CN105136928A - Method for fast detecting whole red blood cell octadecadienoic acid - Google Patents

Abstract

The invention provides a method for rapidly detecting octadecadenoic acid in whole blood erythrocytes, belonging to the technical field of biological detection, comprising the following steps: A) pretreatment of whole blood samples; B) using gas chromatography- mass spectrometer for detection. The detection method provided by the invention has the advantages of less sample consumption, simple pretreatment, high sensitivity, strong specificity, powerful qualitative and quantitative functions, short time in the whole detection process, and high detection throughput.

Description

A method for rapid detection of octadecadienoic acid in whole blood erythrocytes

technical field

The invention belongs to the technical field of biological detection, and relates to a method for rapidly detecting octadecanedienoic acid in whole blood red blood cells.

Background technique

In the human body, a small amount of fatty acids exist in free form, and most of them exist in bound form, such as triglycerides, phospholipids, glycolipids, etc. From a nutritional point of view, fatty acids can be divided into essential fatty acids and non-essential fatty acids. Non-essential fatty acids refer to fatty acids that can be synthesized by the body without relying on food supply; The synthesis rate is slow, unable to meet the needs of the body, and must be supplied by food. Essential fatty acids can be divided into omega (Omega)-6 series fatty acids and omega (Omega)-3 series fatty acids. Dietary deficiencies in essential fatty acids can lead to skin lesions, neurological and visual disorders, heart disease and other problems.

Since the composition of fatty acids on red blood cell membranes can reflect the long-term dietary structure of the population, it will not be affected by short-term diet. Therefore, the determination of human whole blood erythrocyte fatty acids can accurately and objectively reflect the nutritional levels of different fatty acids in the body.

There are many reports on the detection of fatty acids at home and abroad, involving food, environment, medicine and health, clinical applications and other fields. The detection methods include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography. Chromatography-tandem mass spectrometry (LC-MS/MS), the type of detection sample is related to food, water quality, medicine, blood, tissue, etc. However, different application fields and different sample types involve different sample processing and fatty acid detection and analysis procedures, so there is no comparison between fatty acid detection methods in different fields and different sample types.

At present, there are three main methods for the detection of fatty acids in whole blood red blood cells: GC, GC-MS and LC-MS/MS. GC is currently a common method for the detection of red blood cell fatty acids, but the detector of this method cannot achieve accurate qualitative separation and quantitative analysis of similar fatty acids that have not been chromatographically separated, so it cannot perform specific and accurate qualitative and quantitative analysis of various fatty acids. The pretreatment of LC-MS/MS method is relatively complicated, and the reproducibility of analysis results is poor. The GC-MS method can solve the disadvantages of GC and LC-MS/MS because it has the separation function of the gas phase and the specific qualitative and quantitative functions of the mass spectrometer. The amount is lower.

Chinese patent 201210247480.2 discloses a method for detecting fatty acid content by gas chromatography-mass spectrometry. The sample used in this method is animal tissue. The pretreatment of the sample includes a saponification step, and nitrogen protection is also required. The pretreatment of the sample is complicated.

Therefore, those skilled in the art urgently need a method for detecting fatty acids in whole blood red blood cells that is simple in pretreatment, fast in detection, high in sensitivity and strong in specificity.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a method for rapidly detecting octadecadenoic acid in whole blood erythrocytes. The method requires less sample consumption, simple pretreatment, high sensitivity, strong specificity, qualitative, The quantitative function is powerful, and the whole detection process is short and the detection throughput is high. The present invention only relates to the detection of erythrocyte octadecadienoic acid in whole blood samples.

A method for rapidly detecting octadecadienoic acid in whole blood erythrocytes, comprising the steps of:

A) Pretreatment of whole blood samples: separate erythrocytes from whole blood samples, add internal standard solution and hydrochloric acid-methanol solution for derivatization reaction, then add extractant for extraction, take supernatant and blow dry with nitrogen, add complex to the residue Solvent, mixed, to obtain the sample detection solution;

B) The sample detection solution is detected by gas chromatography-mass spectrometer. The gas chromatography conditions include: the chromatographic column adopts a polar chromatographic column, the carrier gas is helium, splitless injection, linear velocity flow control mode, carrier gas flow rate The temperature is 0.80~1.00mL/min, the initial column temperature is 60°C, the temperature is first raised to 180°C, and then the temperature is raised to 220°C, the gradient time of the whole temperature rise program is 8~15min; the mass spectrometry conditions are: electron bombardment ion source, Scanning and Selected Ion Scanning Mode.

Adopting the above-mentioned technical scheme, the pretreatment is simple, and through the process of extraction-drying-reconstitution, the interfering substances are effectively removed, and the octadecadienoic acid in red blood cells is converted into a highly volatile product suitable for detection by gas chromatography-mass spectrometers. Octadecanadienoic acid methyl ester product; the detection method has high sensitivity, strong specificity, strong qualitative and quantitative functions, good accuracy, and the whole detection process is short in time and high in detection throughput.

Preferably, the internal standard solution is selected from heptadecanoic acid. In addition to heptadecanoic acid, pentadecanoic acid or nonadecanoic acid can also be used as the internal standard solution in the present invention.

Preferably, the extractant is selected from n-hexane, and the reconstituted solvent is selected from n-hexane. Using n-hexane as the extractant, the methyl esterification products of various fatty acids can be extracted, the extraction efficiency is high and the interference of other substances is effectively avoided.

Preferably, the volume ratio of the internal standard solution to red blood cells is 1:(1~2); the volume ratio of the red blood cells, hydrochloric acid-methanol solution, extractant and reconstitution solvent is 1:(10~25):(20 ~50): (4~10).

Preferably, the volume ratio of the red blood cells, hydrochloric acid-methanol solution, extraction agent and reconstitution agent is 1:20:40:8.

Preferably, the polar chromatographic column is selected from polar capillary chromatographic columns with specifications of 20m*0.18mm*0.20μm.

Preferably, the gas chromatography conditions further include: the injection volume is 1 μL, the inlet temperature is 220-230°C, the post-run temperature is 240-250°C, the post-run time is 2min, and the purge flow rate is 3.0-5.0mL /min, the total flow rate is 50.0~60.0mL/min, and the average linear velocity is 35.0~45.0cm/s.

Preferably, the mass spectrometry conditions also include: the interface temperature is 210~230°C; the ion source temperature is 220~240°C; the quadrupole temperature is 140~160°C, the solvent delay time is 3.0~4.0min, and the full scan range is m/z60.00~450.00.

Preferably, the detection method further includes the following steps: qualitative judgment and/or quantitative calculation of the detection results.

Compared with the prior art, the beneficial effects of the present invention are: the detection method provided by the present invention can realize the qualitative and quantitative detection of octadecadienoic acid in the red blood cells of the whole blood sample, has the advantages of simple pretreatment, effectively removes interfering substances, High sensitivity, strong specificity, powerful qualitative and quantitative functions, good accuracy, and the whole detection process is short in time and high in detection throughput.

The present invention adopts the scanning mode of full scan and selected ion scan at the same time, and can obtain the total ion current chromatogram and selected ion chromatogram of the sample, and the total ion current chromatogram can be used to characterize the compound and to monitor the Whether the compound at the peak time is the target compound, so as to avoid other interferences. In addition, the selected ion scan scans the characteristic ions of each compound, which solves the problem of inaccurate quantification of fatty acids using gas chromatography alone, and further improves the detection accuracy and specificity of the method. The polar chromatographic column used in the present invention is suitable for the separation and analysis of octadecanedienoic acid. The high column efficiency and low loss of the chromatographic column greatly reduce the background interference during mass spectrometry detection, and the minimum detection limit reaches 0.01mg/L. high sensitivity. According to the detection method provided by the invention, the detection time of a single sample is 12 minutes, and compared with the existing traditional method, the detection time is short and the detection throughput of the instrument is high.

Description of drawings

Figure 1 Standard curve of octadecadedienoic acid methyl ester.

Figure 2 Total ion chromatogram of fatty acid methyl esters in whole blood red blood cell samples.

Figure 3 Selected ion chromatograms of methyl octadecadienoate in red blood cell samples from whole blood.

Figure 4 Selected ion chromatogram of methyl heptadecanoate in whole blood red blood cell samples.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

The pretreatment of embodiment one whole blood sample

Mix the anticoagulated whole blood sample, centrifuge, discard the upper layer of plasma, white blood cells, platelets, etc.; add an equal volume of normal saline to the remaining red blood cells, mix and centrifuge, discard the upper layer of normal saline; then add the remaining red blood cells Add an equal volume of normal saline and mix well to obtain the separated red blood cell sample.

Take 50 μL red blood cell sample, put it in a glass centrifuge tube with screw cap, add heptadecanoic acid as an internal standard solution, add 1 mL of 3mol/L hydrochloric acid-methanol solution, mix well, put it in an oven, react at 90°C for 3 hours in the dark, and derivatize After the reaction was completed, cool to room temperature, add 2 mL of n-hexane to extract the methyl esterification reaction product, take the supernatant and dry it with nitrogen, then add n-hexane to the residue as a reconstitution solvent, mix well, and obtain a sample detection solution. Among them, the volume ratio of heptadecanoic acid to red blood cell sample is 1:1; the volume ratio of red blood cell sample, hydrochloric acid-methanol solution, extraction agent and reconstitution solvent is 1:20:40:8.

Through the above pretreatment, fatty acids in red blood cells are converted into highly volatile fatty acid methyl ester products suitable for detection by gas chromatography-mass spectrometry, and extracted and separated, effectively avoiding the interference of other substances on subsequent detection.

Example 2 Detection of whole blood erythrocyte fatty acids

The sample detection solution is detected by gas chromatography-mass spectrometer.

The chromatographic conditions include: the chromatographic column is a polar capillary column with a specification of 20m*0.18mm (ID)*0.20μm (film) (purchased from Agilent, model DB-23, PartNumber122-2361), and the carrier gas is 99.999% pure Helium, the carrier gas flow rate is 0.90mL/min, the sampling method is splitless injection, the injection volume is 1 μL, the inlet temperature is 230°C, the post-run temperature is 240°C, the post-run time is 2min, and the purge The flow rate is 4.0mL/min, and the total flow rate is 55mL/min. The temperature programming conditions are shown in Table 1.

Mass spectrometry detection adopts electron bombardment ionization mode, and adopts full scan and selected ion scan methods. Mass spectrometry conditions include: ionization source is electron bombardment ion source (EI), 70ev; transfer line temperature is 230°C, ion source temperature is 250°C, quadrupole mass analyzer temperature is 150°C, solvent delay time is 3.70min; scan mode It is a full scan (Scan) and a selected ion scan (SIM), the full scan range is m/z60.00~450.00, and the parameters of the selected ion scan (SIM) mode are shown in Table 2.

Qualitative and quantitative detection of fatty acids in whole blood red blood cells: the retention time of each fatty acid in the sample and the methyl ester product of the standard, the mass spectrum obtained from the full scan, the NIST spectral library search and the comparison with the mass spectrum of the standard substance as the qualitative basis , the retention times of each fatty acid methyl ester are shown in Table 3. If the deviation between the relative abundance ratio of the qualitative and quantitative ions selected in the sample chromatogram and the ion relative abundance ratio of the standard solution does not exceed the preset specified range, it can be determined that there is a corresponding target substance in the sample. Quantitative with internal standard standard curve method, the ratio of the peak area of octadecadienoic acid methyl ester and corresponding internal standard methyl ester peak area is ordinate, the concentration ratio of octadecadienoic acid and internal standard is The abscissa draws a standard curve, the results are shown in Figure 1, and the content of octadecadienoic acid in human erythrocytes is calculated.

Using the above method, the fatty acids in a sample of whole blood red blood cells were detected, and the respective contents of 20 fatty acids were obtained. The total ion chromatogram of fatty acid methyl esters is shown in Fig. 2 . It can be seen from Figure 2 that the peak shapes of fatty acid methyl esters are symmetrical, there is no tailing phenomenon, and the separation effect is very good. In the whole blood red blood cell sample, the selected ion chromatogram of octadecadienoic acid methyl ester is shown in Figure 3, and the selected ion chromatogram of heptadecanoic acid methyl ester is shown in Figure 4.

The minimum detection limit of the detection method of the present invention reaches 0.01 mg/L, and it is obtained that the 20 kinds of fatty acids in the human red blood cell sample are within the determined concentration range, and the linearity is good, and the coefficient of determination R2 is between ^0.996-0.999 .

Reproducibility experiment: 5 whole blood samples from the same source were taken and subjected to the same pretreatment and detection. As a result, the RSDs of the contents of 20 fatty acids were all between 3% and 8%, indicating that the method had good repeatability.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (9)

1. detect a method for whole blood red blood cell octadecane dienoic acid fast, it is characterized in that: comprise the steps:

A) pre-treatment of whole blood sample: separating red corpuscle from whole blood sample, adds inner mark solution and hydrochloric acid-methanol solution carries out derivative reaction, then adds extractant extraction, get supernatant and nitrogen dries up, add double solvents in residue, mixing, obtains sample detection liquid;

B) gas chromatograph-mass spectrometer (GCMS) is adopted to detect to described sample detection liquid, GC conditions comprises: chromatographic column adopts polarity chromatographic column, carrier gas is helium, Splitless injecting samples, linear velocity flow control mode, carrier gas flux is 0.80 ~ 1.00mL/min, initial column temperature 60 DEG C, first be warming up to 180 DEG C, be then warming up to 220 DEG C, the gradient timetable of whole heating schedule is 8 ~ 15min; Mass Spectrometry Conditions is: adopt electron impact ion source, adopts full scan and Selective ion mode scan mode.

2. the method for quick detection whole blood red blood cell octadecane dienoic acid according to claim 1, is characterized in that: described inner mark solution is selected from Heptadecanoic acide.

3. the method for quick detection whole blood red blood cell octadecane dienoic acid according to claim 1, it is characterized in that: described extractant is selected from normal hexane, described double solvents is selected from normal hexane.

4. the method for quick detection whole blood red blood cell octadecane dienoic acid according to claim 3, is characterized in that: described inner mark solution and erythrocytic volume ratio are 1:(1 ~ 2); The volume ratio of described red blood cell, hydrochloric acid-methanol solution, extractant and double solvents is 1:(10 ~ 25): (20 ~ 50): (4 ~ 10).

5. the method for quick detection whole blood red blood cell octadecane dienoic acid according to claim 4, is characterized in that: the volume ratio of described red blood cell, hydrochloric acid-methanol solution, extractant and double solvents is 1:20:40:8.

6. the method for quick detection whole blood red blood cell octadecane dienoic acid according to claim 1, is characterized in that: described polarity chromatographic column is selected from the polarity capillary chromatographic column that specification is 20m*0.18mm*0.20 μm.

7. the method for quick detection whole blood red blood cell octadecane dienoic acid according to any one of claim 1 to 6, it is characterized in that: described GC conditions also comprises: sampling volume is 1 μ L, injector temperature is 220 ~ 230 DEG C, rear running temperature is 240 ~ 250 DEG C, rear working time is 2min, purge flow rate is 3.0 ~ 5.0mL/min, and total flow is 50.0 ~ 60.0mL/min, and average linear velocity is 35.0 ~ 45.0cm/s.

8. the method for quick detection whole blood red blood cell octadecane dienoic acid according to any one of claim 1 to 6, is characterized in that: described Mass Spectrometry Conditions also comprises: interface temperature is 210 ~ 230 DEG C; Ion source temperature is 220 ~ 240 DEG C; Quadrupole rod temperature is 140 ~ 160 DEG C, and the solvent delay time is 3.0 ~ 4.0min, and full scan scope is m/z60.00 ~ 450.00.

9. the method for quick detection whole blood red blood cell octadecane dienoic acid according to any one of claim 1 to 6, is characterized in that: also comprise the steps: the qualitative judgement of testing result and/or quantitatively calculate.