# US 10,871,473 B2 · Detection of free mannose and glucose in serum using high performance liquid chromatography

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- Number: US 10,871,473 B2
- Document ID: US10871473B2
- Status: Granted
- Kind: B2
- Application number: 16/618,033
- Filed: 2018-05-29
- Published: 2020-05-21
- Granted: 2020-12-22
- Earliest claimed: 2017-05-29
- Assignees: The Affiliated Hospital of Qingdao University
- Applicants: The Affiliated Hospital of Qingdao University
- Inventors: Lijuan Zhang; Yong Liu
- Web page: https://patentgrep.com/patent/US10871473B2

## CPC

- G01N 30/06: Preparation
- G01N 30/88: Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N 30/74: Optical detectors {(measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J)}
- G01N 30/8631: {Peaks}
- G01N 30/8679: {Target compound analysis, i.e. whereby a limited number of peaks is analysed}
- G01N 30/34: of fluid composition, e.g. gradient (G01N30/36 takes precedence)
- G01N 2030/027: {Liquid chromatography}
- G01N 2030/067: {by reaction, e.g. derivatising the sample}
- G01N 2030/8813: {biological materials}
- G01N 2030/8836: {involving saccharides}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

The method of claim 1, wherein:
the volume of the serum sample in step a) is 10 μL, the volume of the ultrapure water in step a) is 20 μL, the concentration of the rhamnose solution in a) is 0.1 mg/ml, and the volume of the rhamnose solution in a) is 10 μL;
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.

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 1)

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 (4.6×100 mm 2.7 μm); 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 μL; and
a chromatography column flushing solvent of water and acetonitrile containing 0.1% trifluoroacetic acid.

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 5)

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.

### Claim 8 (depends on 1)

The method of claim 1, wherein the external standard method comprises:
c) drawing a standard curve comprising: taking 40 μL 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 μL of 0.3M NaOH solution, adding 60 μL of 0.5M PMP solution, and heating at 70° C. n for 1 hour; taking out the reactant, cooling to room temperature, adding 80 μL of 0.3M HCl, extracting CHCl3 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 μL of serum sample to be detected, adding 20 μL of ultrapure water and 10μL 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).

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

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