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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

A glutamine and microbial enzyme technology, applied in the field of analysis and testing, can solve rare problems and achieve the effect of accurate strain performance and production process

Inactive Publication Date: 2016-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are many methods for the determination of amino acids by HPLC, there are few reports on the determination of dipeptides by HPLC, and the simultaneous determination of oligopeptides, amino acids and amino acid derivatives by HPLC There are no reports of similar substances

Method used

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  • 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
  • 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
  • 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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The selection of embodiment 1 chromatographic conditions

[0032] 1.1 Selection of detection wavelength

[0033] Take the mother solution of five substance standard samples and dilute to make a 0.2mM solution. The excitation wavelength Ex is from 260nm to 400nm, and the emission wavelength Em is from 400nm to 500nm. It is found that when Ex is 338nm and Em is 450nm, all five substances have maximum absorption wavelengths, so the two are selected as detection wavelengths.

[0034] 1.2 Selection of chromatographic column

[0035] Three brands of Hitachi, Agilent, and Waters C18 reversed-phase columns were selected for the test. The results showed that the Agilent ZORBAXSB-AqC18 chromatographic column had a faster peak time, symmetrical peak shape, and high peak resolution, so it was selected as the detection column. .

[0036] 1.3 Selection of column temperature

[0037] The column temperature commonly used in high performance liquid chromatography was investigated, a...

Embodiment 2

[0048] The establishment of embodiment 2 methodology

[0049] 2.1 Linear range

[0050] Accurately measure a certain volume of 4mM mother solution of five kinds of standards, dilute it to 0.4mM with deionized water, and then measure 0.2mL, 0.4mL, 0.5mL, 0.6mL of five kinds of 0.4mM standard solution, respectively, 0.8mL, and finally make up to 1mL with deionized water to make a standard gradient solution. After automatic pre-column derivatization of five standard gradient solutions, quantitative detection was performed, chromatograms were recorded, and peak areas were investigated. The linearity of the standard curves for the five substances was satisfactory. Standard curve and linear range of five substances and their R 2 See Table 1 for the values.

[0051] Table 1: Standard curve, linear range, limit of quantification

[0052]

[0053] 2.2 Recovery rate

[0054] A real sample was used as the standard spiked sample, and three levels of low (about 50%), medium (about...

Embodiment 3

[0073] Embodiment 3 actual sample determination result

[0074] Prepare a mixed solution of L-glutamine and L-alanine methyl ester hydrochloride substrate at a certain concentration, and ferment the L-alanyl-L-glutamine production strain for 36 hours to obtain the fermentation broth or bacteria Add the body as a crude enzyme source to the mixed solution of the two substrates, adjust the pH value of the entire substrate system to about 8, place the entire substrate system at 25°C, and shake for 1-2 hours. Get a certain amount of reaction solution high-speed (≥ 12000g) centrifugation 5-10min, take the supernatant and dilute it to the linear range described in Example 2, after the on-line automatic derivation before the column, according to the chromatographic conditions described in Example 1 Perform quantitative detection, record the peak area, and calculate the yield of each substance according to the standard curve described in Example 2. The comparison chromatograms of actu...

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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 [0001] The invention discloses a high-efficiency liquid phase detection method for simultaneously detecting five substances in a reaction system for producing L-alanyl-L-glutamine by microbial enzyme method, belonging to the technical field of analysis and testing methods. Background technique [0002] L-alanyl-L-glutamine (L-alanyl-L-glutamine, L-Ala-L-Gln) is a dehydration condensation of two amino acids, L-alanine and L-glutamine. A dipeptide, referred to as CG dipeptide. L-glutamine (L-Gln), as a conditionally essential amino acid, is widely used in the pharmaceutical industry. L-glutamine can be used as a raw material for gluconeogenesis and urea synthesis, a precursor of neurotransmitters, and an important intermediate of biochemical metabolic pathways in vivo. Due to its physical and chemical properties different from L-glutamine, glutamine overcomes the disadvantages of L-glutamine's low solubility and thermal instability, so it is often used as a s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/74
CPCG01N30/02G01N30/74G01N2030/027
Inventor 张震宇魏照辉
Owner JIANGNAN UNIV
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