CN115728422A

CN115728422A - N- (5-oxo-L-prolyl) -L-glutamic acid purity detection method

Info

Publication number: CN115728422A
Application number: CN202211629941.2A
Authority: CN
Inventors: 徐红岩; 夏伟冬; 叶敏; 彭丽勤
Original assignee: Shanghai Jifeng Biotechnology Co ltd
Current assignee: Shanghai Jifeng Biotechnology Co ltd
Priority date: 2022-12-19
Filing date: 2022-12-19
Publication date: 2023-03-03

Abstract

The invention discloses a method for detecting the purity of N- (5-oxo-L-prolyl) -L-glutamic acid, which mainly solves the technical problems of complicated steps in the chemical analysis process required by the existing N- (5-oxo-L-prolyl) -L-glutamic acid purity detection and complicated operation caused by sample pretreatment. The determination method comprises the following steps: firstly, preparing a measuring reagent, and secondly, carrying out high performance liquid chromatography measurement, wherein an ultraviolet detector is adopted in the process of liquid chromatography measurement, and a phosphoric acid water solution with the mass percentage concentration of 0.3% is added into a high performance liquid chromatography water phase, so that a sample can be well ensured not to be interfered; the invention realizes the undisturbed synchronous detection of the sample to be detected and provides a reliable and convenient detection means for the purity detection of the N- (5-oxo-L-prolyl) -L-glutamic acid.

Description

N- (5-oxo-L-prolyl) -L-glutamic acid purity detection method

Technical Field

The invention relates to a glutamic acid purity detection method, in particular to an N- (5-oxo-L-prolyl) -L-glutamic acid purity detection method.

Background

In the conventional N- (5-oxo-L-prolyl) -L-glutamic acid purity detection, a sample is pretreated by using a chemical reagent formaldehyde, then a pH indicator is added by using an acidimetry method, a sodium hydroxide standard solution is used for titration, and the end point judgment is carried out and the purity is calculated by controlling the pH value; the link is complex, the time consumption is long, and the error control of the final result is difficult.

Disclosure of Invention

The invention provides a method for detecting the purity of N- (5-oxo-L-prolyl) -L-glutamic acid, which mainly solves the technical problems that a sample needs pretreatment, the link is complex, the time consumption is long, the final calculation result is wrong, and the like.

The invention adopts the following technical scheme: the method for detecting the purity of the N- (5-oxo-L-prolyl) -L-glutamic acid comprises the following steps:

step one, preparing a measuring reagent, wherein the measuring reagent comprises acetonitrile-chromatographic purity, ultrapure water, chemical reagent orthophosphoric acid and analytically pure phosphoric acid.

Step two: and (3) preparing a sample solution, weighing a sample, placing the sample in a volumetric flask, adding pure water, dissolving the sample by ultrasonic waves at room temperature, metering volume by using acetonitrile, and finally filtering the sample by using a polypropylene organic filter membrane and performing HPLC analysis.

And step three, performing high performance liquid chromatography determination, wherein the chromatographic determination process comprises the following steps: the chromatographic condition is that the temperature of the sample room is normal temperature, the sample injection volume of the automatic sample injector is 100 mu L, and the chromatographic column is as follows: sielc Primesep 100, U.S. column size 4.6mm × 250mm, 5.0 μm, column temperature: 40. DEG C, flow rate: 1.0mL/min, mobile phase at 0.3% by mass ₃ PO ₄ An aqueous solution (A) and an acetonitrile solution (B) having a mass percentage concentration of 100%; a gradient elution procedure was used: 0-15min, 5-75% of V (B), 15-15.1 min, 75-5% of V (B), 15.1-20 min, 5% of V (B) balance system; that is, the main component of N- (5-oxo-L-prolyl) -L-glutamic acid was collected from the peak of the chromatogram with a retention time of 3.5 min.

The chromatograph adopted during the liquid chromatogram determination is an Agilent 1260 chromatograph, and the chromatograph adopts an ultraviolet detector.

The invention has the following beneficial effects: the method for detecting the purity of the N- (5-oxo-L-prolyl) -L-glutamic acid has the advantages of simple, convenient, rapid and accurate operation, good stability and good peak symmetry; the invention mainly utilizes the characteristic that Primesep 100 is a chromatographic column with a mixed stationary phase, researches show that the Primesep has hydrophobic action force and electrostatic action force, increases the function group of bonded weak-acid ion pair through cation exchange action, and simultaneously utilizes the characteristic that a reversed-phase chromatographic system can retain neutral compounds, and the production process of the N- (5-oxo-L-prolyl) -L-glutamic acid is to extract in a water phase, sequentially wash by using 1N HCl, saturated NaHCO3 and saturated NaCl, add petroleum ether and ethyl acetate mixed solution with a certain proportion after decompression and evaporation to dryness, and pulp and filter out a product; it is a hydrophobic compound, and is difficult to elute and separate by using a conventional C18 silica gel chromatographic column; therefore, the hydrophilic property of the product is utilized to perform the function of separating the isomer from the compound with similar structure through the cation exchange function, and the combination of the two has unexpected separation effect; meanwhile, the recovery rate can reach the maximum value when the phosphoric acid is separated in a mobile phase of a phosphoric acid system with the mass percentage concentration of 0.3 percent, and the recovery effect is good.

Drawings

FIG. 1 spectrum of detection on a Sielc Primesep 100 (4.6 mm. Times.250 mm, 5.0 μm) column, USA.

FIG. 2 is a graph of the linear range, linear equation and correlation coefficient of N- (5-oxo-L-prolyl) -L-glutamic acid according to the present invention.

Detailed Description

The following examples further illustrate the assay procedure of the present invention.

The main apparatus is as follows:

an Agilent 1260 type high performance liquid chromatograph equipped with an ultraviolet detector, american Sielc Primesep 100 chromatographic column size of 4.6mm × 250mm, 5.0 μm; an ultrasonic extractor (KQ 5200E, zhengzhou Ketai laboratory instruments Co., ltd.), a 0.22 μm polypropylene organic filter membrane (Shanghai Ann spectral science instruments Co., ltd.), and an electronic analytical balance (CPA 225D, sadoris scientific instruments (Shanghai) Co., ltd.).

The main reagents are as follows:

the reagents measured included acetonitrile-chromatographically pure, reagent grade orthophosphoric acid, ultrapure water with a resistivity of 18.2M Ω. Cm; acetonitrile (chromatographically pure, merck, germany), orthophosphoric acid (analytically pure, shanghai national pharmaceuticals); ultrapure water (resistivity 18.2M Ω. Cm, dou.S.Dreher).

Pretreatment of a sample:

weighing about 25mg of an N- (5-oxo-L-prolyl) -L-glutamic acid sample, placing the sample in a 10 mL volumetric flask, adding 8 mL of pure water, ultrasonically dissolving the sample for 3 min at room temperature, then diluting the volume to 10 mL by using acetonitrile, and finally filtering the sample by using a 0.22 mu m polypropylene organic filter membrane and performing HPLC analysis;

chromatographic conditions are as follows:

the temperature of the sample room is room temperature, the sample injection volume of the automatic sample injector is 10 mu L, and the chromatographic column is as follows: sielc Primesep 100, U.S. A size of 4.6mm X250 mm, 5.0 μm, column temperature: 40. DEG C, flow rate: 1.0mL/min, wherein the mobile phase comprises orthophosphoric acid with the mass percentage concentration of 0.3 percent, a water solution (A) and acetonitrile (B) with the mass percentage concentration of 100 percent; a gradient elution procedure was used: 0 to 15min, 5 to 75 percent of V (B), 15 to 15.1 min, 75 to 5 percent of V (B) in initial proportion, 15.1 to 20 min, 5 percent of V (B) in balance system.

Selecting and optimizing a sample pretreatment method:

currently, N- (5-oxo-L-prolyl) -L-glutamic acid is in a solid crystal form on the market, and the sample to be measured by the invention is N- (5-oxo-L-prolyl) -L-glutamic acid produced by Gill Biochemical (Shanghai) Co.

Optimization of chromatographic conditions:

in the present invention, the separation effects of 0.1% by mass of TFA water-0.1% by mass of TFA acetonitrile and 0.3% by mass of orthophosphoric acid-100% by mass of acetonitrile as mobile phases were compared in the selection. When the TFA water-based assay was carried out in the TFA acetonitrile system in a mass percentage of 0.1% or 0.1%, the chromatographic peak shape was asymmetric and the tailing was observed; when the orthophosphoric acid with the mass percentage concentration of 0.3 percent and the acetonitrile with the mass percentage concentration of 100 percent are adopted, the peak type symmetry degree of the N- (5-oxo-L-prolyl) -L-glutamic acid reaches the standard, and the separation degree, the sensitivity and the peak type of the target compound are superior to those of other leaching systems. Therefore, in the experiment, orthophosphoric acid with the mass percentage concentration of 0.3 percent and acetonitrile with the mass percentage concentration of 100 percent are used as leacheate, and chromatographic separation is carried out by gradient elution.

The invention also compares the U.S. Waters spuresil C18 (4.6 mm. Times.150 mm, 5.0 μm) column with the U.S. Sielc Primesep 100 (4.6 mm. Times.250 mm, 5.0 μm) column in selecting the column, and the experimental results show that the previous column can also separate peaks, but the response is small, the half-peak width is large, the tailing is serious, while the U.S. Sielc Primesep 100 (4.6 mm. Times.250 mm, 5.0 μm) column can completely separate the main peak of N- (5-oxo-L-prolyl) -L-glutamic acid from impurities, the peaks are symmetrical and the peaks are narrow, and the detection limit of the target compound can be reduced (see FIG. 1). Therefore, the present invention uses a Sielc Primesep 100 (4.6 mm. Times.250 mm, 5.0 μm) chromatography column in the United states.

The temperature of the sample chamber is set to be room temperature, so that the acetonitrile solvent in the sample chamber can be stored conveniently, and the temperature of the column incubator is set to be 40 ℃ in the invention.

Linear range:

the method is characterized in that solutions with different volumes of Sielc Primesep 100 (4.6 mm multiplied by 250mm, 5.0 mu m) in the United states are prepared into 1mg/ml, 2mg/ml, 3mg/ml and 4mg/ml, and the measurement is carried out according to the chromatographic conditions, wherein the mass concentration (x, mu g/L) of the components is used as a horizontal coordinate, and the peak area (y) of the corresponding mass spectrum peak is used as a vertical coordinate to obtain a standard curve. The results show that: n- (5-oxo-L-prolyl) -L-glutamic acid has a good linear relationship within a certain range. See fig. 2.

Precision and recovery:

for comparison of precision and recovery according to the invention, N- (5-oxo-L-prolyl) -L-glutamic acid recovery was chosen, and was determined within 24 hours according to the method described above, with each concentration level being determined 2 times separately, and the recovery and the relative standard deviation are given in the table below. The following table shows that the recovery rate of N- (5-oxo-L-prolyl) -L-glutamic acid is 99.1-99.8%, and the relative standard deviation is 0.1-0.4%, which indicates that the method has better recovery rate and repeatability for N- (5-oxo-L-prolyl) -L-glutamic acid and can meet the analysis requirements of actual samples.

Determination of the actual sample:

the detection result of the method for the N- (5-oxo-L-prolyl) -L-glutamic acid sample shows that the method is quick and accurate, has high sensitivity, good selectivity and short detection period, and has practical application value.

And (4) conclusion:

the invention establishes a high performance liquid chromatography for measuring N- (5-oxo-L-prolyl) -L-glutamic acid. The method is simple, convenient, rapid and accurate to operate, has good stability, is suitable for detecting the purity of the N- (5-oxo-L-prolyl) -L-glutamic acid, provides a new method, and has certain practical significance.

Claims

The method for detecting the purity of the N- (5-oxo-L-prolyl) -L-glutamic acid is characterized by comprising the following steps: the method comprises the following steps:

step one, preparing a measuring reagent: the measured reagents comprise acetonitrile-chromatographic purity, ultrapure water, chemical reagent orthophosphoric acid and analytically pure phosphoric acid;

step two, preparing a sample solution, weighing a sample, placing the sample in a volumetric flask, adding pure water, dissolving the sample by ultrasonic waves at room temperature, adding acetonitrile to a constant volume, and finally filtering the sample by a polypropylene organic filter membrane and performing HPLC analysis;

step three, performing high performance liquid chromatography determination: the chromatographic determination process comprises the following steps: the chromatographic conditions are that the temperature of a sample room is normal temperature, an automatic sample injector is used, and a chromatographic column is as follows: column temperature of Sielc corporation, usa: 40. DEG C, flow rate: 1.0mL/min, wherein the mobile phase A is orthophosphoric acid aqueous solution with the mass percentage concentration of 0.3 percent; the mobile phase B is acetonitrile solution with the mass percentage concentration of 100 percent, and a gradient elution procedure is adopted.
2. The method for detecting the purity of N- (5-oxo-L-prolyl) -L-glutamic acid according to claim 1, wherein: the chromatograph adopted during the liquid chromatogram determination is an Agilent 1260 liquid chromatograph which adopts an ultraviolet detector.
3. The method for detecting the purity of N- (5-oxo-L-prolyl) -L-glutamic acid according to claim 1, wherein: the American Sielc column was Primesep 100, with column dimensions of 4.6mm by 250mm, 5.0 μm.
4. The method for detecting the purity of N- (5-oxo-L-prolyl) -L-glutamic acid according to claim 1, wherein: the gradient elution procedure is as follows: 0 to 15min, 5 to 75 percent of V (B), 15 to 15.1 min, 75 to 5 percent of V (B) in initial proportion, 15.1 to 20 min, 5 percent of V (B) in balance system.