CN113466384B - Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood - Google Patents
Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood Download PDFInfo
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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 1c Dipeptide and non-glycosylated HbA 0 Dipeptides, which were separated and quantified by HPLC-MS/MS, and HbA1c dipeptide standards and HbA 0 Separating and quantifying dipeptide standard mixture as calibrator to obtain standard curve based on HbA1c dipeptide and HbA 0 The 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
Technical Field
The invention relates to the field of medical inspection, in particular to a liquid chromatography tandem mass spectrometry quantitative detection method for the content of glycosylated hemoglobin in whole blood.
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 1C Prepared from grapesThe free aldehyde group of the sugar and the amino group of the valine at the N terminal of the beta chain of HbA are subjected to non-enzymatic binding reaction to form unstable Schiff base (aldimine), then Amadori (glucosamine) is subjected to rearrangement, and finally a stable ketoamine compound is formed, wherein the content of the stable ketoamine compound is mainly determined by blood glucose concentration and contact time of blood glucose and hemoglobin, and can reflect the average blood glucose level of 120 days before measurement. At present, hbA is used for clinical quantitative determination and application 1C Results [1] 。
International Federation of Clinical Chemistry and medical Laboratory Medicine (IFCC): measurement of HbA 1C The 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) 1c Hexapeptide, hbA 0 Hexapeptides); finally, high performance liquid chromatography is adopted to be connected with electrospray ionization primary mass spectrum or high performance liquid chromatography is adopted to be connected with capillary electrophoresis to HbA 1c Hexapeptides and HbA 0 The hexapeptide was subjected to quantitative analysis. Using a standard substance IRMM/IFCC-466HbA 1c And IRMM/IFCC-467HbA 0 The mixture of (A) is used as a calibrator, and is subjected to enzymolysis and analysis synchronously to obtain a standard curve according to HbA 1c Hexapeptide, hbA 0 HbA is calculated by the peak area ratio of hexapeptide 1c In 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 1C Hexapeptides and non-glycosylated HbA 0 Hexapeptides, namely: c 4 H 9 O 4 -CO-CH 2 -NH-Val-His-Leu-Thr-Pro-Glu-COOH and NH 2 -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:
[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 of whole blood, the invention provides a liquid chromatography tandem mass spectrometry quantitative detection method for the glycosylated hemoglobin content of whole blood, 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 HbA 0 Dipeptide standards and HbA 1c Using 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 1c Dipeptide and HbA 0 The peak area value of the dipeptide is calculated through a standard curve to obtain HbA in the whole blood sample to be detected 1c And HbA 0 The concentration of dipeptide is calculated by the following calculation formula to obtain HbA in the whole blood sample to be detected 1c The unit is mass percentage, and the calculation formula is as follows: hbA in whole blood sample to be measured 1c Content = HbA 1c Dipeptide concentration/(HbA) 1c Dipeptide concentration + HbA 0 Dipeptide concentration). Times.100%, in the formula, hbA 1c Dipeptide concentration represents HbA in the whole blood sample to be assayed 1c Dipeptide concentration, hbA 0 Dipeptide concentration representing HbA in a whole blood sample to be assayed 0 The 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, homogenizing at 1500r/min for 30s, adding 200 mu L of reserve buffer solution, and uniformly mixing at 1500r/min for 1min to obtain 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 pipettor 2 EDTA solution, adding pure water to a constant volume of 100mL, and mixing to obtain betaFinal concentration of morpholine ethanesulfonic acid is 50mmol/L, naHCO 3 Final concentration of 25mmol/L, na 2 The 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 mul of recombinant carboxypeptidase B solution with the concentration of 200 mu g/mL and 230 mul of digestion buffer solution into each centrifuge tube, mixing uniformly, and then adding HbA containing different concentrations into each test tube respectively 0 Dipeptide standards and HbA 1c Stock of standard dipeptide stock 5. Mu.l to HbA 0 Dipeptide standards and HbA 1c The 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/mL 0 Dipeptide Standard and HbA at 0.03125. Mu.g/mL 1c Standard yeast working solution of the dipeptide standard product;
calibration product 2: hbA containing 0.625. Mu.g/mL 0 Dipeptide Standard and HbA at 0.0625. Mu.g/mL 1c Standard yeast working solution of the dipeptide standard product;
the calibrator 3: hbA containing 1.25. Mu.g/mL 0 Dipeptide Standard and HbA at 0.125. Mu.g/mL 1c Standard yeast working solution of the dipeptide standard product;
calibration product 4: hb containing 2.5. Mu.g/mLA 0 Dipeptide Standard and HbA at 0.25. Mu.g/mL 1c A standard working solution of a dipeptide standard product;
and (5) calibration product: hbA containing 5. Mu.g/mL 0 Dipeptide Standard and HbA at 0.5. Mu.g/mL 1c Standard yeast working solution of the dipeptide standard product;
and 6, calibration product: hbA containing 10. Mu.g/mL 0 Dipeptide Standard and HbA at 1. Mu.g/mL 1c A standard working solution of a dipeptide standard product.
The digestion buffer solution is the same as described above.
Further, the different concentrations of HbA-containing substance 0 Dipeptide standards and HbA 1c The 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/mL 0 Dipeptide Standard stock solution and HbA at a concentration of 1000. Mu.g/mL 1c Mixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution 0 Dipeptide standards and HbA 1c 6 parts of standard yeast stock solutions of the dipeptide standard product with different final concentrations are respectively as follows:
containing 500. Mu.g/mL HbA 0 Dipeptide Standard and 50. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
containing 250. Mu.g/mL HbA 0 Dipeptide Standard and 25. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
containing 125. Mu.g/mL HbA 0 Dipeptide Standard and 12.5. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
containing 62.5. Mu.g/mL HbA 0 Dipeptide Standard and 6.25. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
containing 31.25. Mu.g/mL HbA 0 Dipeptide Standard and 3.125. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
containing 15.625 mu g/mL HbA 0 Dipeptide Standard and 1.5625. Mu.g/mL HbA 1c Standard 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.150mm, 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: 95% by weight of B,5% by weight of A isocratic elution, with an elution time of 4.5min;
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 1c Dipeptide (C) 4 H 9 O 4 -CO-CH 2 -NH-Val-His-COOH) and non-glycosylated HbA 0 Dipeptide (NH) 2 Val-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 very 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 can analyze a single sample for 23min, while the method can analyze a single sample only needing 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 available at a price of 399 RMB per mg, whereas 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 is a schematic diagram: hbA 0 Spectrum of standard curve of dipeptide standard, hbA is plotted on abscissa in FIG. 2 0 Concentration of dipeptide Standard (. Mu.g/mL), the ordinate is HbA 0 Chromatographic peak area of dipeptide standard.
FIG. 3: hbA 1c Spectrum of standard curve of dipeptide standard, hbA is plotted on abscissa in FIG. 2 1c Concentration of dipeptide Standard (. Mu.g)/mL), ordinate is HbA 1c Chromatographic peak area of dipeptide standard.
FIG. 4 is a schematic view of: hbA in whole blood sample to be detected 1c Dipeptide 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 is a schematic view of: hbA in whole blood sample to be detected 0 Dipeptide chromatogram. FIG. 5 shows the horizontal axis of HbA in the whole blood sample to be measured 0 Dipeptide analysis time (min), ordinate HbA in whole blood sample to be tested 0 Dipeptide instrument response value (mv).
FIG. 6: hbA in whole blood sample to be detected 1c Dipeptide 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 is a schematic view of: hbA in whole blood sample to be detected 0 Dipeptide mass spectrum. FIG. 7 shows the horizontal axis of HbA in the whole blood sample to be measured 0 Dipeptide Mass/Charge ratio (m/z), with the ordinate being HbA in the whole blood sample to be assayed 0 Dipeptide instrument response value (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 1c is glycosylated hemoglobin.
HbA 0 Is non-glycated hemoglobin.
HbA 1c The dipeptide is a dipeptide compound in which glycated hemoglobin is cleaved at the 2 nd amino acid residue (His) at the N-terminal end of the beta chain.
HbA 0 Dipeptides 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 the invention provides a method for quantitatively detecting the 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 preparing standard yeast stock solution:
1.1.1 preparation of standard stock solution:
accurately weighing 3mg HbA 0 Dipeptide Standard and 3mg HbA 1c Dipeptide Standard products were dissolved in 3mL of pure water and prepared into HbA solutions having a concentration of 1000. Mu.g/mL 0 Dipeptide Standard stock solution and HbA at a concentration of 1000. Mu.g/mL 1c The 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/mL 0 Dipeptide Standard stock solution and HbA with concentration of 1000 mug/mL 1c Mixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution 0 Dipeptide standards and HbA 1c 6 parts of standard stock solutions of the dipeptide standard are respectively marked as L6, L5, L4, L3, L2 and L1:
l6: containing 500. Mu.g/mL HbA 0 Dipeptide Standard and 50. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
l5: containing 250. Mu.g/mL HbA 0 Dipeptide Standard and 25. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
l4: containing 125. Mu.g/mL HbA 0 Dipeptide Standard and 12.5. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
l3: containing 62.5. Mu.g/mL HbA 0 Dipeptide Standard and 6.25. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
l2: containing 31.25. Mu.g/mL HbA 0 Dipeptide Standard and 3.125. Mu.g/mL HbA 1c A standard stock solution of a dipeptide standard;
l1: containing 15.625 mu g/mL HbA 0 Dipeptide Standard and 1.5625. Mu.g/mL HbA 1c Standard stock solutions of dipeptide standards.
1.1.3 other solution preparation:
1.1.3.1 digestion buffer solution (50 mmol/L NaHCO) 3 ) Preparation:
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 pure water to the 2mol/L sodium bicarbonate solution until the volume is 100mL, uniformly mixing the sodium bicarbonate and the pure water, measuring the pH of the mixture, adjusting the pH value of the mixture to 8.0 by using acetic acid to obtain a digestion buffer solution (50 mmol/L NaHCO) 3 ) And storing at 4 ℃ for later use.
1.1.3.2 Preparation of 200. Mu.g/mL recombinant carboxypeptidase B solution: 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: taking 5mL 1mol/L beta morpholine ethanesulfonic acid (MES) solution, 5mL 0.5mol/L sodium bicarbonate solution and 100 mu.L 1mol/L Na by using a pipettor 2 EDTA 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/L 3 、1mmol/L Na 2 EDTA, 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 mu l of standard curve stock solutions with different concentrations in the step 1.1.2, respectively placing the stock solutions into different 1.5mL centrifuge tubes, respectively adding 15 mu l of recombinant carboxypeptidase B solution with the concentration of 200 mu g/mL and 230 mu l of digestion buffer solution into each centrifuge tube, uniformly mixing, and respectively preparing the mixture into the mixture containing HbA 0 Dipeptide standards and HbA 1c The final concentration of the dipeptide standard product is as follows, and the standard working solution is a calibrator:
calibration product 1: hbA containing 0.3125. Mu.g/mL 0 Dipeptide Standard and HbA at 0.03125. Mu.g/mL 1c Working solutions of the standard dipeptide koji (correspondingly prepared from stock solutions of the standard dipeptide koji designated L1) were used.
Calibration article 2: hbA containing 0.625. Mu.g/mL 0 Dipeptide Standard and HbA at 0.0625. Mu.g/mL 1c Working solutions of the standard dipeptide standard were prepared (correspondingly with stock solutions of the standard dipeptide labeled L2).
Calibration product 3: hbA containing 1.25. Mu.g/mL 0 Dipeptide Standard and HbA at 0.125. Mu.g/mL 1c Working solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji designated L3 above).
The calibrator 4: hbA containing 2.5. Mu.g/mL 0 Dipeptide Standard and HbA at 0.25. Mu.g/mL 1c Working solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard koji designated L4).
And (5) calibration product: hbA containing 5. Mu.g/mL 0 Dipeptide Standard and HbA at 0.5. Mu.g/mL 1c Working solutions of the standard dipeptide standard were prepared (correspondingly with stock solutions of the standard dipeptide labeled L5).
And 6, calibration product: hbA containing 10. Mu.g/mL 0 Dipeptide Standard and HbA at 1. Mu.g/mL 1c Working solutions of the standard dipeptide standards were prepared (correspondingly from stock solutions of the standard transcripts indicated above with L6).
1.3 drawing a standard curve:
respectively using the calibrators in the step 1.2 to respectively operate on an Shimadzu high performance liquid chromatography tandem mass spectrometer 8045 instrument to carry out separation quantitative detection by using a high performance liquid chromatography tandem mass spectrometry, wherein the detection parameter conditions of chromatography and mass spectrometry are shown in tables 2 and 3, and HbA of each calibrator is respectively obtained 0 Peak area value and HbA of dipeptide Standard 1c Peak area values of the dipeptide standard. The HbA content of each calibrator was measured 0 Concentration of dipeptide Standard, hbA 1c The concentration of the dipeptide standards was plotted on the abscissa, and HbA in each calibrator was used in accordance with the concentration 0 Peak area value and HbA of dipeptide Standard 1c HbA is respectively drawn by taking the peak area value of the dipeptide standard product as a vertical coordinate 0 Standard Curve spectrogram (FIG. 2) and HbA of dipeptide Standard substance 1c Spectrum 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 3 Q3 SIM parameter setup 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 solution 0 Dipeptide Standard and HbA 1c Except 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 submission 2 2mL 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 uniformly 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 by the hydrolysis of the recombinant carboxypeptidase B, the hemoglobin in the hemolytic solution of the whole blood sample to be detected is broken at the 2 nd amino acid residue (His) at the N tail end of a beta chain to obtain the glycosylated HbA 1c Dipeptide (C) 4 H 9 O 4 –CO-CH 2 -NH-Val-His-COOH) and non-glycosylated HbA 0 Dipeptide (NH) 2 Val-His-COOH) which were separated and quantified by HPLC-MS/MS, using HbA1c dipeptide standard and HbA 0 The 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 1c Dipeptide chromatographic Peak area value and HbA 0 Chromatographic peak area of dipeptide (FIGS. 4 and 5) HbA in whole blood sample to be measured was calculated from the standard curve 1c Dipeptide and HbA 0 The 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 1c And the unit of the reported result is mass percent. Specifically, as shown in FIG. 4 and FIG. 5, hbA in the whole blood sample to be measured 1c The dipeptide chromatographic peak area value is 1488657 (shown by an instrument), and HbA in the whole blood sample to be detected 0 The peak area value of the dipeptide is 62527081 (shown by instrument), and the peak area value is shown by a standard curve chart 2,FIG. 3 shows the calculation (automatic calculation) of HbA in the whole blood sample to be measured by inputting the chromatographic peak area value 1c Dipeptide concentration was 0.150. Mu.g/mL and HbA 0 The 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 1c The content was 4.93% (mass percent).
Calculating the formula: hbA in whole blood sample to be detected 1c Content (%) = HbA 1c Dipeptide concentration/(HbA) 1c Dipeptide concentration + HbA 0 Dipeptide concentration) × 100%, hbA in the formula 1c Dipeptide concentration representing HbA in a whole blood sample to be assayed 1c Dipeptide concentration (in. Mu.g/mL, calculated from a standard curve); hbA 0 Dipeptide concentration represents HbA in the whole blood sample to be assayed 0 Dipeptide concentration (in. Mu.g/mL, calculated from a standard curve).
3 methodological validation
Daytime precision within 3.1 days
3.1.1 Experimental purposes
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 Experimental results (Table 4)
TABLE 4 method precision measurement results
3.1.4 conclusion 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 measurement RSD is less than 15 percent is met.
3.2 recovery rate of the added 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 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 yeast stock solution, 8 mu L of the L3 standard yeast stock solution and 8 mu L of the L4 standard yeast 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 3.2.2.1 into the three centrifuge tubes, uniformly mixing the three centrifuge tubes 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 ) And respectively taking 5 parts of each labeled parallel sample, respectively taking 5 mu L of labeled solution, putting the labeled solution into a 1.5mL plastic centrifuge tube, adding 15 mu L of 200 mu g/mL recombinant carboxypeptidase B and 200 mu L digestion buffer solution, uniformly mixing at 1500r/min for 1min, then carrying out water bath at 37 ℃ for 2h, taking out, freezing and storing at-20 ℃ for 10min, taking 100 mu L of each sample, and analyzing by HPLC-MS/MS, wherein the sample feeding amount is 1 mu L, and the chromatographic and mass spectrum parameter conditions are shown in tables 2 and 3.
3.2.3 Experimental results (Table 5)
TABLE 5 recovery test results
Table 5 the white sample is a 3.2.2.1 normal human whole blood sample.
3.2.4 conclusions of the experiment
The experimental result shows that the recovery rates of the three standard adding concentrations are all between 95 and 115 percent, the relative standard deviation RSD is less than 6.0 percent, and the requirements that the recovery rate is between 85 and 120 percent and the RSD is less than 15.0 percent are met through methodology verification.
3.3 Standard Curve
3.3.1 Experimental purposes
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 verification of standard curve
3.3.4 conclusion of the experiment
Standard curve range: hbA is shown by 1.2, 1.3 and 3.3.3 0 The dipeptide standard product has good linearity and correlation coefficient R within the range of 0.3125 mu g/mL to 10 mu g/mL 2 ﹥0.999；HbA 1c The 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 is 2 More than 0.999; satisfies the methodology R 2 > 0.990.
3.4 lower limit of quantitation
3.4.1 Experimental purposes
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): hbA 0 Dipeptide 0.12 mug/mL，HbA 1c Dipeptide 0.024 μ g/mL; linear range: hbA 0 Dipeptide in the range of 0.3125. Mu.g/mL to 10. Mu.g/mL, hbA 1c The 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 detection of each sample is 4.5 min), 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), and good stability (the recovery rates are all between 95 and 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) 0 Dipeptide parent ion 288.6m/z, daughter ion 110.1m/z, hbA 1c Dipeptide parent ion 408.3m/z, daughter ion 263.1 m/z), thereby meeting the requirement of clinical glycosylated hemoglobin determination.
Claims (5)
1. A method for quantitatively detecting the content of glycosylated hemoglobin in whole blood by liquid chromatography-tandem mass spectrometry 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 and quantitative analysis are carried out by HPLC-MS/MS, and HbA is used 0 Dipeptide standards and HbA 1c Taking the mixture of the dipeptide standard products as a calibrator, performing separation quantitative analysis by HPLC-MS/MS, drawing a standard curve, and using HbA in a whole blood sample to be tested 1c Dipeptide and HbA 0 The peak area value of the dipeptide is calculated through a standard curve to obtain HbA in the whole blood sample to be detected 1c And HbA 0 The concentration of dipeptide is calculated by the following calculation formula to obtain HbA in the whole blood sample to be detected 1c The unit is mass percentage, and the calculation formula is as follows: hbA in whole blood sample to be measured 1c Content of = HbA 1c dipeptide/(HbA) 1c Dipeptide + HbA 0 Dipeptide) × 100%, formula, hbA 1c Dipeptide represents HbA in whole blood sample to be tested 1c Dipeptide concentration, hbA 0 Dipeptide represents HbA in a whole blood sample to be tested 0 The concentration of dipeptide;
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 X150mm, 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: 95% by volume B,5% by volume A isocratic elution, with an elution time of 4.5min;
mass spectrum conditions: the ion source mode is an ESI source positive ion mode, and the scanning mode is as follows: RMR, atomizing airflow: 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.
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 removing plasma, adding 10 mu L of pure water, homogenizing at 1500r/min for 30s, adding 200 mu L of reserve buffer solution, and uniformly 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 liquid transfer machine 2 EDTA solution, adding pure water to constant volume of 100mL, mixing well to make the final concentration of beta morpholine ethanesulfonic acid 50mmol/L, naHCO 3 Final concentration of 25mmol/L, na 2 -a final EDTA concentration of 1mmol/L, P the H 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 supernatant liquid, adding 15 mu L of recombinant carboxypeptidase B solution with the concentration of 200 mu g/mL into another new 1.5mL plastic centrifuge tube, adding 200 mu L of digestion buffer solution in water bath at 37 ℃ for 2h, freezing at-20 ℃ for 10min, and 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 until the volume is 100mL, uniformly mixing the solution, measuring the volume P H, conditioning it with acetic acid P When the H value reaches 8.0, the digestion buffer solution is obtained, and the solution is stored at 4 ℃.
4. The method for quantitative measurement of glycated hemoglobin content in whole blood according to claim 3, wherein the preparation of the calibrator comprises: taking 6 centrifugal tubes, adding 15 mul of recombinant carboxypeptidase B solution with the concentration of 200 mug/mL and 230 mul of digestion buffer solution into each centrifugal tube, uniformly mixing, and adding HbA containing different concentrations into each test tube 0 Dipeptide standards and HbA 1c 5 mul of standard yeast stock solution of dipeptide standard product to enable HbA 0 Dipeptide standards and HbA 1c The 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 μ g/mL 0 Dipeptide Standard and HbA of 0.03125 μ g/mL 1c A standard working solution of a dipeptide standard product;
calibration product 2: hbA containing 0.625 mug/mL 0 Dipeptide Standard and HbA of 0.0625 [ mu ] g/mL 1c A standard working solution of a dipeptide standard product;
the calibrator 3: hbA containing 1.25 mug/mL 0 Dipeptide Standard and HbA of 0.125 microgram/mL 1c Standard yeast working solution of the dipeptide standard product;
the calibrator 4: hbA containing 2.5 mug/mL 0 Dipeptide Standard and HbA of 0.25 [ mu ] g/mL 1c Standard yeast working solution of the dipeptide standard product;
and (5) calibration product: hbA containing 5 microgram/mL 0 Dipeptide Standard and HbA of 0.5 [ mu ] g/mL 1c A standard working solution of a dipeptide standard product;
calibration article6: hbA containing 10 mug/mL 0 Dipeptide standard and HbA of 1 microgram/mL 1c A standard working solution of a dipeptide standard product.
5. The method of claim 4, wherein the HbA-containing blood of different concentrations is measured by liquid chromatography-tandem mass spectrometry 0 Dipeptide Standard and HbA 1c The standard yeast stock solution of the dipeptide standard product is prepared by the following method: hbA with the concentration of 1000 mug/mL is respectively taken 0 Dipeptide standard product stock solution and HbA with concentration of 1000 mug/mL 1c Mixing the dipeptide standard stock solution and diluting with pure water to obtain HbA-containing solution 0 Dipeptide Standard and HbA 1c 6 parts of standard yeast stock solutions of the dipeptide standard product with different final concentrations are respectively as follows:
HbA containing 500 mug/mL 0 Dipeptide Standard and 50 microgram/mL HbA 1c A standard stock solution of a dipeptide standard;
HbA containing 250 microgram/mL 0 Dipeptide Standard and 25 microgram/mL HbA 1c A standard stock solution of a dipeptide standard;
HbA containing 125 microgram/mL 0 Dipeptide Standard and 12.5 mug/mL HbA 1c A standard stock solution of a dipeptide standard;
HbA containing 62.5 mug/mL 0 Dipeptide Standard and 6.25 mug/mL HbA 1c A standard stock solution of a dipeptide standard;
HbA containing 31.25 microgram/mL 0 Dipeptide Standard and 3.125 mug/mL HbA 1c A standard stock solution of a dipeptide standard;
HbA containing 15.625 mug/mL 0 Dipeptide Standard and 1.5625 mug/mL HbA 1c Standard stock solutions of dipeptide standards.
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