CN105675756A - HPLC (high performance liquid chromatography) detection method for simultaneously determining five substances in reaction system for producing L-Ala-L-Gln (L-alanyl-L-glutamine) with microbial enzyme method - Google Patents
HPLC (high performance liquid chromatography) detection method for simultaneously determining five substances in reaction system for producing L-Ala-L-Gln (L-alanyl-L-glutamine) with microbial enzyme method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses an HPLC (high performance liquid chromatography) detection method for simultaneously determining five substances (L-Ala-L-Gln (L-alanyl-L-glutamine), L-Gln, L-AlaOMe, L-Glu and L-alanyl-L-alanine) in a reaction system for producing L-Ala-L-Gln with a microbial enzyme method. The five substances are subjected to pre-column automatic derivatization treatment by ortho-phthaladehyde, so that the five substances have fluorescence groups and are detected by a fluorescence detector. The chromatographic condition is as follows: a high performance liquid chromatograph Agilent 1260 Infinity is adopted; an Agilent ZORBAX SB-Aq, 5 mu m, 4.6*250 mm C18 column is adopted as a chromatographic column; an Agilent 1260 Infinity fluorescence detector is adopted; a mobile phase comprises acetonitrile and a phosphate buffer solution in the volume ratio being 12:88, and the pH of the mobile phase is adjusted to 7.4 by the aid of phosphoric acid; the flow velocity of the mobile phase is 1.0 ml/min; the column room temperature is 40 DEG C; detection wavelength comprises excitation wavelength Ex 338 nm and emission wavelength Em 450 nm. The method realizes simultaneous determination of oligopeptide, amino acid and amino derivatives, is simple to operate and high in sensitivity and has important application value.
Description
Technical field
Detect the efficient liquid phase detection method that microbial enzyme method produces five kinds of materials in Ala-Gln reaction system simultaneously, belong to analysis test method technical field.
Background technology
Ala-Gln (L-alanyl-L-glutamine, L-Ala-L-Gln) is a kind of a kind of dipeptides formed by ALANINE and L-glutaminate two seed amino acid dehydrating condensation, is called for short glutamine dipeptide. L-glutaminate (L-Gln) is applied extremely extensive as a kind of conditionally essential amino acid in pharmaceutical industries. L-glutaminate can be used as the raw material of glyconeogenesis and urea synthesis, the precursor substance of neurotransmitter, the important intermediate of biochemical metabolism approach simultaneously or in body. The physico-chemical property being different from L-glutaminate owing to having, glutamine dipeptide overcomes the shortcomings such as the solubleness of L-glutaminate is low, thermolability, and the substitute being therefore usually used as L-glutaminate is applied to clinical with the form of injection liquid.
Glutamine dipeptide is produced at present mainly through chemical synthesis; chemical synthesis usually needs to add and removes poisonous this process of protecting group; usually there is building-up process complexity, production cost height, easily generate the shortcomings such as by product, object purification of products difficulty, synthesis condition is harsh, agents useful for same is harmful in the method, thus chemical synthesis can not large-scale application in commercial field. For above-mentioned present situation, this laboratory takes the lead in having carried out the research that relevant microbial enzyme method produces glutamine dipeptide at home. A series of microbial strains possessing production Ala-Gln ability has been constructed in this laboratory at present, and these bacterial classifications can have been utilized to realize the biological preparation of Ala-Gln. Therefore, we are badly in need of a kind of stable, and accurately, detection method carrys out the content of each material in quantitative analysis reaction system fast so that we to constructed go out bacterial classification carry out Performance Evaluation, and the groping of optimum fermentation condition and optimum reaction condition.
ALANINE methyl ester hydrochloride (L-AlaOMe Hcl) exists with the form of ALANINE methyl esters (L-AlaOMe) in aqueous.The bacterial classification gone out constructed by this laboratory can express α-aminoacidesters acyltransferase; under the effect of this enzyme, the ester acyl group of ALANINE methyl esters (L-AlaOMe) can take off except and then form object product glutamine dipeptide with the amino condensation of L-glutaminate. Meanwhile, take off the ALANINE methyl esters after except ester acyl group and can form L-Ala-L-Ala (L-Ala-L-Ala) this by product with the amino condensation of himself equally. Therefore two kinds of substrates completely consumed can not have residue in reaction system. In addition, L-glutaminate is unstable, and having Partial digestion in reaction process is Pidolidone (L-Glu). Therefore, whole reaction system co-exists in Ala-Gln and L-Ala-L-Ala two kinds of dipeptides, L-glutaminate and Pidolidone two seed amino acid, a kind of these five kinds of materials of amino acid derivative of ALANINE methyl esters. A lot of method has been had to realize although measuring amino acid by high-efficient liquid phase technique, but about actually rare with the report of high-efficient liquid phase technique mensuration dipeptides, and with this three classes material of high-efficient liquid phase technique Simultaneously test oligopeptides, amino acid and amino acid derivative more without any report.
Detecting compared to ultraviolet and parallax, fluoroscopic examination has sensitivity and response value height, quantitatively accurate advantage. O-phthalaldehyde(OPA) (OPA) can combine with amino under the protection of mercaptoethanol thus form the group with hyperfluorescenceZeng Yongminggaoyingguang, can be used for fluoroscopic examination. In reaction system of the present invention, five kinds of materials all also have free amino, therefore can be derived by OPA and form the product with hyperfluorescenceZeng Yongminggaoyingguang, thus smoothly by the detection of fluorimetric detector. And automatic online is derivative manually derivative compared to traditional before post, operates more specification, efficiently avoid personal errors, it is possible to improve measuring accuracy greatly.
Summary of the invention
It is an object of the invention to provide a kind of efficient liquid phase detection method producing five kinds of materials in Ala-Gln reaction system for detecting microbial enzyme method simultaneously. The method achieve micro bioenzyme catalysis method and produce a kind of main product thing Ala-Gln in Ala-Gln reaction system, a kind of by product L-Ala-L-Ala, two kinds of substrate ALANINE methyl esters and L-glutaminate, and the Simultaneously test of a kind of chaff interference Pidolidone. The method is that before standard substance and reaction solution are carried out post by derivatization reagent, automatic online derives taking OPA, is detected by fluorimetric detector. The method have can Simultaneously test five kinds of materials, each material peak resolution height, repeatability good, measuring accuracy and accuracy height, sample working time short (20min), isocratic elution simplicity etc. advantage.
The present invention is a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system, it is characterised in that this detection method comprises the steps:
(1) chromatographic condition:
High performance liquid chromatograph: Agilent1260Infinity;
Chromatographic column: AgilentZORBAXSB-Aq, 5 μm, 4.6 × 250mmC18 post;
Detector: Agilent1260Infinity fluorimetric detector;
Moving phase forms: the volume ratio of acetonitrile and phosphate buffered saline buffer is 12:88, and phosphoric acid regulates moving phase pH to 7.4;
Flow rate of mobile phase: 1.0ml/min;
Post chambers temp: 40 DEG C;
Determined wavelength: excitation wavelength Ex338nm, emission wavelength Em450nm;
Working time: 20min.
(2) standard substance preparation:
Accurately taking the Ala-Gln of certain mass on analytical balance respectively, L-glutaminate, ALANINE methyl ester hydrochloride, Pidolidone, L-Ala-L-Ala, unification 10mL volumetric flask is mixed with the mother liquor that concentration is 4mM. Mark product needed for follow-up test all obtain with the collocation dilution of 4mM mother liquor.
(3) preparation of moving phase
The preparation of phosphate buffered saline buffer: compound concentration is all dipotassium hydrogen phosphate and the potassium dihydrogen phosphate of 12.5mM respectively, the two intermodulation pH to 7.4. Above-mentioned phosphate buffered saline buffer and trifluoroacetic acid aqueous solution are carried out isocratic elution in 88:12 ratio.
(4) derivating agent preparation:
50mg o-phthalaldehyde(OPA), 100 μ L beta-mercaptoethanols, add 300 μ L dissolve with methanol, add 1.6mL borate buffer solution (pH10.0) mixed even both. The borate buffer solution preparation of pH10.0: prepare 0.05M borax soln and 0.2M sodium hydroxide solution respectively, accurately pipettes 50mL borax soln and 23mL sodium hydroxide solution in 200mL volumetric flask, mixes, and be diluted with water to scale marks and both must.
(5) the derivative program of automatic online before post:
Drawing 2 μ l samples, 2 μ l derivating agents mix 3 times, draw 16 μ l borate buffers and mix 15 times, finally mix 5 laggard samples drawing 20 μ l moving phases. This program can represent: 2 μ l sample+2 μ l derivating agent+16 μ l borate buffer+20 μ l moving phases. After drawing, all need operation to wash pin program every time.
The main points of the present invention are, before utilizing OPA that the amino of standard substance carries out post, automatic online derives, thus obtains the fluorophor that can be detected by fluorimetric detector. Establishing a kind of working time by the optimization of chromatographic condition short, resolution height, quantitatively accurately, repeatability is good, the efficient liquid phase detection method that tolerance is strong.
Due to domestic still not by the report of microbial enzyme method production glutamine dipeptide at present, this laboratory takes the lead in having carried out this research on the one hand, and achieve the biological preparation of Ala-Gln, so the invention of this measuring method can each material in the quantitative enzymic catalytic reaction system of real-time analysis, thus more adequately assess bacterial classification performance and production technique, also for industry's enlarging production provides a kind of technical support.
Accompanying drawing explanation
Fig. 1 is five kinds of material HPLC-UV detection. Material peak corresponding in this color atlas is: 1.L-L-glutamic acid, 2.L-glutamine, 3.L-alanine methyl ester, 4.L-alanyl-L-glutamine, 5.L-alanyl-ALANINE.
Fig. 2 is that five kinds of material repeatability investigate color atlas. Material peak corresponding in this color atlas is: 1.L-L-glutamic acid, 2.L-glutamine, 3.L-alanine methyl ester, 4.L-alanyl-L-glutamine, 5.L-alanyl-ALANINE.
Fig. 3 is the contrast color spectrogram of actual sample and standard model. Material peak corresponding in this color atlas is: 1.L-L-glutamic acid, 2.L-glutamine, 3.L-alanine methyl ester, 4.L-alanyl-L-glutamine, 5.L-alanyl-ALANINE.
Embodiment
General explanation: the high performance liquid chromatograph used by the present invention is Agilent1260Infinity and Waters2695, wherein Agilent1260Infinity is equipped with Agilent fluorimetric detector, and Waters2695 is equipped with Waters2475 fluorimetric detector. Chromatographic column is AgilentZORBAXSB-Aq, 5 μm, 4.6 × 250mmC18 post.Ala-Gln is purchased from TCI; O-phthalaldehyde(OPA), mercaptoethanol, L-Ala-L-Ala is purchased from Sigma; ALANINE methyl ester hydrochloride is purchased from An Naiji; Pidolidone, L-glutaminate, dipotassium hydrogen phosphate, potassium primary phosphate, acetonitrile, methyl alcohol etc. are purchased from traditional Chinese medicines group.
The selection of embodiment 1 chromatographic condition
The selection of 1.1 determined wavelength
Get five kinds of material mark product mother liquor dilutions and make 0.2mM solution. At excitation wavelength Ex from 260nm to 400nm, emission wavelength Em from 400nm to 500nm detect, found that five kinds of materials all have maximum absorption wavelength, so electing the two as determined wavelength when Ex be 338nm, Em is 450nm.
The selection of 1.2 chromatographic columns
The C18 reversed-phase column choosing Hitachi, Agilent, Waters tri-kinds of brands respectively is tested, and result shows, and AgilentZORBAXSB-AqC18 chromatographic column appearance time is very fast, and peak shape is symmetrical, and peak resolution is higher, so being elected as detection post.
The selection of 1.3 post temperature
The post temperature that high performance liquid chromatography is conventional is investigated, and choosing temperature range is 20 DEG C-45 DEG C, and result shows, and main peak retention time shortens with the rising of post temperature, and resolution is all qualified, and content detection result is normal. Consider the factor such as chromatographic column life-span and post effect, and shorten sample working time as far as possible, so post temperature is chosen between 35 DEG C-40 DEG C.
The selection of 1.4 moving phases
Choose phosphate buffered saline buffer as the inorganic phase in moving phase, choose acetonitrile as the organic phase in moving phase.
1.4.1 the selection of phosphate buffering liquid concentration
Being test between 5mM-20mM at phosphate concn, result shows, and when phosphate concn is lower, chromatographic peak is unstable; And concentration higher time, peak shape is poor, and resolution is not high. Can analyze when phosphate buffering liquid concentration is 10mM-15mM. But consider that buffer salinity is too high, it is easy to precipitate out from moving phase, it is possible to damage chromatographic column and instrument. So choosing phosphate buffering liquid concentration is 12.5mM.
1.4.2 the selection of phosphate buffered saline buffer pH
The different pH of phosphate buffered saline buffer are investigated, pH scope chooses 4-8, result display retention time increasing and shorten with pH, it is contemplated that the resolution between selected chromatographic column pH tolerance range (2-8) and each material peak, finally choosing phosphate buffered saline buffer pH is 7.4.
1.4.3 moving phase wash-out way choice
In the present invention, isocratic elution has achieved good peak separating effect, and assorted peak is less, again because gradient elution usually can cause baseline wander thus affect quantitatively, so choosing isocratic elution mode.
1.4.4 the selection of inorganic phase and organic phase proportioning in moving phase
Test 12.5mM phosphate buffered saline buffer adjusts different proportionings to prepare moving phase from acetonitrile, and selected ratio range is phosphate buffered saline buffer: acetonitrile=60:40 to 95:5. Found that, when phosphate buffered saline buffer consumption less than cumulative volume 83% time, Ala-Gln can not be separated completely with both ALANINE methyl ester hydrochlorides peak, even occurs folding peak phenomenon completely; When phosphate buffered saline buffer consumption is greater than the 86% of cumulative volume, resolution therebetween reaches 1.5, it is possible to for quantitative assay; When phosphate buffered saline buffer consumption is greater than the 95% of cumulative volume, moving phase almost loses elutive power, and each material peak conditions of streaking is serious, can not be better separated.Considering, inorganic phase and organic phase proportioning can be chosen between 86:14 to 90:10, preferentially choose 88:12. Five kinds of standard specimen chromatographic fractionation figures are shown in accompanying drawing 1.
The foundation of embodiment 2 methodology
2.1 linearity range
Accurately measure the 4mM mother liquor of certain volume five kinds of standard substance, it is diluted to 0.4mM with deionized water, then the standard solution by five kinds of 0.4mM, measure 0.2mL respectively, 0.4mL, 0.5mL, 0.6mL, 0.8mL, finally mends with deionized water neat to 1mL, is namely made into normal gradients solution. Being carried out by five kinds of normal gradients solution before post after automated derivative, detection by quantitative, record color atlas, investigates peak area. The typical curve of five kinds of materials is linearly satisfactory. The typical curve of five kinds of materials and linearity range and R thereof2Value is in table 1.
Table 1: detection thing typical curve, linearity range, quantitative limit
2.2 the rate of recovery
Using a true sample as adding base number of a tender sample, choose low (about 50%), in (about 100%), high (about 200%) three levels carry out adding mark experiment, and each level repeats 3 times. To add standard specimen carries out before post after automated derivative equally, detection by quantitative, and record color atlas, investigates peak area. After measured, calculation result shows, and the average recovery rate of five kinds of materials is between 90%-110%, and RSD value can control within 7%, illustrates that accuracy is good, and concrete outcome refers to table 2.
Table 2: experimental result (n=3) is marked in adding of detection thing
2.3 study on the stability
By hybrid standard product solution 1 part, each 2h measures, and carries out 6 times altogether and measures, the retention time at observation five kinds of material peaks, record peak area. The appearance time RSD value that five kinds of materials enter sample for six times is respectively: L-Ala-L-Gln, 0.05%; L-Gln, 0.05%; L-AlaOMe, 0.07%; L-Glu, 0.12%; L-Ala-L-Ala, 0.04%. The peak area RSD value that five kinds of materials enter sample for six times is respectively: L-Ala-L-Gln, 0.61%; L-Gln, 0.30%; L-AlaOMe, 1.19%; L-Glu, 0.43%; L-Ala-L-Ala, 0.60%. This result is satisfactory, shows that this detection method stability is better, and in a few days error is little. Five kinds of material repeatability are investigated color atlas and are seen accompanying drawing 2.
2.4 quantitative limit measure
Determining quantitative limit with 10 times of signal to noise ratios, product assay condition enters sample in the same old way, and record color atlas, the quantitative limit of five kinds of materials refers to table 1.
2.5 wearing quality
2.5.1 different chromatographic column measures
Again choose the C18 post of two phase same specifications of other brands (Hitachi, Waters), carry out standard substance determination experiment, except the retention time of entirety can offset backward, do not find that concrete measurement result is had impact.
2.5.2 proportion of mobile phase changes
Suitably change proportion of mobile phase (86:14-90:10), carry out standard substance determination experiment, except offseting backward along with the retention time of the increase entirety of inorganic Phase Proportion and material peak resolution has and slightly changes except these two features, do not find that concrete measurement result is had impact.
2.5.3 moving phase pH value changes
Suitably change moving phase pH value (7.0-7.6), standard substance are measured, except the retention time of the rising entirety along with pH value can slightly backward offset except this feature, do not find that concrete measurement result is had impact.
2.5.4 the change of post chambers temp
Standard substance are measured by suitable adjustment column chambers temp (35 DEG C-40 DEG C), except offseting backward except this feature along with the retention time of the reduction entirety of post chambers temp, do not find that concrete measurement result is had impact.
2.5.5 the change of determined wavelength
Suitably adjust determined wavelength (Ex:320nm-350nm; Em:420nm-480nm) standard substance are measured, except (Ex:338nm under overall peak area and peak height relatively most optimum wavelengths; Em:450nm) decrease outside this feature, do not find that concrete measurement result is had impact.
Embodiment 3 actual sample measurement result
Configure certain density L-glutaminate and ALANINE methyl ester hydrochloride substrate mixing solutions, the fermented liquid obtained after Ala-Gln is produced strain fermentation 36h or thalline add in the mixing solutions of two kinds of substrates as thick enzyme source, the pH value adjusting whole substrate system is about 8, under whole substrate system is placed in 25 DEG C of conditions, oscillatory reaction 1-2h. Get a certain amount of reaction solution (>=12000g) centrifugal 5-10min at a high speed, getting supernatant is diluted in the linearity range described in embodiment 2, after before post, on-line automaticization derives, detection by quantitative is carried out according to the chromatographic condition described in embodiment 1, record peak area, calculates each substance production respectively according to the typical curve described in embodiment 2. The contrast color spectrogram of actual sample and standard substance is shown in accompanying drawing 3.
Claims (7)
1. a Simultaneously test microbial enzyme method produces five kinds of material (Ala-Glns in Ala-Gln reaction system, L-glutaminate, ALANINE methyl esters, Pidolidone, L-Ala-L-Ala) efficient liquid phase detection method, it is characterised in that this detection method comprises the steps:
(1) chromatographic condition:
High performance liquid chromatograph: Agilent1260Infinity;
Chromatographic column: AgilentZORBAXSB-Aq, 5 μm, 4.6 × 250mmC18 post;
Detector: Agilent1260Infinity fluorimetric detector;
Moving phase forms: the volume ratio of acetonitrile and phosphate buffered saline buffer is 12:88, and phosphoric acid regulates moving phase pH to 7.4;
Flow rate of mobile phase: 1.0ml/min;
Post chambers temp: 40 DEG C;
Determined wavelength: excitation wavelength Ex338nm, emission wavelength Em450nm;
Working time: 20min.
(2) standard substance preparation:
Accurately taking the Ala-Gln of certain mass on analytical balance respectively, L-glutaminate, ALANINE methyl ester hydrochloride, Pidolidone, L-Ala-L-Ala, unification 10mL volumetric flask is mixed with the mother liquor that concentration is 4mM. Mark product needed for follow-up test all obtain with the collocation dilution of 4mM mother liquor.
(3) preparation of moving phase
The preparation of phosphate buffered saline buffer: compound concentration is all dipotassium hydrogen phosphate and the potassium dihydrogen phosphate of 12.5mM respectively, the two intermodulation pH to 7.4. Above-mentioned phosphate buffered saline buffer and trifluoroacetic acid aqueous solution are carried out isocratic elution in 88:12 ratio.
(4) derivating agent preparation:
50mg o-phthalaldehyde(OPA), 100 μ L beta-mercaptoethanols, add 300 μ L dissolve with methanol, add 1.6mL borate buffer solution (pH10.0) mixed even both. The borate buffer solution preparation of pH10.0: prepare 0.05M borax soln and 0.2M sodium hydroxide solution respectively, accurately pipettes 50mL borax soln and 23mL sodium hydroxide solution in 200mL volumetric flask, mixes, and be diluted with water to scale marks and both must.
(5) the derivative program of automatic online before post:
Drawing 2 μ l samples, 2 μ l derivating agents mix 3 times, draw 16 μ l borate buffers and mix 15 times, finally mix 5 laggard samples drawing 20 μ l moving phases. This program can represent: 2 μ l sample+2 μ l derivating agent+16 μ l borate buffer+20 μ l moving phases.After drawing, all need operation to wash pin program every time.
2. a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system according to claim 1, it is characterized in that: in step (1), described high performance liquid chromatograph is including, but not limited to Agilent, Waters, Hitachi, the main brand machines such as Dionex, but preferentially choose the Agilent1260Infinity that can carry out automatic derivatization; Described chromatographic column is including, but not limited to main flow chromatographic columns such as Agilent, Waters, Hitachi, Thermo, but preferentially chooses AgilentZORBAXSB-Aq, 5 μm, 4.6 × 250mmC18 post; Described chromatographic column is C18 reverse-phase chromatographic column, including, but not limited to 250mm, 150mm two kinds of post long sizes, but preferentially chooses 250mm specification; Described detector is including, but not limited to main brand detectors such as Agilent, Waters, Hitachi, Dionex; Described flow velocity can control at 0.8-2.0ml/min, but preferentially chooses 1.0ml/min; Described post temperature can control at 30-50 DEG C, but preferentially chooses 40 DEG C; Described determined wavelength Ex controls to control at 420-480nm at 350-400nm, Em, but preferentially to choose Ex be 338nm, Em is 450nm; Described working time and sample size can adjust flexibly with practical situation.
3. a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system according to claim 1, it is characterized in that: in step (2), described standard solution, the concentration of Ala-Gln and L-alanyl methyl esters is 0.08-0.4mM, preferentially chooses 0.2mM; The concentration of L-glutaminate is 0.04-0.2mM, preferentially chooses 0.1mM; The concentration of Pidolidone is 0.04-0.2mM, preferentially chooses 0.1mM; L-Ala-L-Ala concentration is 0.08-0.4mM, preferentially chooses 0.2mM.
4. a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system according to claim 1, it is characterized in that: in step (1) with (3), the volume ratio of described moving phase composition acetonitrile and phosphate buffered saline buffer controls between 14:86-10:90, but preferentially chooses 12:88; Described phosphate buffered saline buffer is selected from dipotassium hydrogen phosphate, potassium primary phosphate, Sodium phosphate dibasic, one or more of SODIUM PHOSPHATE, MONOBASIC, but preferentially chooses sylvite, and phosphate concentration controls at 5-20mM; Described phosphate buffered saline buffer pH should control between 7.0-7.6, but preferentially chooses 7.4, and the phosphate buffered liquid making method under this pH value condition is including, but not limited to this one method above-mentioned.
5. a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system according to claim 1, it is characterized in that: in step (4), OPA consumption and standard substance consumption mol ratio control more than 200, but preferentially choose 400; The configuration of borate buffer solution joins method including, but not limited to above-mentioned, and borate buffer solution pH value controls between 9.0-11, preferentially chooses 10.0; Methanol usage to be ensured to dissolve OPA, can adjust flexibly.
6. a kind of efficient liquid phase detection method simultaneously detecting five kinds of materials in microbial enzyme method product Ala-Gln reaction system according to claim 1, it is characterized in that: in step (5), the derivatize of standard substance can be chosen machine automatic derivatization can also be chosen manually derivative, but preferentially chooses machine automatic derivatization;Derivating agent and standard substance volume ratio can adjust according to the two concentration separately, preferentially choose 1:1; The consumption of borate buffer solution wants enough, ensures the pH value of whole derivative system, and cumulative volume and the borate buffer solution volume ratio of derivating agent and sample preferentially choose 1:4; The volume ratio of moving phase and whole derivative system is without specific requirement, and moving phase plays diluting effect, can adjust flexibly but preferentially choose 1:1. The mixed even number of times of automatic derivatization and sample size can adjust flexibly according to particular case.
7. a kind of Simultaneously test microbial enzyme method according to claim 1,2,3,4,5,6 produces the efficient liquid phase detection method of five kinds of materials in Ala-Gln reaction system, it is characterized in that detected material is including, but not limited to Ala-Gln, L-glutaminate, ALANINE methyl esters, Pidolidone, these five kinds of materials of L-Ala-L-Ala, as long as good separation can be realized under this chromatographic condition, just can be used for having the oligopeptides of free amine group with other kinds of mensuration, amino acid, and amino acid derivative.
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