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Method for simultaneously detecting five fat-soluble vitamins, and application thereof

A fat-soluble vitamin and tocopherol technology, applied in the field of analysis and detection, can solve the problems that the specificity and sensitivity of high performance liquid chromatography cannot meet the clinical detection requirements, cannot comprehensively and effectively evaluate the nutritional level of vitamin E, and limit the scope of application, etc. Achieve the effect of low cost, high sensitivity and short analysis time

Inactive Publication Date: 2018-10-12
北京爱普益医学检验中心有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Albahrani et al. used two types of triple quadrupole liquid chromatography tandem mass spectrometry to detect vitamins A, D and E in human serum (Albahrani A A, Rotarou V, Roche P J, et al. A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.Journal of Steroid Biochemistry&Molecular Biology, 2016,159:41.), however, the pretreatment process of this method uses a large amount of organic solvents, including 200μL methanol, 1.5mL n-hexane and 250 μL of methanol, and the single run time of the detection method is 42-45 minutes, which takes a long time, which limits the scope of application of the method
Sadilek et al. used column-switching high-performance liquid chromatography to detect vitamin A and vitamin E in human plasma (Sadilek P, Satinsky D, Otapka M, et al. Rapid and simple determination of Vitamin A and Vitamin E in human plasma by column-switching high -performanceliquid chromatography.Current Analytical Chemistry, 2009,5:311.), the way of column switching simplifies the pretreatment process, but it has high requirements on the instrument, and this method requires a large amount of plasma, and the single operation time is 15 minutes, which is time-consuming Longer, the specificity and sensitivity of high performance liquid chromatography cannot meet the clinical detection requirements
It cannot be ignored that these two methods only detect α-tocopherol for vitamin E, which cannot comprehensively and effectively evaluate the nutritional level of vitamin E in the human body.

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  • Method for simultaneously detecting five fat-soluble vitamins, and application thereof
  • Method for simultaneously detecting five fat-soluble vitamins, and application thereof
  • Method for simultaneously detecting five fat-soluble vitamins, and application thereof

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Experimental program
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Effect test

Embodiment 1

[0059] Configuration and calibration of embodiment 1 solution

[0060] (1) Preparation of standard products

[0061] Take 200 µL of α-tocopherol at a concentration of 5 mg / mL, 20 µL of β-tocopherol at a concentration of 50 mg / mL, 200 µL of γ-tocopherol at a concentration of 1 mg / mL, 20 µL of δ-tocopherol at a concentration of 50 mg / mL, and 560 µL BHT chromatographic methanol with a concentration of 0.1%, mixed evenly to obtain a 1mg / mL vitamin E standard;

[0062] Take 50 μL of retinol with a concentration of 1 mg / mL, 500 μL of vitamin E standard with a concentration of 1 mg / mL, and 450 μL of BHT chromatographic methanol with a concentration of 0.1%, vortex and mix well to obtain a solution containing 50 μg / mL vitamin A and 500 μg / mL Mixed standard of vitamin E.

[0063] Store at -80°C in the dark for 3 months and avoid repeated freezing and thawing.

[0064] (2) Calibration of standard products

[0065] Draw 20 μL of a mixed standard substance containing 50 μg / mL vitamin ...

Embodiment 2

[0073] Embodiment 2 sample pretreatment

[0074] Add the internal standard working solution containing 0.125 μg / mL pentadeuterated retinol and 1.25 μg / mL hexadeuterated α-tocopherol to the sample to be tested, dilute with pure water or aqueous formic acid, add ethanol, methanol or acetonitrile solution, vortex Vortex and mix at high speed to remove protein, add n-hexane for extraction, vortex mix and centrifuge to get the supernatant, dry the supernatant with nitrogen at room temperature, add reconstitution solution, vortex mix and centrifuge to obtain the supernatant is the pretreated sample.

Embodiment 3

[0075] Embodiment 3 liquid chromatography-mass spectrometry analysis

[0076] Add 7 μL of the pretreated sample into the injection bottle of the liquid chromatography-mass spectrometry instrument. The chromatographic column uses a Shimadzu C18 column with a specification of 5 μm×2.1 μm×50 mm. The temperature of the chromatographic column is 25 ° C. The chromatographic column adopts a gradient Elution method, the mobile phase of gradient elution adopts ammonium formate-acetonitrile mixed solution, the flow rate of mobile phase is 0.5mL / min, the time of gradient elution is 6min, wherein, the condition of gradient elution is as shown in Table 1, specific It is: within the first 0-0.3min, the mobile phase is constant at 50% ammonium formate and 50% acetonitrile; within the first 0.3-0.6min, the volume percentage of ammonium formate is continuously reduced from 50% to 5%, and the volume percentage of acetonitrile is from 50% % continuously increased to 95%; within 0.6-1.8min, the m...

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Abstract

The invention provides a method for simultaneously detecting five fat-soluble vitamins, and an application thereof. The method comprises the following steps: (1) analyzing a pretreated sample by a liquid chromatograph-mass spectrometer; and (2) calculating the concentrations of the five fat-soluble vitamins in the sample to be tested according to a standard curve, wherein the fat-soluble vitaminscomprise retinol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol. A chromatographic column in the liquid chromatograph-mass spectrometer adopts gradient elution, and the mobile phase for the gradient elution is an ammonium formate-acetonitrile mixed solution. The recovery rate, the detection limit, the precision and other technical indicators of the method meet requirements, and isotope labels are used as internal standard substances, so the target compounds can be recognized in the manner of strong specificity, high sensitivity, good accuracy and short analysis time,and the method has a good reproducibility and a small error.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and relates to a method and application for simultaneously detecting five kinds of fat-soluble vitamins. Background technique [0002] Vitamin A and vitamin E are fat-soluble vitamins necessary for the human body, mainly from food, and the lack of vitamin A and vitamin E will affect the growth and development of the human body. Vitamin A is mainly retinol, which participates in the synthesis of rhodopsin, and has effects on the pleiotropic behavior of various physiological functions such as growth, reproduction, hematopoiesis and immunity of the human body (Albarhani A A, Collier F, Greaves R F, et al. D and A can be successfully measured by LC-MS / MS in cord blood diluted plasma.Clinical Biochemistry,2015,48:1105;Villamor E,Fawzi W W.Effects of vitamin A supplementation on immune responses and correlation with clinical outcomes.Clinical Microbiology Reviews ,2005,18:446.), lack of...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 王玲申童王凯张莉周亚琼
Owner 北京爱普益医学检验中心有限公司
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