阅读说明：本技术 同时检测血液中视黄醇和α-生育酚含量的方法 (Method for simultaneously detecting contents of retinol and α -tocopherol in blood ) 是由 贾永娟 雒琴 倪君君 于 2019-12-02 设计创作，主要内容包括：本发明提供了同时检测血液中视黄醇和α-生育酚含量的方法,该方法包括：基于特定的液相条件,利用高效液相色谱仪,分别检测至少三个标准溶液,得到各个标准溶液的色谱图,各标准溶液中均含有浓度已知的视黄醇和α-生育酚的标准品及内标物；根据各个标准溶液的色谱图,拟合得到视黄醇和α-生育酚的标准曲线方程；将混合内标工作液添加到经处理至少2mL待检测血液而得到的血液样本中,经样本前处理以得到待测样本；同样检测待测样本得到其色谱图；根据待测样本的色谱图和各个标准曲线方程,计算血液样本中视黄醇和α-生育酚的含量。本发明能够更加快速的同时检测血液中视黄醇和α-生育酚的含量。(The invention provides a method for simultaneously detecting contents of retinol and α -tocopherol in blood, which comprises the steps of respectively detecting at least three standard solutions by utilizing a high performance liquid chromatograph based on specific liquid phase conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains standard products and internal standard substances of the retinol and α -tocopherol with known concentrations, fitting the standard curve equations of the retinol and α -tocopherol according to the chromatograms of the standard solutions, adding mixed internal standard working solution into a blood sample obtained by processing at least 2mL of blood to be detected, preprocessing the sample to be detected to obtain a sample to be detected, detecting the sample to be detected to obtain the chromatogram thereof, and calculating the contents of the retinol and α -tocopherol in the blood sample according to the chromatograms and each standard curve equation.)

1. A method for simultaneously detecting the contents of retinol and α -tocopherol in blood, comprising:

respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different;

fitting to obtain a standard curve equation of retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution;

adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol and an internal standard substance of α -tocopherol, wherein the concentration of the internal standard substance is known;

detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the samples to be detected;

calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;

wherein the liquid phase condition in the detection conditions comprises: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.

2. The method of claim 1,

the detector switching mode of the fluorescence detector in the high performance liquid chromatograph comprises the following steps:

3. the method of claim 1,

the liquid phase conditions include: the elution mode is gradient elution;

the elution process comprises the following steps:

time (min) Methanol (%) Water (%) 0 92 8 0.2 92 8 0.3 99 1 1.9 99 1 2 92 8 3 92 8

。

4. The method of claim 1,

the liquid phase conditions include: the column temperature was 35 ℃ and the amount of sample was 5. mu.L at a flow rate of 0.8 mL/min.

5. The method according to any one of claims 1 to 4,

the internal standard of retinol is retinol acetate, α -internal standard of tocopherol, α -tocopherol acetate.

Technical Field

The invention relates to the technical field of clinical chemistry, in particular to a method for simultaneously detecting the contents of retinol and α -tocopherol in blood.

Background

Retinol, also known as vitamin a (vitamine a), or anti-xerophthalmia factor, is an alicyclic unsaturated monohydric alcohol, belongs to fat-soluble vitamins, and is a constituent of rhodopsin which is sensitive to weak light in visual cells. Vitamin A can maintain normal visual function and normal growth and development of bones of human bodies, maintain the health of epithelial tissue cells, promote the synthesis of immunoglobulin, promote growth and reproduction and the like. When a human body lacks vitamin A, symptoms such as dry skin, desquamation, alopecia and the like can occur, night blindness can occur when the human body is seriously deficient, and normal growth and development of bones are influenced by destroying the balance between osteoblasts and osteoclasts, or enabling bone to be excessively proliferated, or enabling formed bone not to be absorbed. If the pregnant women lack the vitamin A, the development of the fetus can be directly influenced, and even dead fetus can occur.

α -tocopherol is the most widely distributed, abundant and active vitamin E form in nature, and is also a fat-soluble vitamin, which can increase the activity and quantity of sperms of men by promoting sex hormone secretion, so that the concentration of female estrogen of women is increased, the fertility is improved, and abortion is prevented, symptoms such as testicular atrophy, epithelial cell degeneration, abnormal pregnancy and the like can be caused when vitamin E is deficient.

For example, a method for simultaneously detecting the contents of vitamin A and vitamin E in blood, which is disclosed in publication No. CN106442754A, has retention times of vitamin A, an internal standard of vitamin A, vitamin E and an internal standard of vitamin E of 0.57min, 0.90min, 2.25min and 2.90min respectively, and an analysis time of 4.2 min.

Disclosure of Invention

The invention provides a method for simultaneously detecting the contents of retinol and α -tocopherol in blood, which can more quickly and simultaneously detect the contents of retinol and α -tocopherol in blood.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a method for simultaneously detecting the content of retinol and α -tocopherol in blood, which comprises the following steps:

respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different;

fitting to obtain a standard curve equation of retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution;

adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol and an internal standard substance of α -tocopherol, wherein the concentration of the internal standard substance is known;

detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain a chromatogram of the samples to be detected;

calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting;

wherein the liquid phase condition in the detection conditions comprises: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.

Preferably, the detector switching mode of the fluorescence detector in the high performance liquid chromatograph comprises the following steps:

preferably, the liquid phase conditions include: the elution mode is gradient elution;

the elution process comprises the following steps:

time (min)	Methanol (%)	Water (%)
			0	92	8
0.2	92	8
			0.3	99	1
1.9	99	1
			2	92	8
3	92	8

。

Preferably, the liquid phase conditions include: the column temperature was 35 ℃ and the amount of sample was 5. mu.L at a flow rate of 0.8 mL/min.

Preferably, the internal standard for retinol is retinol acetate, α -internal standard for tocopherol, α -tocopheryl acetate.

The invention provides a method for simultaneously detecting contents of retinol and α -tocopherol in blood, which comprises the steps of respectively detecting at least three standard solutions by utilizing a high performance liquid chromatograph based on specific liquid phase conditions to obtain chromatograms of the standard solutions, wherein each standard solution contains standard products and internal standard substances of the retinol and α -tocopherol with known concentrations, fitting the standard curve equations of the retinol and α -tocopherol according to the chromatograms of the standard solutions, adding mixed internal standard working solution into a blood sample obtained by processing at least 2mL of blood to be detected, preprocessing the sample to be detected to obtain a sample to be detected, detecting the sample to be detected to obtain the chromatogram thereof, and calculating the contents of the retinol and α -tocopherol in the blood sample according to the chromatograms and each standard curve equation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a flow chart of a method for simultaneously detecting retinol and α -tocopherol in blood according to an embodiment of the present invention;

FIG. 2 is a chemical formula of retinol provided in accordance with one embodiment of the present invention;

FIG. 3 is a chemical structural formula of α -tocopherol provided by an embodiment of the present invention;

FIG. 4 is a chromatogram of a standard solution of retinol and α -tocopherol standard;

FIG. 5 is a chromatogram of an internal standard for retinol and an internal standard for α -tocopherol in a standard solution provided in accordance with an embodiment of the present invention;

FIG. 6 is a chromatogram of retinol and α -tocopherol in a sample provided by an embodiment of the present invention;

FIG. 7 is a chromatogram of an internal standard for retinol and an internal standard for α -tocopherol in a sample provided in accordance with an embodiment of the present invention;

FIG. 8 is a graph of the linear relationship of retinol provided in accordance with one embodiment of the present invention;

FIG. 9 is a graph of α -tocopherol linearity provided by an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.

As shown in fig. 1, an embodiment of the present invention provides a method for simultaneously detecting the contents of retinol and α -tocopherol in blood, which may include the following steps:

and 101, respectively detecting at least three standard solutions by using a high performance liquid chromatograph under certain detection conditions to obtain chromatograms of the standard solutions, wherein any standard solution contains a standard product and an internal standard substance of retinol with known concentrations, and a standard product and an internal standard substance of α -tocopherol, and the concentrations of the same standard product in different standard solutions are different.

In an embodiment of the present invention, the liquid phase condition in the detection condition includes: the Poroshell120SB-C18 chromatographic column has a length of 50mm, an inner diameter of 3.0mm, a filler particle size of 2.7 μm, a mobile phase of methanol and water, an analysis time of 2.9-3.5min, a column temperature of 25-35 ℃, a sample injection amount of 0.1-10 μ L and a flow rate of 0.5-1 mL/min.

For example, the value of the analysis time may be 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5, preferably 3.0 min; the column temperature can take the value of 25, 27, 29, 30, 31, 33 or 35; the value of the sample amount can be 0.1, 0.5, 1, 3, 5 or 10; the flow rate may take on the value 0.5, 0.6, 0.7, 0.8, 0.9 or 1.

And 102, fitting to obtain a standard curve equation of the retinol and a standard curve equation of α -tocopherol according to the chromatogram of each standard solution.

103, adding a certain amount of mixed internal standard working solution into a certain amount of blood sample, and performing sample pretreatment to obtain a sample to be detected, wherein the blood sample is obtained by processing at least 2mL of blood to be detected, and the mixed internal standard working solution contains an internal standard substance of retinol with known concentration and an internal standard substance of α -tocopherol.

In detail, the blood to be tested is usually venous blood taken from a human body.

Step 104: and detecting a certain amount of samples to be detected by using a high performance liquid chromatograph under the same detection condition to obtain the chromatogram of the samples to be detected.

And 105, calculating the contents of retinol and α -tocopherol in the blood sample according to the chromatogram of the sample to be detected and each standard curve equation obtained by fitting.

Researches show that the time for separating impurities by using a C8 column and other types of C18 columns is longer, and a Poroshell120SB-C18 chromatographic column which is a core-shell chromatographic column has high separation efficiency and low back pressure and is beneficial to shortening the analysis time. In addition, a Poroshell120SB-C18 column having a size of 3.0 mm. times.50 mm 2.7 μm is preferable in combination with the flow rate, separation effect, analysis time and the like.

In the embodiment of the invention, based on the liquid phase condition, the analysis time can be shortened on the premise of ensuring the separation effect, and the method is particularly suitable for the condition of more blood samples and is beneficial to the rapid and accurate detection of a large number of blood samples.

Preferably, the column temperature is 35 ℃, the sample size is 5 μ L, and the flow rate is 0.8 mL/min.

In the embodiment of the invention, the high performance liquid chromatography is selected to detect the contents of retinol and α -tocopherol in blood, compared with the high performance liquid mass spectrometry, the detection instrument used by the high performance liquid chromatography is a high performance liquid chromatograph, the cost investment of the instrument is greatly reduced, the popularization rate of the instrument is higher, and the detection method is easier to standardize.

In the embodiment of the invention, a high performance liquid chromatography method is used for content detection, the blood consumption can be as low as 2mL, and the method is particularly suitable for people with small blood sampling difficulty, such as most adults. Considering that the number of adults is large, and the analysis time used by the embodiment of the invention is shorter, the application prospect of the embodiment of the invention has certain advantages.

Referring to fig. 2 and 3, fig. 2 shows the chemical structural formula of retinol, and fig. 3 shows the chemical structural formula of α -tocopherol.

Usually, at least three coordinate points are required for establishing the standard curve equation so as to ensure the accuracy of the established equation, so at least three standard solutions are required to be prepared in advance, so that the standard curve equation of retinol and the standard curve equation of α -tocopherol can be fitted according to the chromatogram obtained by detecting each standard solution.

In detail, taking retinol as an example, the standard curve equation of retinol obtained by fitting may be generally that y is k × x + b, wherein, the two variables x and y may be the peak area ratio of the standard product of retinol and the corresponding internal standard substance in the chromatogram of each standard solution, and the concentration ratio of the standard product of retinol and the corresponding internal standard substance in each standard solution.

Preferably, the internal standard for retinol is retinol acetate, α -internal standard for tocopherol, α -tocopheryl acetate.

Generally, after at least 2mL of blood to be detected is taken, the blood to be detected is processed, for example, centrifuged at 3500rpm for 10min, and the supernatant is taken to obtain serum or plasma, thus obtaining the blood sample. Serum or plasma samples were stored frozen at-20 ℃ until needed for analysis.

After the blood sample is obtained, pretreatment can be carried out to obtain a corresponding sample to be detected which can be directly loaded. In one embodiment of the present invention, the implementation process of sample preprocessing may include:

(1) transferring 10 mu L of mixed internal standard working solution into a 1.5mL centrifuge tube by using a liquid transfer gun, then adding 50-200 mu L of blood sample, adding a certain amount of diluent, and carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500-2000 rpm;

(2) adding a certain amount of protein precipitation reagent, and carrying out vortex mixing for 0.5-2min at the rotating speed of 1500-;

(3) adding a certain amount of extractant, carrying out vortex mixing for 3-10min at the rotating speed of 1500-2000rpm, and then carrying out high-speed centrifugation for 8-12min at the rotating speed of 10000-15000 rpm;

(4) transferring a certain amount of centrifuged supernatant into a clean 1.5mL centrifuge tube, transferring the centrifuge tube containing the supernatant into a nitrogen blow-drying device, and blow-drying the supernatant;

(5) transferring a certain amount of redissolution to a 1.5mL centrifuge tube for blowing the supernatant, carrying out vortex mixing for 0.5-1.5min at the rotating speed of 1500 plus 2000rpm, then carrying out high-speed centrifugation for 4-6min at the rotating speed of 10000 plus 15000rpm, and transferring the supernatant, namely the sample to be detected. For example, 80. mu.L of the supernatant can be removed as a sample to be tested.

Preferably, in the sample pretreatment, the diluent is pure water, and the amount of the diluent is 50-200 μ L; the precipitated protein reagent is absolute ethyl alcohol, and the amount of the protein precipitant is 100-; the extraction reagent is n-hexane, and the amount of the extraction reagent is 300-800 mu L; the compound solution is methanol, and the amount of the compound solution is 50-200 μ L.

Considering the strong specificity and high sensitivity of the fluorescence detector in the hplc-coupled detector, in one embodiment of the present invention, the detector switching method of the fluorescence detector in the hplc is shown in table 1 below.

TABLE 1

In the embodiment of the invention, the excitation wavelength corresponding to retinol is 325nm, and the emission wavelength is 470 nm; the excitation wavelength corresponding to the alpha-tocopherol is 295nm, and the emission wavelength is 330 nm. In the embodiment of the invention, the maximum absorption wavelength is selected, and the sensitivity is highest at the wavelength.

In detail, retinol and α -tocopherol can be eluted generally with equal elution with 85% or less of the organic phase, with good separation from interfering substances, but with greatly extended retention time of the target, resulting in extended analysis time.

Based on this, considering the separation degree of the target and the interferent and the shortest possible analysis time, in one embodiment of the present invention, preferably, the liquid phase conditions include: the elution mode is gradient elution; the elution process is shown in Table 2 below.

Based on the elution mode, the separation degree of the target object and the interfering object can be ensured, the analysis time is shortened, and meanwhile, the procedures of washing the chromatographic column and balancing the chromatographic column are added, so that the interference of strongly-retained substances is reduced, the consistency of the sample injection states of the chromatographic columns of a plurality of samples in front and back is ensured, the sample detection accuracy is higher, and the reproducibility is better.

TABLE 2

Time (min)	Methanol (%)	Water (%)
			0	92	8
0.2	92	8
			0.3	99	1
1.9	99	1
			2	92	8
3	92	8

In addition, in the embodiment of the invention, the conversion speed of the elution gradient is slow, so that the influence on the detection stability and the detection accuracy caused by the excessively fast conversion speed when the flow speed is high can be avoided.

In one embodiment of the present invention, before the separately detecting at least three standard solutions, further comprising:

preparing at least three standard working solutions, wherein the standard working solutions contain a known concentration of a retinol standard and a α -tocopherol standard, the concentration of the retinol standard is 0.0625-4.00mg/L, and the concentration of the α -tocopherol standard is 0.625-40.00 mg/L;

using a pipettor to transfer 90 mu L of standard working solution and 10 mu L of the mixed internal standard working solution into a centrifuge tube;

uniformly mixing the centrifugal tube at the rotating speed of 1500-2000rpm for 0.5-2min in a vortex manner, and then transferring supernatant to obtain a standard solution;

wherein, in the at least three standard working solutions, the concentration of the retinol standard substance is at least three of 0.0625mg/L, 0.125mg/L, 0.25mg/L, 0.50mg/L, 1.00mg/L, 2.00mg/L and 4.00mg/L, and the concentration of the α -tocopherol standard substance is at least three of 0.625mg/L, 1.25mg/L, 2.50mg/L, 5.00mg/L, 10.00mg/L, 20.00mg/L and 40.00 mg/L.

In detail, a linear range can be set by combining the population to be tested, the amount of blood to be tested, and the approximate content range of retinol and α -tocopherol in the human body, so as to ensure that most of the test results of the clinical samples fall within a reportable range.

Preferably, the number of standard working fluids is 7.

In summary, the method for simultaneously detecting the contents of retinol and α -tocopherol in blood provided by the embodiment of the invention combines the internal standard method and the high performance liquid chromatography, so that the interference factors are greatly reduced, the specificity is strong, the sensitivity is high, the detection result is accurate, and the analysis time is shortened.

The present invention will be described in detail below by way of examples, but the present invention is not limited to the following examples.