, the experimental selection enzymolysis time is 18-20 h.
2.1.3 Selection of enzyme inactivation method
To ensure stability and accuracy of the analysis method, experiments examined the effect of two different inactivation methods, namely adding methanol after enzymolysis and heating at 90 ℃ for 15-20 minutes, on the extraction and recovery rate of the target (figure 3).
Studies have shown that the use of methanol inactivation results in lower target levels and recovery. The reason may be that, on the one hand, the addition of methanol, while inactivating the enzyme, also precipitates part of the interfering substances, which may also adsorb or encapsulate part of the target substances; another aspect may be the difference in solubility of the target in methanol and water. Thus, after experimental selection of enzymatic hydrolysis, the sample is heated at 90℃for 15-20 minutes to inactivate the enzyme.
2.2 Optimization of analysis conditions
2.2.1 Selection of chromatographic conditions
2.2.1.1 Selection of chromatographic columns
The effect of three columns Waters Acquity UPLC HSS T (2.1X100 mm,1.7 μm), waters Acquity BEH Shield RP (2.1X100 mm,1.7 μm) and AccQ-Tag Ultra C18 (2. mm ×100 mm,1.7 μm) on peak area response and peak shape were experimentally selected. The effect of three columns on the matrix effect (fig. 4) was examined experimentally.
The results show that the peak area response values of the target on the three chromatographic columns are not obviously different, the peak shapes of the Ultra C18 and HSS T3 target are good, but Asp and Glu tail on the Shield RP18. As can be seen from fig. 4, the matrix effect of the 4 amino acids on the Ultra C18 column was significantly lower than HSS T3 and Shield RP18. The Ultra C18 column was selected for this experiment.
2.2.1.2 Selection of mobile phase
Acetonitrile is adopted as an organic phase in the experiment, and a mixed standard solution (10 mu mol/L) of 4 amino acids is adopted to compare the sensitivity and peak shape effects of three different flows of 10 mmol/L ammonium formate, 10 mmol/L ammonium formate (containing 0.1% formic acid) and 10 mmol/L ammonium acetate relative to a target object. Experiments show that the peak shapes of the target objects in the three mobile phases have no obvious difference; but the peak area response value is highest, the sensitivity is highest and the matrix effect is smallest in an acetonitrile-10 mmol/L ammonium formate system. Thus, acetonitrile-10 mmol/L ammonium formate was experimentally chosen as the mobile phase.
The experiment examined the effect of different salt concentrations (5 mmol/L, 10 mmol/L, 20 mmol/L ammonium formate solution) on the peak area response values and peak shapes of the 4 amino acids. The results show that the salt concentration has no obvious difference on the sensitivity of the target compound, but the peak shape of the target compound has tailing phenomenon when the concentration is 5 mmol/L. Considering the influence of salt concentration on the instrument and chromatographic column, the concentration of ammonium formate solution was experimentally selected to be 10 mmol/L.
2.2.2 Optimization of mass spectrometry conditions
Amino acid derivatives are readily ionized to form positive ions under ESI sources. Under the electrospray positive ion mode, the standard solution of each target compound with the concentration of 500 mu mol/L is derived, corresponding parent ions and optimized ion source parameters are obtained through primary mass spectrum scanning, the Fragmentor voltage is optimized, then characteristic fragment ions with strong and stable signals are selected through sub-ion scanning, qualitative ions and quantitative ions are determined, and parameters such as collision energy and the like are further optimized, and the specific parameters are shown in Table 2. Under optimized HPLC-MS/MS conditions, 4 amino acids were mixed with standard solution (10. Mu. Mol/L) and internal standard (glycine- 13 C- 15 The MRM chromatogram of a standard solution with an N concentration of 25. Mu. Mol/L and other labeled amino acids at a concentration of 5. Mu. Mol/L) is shown in FIG. 5.
The derivatives generated by the reaction of the AQC and the primary amine or the secondary amine have good reversed-phase chromatography and mass spectrum characteristics. Monoamino amino acid derivatives formation [ M+Acq+H] + Molecular ion peaks. After derivatization of the amino acids with the AQC reagent, a common fragment ion 171 is generated, which is largely generated by the loss of the Aminoquinoline (AMQ) group from the target analyte.
2.3 Investigation of matrix Effect
Amino acid is an important nutrient substance in infant formula milk powder and prepared milk powder products, a blank matrix is difficult to obtain, a matrix effect cannot be evaluated by adopting a conventional post-extraction adding method and a post-column injection method, and a matrix correction curve cannot be used for quantification. Because the isotope internal standard has the same chemical property as the target object, the experiment adopts the isotope internal standard to replace the target object for matrix effect evaluation, thereby effectively eliminating the influence of matrix effect, improving the accuracy and stability of the analysis method and realizing matrix effect evaluation on each sample. Experiments show that the matrix of the method is between-15.0% and 15.0%, and the influence of matrix effect is small.
2.4 Methodology evaluation
2.4.1 Linear range, detection limit and quantification limit
After the standard series working solution prepared in the step of section 1.3.1 is derived according to section 1.3.3.1, the mass concentration is sequentially measured from low to high, the molar concentration (X, mu mol/L) of each target compound is taken as an abscissa, the ratio (Y) of the peak area of each target compound to the internal standard area is taken as an ordinate, and a standard curve is drawn. The detection limit of the target compound was obtained with RSN.gtoreq.3, and the quantitative limit of the target compound was obtained with RSN.gtoreq.10 (Table 3). The results show that the target compounds have good linear relationship in the respective linear range, and the linear correlation coefficient is larger than 0.9991.
TABLE 3 Linear regression equation, linear range, correlation coefficient, detection limit and quantitative limit for amino acids
2.4.2 Recovery rate and precision
And selecting certain infant formula milk powder as a representative for carrying out a recovery rate test. The recovery rate experiments were performed by adding standard solutions of 3 different levels of concentration, i.e., low, medium and high, to the samples at 50%, 100% and 150% of the target compound, and measuring the respective levels in parallel 6 times (table 4). As can be seen from Table 4, the standard recovery rate was 92.5% to 103.4%, and the relative standard deviation was 1.79% to 3.25%.
Table 4 recovery and precision of the method
Note that: ND represents not detected
2.5 Determination of actual samples
The method was used to analyze the aspartic acid and glutamic acid in infant formulas and modified milk powders of different manufacturers (Table 5).
TABLE 5 infant formula and modified milk powder contents of aspartic acid and glutamic acid
The results show that the aspartic acid and glutamic acid contents in different manufacturers or different formulas are greatly different, and the result difference is probably caused by the difference of materials.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (4)
1. A method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder, which is characterized by comprising the following steps:
(1) Adding a Streptomyces griseus protease solution into a sample for enzymolysis to extract asparagine and glutamine, and deriving through AQC;
the enzymolysis comprises the following specific steps: after uniformly mixing the samples, weighing 0.1g into a 50 mL threaded centrifuge tube with a cover, adding 0.5 mL of 10mg/mL Streptomyces griseus protease solution, 0.1mol/L of 3.0 mL Tris buffer solution with pH of 8.5 and 200 mu L of methanol, uniformly mixing by vortex, carrying out ultrasonic treatment for 20 min, oscillating for 18 h-20 h in a 50 ℃ constant temperature water bath oscillator, and then placing the samples at 90 ℃ for 15-20 min; taking out the sample, cooling to room temperature, transferring the sample solution into a 25 mL volumetric flask, and fixing the volume to the scale with water; after shaking fully and evenly, accurately measuring 100 mu L to 5 mL volumetric flask, adding 50 mu L of amino acid mixed standard solution internal standard, then using ultrapure water to fix the volume to 5 mL, and filtering by a 0.22 mu m organic phase microporous filter membrane for later use;
(2) Hydrochloric acid is added into the sample to hydrolyze and extract aspartic acid and glutamic acid, and the sample is derivatized by AQC;
(3) Preparing a standard solution:
standard stock solution 10.0 mmol/L: accurately weighing a proper amount of standard substances in a 10 mL volumetric flask respectively, and fixing the volume to the scale by using 0.1mol/L hydrochloric acid respectively;
standard working solution: diluting the standard stock solutions to prepare mixed standard solutions, and gradually diluting until the components are serial standard working solutions of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 100.0 and 200.0 mu mol/L before use;
(4) Liquid chromatography-mass spectrometry was used: the aspartic acid content is calculated by subtracting the molar concentration of the asparagine measured in the step (1) from the molar concentration of the aspartic acid in the step (2); the glutamic acid content is calculated by subtracting the molar concentration of the glutamine measured in the step (1) from the molar concentration of the glutamic acid in the step (2);
the conditions of the liquid chromatography were: chromatographic column: accQ-Tag Ultra C18,2.1 mm ×100 mm,1.7 μm; mobile phase: A. 10 mmol/L ammonium formate buffer solution, B, acetonitrile; sample injection volume is 1 mu L; column temperature 40 ℃; gradient elution procedure is shown in table:
。
2. the method for indirectly measuring aspartic acid and glutamic acid in infant formula milk powder and prepared milk powder according to claim 1, wherein mass spectrometry conditions are as follows: ion source: a jet electrospray ion source; ionization mode: a positive ion mode; the temperature of the drying gas is 250 ℃; the dry air flow is 7L/min; atomization air pressure 45 psi; sheath gas temperature is 350 ℃; sheath air flow, 12L/min; capillary voltage 4000V; nozzle voltage 0V; acquisition mode: multiple reaction monitoring.
3. The method for indirectly determining aspartic acid and glutamic acid in infant formula and formula according to claim 1, wherein the deriving step of step (1) is as follows:
transferring 10 mu L of standard working solution or the sample extracting solution to the bottom of a 6 mm multiplied by 50 mm test tube, adding 70 mu L of AccQ-Fluor borate buffer solution, mixing by vortex, adding 20 mu L of AccQ-Fluor derivative 6-aminoquinolinyl-N-hydroxy-succinimidyl formate, mixing by vortex immediately, standing at room temperature for 1 min, transferring the sample solution to a micro automatic sample injection bottle, sealing by a cover, heating on a 55 ℃ heating device for 10 min, cooling to room temperature and measuring by a machine.
4. The method for indirectly measuring aspartic acid and glutamic acid in infant formula and prepared milk powder according to claim 1, wherein the specific steps of the step (2) are as follows: after uniformly mixing the samples, weighing 0.1g of the samples into a hydrolysis tube, adding 10 mL of 6mol/L hydrochloric acid, filling high-purity nitrogen, sealing, putting the sealed hydrolysis tube into an electrothermal blowing incubator at 110 ℃, hydrolyzing 22 h, taking out, and cooling to room temperature; transferring all liquid of the sample solution in the hydrolysis tube into a 25 mL volumetric flask, and fixing the volume to a scale by using water; after shaking fully and evenly, accurately measuring 100 mu L to 5 mL volumetric flask, adding 50 mu L of 12 amino acid mixed standard solution internal standard, then using ultrapure water to fix the volume to 5 mL, and filtering by a 0.22 mu m organic phase microporous filter membrane for later use;
and (3) derivatization: transferring 10 mu L of standard working solution or the sample extracting solution to the bottom of a 6 mm multiplied by 50 mm test tube, adding 70 mu L of AccQ-Fluor borate buffer solution, mixing by vortex, adding 20 mu L of AccQ-Fluor derivative 6-aminoquinolinyl-N-hydroxy-succinimidyl formate, mixing by vortex immediately, standing at room temperature for 1 min, transferring the sample solution to a micro automatic sample injection bottle, sealing by a cover, heating on a 55 ℃ heating device for 10 min, cooling to room temperature and measuring by a machine.
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Citations (7)
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