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A method for the detection of cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry

A cyclic adenosine monophosphate and liquid chromatography-mass spectrometry technology is applied in the field of cyclic adenosine monophosphate liquid chromatography tandem mass spectrometry detection in human urine, and achieves the effects of safe and simple operation, cost reduction and good separation effect

Active Publication Date: 2021-09-03
WUXI APPTEC ZK SUZHOU BIOSCIENCE CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, there is no report on cAMP detection in urine by liquid chromatography tandem mass spectrometry

Method used

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  • A method for the detection of cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry
  • A method for the detection of cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry
  • A method for the detection of cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1. The setting of chromatographic conditions and mass spectrometry parameters

[0061] Column: ACQUITY HSS T3 1.8μm, 2.1mm×50mm;

[0062] Column temperature: 25°C;

[0063] Injection volume: 2μL;

[0064] Mobile phase A: 0.01mM ammonium acetate aqueous solution, mobile phase B: acetonitrile;

[0065] Gradient elution program: mobile phase A+mobile phase B=100%; 0~1.00min, the volume percentage of mobile phase B is maintained at 20%; 1.00~1.5min, the volume percentage of mobile phase B is increased from 20% to 40%; 1.50~ 1.80min, the volume percentage of mobile phase B is increased from 40% to 90%; 1.80~1.81min, the volume percentage of mobile phase B is decreased from 90% to 20%; 1.81~3.00min, the volume percentage of mobile phase B is kept at 20%;

[0066] The mass spectrometry parameters are set as follows: ion source: electrospray ion source, ion mode: positive ion mode, monitoring mode: multiple reaction monitoring, dwell time: 0.079s, cone voltage: ...

Embodiment 2

[0068] Embodiment 2. Sample pretreatment procedure

[0069] 1) Sample loading: Add 50 μL of sample and 10 μL of 0.005 nmol / mL cAMP internal standard (8-methylaminocyclic adenosic acid) working solution to a solid-phase extraction 96-well plate with strong anion-exchange reversed-phase sorbent, and use positive pressure to Drain the device;

[0070] 2) Rinse: Add 50 μL of pure water to the corresponding position of the above 96-well solid phase extraction plate, drain with positive pressure device, add 50 μL of L methanol, and drain with positive pressure device;

[0071] 3) Elution: add 50 μL of 20% formic acid water to the corresponding position of the above-mentioned 96-well solid-phase extraction plate for elution, and collect the eluate with another 96-well plate;

[0072] 4) Neutralization: Add 1000 μL of 10% ammonia water to the corresponding position of the above-mentioned 96-well plate containing the eluent, shake and mix evenly (800 rpm, 5 min) after centrifugation. ...

Embodiment 3

[0074] Embodiment 3. The investigation of matrix effect

[0075] The T-pillar perfusion experiment was used to investigate the matrix effect, and the results were as follows: Figure 4 As shown, there was no apparent ion suppression or enhanced response. This shows that the pretreatment method of the present invention can effectively eliminate components that interfere with cAMP detection in urine samples.

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Abstract

The invention discloses a method for detecting cyclic adenosine monophosphate in urine by liquid chromatography-mass spectrometry, which comprises the following steps: (1) sample pretreatment: the urine sample is pretreated by anion-exchange solid-phase extraction to obtain a test solution; (2) Adopt liquid chromatography-mass spectrometry to detect described need testing solution: chromatographic condition adopts reverse phase C18 hydrophilic chromatographic column; Adopt gradient elution; Mass spectrometry condition is: ion source is electrospray ion source; Ion mode is positive ion mode ; The monitoring mode is multiple reaction monitoring. The invention also discloses an internal standard quantitative detection method for detecting cyclic adenosine monophosphate in urine by liquid mass spectrometry. The method of the present invention is the first liquid chromatography tandem mass spectrometry detection method for cAMP in urine samples, which can effectively detect cAMP in urine samples, has good separation effect, stable baseline, short detection time, good linearity, and high accuracy. Good reproducibility, high sensitivity and high precision.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a liquid chromatography tandem mass spectrometry detection method for cyclic adenosine monophosphate in human urine. Background technique [0002] Cyclic adenosine monophosphate (cAMP) acts as a "second messenger" to regulate the activity of intracellular enzymes or proteins in response to changes in the levels of various hormones (such as parathyroid hormone). The content of cAMP in normal cells is less, but it can be significantly increased under the action of hormones. Most peptide hormones, such as glucagon, parathyroid hormone, calcitonin, vasopressin, and oxytocin, can activate adenylate cyclase on the target cell membrane through the corresponding receptors, thereby making intracellular cAMP concentration increased. [0003] At present, the most commonly used methods for detecting cAMP in body fluids (plasma, urine, cerebrospinal fluid, etc.) mainly include enz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 李月杰李伟
Owner WUXI APPTEC ZK SUZHOU BIOSCIENCE CO LTD
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