Detection of free mannose and glucose in serum using high performance liquid chromatography

Abstract

Provided is a method of detecting free mannose and glucose in serum using high performance liquid chromatography. Compared to existing technology, the pretreatment process of samples is simpler, the detection time is shortened, and the detection efficiency is greater. When detection of serum samples is carried out, mannose, rhamnose and glucose may be completely separated, and mannose and glucose will not affect each other during quantification, thereby ensuring the accuracy of detection results.

Claims

1. A method of detecting free mannose and glucose in serum, using high performance liquid chromatography comprising: a) generating serum free monosaccharide comprising: (i) sequentially adding: ultrapure water, rhamnose solution, NaOH solution and 1-phenyl-3-methyl-5-pyrazolone solution into a serum sample and mixing to obtain a solution with a pH between 7-14, centrifuging the solution, and heating the solution for a predetermined time to obtain a sample A; (ii) cooling sample A to room temperature, adding HCl solution, mixing to obtain a solution with a pH between 1-7, centrifuging the solution, extracting with chloroform, wherein the supernatant becomes sample B; and (iii) centrifuging sample B at high-speed to obtain a sample C; and b) detecting sample C using high performance liquid chromatography; wherein the detection method is an external standard method, and rhamnose is used as internal reference monosaccharide.

2. The method of claim 1, wherein: NaOH and 1-phenyl-3 methyl-5-pyrazolone in step a) are in a molar ratio of 2:5; and HCl: and NaOH in a) are in a molar ratio of 1.5-2.5: 1.

3. The method of claim 1, wherein: the volume of the serum sample in step a) is 10 L, the volume of the ultrapure water in step a) is 20 L, the concentration of the rhamnose solution in a) is 0.1 mg/ml, and the volume of the rhamnose solution in a) is 10 L; the solution in step (i) is heated to 70 C., for 1 hour; and high-speed in step (iii) is a speed of 13000 r/min.

4. The method of claim 3, wherein sample B is centrifuged for 10 minutes.

5. The method of claim 1, wherein, the high performance liquid chromatography is performed on an Agilent 1260 high performance liquid system, an Agilent Porohell EC-C18 chromatography column (4.6100 mm 2.7 m); and wherein the parameters of the high performance liquid chromatography comprise: a detection wavelength of 254 nm; a reference wavelength of 360 nm; a mobile phase of acetate with pH=5.5 as salt phase, acetonitrile as organic phase; a gradient elution; a column temperature of 25-50 C.; a flow rate of 1.0 mL/min; a sample volume of 20 L; and a chromatography column flushing solvent of water and acetonitrile containing 0.1% trifluoroacetic acid.

6. The method of claim 5, wherein gradient mode parameters of the gradient elution comprise: a time gradient of 0.fwdarw.10.fwdarw.15.fwdarw.20 min; a concentration gradient of acetonitrile of 15%.fwdarw.22%.fwdarw.24%.fwdarw.15%; and a concentration gradient of 0.10 mol/L PH=5.5 ammonium acetate buffer solution of 85%.fwdarw.78%.fwdarw.76%.fwdarw.85%.

7. The method of claim 5, wherein the parameters of the high performance liquid chromatography further comprises: a detection wavelength bandwidth of 4 nm; a reference wavelength bandwidth of 100 nm; a column temperature of 37 C.; and a flow rate of 1 mL/min.

8. The method of claim 1, wherein the external standard method comprises: c) drawing a standard curve comprising: taking 40 L of a mixed solution of mannose and glucose standard substances with the concentration of 0.5, 0.25, 0.10, 0.05, 0.025, 0.01, 0.005, 0.0025, 0.001 and 0.0005 mg/mL, adding 40 L of 0.3M NaOH solution, adding 60 L of 0.5M PMP solution, and heating at 70 C. n for 1 hour; taking out the reactant, cooling to room temperature, adding 80 L of 0.3M HCl, extracting CHCl.sub.3 three times; centrifuging at 13000 r/min for 10 min; using high performance liquid chromatography to detect and analyze mannose and glucose respectively, and drawing a standard curve by taking the concentration of monosaccharide as a horizontal coordinate and taking the corresponding peak area as a vertical coordinate; and d) detecting the amount of mannose and glucose in a serum sample comprising: taking 10 L of serum sample to be detected, adding 20 L of ultrapure water and 10L of 0.1 mg/ml rhamnose solution, and deriving and sampling; calculating the content of free mannose and glucose in the serum sample to be detected by using the standard curve obtained in the step c).

9. The method of claim 1, wherein the serum sample is from a patient with a disease is characterized by abnormal protein glycosylation.

10. The method of claim 9 wherein the disease is diabetes or gastric cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a high performance liquid chromatography peak of mannose, rhamnose and glucose.

DETAILED DESCRIPTION

(2) The present invention will be further illustrated with reference to the following examples.

Embodiment 1: Drawing Standard Curve of External Standard Method.

(3) (1) Monosaccharide derivation: taking 40 L of a mixed solution of mannose and glucose standard substances with the concentration of 0.5, 0.25, 0.10, 0.05, 0.025, 0.01, 0.005, 0.0025, 0.001 and 0.0005 mg/mL, adding 40 L of 0.3M NaOH solution, adding 60 L of 0.5M PMP solution, and reacting in an oven at 70 C. for 1 hour; taking out the reactant, cooling to room temperature, adding 80 L of 0.3M HCl, extracting CHCl.sub.3 for three times; centrifuging at 13000 r/min for 10 min;

(4) (2) Detecting and analyzing by high performance liquid chromatography, and drawing a standard curve (see Table 3) by taking the concentration of monosaccharide as a horizontal coordinate and taking the corresponding peak area as a vertical coordinate.

(5) The high performance liquid chromatography is an Agilent 1260 high performance liquid system, an Agilent Porohell EC-C18 chromatography column: (4.6100 mm 2.7 m). The conditions of high performance liquid chromatography are as follows: (1) detection wavelength: 254 nm, bandwidth 4 nm; reference wavelength: 360 nm; bandwidth 100 nm; (2) mobile phase: acetate with PH=5.5 as salt phase, acetonitrile as organic phase; the acetate with pH=5.5 has no impurity precipitation under the condition, so that the blockage of a chromatography column is avoided, and the perfect peak shape in the linear range of an external standard method is ensured. Gradient elution: 0.fwdarw.10.fwdarw.15.fwdarw.20 min; concentration gradient of acetonitrile: 15%.fwdarw.22%.fwdarw.24%.fwdarw.15%; concentration gradient of 0.10 mol/L ammonium acetate buffer solution (PH=5.5): 85%.fwdarw.78%.fwdarw.76%.fwdarw.85%; the gradient mode can perfectly separate 8 monosaccharides and shorten the analysis time as much as possible; (3) column temperature:37 C.; (4) flow rate: 1 mL/min; (5) sample volume: 20 L; (7) chromatography column flushing solvent: flushing the chromatography column with water and acetonitrile containing 0.1% trifluoroacetic acid. By adding 0.1% trifluoroacetic acid into the mobile phase, the pH value of the flushed mobile phase is adjusted to acidic, so that the precipitation of protein and polypeptide substances in a serum sample under the conditions of near neutrality and alkalinity is avoided, and the blockage of a chromatography column is avoided.

(6) TABLE-US-00006 TABLE 3 Standard Curve of Mannose and Glucose External Standard Method Detection Linear Correlation Range/ Monosaccharide Regression Equation Coefficient r.sup.2 mg/mL Man y = 27993x 4.606 0.9999 0.0005-0.5 Glc y = 23604x + 7.285 0.9998 0.0005-0.5

(7) As shown in FIG. 1, the high-performance liquid chromatography method using the above conditions, mannose, rhamnose and glucose can be separated completely, and the chromatographic peak shape is excellent. Peak 1, peak 2, peak 3, peak 4 is the peak of PMP, Man, Rha and Glc respectively.

Embodiment 2: Detection of Free Mannose and Glucose in Normal Human Serum.

(8) (1) serum free monosaccharide derivation:

(9) {circle around (1)} taking 10 L serum samples from 13 normal persons, sequentially adding 20 L of ultrapure water, 10 L of 0.1 mg/ml rhamnose, 40 L of 0.3M NaOH, 60 L of 0.5M 1-phenyl-3-methyl-5-pyrazolone solution, centrifuging after mixing, and reacting in an oven at 70 C. for 1 hour to obtain a sample A;

(10) {circle around (2)} taking out the reactant from step (1), cooling to room temperature, adding 80 L of 0.3M HCl, and extracting with CHCl.sub.3 for three times to obtain sample B. By adjusting the derived solution to acidity and extracting residual PMP, therefore eliminating the influence of impurity peak on monosaccharide quantification and reducing the loss rate of monosaccharide.

(11) {circle around (3)} centrifuging the sample B at the speed of 13000 r/min for 10 min to obtain a sample C.

(12) (2) detecting the sample C by high performance liquid chromatography: calculating the contents of free mannose and glucose in the serum sample to be measured according to the peak area obtained by analysis and the standard curve obtained in embodiment 1.

(13) The high performance liquid chromatography is an Agilent 1260 high performance liquid system, Agilent Porohell EC-C18 chromatography column (4.6100 mm 2.7 m). The conditions of high performance liquid chromatography are as follows: (1) detection wavelength: 254 nm, bandwidth 4 nm; reference wavelength: 350 nm, bandwidth 100 nm; (2) mobile phase: acetate with PH=5.5 as salt phase, acetonitrile as organic phase; the acetate with pH=5.5 has no impurity precipitation under this condition, so that the blockage of the chromatography column is avoided, and the perfect peak shape in the linear range of the external standard method is ensured. Gradient elution: 0.fwdarw.10.fwdarw.15.fwdarw.20 min; concentration gradient of acetonitrile: 15%.fwdarw.22%.fwdarw.24%.fwdarw.15%; concentration gradient of 0.10 mol/L ammonium acetate buffer solution (PH=5.5): 85%.fwdarw.78%.fwdarw.76%.fwdarw.85%; the gradient mode can perfectly separate 8 monosaccharides and shorten the analysis time as much as possible; (3) column temperature: 37 C.; (4) flow rate: 1.0 mL/min; (5) sample volume: 20 L; (7) chromatography column flushing solvent: flushing the chromatography column with water and acetonitrile containing 0.1% trifluoroacetic acid. By adding 0.1% trifluoroacetic acid into the mobile phase, the pH value of the flushed mobile phase is adjusted to acidtity, so that the precipitation of protein and polypeptide substances in a serum sample under the conditions of near neutrality and alkalinity is avoided, and the blockage of a chromatography column is avoided.

Embodiment 3: Detecting Free Mannose and Glucose in Normal Human Serum.

(14) The differences with embodiment 2 are:

(15) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 60 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(16) (2) detecting the sample C by high performance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 25 C.

Embodiment 4: Detecting Free Mannose and Glucose in Normal Human Serum.

(17) The differences with embodiment 2 are:

(18) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 85 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(19) (2) detecting the sample C by high performance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 30 C.

Embodiment 5: Detecting Free Mannose and Glucose in Serum of Diabetic Patients.

(20) (1) serum free monosaccharide derivation:

(21) {circle around (1)} taking 10 L serum samples from 13 diabetic patients, sequentially adding 20 L of ultrapure water, 10 L of 0.1 mg/ml rhamnose, 40 L of 0.3M NaOH, 60 L of 0.5M 1-phenyl-3-methyl-5-pyrazolone solution, centrifuging after mixing, and reacting in an oven at 70 C. for 1 hour to obtain a sample A;

(22) {circle around (2)} taking out the reactant from oven in step {circle around (1)}, cooling to room temperature, adding 80 L of 0.3M HCl, and extracting with CHCl.sub.3 for three times to obtain sample B.

(23) {circle around (3)} centrifuging the sample B at the speed of 13000 r/min for 10 min to obtain a sample C.

(24) (2) detecting the sample C by high performance liquid chromatography: calculating the contents of free mannose and glucose in the serum sample to be measured according to the peak area obtained by analysis and the standard curve obtained in embodiment 1. The high performance liquid chromatography is an Agilent 1260 high performance liquid system, Agilent Porohell EC-C18 chromatography column (4.6100 mm 2.7 m). The conditions of high performance liquid chromatography are as follows: (1) detection wavelength: 254 nm; reference wavelength: 360 nm; (2) mobile phase: acetate with PH=5.5 as salt phase, acetonitrile as organic phase; gradient elution: 0.fwdarw.10.fwdarw.15.fwdarw.20 min; concentration gradient of acetonitrile: 15%.fwdarw.22%.fwdarw.24%.fwdarw.15%; concentration gradient of 0.10 mol/L ammonium acetate buffer solution (PH=5.5): 85%.fwdarw.78%.fwdarw.76%.fwdarw.85%; (3) column temperature: 37 C.; (4) flow rate: 1.0 mL/min; (5) sample volume: 20 L; (7) chromatography column flushing solvent: flushing the chromatography column with water and acetonitrile containing 0.1% trifluoroacetic acid.

Embodiment 6: Detecting Free Mannose and Glucose in Serum of Diabetic Patients.

(25) The differences with embodiment 5 are:

(26) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 90 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(27) (2) detecting the sample C by high performance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 35 C.

Embodiment 7: Detecting Free Mannose and Glucose in Serum of Diabetic Patients.

(28) The differences with embodiment 5 are:

(29) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 70 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(30) (2) detecting the sample C by high performance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 40 C.

Embodiment 8: Detecting Free Mannose and Glucose in Serum of Gastric Cancer Patients.

(31) (1) serum free monosaccharide derivation:

(32) {circle around (1)} taking 10 L serum samples from 13 gastric cancer patients, sequentially adding 20 L of ultrapure water, 10 L of 0.1 mg/ml rhamnose, 40 L of 0.3M NaOH, 60 L of 0.5M 1-phenyl-3-methyl-5-pyrazolone solution, centrifuging after mixing, and reacting in an oven at 70 C. for 1 hour to obtain a sample A;

(33) {circle around (2)} taking out the reactant from oven in step {circle around (1)}, cooling to room temperature, adding 80 L of 0.3M HCl, and extracting with CHCl.sub.3 for three times to obtain sample B.

(34) {circle around (3)} centrifuging the sample B at the speed of 13000 r/min for 10 min to obtain a sample C.

(35) (2) detecting the sample C by high performance liquid chromatography: calculating the contents of free mannose and glucose in the serum sample to be measured according to the peak area obtained by analysis and the standard curve obtained in embodiment 1.

(36) The high performance liquid chromatography is an Agilent. 1260 high performance liquid system, Agilent Porohell EC-C18 chromatography column (4.6100 mm 2.7 m). The conditions of high performance liquid chromatography are as follows: (1) detection wavelength: 254 nm; reference wavelength: 360 nm; (2) mobile phase: acetate with PH=5.5 as salt phase, acetonitrile as organic phase; gradient elution: 0.fwdarw.10.fwdarw.15.fwdarw.20 min; concentration gradient of acetonitrile: 15%.fwdarw.22%.fwdarw.24%.fwdarw.15%; concentration gradient of 0.10 mol/L ammonium acetate buffer solution (PH=5.5): 85%.fwdarw.78%.fwdarw.76%.fwdarw.85%; (3) column temperature: 37 C.; (4) flow rate: 1.0 mL/min; (5) sample volume: 20 L; (7) chromatography column flushing solvent: flushing the chromatography column with water and acetonitrile containing 0.1% trifluoroacetic acid.

Embodiment 9: Detecting Free Mannose and Glucose in Serum of Gastric Cancer Patients.

(37) The differences with embodiment 8 are:

(38) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 100 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(39) (2) detecting the sample C by high pedal' lance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 45 C.

Embodiment 10: Detecting Free Mannose and Glucose in Serum of Gastric Cancer Patients.

(40) The differences with embodiment 8 are:

(41) (1) serum free monosaccharide derivation: {circle around (2)} taking out the reactant treated in the oven in the step {circle around (1)}, cooling to room temperature, adding 75 L of 0.3M HCl, and extracting with CHCl.sub.3 for 3 times to obtain the sample B.

(42) (2) detecting the sample C by high performance liquid chromatography: the conditions of high performance liquid chromatography are as follows: (3) column temperature 50 C.

(43) TABLE-US-00007 TABLE 4 Detected content of serum free glucose and mannose of embodiment 2-10 Sample Mannose Glucose Analysis time Type Embodiment (mol/L) (mol/L) (min) Normal Embodiment 2 52.5 4674.8 20.0 Person (13 samples) Embodiment 3 53.9 4627.3 20.3 (13 samples) Embodiment 4 52.6 4608 20.2 (13 samples) Diabetic Embodiment 5 102.7 7854.5 20.0 Patients (13 samples) Embodiment 6 101.9 7882.1 20.0 (13 samples) Embodiment 7 102.3 7881.8 20.0 (13 samples) Gastric Embodiment8 89.7 4813.0 20.0 cancer (13 samples) patients Embodiment 9 87.8 4746.0 19.9 (13 samples) Embodiment 10 90.2 4750.1 19.8 (13 samples)

(44) According to embodiment 2-10, the serum free mannose and glucose contents of 39 normal persons, 39 diabetes patients and 39 gastric cancer patients were detected by the method of the present invention, and the results are shown in table 4. From the results of embodiment 2 to 4, it was found that the serum mannose concentration of normal persons was much lower than diabetic patients and gastric cancer patients. It fully explains that the concentration of serum free mannose is indeed related to diseases with abnormal glycosylation of proteins such as diabetes, gastric cancer, ect; therefore, the method of the present invention has very important significance for researching the relation between the free monosaccharide in the serum and the disease and searching the marker for clinical detection of the diseases.

(45) In addition, as shown in table 4, compared with the existing monosaccharide derived HPLC detection method, the method provided by the present invention shortens the detection time to about 20 min, and improves the detection efficiency; and the data parallelism is good, and the precision, the accuracy and the repeatability of the method are fully proved.