Method for simultaneously detecting multiple fat-soluble vitamins

Abstract

The invention discloses the technical field of fat-soluble vitamin detection and particularly relates to a method for simultaneously detecting multiple fat-soluble vitamins. The detection method comprises the following steps: step 1, fully and uniformly mixing 10-200 microlitres of sample plasma with a protein precipitation solution and a vitamin deuterated internal standard substance at 100-3000rpm for 1-5 min to obtain a solution A, and centrifuging the solution A at 0-8 DEG C at 200-12000 rpm to obtain a supernatant; and step 2, analyzing the fat-soluble vitamin of the supernatant by usinga liquid chromatograph-mass spectrometer, and calculating the content of the fat-soluble vitamin in the sample plasma by using an internal standard method. Compared with the prior art, the method hasthe advantages that more types of vitamins are detected, the sample can be enriched without the assistance of SLE, SPE and other materials, the detection cost is effectively reduced, the required blood volume is less than that of the existing method, the compliance of patients, especially infants, is better due to the reduction of the blood volume of the sample, and the method has a wide application prospect.

Description

technical field

[0001] The invention relates to the technical field of detection of fat-soluble vitamins, and specifically relates to a method for simultaneously detecting six kinds of fat-soluble vitamins in blood plasma (clear). Background technique

[0002] Vitamins, divided into fat-soluble vitamins and water-soluble vitamins, are a type of organic substances necessary to maintain human life activities, and are also important active substances to maintain human health. Vitamin deficiency or excess will lead to the occurrence of nutritional diseases;

[0003] Vitamin A has various physiological functions such as promoting growth and reproduction, maintaining bone, epithelial tissue, vision and normal secretion of mucosal epithelium; vitamin D, also known as anti-rickets vitamin, is a sterol derivative, and the most important member of its family members is 25 hydroxy-vitamin D3 (cholecalciferol) and 25 hydroxy-vitamin D2 (ergocalciferol), both are fat-soluble vitamins, w...

Examples

Embodiment 1

[0029] Step 1: Preparation of standard solution: preparation containing retinol (VA), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (VE) and VK1, VK2 concentrations were 1000, 40, 160 . Take 200 μL of the solution to 2# tube, mix well, then pipette 200 μL to 3#, mix well until the 9# tube is completely mixed, and get 9 standard mixed solutions with different concentrations.

[0030] Step 2: Preparation of internal standard solution: preparation containing d3-retinol, d6-α tocopherol, d3-25-hydroxyvitamin D2, d6-25-hydroxyvitamin D3, d7-VK1, d7-K1, d7-K2 Mixed internal standard solutions with concentrations of 3750, 5000, 25, 25, 833, and 208 ng / mL respectively, and the solvent is chromatographic ethanol.

[0031] Step 3: Pretreatment: Use a pipette gun to pipette 20 μL of 1#-9# standard solution into a 2.0mL plastic centrifuge tube, add 20 μL of internal standard solution, 180 μL of protein precipitation solution (methanol: acetonitrile = 1:1, v / v), vortexed at 20...

Embodiment 2

[0054] Step 1: Collection of blood samples: Collect venous blood from 64 healthy adults using yellow cap blood collection tubes with gel-promoting gel. The blood is centrifuged at 4000rpm for 10 minutes to obtain serum. Mix the sera of 64 persons evenly to obtain mixed serum and store in separate packages. at -80°C. Take another 50mL of mixed serum, add 32.5, 32.5, 10.35, 20.5, 0.5, 0.5μL of vitamin A, E, D2, D3, K1, K2 solutions with concentrations of 1000, 20000, 100, 100, 10, 10ug / mL ( That is, 0.65, 13, 0.0207, 0.04106, 0.0001, 0.0001ug of vitamins A, E, D2, D3, K1, K2) were added to each 1mL of mixed serum, and mixed thoroughly to obtain standard supplemented serum, which was stored in -80 ℃.

[0055] Step 2: Preparation of internal standard solution: preparation containing d3-retinol, d6-α tocopherol, d3-25-hydroxyvitamin D2, d6-25-hydroxyvitamin D3, d7-VK1, d7-K1, d7-K2 Mixed internal standard solutions with concentrations of 3750, 5000, 25, 25, 833, and 208 ng / mL res...

technical field

[0001] The invention relates to the technical field of detection of fat-soluble vitamins, and specifically relates to a method for simultaneously detecting six kinds of fat-soluble vitamins in blood plasma (clear). Background technique

[0002] Vitamins, divided into fat-soluble vitamins and water-soluble vitamins, are a type of organic substances necessary to maintain human life activities, and are also important active substances to maintain human health. Vitamin deficiency or excess will lead to the occurrence of nutritional diseases;

[0003] Vitamin A has various physiological functions such as promoting growth and reproduction, maintaining bone, epithelial tissue, vision and normal secretion of mucosal epithelium; vitamin D, also known as anti-rickets vitamin, is a sterol derivative, and the most important member of its family members is 25 hydroxy-vitamin D3 (cholecalciferol) and 25 hydroxy-vitamin D2 (ergocalciferol), both are fat-soluble vitamins, w...

Examples

Embodiment 1

[0029] Step 1: Preparation of standard solution: preparation containing retinol (VA), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, α-tocopherol (VE) and VK1, VK2 concentrations were 1000, 40, 160 . Take 200 μL of the solution to 2# tube, mix well, then pipette 200 μL to 3#, mix well until the 9# tube is completely mixed, and get 9 standard mixed solutions with different concentrations.

[0030] Step 2: Preparation of internal standard solution: preparation containing d3-retinol, d6-α tocopherol, d3-25-hydroxyvitamin D2, d6-25-hydroxyvitamin D3, d7-VK1, d7-K1, d7-K2 Mixed internal standard solutions with concentrations of 3750, 5000, 25, 25, 833, and 208 ng / mL respectively, and the solvent is chromatographic ethanol.

[0031] Step 3: Pretreatment: Use a pipette gun to pipette 20 μL of 1#-9# standard solution into a 2.0mL plastic centrifuge tube, add 20 μL of internal standard solution, 180 μL of protein precipitation solution (methanol: acetonitrile = 1:1, v / v), vortexed at 20...

Embodiment 2

[0054] Step 1: Collection of blood samples: Collect venous blood from 64 healthy adults using yellow cap blood collection tubes with gel-promoting gel. The blood is centrifuged at 4000rpm for 10 minutes to obtain serum. Mix the sera of 64 persons evenly to obtain mixed serum and store in separate packages. at -80°C. Take another 50mL of mixed serum, add 32.5, 32.5, 10.35, 20.5, 0.5, 0.5μL of vitamin A, E, D2, D3, K1, K2 solutions with concentrations of 1000, 20000, 100, 100, 10, 10ug / mL ( That is, 0.65, 13, 0.0207, 0.04106, 0.0001, 0.0001ug of vitamins A, E, D2, D3, K1, K2) were added to each 1mL of mixed serum, and mixed thoroughly to obtain standard supplemented serum, which was stored in -80 ℃.

[0055] Step 2: Preparation of internal standard solution: preparation containing d3-retinol, d6-α tocopherol, d3-25-hydroxyvitamin D2, d6-25-hydroxyvitamin D3, d7-VK1, d7-K1, d7-K2 Mixed internal standard solutions with concentrations of 3750, 5000, 25, 25, 833, and 208 ng / mL res...

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