CN108445071B - High-accuracy glycosylated hemoglobin standard substance valuing method - Google Patents
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Abstract
The invention discloses a high-accuracy glycosylated hemoglobin standard substance valuing method, which comprises the following steps: (1) synthesizing characteristic peptide fragments for quantifying hemoglobin and glycosylated hemoglobin; (2) measuring the purity of the characteristic peptide fragment; (3) enzymolysis of the glycosylated hemoglobin; (4) capillary electrophoresis-isotope dilution mass spectrometry of the sample enzymolysis liquid; (5) calculating the content of the specific peptide fragment in the enzymolysis liquid; (6) and (4) calculating the content of the glycosylated hemoglobin. The method can effectively separate biomacromolecules such as peptide fragments, proteins and the like, so that the specific peptide fragments for quantification are not easily influenced by interference components, and the accuracy of the quantification result is improved. And the sample injection amount of the capillary electrophoresis is only a few nanoliters, thereby greatly saving samples.
Description
Technical Field
The invention relates to the technical field of substance content determination, in particular to a method for valuing a glycosylated hemoglobin standard substance.
Background
The measurement is the activity of realizing unity of units and accurate and reliable magnitude, and the research and establishment of a high-accuracy measurement method is one of the important contents of metrological research. The content (concentration) describes the number of measured objects in unit mass or volume, and is a basic value in chemical and biological measurement, and in order to realize accurate measurement of the content (concentration), the isotope dilution mass spectrometry method is widely adopted as a measuring method in the chemical and biological measurement. The isotope dilution mass spectrometry is a special internal standard method, which takes a stable isotope label of a measured object as an internal standard to be added into a sample, and the isotope internal standard is close to the physical and chemical properties of a compound to be measured, so that the isotope internal standard can correct the system error and the random error in the whole analysis process in real time after being fully mixed, and the measurement with high accuracy is realized. After the isotope internal standard is added, the reaction efficiency, the loss rate and the like of the isotope label are consistent in the pretreatment process because the physical and chemical properties of the isotope label are close to those of the target compound to be detected; in the separation process of the high performance liquid chromatography, the isotope internal standard and the target compound to be detected are always together, and the disturbance applied to the target compound to be detected at any time is also applied to the isotope internal standard to the same extent, so that the random error and the system error in the separation process are corrected; in the ionization process, the target compound to be detected and the isotope label have the same ionization efficiency, the target compound and the isotope label are ionized with the same efficiency and enter a mass spectrometer for detection, and finally, the target compound and the isotope label are detected in channels with different mass-to-charge ratios of the mass spectrometer. And finally, calculating according to the proportion of the target compound to be detected and the isotope label and the weighed mass. Because the proportion of various influencing factors in the whole analysis process can not be changed after the isotope internal standard is added, the system error and the random error in the whole analysis process can be eliminated, and the high-accuracy quantitative result can be obtained. This makes isotope dilution mass spectrometry also one of the common measurement benchmark methods in chemistry and biology.
For the analysis of biological samples, the accurate determination of the amount of chemical components in the sample is usually performed by means of high performance liquid chromatography-isotope dilution mass spectrometry. High performance liquid chromatography is a common separation means, and can be better used for separating molecules such as organic molecules, peptide fragments, proteins, oligomeric nucleic acids, nucleic acids and the like. Accurate quantification of inorganic and organic micromolecules, nucleic acid, protein and other biological macromolecules is successfully achieved through a mode of combining high performance liquid chromatography with isotope dilution mass spectrometry, and system errors in sample pretreatment, separation, ionization and mass spectrometry detection processes are overcome through the use of isotope internal standards, so that high measurement accuracy is achieved, and the method becomes one of chemical and biological metering reference methods. Although high performance liquid chromatography-isotope dilution mass spectrometry has been successful, the application of high performance liquid chromatography-isotope dilution mass spectrometry in accurate quantification of biomacromolecules such as nucleic acid and protein is limited due to the limitation of high performance liquid chromatography in separation efficiency of biomacromolecules such as nucleic acid and protein. The capillary electrophoresis technology is a novel separation and analysis technology developed in recent decades and has the advantages of high separation efficiency, simplicity, economy, rapidness, trace quantity, high automation degree and the like. Capillary electrophoresis adopts electroosmotic flow to drive the separation of compounds, overcomes the peak broadening caused by uneven flow velocity of each layer in high performance liquid chromatography by adopting pressure drive, thereby reaching extremely high theoretical plate number and showing column efficiency and separation capability far superior to the high performance liquid chromatography. The improvement of the separation capacity is beneficial to the removal of interference components, and the capillary electrophoresis-isotope dilution mass spectrometry is enabled to become a more accurate quantitative method by combining the excellent system error overcoming capacity of the isotope dilution mass spectrometry.
Disclosure of Invention
The invention aims to provide a high-accuracy glycosylated hemoglobin standard substance value determination method based on capillary electrophoresis-isotope dilution mass spectrometry.
A high-accuracy glycosylated hemoglobin standard substance value determination method comprises the following steps:
(1) synthesizing characteristic peptide fragments for quantifying hemoglobin and glycosylated hemoglobin;
(2) measuring the purity of the characteristic peptide fragment;
(3) enzymolysis of the glycosylated hemoglobin;
(4) capillary electrophoresis-isotope dilution mass spectrometry of the sample enzymolysis liquid;
(5) calculating the content of the specific peptide fragment in the enzymolysis liquid;
(6) and (4) calculating the content of the glycosylated hemoglobin.
The method for determining the value of the high-accuracy glycosylated hemoglobin standard substance comprises the following steps of (1) specifically, obtaining a specific peptide segment VHLTPE for hemoglobin quantification and a corresponding isotope labeled peptide segment VH-LTPE thereof in a chemical synthesis mode; specific peptide fragments Glc-VHLTPE and corresponding isotopically labeled peptide fragments Glc-VH LTPE for quantification of glycated hemoglobin are obtained by chemical synthesis.
The high-accuracy glycosylated hemoglobin standard substance value determination method comprises the following steps of (2) hydrolyzing a synthesized peptide fragment into amino acid, quantitatively adding isotope-labeled amino acid, determining the content of stable amino acid in hydrolysate by using a high performance liquid chromatography-isotope dilution mass spectrometry method established by taking national amino acid standard substances as standards, and calculating the purity of the characteristic peptide fragment according to the content; for the specific peptide segment VHLTPE, the amino acids used for quantification were valine V, leucine L and proline P; for the specific peptide fragment Glc-VHLTPE, the amino acids used for quantification were leucine L and proline P.
The high-accuracy glycosylated hemoglobin standard substance value determination method comprises the specific steps of (3) dissolving a proper amount of glycosylated hemoglobin in an ammonium acetate buffer solution with the pH value of 4.0, adding a proper amount of Glu-C according to the ratio of the amount of hemoglobin to the amount of Glu-C of 100:1 according to the amount of a standard substance candidate, and incubating for 24 hours at the temperature of 60 ℃ for enzyme digestion.
The method for determining the value of the high-accuracy glycosylated hemoglobin standard substance comprises the following steps of (4) adding isotope-labeled VH-LTPE and Glc-VH-LTPE into enzymatic hydrolysate after enzymolysis, uniformly mixing, and filtering by using a 0.22 mu m filter membrane to perform capillary electrophoresis-isotope dilution mass spectrometry;
the separation capillary used was a 57cm × 75 μm polyimide-coated quartz capillary, an ultraviolet detector, and an ammonium acetate buffer solution with a detection wavelength of 200nm and a pH of 50mM ═ 5.0 as a separation medium;
pressure sample introduction is carried out, the separation voltage is 20kV, and mass spectrum detection adopts the following ion peaks in selective detection: m/z 695(VHLTPE), m/z 702(D7-VHL TPE), m/z 857(G-VHLTPE), m/z 864(D7-G-VHLTPE), m/z 623 (VHLTPG); meanwhile, preparing low and high standards by using synthesized VHLTPE, VH LTPE, Glc-VHGLTPE and Glc-VH LTPE, and accurately determining the VHLTPE and the Glc-VHLTPE in the enzymolysis solution by using a bracket method.
The high-accuracy glycosylated hemoglobin standard substance value determination method comprises the following steps of (5) calculating the content of VHLTPE and Glc-VHLTPE in the enzymolysis solution according to the following formula,
in the formula: c-is the content of VHLTPE (Glc-VHLTPE) in the sample, mg/g;
ms-mass of VH LTPE (Glc-VH LTPE), mg, in the sample solution;
Rs-is the ratio of the peak areas of VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE) in the sample solution;
I1-mass ratio of spiking solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
I2-mass ratio of low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R1-the peak area ratio of the spiked solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R2-the peak area ratio of the low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
m-is sample mass, g;
p-is the purity of VHLTPE (Glc-VHLTPE).
The high-accuracy method for quantifying the glycated hemoglobin standard substance comprises the following steps (6) of calculating the content of glycated hemoglobin in a sample according to the following formula;
in the formula (I), the compound is shown in the specification,
mGlc-VE6-the content of Glc-VHLTPE in the enzymatic hydrolysate, g/g;
MWGlc-VE6-molecular weight of Glc-VHLTPE;
mVE6-the content of VHLTPE in the enzymatic hydrolysate, g/g;
MWVE6molecular weight of VHLTPE.
Compared with the traditional high performance liquid chromatography-isotope dilution mass spectrometry, the high-accuracy glycosylated hemoglobin standard substance value determination method based on capillary electrophoresis-isotope dilution mass spectrometry has the following advantages:
(1) the traditional high performance liquid chromatography-isotope dilution mass spectrometry method is limited by the separation performance of the high performance liquid chromatography, and the separation degree of complex components is not as good as that of capillary electrophoresis. Therefore, the capillary electrophoresis-isotope dilution mass spectrometry method created by the invention can effectively separate biomacromolecules such as peptide fragments, proteins and the like, so that the specific peptide fragments for quantification are not easily influenced by interference components, and the accuracy of the quantification result is improved.
(2) The sample volume of the traditional high performance liquid chromatography is a few microlitres, while the sample volume of the capillary electrophoresis is only a few nanoliters, thereby greatly saving samples.
The method for quantifying a glycated hemoglobin standard substance with high accuracy according to the present invention will be described in detail with reference to the following examples.
Detailed Description
Example 1
A glycosylated hemoglobin standard substance value determination method based on capillary electrophoresis-isotope dilution mass spectrometry comprises the following steps:
(1) Synthesis of a characteristic peptide fragment for quantification of hemoglobin and glycated hemoglobin
Obtaining a specific peptide segment VHLTPE for quantifying hemoglobin and a corresponding isotope labeling peptide segment VH-LTPE by a chemical synthesis mode; specific peptide fragments Glc-VHLTPE and corresponding isotopically labeled peptide fragments Glc-VH LTPE for quantification of glycated hemoglobin are obtained by chemical synthesis.
(2) Accurate determination of the purity of characteristic peptide fragments
Hydrolyzing the synthesized peptide segment into amino acid, quantitatively adding isotope-labeled amino acid, establishing a high performance liquid chromatography-isotope dilution mass spectrometry method by taking national amino acid standard substances as standards to determine the content of the stable amino acid in the hydrolysate, and calculating the purity of the characteristic peptide segment according to the content. For the specific peptide segment VHLTPE, the amino acids used for quantification are valine (V), leucine (L) and proline (P); for the specific peptide fragment Glc-VHLTPE, the amino acids used for quantification were L and P.
(3) Enzymatic hydrolysis of glycated hemoglobin
Dissolving an appropriate amount of glycosylated hemoglobin in an ammonium acetate buffer solution with the pH value of 4.0, adding an appropriate amount of Glu-C according to the ratio of the amount of hemoglobin to the amount of Glu-C to 100:1 according to the amount of a candidate substance added with a standard substance, and incubating for 24 hours at 60 ℃ for enzyme digestion.
(4) Capillary electrophoresis-isotope dilution mass spectrometry of sample enzymolysis liquid
Adding isotope-labeled VH LTPE and Glc-VH LTPE into the enzymolysis solution after enzymolysis, mixing uniformly, filtering with a 0.22 μm filter membrane, and performing capillary electrophoresis-isotope dilution mass spectrometry. The separation capillary used was a 57cm × 75 μm polyimide-coated quartz capillary, an ultraviolet detector, and an ammonium acetate buffer solution with a detection wavelength of 200nm and a pH of 50mM ═ 5.0 as a separation medium. Pressure sample introduction is carried out, the separation voltage is 20kV, and mass spectrum detection adopts the following ion peaks in selective detection: m/z 695(VHLTPE), m/z 702(D7-VHL TPE), m/z 857(G-VHLTPE), m/z 864(D7-G-VHLTPE), m/z 623 (VHLTPG). Meanwhile, preparing low and high standards by using synthesized VHLTPE, VH LTPE, Glc-VHGLTPE and Glc-VH LTPE, and accurately determining the VHLTPE and the Glc-VHLTPE in the enzymolysis solution by using a bracket method.
(5) Calculation of content of specific peptide in enzymolysis liquid
Calculating the content of VHLTPE and Glc-VHLTPE in the enzymolysis solution according to the following formula,
in the formula: c-is the amount of VHLTPE (Glc-VHLTPE) in the sample (mg/g);
ms-mass (mg) of VH LTPE (Glc-VH LTPE) in the sample solution;
Rs-is the ratio of the peak areas of VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE) in the sample solution;
I1-mass ratio of spiking solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
I2-mass ratio of low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R1-the peak area ratio of the spiked solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R2——peak area ratio of low standard solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
m-is sample mass (g);
p-is the purity of VHLTPE (Glc-VHLTPE).
(6) Calculation of glycated hemoglobin content
The content of glycated hemoglobin in the sample was calculated according to the following formula.
In the formula (I), the compound is shown in the specification,
mGlc-VE6-the content of Glc-VHLTPE in the enzymatic hydrolysate, g/g;
MWGlc-VE6-molecular weight of Glc-VHLTPE;
mVE6-the content of VHLTPE in the enzymatic hydrolysate, g/g;
MWVE6molecular weight of VHLTPE.
Example 2
A specific embodiment of a glycosylated hemoglobin standard substance quantitative method based on capillary electrophoresis-isotope dilution mass spectrometry comprises the following steps:
(1) synthesis of a characteristic peptide fragment for quantification of hemoglobin and glycated hemoglobin
Obtaining a specific peptide segment VHLTPE for haemoglobin quantification and a corresponding isotope labeling peptide segment VH-LTPE by entrusted biotechnology service company in a solid phase synthesis mode; and simultaneously synthesizing specific peptide fragment Glc-VHLTPE for quantifying glycosylated hemoglobin and corresponding isotope labeled peptide fragment Glc-VH LTPE.
(2) Accurate determination of the purity of characteristic peptide fragments
The determination of the characteristic peptide purity used as a standard uses two technical approaches: isotope dilution mass spectrometry and mass balance.
The isotope dilution mass spectrometry method comprises the following determination processes: firstly, analyzing the purity of VHLTPE and Glc-VHLTPE by adopting high performance liquid chromatography, wherein the analysis conditions of the purity of the high performance liquid chromatography are as follows:
a chromatographic column: 250mm × 4.6mm, Kromasil-C18-5 μm
Mobile phase A: 0.1% TFA-Water
Mobile phase B: 0.1% TFA-acetonitrile
Flow rate: 1.0mL/min
Detection wavelength: 220nm
Sample introduction amount: 10 μ L
The purity of VHLTPE and Glc-VHLTPE was 99.8% and 99.5% respectively by high performance liquid analysis.
Then, preparing a stock solution with the concentration of 0.1mg/g from VHLTPE and Glc-VHLTPE, adding 20 mu L of the stock solution into different ampoules, adding an equal amount of isotope labeled valine (Val), leucine (Leu) and proline (Pro) solution into the VHLTPE solution according to the theoretical concentration of hydrolyzed amino acid, fully mixing uniformly, adding 500 mu L of 6mol/L hydrochloric acid solution, filling nitrogen for 2min, sealing, and putting into a 110 ℃ oven for hydrolysis for 48 hr. Blowing the hydrolyzed solution by using nitrogen, adding 0.01mol/L hydrochloric acid for redissolving, filtering the redissolved sample by using a machine for measuring after 0.45 mu m. The Glc-VHLTPE was treated in a similar manner to VHLTPE except that no isotopic label for Val was added. National amino acid standard substances (Val, Leu and Pro) are adopted as standards, corresponding isotope-labeled amino acids are used as internal standards, high-standard and low-standard solutions are prepared, and isotope dilution mass spectrometry is carried out on the corresponding amino acid concentration in the hydrolysate by adopting a bracket method, so that the mass fractions of VHLTPE and Glc-VHLTPE are respectively 0.890g/g and 0.883 g/g.
The measurement conditions of the mass balance method are as follows:
firstly, the purity of VHLTPE and Glc-VHLTPE is determined by high performance liquid chromatography, the method is the same isotope dilution mass spectrometry, and the results of 5 repeated analyses are 99.8% and 99.5% respectively. Then, the moisture in VHLTPE and Glc-VHLTPE is measured by a Karl Fischer method, and the results of 3 times of repeated measurement are respectively 10.2% and 11.3%; then, inorganic ions in the sample are measured by a residue burning method, and the results are respectively 0.12% and 0.13%; and (3) measuring the organic solvent residue in the peptide fragment by adopting high-performance gas chromatography headspace sample injection, wherein the organic solvent residue components such as methanol, ethanol, acetonitrile, acetone and the like are not detected within the detection limit of an instrument. The fixed value results of the standard mass balance method of the two peptide fragments are calculated according to the following formula:
c＝P×(1-wwater-wresidue-wVOC)
wherein:
c-mass fraction of two peptide fragments, g/g;
p — high performance liquid chromatography purity,%;
wwater-water content,%;
wresidue-content of ignition residue,%;
wVOC-content of volatile organic components,%.
According to the formula, the detection results of the two peptide fragment mass balance methods are respectively as follows: 0.895g/g and 0.881 g/g. The average values of the isotope dilution mass spectrometry method and the mass balance method are taken as the purity determination results of the two peptide fragments and are respectively 0.893g/g and 0.882 g/g.
(3) Characterization of isotopically labeled peptide fragments
Firstly, the synthesized VH and VH LTPE and Glc-VH and LTPE are characterized by high performance liquid chromatography, the purity of the high performance liquid chromatography is 99.1 percent and 99.0 percent respectively, and then the non-labeling peptide segments VHLTPE and Glc-VHLTPE contained in the VH and Glc-VH and LTPE are measured by using a liquid chromatography-mass spectrometry technology. Respectively preparing peptide fragments of VH and LTPE and Glc-VH and LTPE into solutions of 100ng/g by ultrapure water for high performance liquid chromatography-mass spectrometry, wherein the analysis conditions are as follows:
chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-Aq 3.5 μm, 2.1X 150mm
Mobile phase: a: 0.1% formic acid + water
B: 0.1% formic acid + acetonitrile
Sample introduction amount: 20 μ L
Flow rate: 0.2mL/min
Column temperature: 40 deg.C
Gradient: see table 1 below.
TABLE 1 mobile phase gradiometer
Mass spectrometry was performed in Agilent 6410 using electrospray ionization source (ESI) and positive ion selective ion monitoring mode (SIM); the flow rate of the dryer is 9L/min, and the temperature of the capillary is 350 ℃; the capillary voltage was 4000V. The following ion peaks were selectively detected: m/z 695(VHLTPE), m/z 702 (D)7-VHL*TPE)，m/z＝857(Glc-VHLTPE)，m/z＝864(Glc-VH*LTPE)。
Through detection, the target peptide fragments all generate peaks in the channels consistent with the theoretical mass-to-charge ratio of the target peptide fragments, and no peak is detected in the other channels, which indicates that the peptide fragments as the isotope internal standard do not contain corresponding non-labeled peptide fragments.
(3) Enzymatic hydrolysis of glycated hemoglobin
Hemoglobin in the purified erythrocyte lysate was measured to have a concentration of about 40g/L, and 10. mu.L of the solution was diluted with 500. mu.L of ultrapure water and mixed well. 1mL of diluted glycated hemoglobin solution was added with 20. mu.L of 8mol/L urea, 50mmol/L Tris-HCl solution, and 15. mu.L of 45mmol/L dithiothreitol solution, and heated in a water bath at 60 ℃ for 40 min. Then 15. mu.L of 100mmol/L iodoacetamide solution is added, and the mixture is placed in the dark for 45 min. After taking out, 45 mu.L of 45mmol/L dithiothreitol solution is added. Dissolving an appropriate amount of glycosylated hemoglobin in an ammonium acetate buffer solution with the pH value of 4.0, adding an appropriate amount of Glu-C according to the amount of a candidate substance added with a standard substance according to the ratio of the amount of the hemoglobin to the amount of Glu-C of 100:1, incubating at 37 ℃ for 48 hours for enzyme digestion, and supplementing enzyme with the same ratio after 24 hours.
(4) Capillary electrophoresis-isotope dilution mass spectrometry of sample enzymolysis liquid
After crude measurement, isotope-labeled VH LTPE and Glc-VH LTPE with the concentrations approximately same as those of VHLTPE and Glc-VHLTPE are added into the enzymolysis solution after enzymolysis, and after uniform mixing, the mixture is filtered by a 0.22 mu m filter membrane for capillary electrophoresis-isotope dilution mass spectrometry analysis. The separation capillary used was a 57cm × 75 μm polyimide-coated quartz capillary, an ultraviolet detector, a detection wavelength of 200nm, and a 50mM ammonium acetate buffer solution having a pH of 5.0 was used as a separation medium and as a sheath solution at a flow rate of 0.2 mL/min. Adopting pressure to sample, the sample introduction time is 10s, the separation voltage is 20kV, and the mass spectrometry adopts a selective ion detection mode to detect the following ion peaks: m/z 695(VHLTPE), m/z 702(D7-VHL TPE), m/z 857(Glc-VHLTPE), m/z 864(Glc-VH LTPE). Meanwhile, preparing low and high standards by using synthesized VHLTPE, VH LTPE, Glc-VHGLTPE and Glc-VH LTPE, and accurately determining the VHLTPE and the Glc-VHLTPE in the enzymolysis solution by using a bracket method. After the measurement is completed, the peak areas of the labeled and unlabeled peptide fragments in the bracket method are integrated.
(5) Calculation of content of specific peptide in enzymolysis liquid
Calculating the content of VHLTPE and Glc-VHLTPE in the enzymolysis solution according to the following formula,
in the formula: c-is the amount of VHLTPE (Glc-VHLTPE) in the sample (mg/g);
Ms-mass (mg) of VH LTPE (Glc-VH LTPE) in the sample solution;
Rs-is the ratio of the peak areas of VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE) in the sample solution;
I1-mass ratio of spiking solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
I2-mass ratio of low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R1the high-standard solution VHLTPE (Glc-VHLTPE) andpeak area ratio of VH LTPE (Glc-VH LTPE);
R2-the peak area ratio of the low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
m-is sample mass (g);
p-is the purity of VHLTPE (Glc-VHLTPE).
Next, the content of glycated hemoglobin in the sample is calculated according to the following formula.
In the formula (I), the compound is shown in the specification,
mGlc-VE6-the content of Glc-VHLTPE in the enzymatic hydrolysate, g/g;
MWGlc-VE6-molecular weight of Glc-VHLTPE;
mVE6-the content of VHLTPE in the enzymatic hydrolysate, g/g;
MWVE6molecular weight of VHLTPE.
The content of glycated hemoglobin in the sample was calculated to be 5.1%, and the relative standard deviation was 1.2% in 6 replicates.
In order to highlight the beneficial effects of the present invention, the following comparative example experiment was also performed.
Comparative example 1
Comparative example glycated hemoglobin content in a sample was determined using classical high performance liquid chromatography-isotope dilution mass spectrometry.
(1) Synthesis of a characteristic peptide fragment for quantification of hemoglobin and glycated hemoglobin
Obtaining a specific peptide segment VHLTPE for haemoglobin quantification and a corresponding isotope labeling peptide segment VH-LTPE by entrusted biotechnology service company in a solid phase synthesis mode; and simultaneously synthesizing specific peptide fragment Glc-VHLTPE for quantifying glycosylated hemoglobin and corresponding isotope labeled peptide fragment Glc-VH LTPE.
(2) Accurate determination of the purity of characteristic peptide fragments
The determination of the characteristic peptide purity used as a standard uses two technical approaches: isotope dilution mass spectrometry and mass balance.
The isotope dilution mass spectrometry method comprises the following determination processes: firstly, analyzing the purity of VHLTPE and Glc-VHLTPE by adopting high performance liquid chromatography, wherein the analysis conditions of the purity of the high performance liquid chromatography are as follows:
a chromatographic column: 250mm × 4.6mm, Kromasil-C18-5 μm
Mobile phase A: 0.1% TFA-Water
Mobile phase B: 0.1% TFA-acetonitrile
Flow rate: 1.0mL/min
Detection wavelength: 220nm
Sample introduction amount: 10 μ L
The purity of VHLTPE and Glc-VHLTPE was 99.8% and 99.5% respectively by high performance liquid analysis.
Then, preparing a stock solution with the concentration of 0.1mg/g from VHLTPE and Glc-VHLTPE, adding 20 mu L of the stock solution into different ampoules, adding an equal amount of isotope labeled valine (Val), leucine (Leu) and proline (Pro) solution into the VHLTPE solution according to the theoretical concentration of hydrolyzed amino acid, fully mixing uniformly, adding 500 mu L of 6mol/L hydrochloric acid solution, filling nitrogen for 2min, sealing, and putting into a 110 ℃ oven for hydrolysis for 48 hr. Blowing the hydrolyzed solution by using nitrogen, adding 0.01mol/L hydrochloric acid for redissolving, filtering the redissolved sample by using a machine for measuring after 0.45 mu m. The Glc-VHLTPE was treated in a similar manner to VHLTPE except that no isotopic label for Val was added. National amino acid standard substances (Val, Leu and Pro) are adopted as standards, corresponding isotope-labeled amino acids are used as internal standards, high-standard and low-standard solutions are prepared, and isotope dilution mass spectrometry is carried out on the corresponding amino acid concentration in the hydrolysate by adopting a bracket method, so that the mass fractions of VHLTPE and Glc-VHLTPE are respectively 0.890g/g and 0.883 g/g.
The measurement conditions of the mass balance method are as follows:
Firstly, the purity of VHLTPE and Glc-VHLTPE is determined by high performance liquid chromatography, the method is the same isotope dilution mass spectrometry, and the results of 5 repeated analyses are 99.8% and 99.5% respectively. Then, the moisture in VHLTPE and Glc-VHLTPE is measured by a Karl Fischer method, and the results of 3 times of repeated measurement are respectively 10.2% and 11.3%; then, inorganic ions in the sample are measured by a residue burning method, and the results are respectively 0.12% and 0.13%; and (3) measuring the organic solvent residue in the peptide fragment by adopting high-performance gas chromatography headspace sample injection, wherein the organic solvent residue components such as methanol, ethanol, acetonitrile, acetone and the like are not detected within the detection limit of an instrument. The fixed value results of the standard mass balance method of the two peptide fragments are calculated according to the following formula:
c＝P×(1-wwater-wresidue-wVOC)
wherein:
c-mass fraction of two peptide fragments, g/g;
p — high performance liquid chromatography purity,%;
wwater-water content,%;
wresidue-content of ignition residue,%;
wVOC-content of volatile organic components,%.
According to the formula, the detection results of the two peptide fragment mass balance methods are respectively as follows: 0.895g/g and 0.881 g/g. The average values of the isotope dilution mass spectrometry method and the mass balance method are taken as the purity determination results of the two peptide fragments and are respectively 0.893g/g and 0.882 g/g.
(4) Characterization of isotopically labeled peptide fragments
Firstly, the synthesized VH and VH LTPE and Glc-VH and LTPE are characterized by high performance liquid chromatography, the purity of the high performance liquid chromatography is 99.1 percent and 99.0 percent respectively, and then the non-labeling peptide segments VHLTPE and Glc-VHLTPE contained in the VH and Glc-VH and LTPE are measured by using a liquid chromatography-mass spectrometry technology. Respectively preparing peptide fragments of VH and LTPE and Glc-VH and LTPE into solutions of 100ng/g by ultrapure water for high performance liquid chromatography-mass spectrometry, wherein the analysis conditions are as follows:
chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-Aq 3.5 μm, 2.1X 150mm
Mobile phase: a: 0.1% formic acid + water
B: 0.1% formic acid + acetonitrile
Sample introduction amount: 20 μ L
Flow rate: 0.2mL/min
Column temperature: 40 deg.C
Gradient: see table 2 below.
TABLE 2 mobile phase gradiometer
Mass spectrometry was performed in Agilent 6410 using electrospray ionization source (ESI) and positive ion selective ion monitoring mode (SIM); the flow rate of the dryer is 9L/min, and the temperature of the capillary is 350 ℃; the capillary voltage was 4000V. The following ion peaks were selectively detected: m/z 695(VHLTPE), m/z 702 (D)7-VHL*TPE)，m/z＝857(Glc-VHLTPE)，m/z＝864(Glc-VH*LTPE)。
Through detection, the target peptide fragments all generate peaks in the channels consistent with the theoretical mass-to-charge ratio of the target peptide fragments, and no peak is detected in the other channels, which indicates that the peptide fragments as the isotope internal standard do not contain corresponding non-labeled peptide fragments.
(5) Enzymatic hydrolysis of glycated hemoglobin
Hemoglobin in the purified erythrocyte lysate was measured to have a concentration of about 40g/L, and 10. mu.L of the solution was diluted with 500. mu.L of ultrapure water and mixed well. 1mL of diluted glycated hemoglobin solution was added with 20. mu.L of 8mol/L urea, 50mmol/L Tris-HCl solution, and 15. mu.L of 45mmol/L dithiothreitol solution, and heated in a water bath at 60 ℃ for 40 min. Then 15. mu.L of 100mmol/L iodoacetamide solution is added, and the mixture is placed in the dark for 45 min. After taking out, 45 mu.L of 45mmol/L dithiothreitol solution is added. Dissolving an appropriate amount of glycosylated hemoglobin in an ammonium acetate buffer solution with the pH value of 4.0, adding an appropriate amount of Glu-C according to the amount of a candidate substance added with a standard substance according to the ratio of the amount of the hemoglobin to the amount of Glu-C of 100:1, incubating at 37 ℃ for 48 hours for enzyme digestion, and supplementing enzyme with the same ratio after 24 hours.
(6) High performance liquid chromatography-isotope dilution mass spectrometry analysis of sample enzymolysis liquid
After rough measurement, isotope-labeled VH LTPE and Glc-VH LTPE with the concentrations approximately same as those of VHLTPE and Glc-VHLTPE are added into the enzymolysis solution after enzymolysis, and after uniform mixing, the mixture is filtered by a 0.22 mu m filter membrane for high performance liquid chromatography-isotope dilution mass spectrometry analysis. The chromatographic conditions and mass spectrometric conditions for the measurement were the same as in (4):
Chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX SB-Aq 3.5 μm, 2.1X 150mm
Mobile phase: a: 0.1% formic acid + water
B: 0.1% formic acid + acetonitrile
Sample introduction amount: 20 μ L
Flow rate: 0.2mL/min
Column temperature: 40 deg.C
Gradient: see table 3 below.
TABLE 3 mobile phase gradiometer
Mass spectrometry was performed in Agilent 6410 using electrospray ionization source (ESI) and positive ion selective ion monitoring mode (SIM); the flow rate of the dryer is 9L/min, and the temperature of the capillary is 350 ℃; the capillary voltage was 4000V. The following ion peaks were selectively detected: m/z 695(VHLTPE), m/z 702 (D)7-VHL*TPE)，m/z＝857(Glc-VHLTPE)，m/z＝864(Glc-VH*LTPE)。
After the measurement is completed, the peak areas of the labeled and unlabeled peptide fragments in the bracket method are integrated.
(7) Calculation of content of specific peptide in enzymolysis liquid
Calculating the content of VHLTPE and Glc-VHLTPE in the enzymolysis solution according to the following formula,
in the formula: c-is the amount of VHLTPE (Glc-VHLTPE) in the sample (mg/g);
Ms-mass (mg) of VH LTPE (Glc-VH LTPE) in the sample solution;
Rs-is the ratio of the peak areas of VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE) in the sample solution;
I1-mass ratio of spiking solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
I2-mass ratio of low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R1-the peak area ratio of the spiked solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
R2-the peak area ratio of the low label solution VHLTPE (Glc-VHLTPE) to VH LTPE (Glc-VH LTPE);
m-is sample mass (g);
p-is the purity of VHLTPE (Glc-VHLTPE).
Next, the content of glycated hemoglobin in the sample is calculated according to the following formula.
In the formula (I), the compound is shown in the specification,
mGlc-VE6-the content of Glc-VHLTPE in the enzymatic hydrolysate, g/g;
MWGlc-VE6-molecular weight of Glc-VHLTPE;
mVE6-the content of VHLTPE in the enzymatic hydrolysate, g/g;
MWVE6molecular weight of VHLTPE.
The content of glycated hemoglobin in the sample was calculated to be 5.1%, and the relative standard deviation was 1.1% in 6 replicates.
Comparing the results obtained by the capillary electrophoresis-isotope dilution mass spectrometry with the results obtained by the high performance liquid chromatography-isotope dilution mass spectrometry, the two results have the same mean value of the measurement results of the same sample and are close to the precision, but the high performance liquid chromatography analysis needs the sample injection amount of 20 mu L, and the capillary electrophoresis analysis only needs the sample injection amount of tens of nanoliters, so that the sample is greatly saved, and the accuracy and the precision of the method are maintained.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (4)
1. A high-accuracy glycosylated hemoglobin standard substance value determination method is characterized by comprising the following steps:
(1) synthesizing characteristic peptide fragments for quantifying hemoglobin and glycosylated hemoglobin;
(2) measuring the purity of the characteristic peptide fragment;
(3) enzymolysis of the glycosylated hemoglobin;
(4) capillary electrophoresis-isotope dilution mass spectrometry of the sample enzymolysis liquid: adding isotope-labeled VH LTPE and Glc-VH LTPE into the enzymolysis solution after enzymolysis, mixing uniformly, and filtering with a 0.22 μm filter membrane for capillary electrophoresis-isotope dilution mass spectrometry;
the separation capillary used was a 57cm × 75 μm polyimide-coated quartz capillary, an ultraviolet detector, and an ammonium acetate buffer solution with a detection wavelength of 200nm and a pH of 50mM ═ 5.0 as a separation medium; meanwhile, the flow rate of the sheath fluid is 0.2 mL/min;
pressure sample introduction is carried out, the separation voltage is 20kV, and mass spectrum detection adopts the following ion peaks in selective detection: m/z 695, VHLTPE; 702, D7-VHL TPE; 857, G-VHLTPE; 864, D7-G-VHLTPE; simultaneously, preparing low and high standards by using synthesized VHLTPE, VH LTPE, Glc-VHGLTPE and Glc-VH LTPE, and accurately determining the VHLTPE and the Glc-VHLTPE in the enzymolysis solution by using a bracket method; after the measurement is finished, integrating the peak areas of the marked and unmarked peptide segments in the bracket method high and low markers and the sample;
(5) Calculating the content of the specific peptide fragment in the enzymolysis solution: calculating the content of VHLTPE and Glc-VHLTPE in the enzymolysis solution according to the following formula,
in the formula: c-is the content of VHLTPE or Glc-VHLTPE in the sample, mg/g;
ms-mass of VH LTPE or Glc-VH LTPE, mg, in the sample solution;
Rs-is the peak area ratio of VHLTPE or Glc-VHLTPE to VH LTPE or Glc-VH LTPE in the sample solution;
I1-mass ratio of the spiking solution VHLTPE or Glc-VHLTPE to VH LTPE or Glc-VH LTPE;
I2-mass ratio of low label solution VHLTPE or Glc-VHLTPE to VH LTPE or Glc-VH LTPE;
R1-the peak area ratio of the spiking solution VHLTPE or Glc-VHLTPE to VH LTPE or Glc-VH LTPE;
R2-the peak area ratio of the low label solution VHLTPE or Glc-VHLTPE to VH LTPE or Glc-VH LTPE;
m-is sample mass, g;
p-is the purity of VHLTPE or Glc-VHLTPE;
(6) and (3) calculating the content of the glycosylated hemoglobin: the content of glycated hemoglobin in the sample is calculated according to the following formula,
in the formula (I), the compound is shown in the specification,
mGlc-VE6-the content of Glc-VHLTPE in the enzymatic hydrolysate, g/g;
MWGlc-VE6-molecular weight of Glc-VHLTPE;
mVE6-the content of VHLTPE in the enzymatic hydrolysate, g/g;
MWVE6molecular weight of VHLTPE.
2. The method for high-accuracy glycated hemoglobin standard substance quantitation of claim 1, wherein: specifically, the step (1) is to obtain a specific peptide segment VHLTPE for hemoglobin quantification and a corresponding isotope labeling peptide segment VH LTPE thereof by a chemical synthesis mode; specific peptide fragments Glc-VHLTPE and corresponding isotopically labeled peptide fragments Glc-VH LTPE for quantification of glycated hemoglobin are obtained by chemical synthesis.
3. The method for high-accuracy glycated hemoglobin standard substance quantitation of claim 2, wherein: hydrolyzing the synthesized peptide segment into amino acid, quantitatively adding isotope-labeled amino acid, determining the content of stable amino acid in the hydrolysate by using a high performance liquid chromatography-isotope dilution mass spectrometry method established by taking national amino acid standard substances as standards, and calculating the purity of the characteristic peptide segment according to the content; for the specific peptide segment VHLTPE, the amino acids used for quantification were valine V, leucine L and proline P; for the specific peptide fragment Glc-VHLTPE, the amino acids used for quantification were leucine L and proline P.
4. The method for high-accuracy glycated hemoglobin standard substance quantitation of claim 3, wherein: the step (3) is specifically to dissolve a proper amount of glycosylated hemoglobin in an ammonium acetate buffer solution with the pH value of 4.0, add a proper amount of Glu-C according to the ratio of the amount of hemoglobin to the amount of Glu-C of 100:1 according to the amount of the standard substance added, and incubate for 24 hours at 60 ℃ for enzyme digestion.
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