CN113759039A

CN113759039A - Synchronous detection method and device for beta-carotene and vitamin A

Info

Publication number: CN113759039A
Application number: CN202110989339.9A
Authority: CN
Inventors: 杨艳玲; 董慧; 陈泽辉; 马乐; 杨元凯
Original assignee: Shenzhen Aonelab Testing Laboratory
Current assignee: Shenzhen Aonelab Testing Laboratory
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2021-12-07

Abstract

The embodiment of the invention provides a synchronous detection method and a synchronous detection device for beta-carotene and vitamin A. The method comprises the following steps: establishing a standard curve of beta-carotene and vitamin A; adding an internal standard substance into a biological sample to be detected; adding a protein precipitator, fully and uniformly mixing and oscillating to precipitate protein to form a solid-liquid mixed sample; extracting target components (beta-carotene and vitamin A); carrying out high performance liquid chromatography detection on the target component; and calculating the concentration of the beta-carotene and the vitamin A in the biological sample to be detected according to the standard curve and the detection result. The method combines extraction and high performance liquid chromatography detection, can realize synchronous analysis of vitamin A and beta-carotene in biological samples, and does not need to be eluted respectively. In addition, by applying a suitable pre-treatment to the biological sample, clogging of the column by impurities in the biological sample is avoided.

Description

Synchronous detection method and device for beta-carotene and vitamin A

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of organic matter detection, in particular to a synchronous detection method and a synchronous detection device for beta-carotene and vitamin A.

[ background of the invention ]

Vitamin a, also known as retinol, is one of the 13 vitamins essential to humans. It has important significance in human growth and development, maintenance of immune system, inhibition of tumor cell growth and visual system. Vitamin a in the human body is usually obtained from ingestion of food containing vitamin a.

Among them, in animal foods, vitamin a exists mainly as retinol ester and can be degraded into retinol in the small intestine. In vegetable foods, vitamin a is mainly present in the form of carotenoids, most of which are in the form of β -carotene, which can be converted to retinol by the catalytic action of enzymes.

When a human suffers from certain diseases, the intake of vitamin a may be reduced, or the consumption of vitamin a may be increased, resulting in vitamin a deficiency. In addition, certain diseases may lead to a decrease in vitamin a consumption, causing vitamin a to accumulate in certain tissues of the human body, resulting in vitamin a overload.

Beta-carotene is a dimer consisting of 2 vitamin A molecules, can generate two molecules of vitamin A in a human body through enzyme catalysis, and greatly contributes to the level of the vitamin A in the human body. Although there is no temporary evidence that excessive intake of beta-carotene can lead to vitamin a overdose, excessive intake of food rich in beta-carotene can cause carotenoemia, manifested as yellow skin staining.

When a human body suffers from certain diseases, beta-carotene cannot be degraded into vitamin A, so that the beta-carotene is accumulated in the human body, particularly severe yellow skin dyeing is shown. Therefore, monitoring the nutrient level of vitamin A and beta-carotene in human body or organism has certain positive significance.

Many analytical detection methods based on high performance liquid chromatography or chemiluminescence are currently provided for the detection of vitamin a concentrations. However, beta-carotene is characterized by low polarity in chromatographic behavior and is difficult to elute compared to other organic compounds. Moreover, it is also characterized by susceptibility to degradation by light and to oxidation by exposure to air, which necessitates careful handling during the pretreatment steps of the analytical test method to ensure stability, precision and reproducibility of the method.

[ summary of the invention ]

The embodiment of the invention provides a synchronous detection method and a synchronous detection device for beta-carotene and vitamin A, and aims to overcome one or more defects in the prior art.

In order to solve the above technical problems, embodiments of the present invention provide the following technical solutions: a synchronous detection method of beta-carotene and vitamin A. The synchronous detection method comprises the following steps:

establishing a standard curve of beta-carotene and vitamin A;

adding an internal standard substance to a biological sample to be detected to form a first mixture;

adding a protein precipitant into the first mixture, and fully and uniformly mixing to form a second mixture;

oscillating the second mixture to precipitate the proteins in the second mixture to form a solid-liquid mixed sample;

extracting target components in the solid-liquid mixed sample, wherein the target components comprise: beta-carotene and vitamin a;

performing high performance liquid chromatography detection on the target component to obtain detection results under a first detection wavelength, a second detection wavelength and a third detection wavelength;

calculating the concentration of the beta-carotene in the biological sample to be detected according to the detection results of the beta-carotene under the standard curve, the first detection wavelength and the third detection wavelength; and is

And calculating the concentration of the vitamin A in the biological sample to be detected according to the detection results of the standard curve of the vitamin A, the second detection wavelength and the third detection wavelength.

Optionally, the protein precipitating agent is taken from one or more of methanol or acetonitrile; the shaking time of the first mixture was 10 minutes.

Optionally, the extracting a target component in the solid-liquid mixed sample specifically includes:

adding an extracting agent into the solid-liquid mixed sample, and fully and uniformly mixing to form a third mixture;

shaking the third mixture to form a fourth mixture;

centrifuging the fourth mixture, and separating and taking out an extract phase containing the target component;

drying the extract phase under nitrogen atmosphere to obtain dry target component.

Optionally, the extractant comprises: an alkane having 4 to 8 carbon atoms;

the centrifugal force of the centrifugal treatment was 10000-15000g, and the centrifugation time was 10 minutes.

Optionally, the drying the extract phase in a nitrogen environment specifically includes:

placing the extract phase in a metal dry bath type nitrogen blowing instrument, and drying in a nitrogen blowing mode; the temperature of the metal dry bath type nitrogen blowing instrument is set to be 20-50 ℃, and the nitrogen blowing time is 10 minutes.

Optionally, the performing high performance liquid chromatography detection on the target component specifically includes:

adding a double solvent to the dried target component to re-dissolve the target component to form a fifth mixture; the double solvent is a binary organic solvent;

putting the fifth mixture on a machine, and carrying out high performance liquid chromatography detection;

and acquiring detection results of the third mixture at the first detection wavelength, the second detection wavelength and the third detection wavelength by using a diode array detector.

Optionally, the double solvent is selected from one or more of methanol, acetonitrile, acetone or dichloromethane;

the mobile phase detected by the high performance liquid chromatography is selected from one or more of acetonitrile, water or isopropanol; the acquisition time of the high performance liquid chromatography detection is 20 minutes;

the internal standard substance is Sudan I, the first detection wavelength is 455nm, and the second detection wavelength is 325 nm; the third detection wavelength is 477 nm.

Optionally, the establishing a standard curve of β -carotene and vitamin a specifically includes:

preparing a plurality of first standard solutions and a plurality of second standard solutions according to a preset concentration gradient;

wherein, the first standard solution takes bovine serum albumin solution as a blank matrix and contains an internal standard substance with known concentration and a beta-carotene standard substance with known concentration;

the second standard solution takes bovine serum albumin solution as a blank matrix and contains an internal standard substance with known concentration and the vitamin A standard substance with known concentration;

adding a protein precipitator into the first standard solution and the second standard solution to remove proteins in the first standard solution and the second standard solution;

adding an extracting agent into the first standard solution and the second standard solution after the protein is removed, and extracting to obtain an extract phase containing a target component;

drying the extract phase in a nitrogen environment to obtain a dried target component;

redissolving the dried target component by adding a binary organic solvent;

performing high performance liquid chromatography detection on the redissolved target component to obtain the peak area ratio of the beta-carotene to the internal standard substance in the first standard solution; and the peak area ratio of the vitamin A and the internal standard substance in the second standard solution;

fitting to generate a standard curve of the beta-carotene according to the corresponding relation between the concentration ratio of the beta-carotene to the internal standard substance and the peak area ratio; and is

And fitting to generate a standard curve of the vitamin A according to the corresponding relation between the concentration ratio of the vitamin A to the internal standard substance and the peak area ratio.

Optionally, the concentration of bovine serum albumin is 20-60 mg/mL;

the set number of the first standard solution and the second standard solution is 2-8 groups; the beta-carotene standard and the vitamin A standard are selected from the following concentrations:

0.05,0.1,0.2,0.4,0.6,0.8,1.2,1.6,2.4,3.2 or 6.4 μ g/mL;

the internal standard was selected from the following concentrations:

15,20,25,30,35 or 40. mu.g/mL.

In order to solve the above technical problems, embodiments of the present invention further provide the following technical solutions: a synchronous detection device for beta-carotene and vitamin A.

Wherein, this synchronous detection device includes:

a memory for recording a standard curve for beta-carotene, vitamin A, and an internal standard;

a sample processing kit, comprising: protein precipitant, extractant, double solvent and internal standard solution;

a high performance liquid chromatography apparatus that uses a diode array detector to simultaneously detect at a plurality of different detection wavelengths.

Compared with the prior art, the embodiment of the invention provides a synchronous detection method of vitamin A and beta-carotene, which adopts a mode of combining extraction and high performance liquid chromatography detection, can realize synchronous analysis of the vitamin A and the beta-carotene in a biological sample, and does not need to be eluted respectively. In addition, by applying a suitable pre-treatment to the biological sample, clogging of the column by impurities in the biological sample is avoided.

[ description of the drawings ]

One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the figures in which like reference numerals refer to similar elements and which are not to scale unless otherwise specified.

FIG. 1 is a flow chart of a method for simultaneous detection of beta-carotene and vitamin A according to example 1 of the present invention;

FIG. 2 is a flow chart of a method for extracting a target component according to example 1 of the present invention;

FIG. 3 is a flowchart of a method for constructing a standard curve according to embodiment 2 of the present invention;

FIG. 4a is a schematic representation of a standard curve for beta-carotene provided in example 2 of the present invention;

FIG. 4b is a schematic representation of a standard curve of vitamin A provided in example 2 of the present invention;

FIG. 5 is a schematic diagram of the operation steps of a biological sample provided in example 3 of the present invention before on-line detection;

FIG. 6a is a graph of the UV absorption spectrum at a detection wavelength of 325nm as provided in example 3 of the present invention;

FIG. 6b is a graph of the ultraviolet absorption spectrum at 455nm detection wavelength provided in example 3 of the present invention;

FIG. 6c is a graph of the UV absorption spectrum at detection wavelength 477nm provided in example 3 of the present invention;

fig. 7 is a schematic structural diagram of a detection apparatus provided in embodiment 4 of the present invention.

[ detailed description ] embodiments

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may be present. As used in this specification, the terms "upper," "lower," "inner," "outer," "bottom," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the invention and simplicity in description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Furthermore, the technical features mentioned in the different embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

Example 1:

fig. 1 is a flowchart of a method for synchronously detecting beta-carotene and vitamin a according to an embodiment of the present invention. In this example, quantitative analysis of β -carotene and vitamin a can be accomplished using an internal standard method. As shown in fig. 1, the synchronization detection method may include the steps of:

s100, establishing a standard curve of beta-carotene and vitamin A.

The standard curve is a concentration-peak area curve established by using a least square method and a similar regression analysis method according to standard substances with different concentrations and peak areas corresponding to the standard substances as reference points.

In this example, the concentration ratio between the internal standard substance of known concentration and β -carotene and the corresponding peak area ratio were used as reference points for constructing the standard curve of β -carotene, and the concentration ratio between the internal standard substance of known concentration and β -carotene and the corresponding peak area ratio were used as reference points for constructing the standard curve of vitamin a.

It should be noted that the step S100 is an off-line processing step, and it may be executed in advance to obtain relevant data, store the data in a suitable database or similar data storage device, and call the data for use in a subsequent synchronous detection method.

And S200, adding an internal standard substance into the biological sample to be detected to form a first mixture.

Wherein the internal standard is a pure substance of known concentration or mass. It can be used as a standard for biological samples to be detected to realize quantitative analysis of beta-carotene and vitamin A.

The biological sample may be any suitable type of sample taken from a human body or other organisms, such as blood plasma, etc., and may be determined according to the needs of different application scenarios.

Specifically, after the internal standard solution with a known concentration is added, the internal standard solution can be mixed uniformly and then kept stand for 1 to 5 minutes, and then the operation of removing the protein can be carried out.

And S300, adding a protein precipitator into the first mixture, and fully and uniformly mixing to form a second mixture.

The protein precipitant is a reagent for denaturing and coagulating proteins and precipitating the proteins from a biological sample. Specifically, the protein precipitant may be selected from methanol, acetonitrile, or a combination thereof.

S400, oscillating the second mixture to precipitate the protein in the second mixture to form a solid-liquid mixed sample.

Wherein, the second mixture after being fully and uniformly mixed is oscillated (for example, the second mixture is oscillated on a vortex oscillator), so that the protein in the second mixture is precipitated, the protein is separated out from the sample solution, and the protein precipitate is generated. In this example, the sample solution containing the protein precipitate is referred to as a "solid-liquid mixed sample".

Specifically, the oscillation time of the second mixture may be 10 minutes to ensure sufficient protein precipitation and achieve good protein removal.

And S500, extracting the target component in the solid-liquid mixed sample.

The "extraction" is a separation operation based on the principle of similar solubility, which retains target components that can be dissolved in an organic solvent and eliminates components (such as amino acids) that cannot be dissolved in the organic solvent. It may also be commonly referred to as "liquid-liquid extraction" or "extraction".

In this embodiment, the target components to be separated and extracted from the biological sample to be detected include: beta-carotene and vitamin A.

In some embodiments, as shown in fig. 2, the operation of extracting the target component may specifically include the following steps:

and S510, adding an extracting agent into the solid-liquid mixed sample, and fully and uniformly mixing to form a third mixture.

Wherein the extractant is a solvent added to the sample solution in which the target component has greater solubility. In particular, the extractant may be an alkane containing from 4 to 8 carbon atoms.

In a preferred embodiment, it has surprisingly been found that the best extraction is achieved by selecting an alkane having 6 carbon atoms, which maximizes the extraction and separation of the target component from the biological sample.

And S520, oscillating the third mixture to form a fourth mixture.

Wherein, the third mixture which is added with the extractant and then uniformly mixed can also be placed in a vortex oscillator or other similar equipment for oscillation treatment so as to ensure the extraction effect on the target component. Specifically, the oscillation time of the third mixture may be controlled to be about 10 minutes.

S530, centrifuging the fourth mixture, and separating and taking out an extract phase containing the target component.

The centrifugation process may be carried out in any suitable type of centrifuge, depending on the volume of solution to be centrifuged or whether a low temperature is to be maintained, among other things. After centrifugation, the layers will separate to form an extract phase, and the target component will be mainly dissolved in the extract phase.

Specifically, the centrifugation operation of the fourth mixture can be performed at 10000-.

And S540, drying the extract phase in a nitrogen environment to obtain a dry target component.

Wherein, the extracted phase after centrifugation can be transferred to a proper nitrogen blowing instrument and other equipment, and the extraction phase is dried by blowing in a nitrogen blowing way to obtain a dry target component.

Specifically, the extract phase may be placed in a metal dry bath nitrogen blower, the temperature is set to 20 to 50 ℃, and nitrogen is blown by means of a nitrogen purge for 10 minutes to obtain a dry target component.

S600, performing high performance liquid chromatography detection on the target component to obtain detection results under a first detection wavelength, a second detection wavelength and a third detection wavelength.

Among them, High Performance Liquid Chromatography (HPLC) is a common organic analysis method. The main detection principle is that a sample solution is loaded into a chromatographic column (stationary phase) by a mobile phase. When the components in the sample solution move relatively in the two phases, the components are separated into single components at the end of the chromatographic column and flow out sequentially due to the large difference in moving speed caused by the different partition coefficients in the two phases. When each component passes through the detector, the component is converted into an electric signal and transmitted to a recorder, and a detection result is output.

In this embodiment, detection is performed at three different detection wavelengths (i.e., a first detection wavelength, a second detection wavelength, and a third detection wavelength), thereby enabling simultaneous analysis of β -carotene and vitamin a.

Specifically, the detection result of the first detection wavelength corresponds to beta-carotene in the biological sample, the detection result of the second detection wavelength corresponds to vitamin A in the biological sample, and the detection result of the third detection wavelength corresponds to an internal standard substance added to the biological sample.

In some embodiments, the dry target component (S540) obtained after nitrogen blowing the extract phase may be redissolved by adding a suitable re-solvent to obtain a liquid fifth mixture, which is then tested on the machine.

Specifically, the double solvent may be a binary organic solvent selected from methanol, acetonitrile, acetone, dichloromethane or a combination thereof. And the mobile phase of the high performance liquid chromatography detection can be selected from one or more of acetonitrile, water or isopropanol, and the collection time of the high performance liquid chromatography detection is 20 minutes.

S700, calculating the concentration of the beta-carotene in the biological sample to be detected according to the standard curve of the beta-carotene, the detection results under the first detection wavelength and the third detection wavelength.

Wherein, firstly, the peak area ratio of the internal standard substance to the beta-carotene can be determined through the detection results of the first detection wavelength and the third detection wavelength. Then, the concentration ratio corresponding to the peak area ratio is found in the standard curve of beta-carotene. Finally, since the concentration of the internal standard added to the biological sample is known, the concentration of β -carotene in the biological sample can be solved accordingly.

S800, calculating the concentration of the vitamin A in the biological sample to be detected according to the detection results of the vitamin A under the standard curve, the second detection wavelength and the third detection wavelength

Similarly to the calculation of the concentration of beta-carotene, when the concentration of vitamin A is calculated, the corresponding concentration ratio is found on the standard curve of vitamin A according to the detection results of the second detection wavelength and the third detection wavelength. Then, the concentration of vitamin A in the biological sample is solved based on the concentration of the internal standard substance added to the biological sample.

In particular, the detector for high performance liquid chromatography detection may use a diode array detector or similar device capable of detecting at a plurality of different detection wavelengths simultaneously.

In some embodiments, sudan i may be selected for use as an internal standard. Accordingly, the first detection wavelength corresponding to β -carotene was 455nm, the second detection wavelength corresponding to vitamin A was 325nm, and the third detection wavelength corresponding to Sudan I (internal standard) was 477 nm.

The synchronous detection method of the beta-carotene and the vitamin A, provided by the embodiment of the application, can realize synchronous analysis and detection of the beta-carotene and the vitamin A in a biological sample, and has wide application prospects in the fields of nutrition analysis and the like. In addition, through proper pretreatment of the biological sample, impurities in the sample can be prevented from blocking the chromatographic column, and the detection reliability is improved.

Example 2:

FIG. 3 is a flowchart of a method for establishing a standard curve of beta-carotene and vitamin A according to an embodiment of the present invention. In this example, for the sake of convenience of presentation, the standard curve for β -carotene is referred to as "first standard curve", and the standard curve for vitamin a is referred to as "second standard curve". As shown in fig. 3, the standard curve is established as follows:

s110, preparing a plurality of first standard solutions and second standard solutions with different concentrations.

Wherein the first standard solution is a solution containing a known concentration of beta-carotene and sudan i (internal standard). The second standard solution is a solution containing known concentrations of vitamin a and sudan i (internal standard).

In a preferred embodiment, the first standard solution and the second standard solution can use bovine serum albumin solution as a blank matrix. The standard curve drawn in such a way is more accurate, and the interference and influence of endogenous beta-carotene and vitamin A in the biological sample can be avoided. Surprisingly, it was found that the best results were obtained when bovine serum albumin was selected at a concentration of 20-60 mg/mL.

Specifically, the standard solutions of beta-carotene, vitamin a and sudan i (internal standard substance) with appropriate amount and different concentrations can be determined and prepared according to the needs of actual conditions, and then the standard solutions are combined to form corresponding first standard solutions and second standard solutions.

Wherein, the first standard solution and the second standard solution can be arranged in 2-8 groups (wherein, the standard curve shown in figure 3 is 6 groups). Each set of standard solutions may provide a reference point for fitting.

The beta-carotene standard and the vitamin a standard were selected from the following concentrations: 0.05,0.1,0.2,0.4,0.6,0.8,1.2,1.6,2.4,3.2 or 6.4 μ g/mL; sudan I (internal standard) standards were selected from the following concentrations: 15,20,25,30,35 or 40. mu.g/mL.

After the plurality of first standard solutions and the plurality of second standard solutions are prepared, the following operation steps are carried out on each prepared standard solution, and a corresponding high performance liquid chromatography detection result is obtained. For simplicity, the following description of specific operating steps is given by way of example of a "standard solution":

and S120, adding a protein precipitator into the standard solution, and removing the protein in the standard solution.

The protein precipitator can select methanol, acetonitrile or the combination of the methanol and the acetonitrile to achieve the effect of separating out the protein in the standard solution and avoid causing the blockage of the chromatographic column.

And S130, adding an extracting agent into the standard solution after the protein is removed, mixing uniformly, and oscillating.

Wherein, the target component to be detected can be separated and extracted from the standard solution by using a liquid-liquid extraction mode. Specifically, the extractant may be an alkane containing 4 to 8 carbon atoms, and is mixed well and shaken for about 10 minutes.

And S140, carrying out centrifugal treatment, and separating to obtain an extract phase containing the target component.

Wherein, after a certain centrifugal force and proper centrifugal time, the separated extract phase (liquid) can be taken out to complete the separation and extraction of the target component. Specifically, the centrifugation parameter of the centrifugation treatment in the step S140 is 10000-15000g, and the centrifugation time is 10 minutes.

S150, drying the extract phase in a nitrogen environment to obtain a dry target component.

Wherein the extraction phase can be dried by blowing nitrogen for 10 minutes at the temperature of 20-50 ℃ through a metal dry bath type nitrogen blowing instrument to obtain a dry target component.

And S160, adding a binary organic solvent, and redissolving the dried target component.

Specifically, the binary organic solvent (double solvent) may be selected from methanol, acetonitrile, acetone, dichloromethane or a combination thereof.

S170, feeding the re-dissolved target component on a machine for sample injection, and carrying out high performance liquid chromatography detection to obtain detection results under different detection wavelengths.

The mobile phase for high performance liquid chromatography detection can adopt acetonitrile, water and isopropanol or a mixed solution thereof. The acquisition time of the high performance liquid chromatography is controlled to be 20 minutes, and the absorbance conditions under different detection wavelengths (channels) are detected simultaneously by a diode array detector.

Specifically, the detection wavelength of the vitamin A is 325nm, the detection wavelength of the beta-carotene is 455nm, and the detection wavelength of the Sudan I (internal standard substance) is 477 nm.

Through the above-described operation steps (S120 to S170), the peak area ratio of β -carotene to the internal standard substance in the first standard solution and the peak area ratio of vitamin a to the internal standard substance in the second standard solution can be obtained.

Since the concentrations of beta-carotene, vitamin a and the internal standard in the standard solution are known. Therefore, each standard solution can be used as a reference point, and a first standard curve and a second standard curve which take the concentration ratio as the ordinate and the peak area ratio as the abscissa are fitted to establish so as to represent the relationship between the peak area and the concentration.

Example 2 the first and second standard curves calculated are shown in detail in fig. 4a and 4 b. Wherein, fig. 4a is a standard curve of beta-carotene, and fig. 4b is a standard curve of vitamin A.

Example 3:

based on the synchronous detection methods provided in embodiments 1 and 2, the embodiments of the present invention also provide a synchronous analysis method of beta-carotene and vitamin a based on an internal standard method. The synchronous analysis method may include the steps of:

1. with respect to the internal standards selected:

11) to 200. mu.L of plasma, 20. mu.L of Sudan I (50ppm in methanol) was added and mixed to obtain test samples (experiments with 6 plasma samples were performed).

12) Adding 600 mu L of protein precipitant into the sample to be tested, uniformly mixing, and oscillating for 10 minutes to precipitate the protein in the bio-based sample to be tested to form the sample to be tested in a solid-liquid mixed state.

13) Adding 600 mu L of extracting agent into a sample to be detected in a solid-liquid mixed state, uniformly mixing, and oscillating for 10 minutes.

14) After shaking for 10 minutes, it was centrifuged (15000g,10 minutes) and the extract phase containing vitamin A and beta-carotene was separated.

15) The extract phase was dried (10 minutes at 20 ℃ C.) using a nitrogen blower to obtain samples of dried vitamin A, beta-carotene and Sudan I.

16) Redissolving the dried extract phase with 100. mu.L of a binary organic solvent.

17) The re-solution of the plasma sample extract was analyzed by liquid chromatography (sample size 10 uL).

The results of liquid chromatography analysis of 6 groups of plasma samples (designated sample 1 to sample 6, respectively, in table 1) are shown in table 1 below:

table 1

As shown in table 1, the retention time of sudan I in this example is stable, the peak area RSD% is good, and no significant matrix effect is observed. Thus, sudan I number may be used as an internal standard.

2. As shown in FIG. 5, the processing steps before the on-machine detection of β -carotene and vitamin A are as follows:

21) to 200. mu.L of the biological sample (plasma) was added 20. mu.L of Sudan I solution (50ppm in methanol) and mixed well.

22) Adding 600 mu L of protein precipitant into the biological sample, uniformly mixing, and oscillating for 10 minutes to precipitate the protein in the biological sample to be detected to obtain a sample in a solid-liquid mixed state;

23) to the sample in the solid-liquid mixed state, 600. mu.L of an extractant was added, and after uniform mixing, shaking was carried out for 10 minutes.

24) After shaking, the mixture was centrifuged (15000g,10 min) and the extract phase containing vitamin A and beta-carotene was removed.

25) Drying the extract phase (20 ℃,10 minutes) with a nitrogen blower to obtain dried samples of vitamin A, beta-carotene and Sudan I;

26) redissolving the sample obtained after drying the extract phase with 100. mu.L of binary organic solvent.

3. High performance liquid chromatography analysis:

the redissolved sample can be transferred to a light-proof sample injection bottle for machine detection (the sample injection amount is 10 uL). In the high performance liquid chromatography analysis, the liquid phase can adopt binary solution consisting of acetonitrile and isopropanol as an organic phase, deionized water as a water phase, and the collection time is 20 minutes.

In addition, a diode array detector is used for simultaneously detecting at three different detection wavelengths of 325nm, 455nm and 477nm respectively to obtain ultraviolet absorption spectrums of vitamin A, beta-carotene and Sudan I in a biological sample to be detected, so that synchronous analysis of the vitamin A and the beta-carotene is realized.

Specifically, fig. 6a provides an example of the detection of the diode array detector at 325nm for embodiment 3 of the present invention. Fig. 6b is a detection example of 455nm of the diode array detector provided in embodiment 3 of the present invention. Fig. 6c shows an exemplary detection at 477nm of a diode array detector provided in embodiment 3 of the present invention.

28) Determining the peak area ratio of each component to be detected (vitamin A and beta-carotene) to Sudan I in the biological sample to be detected.

29) And establishing a standard curve and a peak area ratio of the vitamin A and the beta-carotene based on an internal standard method, and calculating and determining the content of the component to be detected in the biological sample to be detected.

Specifically, the concentration of vitamin a and β -carotene in the biological sample to be measured can be calculated by substituting the ratio of the peak area of the collected components to be measured (vitamin a and β -carotene) to the peak area of sudan I (internal standard substance) into the corresponding standard curve equation (y ═ ax + b).

The analysis and detection method provided by the embodiment of the invention is accurate and reliable, the standard recovery rate of the vitamin A and the beta-carotene measured in the experiment is 90.7-105.3%, and the lower detection limits are 0.025 mu g/mL and 0.05 mu g/mL respectively.

50 healthy adult plasma were tested according to the synchronous testing method provided in example 3 of the present invention. The results of the assay showed that the vitamin A and beta-carotene contents in 50 plasma samples were 0.346 + -0.0282 μ g/mL and 0.581 + -0.112 μ g/mL, respectively, consistent with the normal ranges reported in the literature.

Example 4

Based on the steps provided by the above method embodiment, the embodiment of the present invention further provides a device for synchronously detecting vitamin a and β -carotene. Fig. 7 is a schematic structural diagram of a detection apparatus according to an embodiment of the present invention.

As shown in fig. 7, the detecting device 70 includes: a memory 71, a sample processing kit 72, a liquid chromatography device 73.

Wherein the memory 71 is a hardware device for storing standard curves of vitamin a and beta-carotene. It may include any suitable type of non-volatile storage medium, database, or cloud distributed storage platform, as long as the functionality to meet the data storage requirements is provided.

Preferably, the memory 71 is also coupled to one or more user interaction devices (such as a mouse, a keyboard or a touch screen) for receiving user input data and generating a desired standard curve by itself based on the input data.

The sample processing kit 72 contains a plurality of pre-configured reagents for performing the processing steps (e.g., protein removal, separation and extraction, drying, reconstitution, etc.) of the biological sample prior to on-machine analysis and subsequent liquid chromatography analysis.

Specifically, the reagent contained in the sample processing kit 72 may be determined by the actual processing procedure and the liquid chromatography analysis method. Taking the synchronous detection method provided in embodiment 3 as an example, the sample processing kit includes: methanol (HPLC grade), ultrapure water, isopropanol (HPLC grade), acetonitrile (HPLC grade), n-hexane (HPLC grade), dichloromethane (HPLC grade), acetone (HPLC grade), and the like.

In some embodiments, the sample processing kit may further comprise: centrifuge tubes for holding liquid samples and the like are used as experimental consumables related to processing steps and liquid chromatographic analysis.

The liquid chromatography device 73 is a system platform constructed of several devices or equipment capable of liquid chromatography analysis principles. The corresponding equipment or equipment can be selected and used according to the needs of actual conditions. For example, the HPLC apparatus 430 may include a Shimadzu LC-20AD XR pump, a Shimadzu SIL-20AC XR injector, a Shimadzu CTO-20AC column oven, a Shimadzu SPD-M20A detector, a Shimadzu DGU-20A 5R degas unit, Labsolutions chromatographic workstation software, and the like, or components, to perform liquid chromatographic analysis of a sample.

In practical use of the detection apparatus 70, the detector of the liquid chromatography device 73 can provide absorbance of vitamin a, β -carotene, and sudan I (internal standard) in the upper computer sample at the corresponding detection wavelength.

Accordingly, the ratio of the absorption peak areas of the vitamin A and the Sudan I (internal standard substance) can be calculated; ratio of the absorption peak areas of beta-carotene to Sudan I (internal standard).

Then, according to the corresponding standard curve of the two, the corresponding concentration ratio under the ratio of the absorption peak areas can be obtained. Finally, the concentrations of vitamin a and β -carotene in the biological sample were calculated from the concentration ratio and the concentration of sudan I (internal standard) added to the biological sample.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A synchronous detection method of beta-carotene and vitamin A is characterized by comprising the following steps:

establishing a standard curve of beta-carotene and vitamin A;

2. The synchronous detection method according to claim 1, wherein the protein precipitant is selected from one or more of methanol and acetonitrile; the shaking time of the first mixture was 10 minutes.

3. The synchronous detection method according to claim 1, wherein the extracting target components in the solid-liquid mixed sample specifically comprises:

shaking the third mixture to form a fourth mixture;

4. The synchronous detection method according to claim 3, wherein the extraction agent comprises: an alkane having 4 to 8 carbon atoms;

5. The synchronous detection method according to claim 3, wherein the drying the extract phase in a nitrogen environment comprises:

6. The synchronous detection method according to claim 4, wherein the performing high performance liquid chromatography detection on the target component specifically comprises:

7. The synchronous detection method according to claim 6, wherein the double solvent is selected from methanol, acetonitrile, acetone or dichloromethane;

8. The method for synchronous detection according to claim 1, wherein the establishing of the standard curve of β -carotene and vitamin a specifically comprises:

redissolving the dried target component by adding a binary organic solvent;

9. The synchronous detection method according to claim 8, wherein the concentration of bovine serum albumin is 20-60 mg/mL;

0.05,0.1,0.2,0.4,0.6,0.8,1.2,1.6,2.4,3.2 or 6.4 μ g/mL;

the internal standard was selected from the following concentrations:

15,20,25,30,35 or 40. mu.g/mL.

10. A synchronous detection device of beta-carotene and vitamin A is characterized by comprising:

a liquid chromatography apparatus that uses a diode array detector to simultaneously detect at a plurality of different detection wavelengths.