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Sample pretreatment method for detecting fat-soluble vitamins in serum through high performance liquid chromatography-tandem mass spectrometry

A fat-soluble vitamin and serum technology, applied in the field of chemical analysis, can solve the problems of cumbersome process, low recovery rate and high cost of consumables

Pending Publication Date: 2021-09-14
杭州凯莱谱精准医疗检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing high-performance liquid chromatography tandem mass spectrometry detects the sample pretreatment method of fat-soluble vitamin in serum mainly adopts liquid-liquid extraction (as CN106504947A) and solid-liquid extraction method (as CN110763788A), needs through dilution, multiple extraction, rotary evaporation , nitrogen drying, purification and other steps, the process is cumbersome and requires a lot of manual operations, and it is difficult to achieve high-throughput and automation for liquid-liquid extraction. Although solid-liquid extraction can achieve high-throughput, the sample extraction process mainly relies on SLE Board to complete, high cost of consumables
[0004] At present, there are also many related patent applications for sample pretreatment using protein precipitation method for the determination of fat-soluble vitamin samples in serum and plasma, such as CN111999397A, CN110487943B, etc., but there is generally a problem of low recovery rate resulting in low sensitivity, and some even After protein precipitation, further extraction is required for subsequent detection and analysis, which makes clinical detection require more manual operations, which is not conducive to clinical promotion and use

Method used

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  • Sample pretreatment method for detecting fat-soluble vitamins in serum through high performance liquid chromatography-tandem mass spectrometry
  • Sample pretreatment method for detecting fat-soluble vitamins in serum through high performance liquid chromatography-tandem mass spectrometry
  • Sample pretreatment method for detecting fat-soluble vitamins in serum through high performance liquid chromatography-tandem mass spectrometry

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: sample preparation, pretreatment, detection, analysis

[0066] 1. Sample preparation

[0067] 1. Preparation of standard curve and quality control samples

[0068] Prepare vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1, and vitamin K2 standard products into a mixed solution, which is used as the stock solution of the standard working solution and the quality control working solution, and then mixed with the negative blank Serum and negative serum were mixed at a volume ratio of 1:49 to prepare standard curve and quality control samples.

[0069] The six fat-soluble vitamins have 10 serial concentrations (S1-S10) in the standard product, as shown in Table 1:

[0070] Table 1. Concentrations of 10 series of 6 fat-soluble vitamins in standard products (S1~S10)

[0071] ng / ml Vitamin A 25-Hydroxyvitamin D2 25-Hydroxyvitamin D3 Vitamin E Vitamin K1 Vitamin K2 S1 40 2 3 500 0.10 0.10 S2 60 3 ...

Embodiment 2

[0119] Example 2: Comparison of long-term storage stability evaluation results of different internal standard solvent systems

[0120] According to the internal standard solution preparation method provided in Example 1, the present embodiment prepares internal standard solutions containing 6 kinds of fat-soluble vitamin internal standards, and adopts different protein precipitation agent components as shown in Table 7 to carry out stability tests respectively. The stability test refers to putting it in a 48°C incubator for six months to observe whether the internal standard solution has any abnormal phenomena such as turbidity and deterioration, and compare whether the internal standard solution that has been tested for stability is different from the newly prepared internal standard solution. The detection method provided in Example 1 detects whether there is a concentration deviation of the internal standard component, and whether there is a deviation in the recovery rate of...

Embodiment 3

[0125] Example 3: Comparison of detection results after sample pretreatment using different protein precipitation agents

[0126] In this example, according to the preparation method of the protein precipitation agent provided in Example 1, different protein precipitation agents as shown in Table 8 are used, and after the sample preparation and pretreatment are completed according to the steps and methods provided in Example 1, the lowest standard curve is taken. The concentration point (S1) was analyzed by the liquid chromatography-mass spectrometry system, and the peak areas of the five fat-soluble vitamins in the S1 sample were measured as shown in Table 8:

[0127] Table 8. Comparison of detection results after sample pretreatment using different protein precipitants

[0128]

[0129] As can be seen from Table 8, when the protein precipitation agent provided by this embodiment is 50mM ammonium acetate (75% methanol+15% acetonitrile+10% isopropanol), the peaks of vitamin...

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Abstract

The invention provides a sample pretreatment method for detecting fat-soluble vitamins in serum through high performance liquid chromatography-tandem mass spectrometry. The specific protein precipitant is adopted and mixed with the internal standard substance working solution to form the internal standard solution, so that sample pretreatment is simply and efficiently completed, no extra sample enrichment or other operation is needed, the recovery rate of fat-soluble vitamins A, E, K1 and K2 in a serum sample can be remarkably improved, and the pretreatment cost of the sample is reduced, so that the detection sensitivity of the fat-soluble vitamins in the serum is greatly improved, and the accuracy and the stability of a detection result can be ensured. Meanwhile, the invention further provides application of ammonium acetate to preparation of the protein precipitant.

Description

technical field [0001] The invention relates to the technical field of chemical analysis, in particular to a sample pretreatment method for detecting fat-soluble vitamins in serum by high performance liquid chromatography tandem mass spectrometry. Background technique [0002] Vitamin A (VA), also known as retinol, has the functions of maintaining normal vision, maintaining epithelial tissue, and promoting normal growth and development of bones. Insufficient VA in the human body can lead to nutritional deficiencies such as dry eye disease, night blindness, and hyperkeratosis of the skin. 25-Hydroxyvitamin D is an important factor that regulates the metabolism of calcium and phosphorus in the human body, coordinates the mobilization or deposition of bone calcium, the absorption or excretion of urinary calcium, maintains the stability of blood calcium levels in the body, and plays an important role in bone development and shaping, muscle nerve conduction effects and cell regen...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/72G01N30/8634G01N2030/045
Inventor 曾珊珊张琦王媛韩宝玉刘鹏云刘华芬
Owner 杭州凯莱谱精准医疗检测技术有限公司
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