CN103837620A - Method for simultaneously detecting content of multiple amino acids in plasma of human body - Google Patents
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Abstract
The invention relates to a method for simultaneously detecting the content of multiple amino acids in the plasma of the human body. The method comprises the following steps of (1) collecting the plasma of the human body, and removing proteins of the plasma by utilizing methanol to obtain supernatant containing multiple amino acids; (2) facilitating the derivatization reaction between the supernatant containing the amino acids and a derivatization reagent to generate an ultraviolet absorbed derivatization product; and (3) analyzing the derivatization product by adopting a high-efficient liquid phase method. The method is simple to operate, multiple amino acids contained in the blood of the human body can be simultaneously detected, and the accuracy and sensitivity are high.
Description
Technical field
The present invention relates to chemical analysis detection field, especially a kind of simple to operate, testing cost is low, have pin-point accuracy and super-sensitive detection method, is specifically related to the method for several amino acids content in a kind of while human body blood plasma.
Background technology
Amino acid is to form biosome protein and with vital movement the most basic relevant material, is the base unit that forms in vivo protein molecule, has close relationship with biological vital movement.It has special physiological function in antibody, is one of indispensable nutritional labeling in biosome.The most basic material that forms human body, has protein, lipid, carbohydrates, inorganic salts, vitamin, water and food fiber etc.As the amino acid of base unit that forms protein molecule, form beyond doubt one of base substance of human body.
The digestion of protein in body and absorptionly complete by amino acid: as the protein of the first nutritional factors in body, its effect in food nutrition is apparent, it can not directly be utilized in human body, but is utilized by becoming the little molecule of amino acid.
Generation, existence and the extinction of life, none is not relevant with protein, said as Engels: " protein is the material base of life, and life is a kind of form that protein exists.If " lacking protein in human body, the lighter's physique declines, hypoevolutism, resistibility weakens, and anaemia is weak, and severe one forms oedema, even threat to life.Once lose protein, life has not just existed yet, therefore there is people to claim that protein is " carrier of life ".Can say, it is the first element of life.
The base unit of protein is amino acid.If human body lacks any essential amino acid, just can cause physiological function abnormal, affect normally carrying out of organism metabolism, finally cause disease.Equally, if lack some nonessential amino acid in human body, can produce organism metabolism obstacle.Arginine and citrulline are very important to forming urea; Cystine insufficiency of intake will cause that insulin reduces, and blood sugar raises.And for example after wound, cystine and arginic requirement increase, as lacked, even still synthetic protein smoothly of heat energy abundance.In a word, amino acid can be brought into play more following effects by metabolism in human body: 1. synthetic tissue protein; 2. become acid, hormone, antibody, creatine etc. containing ammoniacal substance; 3. change carbohydrates and fat into; 4. be oxidized to carbon dioxide and water and urea, produce power.Therefore, the existence of amino acid in human body, not only provides the important source material of synthetic protein, and for promoting growth, carry out eubolism, sustaining life provides material base.If human body lacks or minimizing is wherein a certain, the normal life metabolism of human body will be subject to obstacle, even causes the generation of various diseases or vital movement to stop.As can be seen here, how amino acid in human life activity needs if seeming.
The detection method using both at home and abroad at present has electrochemical process, spectrophotometric method, high performance liquid chromatography, capillary electrophoresis and vapor-phase chromatography etc.High performance liquid chromatography is the most general method that uses at present, and ion exchange process and reversed phase chromatography in high performance liquid chromatography is most widely used two kinds.But the most compare operation complexity of current method.
Summary of the invention
The object of this invention is to provide the method for several amino acids content in a kind of while human body blood plasma, this detection method is simple to operate, any one or a few in 24 seed amino acids that simultaneously may occur in human body blood plasma or whole, has pin-point accuracy and high sensitivity.
The object of the invention is to be achieved through the following technical solutions:
A method for several amino acids content in while human body blood plasma, described several amino acids is respectively: one or more in aspartic acid, glutamic acid, asparagine, serine, glutamine, glycocoll, histidine, citrulline, taurine, arginine, threonine, alanine, proline, ammonium chloride, tyrosine, valine, methionine, halfcystine, isoleucine, leucine, phenylalanine, tryptophane, ornithine and lysine or whole; Said method comprising the steps of: 1. take human plasma, and blood plasma is taken off to albumen with methyl alcohol, obtain the supernatant that contains several amino acids; 2. the obtained supernatant that contains several amino acids and derivatization reagent are carried out to derivative reaction, generate the derivatization product that has uv absorption; 3. adopt high-efficient liquid phase technique to analyze to derivatization product.
Further, step 1. in Deproteinated detailed process be: get human plasma sample and be placed in centrifuge tube, add methyl alcohol, concussion mixes, use again the centrifugal 5min of rotating speed of 12000r/m, and get supernatant and be placed in clean centrifuge tube, this supernatant is the 1. described supernatant that contains several amino acids of step.
Further, the step 2. detailed process of middle derivative reaction is: in the described supernatant that contains several amino acids, add triethylamine solution, phenyl isothiocyanate solution and inner mark solution, evenly mix, under room temperature, derivative 1h, adds normal hexane, and whirlpool mixes, leave standstill 10min, take off layer solution and cross the filter membrane of 0.2 μ m, sample introduction analysis, with chromatographic peak area inner mark method ration.
Further, described triethylamine solution is triethylamine acetonitrile solution, and described phenyl isothiocyanate solution is phenyl isothiocyanate acetonitrile solution, and described inner mark solution is that with 0.1mol/L hydrochloric acid, internal standard compound to be dissolved and diluted be the inner mark solution of 50 μ mol/L.
Further, described internal standard compound is nor-leucine (Nle).
Further, described human plasma sample with add methyl alcohol volume ratio be: 1: 3.
Further, the volume ratio that contains supernatant, triethylamine acetonitrile solution, phenyl isothiocyanate acetonitrile solution and the inner mark solution of several amino acids described in is: 2: 1: 1: 1.
Further, in analyzing with high-efficient liquid phase technique, mobile phase used comprises mobile phase A and Mobile phase B; Flow velocity 0.8mL/min; Detect wavelength: 254nm.
Further, described mobile phase A is 0.05% sodium acetate solution, and described sodium acetate solution is to add water in sodium acetate, and adjusts PH to 6.35 with glacial acetic acid, shakes up the membrane filtration of rear use 0.45 μ m, ultrasonic degas 10min-15min.
Further, described Mobile phase B is analytically pure acetonitrile.
The invention provides the method for several amino acids content in a kind of while human body blood plasma, its beneficial effect mainly having is: method of the present invention can detect simultaneously in 24 seed amino acids that may exist in blood any one or more or all, the method is simple to operate, have pin-point accuracy and high sensitivity, has clinically important using value.
Accompanying drawing explanation
With reference to the accompanying drawings the present invention is described in further detail below.
Fig. 1 is the hydrochloric acid solution using 0.1mol/L described in the embodiment of the present invention high-efficient liquid phase chromatogram as negative control gained;
Fig. 2 is the high-efficient liquid phase chromatogram of the standard solution containing 24 seed amino acids described in the embodiment of the present invention;
Fig. 3 is the high-efficient liquid phase chromatogram of 24 seed amino acids in the normal human's blood plasma liquid described in the embodiment of the present invention;
Fig. 4 is the high-efficient liquid phase chromatogram of 24 seed amino acids in the abnormal person's blood plasma of the phenylalanine described in the embodiment of the present invention;
In Fig. 1～4: 1, aspartic acid (asp); 2, glutamic acid (glu); 3, asparagine (asn); 4, serine (ser); 5, glutamine (Gln); 6, glycocoll (gly); 7, histidine (his); 8, citrulline (cit); 9, taurine (tau); 10, arginine (arg); 11, threonine (thr); 12, alanine (ala); 13, proline (pro); 14, ammonium chloride (NH4Cl); 15, tyrosine (tyr); 16, valine (val); 17, methionine (met); 18, halfcystine (cys); 19, isoleucine (ile); 20, leucine (leu); 21, phenylalanine (phe); 22, tryptophane (trp); 23, ornithine (orn); 24, lysine (lys).
Embodiment
As Figure 1-4, all 24 seed amino acids of the present invention all refer to following 24 kinds, and its respectively respective figure be labeled as: 1, aspartic acid (asp); 2, glutamic acid (glu); 3, asparagine (asn); 4, serine (ser); 5, glutamine (Gln); 6, glycocoll (gly); 7, histidine (his); 8, citrulline (cit); 9, taurine (tau); 10, arginine (arg); 11, threonine (thr); 12, alanine (ala); 13, proline (pro); 14, ammonium chloride (NH4Cl); 15, tyrosine (tyr); 16, valine (val); 17, methionine (met); 18, halfcystine (cys); 19, isoleucine (ile); 20, leucine (leu); 21, nor-leucine (Nle, is interior mark, does not belong to 24 seed amino acids of the present invention); 22, phenylalanine (phe); 23, tryptophane (trp); 24, ornithine (orn); 25, lysine (lys).
In while human body blood plasma of the present invention, the method for several amino acids content specifically comprises the following steps: 1. take human plasma, and blood plasma is taken off to albumen with methyl alcohol, the sample of getting human plasma 200 μ L is placed in the centrifuge tube of 1.5mL, add the methyl alcohol of 600 μ L, concussion mixes, then uses the centrifugal 5min of rotating speed of 12000r/m; 2. get in step a gained supernatant and get 200 μ L and be placed in another centrifuge tube, then add the each 100 μ L of triethylamine acetonitrile solution, phenyl isothiocyanate acetonitrile solution and interior mark, mix 3min, derivative 1h under room temperature; Add normal hexane 400 μ L, whirlpool mixes, and leaves standstill 10min, takes off a layer solution (being the derivatization product of gained); 3. adopt high-efficient liquid phase technique to carry out sample introduction analysis to derivatization product, with chromatographic peak area inner mark method ration, wherein, mobile phase comprises mobile phase A and Mobile phase B, flow velocity 0.8mL/min; Detect wavelength: 254nm; Described mobile phase A is 0.05% sodium acetate solution, adjusts PH to 6.35 with acetic acid, shakes up the membrane filtration of rear use 0.45 μ m, ultrasonic degas 10min-15min; Described Mobile phase B is analytically pure acetonitrile.The percent by volume of mobile phase A and Mobile phase B and the corresponding relation of time can see table.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 96 | 4 |
| 5 | 92 | 8 |
| 16 | 87 | 13 |
| 17 | 78 | 22 |
| 36 | 66 | 34 |
| 50 | 66 | 34 |
As an example of specific experiment case example, embodiment is described below, should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described:
1, key instrument equipment used and reagent: instrument: Waters2695, Ultrasound Instrument, oscillator, vacuum pump, PH meter etc.; Reagent: acetonitrile, ultrapure water, methyl alcohol, HUL, triethylamine, the sour sodium of anhydrous mistake, glacial acetic acid, normal hexane etc.
2, detection method
1. chromatographic condition: chromatographic column: PntulipsTM, BP-C18,5 μ m, 4.6*250mm, column temperature: 40 ℃; Mobile phase A: acetonitrile, Mobile phase B: 0.05% sodium acetate solution (pH6.35); Gradient elution program is as shown in the table, flow velocity: 0.8mL/min; Detect wavelength: 254nm; Sample size: 10 μ L.
| Time (min) | Mobile phase A | Mobile phase B |
| 0 | 96 | 4 |
| 5 | 92 | 8 |
| 16 | 87 | 13 |
| 17 | 78 | 22 |
| 36 | 66 | 34 |
| 50 | 66 | 34 |
2. the preparation of titer and other solution:
Standard solution preparation: the standard items that accurately take 24 seed amino acids, and dilute with 0.1mol/L hydrochloric acid solution, be mixed with the standard solution containing 24 seed amino acids, its concentration is respectively 5 μ mol/L, 10 μ mol/L, 20 μ mol/L, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L, 500 μ mol/L, 1000 μ mol/L.
Inner mark solution preparation: accurately take again internal standard compound nor-leucine (Nle) appropriate, be made into the Nle inner mark solution of 50 μ mol/L with 0.1mol/L dissolve with hydrochloric acid solution dilution; Then the kilnitamin standard solution preparing and Nle inner mark solution being placed in to 4 ℃ of refrigerators saves backup.
Triethylamine acetonitrile solution preparation: get triethylamine 1.4mL, then add acetonitrile 8.6mL, mix and be triethylamine acetonitrile solution.
Phenyl isothiocyanate acetonitrile solution preparation: get phenyl isothiocyanate 25 μ L, add acetonitrile 2mL, mix and be phenyl isothiocyanate acetonitrile solution.
Sodium acetate solution preparation: get sodium acetate 8.2g, be dissolved in water and add water to 1000mL, then adjusting pH to 6.35 with glacial acetic acid, mix, cross film, the degassed sodium acetate solution that obtains.
3. blood sample processing:
Normal human's plasma sample derivatization treatment: get blood plasma or blood serum sample 200 μ L are placed in centrifuge tube, add methyl alcohol 600 μ L, jolting, the centrifugal 5min of 12000r/min, get supernatant 200 μ L, be placed in plastic centrifuge tube at the bottom of 1.5mL tool plug tip, add the above-mentioned Nle inner mark solution preparing 100 μ L, triethylamine acetonitrile solution 100 μ L and phenyl isothiocyanate acetonitrile solution 100 μ L, mix, room temperature is placed 1h, add normal hexane 400 μ L, vibration, place 10min, and then take off a layer solution, cross 0.2 μ m filter membrane, obtain the solution after normal human's blood plasma derives, for the solution (solution after derivative) that contains 24 seed amino acids, adopt high efficiency liquid phase analysis, sample introduction, records the area at the each peak of chromatogram again, and internal standard method is with peak area quantification.
The abnormal person's plasma sample of phenylalanine derivatization treatment: method, with normal human's plasma sample derivatization treatment method, obtains the abnormal person's blood plasma of phenylalanine liquid.
4. high-efficient liquid phase technique detects:
Under above-mentioned chromatographic condition, hydrochloric acid solution using 0.1mol/L obtains chromatogram 1 as negative control, see Fig. 1, (owing to can not obtaining not containing amino acid whose blank serum, thereby make negative control with 0.1mol/L hydrochloric acid solution, carry out as stated above derivatization treatment by it, to investigate derivatization reagent and the reacted product of this derivatization reagent, whether each derived from amino acid product peak is had to interference); Adopt high efficiency liquid phase to analyze take 24 seed amino acids in 24 seed amino acids, the abnormal person's blood plasma of phenylalanine in the standard solution containing 24 seed amino acids, normal human's blood plasma as experimental subjects respectively again, obtain respectively chromatogram 2,3,4, see Fig. 2, Fig. 3 and Fig. 4.
From accompanying drawing 1～4, each seed amino acid can all go out peak in 50min, good separation, and most of degree of separation is greater than 1.5, and the reagent such as phenyl isothiocyanate is noiseless to measuring.Because the amount of added internal standard compound in Fig. 3 and Fig. 4 is certain, therefore the peak area of various amino acid whose peak areas and internal standard compound 21 can be compared, just can know that various amino acid whose content is high or low compared with standard content, phenomenon can be known the cause of disease that patient may suffer from by inference thus.In Fig. 3, in normal human's blood plasma, the peak area of the peak area ratio internal standard compound 21 of phenylalanine 22 is little, and in Fig. 4, the peak area of the peak area ratio internal standard compound 21 of phenylalanine 22 is obviously much larger, and methionine 17 and serine 4 also have raising slightly, then be phenylketonuria in conjunction with the patient diagnosis in other some illness Fig. 4.
In Fig. 3, the peak area of each seed amino acid and internal standard compound is as shown in the table, wherein Aus can think in the unit of peak area, is unit (Au, the i.e. responsiveness of detecting device of ordinate, for a kind of voltage signal) unit (s, second) that is multiplied by horizontal ordinate forms.
In Fig. 4, the peak area of each seed amino acid and internal standard compound is as shown in the table, wherein Aus can think in the unit of peak area, is unit (Au, the i.e. responsiveness of detecting device of ordinate, for a kind of voltage signal) unit (s, second) that is multiplied by horizontal ordinate forms.
Utilize said method to measure 132 people's 132 routine plasma specimens, in detection, find that there is 3 routine phenylalanine patients with abnormals, the abnormal person's of this phenylalanine HPLC chromatogram as shown in Figure 4.In conjunction with clinical manifestation and other laboratory inspections such as feeblemindedness, convulsions, neuropsychic symptoms, be finally diagnosed as phenylketonuria, make infant obtain timely treatment and intervention.
Embodiment 2: linearity and lowest detectable limit
Get the each 200 μ L of standard solution containing 24 seed amino acids that concentration is respectively 5 μ mol/L, 10 μ mol/L, 20 μ mol/L, 50 μ mol/L, 100 μ mol/L, 200 μ nol/L, 500 μ mol/L and 1000 μ mol/L, totally 8 gradient concentrations, each methyl alcohol 600 μ L that add, carry out derivative reaction disposal methods and measure respectively by the method for the invention again, ratio (y) with the peak area at amino acid peak and the peak area of internal standard compound carries out linear regression to each concentration (x), obtains amino acid regression equation (seeing the following form).The range of linearity of 24 seed amino acids is 5-1000 μ mol/L.Dilution low concentration standard solution, in the time making S/N=3 (signal/noise=3), now amino acid concentration is lowest detectable limit.RSD (Relative Standard Deviation, relative standard deviation) represents the meaning of precision.Following table is that the range of linearity and lowest detectable limit test findings detect composition regression equation (n=7) related coefficient lowest detectable limit, and from following table, this 24 seed amino acid is processed by the method for the invention, can both detect.
Embodiment 3: the recovery and precision.
First, detect children's blood plasma and calculate.In children's blood plasma, add again the standard solution containing 24 seed amino acids, be mixed with respectively containing each 5 parts of basic, normal, high three concentration series of 50 μ mol/L, the 200 μ mol/L of 24 seed amino acids, 600 μ mol/L, take off albumen and derivatization treatment by the method for the invention, the method of pressing is again processed and is measured, with measured value and the ratio calculation recovery that adds value (being the value in the standard solution of 24 seed amino acids that contains adding), and calculate precision, the results are shown in following table.
Embodiment 4: the repeatability of method
Getting concentration is the standard solution containing 24 seed amino acids of 100 μ mol/L, carry out the processing such as derivatization and measure respectively by processing side of the present invention is timid, continuous sample introduction 5 times, the RSD that calculates retention time and peak area is straight, the results are shown in following table, the relative standard deviation of 24 seed amino acid retention times and peak area detects composition.
Following examples 5～7 are the test to plasma sample stability.
Embodiment 5: plasma sample room temperature shelf-stability
Place at ambient temperature with human body health check-up blood plasma, again respectively at 0,2,4,8, take off the processing sample such as albumen and derivatization by the method for the invention when 12h, measure as stated above again, and calculate various amino acid whose concentration, results sample is stable in room temperature is placed 12h, the results are shown in shown in following table.
Embodiment 6: the stability that plasma sample is placed at 2 ℃
With the airtight placement under 2 ℃ of conditions of human body health check-up blood plasma, during again respectively at 0,3,7,10 day (d), take off the processing sample such as albumen and derivatization by the method for the invention, measure as stated above again, and calculate various amino acid whose concentration, results sample airtight placement under 2 ℃ of conditions was stablized in 30 days, the results are shown in shown in following table.
Embodiment 7: the freezing shelf-stability of plasma sample
By human plasma freezing placement under the condition of-20 ℃, during again respectively at 0,10,20,30 day (d), take off the processing sample such as albumen and derivatization by the method for the invention, measure as stated above again, and calculate various amino acid whose concentration, result shows, the freezing placement under-20 ℃ of conditions of this plasma sample was stablized in 30 days, the results are shown in shown in following table.
Known by said determination result, this law is easy and simple to handle, analysis is quick, detection limit is low, reproducible.
Embodiment 8
Itself does not have uv absorption most of amino acid, and also emitting fluorescence not, therefore, needs derivatization just can measure.The amino acid detecting by the method for the invention can separate 24 seed amino acids in 50min, makes it in the clinical practice of hospital, have practicality.The composition of measuring due to this law is many, for guaranteeing to record the reliable of result, should strict Internal Quality Control in mensuration process, and experiment derivatization condition should be consistent, when preferably every batch sample is measured, does quality control standard with amino acid standard solution.Vivo acid concentration is subject to various factors, as food, medicine etc., thereby before blood sample collection, should be appreciated that experimenter eats recently the situations such as food, medicine and (whether taken containing amino acid whose health products, medicine, the medicine of whether having injected Amino Acid Compound Injection or having combined as the acid group such as arginine, lysine containing amino acid acid group), otherwise, the concentration of surveying will not be concentration under its normal condition.In addition, be preferably in sample introduction in 24h when mensuration.
The present invention is not limited to above-mentioned preferred forms, any modification relevant of the present invention or change that anyone does under enlightenment of the present invention, and every have identical with a application or akin technical scheme, within all dropping on protection scope of the present invention.
Claims (10)
1. a method for several amino acids content in while human body blood plasma, is characterized in that: described several amino acids is respectively: one or more in aspartic acid, glutamic acid, asparagine, serine, glutamine, glycocoll, histidine, citrulline, taurine, arginine, threonine, alanine, proline, ammonium chloride, tyrosine, valine, methionine, halfcystine, isoleucine, leucine, phenylalanine, tryptophane, ornithine and lysine; Said method comprising the steps of:
1. take human plasma, and blood plasma is taken off to albumen with methyl alcohol, obtain the supernatant that contains several amino acids;
2. the obtained supernatant that contains several amino acids and derivatization reagent are carried out to derivative reaction, generate the derivatization product that has uv absorption;
3. adopt high-efficient liquid phase technique to analyze to derivatization product.
2. the method for several amino acids content in while human body blood plasma according to claim 1, it is characterized in that: step 1. in Deproteinated detailed process be: get human plasma sample and be placed in centrifuge tube, add methyl alcohol, concussion mixes, use again the centrifugal 5min of rotating speed of 12000r/m, and get supernatant and be placed in clean centrifuge tube, this supernatant is the 1. described supernatant that contains several amino acids of step.
3. the method for several amino acids content in while human body blood plasma according to claim 1 and 2, it is characterized in that: the step 2. detailed process of middle derivative reaction is: in the described supernatant that contains several amino acids, add triethylamine solution, phenyl isothiocyanate solution and inner mark solution, evenly mix, under room temperature, derivative 1h, adds normal hexane, and whirlpool mixes, leave standstill 10min, take off layer solution and cross the filter membrane of 0.2 μ m, sample introduction analysis, with chromatographic peak area inner mark method ration.
4. the method for several amino acids content in while human body blood plasma according to claim 3, it is characterized in that: described triethylamine solution is triethylamine acetonitrile solution, described phenyl isothiocyanate solution is phenyl isothiocyanate acetonitrile solution, and described inner mark solution is that with 0.1mol/L hydrochloric acid, internal standard compound to be dissolved and diluted be the inner mark solution of 50 μ mol/L.
5. the method for several amino acids content in while human body blood plasma according to claim 4, is characterized in that: described internal standard compound is nor-leucine.
6. the method for several amino acids content in while human body blood plasma according to claim 2, is characterized in that: described human plasma sample with the volume ratio of add methyl alcohol is: 1: 3.
7. the method for several amino acids content in while human body blood plasma according to claim 4, is characterized in that: described in contain several amino acids the volume ratio of supernatant, triethylamine acetonitrile solution, phenyl isothiocyanate acetonitrile solution and inner mark solution be: 2: 1: 1: 1.
8. the method for several amino acids content in while human body blood plasma according to claim 1, is characterized in that: in analyzing with high-efficient liquid phase technique, mobile phase used comprises mobile phase A and Mobile phase B; Flow velocity 0.8mL/min; Detect wavelength: 254nm.
9. the method for several amino acids content in while human body blood plasma according to claim 8, it is characterized in that: described mobile phase A is 0.05% sodium acetate solution, described sodium acetate solution is to add water in sodium acetate, and with glacial acetic acid adjust PH to 6.35, shake up the membrane filtration of rear use 0.45 μ m, ultrasonic degas 10min-15min.
10. the method for several amino acids content in while human body blood plasma according to claim 8, is characterized in that: described Mobile phase B is analytically pure acetonitrile.
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