CN113466384A - Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood - Google Patents

Abstract

A whole blood glycosylated hemoglobin content liquid chromatography tandem mass spectrometry quantitative detection method includes obtaining blood cells through whole blood sample centrifugal separation, preparing the blood cells into hemolytic liquid, and obtaining glycosylated HbA through the hydrolysis of recombinant carboxypeptidase B and the disconnection of hemoglobin at a second amino acid residue (His) at the N tail end of a beta chain_1cDipeptide and non-glycosylated HbA₀Dipeptides, which were separated and quantified by HPLC-MS/MS, and HbA1c dipeptide standards and HbA₀Separating and quantifying dipeptide standard mixture as calibrator to obtain standard curve according to HbA1c dipeptide and HbA₀The content of the dipeptide was calculated from the peak area of the dipeptide. The detection method has the advantages of small sample amount, high sensitivity, strong specificity, good reproducibility and short analysis time, and can provide reliable basis for clinical diagnosis.

Description

Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood

Technical Field

The invention relates to the field of medical inspection, in particular to a quantitative detection method of whole blood glycosylated hemoglobin content by liquid chromatography tandem mass spectrometry.

Background

Glycated Hemoglobin (Hemoglobin A)_1c；HbA_1c) Is a stable compound formed by combining glucose and N-terminal valine residue of beta chain of hemoglobin in human blood by covalent bond, and is totally called as: hemoglobin beta chain (blood) -N- (1-deoxyfructose-1-yl) hemoglobin beta chain. HbA_1CNon-enzymatic combination reaction is carried out on free aldehyde group of glucose and amino group of N-terminal valine of beta chain of HbA, unstable Schiff base (aldimine) is firstly formed, then Amadori (glucosamine) is rearranged, and finally stable ketoamine compound is formed, the content of the stable ketoamine compound is mainly determined by blood glucose concentration and contact time of blood glucose and hemoglobin, and the average blood glucose level of 120d before measurement can be reflected. At present, HbA is used for clinical quantitative determination and application_1CResults^[1]。

International Federation of Clinical Chemistry and medical Laboratory Medicine (IFCC): measurement of HbA_1CThe internationally recognized reference method is IFCC recommended high performance liquid chromatography-electrospray ionization-tandem mass spectrometry or high performance liquid chromatography-capillary electrophoresis, and the two methods have consistent results. The determination method mainly comprises three steps: firstly, preparing hemolytic liquid; then adopting endoprotease Glu-C to carry out enzymolysis digestion on the hemolytic solution to obtain glycosylated and non-glycosylated beta chain N-terminal hexapeptide (HbA)_1cHexapeptide, HbA₀Hexapeptides); finally adopting high performance liquid chromatography to connect with electrospray ionization in seriesFirst-order mass spectrum or high performance liquid chromatography tandem capillary electrophoresis (HPLC) to HbA_1cHexapeptides and HbA₀The hexapeptide was quantitatively analyzed. Using a standard substance IRMM/IFCC-466HbA_1cAnd IRMM/IFCC-467HbA₀The mixture of (A) is used as a calibrator, and is subjected to enzymolysis and analysis synchronously to obtain a standard curve according to HbA_1cHexapeptide, HbA₀HbA is calculated by the peak area ratio of hexapeptide_1cIn an amount of^[1]. The hydrolysis of Glu-C endonuclease breaks the hemoglobin at the 6 th amino acid residue (Glu) at the N-terminal of the beta chain to obtain the glycosylated HbA_1CHexapeptides and non-glycosylated HbA₀Hexapeptides, namely: c₄H₉O₄-CO-CH₂-NH-Val-His-Leu-Thr-Pro-Glu-COOH and NH₂-Val-His-Leu-Thr-Pro-Glu-COOH^[2]. The reference method has the advantages of high accuracy, good repeatability and the like, but the pretreatment operation is complex, the time consumption is long, and the measurement cost is high.

Reference documents:

[1] sanitary industry standard WS/T461-2015 of the people's republic of China

[2] Wandong Ring, etc., pre-column derivatization of glycosylated hemoglobin by HPLC, Chinese laboratory diagnostics, 2015 12 months at the end 19 and 12 th period

Disclosure of Invention

In order to solve the technical problems of complex operation, long time consumption and high cost of the existing mass spectrometry for measuring the glycosylated hemoglobin in whole blood, the invention provides a quantitative detection method of the glycosylated hemoglobin content in whole blood by liquid chromatography-tandem mass spectrometry, which comprises the following steps: preparing a whole blood sample to be detected into a hemolytic solution, carrying out enzymolysis on the hemolytic solution by using recombinant carboxypeptidase B, then carrying out separation quantitative analysis by using HPLC-MS/MS, and using HbA₀Dipeptide standards and HbA_1cUsing the mixture of the dipeptide standard products as a calibrator, performing separation and quantitative analysis by HPLC-MS/MS, drawing a standard curve, and using HbA in a whole blood sample to be detected_1cDipeptide and HbA₀The peak area value of the dipeptide is calculated through a standard curve to obtain HbA in the whole blood sample to be detected_1cAnd HbA₀The concentration of the dipeptide is calculated by the following calculation formula to obtain HbA in the whole blood sample to be measured_1cThe content of (a) in (b),the unit is mass percentage, and the calculation formula is as follows: HbA in whole blood sample to be measured_1cContent of HbA_1cDipeptide concentration/(HbA)_1cDipeptide concentration + HbA₀Dipeptide concentration). times.100%, in the formula, HbA_1cDipeptide concentration representing HbA in a whole blood sample to be assayed_1cDipeptide concentration, HbA₀Dipeptide concentration representing HbA in a whole blood sample to be assayed₀The concentration of the dipeptide.

Further, the process for preparing the hemolysis solution from the whole blood sample to be detected comprises the following steps: transferring 100 mu L of whole blood sample into a 1.5mL plastic centrifuge tube by using a pipette, centrifuging at 5600r/min at 8 ℃ for 10min, removing plasma, taking out 10 mu L of blood cells after plasma removal, adding 10 mu L of pure water into a new 1.5mL plastic centrifuge tube, homogenizing at 1500r/min for 30s, adding 200 mu L of reserve buffer solution, and rotationally mixing at 1500r/min for 1min to prepare the hemolytic solution.

The stock buffer solution is prepared by the following method: taking 1mol/L beta morpholine ethanesulfonic acid solution, 0.5mol/L sodium bicarbonate solution and 1mol/L Na by a pipettor₂Adding pure water into EDTA solution, fixing the volume to 100mL, and uniformly mixing until the final concentration of the beta morpholine ethanesulfonic acid is 50mmol/L, NaHCO₃The final concentration is 25mmol/L, Na₂The final concentration of EDTA is 1mmol/L, and the pH value is 6.2, namely the stock buffer solution.

Further, the enzymolysis process of the hemolytic liquid by using the recombinant carboxypeptidase B comprises the following steps: centrifuging the plastic centrifuge tube containing the hemolytic solution at 12000r/min for 10min at high speed, taking out 5 μ L of supernatant, placing in another new 1.5mL plastic centrifuge tube, adding 15 μ L of 200 μ g/mL recombinant carboxypeptidase B solution, adding 200 μ L digestion buffer solution, placing in water bath at 37 deg.C for 2h, freezing at-20 deg.C for 10min, and taking 100 μ L solution for HPLC-MS/MS separation and quantitative analysis, wherein the sample amount is 1 μ L.

The digestion buffer solution is prepared by the following method:

precisely weighing 1.68g of sodium bicarbonate, placing the sodium bicarbonate into a 15mL centrifuge tube, adding 10mL of pure water to dissolve the sodium bicarbonate, oscillating and uniformly mixing the sodium bicarbonate and the pure water to obtain a 2mol/L sodium bicarbonate solution, taking 2.5mL of the 2mol/L sodium bicarbonate solution by a pipettor, adding the pure water to the solution to a constant volume of 100mL, uniformly mixing the solution, measuring the pH value of the solution, adjusting the pH value of the solution to 8.0 by using acetic acid to obtain a digestion buffer solution, and storing the digestion buffer solution at 4 ℃.

Further, the calibrator formulation comprises: taking 6 centrifuge tubes, adding 15 μ l of 200 μ g/mL recombinant carboxypeptidase B solution and 230 μ l digestion buffer solution into each centrifuge tube, mixing, and adding HbA containing different concentrations into each test tube₀Dipeptide standards and HbA_1cStock of standard dipeptide stock 5. mu.l to HbA₀Dipeptide standards and HbA_1cThe final concentration of the dipeptide standard product is as follows, 6 parts of standard working solution are 6 parts of calibrator:

calibration product 1: HbA containing 0.3125. mu.g/mL₀Dipeptide Standard and HbA at 0.03125. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 2: HbA containing 0.625. mu.g/mL₀Dipeptide Standard and HbA at 0.0625. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 3: HbA containing 1.25. mu.g/mL₀Dipeptide Standard and HbA at 0.125. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 4: HbA containing 2.5. mu.g/mL₀Dipeptide Standard and HbA at 0.25. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

and (5) calibration product: HbA containing 5. mu.g/mL₀Dipeptide Standard and HbA at 0.5. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

and 6, calibration product: HbA containing 10. mu.g/mL₀Dipeptide Standard and HbA at 1. mu.g/mL_1cAnd (3) standard yeast working solution of a dipeptide standard product.

The digestion buffer solution is the same as described above.

Further, the different concentrations of HbA-containing substance₀Dipeptide standards and HbA_1cThe standard yeast stock solution of the dipeptide standard product is prepared by the following method: HbA was collected at a concentration of 1000. mu.g/mL₀Dipeptide Standard stock solution and HbA with concentration of 1000 mug/mL_1cMixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution₀Dipeptide standards and HbA_1c6 parts of standard yeast stock solutions of the dipeptide standard product with different final concentrations are respectively as follows:

containing 500. mu.g/mL HbA₀Dipeptide Standard and 50. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 250. mu.g/mL HbA₀Dipeptide Standard and 25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 125. mu.g/mL HbA₀Dipeptide Standard and 12.5. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 62.5. mu.g/mL HbA₀Dipeptide Standard and 6.25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 31.25. mu.g/mL HbA₀Dipeptide Standard and 3.125. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 15.625. mu.g/mL HbA₀Dipeptide Standard and 1.5625. mu.g/mL HbA_1cStandard stock solutions of dipeptide standards.

Further, the following chromatographic and mass spectrometric conditions were used in the HPLC-MS/MS analysis:

chromatographic conditions are as follows: a chromatographic column: ZORBAX SB-CN (2.1 mm. times.150 mm,5 μm), column temperature: 35 ℃, mobile phase a: 0.2% aqueous formic acid, mobile phase B: acetonitrile, sample introduction amount: 1 μ L, flow rate: 0.35mL/min, mobile phase ratio: isocratic elution of 95% B and 5% A for 4.5 min;

mass spectrum conditions: the ion source mode is an ESI source positive ion mode, and the scanning mode is as follows: RMR, atomizing gas flow rate: 3L/min; heating air flow: 10L/min; interface temperature: 350 ℃; DL temperature: 150 ℃; temperature of the heating block: 350 ℃; flow rate of drying gas: 10L/min.

Compared with the prior art, the invention has the beneficial effects that:

the method of the invention has the advantages of few required sample amount, high sensitivity, strong specificity, good reproducibility, short analysis time and simple operation, and can provide reliable basis for clinical diagnosis. The concrete embodiment is as follows:

(1) according to the invention, glycosylated hemoglobin is broken at the 2 nd amino acid residue (His) at the N terminal of a hemoglobin beta chain by the specific hydrolysis of recombinant carboxypeptidase B, so that glycosylated HbA is obtained_1cDipeptide (C)₄H₉O₄-CO-CH₂-NH-Val-His-COOH) and non-glycosylated HbA₀Dipeptide (NH)₂Val-His-COOH) and reasonable HPLC-MS/MS quantitative analysis parameters and technical means such as standard substances, pretreatment and the like, the mass percentage content of the whole blood glycosylated hemoglobin to be detected can be rapidly determined, the required sample amount is extremely small, and 10 mu l of blood cells can meet the requirements.

(2) According to the invention, through the specific hydrolysis action of the recombinant carboxypeptidase B, the hydrolysis efficiency is greatly improved, complete hydrolysis can be carried out at 37 ℃ for 2h, the pretreatment time is shortened, the detection time is greatly shortened, and the inspection efficiency is improved. Compared with other methods for measuring glycated hemoglobin by mass spectrometry, the method has the advantages of short pretreatment time, short analysis time, high detection efficiency and low consumable cost, and is more suitable for batch operation in clinical laboratory.

(3) The reasonable separation parameters of the high performance liquid chromatography column and the selection of the mass spectrum characteristic ions adopted by the invention reduce the interference in the experimental process, have good specificity, can ensure the detection precision and provide reliable basis for clinical diagnosis.

Compared with the IFCC reference method, the method has the following advantages:

1. the pretreatment steps are simple: the pretreatment of the IFCC method needs to be carried out by warm bath for 4 hours to wash the red blood cells twice, the V8 protease is added to hydrolyze the red blood cells and needs to be carried out by warm bath for 18 hours, but the invention does not need to wash the red blood cells by warm bath, and the hydrolysis of the red blood cells by the recombinant carboxypeptidase B only needs to be carried out for 2 hours. The pretreatment steps are simple and the required time is greatly shortened.

2. The analysis time on the computer is short: the IFCC method has the advantage that the analysis time of a single sample is as long as 23min, but the method can analyze the single sample only by 4.5 min.

3. The precision is high: the IFCC method adopts a primary mass spectrum (HPLC-MS) for analysis and a parent ion scanning mode, and the invention adopts a secondary mass spectrum (HPLC-MS/MS) for analysis and an RMR mode for scanning, thereby improving the experimental precision and sensitivity.

4. The detection cost is low: the price of recombinant carboxypeptidase B used in the present invention is lower than that of V8 protease (currently commercially available recombinant carboxypeptidase B is 399 RMB per mg, while V8 protease is 1800 RMB per mg).

(4) The method is suitable for measuring the content of the glycated hemoglobin in the human whole blood sample, and can accurately reflect the ratio of the glycated hemoglobin in the human whole blood sample to the hemoglobin after being verified in normal people and diabetics so as to assist in diagnosis of related diseases.

(5) The invention is not affected by sample hemolysis, sample transportation time and transportation conditions, and the sample can be stored for 7 days at normal temperature and can still reflect the real concentration of the sample after being placed for more than three months at-20 ℃.

Drawings

FIG. 1: reagent blank mass spectrum.

FIG. 2: HbA₀Spectrum of standard curve of dipeptide standard, HbA is plotted on abscissa in FIG. 2₀Concentration of dipeptide Standard (. mu.g/mL), the ordinate is HbA₀Chromatographic peak area of dipeptide standard.

FIG. 3: HbA_1cSpectrum of standard curve of dipeptide standard, HbA is plotted on abscissa in FIG. 2_1cConcentration of dipeptide Standard (. mu.g/mL), the ordinate is HbA_1cChromatographic peak area of dipeptide standard.

FIG. 4: HbA in whole blood sample to be detected_1cDipeptide chromatogram. In FIG. 4, the abscissa represents the HbA1c dipeptide analysis time (min) in the whole blood sample to be tested, and the ordinate represents the HbA1c dipeptide instrument response value (mv) in the whole blood sample to be tested.

FIG. 5: HbA in whole blood sample to be detected₀Dipeptide chromatogram. FIG. 5 shows the horizontal axis of HbA in the whole blood sample to be measured₀Dipeptide analysis time (min), ordinate HbA in whole blood sample to be tested₀Dipeptide instrument response value (mv).

FIG. 6: HbA in whole blood sample to be detected_1cDipeptide mass spectrum. FIG. 6 shows the HbA1c dipeptide mass-to-charge ratio (m/z) in the whole blood sample to be tested on the abscissa and the HbA1c dipeptide instrument response value (mv) in the whole blood sample to be tested on the ordinate.

FIG. 7: HbA in whole blood sample to be detected₀Dipeptide mass spectrum. FIG. 7 shows the horizontal axis of HbA in the whole blood sample to be measured₀Dipeptide Mass-to-Charge (m/z), with the ordinate being HbA in the whole blood sample to be assayed₀Dipeptide instrument response values(mv)。

Detailed Description

The present invention is further illustrated by the following examples, in which reagents and equipment are commercially available, and conventional methods in the art are not specifically described in the examples.

The terms:

HbA_1cis glycosylated hemoglobin.

HbA₀Is non-glycated hemoglobin.

HbA_1cThe dipeptide is a dipeptide compound in which glycated hemoglobin is cleaved at the 2 nd amino acid residue (His) at the N-terminal of the beta chain.

HbA₀Dipeptides are dipeptide compounds in which hemoglobin is cleaved at the 2 nd amino acid residue (His) at the N-terminal end of the beta chain.

HPLC-MS/MS: is short for high performance Liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Reagent and main instrument

Reagents (see table 1):

TABLE 1 reagent information

The main apparatus comprises:

the detection device comprises: shimadzu high performance liquid chromatography tandem mass spectrometer 8045(LCMS-8045)

Detecting environmental requirements: the humidity is 20-80%; the temperature is 15-28 DEG C

The detection reagent is pure in chromatography if no special requirement exists.

Embodiment of the invention provides a method for quantitatively detecting content of glycated hemoglobin in whole blood by liquid chromatography tandem mass spectrometry

The embodiment is suitable for measuring the content of the glycosylated hemoglobin in the whole blood. Preparation of the whole blood sample, the test equipment and the test reagents is required prior to the test. The specific operation is as follows:

1 drawing a standard curve

1.1 preparation of standard yeast stock solution:

1.1.1 preparation of standard stock solution:

accurately weighing 3mg HbA₀Dipeptide Standard and 3mg HbA_1cThe dipeptide standards were dissolved in 3mL of pure water and prepared into HbA solutions of 1000. mu.g/mL concentrations in pure water₀Dipeptide Standard stock solution and HbA at a concentration of 1000. mu.g/mL_1cThe dipeptide standard stock solution (the pure concentration without salt and crystal water according to the conversion of the purity of the standard product) is preserved at the temperature of 80 ℃.

1.1.2 preparation of standard yeast stock solution:

HbA was collected at a concentration of 1000. mu.g/mL₀Dipeptide Standard stock solution and HbA with concentration of 1000 mug/mL_1cMixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution₀Dipeptide standards and HbA_1c6 parts of standard dipeptide stock solutions with different final concentrations are respectively marked as L6, L5, L4, L3, L2 and L1:

l6: containing 500. mu.g/mL HbA₀Dipeptide Standard and 50. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

l5: containing 250. mu.g/mL HbA₀Dipeptide Standard and 25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

l4: containing 125. mu.g/mL HbA₀Dipeptide Standard and 12.5. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

l3: containing 62.5. mu.g/mL HbA₀Dipeptide Standard and 6.25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

l2: containing 31.25. mu.g/mL HbA₀Dipeptide Standard and 3.125. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

l1: containing 15.625. mu.g/mL HbA₀Dipeptide Standard and 1.5625. mu.g/mL HbA_1cStandard stock solutions of dipeptide standards.

1.1.3 other solution preparation:

1.1.3.1 digestion buffer solution (50mmol/L NaHCO)₃) Preparation:

sodium bicarbonate 1.68g was precisely weighed and placed in a 15mL containerAdding 10mL of pure water into a heart tube for dissolving, oscillating and uniformly mixing to obtain a 2mol/L sodium bicarbonate solution, taking 2.5mL of the 2mol/L sodium bicarbonate solution by a pipettor, adding pure water to a constant volume of 100mL, uniformly mixing, measuring the pH value of the mixture, and adjusting the pH value of the mixture to 8.0 by using acetic acid to obtain a digestion buffer solution (50mmol/L NaHCO)₃) And storing at 4 ℃ for later use.

1.1.3.2200 μ g/mL recombinant carboxypeptidase B solution preparation: precisely weighed analytically pure recombinant carboxypeptidase B was dissolved and diluted with pure water to prepare a recombinant carboxypeptidase B solution having a concentration of 200. mu.g/mL.

1.1.3.3 stock buffer solution preparation: the pipettor took 5mL of 1mol/L beta morpholine ethanesulfonic acid (MES) solution, 5mL of 0.5mol/L sodium bicarbonate solution and 100. mu.L of 1mol/L Na₂EDTA solution, adding pure water to constant volume of 100mL, and mixing, wherein the solution contains beta morpholine ethanesulfonic acid (MES) with final concentration of 50mmol/L and NaHCO with final concentration of 25mmol/L₃、1mmol/L Na₂EDTA, measured by a pH meter, should have a pH of 6.2, this solution being a stock buffer solution.

1.2 preparation of standard yeast working solution (namely, calibrator):

respectively taking 5 mul of the standard curve stock solutions with different concentrations in the step 1.1.2, respectively placing the standard curve stock solutions into different 1.5mL centrifuge tubes, respectively adding 15 mul of the recombinant carboxypeptidase B solution with the concentration of 200 mu g/mL and 230 mul of the digestion buffer solution into each centrifuge tube, uniformly mixing the solution and the digestion buffer solution to respectively prepare the target product containing HbA₀Dipeptide standards and HbA_1cThe final concentration of the dipeptide standard product is as follows, and the standard yeast working solution is a calibrator:

calibration product 1: HbA containing 0.3125. mu.g/mL₀Dipeptide Standard and HbA at 0.03125. mu.g/mL_1cWorking solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji marked L1 described above).

Calibration product 2: HbA containing 0.625. mu.g/mL₀Dipeptide Standard and HbA at 0.0625. mu.g/mL_1cWorking solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji marked L2 described above).

Calibration product 3: HbA containing 1.25. mu.g/mL₀Dipeptide Standard and HbA at 0.125. mu.g/mL_1cStandard dipeptide Standard A working solution (corresponding to the above-mentioned standard A stock of Standard A. RTM. with the reference L3Preparation of a stock solution).

Calibration product 4: HbA containing 2.5. mu.g/mL₀Dipeptide Standard and HbA at 0.25. mu.g/mL_1cWorking solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji marked L4 described above).

And (5) calibration product: HbA containing 5. mu.g/mL₀Dipeptide Standard and HbA at 0.5. mu.g/mL_1cWorking solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji marked L5 described above).

And 6, calibration product: HbA containing 10. mu.g/mL₀Dipeptide Standard and HbA at 1. mu.g/mL_1cWorking solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji marked L6 described above).

1.3 drawing a standard curve:

respectively using the calibrators in the step 1.2 to perform separation and quantitative detection on the Shimadzu high performance liquid chromatography tandem mass spectrometer 8045 instrument by using the high performance liquid chromatography tandem mass spectrometry, wherein the detection parameter conditions of the chromatogram and the mass spectrometry are shown in tables 2 and 3, and obtaining HbA of each calibrator respectively₀Peak area value and HbA of dipeptide Standard_1cPeak area values of the dipeptide standard. The HbA content of each calibrator was measured₀Concentration of dipeptide Standard, HbA_1cThe concentration of the dipeptide standards was plotted on the abscissa, and HbA in each calibrator was used in accordance with the concentration₀Peak area value and HbA of dipeptide Standard_1cHbA is respectively drawn by taking peak area value of dipeptide standard product as ordinate₀Standard Curve spectrogram (FIG. 2) and HbA of dipeptide Standard substance_1cSpectrum of standard curve of dipeptide standard (fig. 3).

TABLE 2 chromatographic and Mass Spectrometry conditions for Shimadzu high Performance liquid chromatography tandem Mass spectrometer 8045

Mass spectrometry scan parameters: (see Table 3)

Table 3Q 3 SIM parameter setting table

Simultaneously detecting each calibrator, and performing reagent blank detection under the same chromatographic and mass spectrum conditions of each calibrator, wherein the reagent is HbA except for all related HbA in standard working solution₀Dipeptide standards and HbA_1cExcept that pure water is used for replacing the dipeptide standard product, the other reagent components, the dosage and the concentration are the same as those of the standard yeast working solution. The blank mass spectrum of the reagent is shown in figure 1.

2 liquid chromatography tandem mass spectrometry quantitative detection of glycosylated hemoglobin content in whole blood sample to be detected

2.1 treatment of whole blood sample to be tested:

2.1.1 Collection of Whole blood samples: before the whole blood sample is collected, the nervous psychology of a patient needs to be eliminated, the operation needs to be standardized during blood collection, and the whole blood sample is mixed in time. EDTA-Na is selected for whole blood sample for submission₂2mL of blood of the patient is extracted by a purple cap blood sampling vacuum tube. Selecting a sample container: sterile disposable vacuum blood collection tubes (bidi medical devices (shanghai) ltd.).

2.1.2 preparation of hemolysis solution from Whole blood sample to be tested

And transferring 100 mu L of whole blood sample into a 1.5mL plastic centrifuge tube by using a pipette, centrifuging at 5600r/min at 8 ℃ for 10min, then discarding plasma, taking 10 mu L of blood cells from the plastic centrifuge tube into a new 1.5mL plastic centrifuge tube, adding 10 mu L of pure water, homogenizing at 1500r/min for 30s, adding 200 mu L of reserve buffer solution, and then rotationally mixing at 1500r/min for 1min to prepare the hemolytic solution.

2.1.3 enzymolysis and detection on computer

Centrifuging the plastic centrifuge tube containing the hemolytic solution at high speed of 12000r/min for 10min, taking out 5 μ L of supernatant, placing the supernatant in another new 1.5mL plastic centrifuge tube, adding 15 μ L of 200 μ g/mL recombinant carboxypeptidase B solution, adding 200 μ L digestion buffer solution, placing in a water bath at 37 ℃ for 2h, freezing at-20 ℃ for 10min, taking 100 μ L of solution, and performing HPLC-MS/MS separation quantitative analysis, wherein the sample amount is 1 μ L, and the chromatographic and mass spectrum parameter conditions are shown in Table 2 and Table 3.

2.2 calculating the concentration, and the invention uses the hydrolysis of recombinant carboxypeptidase B to detect the 2 nd amino group of the hemoglobin in the haemolysis solution of the whole blood sample to be detected at the N tail end of the beta chainCleavage at acid residue (His) to give glycosylated HbA_1cDipeptide (C)₄H₉O₄–CO-CH₂-NH-Val-His-COOH) and non-glycosylated HbA₀Dipeptide (NH)₂Val-His-COOH) which were separated and quantified by HPLC-MS/MS, using HbA1c dipeptide standard and HbA₀The dipeptide standard substance mixture is used as a calibrator, HPLC-MS/MS is used for carrying out separation and quantitative analysis on the calibrator, a standard curve is drawn, and HbA in a whole blood sample to be detected is used_1cDipeptide chromatographic Peak area value and HbA₀Chromatographic peak area of dipeptide (FIGS. 4 and 5) HbA in whole blood sample to be measured was calculated from the standard curve_1cDipeptide and HbA₀The concentration unit of the dipeptide is ug/ml, and then HbA in the whole blood sample to be detected is obtained by calculating according to the following calculation formula_1cAnd the content and the reported result unit are mass percent. Specifically, as shown in FIG. 4 and FIG. 5, HbA in the whole blood sample to be measured_1cThe peak area value of the dipeptide chromatogram is 1488657 (shown by an instrument), and HbA in the whole blood sample to be detected₀The chromatographic peak area value of the dipeptide is 62527081 (shown by the instrument), and HbA in the whole blood sample to be measured is calculated (automatically calculated by the instrument) by correspondingly inputting the chromatographic peak area value through a standard curve chart 2 and a figure 3_1cDipeptide concentration was 0.150. mu.g/mL and HbA₀The dipeptide concentration is 2.893 mug/mL, and HbA in the whole blood sample to be detected is obtained by calculating according to the following calculation formula_1cThe content was 4.93% (mass percent).

Calculating the formula: HbA in whole blood sample to be detected_1cContent (%) ═ HbA_1cDipeptide concentration/(HbA)_1cDipeptide concentration + HbA₀Dipeptide concentration). times.100%, HbA in the formula_1cDipeptide concentration representing HbA in a whole blood sample to be assayed_1cDipeptide concentration (in. mu.g/mL, calculated from a standard curve); HbA₀Dipeptide concentration representing HbA in a whole blood sample to be assayed₀Dipeptide concentration (in. mu.g/mL, calculated from a standard curve).

3 methodological validation

Daytime precision within 3.1 days

3.1.1 purposes of the experiment

Stability of the investigation experiment

3.1.2 Experimental methods

Taking 3 different human whole blood samples, marking the samples as a sample 1, a sample 2 and a sample 3, processing the whole blood sample to be detected according to the step 2.1, carrying out parallel processing on 5 parts of each sample, and measuring three batches on the same day to calculate the in-day precision; the 3 samples were processed 5 in parallel per sample and the day precision calculated for three consecutive days, and the chromatographic and mass spectral parameter conditions are shown in tables 2 and 3.

3.1.3 results (Table 4)

TABLE 4 method precision measurement results

3.1.4 conclusions of the experiment

The relative standard deviation RSD of the precision in the day time in the experimental result day is less than 6.5 percent, and the requirement of the biological sample for measuring the RSD is less than 15 percent is met.

3.2 recovery rate by adding standard

3.2.1 purpose of the experiment

The accuracy of the experimental results was investigated by the recovery of the spiked samples.

3.2.2 Experimental methods

3.2.2.1 preparation of hemolytic solution, taking 200 μ l of normal human whole blood sample, centrifuging at 5600r/min at 8 ℃ for 10min to remove plasma, taking 50 μ l of blood cells, adding 50 μ l of pure water, mixing uniformly, adding 1mL of storage buffer solution, mixing uniformly, and centrifuging at 12000r/min8 ℃ for 10min to obtain hemolytic solution.

3.2.2.2 labeling, namely taking three 1.5mL centrifuge tubes, respectively adding 8 mu L of the L1 standard curve stock solution, 8 mu L of the L3 standard curve stock solution and 8 mu L of the L4 standard curve stock solution which are described in the step 1.1.2, then adding 192 mu L of the hemolytic solution which is described in the step 3.2.1 into the three centrifuge tubes, uniformly mixing the mixture at 1500r/min for 1min to obtain 3 labeling solutions, wherein the theoretical labeling solution concentration is low (0.625 mu g/mL HbA A)₀、0.0625μg/mL HbA_1c) Medium concentration (2.5. mu.g/mL HbA)₀、0.25μg/mL HbA_1c) And high concentration (5. mu.g/mL HbA)₀、0.5μg/mL HbA_1c) 5 parts of each labeled parallel sample are respectively taken out, 5 mu L of labeled liquid is respectively put in a 1.5mL plastic centrifuge tubeAdding 15 mu L of 200 mu g/mL recombinant carboxypeptidase B and 200 mu L of digestion buffer solution, uniformly mixing at 1500r/min for 1min, then placing in a water bath at 37 ℃ for 2h, taking out and freezing at-20 ℃ for 10min, taking 100 mu L of each component, and carrying out HPLC-MS/MS analysis, wherein the sample amount is 1 mu L, and the chromatographic and mass spectrum parameter conditions are shown in tables 2 and 3.

3.2.3 results (Table 5)

TABLE 5 recovery test results

Table 5 the white-in-air samples are 3.2.2.1 normal human whole blood samples.

3.2.4 conclusions of the experiment

According to experimental results, the recovery rates of the three standard adding concentrations are all 95-115%, the relative standard deviation RSD is less than 6.0%, and the requirements that the recovery rate is 85-120% and the RSD is less than 15.0% are met through methodology verification.

3.3 Standard Curve

3.3.1 purpose of the experiment

Verification of the Linear Range of the method

3.3.2 Experimental methods

And (4) drawing a standard curve according to the method in the step 1, carrying out sample injection analysis, and analyzing in total.

3.3.3 results of the experiment (Table 6)

TABLE 6 Standard Curve validation results

3.3.4 conclusions of the experiment

Standard curve range: HbA is shown by 1.2, 1.3 and 3.3.3₀The dipeptide standard product has good linearity and correlation coefficient R in the range of 0.3125 mu g/mL to 10 mu g/mL²﹥0.999；HbA_1cThe dipeptide standard product is in the range of 0.03125 mu g/mL to 1 mu g/mL, the linearity is good, and the correlation coefficient R is²More than 0.999; satisfies the methodology R²> 0.990.

3.4 lower limit of quantitation

3.4.1 purpose of the experiment

Examining the accuracy of detection of a sample at around the lower limit of quantitation

3.4.2 Experimental methods

Taking a normal human whole blood sample according to the method of the steps 2.1.1-2.1.3, processing five parts in parallel, calculating the average concentration value and the corresponding S/N (signal to noise ratio) value of the five parts of the sample, diluting the whole blood sample according to the known S/N value, processing five parts in parallel, and enabling the S/N value corresponding to the diluted sample to be equal to about 10, wherein the corresponding concentration value is the quantification limit.

3.4.3 results (Table 7)

TABLE 7 quantitative limit verification results

3.4.4 conclusions of the experiment

Limit of quantitation (LOQ): HbA₀Dipeptide 0.12. mu.g/mL, HbA_1cDipeptide 0.024 μ g/mL; linear range: HbA₀Dipeptide in the range of 0.3125 μ g/mL to 10 μ g/mL, HbA_1cThe dipeptide is in the range of 0.03125 mug/mL to 1 mug/mL, has good linearity, and meets the requirement of taking 10 times of signal-to-noise ratio (10S/N) as a limit of quantitation (LOQ).

By combining the verification experiments, the method has the advantages of short pretreatment time (the whole pretreatment time is 2.5-3.0 hours), short on-machine detection and analysis time (the time required by each sample for detection is 4.5min), high precision (the maximum value of the precision RSD in each day is 6.41 percent, and the maximum value of the precision RSD in each day is 4.48 percent), good stability (the recovery rates are 95-115 percent, and the relative standard deviation RSD is<6.0 percent), high sensitivity (the limit of quantitation is 0.12 mu g/mL), and strong specificity (HbA)₀Dipeptide parent ion 288.6m/z, daughter ion 110.1m/z, HbA_1cDipeptide parent ion 408.3m/z, daughter ion 263.1m/z) to meet the requirement of clinical glycosylated hemoglobin determination.

Claims (6)

1. A method for quantitatively detecting the content of glycosylated hemoglobin in whole blood by liquid chromatography-tandem mass spectrometry,the method is characterized in that a whole blood sample to be detected is prepared into a hemolytic solution, the hemolytic solution is subjected to enzymolysis by recombinant carboxypeptidase B, then separation quantitative analysis is carried out by HPLC-MS/MS, and HbA is used₀Dipeptide standards and HbA_1cUsing the mixture of the dipeptide standard products as a calibrator, performing separation and quantitative analysis by HPLC-MS/MS, drawing a standard curve, and using HbA in a whole blood sample to be detected_1cDipeptide and HbA₀The peak area value of the dipeptide is calculated through a standard curve to obtain HbA in the whole blood sample to be detected_1cAnd HbA₀The concentration of the dipeptide is calculated by the following calculation formula to obtain HbA in the whole blood sample to be measured_1cThe unit is mass percentage, and the calculation formula is as follows: HbA in whole blood sample to be measured_1cContent of HbA_1cDipeptide concentration/(HbA)_1cDipeptide concentration + HbA₀Dipeptide concentration). times.100%, in the formula, HbA_1cDipeptide concentration representing HbA in a whole blood sample to be assayed_1cDipeptide concentration, HbA₀Dipeptide concentration representing HbA in a whole blood sample to be assayed₀The concentration of the dipeptide.

2. The method for the quantitative determination of the glycated hemoglobin content in whole blood by liquid chromatography-tandem mass spectrometry of claim 1, wherein the step of preparing the whole blood sample to be tested into the hemolysis solution comprises the steps of: transferring 100 mu L of whole blood sample into a 1.5mL plastic centrifuge tube by using a pipette, centrifuging at 5600r/min at 8 ℃ for 10min, removing plasma, taking out 10 mu L of blood cells after plasma removal, adding 10 mu L of pure water into a new 1.5mL plastic centrifuge tube, homogenizing at 1500r/min for 30s, adding 200 mu L of reserve buffer solution, and rotationally mixing at 1500r/min for 1min to prepare the hemolytic solution;

the stock buffer solution is prepared by the following method: taking 1mol/L beta morpholine ethanesulfonic acid solution, 0.5mol/L sodium bicarbonate solution and 1mol/L Na by a pipettor₂Adding pure water into EDTA solution, fixing the volume to 100mL, and uniformly mixing until the final concentration of the beta morpholine ethanesulfonic acid is 50mmol/L, NaHCO₃The final concentration is 25mmol/L, Na₂The final concentration of EDTA is 1mmol/L, and the pH value is 6.2, namely the stock buffer solution.

3. The method for the quantitative determination of glycated hemoglobin content in whole blood by liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the enzymatic hydrolysis of the hemolysis solution with recombinant carboxypeptidase B comprises: centrifuging the plastic centrifuge tube containing the hemolytic solution at a high speed of 12000r/min for 10min, taking out 5 mu L of upper layer solution, putting the upper layer solution into another new plastic centrifuge tube with the volume of 1.5mL, adding 15 mu L of recombinant carboxypeptidase B solution with the concentration of 200 mu g/mL, adding 200 mu L of digestion buffer solution, carrying out water bath at 37 ℃ for 2h, freezing at-20 ℃ for 10min, and then taking 100 mu L of solution for HPLC-MS/MS separation quantitative analysis, wherein the sample injection amount is 1 mu L;

the digestion buffer solution is prepared by the following method:

precisely weighing 1.68g of sodium bicarbonate, placing the sodium bicarbonate into a 15mL centrifuge tube, adding 10mL of pure water to dissolve the sodium bicarbonate, oscillating and uniformly mixing the sodium bicarbonate and the pure water to obtain 2mol/L sodium bicarbonate solution, taking 2.5mL of the 2mol/L sodium bicarbonate solution by a pipettor, adding the pure water to the solution to a constant volume of 100mL, uniformly mixing the solution, measuring the pH value of the solution, adjusting the pH value of the solution to 8.0 by using acetic acid to obtain a digestion buffer solution, and storing the digestion buffer solution at 4 ℃.

4. The method for quantitative measurement of glycated hemoglobin content in whole blood according to claim 1, wherein the preparation of the calibrator comprises: taking 6 centrifuge tubes, adding 15 μ l of 200 μ g/mL recombinant carboxypeptidase B solution and 230 μ l digestion buffer solution into each centrifuge tube, mixing, and adding HbA containing different concentrations into each test tube₀Dipeptide standards and HbA_1cStock of standard dipeptide stock 5. mu.l to HbA₀Dipeptide standards and HbA_1cThe final concentration of the dipeptide standard product is as follows, 6 parts of standard working solution are 6 parts of calibrator:

calibration product 1: HbA containing 0.3125. mu.g/mL₀Dipeptide Standard and HbA at 0.03125. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 2: HbA containing 0.625. mu.g/mL₀Dipeptide Standard and HbA at 0.0625. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 3: HbA containing 1.25. mu.g/mL₀Dipeptide Standard and HbA at 0.125. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

calibration product 4: HbA containing 2.5. mu.g/mL₀Dipeptide Standard and HbA at 0.25. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

and (5) calibration product: HbA containing 5. mu.g/mL₀Dipeptide Standard and HbA at 0.5. mu.g/mL_1cStandard yeast working solution of the dipeptide standard product;

and 6, calibration product: HbA containing 10. mu.g/mL₀Dipeptide Standard and HbA at 1. mu.g/mL_1cAnd (3) standard yeast working solution of a dipeptide standard product.

The digestion buffer solution is the same as the digestion buffer solution described in claim 3.

5. The method of claim 4, wherein the HbA-containing blood of different concentrations is measured by liquid chromatography-tandem mass spectrometry₀Dipeptide standards and HbA_1cThe standard yeast stock solution of the dipeptide standard product is prepared by the following method: HbA was collected at a concentration of 1000. mu.g/mL₀Dipeptide Standard stock solution and HbA with concentration of 1000 mug/mL_1cMixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution₀Dipeptide standards and HbA_1c6 parts of standard yeast stock solutions of the dipeptide standard product with different final concentrations are respectively as follows:

containing 500. mu.g/mL HbA₀Dipeptide Standard and 50. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 250. mu.g/mL HbA₀Dipeptide Standard and 25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 125. mu.g/mL HbA₀Dipeptide Standard and 12.5. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 62.5. mu.g/mL HbA₀Dipeptide Standard and 6.25. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 31.25. mu.g/mL HbA₀Dipeptide Standard and 3.125. mu.g/mL HbA_1cA standard stock solution of a dipeptide standard;

containing 15.625. mu.g/mL HbA₀Dipeptide Standard and 1.5625. mu.g/mL HbA_1cStandard mark of dipeptide standard productAnd (4) stock solution.

6. The method for the quantitative determination of glycated hemoglobin content in whole blood by liquid chromatography-tandem mass spectrometry according to claim 1, wherein the following chromatographic conditions and mass spectrometry conditions are used in the HPLC-MS/MS analysis:

chromatographic conditions are as follows: a chromatographic column: ZORBAX SB-CN (2.1 mm. times.150 mm,5 μm), column temperature: 35 ℃, mobile phase a: 0.2% aqueous formic acid, mobile phase B: acetonitrile, sample introduction amount: 1 μ L, flow rate: 0.35mL/min, mobile phase ratio: isocratic elution of 95% B and 5% A for 4.5 min;

mass spectrum conditions: the ion source mode is an ESI source positive ion mode, and the scanning mode is as follows: RMR, atomizing gas flow rate: 3L/min; heating air flow: 10L/min; interface temperature: 350 ℃; DL temperature: 150 ℃; temperature of the heating block: 350 ℃; flow rate of drying gas: 10L/min.

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