CN105158390B - A kind of method of quick detection whole blood red blood cell lignocerane monoenoic acid - Google Patents
A kind of method of quick detection whole blood red blood cell lignocerane monoenoic acid Download PDFInfo
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- CN105158390B CN105158390B CN201510545239.1A CN201510545239A CN105158390B CN 105158390 B CN105158390 B CN 105158390B CN 201510545239 A CN201510545239 A CN 201510545239A CN 105158390 B CN105158390 B CN 105158390B
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Abstract
The present invention provides a kind of method of quick detection whole blood red blood cell lignocerane monoenoic acid, belongs to technical field of biological, comprises the following steps：A）The pre-treatment of whole blood sample；B）The sample detection liquid is detected using gas chromatography mass spectrometer.The advantages of detection method that the present invention is provided has amount of samples few, and pre-treatment is simple, and sensitivity is high, high specificity, and qualitative, quantitative function is powerful, and the whole detection process time is short, detection flux is high.
Description
Technical field
The invention belongs to technical field of biological, it is related to a kind of quick detection whole blood red blood cell lignocerane monoenoic acid
Method.
Background technology
In human body, a small amount of aliphatic acid exists in a free form, and major part is with combining form, such as triglycerides, phosphatide, sugar
Fat etc. is present.From nutrition angle, aliphatic acid can be divided into essential fatty acid and non-essential fatty acid, and non-essential fatty acid refers to machine
Body can voluntarily synthesize, it is not necessary to by the aliphatic acid of food supply；Essential fatty acid refers to that human body maintains body eubolism not
Can lack and itself can not synthesize or aggregate velocity is slow, it is impossible to meet body requirement, it is necessary to by the aliphatic acid of food supply.
Essential fatty acid can be divided into ω（Omega）- 6 series fatty acids and ω（Omega）- 3 series fatty acids.Required fat in meals
Acid heat will cause cutaneous lesions, nerve and the problems such as vision difficult disease, heart disease.
The chronic dietary structure of crowd can be reflected due to the composition of aliphatic acid on erythrocyte membrane, without by a short time
The influence of diet.Therefore, people's whole blood Erythrocyte Fatty Acids are determined, can accurately, objectively reflects different aliphatic acid in body
Trophic level.
It is abundant about the relevant report of fatyy acids both at home and abroad, it is related to food, environment, medical and health, clinical practice
Etc. multiple fields, detection method includes gas chromatography（GC）, gas chromatography-mass spectrography（GC-MS）With liquid chromatography-tandem matter
Spectrometry（LC-MS/MS）, detect that sample type is even more and be related to food, water quality, medicine, blood, tissue etc..But, different applications
Field and different sample types, involved sample treatment vary with fatyy acids analysis process, therefore different necks
Without comparativity between the fatyy acids method of domain and different sample types.
At present, the detection of whole blood Erythrocyte Fatty Acids mainly has tri- kinds of GC, GC-MS and LC-MS/MS.GC is red at present thin
The common method of born of the same parents' fatyy acids, but the detector of the method is to the similar aliphatic acid of unrealized chromatographic isolation, it is impossible to it is real
Now accurate qualitative separation and quantitative analysis, therefore cannot specific to various aliphatic acid, accurate qualitative and quantitative analysis.LC-
MS/MS method pre-treatments are complex, and reproducibility of analysis results is poor.And GC-MS methods are due to having dividing for gas phase simultaneously
From function and mass spectrographic specificity be qualitative, quantitative function, it is possible to the drawbacks of solving GC and LC-MS/MS, but it is existing
GC-MS method analysis times are more long, and detection flux is relatively low.
Chinese patent 201210247480.2 discloses the side that a kind of gas chromatography-mass spectrography detects content of fatty acid
Method, the sample that the method is directed to is animal tissue, and the pre-treatment of sample includes saponification step, in addition it is also necessary to nitrogen charging gas shielded, sample
Complex pretreatment.
Therefore, those skilled in the art are in the urgent need to a kind of pre-treatment is simple, detection is quick, method sensitivity is high, special
The strong detection method suitable for whole blood Erythrocyte Fatty Acids of property.
The content of the invention
In order to solve problems of the prior art, the present invention provides a kind of quick detection whole blood red blood cell lignocerane
The method of monoenoic acid, the amount of samples of the method is few, and pre-treatment is simple, and sensitivity is high, high specificity, and qualitative, quantitative function is strong
Greatly, and entirely the detection process time is short, detect that flux is high.Present invention relates solely to whole blood sample red blood cell lignocerane monoenoic acid
Detection.
A kind of method of quick detection whole blood red blood cell lignocerane monoenoic acid, comprises the following steps：
A）The pre-treatment of whole blood sample：The separating red corpuscle from whole blood sample, adds inner mark solution and hydrochloric acid-methanol solution
Reaction is performed the derivatization, extractant extraction is subsequently adding, supernatant and nitrogen drying is taken, double solvents is added in residue, mixed, obtained
To sample detection liquid；
B）GC conditions include to be detected using gas chromatograph-mass spectrometer to the sample detection liquid：Chromatographic column
Using polarity chromatographic column, carrier gas is helium, Splitless injecting samples, linear velocity flow control mode, carrier gas flux is 0.80 ~
1.00mL/min, 60 DEG C of initial column temperature, is first warming up to 180 DEG C, then heats to 220 DEG C, the gradient timetable of whole heating schedule
It is 8 ~ 15min；Mass Spectrometry Conditions are：Using electron impact ion source, using full scan and selection ion scan mode.
Using above-mentioned technical proposal, pre-treatment is simple, by the process of extraction-drying-redissolution, effectively eliminates chaff interference
Matter, makes the lignocerane monoenoic acid in red blood cell be converted into volatility is detected relatively by force, suitable for gas chromatography mass spectrometer 20
Four alkane monoenoic acid methyl ester products；The sensitivity of detection method is high, high specificity, and qualitative, quantitative function is powerful, and accuracy is good, and
And entirely the detection process time is short, detect that flux is high.
Preferably, the inner mark solution is selected from Heptadecanoic acide.Except Heptadecanoic acide, the outer present invention can also select pentadecanoic acid
Or nonadecylic acid is used as inner mark solution.
Preferably, the extractant is selected from n-hexane, and the double solvents is selected from n-hexane.Using n-hexane as extraction
Agent, can extract the esterification product of various aliphatic acid, and extraction efficiency is high and effectively prevent the interference of other materials.
Preferably, the inner mark solution and the volume ratio of red blood cell are 1：(1~2)；The red blood cell, hydrochloric acid-methanol are molten
The volume ratio of liquid, extractant and double solvents is 1：(10~25)：(20~50)：(4~10).
Preferably, the volume ratio of the red blood cell, hydrochloric acid-methanol solution, extractant and double solvents is 1：20：40：8.
Preferably, the polarity chromatographic column is selected from the polarity capillary chromatographic column that specification is for 20m*0.18mm*0.20 μm.
Preferably, the GC conditions also include：Sampling volume is 1 μ L, and injector temperature is 220 ~ 230 DEG C, after
Running temperature be 240 ~ 250 DEG C, rear run time be 2min, purge flow rate be 3.0 ~ 5.0mL/min, total flow be 50.0 ~
60.0 mL/min, average linear velocity is 35.0 ~ 45.0cm/s.
Preferably, the Mass Spectrometry Conditions also include：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/z 60.00 ~ 450.00.
Preferably, the detection method also comprises the following steps：The qualitative judgement of testing result and/or quantitative calculating.
Compared with prior art, the beneficial effects of the invention are as follows：The detection method that the present invention is provided can realize whole blood
The qualitative and quantitative determination of lignocerane monoenoic acid in pinkish red cell, it is simple with pre-treatment, interfering material is effectively eliminated, spirit
Sensitivity is high, high specificity, and qualitative, quantitative function is powerful, and accuracy is good, and the whole detection process time is short, detect that flux is high.
The scan pattern that the present invention is carried out simultaneously using full scan and selection ion scan, can obtain total ion of sample
Flow chromatography figure and selection chromatography of ions figure, TIC can be used for carrying out compound it is qualitative, for detection process
Whether the compound at middle monitoring appearance time is target compound, in order to avoid there are other interference.Additionally, selection ion scan pair
The characteristic ion of each compound is scanned, solve exclusive use gas-chromatography aliphatic acid is carried out it is quantitatively not accurate enough
Problem, further increases the specificity of accuracy in detection and method.Polarity chromatographic column used in the present invention is adapted to 24
The separation analysis of alkane monoenoic acid, the Gao Zhuxiao and low-bleed of chromatographic column greatly reduce ambient interferences during Mass Spectrometer Method, most
Low detection limits have reached 0.01mg/L, and detection sensitivity is high.By the detection method that the present invention is provided, single sample detection time is
12min, compared with existing conventional method, detection time is short, and instrument detection flux is high.
Brief description of the drawings
The standard curve of Fig. 1 lignocerane monoenoic acid methyl esters.
The TIC of fatty acid methyl ester in Fig. 2 whole blood red blood cell samples.
The selection chromatography of ions figure of lignocerane monoenoic acid methyl esters in Fig. 3 whole blood red blood cell samples.
The selection chromatography of ions figure of methyl margarate in Fig. 4 whole blood red blood cell samples.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
The pre-treatment of the whole blood sample of embodiment one
Anticoagulated whole blood sample is mixed, centrifugation, reject upper plasma, leucocyte, blood platelet etc.；Add in remaining red blood cell
Enter isometric physiological saline, be centrifuged after mixing, the physiological saline on reject upper strata；It is isometric to being added in remaining red blood cell again
Physiological saline, the red blood cell sample of separator well is obtained after mixing.
50 μ L red blood cell samples are taken, is placed in screw-cap glass centrifuge tube, add Heptadecanoic acide as inner mark solution, plus
Enter the hydrochloric acid-methanol solution of 1mL 3mol/L, mix in rearmounted baking oven, 90 DEG C of lucifuges react 3h, cold after derivative reaction is finished
But to room temperature, 2mL n-hexane extraction esterification reaction of organic acid products are added, takes supernatant and carry out nitrogen drying, then in residue
Add n-hexane as double solvents, mix, obtain sample detection liquid.Wherein, Heptadecanoic acide is with the volume ratio of red blood cell sample
1：1；The volume ratio of red blood cell sample, hydrochloric acid-methanol solution, extractant and double solvents is 1：20：40：8.
By above-mentioned pre-treatment, by the aliphatic acid in red blood cell be converted into volatility it is relatively strong, suitable for gas chromatography-mass spectrum
The fatty acid methyl ester products of instrument detection, and extract and separate is out, effectively prevent interference of other materials to subsequent detection.
The detection of the whole blood Erythrocyte Fatty Acids of embodiment two
Sample detection liquid is detected using gas chromatograph-mass spectrometer.
Chromatographic condition includes：Chromatographic column is that specification is the polarity capillary of * 0.20 μm of 20m*0.18mm (ID) (film)
Chromatographic column（Purchased from Agilent, model DB-23, Part Number 122-2361）, carrier gas is the helium of 99.999% purity, is carried
Gas velocity is 0.90mL/min, and input mode is Splitless injecting samples, and sampling volume is 1 μ L, and injector temperature is 230 DEG C, is transported afterwards
Trip temperature is 240 DEG C, and rear run time is 2min, and purge flow rate is 4.0mL/min, and total flow is 55mL/min.Temperature programming
Condition is as shown in table 1.
Mass Spectrometer Method uses electron impact ionization pattern, using full scan and selection ion scan mode.Mass Spectrometry Conditions bag
Include：Ionization source is electron impact ion source（EI）, 70ev；Transmission line temperature is 230 DEG C, and ion source temperature is 250 DEG C, quadrupole rod
Mass analyzer temperature is 150 DEG C, and the solvent delay time is 3.70min；Scan pattern is full scan（Scan）With selection ion
Scanning（SIM）, full scan scope is m/z 60.00 ~ 450.00, selects ion scan（SIM）Mode parameter is as shown in table 2.
The qualitative and quantitative analysis of aliphatic acid in whole blood red blood cell：With each aliphatic acid in sample and the methyl ester product of standard items
Mass spectrogram obtained by retention time, full scan carries out NIST library searchings and compares conduct with the mass spectrogram of standard substance
Qualitative foundation, the retention time of each fatty acid methyl ester is as shown in table 3.The phase of the qualitative, quantitative ion selected in sample chromatogram figure
To the ion relative abundance of abundance ratio and standard liquid than deviation be no more than preset rules scope, then may determine that and deposit in sample
In corresponding target substance.Quantitative, peak area and the corresponding internal standard compound of lignocerane monoenoic acid methyl esters with Internal standard curve method
The ratio of methyl esters peak area is ordinate, and the concentration of lignocerane monoenoic acid is abscissa with the concentration proportion of internal standard compound, is drawn
Standard curve, as a result as shown in figure 1, calculating the content of HRBC's lignocerane monoenoic acid.
Using the above method, the aliphatic acid in 1 whole blood red blood cell sample is detected, drawn 20 kinds of aliphatic acid
Respective content, the TIC of fatty acid methyl ester is as shown in Figure 2.Figure it is seen that each fatty acid methyl ester peak type
Symmetrically, there is no conditions of streaking, separating effect is fine.In whole blood red blood cell sample, the selection ion of lignocerane monoenoic acid methyl esters
Chromatogram is as shown in figure 3, the selection chromatography of ions figure of methyl margarate is as shown in Figure 4.
Detection method minimum detectability of the invention has reached 0.01mg/L, draws 20 kinds of aliphatic acid in HRBC's sample
It is linear good in the concentration range for determining, coefficient of determination R2Between 0.996-0.999.
Repeated experiment：5 parts of the whole blood sample of same source is taken, identical pre-treatment and detection is carried out, as a result 20 kinds of fat
The RSD of fat acid content shows the reproducible of the method between 3% ~ 8%.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
Specific implementation of the invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should be all considered as belonging to of the invention
Protection domain.
Claims (3)
1. a kind of method of quick detection whole blood red blood cell lignocerane monoenoic acid, it is characterised in that：Comprise the following steps：
A) the pre-treatment of whole blood sample：The separating red corpuscle from whole blood sample, adds inner mark solution and hydrochloric acid-methanol solution to carry out
Derivative reaction, is subsequently adding extractant extraction, takes supernatant and nitrogen drying, and double solvents is added in residue, mixes, and obtains sample
Liquid is surveyed in product examine；
B GC conditions include) to be detected using gas chromatograph-mass spectrometer to the sample detection liquid：Chromatographic column is used
Polarity chromatographic column, carrier gas is helium, Splitless injecting samples, linear velocity flow control mode, and carrier gas flux is 0.80~1.00mL/
Min, 60 DEG C of initial column temperature, is first warming up to 180 DEG C, then heats to 220 DEG C, the gradient timetable of whole heating schedule for 8~
15min；Mass Spectrometry Conditions are：Using electron impact ion source, using full scan and selection ion scan mode；
The inner mark solution is selected from Heptadecanoic acide；The polarity chromatographic column is selected from the polarity that specification is for 20m*0.18mm*0.20 μm
Capillary chromatographic column；
The volume ratio of the red blood cell, hydrochloric acid-methanol solution, extractant and double solvents is 1：20：40：8；The gas-chromatography bar
Part also includes：Sampling volume is 1 μ L, and injector temperature is 220~230 DEG C, and rear running temperature is 240~250 DEG C, when running afterwards
Between be 2min, purge flow rate be 3.0~5.0mL/min, total flow be 50.0~60.0mL/min, average linear velocity be 35.0~
45.0cm/s；
The Mass Spectrometry Conditions also include：Interface temperature is 210~230 DEG C；Ion source temperature is 220~240 DEG C；Quadrupole rod temperature
It it is 140~160 DEG C, the solvent delay time is 3.0~4.0min, full scan scope is m/z60.00~450.00.
2. the method for quick detection whole blood red blood cell lignocerane monoenoic acid according to claim 1, it is characterised in that：Institute
State extractant and be selected from n-hexane, the double solvents is selected from n-hexane.
3. the method for quick detection whole blood red blood cell lignocerane monoenoic acid according to claim 1, it is characterised in that：Also
Comprise the following steps：The qualitative judgement of testing result and/or quantitative calculating.
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Effective date of registration: 20170628 Address after: 710065 Shaanxi city in Xi'an Province Economic Development Zone No. 8989 is Jilu Xi'an Service Outsourcing Industrial Park Innovation Incubation Center C block 11-15 Patentee after: Xi'an Kingmed Medical Institute Co., Ltd. Address before: 510330 Haizhuqu District, Guangdong, Xingang East Road, No. 2429, on the third floor Patentee before: Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd. |