A method for detecting enantiomers in (s)-(1-hydroxypropan-2-yl) tert-butyl carbamate by high performance liquid chromatography

Abstract

The application discloses a method for detecting enantiomers in (S)-(1-hydroxypropan-2-yl) tert-butyl carbamate by high performance liquid chromatography. Specifically, the test product is dissolved in a diluent, and detection is performed by using a high performance liquid chromatograph, wherein a CHIRALPAK IG is used as the chromatographic column, the specification of the chromatographic column is an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 microns; a mobile phase is water-acetonitrile-tetrahydrofuran, and the volume ratio is 900:75:25; and isocratic elution is adopted. The method is simple and rapid, does not need derivatization treatment, has high sensitivity, good sample solubility, and accurate results, and is suitable for quality control of (S)-(1-hydroxypropan-2-yl) tert-butyl carbamate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the enantiomer (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester in (S)-(1-hydroxypropyl-2-yl)carbamate using high performance liquid chromatography. Background Technology

[0002] (S)-(1-Hydroxypropyl-2-yl)carbamate, with its common amino acid configuration found in nature, is directly used in the synthesis of bioactive peptide analogs, enzyme inhibitors, etc., and is a key intermediate in the synthesis of many drugs. Its chemical structure is as follows: .

[0003] As an enantiomer of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester, the effective control of its content plays a crucial role in the quality of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester. Its chemical structural formula is as follows: .

[0004] Because amino acid compounds generally have low UV response, common amino acid compounds typically employ pre-column derivatization to control their enantiomers. For example, the invention patent with patent number 2024107825719 uses Fmoc-OSU derivatization followed by chromatographic analysis, a relatively complex procedure. Furthermore, conventional normal-phase chromatography often suffers from low enantiomer response and poor solubility in high-concentration samples, making accurate determination of trace enantiomer impurities difficult. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for detecting the enantiomer (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester without complex derivatization. This method aims to assess the accuracy of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester detection, thereby enabling more precise control of the enantiomer content in the product.

[0006] The present invention is implemented using the following technical solutions: A method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography, the method comprising the following steps: 1) Dissolve and dilute the test sample (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester with a diluent to obtain the test sample solution; 2) The test solution was detected by high performance liquid chromatography with an ultraviolet detector. The chromatographic conditions were as follows: CHIRALPAK IG column, water-acetonitrile-tetrahydrofuran mixture as the mobile phase, column temperature of 30-40℃, flow rate of mobile phase of 0.9 ml / min-1.1 ml / min, injection volume of 10 μl, and isocratic elution mode.

[0007] Further, in step 1), the diluent is acetonitrile-tetrahydrofuran-water, with a volume ratio of 15:5:80.

[0008] Further, in step 1), the concentration of the test solution is 100 mg of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester per 1 ml.

[0009] Furthermore, in step 2), the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

[0010] Furthermore, in step 2), the volume ratio of water-acetonitrile-tetrahydrofuran is 900:75:25.

[0011] Furthermore, in step 2), the column temperature is 35°C and the flow rate of the mobile phase is 1.0 ml / min.

[0012] Furthermore, in step 2), the ultraviolet detection wavelength is 210 nm.

[0013] The method of this invention employs a specific reversed-phase chromatography system (water-acetonitrile-tetrahydrofuran) in conjunction with a CHIRALPAK IG chiral column, achieving effective separation of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester and its enantiomers without complex derivatization. The method is simple and rapid, and its high sensitivity meets the requirements for detecting trace enantiomeric impurities, providing accurate detection results. Attached Figure Description

[0014] Figure 1 This is the chromatogram of the test sample solution in Example 1 of the present invention; Figure 2 This is a chromatogram of the enantiomer solution in Example 1 of the present invention; Figure 3 This is the chromatogram of the test sample solution in Comparative Example 1 of the present invention; Figure 4 This is a chromatogram of the enantiomer solution in Comparative Example 1 of the present invention; Figure 5 The UV absorption spectrum of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester in Example 1 of this invention is shown. Detailed Implementation

[0015] The invention will be further described below with reference to the accompanying drawings to provide a better understanding of the technical solution. Example

[0016] Chromatographic column: CHIRALPAK IG, 250 × 4.6 mm, 5 μm Mobile phase: water-acetonitrile-tetrahydrofuran (900:75:25) Flow rate: 1.0 ml / min Column temperature: 35℃ Injection volume: 10µl Preparation of test solution: Take an appropriate amount of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester test sample, accurately weigh it, add diluent acetonitrile-tetrahydrofuran-water (15:5:80) to dissolve and quantitatively dilute to prepare a solution containing approximately 100 mg per ml.

[0017] Preparation of enantiomer solution: Weigh an appropriate amount of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester reference standard accurately, dissolve and quantitatively dilute it in acetonitrile-tetrahydrofuran-water (15:5:80) to prepare a solution containing approximately 0.2 mg per ml.

[0018] The chromatogram of the test sample in this embodiment is as follows: Figure 1 As shown, the signal-to-noise ratio of the enantiomer peak at an area percentage of 0.05% is greater than the limit of quantitation. The enantiomer solution chromatogram is shown below. Figure 2 As shown, the enantiomer exhibits good peak shape, and at a concentration of 0.2 mg / ml (equivalent to 0.2% of the test sample solution concentration), the signal-to-noise ratio is approximately 95, which can be used for the control of this isomer. The UV spectrum of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester under the detection conditions of this invention is shown below. Figure 5 As shown, the substance has good ultraviolet absorption at around 210 nm. The chromatogram shows that the method of the present invention has good sensitivity and can be used to detect the content of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester.

[0019] Comparative Example 1 Detection by reference normal phase chromatography Column: CHIRALPAK AD-H, 250 × 4.6 mm, 5 μm Mobile phase: n-Heptane:Ethanol:Ethanolamine = 95:5:0.1 Flow rate: 1.5 ml / min Column temperature: 30℃ Injection volume: 50µl Detection wavelength: 210nm Preparation of test solution: Weigh an appropriate amount of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester test sample accurately, add diluent n-heptane-ethanol-ethanolamine (95:5:0.1) to dissolve and quantitatively dilute to prepare a solution containing approximately 5 mg per ml.

[0020] Preparation of enantiomer solution: Weigh an appropriate amount of (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester reference standard accurately, add diluent n-heptane-ethanol-ethanolamine (95:5:0.1) to dissolve and quantitatively dilute to prepare a solution containing approximately 0.5 mg per ml.

[0021] Under the above detection conditions, 50 μl each of the test sample solution and the enantiomer solution were injected into the liquid chromatograph, and the chromatograms were recorded. The results are as follows: Figure 3 , Figure 4 As shown, (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester and its enantiomer (R)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester showed good separation, but the enantiomer response value was low. Increasing the sample concentration resulted in incomplete dissolution, and adjusting the additives did not improve its detection.

Claims

1. A method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography, characterized in that, The method includes the following steps: 1) Dissolve and dilute the test sample (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester with a diluent to obtain the test sample solution; 2) The test solution was detected by high performance liquid chromatography with an ultraviolet detector. The chromatographic conditions were as follows: CHIRALPAK IG column, water-acetonitrile-tetrahydrofuran mixture as the mobile phase, column temperature of 30-40℃, flow rate of mobile phase of 0.9 ml / min-1.1 ml / min, injection volume of 10 μl, and isocratic elution mode.

2. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, In step 1), the diluent is acetonitrile-tetrahydrofuran-water, with a volume ratio of 15:5:

80.

3. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, In step 1), the concentration of the test solution is 100 mg of (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester per 1 ml.

4. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, In step 2), the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

5. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, In step 2), the volume ratio of water-acetonitrile-tetrahydrofuran is 900:75:

25.

6. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, Step 2) The column temperature is 35℃ and the flow rate of the mobile phase is 1.0 ml / min.

7. The method for detecting enantiomers in (S)-(1-hydroxypropyl-2-yl)carbamate tert-butyl ester by high performance liquid chromatography as described in claim 1, characterized in that, In step 2), the ultraviolet detection wavelength is 210nm.

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