The molecular weight of N-FMOC-Napos is 100-200; method for detecting purity of-[1-(4, 4-dimethyl-2, 6-dioxocyclohexyl) ethyl]-L-lysine

Abstract

The invention discloses a method for detecting the purity of N-FMOC-N '-[1-(4, 4-dimethyl-2, 6-dioxocyclohexyl) ethyl]-L-lysine, and mainly solves the technical separation of related byproducts generated in the production process. The determination method comprises the following steps: 1, preparation of a measurement reagent; 2, pretreatment of a sample; 3, high performance liquid chromatography determination is carried out, a chromatographic instrument is an Agilent 1260 chromatographic instrument, a VWD ultraviolet detector is adopted, a chromatographic column adopts Waters Sun Fire C18, and a mobile phase is a methyl aqueous solution (A) with the molar concentration being 0.05% and a formic acid acetonitrile solution (B) with the mass percentage concentration being 0.05%. According to the invention, a reliable and convenient detection means is provided for the field of purity detection of N-FMOC-N '-[1-(4, 4-dimethyl-2, 6-dioxocyclohexyl) ethyl]-L-lysine.

Description

Technical Field

[0001] The present invention relates to a method for detecting the purity of substituted lysine, in particular to a method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine. Background Art

[0002] As an important intermediate of API, N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine has strict control over the by-products Fmoc-Lys(Fmoc)-OH, H-Lys(ivdde)-OH and ivdde-OH produced in the production. Conventional HPLC detection methods cannot accurately separate the by-products Fmoc-Lys(Fmoc)-OH, H-Lys(ivdde)-OH and ivdde-OH impurities produced in the production, and there is currently no good solution. Summary of the invention

[0003] The present invention provides a method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine, which mainly solves the technical problems of baseline separation of by-products Fmoc-Lys(Fmoc)-OH, H-Lys(ivdde)-OH and ivdde-OH generated in production, thereby accurately controlling the residual amount of by-products and the like.

[0004] The present invention adopts the following technical scheme: a method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine, comprising the following steps: Step 1: Prepare the reagents for measurement: acetonitrile-chromatographic grade, ultrapure water, chromatographic grade-formic acid.

[0005] Step 2: Preparation of sample solution: Weigh the sample into a volumetric flask, add acetonitrile, dissolve it by ultrasonic at room temperature, and then make up to volume with water. Finally, filter the sample with a polypropylene organic filter membrane and perform HPLC analysis.

[0006] Step 3, perform high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature ℃, the automatic sampler injection volume is 10 µL, the chromatographic column is Waters Sun Fire C18, the column temperature is 35 ℃, the flow rate is 1.0 mL / min, the mobile phase A is a molar concentration of 0.05% formic acid aqueous solution; the mobile phase B is a mass percentage concentration of 0.05% formic acid acetonitrile solution, and a gradient elution program is used.

[0007] The chromatograph used in the liquid chromatography determination is an Agilent 1260 chromatograph, which uses a VWD ultraviolet detector. The size of the chromatographic column is 4.6mm × 150mm, 3.5μm. The gradient elution program is: 0-12min, the volume ratio of mobile phase B is from 50% to 85%, 12-12.1 min, the volume ratio of mobile phase B is from 85% to the initial ratio of 50%, 12.1-20min, the volume ratio of mobile phase B is from 50% to the equilibrium system.

[0008] The invention has the following beneficial effects: the invention utilizes the accurate quantification of the automatic sample injector of the high performance liquid chromatograph, and the VWD ultraviolet detector can have a good response value for N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine, thereby solving the problem that the conventional HPLC detection method cannot accurately separate the impurities such as the by-products Fmoc-Lys(Fmoc)-OH, H-Lys(ivdde)-OH, ivdde-OH and the like generated in the production, thereby efficiently detecting the purity of the product, and achieving the effects of simple, rapid, accurate and good stability in operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Product positioning map.

[0010] Figure 2 H-Lys(ivdde)-OH impurity localization map.

[0011] Figure 3 Fmoc-Lys(Fmoc)-OH impurity localization map.

[0012] Figure 4 ivdde-OH impurity localization map. DETAILED DESCRIPTION

[0013] Main instruments: Agilent 1260 high performance liquid chromatograph, equipped with VWD UV detector, American Waters Sun Fire C18 (4.6mm ×150mm, 3.5μm); ultrasonic extractor (KQ5200E, Zhengzhou Ketai Experimental Equipment Co., Ltd.), 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instrument Co., Ltd.), electronic analytical balance (CPA225D, Sartorius Scientific Instrument (Shanghai) Co., Ltd.).

[0014] Main reagents: The reagents used for the measurement include ultrapure water with a resistivity of 18.2 MΩ.cm; acetonitrile (chromatographically pure, Merck, Germany), formic acid (chromatographically pure, Shanghai Sinopharm Company); ultrapure water (resistivity 18.2 MΩ.cm, Chlorhexidine, USA).

[0015] Sample pretreatment: Weigh N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine sample and place it in a 10ml volumetric flask, add 7ml acetonitrile, dissolve it by ultrasonic at room temperature, then add ultrapure water to make up the volume, and finally filter the sample with a polypropylene organic filter membrane for chiral detection;.

[0016] Chromatographic conditions: The sample chamber temperature is room temperature, the autosampler injection volume is 10 µL, Waters SunFire C18, column temperature: 35°C, flow rate: 1.0 mL / min, mobile phase A is a 0.05% molar concentration of formic acid aqueous solution; mobile phase B is a 0.05% mass percentage concentration of formic acid acetonitrile solution, and a gradient elution program is used. The gradient elution program is: 0-12min, the volume ratio of mobile phase B is from 50% to 85%, 12-12.1 min, the volume ratio of mobile phase B is from 85% to the initial ratio of 50%, 12.1-20min, the volume ratio of mobile phase B is from 50% to the equilibrium system.

[0017] Selection and optimization of sample pretreatment methods: Currently, N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine is a powdered solid on the market. The sample and by-product measured in the present invention are products produced by Jier Biochemical (Shanghai) Co., Ltd.

[0018] Optimization of chromatographic conditions: The present invention compares the separation effects of 0.05% molar concentration formic acid aqueous solution-0.05% mass percentage formic acid acetonitrile and 0.05% mass percentage TFA aqueous solution-100% mass percentage acetonitrile as mobile phases. When the mass percentage concentration of 0.05% TFA aqueous solution-100% mass percentage acetonitrile system is used, it is found that the chromatographic peak shape is asymmetric and easy to tail; when the molar concentration of 0.05% formic acid aqueous solution-0.05% mass percentage formic acid acetonitrile is used as the eluent system, the peak shape symmetry of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine reaches the standard, and the separation, sensitivity and peak shape of the target compound are better than other eluent systems. Therefore, this experiment uses 0.05% molar concentration formic acid aqueous solution-0.05% mass percentage formic acid acetonitrile as the eluent, and gradient elution is used for chromatographic separation.

[0019] When selecting the chromatographic column, the present invention uses Agilent Eclipse XDB-C18 (4.6mm × 150mm, 3.5μm) and waters SunFire C18 (4.6mm × 150mm, 3.5μm) for relevant comparison, and there are obvious differences in the separation degree between the main product and the related by-products, especially the by-product H-Lys(ivdde)-OH, which is not separated from the main product on the chromatographic column Agilent EclipseXDB-C18 (4.6mm × 150mm, 3.5μm), but is clearly separated on the chromatographic column of waters Sun Fire C18 (4.6mm × 150mm, 3.5μm). Therefore, this experiment uses waters Sun Fire C18 (4.6mm × 150mm, 3.5μm) for chromatographic detection.

[0020] The temperature of the sample chamber of the present invention is set to room temperature, which is beneficial to the preservation of the methanol solvent in the sample chamber. The temperature of the column oven is set to 35°C in the present invention.

[0021] Determination of actual samples: HPLC spectrum of the product is as follows Figure 1 As shown, the H-Lys(ivdde)-OH impurity localization map is shown in Figure 2 , Fmoc-Lys(Fmoc)-OH impurity localization map is shown in Figure 3 , ivdde-OH impurity localization map see Figure 4The results of chemical purity test of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine product using this method showed that this method was rapid, accurate, highly sensitive, with a symmetrical main peak shape, baseline separation from the three by-product impurity peaks, good stability, and had practical application value.

Claims

1. A method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine; wherein: The following steps are involved: Step 1: Prepare the reagents for measurement: acetonitrile-chromatographic grade, ultrapure water, chromatographic grade-formic acid; Step 2, preparation of sample solution: weigh the sample and place it in a volumetric flask, add acetonitrile, dissolve it by ultrasonic at room temperature, add ultrapure water to make up the volume, and finally filter the sample with a polypropylene organic filter membrane for chiral detection; Step three, perform high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature, automatic sample injector, chromatographic column: Waters Sun Fire C18 chromatographic column, column temperature: 35 °C, flow rate: 1.0 mL / min, mobile phase A is 0.05% molar concentration of formic acid aqueous solution; mobile phase B is 0.05% mass percentage concentration of formic acid acetonitrile solution, and a gradient elution program is used.

2. The method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine according to claim 1, characterized in that: The chromatograph used in the liquid chromatography determination is an Agilent 1260 liquid chromatograph, which uses a VWD ultraviolet detector.

3. The method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine according to claim 1, characterized in that: The size of the chromatographic column is 4.6 mm × 150 mm, 3.5 μm.

4. The method for detecting the purity of N-FMOC-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine according to claim 1, characterized in that: The gradient elution program is as follows: 0-12 min, the volume ratio of mobile phase B increases from 50% to 85%, 12-12.1 min, the volume ratio of mobile phase B decreases from 85% to an initial ratio of 50%, and 12.1-20 min, the volume ratio of mobile phase B decreases from 50% to a balanced system.

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