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Liquid chromatography-tandem mass spectrometry detection method for glucocorticoid in biological fluid

A technology of liquid chromatography mass spectrometry and glucocorticoids, which is applied in the field of liquid chromatography tandem mass spectrometry detection of glucocorticoids in biological fluids, and can solve problems such as difficult batch processing, time-consuming analysis methods, poor accuracy and reliability, etc.

Pending Publication Date: 2021-01-01
WUXI DIAGNOSTICS LAB SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before LC-MS detection, it is usually necessary to use an appropriate sample pretreatment method to remove the matrix from the sample. The existing LC-MS detection methods have the disadvantages of long analysis methods, poor accuracy and reliability, or low precision and insufficiency. Disadvantages such as easy batch processing

Method used

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  • Liquid chromatography-tandem mass spectrometry detection method for glucocorticoid in biological fluid
  • Liquid chromatography-tandem mass spectrometry detection method for glucocorticoid in biological fluid
  • Liquid chromatography-tandem mass spectrometry detection method for glucocorticoid in biological fluid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1. The setting of chromatographic conditions and mass spectrometry parameters

[0054] Column: Phenomenex Synergi TM 4μm Max-RP 50x4.6mm, 4μm;

[0055] Column temperature: 50°C;

[0056] Injection volume: 10μL;

[0057] Mobile phase A: water containing 0.1mM ammonium acetate: acetonitrile (9:1) solution;

[0058] Mobile phase B: water containing 0.1mM ammonium acetate: acetonitrile (1:9) solution;

[0059] Gradient elution program: mobile phase A+mobile phase B=100%; 0~6.50min, the volume percentage of mobile phase B is maintained at 25%; 6.50~6.60min, the volume percentage of mobile phase B is increased from 25% to 60%; 6.60~ 8.68min, the volume percentage of mobile phase B increased from 60% to 90%; 8.68~9.18min, the volume percentage of mobile phase B remained at 90%; 9.18~9.20min, the volume percentage of mobile phase B decreased from 90% to 25%; 9.20 ~10.00, the volume percentage of mobile phase B was kept at 25%; the flow rate of mobile phase was...

Embodiment 2

[0062] Embodiment 2. Sample pretreatment procedure

[0063] 1) Protein precipitation: Add 300 μL sample, 35 μL Bude-d8 internal standard (3.13 μg / dL) and 25 μL (25 μg / dL) of other 6 mixed internal standard solutions to a 1.5 mL centrifuge tube, then add 60 μL water, shake and mix ( 1000r / min, 3min), transfer the sample tube to a centrifuge, and centrifuge at 14000rpm for 2min;

[0064] 2) SLE: Transfer the above liquid to the SLE plate on the 96-well deep-well plate. After all the liquid enters the filler, add 700 μL of dichloromethane to each well for elution 3 times, and collect the eluate with another 96-well plate;

[0065] 3) Nitrogen blowing: Transfer all the organic liquid collected in the 96-well plate to a plate nitrogen blowing apparatus, and blow it to dryness with nitrogen at 40±2°C;

[0066] 4) Reconstitution: add 150 μL of 0.1% estriol and 70% methanol aqueous solution to the corresponding position of the above-mentioned 96-well plate containing the eluent for r...

Embodiment 3

[0068] Embodiment 3. Determination of linear range

[0069] Prepare a series of 12 synthetic glucocorticoid standard solutions with concentration gradients: 0, 0.1, 0.5, 2.5, 5 and 10 μg / dL. The sample processing method is the same as in Example 2, and the chromatographic conditions and mass spectrometry parameters are the same as in Example 1, and are measured once a day for 5 consecutive days. Quantitatively by the internal standard method described in the technical scheme, take the theoretical concentration as the abscissa, and take the measured concentration as the ordinate to carry out linear regression, the results are as follows image 3 As shown, the linear slopes and correlation coefficients of the 12 synthetic glucocorticoids are respectively: Slope=1.0037, R 2 = 0.9989 (Dexa); Slope = 1.0091, R 2 = 0.9988 (Beta); Slope = 1.0385, R 2= 0.9989 (Bude); Slope = 1.0024, R 2 = 0.9999 (Cortisol); Slope = 1.0275, R 2 = 0.9998 (Cortisone); Slope = 0.9852, R 2 = 0.9989 (...

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Abstract

The invention discloses a liquid chromatography-mass spectrometry detection method for glucocorticoid in biological fluid; the method comprises the following steps: (1), carrying out protein precipitation and solid-liquid extraction plate pretreatment on a sample to obtain a test solution and a reference solution; and (2), performing liquid chromatography-mass spectrometry combined detection: using a reversed-phase C12 chromatographic column as a chromatographic column, the mobile phase A being composed of an organic acid salt aqueous solution and acetonitrile in a ratio of 9: 1, and the mobile phase B being composed of an organic acid salt aqueous solution and acetonitrile in a ratio of 1: 9; and performing gradient elution. The method provided by the invention can effectively detect glucocorticoid in human urine and serum, and has the characteristics of good separation effect, stable baseline, short detection time, good linearity, high accuracy, good reproducibility, high sensitivityand 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 glucocorticoids in biological fluids. Background technique [0002] Glucocorticoids belong to the family of steroids, which have important functions in glucose, protein and calcium metabolism. Synthetic glucocorticoids are also widely used, and they have important clinical application value as anti-inflammatory drugs and immunosuppressants. However, the application of these drugs may also have some other unexpected clinical manifestations. Patients exposed to these steroids may develop the clinical features of Cushing's syndrome but with decreased cortisol levels and suppression of the hypothalamic-pituitary-adrenal axis. The activity of glucocorticoids is largely dependent on substituents on the nuclide, which have been found to significantly increase the activity of glucocorticoids and mineralocor...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/14G01N30/34G01N30/54G01N30/60G01N30/72
CPCG01N30/02G01N30/06G01N30/14G01N30/34G01N30/54G01N30/60G01N30/72G01N2030/045G01N2030/062
Inventor 王俊霞刘浩浩李月杰刘釜均
Owner WUXI DIAGNOSTICS LAB SHANGHAI CO LTD
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