阅读说明：本技术 一种人体内血液中dha浓度的检测方法 (Method for detecting concentration of DHA in blood of human body ) 是由 蒙晋宁 邹皓月 王贯宇 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种人体内血液中DHA浓度的检测方法,包括如下步骤：S1、血液样品前处理；S2、标准储备液和标准溶液的配制；S3、LC-MS/MS分析检测,步骤S1的具体操作方法为：准确称取0.3g血液,置于10mL离心管中,依次加入40μL质量浓度为40μg/mL的C23∶0内标液、1.5mL蒸馏水、400μL含有0.05％抗氧化剂(BHT)的甲醇溶液、2mL氯仿,振荡均匀,以2500r/min的转速离心10min,取氯仿层,重复以上提取过程,合并氯仿层至20mL顶空进样瓶中,氮气吹干备用。本发明采用杂环衍生化结合下对DHA进行分析,并将该方法应用到人血液中DHA的检测,采用内标法定量,而不是归一化法确定其相对含量,该方法操作简便,准确度、灵敏度、精密度均达到满意效果,实用性高,适用于人血液中DHA含量的分析检测。(The invention discloses a method for detecting the concentration of DHA in blood of a human body, which comprises the following steps: s1, preprocessing a blood sample; s2, preparing a standard stock solution and a standard solution; s3, LC-MS/MS analysis and detection, wherein the specific operation method in the step S1 is as follows: accurately weighing 0.3g of blood, placing the blood into a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layers to a 20mL headspace sampling bottle, and blowing the headspace sampling bottle with nitrogen for later use. The invention adopts the heterocyclic derivatization to analyze the DHA under the combination, applies the method to the detection of the DHA in human blood, adopts the internal standard method for quantification instead of the normalization method to determine the relative content of the DHA, has simple and convenient operation, achieves satisfactory effects on accuracy, sensitivity and precision, has high practicability, and is suitable for the analysis and detection of the DHA content in the human blood.)

1. A method for detecting the concentration of DHA in blood of a human body is characterized by comprising the following steps: the method comprises the following steps:

s1, preprocessing a blood sample;

s2, preparing a standard stock solution and a standard solution;

and S3, and LC-MS/MS analysis and detection.

2. The method for detecting the concentration of DHA in human blood according to claim 1, wherein: the specific operation method of step S1 is as follows: accurately weighing 0.3g of blood, placing the blood in a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layer to a 20mL headspace sample feeding bottle, blowing the chloroform layer to dry by nitrogen for later use, adding 200 μ LAMP into the blow-dried headspace sample feeding bottle under the protection of nitrogen, filling the blow-dried headspace sample feeding bottle with nitrogen, sealing, reacting in a constant-temperature oil bath at 210 ℃ for 75min, cooling to room temperature, adding methanol to fix the volume to 1mL, filtering the sample by a 0.22 μm filter membrane, and analyzing and detecting by LC-MS/MS.

3. The method for detecting the concentration of DHA in human blood according to claim 1, wherein: the preparation method of the standard stock solution in the step S2 comprises the following steps: taking 12.5mg (accurate to 0.1mg) of DHA standard substance into a 50mL brown volumetric flask, adding BHT25mg, dissolving with methanol and fixing the volume to the scale, preparing 250 mug/mL standard stock solution, and placing in a refrigerator at-20 ℃ for later use.

4. The method for detecting the concentration of DHA in human blood according to claim 1, wherein: the preparation method of the standard solution in the step S2 comprises the following steps: taking 7, 10, 50, 100, 300, 600 and 1000 mu L of standard stock solutions respectively, diluting to 25mL with methanol, adding 40 mu L of C23: 0 internal standard solution with the mass concentration of 40 mu g/mL respectively to prepare 0.07, 0.1, 0.5, 1, 3, 6 and 10 mu g/mL series of standard solutions.

5. The method for detecting the concentration of DHA in human blood according to claim 1, wherein: the LC-MS chromatographic conditions in the step S3 are as follows: liquid chromatography column: varian Polarisc 18-A100X 2.1mm, 5 μm; column temperature: room temperature; mobile phase: methanol A and water B; gradient conditions: 50% A-90% A at 0-3min, 90% A at 3-6min, 50% A at 6-12 min; flow rate: 0.2-0.5 mL/min.

6. The method for detecting the concentration of DHA in human blood according to claim 1, wherein: the MS chromatographic conditions in the step S3 are as follows: spray voltage 3500V, sheath gas 40arb, auxiliary gas 7arb, capillary temperature 330 ℃, scanning window width: q10.7 and Q30.7.

Technical Field

The invention relates to the technical field of unsaturated fatty acid detection, in particular to a method for detecting the concentration of DHA in human blood.

Background

Docosahexaenoic acid (DHA), commonly known as NAOHUANGJIN, belongs to n-3 series polyunsaturated fatty acid, is the main lipid component in human brain nerve cell membrane, and can promote infantile central nervous system development and protect eyesight. DHA also has effects of preventing cardiovascular disease and cerebrovascular disease, inhibiting tumor growth, resisting inflammation, and inhibiting anaphylaxis. Epidemiological investigation shows that newborns, infants, pregnant women and old people are the most prone to DHA deficiency in the population, but the more DHA is ingested, the better, the bleeding tendency is generated by the ingestion of a large amount, and patients with blood coagulation dysfunction, severe hypertension and autoimmune diseases must take care. Therefore, the establishment of a rapid and accurate detection method for determining the DHA content in human blood has certain practical significance. In the literature, thin-layer chromatography is adopted to analyze DHA, most of the methods use gas chromatography or gas chromatography-mass spectrometry combined technology, because DHA has strong polarity and low volatility and stability, the gas chromatography detection is carried out after derivatization treatment, most of the derivatization methods are methyl ester derivatization methods, which can be divided into two categories, namely acid catalysis and alkali catalysis, and related researches find that acid catalysis is more suitable for esterifying free fatty acids such as DHA than alkali catalysis, and the acid catalysis mainly adopts a hydrogen chloride-methanol esterification method, a sulfuric acid-methanol esterification method and a boron trifluoride-diethyl ether-methanol esterification method, wherein the adsorption concentration of hydrogen chloride-methanol in the hydrogen chloride-methanol esterification method is difficult to control, and the measurement accuracy can be influenced; the DHA esterification rate in the sulfuric acid-methanol esterification method is not high enough; in addition, the position of double bonds of unsaturated fatty acid methyl ester is difficult to correctly position under electron bombardment, and therefore, the method for detecting the concentration of DHA in blood in a human body is provided.

Disclosure of Invention

The present invention aims to provide a method for detecting the concentration of DHA in blood of a human body to solve the above problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting the concentration of DHA in blood of a human body comprises the following steps:

s1, preprocessing a blood sample;

s2, preparing a standard stock solution and a standard solution;

and S3, and LC-MS/MS analysis and detection.

Preferably, the specific operation method of step S1 is: accurately weighing 0.3g of blood, placing the blood in a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layer to a 20mL headspace sample feeding bottle, blowing the chloroform layer to dry by nitrogen for later use, adding 200 μ LAMP into the blow-dried headspace sample feeding bottle under the protection of nitrogen, filling the blow-dried headspace sample feeding bottle with nitrogen, sealing, reacting in a constant-temperature oil bath at 210 ℃ for 75min, cooling to room temperature, adding methanol to fix the volume to 1mL, filtering the sample by a 0.22 μm filter membrane, and analyzing and detecting by LC-MS/MS.

Preferably, the preparation method of the standard stock solution in step S2 is as follows: taking 12.5mg (accurate to 0.1mg) of DHA standard substance into a 50mL brown volumetric flask, adding BHT25mg, dissolving with methanol and fixing the volume to the scale, preparing 250 mug/mL standard stock solution, and placing in a refrigerator at-20 ℃ for later use.

Preferably, the preparation method of the standard solution in step S2 is as follows: taking 7, 10, 50, 100, 300, 600 and 1000 mu L of standard stock solutions respectively, diluting to 25mL with methanol, adding 40 mu L of C23: 0 internal standard solution with the mass concentration of 40 mu g/mL respectively to prepare 0.07, 0.1, 0.5, 1, 3, 6 and 10 mu g/mL series of standard solutions.

Preferably, the LC-MS chromatographic conditions in step S3 are: liquid chromatography column: varian Polarisc 18-A100X 2.1mm, 5 μm; column temperature: room temperature; mobile phase: methanol A and water B; gradient conditions: 50% A-90% A at 0-3min, 90% A at 3-6min, 50% A at 6-12 min; flow rate: 0.2-0.5 mL/min.

Preferably, the MS chromatographic conditions in step S3 are: spray voltage 3500V, sheath gas 40arb, auxiliary gas 7arb, capillary temperature 330 ℃, scanning window width: q10.7 and Q30.7.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, DHA is analyzed under the combination of heterocyclic derivatization, the method is applied to the detection of DHA in human blood, the internal standard method is adopted for quantification, and the relative content of DHA in human blood is determined by a normalization method, so that the method is simple and convenient to operate, and the accuracy, sensitivity and precision of DHA in human blood can reach satisfactory effects, and the method is suitable for the analysis and detection of DHA content in human blood.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a method for detecting the concentration of DHA in blood of a human body comprises the following steps:

s1, preprocessing a blood sample;

s2, preparing a standard stock solution and a standard solution;

and S3, and LC-MS/MS analysis and detection.

The first embodiment is as follows:

s1, pretreatment of the blood sample:

(1) weighing 0.3g of blood, placing the blood in a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layers to a 20mL headspace sample feeding bottle, and drying the chloroform layer by nitrogen for later use;

(2) adding 200 mu LAMP into the blow-dried headspace sample injection bottle under the protection of nitrogen, sealing after being filled with nitrogen, reacting in a constant-temperature oil bath at 180 ℃ for 120min, cooling to room temperature, adding methanol to fix the volume to 1mL, filtering a sample through a 0.22 mu m filter membrane, and analyzing and detecting by LC-MS/MS.

S2, preparation of standard stock solution and standard solution:

(1) taking 12.5mg (accurate to 0.1mg) of DHA standard substance into a 50mL brown volumetric flask, adding BHT25mg, dissolving with methanol, metering to a certain volume to obtain a standard stock solution of 250 mug/mL, and placing in a refrigerator at-20 ℃ for later use;

(2) taking 7, 10, 50, 100, 300, 600 and 1000 mu L of standard stock solution, diluting to 25mL with methanol, adding 40 mu L of C23: 0 internal standard solution with the mass concentration of 40 mu g/mL respectively to prepare 0.07, 0.1, 0.5, 1, 3, 6 and 10 mu g/mL series of standard solutions.

S3, LC-MS/MS analysis and detection:

(1) the LC-MS chromatographic conditions are as follows: liquid chromatography column: varian Polarisc 18-A100X 2.1mm, 5 μm; column temperature: room temperature; mobile phase: methanol A and water B; gradient conditions: 50% A-90% A at 0-3min, 90% A at 3-6min, 50% A at 6-12 min; flow rate: 0.2-0.5 mL/min;

(2) the MS chromatographic conditions are as follows: spray voltage 3500V, sheath gas 40arb, auxiliary gas 7arb, capillary temperature 330 ℃, scanning window width: q10.7 and Q30.7.

Example two:

s1, pretreatment of the blood sample:

(1) weighing 0.3g of blood, placing the blood in a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layers to a 20mL headspace sample feeding bottle, and drying the chloroform layer by nitrogen for later use;

(2) adding 200 mu LAMP into the blow-dried headspace sample injection bottle under the protection of nitrogen, sealing after being filled with nitrogen, reacting in a constant-temperature oil bath at 180 ℃ for 100min, cooling to room temperature, adding methanol to fix the volume to 1mL, filtering a sample through a 0.22 mu m filter membrane, and analyzing and detecting by LC-MS/MS.

S2, preparation of standard stock solution and standard solution:

(1) taking 12.5mg (accurate to 0.1mg) of DHA standard substance into a 50mL brown volumetric flask, adding BHT25mg, dissolving with methanol, metering to a certain volume to obtain a standard stock solution of 250 mug/mL, and placing in a refrigerator at-20 ℃ for later use;

(2) taking 7, 10, 50, 100, 300, 600 and 1000 mu L of standard stock solution, diluting to 25mL with methanol, adding 40 mu L of C23: 0 internal standard solution with the mass concentration of 40 mu g/mL respectively to prepare 0.07, 0.1, 0.5, 1, 3, 6 and 10 mu g/mL series of standard solutions.

S3, LC-MS/MS analysis and detection:

(1) the LC-MS chromatographic conditions are as follows: liquid chromatography column: varian Polarisc 18-A100X 2.1mm, 5 μm; column temperature: room temperature; mobile phase: methanol A and water B; gradient conditions: 50% A-90% A at 0-3min, 90% A at 3-6min, 50% A at 6-12 min; flow rate: 0.2-0.5 mL/min;

(2) the MS chromatographic conditions are as follows: spray voltage 3500V, sheath gas 40arb, auxiliary gas 7arb, capillary temperature 330 ℃, scanning window width: q10.7 and Q30.7.

Example three:

s1, pretreatment of the blood sample:

(1) weighing 0.3g of blood, placing the blood in a10 mL centrifuge tube, sequentially adding 40 μ L of C23: 0 internal standard solution with the mass concentration of 40 μ g/mL, 1.5mL of distilled water, 400 μ L of methanol solution containing 0.05% of antioxidant (BHT) and 2mL of chloroform, oscillating uniformly, centrifuging at the rotating speed of 2500r/min for 10min, taking a chloroform layer, repeating the extraction process, combining the chloroform layers to a 20mL headspace sample feeding bottle, and drying the chloroform layer by nitrogen for later use;

(2) adding 200 mu LAMP into the blow-dried headspace sample injection bottle under the protection of nitrogen, sealing after being filled with nitrogen, reacting in a constant-temperature oil bath at 210 ℃ for 75min, cooling to room temperature, adding methanol to reach a constant volume of 1mL, filtering a sample through a 0.22 mu m filter membrane, and analyzing and detecting by LC-MS/MS.

S2, preparation of standard stock solution and standard solution:

(1) taking 12.5mg (accurate to 0.1mg) of DHA standard substance into a 50mL brown volumetric flask, adding BHT25mg, dissolving with methanol, metering to a certain volume to obtain a standard stock solution of 250 mug/mL, and placing in a refrigerator at-20 ℃ for later use;

(2) taking 7, 10, 50, 100, 300, 600 and 1000 mu L of standard stock solution, diluting to 25mL with methanol, adding 40 mu L of C23: 0 internal standard solution with the mass concentration of 40 mu g/mL respectively to prepare 0.07, 0.1, 0.5, 1, 3, 6 and 10 mu g/mL series of standard solutions.

S3, LC-MS/MS analysis and detection:

(1) the LC-MS chromatographic conditions are as follows: liquid chromatography column: varian Polarisc 18-A100X 2.1mm, 5 μm; column temperature: room temperature; mobile phase: methanol A and water B; gradient conditions: 50% A-90% A at 0-3min, 90% A at 3-6min, 50% A at 6-12 min; flow rate: 0.2-0.5 mL/min;

(2) the MS chromatographic conditions are as follows: spray voltage 3500V, sheath gas 40arb, auxiliary gas 7arb, capillary temperature 330 ℃, scanning window width: q10.7 and Q30.7.

After the implementation operation of the invention, the data conclusion obtained is as follows:

	DHA content RSD (n ═ 6)	DHA concentration at room temperature	DHA concentration at-20 ℃
				Example one	3.22％	0.04％	0.08％
Example two	3.22％	2.87％	1.47％
				EXAMPLE III	3.22％	4.63％	2.42％

According to the invention, DHA is analyzed under the combination of heterocyclic derivatization, the method is applied to the detection of DHA in human blood, the internal standard method is adopted for quantification, and the relative content of DHA in human blood is determined by a normalization method, so that the method is simple and convenient to operate, and the accuracy, sensitivity and precision of DHA in human blood can reach satisfactory effects, and the method is suitable for the analysis and detection of DHA content in human blood.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.