Chiral detection method for N-methyl-D-valine

Abstract

The invention discloses a chiral detection method for N-methyl-D-valine, and mainly solves the technical problems that a sample ultraviolet detector is weak in absorption, isomeride is difficult to separate and the like. The detection method comprises the following steps: 1, derivation preparation, and 2, high performance liquid chromatography chiral detection, a derivation method is mainly utilized, on the basis of not changing the nature of the product, the derivation product can have strong ultraviolet absorption on a liquid chromatograph-diode array detector (DAD), so that the isomeride is detected and separated, and the detection accuracy is high. According to the N-methyl-D-valine chiral detection method disclosed by the invention, synchronous detection of the sample to be detected without interference is realized, and a reliable and convenient detection means is provided for N-methyl-D-valine chiral detection.

Description

Technical Field

[0001] The invention relates to a method for detecting the chirality of N-methyl-D-valine. Background Art

[0002] N-methyl-D-valine is an important pharmaceutical intermediate and one of the main raw materials for synthesizing 20 major amino acids. N-methyl-D-valine is also a chiral compound with L-type and D-type, but there is no conjugated group in its molecular structure. Its absorption in the UV detector is very weak, and its ee value content determines the ee value of the finished API. In the existing chiral purity detection process, due to the weak UV absorption of the product itself, the product cannot be distinguished from the isomers during the chiral detection process, resulting in the inability to determine whether the product has achieved 100% separation during the splitting process; therefore, accurate detection of chiral N-methyl-D-valine is crucial. Summary of the invention

[0003] The present invention provides a method for detecting the chirality of N-methyl-D-valine, which mainly solves the technical problems of weak absorption of the sample by the ultraviolet detector and difficulty in separating isomers. Since the N-methyl-D-valine product has weak absorption in the ultraviolet detector and cannot completely separate isomers, the idea of ​​the present invention is to use a derivatization reaction to make a chemical transformation to convert the compound into a substance with a similar chemical structure, and the new chemical properties thus generated can be used for quantification or separation. The function of the derivatization of the sample is mainly to convert the difficult-to-analyze substance into a substance with a similar chemical structure but easy to analyze, which is convenient for quantification and separation, and provides reliable data for the chiral purity of the final N-methyl-D-valine.

[0004] The present invention adopts the following technical scheme: a method for detecting the chirality of N-methyl-D-valine, which comprises the following steps: Step 1: Add N-methyl-D-valine sample to the reaction bottle, add sodium bicarbonate aqueous solution until the pH value is alkaline, then add derivatization reagent NA-(2,4-dinitro-5-fluorophenyl)-L-alanine, heat to 50°C, keep at 50°C for 30 minutes, after the reaction is complete, confirm the reaction endpoint by TLC spot plate, and finally filter and concentrate to obtain the derivatized product; Step 2: Prepare the reagents for measurement, including acetonitrile-chromatographic grade, formic acid-chromatographic grade, derivatization reagent-NA-(2,4-dinitro-5-fluorophenyl)-L-alanine, sodium bicarbonate-industrial grade, and distilled water-Watsons.

[0005] Step 3: Weigh the product sample of step 1 and place it in a volumetric flask, add pure acetonitrile, dissolve it by ultrasonic at room temperature, and finally filter the sample with a polypropylene organic filter membrane and perform HPLC analysis.

[0006] Step 4, high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature, the automatic sample injector, the chromatographic column is: a chiral chromatographic column of Daicel, the column temperature is 30°C, the flow rate is 1.0mL / min, the mobile phase A is a 0.02% formic acid aqueous solution by mass percentage concentration; the mobile phase B is a 100% acetonitrile solution by mass percentage concentration, and a gradient elution program is used. The gradient elution program is: 0-30min, the volume ratio of the mobile phase B is 25% to 65%, 30-30.1 min, the volume ratio of the mobile phase B is from 65% to the initial ratio of 25%, 30.1-40.0 min, the volume ratio of the mobile phase B is from 25% to the equilibrium system.

[0007] The chromatograph used in the liquid chromatography determination is an Agilent 1260 chromatograph, which uses an ultraviolet detector.

[0008] The present invention has the following beneficial effects: the purity of the chiral detection method of N-methyl-D-valine is determined by the present invention, the method is reliable, accurate, has good stability, the derivatization process is relatively simple, and the chiral compound peak has good symmetry; the present invention has found that the reaction of N-methyl-D-valine with the derivatization reagent-NA-(2,4-dinitro-5-fluorophenyl)-L-alanine at 50°C under alkaline conditions during the derivatization preparation process is a chemical transformation that converts the compound into a substance with a similar chemical structure. Thus, there is a strong absorption on the diode array detector, and at the same time, the normal phase chiral column CHIRALPAK® OJ of Daicel is used as a reverse phase coated chiral chromatographic column of polysaccharide derivatives, and the characteristic of cellulose-tri(4-methylbenzoate coated on the surface of silica gel can well separate the isomers of the derivatives, which provides a basis for the detection of the chiral purity of N-methyl-D-valine. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Spectrum of mixed sample detected by Agilent 1260 DAD detector.

[0010] Figure 2 Chiral spectrum of a single sample after derivatization with N-methyl-D-valine detected by Agilent 1260 DAD detector.

[0011] Figure 3 Chiral spectra of single samples of N-methyl-D-valine derivatized with Agilent 1260 DAD detector. DETAILED DESCRIPTION

[0012] The measuring process of the present invention is further described below by way of examples.

[0013] Main instruments: Agilent 1260 high performance liquid chromatograph, equipped with a diode array detector (DAD), a chiral CHIRALPAK® OJ column of 4.6 mm × 150 mm, 5.0 μm; an ultrasonic extractor (KQ5200E, Zhengzhou Ketai Experimental Equipment Co., Ltd.), a 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instrument Co., Ltd.), and an electronic analytical balance (CPA225D, Sartorius Scientific Instrument (Shanghai) Co., Ltd.).

[0014] Main reagents: The reagents used for the measurement included acetonitrile (chromatographic grade, Merck, Germany), formic acid (chromatographic grade, Shanghai Sinopharm), sodium bicarbonate (industrial grade, Shanghai Sinopharm), derivatization reagent-NA-(2,4-dinitro-5-fluorophenyl)-L-alanine (reagent grade, Shanghai McLean Biochemical Technology Co., Ltd.), and distilled water (Chlorhexidine).

[0015] Sample derivatization reaction: add 10g N-methyl-D-valine sample to the reaction bottle, add 50ml sodium bicarbonate aqueous solution until the pH value is alkaline, then add 25ml derivatization reagent-NA-(2,4-dinitro-5-fluorophenyl)-L-alanine acetone solution, heat to 50℃, keep at 50℃ for 30 minutes, after the reaction is complete, confirm the reaction endpoint by TLC spot plate, and finally filter and concentrate to obtain the derivatized product; Sample pretreatment: Weigh about 10 mg of the N-methyl-D-valine derivatized sample, place it in a 5 mL volumetric flask, add pure water, and dissolve it by ultrasonic at room temperature for 3 min. Finally, filter the sample with a 0.22 µm polypropylene organic filter membrane and perform HPLC analysis. Chromatographic conditions: The sample chamber temperature is room temperature, the injection volume of the automatic sampler is 10 µL, the chromatographic column is: Daicel's chiral, CHIRALPAK® OJ chromatographic column size is 4.6mm ×150mm, 5.0μm; column temperature: 30 °C, flow rate: 1.0mL / min, the mobile phase is a 0.02% by mass concentration of formic acid aqueous solution (A) and a 100% by mass concentration of acetonitrile solution (B); a gradient elution program is used, and the gradient elution program is: 0-30min, the volume ratio of mobile phase B is 25% to 65%, 30-30.1min, the volume ratio of mobile phase B is from 65% to the initial ratio of 25%, 30.1-40.0 min, the volume ratio of mobile phase B is from 25% to the equilibrium system.

[0016] Selection and optimization of sample pretreatment methods: Currently, N-methyl-D-valine is in solid crystal form on the market. The sample measured in the present invention is N-methyl-D-valine produced by Jier Biochemical (Shanghai) Co., Ltd.

[0017] Optimization of chromatographic conditions: The present invention uses the reverse phase system A phase-0.1% TFA water-mass percentage concentration, B phase-0.1% TFA acetonitrile, A phase-mass percentage concentration 0.02% formic acid-mass percentage concentration, B phase-100% acetonitrile for gradient elution, and compares the separation effect during elution. Using A phase-mass percentage concentration 0.02% formic acid-mass percentage concentration, B phase-100% acetonitrile for gradient elution, the peak symmetry of the isomers meets the standard, and the separation, sensitivity and peak shape of the target compound are better than other elution systems. Therefore, this experiment uses a mass percentage concentration of 0.02% formic acid aqueous solution (A) and a mass percentage concentration of 100% acetonitrile solution (B) for gradient elution.

[0018] The present invention also compares the chromatographic column of Daicel CHIRALPAKR IB-3 (4.6mm×150mm, 5.0μm) and the chromatographic column of Daicel CHIRALPAKR OJ (4.6mm×150mm, 5.0μm). The experimental results show that the separation degree of the former chromatographic column does not reach the baseline level separation, and there is a tailing phenomenon, while the chiral chromatographic column of Daicel CHIRALPAKR OJ (4.6mm×150mm, 5.0μm) can completely separate the main peak and isomers of the derivatized sample of N-methyl-D-valine, and the peaks are symmetrical and narrow. Therefore, the present invention adopts the chiral chromatographic column of Daicel CHIRALPAKR OJ (4.6mm×150mm, 5.0μm).

[0019] The temperature of the sample chamber of the present invention is set to room temperature, which is beneficial to the preservation of the solvent in the sample chamber. The temperature of the column oven is set to 30°C in the present invention.

[0020] Determination of actual samples: The results of the detection of N-methyl-D-valine derivative samples using this method show that this method is rapid, accurate, highly sensitive, and has good selectivity, and has practical application value. Figure 1 , the chiral spectrum of a single sample after derivatization with N-methyl-D-valine is shown in Figure 2 The chiral spectra of the corresponding isomers derived from N-methyl-D-valine are shown in Figure 3 .

[0021] in conclusion: The present invention establishes a method for determining the chirality of N-methyl-D-valine by detecting the chirality of a derivative sample of N-methyl-D-valine. The method successfully separates isomer chiral compounds after derivatization, and is accurate and stable.

Claims

1. A method for detecting the chirality of N-methyl-D-valine; characterized in that: The following steps are involved: Step 1, add N-methyl-D-valine sample to the reaction bottle, add sodium bicarbonate aqueous solution until the pH value is alkaline, then add derivatization reagent NA-(2,4-dinitro-5-fluorophenyl)-L-alanine, heat to 50°C, keep warm for 30 minutes, after the reaction is complete, confirm the reaction endpoint by TLC spot plate, and finally filter and concentrate to obtain the derivatized product; Step 2: Preparation of sample solution: weigh the product sample and place it in a volumetric flask, add pure acetonitrile, dissolve it by ultrasonic at room temperature, and finally filter the sample with a polypropylene organic filter membrane and perform HPLC analysis; Step three, perform high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature, automatic sample injector, the chromatographic column is: Daicel chiral chromatographic column, column temperature: 30°C, flow rate: 1.0 mL / min, mobile phase A is 0.02% by mass concentration of formic acid aqueous solution; mobile phase B is 100% by mass concentration of acetonitrile solution, and a gradient elution program is used.

2. The method for detecting chiral N-methyl-D-valine according to claim 1, characterized in that: The chromatograph used in the liquid chromatography determination is an Agilent 1260 liquid chromatograph, which uses a diode array detector.

3. The method for detecting the chirality of N-methyl-D-valine according to claim 1, characterized in that: The Daicel chiral chromatographic column is CHIRALPAKR OJ, and the size of the chromatographic column is 4.6 mm × 150 mm, 5.0 μm.

4. The method for detecting chiral N-methyl-D-valine according to claim 1, characterized in that: The gradient elution program is as follows: 0-30 min, the volume ratio of mobile phase B is 25% to 65%, 30-30.1 min, the volume ratio of mobile phase B is from 65% to the initial ratio of 25%, 30.1-40.0 min, the volume ratio of mobile phase B is from 25% to the equilibrium system.

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