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Method for simultaneously detecting serum 24,25(OH)2D and 25OHD

A serum and stable isotope technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of large sample consumption, complex and time-consuming operation, etc., and achieve the effect of less serum consumption, simple pretreatment and accurate measurement

Active Publication Date: 2018-09-28
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However for 24,25(OH) 2 At present, there is no immunological method for detection of D, and foreign reports use liquid chromatography tandem mass spectrometry for determination, but the derivatization method is required (Mamoru Satoh, et al. Development and validation of the simultaneous measurement of four vitaminD metabolites in serum by LC– MS / MS for clinical laboratory applications. AnalBioanal Chem (2016) 408:7617–7627), the operation is complicated and time-consuming, and some methods without derivatization methods use a large amount of samples (at least 2ml serum), or cannot be used for 24,25( Oh) 2 D. 3 and 24,25(OH) 2 D. 2 Simultaneous measurement

Method used

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  • Method for simultaneously detecting serum 24,25(OH)2D and 25OHD
  • Method for simultaneously detecting serum 24,25(OH)2D and 25OHD
  • Method for simultaneously detecting serum 24,25(OH)2D and 25OHD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 sample preparation

[0033] Take 200ul serum sample or calibrator into a 5ml glass tube, add 50ul isotope internal standard solution, mix and shake, add 200ul methanol, 500ul 0.1M zinc sulfate solution in sequence; then add 1ml n-hexane-ethyl acetate mixed in equal volume ratio solution, mixed and oscillated, took 800ul of the supernatant, blown dry with nitrogen, added 100ul of reconstitution solution (methanol-water volume ratio 8:1) to redissolve, and waited for the test on the machine.

[0034] The isotope internal standard solution contains 5ng / mL 24,25(OH) 2 D. 3 -d6 internal standard, 40ng / mL 25OHD 2 -d3 internal standard and 50ng / mL 25OHD 3 -d3 internal standard.

Embodiment 2

[0035] Embodiment 2 chromatographic separation

[0036] The liquid chromatography column used in this example is an Ascentis Express F5 column (2.1 mm×100 mm, 2.7 μm). Use methanol solution containing 0.01%-0.5% formic acid as mobile phase A, and aqueous solution containing 0.01%-0.5% formic acid as mobile phase B, using gradient elution: 0-0.2min 25%A, 0.2-1.0min 72%A , 1.0-3.8min, 72%A, 3.8-3.9min80%A, 3.9-6.5min 80%A, 6.5-7.0min 95%A, 7.0-7.5min 25%A, flow rate 0.3ml / min, column temperature is 25°C, the injection volume is 20 μl. HPLC is mainly to remove the interfering substance 3-epi 24,25(OH) 2 D. 3 and 3-epi 25(OH)D 3 For target 24,25(OH) 2 D. 3 、24,25(OH) 2 D. 2 , 25OHD 2 , 25OHD 3 The impact of mass spectrometry detection.

Embodiment 3

[0037] Embodiment 3 tandem mass spectrometry analysis

[0038] In this embodiment, the detection is accomplished by a triple quadrupole mass spectrometry system (AB sciex 4000Qtrap).

[0039] Mass spectrometry selects multiple ion monitoring reaction mode, positive ion mode, electrospray ionization, ion source temperature 300-600 ° C, this method is selected as 550 ° C, GS1: select 45 (optional 30-80), GS2: select 60 ( Optional 30-80), specific monitored ion pairs, collision energy (CE), declustering voltage (DP), etc. are shown in Table 1 (DP, EP, CE, CXP are not fixed, and should be adjusted according to the instrument).

[0040] Table 1 Mass Spectrometry Parameters

[0041]

[0042] The typical spectra obtained from the detection can be seen in figure 1 . This method can completely separate 3-epi 24,25(OH) 2 D. 3 and 3epi25OHD 3 ( figure 2 ).

[0043] Take the concentration of the standard solution as the abscissa, and use the ratio of the signal intensity of th...

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Abstract

The invention provides a method for simultaneously detecting serum 24,25(OH)2D and 25OHD by liquid chromatography tandem mass spectrometry. The method comprises the following steps: 1) preparing a serum sample; 2) separating the sample through liquid chromatogram, and removing an interference substance; 3) obtaining a detection signal through mass spectrometry; 4) drafting a standard curve; and 5)detecting the signal intensity of 24,25(OH)2D3, 24,25(OH)2D2, 25OHD2, and 25OHD3 in the to-be-measured sample, substituting the signal intensity in the above standard curve for calculation, and realizing the quantitative determination of 24,25(OH)2D and 25OHD in serum. The rapid detection method for simultaneously detecting 24,25(OH)2D and 25OHD by using less sample (200 [mu]l of serum) based onliquid chromatography tandem mass spectrometry, and can distinguish the interference of 3-epi 24,25(OH)2D3and 3-epi 25(OH)D3.

Description

technical field [0001] The invention relates to a method for simultaneously detecting serum 24,25(OH)2D and 25OHD by liquid chromatography tandem mass spectrometry. Background technique [0002] Vitamin D not only plays an important role in maintaining bone health, but is also closely related to kidney diseases, tumors, and cardiovascular diseases. Vitamin D synthesized by exogenous supplementation or sunlight exposure to 7-dehydrocholesterol in the skin enters the liver through blood circulation , converted into monohydroxyvitamin D under the action of 25-hydroxylase CYP2R1: 25OHD (25OHD 2 and 25OHD 3 ), and then metabolized into biologically active 1,25(OH) under the action of renal 1α hydroxylase CYP27B1 2 d. 25OHD can also be catalyzed by 24 hydroxylase CYP24A1 (CYP24A1 (cytochrome P450 family member 24A1) in the kidney to generate 24,25(OH) 2 D(24,25(OH) 2 D. 2 and 24,25(OH) 2 D. 3 ), CYP24A1 can also make excess active 1,25(OH) 2 D is inactivated and excreted ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72
Inventor 禹松林邱玲程倩谢少伟
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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