CN106442758A - Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode - Google Patents
Liquid mass spectrometry method for detecting various amino acids in human blood plasma in underivatized mode Download PDFInfo
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Abstract
The invention discloses a liquid mass spectrometry method for detecting various amino acids in human blood plasma in an underivatized mode. The method includes the following steps that firstly, the human blood plasma is obtained and subjected to deproteinization with sulfosalicylic acid, and supernate is obtained; secondly, the supernate is mixed with an internal standard solution containing a volatile ionized reagent, LC-MS/MS is used for analysis, wherein according to chromatographic conditions, 1 mM perfluoroheptanoic acid aqueous solution serves as a mobile phase A, a 1 mM perfluoroheptanoic acid acetonitrile solution serves as a mobile phase B, and gradient elution is conducted; according to mass spectrum conditions, multi-ion reaction monitoring of positive ion electrospray ionization is conducted. A volatile ionized reagent sample is added to a sample and mobile phase system, analysis time is short, analysis efficiency is improved, operation is easy, and accuracy of experiment results is improved. LC-MS/MS is used for detecting twenty amino acids in the human blood plasma, specificity, sensitivity and flux are high, operation is easy, sample processing is easy, detection time is short, and the method is particularly suitable for clinical application and popularization.
Description
Technical field
The present invention relates in a kind of non-derivative efficient detection human plasma several amino acids liquid chromatography mass spectrometric method, belong to
Amino acid detection techniques field.
Background technology
Amine substance is the basis of body metabolism, and its main representative amino acid is even more the base unit constituting protein, tool
There is important biochemical activity.Protein fragments or thing that dipeptides is made up of the amido link that two amino acid dehydrating condensations are formed
Matter, with nutrition, hormone, ferment suppression, adjust immunity, antibacterial, antiviral, anti-oxidant have closely relation.Additionally, research
Show that the methylating of amino acid, acetylate and human health status are closely related, be the key character of tumor development.
Therefore, realize amine substance and carry out metabolic profiling analysis, to research human body physiological state, disease process and therapeutic evaluation etc. are all
There is important Research Significance.
Amine substance polarity is strong, is difficult to retain in reversed-phase liquid chromatography, and ion suppression is serious, quantitative analysis difficult.Mesh
Before, conventional quantitative analysis method is by liquid chromatography-tandem mass spectrometry many reaction detection method (LC-MRM).This method speed
Degree is fast, and sample pretreatment is simple.But because different amine substance polarity differences is larger, many amine substance chromatograms retain energy
Power is weak, and differing greatly in ionization process, and Ionization Efficiency in electric spray ion source for many amines is very low, causes
Its detection sensitivity is poor.Therefore in actually detected, typically can only carry out qualitative analysis, or the amino acid for high-load at present
Enter quantitative analysis.In order to solve this problem, researcher is had to improve Ionization Efficiency using derivatization method, thus reach determining
The purpose of amount analysis, but the complex operation due to Derivative, time consuming nature and shortage specificity are it is impossible to be applied to clinical blood
The high throughput analysis of all.
Content of the invention
For above-mentioned prior art, the invention provides a kind of non-derivative detects the liquid of several amino acids in human plasma
Phase mass spectrometry method.The present invention passes through continuous research and discovery, using volatility ion-pairing agent it is not necessary to carry out to amino acid
Derivatization just can carry out Direct Analysis to it, in the present invention, adds ion-pairing agent in flow visualizing, need not be to right
Blood sample derivatization it is possible to reach higher detection sensitivity, specificity, because sample pre-treatments are simple, analysis time
Also very short (within 20 minutes), such that it is able to meet the high throughput analysis of clinical blood sample sample.
The present invention is achieved by the following technical solutions：
A kind of non-derivative detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and described several amino acids are selected from
Aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, alanine,
Proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, tryptophan and bad ammonia
Two or more or whole in acid；Comprise the following steps：
(1) take human plasma, carry out de- albumen with sulfosalicylic acid, obtain the supernatant containing several amino acids；
(2) above-mentioned supernatant is mixed with the inner mark solution containing volatility ionization reagent, carried out point using LC-MS/MS
Analysis.
Further, described step (1) is specially：Take human plasma sample, be placed in centrifuge tube, add isopyknic matter
Amount concentration is the inner mark solution of the sulfosalicylic acid of 1%～8% (preferably 6%), and concussion mixes, and is centrifuged 5min under 13000rpm,
Take supernatant, be placed in clean centrifuge tube, standby.
Further, in described step (2), volatility ionization reagent is selected from hyptafluorobutyric acid, nine fluorine valeric acids or perfluor heptan
Acid, preferably perfluoro-heptanoic acid；The concentration of volatility ionization reagent is 1.5～2.5mM；Described inner mark solution is by 0.375uM ammonia
(glycine, serine, asparagus fern acyl that base gluconic acid solution and 0.25uM S-2 aminoethyl cysteine solution mix
The internal standard compound of amine, glutamine and glutamic acid is glucosaminicacid；The internal standard compound of remaining amino acid is S-2 amino-ethyl half
Cystine), the two volume ratio is 0.8～1.2:0.8～1.2；Supernatant and the inner mark solution containing volatility ionization reagent
Volume ratio is 1:75～85.
The condition of described LC-MS/MS is：
Chromatographic condition：Mobile phase A：The 1mM perfluoro-heptanoic acid aqueous solution；Mobile phase B：1mM perfluoro-heptanoic acid acetonitrile solution；Gradient is washed
De-；Chromatographic column：Pre-column：Thermo Fisher, 3um Hypercarb, 4.6mm*50mm, column temperature：40℃；Analytical column：
Avanced Materials, 2.7um HaloC18,2.1mm*100mm, column temperature：65℃；Pre-column and analytical column switching cut down (ten
Port valve) connect (add a switching to cut down it is therefore an objective to remove the impurity in blood plasma as far as possible in the middle of pre-column and analytical column,
Make it not enter detector, on the one hand reduce to the impact measuring, on the other hand reduce the pollution to instrument)；
Mass Spectrometry Conditions：Using the polyion reaction monitoring pattern of positive ionization electrospray ionization, gasification temperature：300℃；
NEB:60psi；Spray voltage：2100V；Remove solvent temperature：350℃.
Preferably, condition of gradient elution is as shown in table 1.
Table 1 condition of gradient elution
Remarks：The liquid flowing out from pre-column before 1.7 minutes cuts down entrance waste liquid bottle through switching, and switching when 1.7 minutes is cut down and cut
Change, the liquid flowing out from pre-column enters analytical column and mass detector analysis detection.
Preferably, MRM mass spectrometry parameters are as shown in table 2.
2 two ten kinds of amino acid of table and interior target Mass Spectrometry Conditions condition
Preferably, a kind of non-derivative detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and step is as follows：
(1) sample pre-treatments：Add the inner mark solution of isopyknic 6% sulfosalicylic acid in serum sample, be vortexed shake
Swing rear 13000rpm rotating speed to be centrifuged 5 minutes, take supernatant 10ul, add the inner mark solution of 800ul 2mM perfluoro-heptanoic acid, uniformly mix
Close, sample introduction is analyzed；
The beneficial effect carrying out sample pre-treatments is：By this process, efficiently separate protein precipitation, go the removal of impurity, subtract
Few interference, reduces matrix effect, farthest can retain amino acid simultaneously, and add volatility ionization reagent, improves
The sensitivity of Mass Spectrometer Method；
(2) preparation of standard working solution：Each amino acid stock is prepared with 0.1N HCL, then uses concentration to be 5% (unit
G/ml BSA (bovine serum albumin(BSA)) methanol solution) prepare 6 gradients (10,50,100,250,500,1000uM) mixing mark
Quasi- solution, is sub-packed in 1.5mL brown bottle, -20 DEG C save backup；
(3) LC-MS/MS detection：Sample to be tested is entered after chromatogram post separation by gradient elution mode, using cation
The polyion reaction monitoring pattern of electro-spray ionization, test substance is carried out qualitative (with the ion pair of various amino acid and phase
To retention time as qualitative foundation) and quantitative determination (making calibration curve with standard items quantitative).
Further, when carrying out quantitative determination, application standard items make calibration curve, with concentration of standard solution as X-axis, mark
Quasi- product peak area and internal standard peak area ratio are Y-axis, carry out linear regression analysis and obtain regression equation, by corresponding vitamin peak area
Substitute into calibration curve equation, calculate the concentration of amino acid in blood serum sample respectively.
Present invention also offers a kind of non-derivative detects the detection kit of several amino acids in human plasma, have not
With each amino acid standard liquid of gradient concentration, mass concentration is the inner mark solution of 1%～8% sulfosalicylic acid, containing volatilization
The inner mark solution of property ionization reagent；Described volatility ionization reagent is selected from hyptafluorobutyric acid, nine fluorine valeric acids or perfluoro-heptanoic acid, waves
The concentration of the property sent out ionization reagent is 1.5～2.5mM；Described inner mark solution be by 0.375uM Glucosamine acid solution and
0.25uM S-2 aminoethyl cysteine solution mixes, and the two volume ratio is 0.8～1.2:0.8～1.2；1mM is complete
The fluorine enanthic acid aqueous solution, 1mM perfluoro-heptanoic acid acetonitrile solution.
Traditional amino acid content detection method has disadvantages that, in order to obtain on a column preferable retention behavior with
And raising detection sensitivity, it usually needs before carrying out the post of sample or post column derivatization, not only operating process is loaded down with trivial details, time-consuming takes
Power, and operating error is larger, the accuracy of impact experimental result.The present invention pass through in flow visualizing add volatility from
Sonization reagent (in order to improve degree of ionization in MS for the amino acid, realizes the accurate quantitative analysis of multiple native amino acids),
Establish a kind of quick, sensitive, easily several amino acids assay method in non-derivative blood sample, especially while measuring
Aspartic acid in serum, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, third
Propylhomoserin, proline, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan and rely
The method of propylhomoserin.The content of 20 kinds of primary amino acids in human serum can be detected using the present invention simultaneously, and carry out accurately determining
Property and quantitative analysis, be that a kind of sample process is simple, quick, flux is high, the detection method of reliable results.
Aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine,
Alanine, proline, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan and
Lysine is important amino acid in human body, and the shortage of any amino acid or rising all can lead to corresponding disease.This
Bright can detect this 20 kinds of amino acid simultaneously, one is reduction of testing cost, and two is time-consuming, three be reduce collection tested
Survey the blood volume of people, four is some medical condition relevant can be diagnosed provide foundation.
The method of the present invention, adds volatility ionization reagent sample in sample and flow visualizing, and analysis time is short,
Improve the efficiency of analysis, ionization reagent replaces conventional derivative reaction, not only simple to operate, and improve experiment knot
The accuracy (because avoiding the human error in derivative reaction) of fruit.The present invention adopts in LC-MS/MS detection human plasma
20 kinds of amino acid, high specificity, sensitivity is high, and flux is high, simple to operate, and sample treatment is simple, and detection time is short, especially suitable
Close clinical popularization and application.
Brief description
Fig. 1：The chromatogram of plasma sample 1.
Fig. 2：The chromatogram of plasma sample 2.
Fig. 3：The calibration curve schematic diagram of alanine.
Fig. 4：Arginic calibration curve schematic diagram.
Fig. 5：The calibration curve schematic diagram of asparagine.
Fig. 6：The calibration curve schematic diagram of aspartic acid.
Fig. 7：The calibration curve schematic diagram of cystine.
Fig. 8：The calibration curve schematic diagram of glutamic acid.
Fig. 9：The calibration curve schematic diagram of glutamine.
Figure 10：The calibration curve schematic diagram of glycine.
Figure 11：The calibration curve schematic diagram of histidine.
Figure 12：The calibration curve schematic diagram of isoleucine.
Figure 13：Leucic calibration curve schematic diagram.
Figure 14：The calibration curve schematic diagram of lysine.
Figure 15：The calibration curve schematic diagram of methionine.
Figure 16：The calibration curve schematic diagram of phenylalanine.
Figure 17：The calibration curve schematic diagram of proline.
Figure 18：The calibration curve schematic diagram of serine.
Figure 19：The calibration curve schematic diagram of threonine.
Figure 20：The calibration curve schematic diagram of tryptophan.
Figure 21：The calibration curve schematic diagram of tyrosine.
Figure 22：The calibration curve schematic diagram of valine.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
Involved instrument, reagent, material etc. in following embodiments, unless otherwise noted, are in prior art existing
Conventional instrument, reagent, material etc., can be either commercially available by regular.Involved experimental technique in following embodiments, inspection
Survey method etc., unless otherwise noted, is existing normal experiment method, detection method etc. in prior art.
Embodiment 1 non-derivative detects the liquid chromatography mass spectrometric method of 20 kinds of amino acid in human plasma
Step is as follows：
(1) prepare sample to be tested：Add the inner mark solution of isopyknic 6% sulfosalicylic acid in serum sample, be vortexed
After concussion, 13000rpm rotating speed is centrifuged 5 minutes, takes supernatant 10ul, adds the inner mark solution of 800ul 2mM perfluoro-heptanoic acid, uniformly
Mixing；Described inner mark solution is by 0.375uM Glucosamine acid solution and 0.25uM S-2 aminoethyl cysteine solution
Mix, the two volume ratio is 1:1.The present embodiment is prepared for sample 1,2 two parts of samples of sample.
(2) LC-MS/MS detection：Sample to be tested enters chromatogram post separation by gradient elution mode, and liquid phase chromatogram condition is such as
Under：
Chromatographic column：Pre-column：Thermo Fisher, 3um Hypercarb, 4.6mm*50mm, column temperature：40℃；Analytical column：
Avanced Materials, 2.7um HaloC18,2.1mm*100mm, column temperature：65℃；Pre-column and analytical column switching cut down (ten
Port valve) connect.
Sampling volume：5ul；
Flow velocity：0.30-0.35ml/min；
Mobile phase A：The 1mM perfluoro-heptanoic acid aqueous solution；
Mobile phase B：1mM perfluoro-heptanoic acid acetonitrile solution；
Condition of gradient elution is as shown in table 1.
Mass Spectrometer Method condition is as shown in table 2.
(3) result of calculation：
The preparation of standard working solution：Each amino acid stock is prepared with 0.1N HCL, then uses concentration to be 5% (unit g/
Ml BSA (bovine serum albumin(BSA)) methanol solution) prepare 6 gradients (10,50,100,250,500,1000uM) hybrid standard
Solution, is sub-packed in 1.5mL brown bottle, -20 DEG C save backup, and standard items participate in sample treatment.
Application standard items make calibration curve, with concentration of standard solution as X-axis, standard items peak area and internal standard compound peak area
Ratio is Y-axis；Carry out linear regression analysis and obtain regression equation.Corresponding amino acid peak area is substituted into calibration curve equation, respectively
Calculate the concentration of 20 kinds of amino acid in blood serum sample, result is as shown in table 3.
Measure the standard items Criterion curve of 20 kinds of amino acid/11 0～1000uM with the inventive method (as Fig. 3～22
Shown), the linear relationship within this range of the amino acid content in human blood sample is good, detection chromatogram see Fig. 1,2, by equation
Understand each amino acid content of this sample, such as table 3 (attached retention time).
20 kinds of amino acid testing results in table 3 Human Blood
Claims (9)
1. a kind of non-derivative detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and described amino acid is selected from asparagus fern ammonia
Acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, alanine, proline,
In tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, tryptophan and lysine two
More than kind or all；It is characterized in that：Comprise the following steps：
(1) take human plasma, carry out de- albumen with sulfosalicylic acid, obtain supernatant；
(2) above-mentioned supernatant is mixed with the inner mark solution containing volatility ionization reagent, be analyzed using LC-MS/MS；
Described volatility ionization reagent is selected from hyptafluorobutyric acid, nine fluorine valeric acids or perfluoro-heptanoic acid；Described inner mark solution be by
0.375uM Glucosamine acid solution and 0.25uM S-2 aminoethyl cysteine solution mix, the two volume ratio
For 0.8～1.2:0.8～1.2；
The chromatographic condition of LC-MS/MS is：Mobile phase A：The 1mM perfluoro-heptanoic acid aqueous solution；Mobile phase B：1mM perfluoro-heptanoic acid acetonitrile is molten
Liquid；Gradient elution；
The Mass Spectrometry Conditions of LC-MS/MS are：The polyion reaction monitoring of positive ionization electrospray ionization.
2. non-derivative according to claim 1 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and it is special
Levy and be：Described step (1) is specially：Take human plasma sample, be placed in centrifuge tube, the isopyknic mass concentration of addition is
The inner mark solution of 1%～8% sulfosalicylic acid, concussion mixes, and is centrifuged 5min, takes supernatant, be placed in centrifugation under 13000rpm
Guan Zhong, standby.
3. non-derivative according to claim 1 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and it is special
Levy and be：In described step (2), the concentration of volatility ionization reagent is 1.5～2.5mM；Supernatant and ion containing volatility
The volume ratio changing the inner mark solution of reagent is 1:75～85.
4. non-derivative according to claim 1 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and it is special
Levy and be：The chromatographic condition of described LC-MS/MS is：Mobile phase A：The 1mM perfluoro-heptanoic acid aqueous solution；Mobile phase B：1mM perfluoro-heptanoic acid
Acetonitrile solution；Gradient elution；Chromatographic column：Pre-column：Thermo Fisher, 3um Hypercarb, 4.6mm*50mm, column temperature：40
℃；Analytical column：Avanced Materials, 2.7um HaloC18,2.1mm*100mm, column temperature：65℃；Pre-column and analytical column
Cut down connection with switching.
5. the non-derivative according to claim 1 or 4 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, its
It is characterised by：Condition of gradient elution is as shown in table 1；
Table 1 condition of gradient elution
Remarks：The liquid flowing out from pre-column before 1.7 minutes cuts down entrance waste liquid bottle through switching, and switching is cut down in switching when 1.7 minutes, from
The liquid that pre-column flows out enters analytical column and mass detector analysis detection.
6. non-derivative according to claim 1 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and it is special
Levy and be：The Mass Spectrometry Conditions of described LC-MS/MS are：Using the polyion reaction monitoring pattern of positive ionization electrospray ionization, gas
Change temperature：300℃；NEB:60psi；Spray voltage：2100V；Remove solvent temperature：350℃.
7. the non-derivative according to claim 1 or 6 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, its
It is characterised by：Described mass spectrographic parameter is as shown in table 2；
2 two ten kinds of amino acid of table and interior target Mass Spectrometry Conditions condition
.
8. the non-derivative according to any one of claim 1～7 detects the liquid phase matter of several amino acids in human plasma
Spectral method it is characterised in that：Step is as follows：
(1) sample pre-treatments：Add the inner mark solution of isopyknic 6% sulfosalicylic acid in serum sample, after the concussion that is vortexed
13000rpm rotating speed is centrifuged 5 minutes, takes supernatant 10ul, adds the inner mark solution of 800ul 2mM perfluoro-heptanoic acid, uniformly mixes,
Sample introduction is analyzed；
(2) preparation of standard working solution：Prepare the standard liquid of each amino acid with 0.1N HCL, standby；
(3) LC-MS/MS detection：Sample to be tested is entered after chromatogram post separation by gradient elution mode, using cation EFI
The polyion reaction monitoring pattern of mist ionization, carries out qualitative and quantitative detection to test substance.
9. non-derivative according to claim 8 detects the liquid chromatography mass spectrometric method of several amino acids in human plasma, and it is special
Levy and be：The manner of formulation of standard working solution is：Each amino acid stock is prepared with 0.1N HCL, is then 5% with concentration
The mixed standard solution of the different gradient of BSA methanol solution preparation, standby.
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