Method for detecting purity of D-tryptophan enantiomer

Abstract

The invention discloses a method for detecting the purity of a D-tryptophan enantiomer, which comprises the following steps: S1, taking D-tryptophan and a D-tryptophan enantiomer standard substance with the same mass, adding water, and dissolving to obtain a system adaptive solution; s2, adjusting chromatographic conditions by using the system adaptive solution until the separation degree of the D-tryptophan enantiomer and the D-tryptophan is greater than or equal to 2 to obtain adaptive chromatographic conditions; and S3, carrying out high performance liquid chromatography analysis on the sample solution according to the adaptive chromatographic conditions, and calculating according to the percentage of the peak area to obtain the purity of the D-tryptophan enantiomer. The method disclosed by the invention is simple in pretreatment method, short in analysis time, good in system adaptability, accurate and reliable in result and good in repeatability, can be used for effectively detecting the purity of the D-tryptophan enantiomer, and has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical detection, and specifically relates to a method for detecting the enantiomeric purity of D-tryptophan. Background Art

[0002] D-tryptophan, also known as aminoindolepropionic acid, is a white or off-white to slightly yellow crystalline powder, odorless and sweet, slightly soluble in water, insoluble in ethanol, soluble in dilute acids or alkalis, colored upon long-term light exposure, and produces a small amount of indole when heated with water. As an important essential amino acid for the human body, D-tryptophan is widely used in medical and food applications, such as in amino acid infusions, comprehensive amino acid preparations, nutritional supplements for pregnant women and special infant milk powders, food additives, etc.

[0003] In the pharmaceutical industry, D-tryptophan is an important component of many peptide-based drugs, such as tadalafil (FDA-approved for the treatment of erectile dysfunction and benign prostatic hyperplasia), macimorelin (FDA-approved for the diagnosis of adult growth hormone deficiency), triptorelin (FDA-approved for the treatment of advanced prostate cancer), parsireotide (FDA-approved orphan drug for the treatment of patients with Cushing's disease who are not suitable for surgical treatment), lanreotide (FDA-approved for the treatment of acromegaly), and octreotide (FDA-approved for the treatment of acromegaly and diarrhea associated with certain types of tumors). The purity of D-tryptophan can directly affect its efficacy or physiological effects.

[0004] D-tryptophan and its enantiomers have the same physical, thermodynamic, and chemical properties, and only show differences in a chiral environment. Due to the limitations of the production process, more or less D-tryptophan enantiomeric impurities will be present in the product of D-tryptophan production. It is difficult to effectively separate D- and L-tryptophan under conventional reversed-phase chromatography conditions, consuming a long time in detection, showing severe tailing, and having poor peak symmetry. Therefore, there is currently a need for a simple and accurate analytical detection method to meet the determination of the enantiomeric content of D-tryptophan. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for detecting the enantiomeric purity of D-tryptophan, comprising the following steps:

[0006] S1. Take equal masses of D-tryptophan and a D-tryptophan enantiomer standard product, dissolve them in water to obtain a system suitability solution;

[0007] S2. Use the system suitability solution to adjust the chromatographic conditions until the resolution of D-tryptophan enantiomers and D-tryptophan is ≥2 to obtain suitable chromatographic conditions;

[0008] S3. Perform high performance liquid chromatography (HPLC) analysis on the sample solution under the described suitability chromatographic conditions, and calculate the enantiomeric purity of D-tryptophan based on the percentage of the peak area.

[0009] In one or more embodiments, the injection sequence during HPLC analysis is ultrapure water blank control, system suitability solution, and sample solution.

[0010] In one or more embodiments, the concentrations of D-tryptophan and its enantiomer in the system suitability solution are both 1 mg / mL.

[0011] In one or more embodiments, the concentration of the sample in the sample solution is 5 mg / mL.

[0012] In one or more embodiments, the suitability chromatographic conditions are as follows:

[0013] Chromatographic column: DAICEL CROWNPAK CR(+) 4.0 mm × 150 mm, 5 μm;

[0014] Mobile phase: 0.95% perchloric acid aqueous solution with a pH of 1.0 - 1.3;

[0015] Flow rate: 1.0 mL / min;

[0016] Wavelength: 200 nm;

[0017] Column temperature: 35 °C;

[0018] Injection volume: 1 μL;

[0019] Run time: 45 min.

[0020] In one or more embodiments, under the described suitability chromatographic conditions, the theoretical plate number of D-tryptophan ≥ 4000; the theoretical plate number of the D-tryptophan enantiomer ≥ 5000.

[0021] In one or more embodiments, under the described suitability chromatographic conditions, the tailing factor of D-tryptophan is 1.0 - 1.5; the tailing factor of the D-tryptophan enantiomer is 0.8 - 1.3.

[0022] In one or more embodiments, under the described suitability chromatographic conditions, the detection limit of the D-tryptophan enantiomer is 0.009%, and the quantitation limit is 0.03%.

[0023] In one or more embodiments, under the described suitability chromatographic conditions, the retention time of D-tryptophan is approximately 31.166 min, and the retention time of the D-tryptophan enantiomer is approximately 38.823 min.

[0024] In one or more embodiments, when performing high performance liquid chromatography (HPLC) analysis on a sample solution under the described adaptable chromatographic conditions, parallel samples are also set, and the absolute value of the error in the calculated results of parallel determination is not greater than 0.03%.

[0025] The method of the present invention has a simple pretreatment method, a short analysis time, good system adaptability, accurate and reliable results, and good repeatability. It can effectively detect the purity of D-tryptophan enantiomers and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the liquid chromatogram of the D-tryptophan standard solution in Example 1.

[0027] Figure 2 It is the liquid chromatogram of the D-tryptophan enantiomer standard solution in Example 1.

[0028] Figure 3 It is the liquid chromatogram of the sample #1 solution in Example 3.

[0029] Figure 4 It is the liquid chromatogram of the sample #2 solution in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] To overcome the shortcomings of the prior art, the present invention provides a method for detecting the purity of D-tryptophan enantiomers, which includes the following steps:

[0031] S1. Take equal masses of D-tryptophan and D-tryptophan enantiomer standard and dissolve them in water to obtain a system suitability solution.

[0032] S2. Use the system suitability solution to adjust the chromatographic conditions until the resolution between D-tryptophan enantiomers and D-tryptophan is ≥2 to obtain adaptable chromatographic conditions.

[0033] S3. Perform high performance liquid chromatography analysis on the sample solution under the described adaptable chromatographic conditions and calculate the purity of D-tryptophan enantiomers as a percentage of the peak area.

[0034] In one or more embodiments, the injection sequence during high performance liquid chromatography analysis is ultrapure water blank control, system suitability solution, and sample solution.

[0035] In one or more embodiments, the concentrations of D-tryptophan and D-tryptophan enantiomers in the system suitability solution are both 1 mg / mL.

[0036] In one or more embodiments, the method for preparing the system suitability solution is as follows: Weigh 0.025 g ± 0.001 g of D-tryptophan standard (accurate to 0.0001 g) and 0.025 g ± 0.001 g of D-tryptophan enantiomer (accurate to 0.0001 g) into a 25-mL volumetric flask, dissolve with ultrapure water, dilute to the mark, and filter through a 0.22-μm water-based syringe filter for standby.

[0037] In one or more embodiments, the concentration of the sample in the sample solution is 5 mg / mL.

[0038] In one or more embodiments, the method for preparing the sample solution is as follows: Weigh 0.25 g ± 0.01 g of the sample (accurate to 0.0001 g) into a 50-mL volumetric flask, dissolve with ultrapure water, dilute to the mark, and filter through a 0.22-μm water-based syringe filter for standby.

[0039] In one or more embodiments, the system suitability chromatographic conditions are as follows:

[0040] Chromatographic column: DAICEL CROWNPAK CR(+) 4.0 mm × 150 mm, 5 μm;

[0041] Mobile phase: 0.95% perchloric acid aqueous solution with a pH of 1.0 - 1.3;

[0042] Flow rate: 1.0 mL / min;

[0043] Wavelength: 200 nm;

[0044] Column temperature: 35 °C;

[0045] Injection volume: 1 μL;

[0046] Running time: 45 min.

[0047] In one or more embodiments, under the system suitability chromatographic conditions, the theoretical plate number of D-tryptophan ≥ 4000; the theoretical plate number of D-tryptophan enantiomer ≥ 5000.

[0048] In one or more embodiments, under the system suitability chromatographic conditions, the tailing factor of D-tryptophan is 1.0 - 1.5; the tailing factor of D-tryptophan enantiomer is 0.8 - 1.3.

[0049] In one or more embodiments, under the system suitability chromatographic conditions, the detection limit of D-tryptophan enantiomer is 0.009%, and the quantitation limit is 0.03%.

[0050] In one or more embodiments, under the described adaptive chromatographic conditions, the retention time of D-tryptophan is approximately 31.166 min, and the retention time of the D-tryptophan enantiomer is approximately 38.823 min.

[0051] In the present invention, during the actual detection process, there will be some variations in the retention times of D-tryptophan and the D-tryptophan enantiomer, but these are all acceptable within the art. The specific reasons are as follows: The chromatographic column used is a chiral column and is very sensitive to slight changes in pH. Although the pH value is detected with a pH meter each time the mobile phase is prepared to make it basically consistent, there are still slight differences; moreover, different temperatures of the mobile phase will also cause this situation to occur. For example, there will be slight differences in the peak emergence when preparing samples in spring and summer. Therefore, when using the detection method of the present invention, an isomer standard will be run together each time, the position of the isomer peak can be found, and this situation is acceptable using the peak area percentage method for the isomer chromatogram.

[0052] In one or more embodiments, when performing high performance liquid chromatography analysis on the sample solution under the described adaptive chromatographic conditions, parallel samples are also set, and the absolute value of the error in the calculation results of the parallel determination is not greater than 0.03%. The injection sequence in this case is: 1 blank injection, 1 injection of the system suitability solution, 1 injection each of the sample solution and the parallel test, and then the chromatogram is recorded.

[0053] The method of the present invention has a simple pretreatment method, a short analysis time, good system suitability, accurate and reliable results, and good repeatability. It can effectively detect the purity of the D-tryptophan enantiomer and has very good application prospects.

[0054] Embodiment

[0055] The following further describes the present invention in detail with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0056] The embodiments involve the addition amounts, contents, and concentrations of various substances. Among them, the percentage content, unless otherwise specified, all refers to the mass percentage content.

[0057] The instruments, reagents, and related conditions used in the following examples and comparative examples:

[0058] (1) Instruments:

[0059] Agilent high performance liquid chromatograph 1260 (or similar type), electronic balance (accurate to 0.0001 g).

[0060] (2) Reagents: Perchloric acid (AR or GR), ultrapure water.

[0061] Preparation of 0.95% perchloric acid aqueous solution: Add 9.5 mL of 70% perchloric acid dropwise to 1000 mL of ultrapure water under stirring. Measure its pH with a pH meter, which is about 1.0 - 1.3. After standing for 15 min, shake it again and measure the pH.

[0062] (3) Samples

[0063] Sample #1: A mixture containing D-tryptophan and its enantiomer (the content of D-tryptophan is 99.98%), sourced from a sample manufactured by Luoyang Huarong Biotechnology Co., Ltd.

[0064] Sample #2: A mixture containing D-tryptophan and its enantiomer (the content of D-tryptophan is 99.94%), sourced from a sample manufactured by Luoyang Huarong Biotechnology Co., Ltd.

[0065] The detection method for the content of D-tryptophan in the above Sample #1 and Sample #2 is as follows:

[0066] Use an Agilent SB Aq chromatographic column, with a specification of 5um, 4.6mm * 250mm. The mobile phase is 0.1% perchloric acid aqueous solution: acetonitrile = 75:25, the flow rate is 1 ml / min, the wavelength is 220 nm, the column temperature is 30 °C, and the external standard method is used for calculation. In the formula:

[0067] δ—Content of D-tryptophan in the sample (%);

[0068] m b —Mass of the D-tryptophan standard sample weighed (g);

[0069] δ b —Content of the D-tryptophan standard sample (%);

[0070] S—Peak area of D-tryptophan in the sample (mv·s);

[0071] m—Mass of the D-tryptophan sample weighed (g);

[0072] S b —Peak area of the D-tryptophan standard sample (mv·s);

[0073] h b —Loss on drying content of the D-tryptophan standard sample (%);

[0074] h—Loss on drying content of the D-tryptophan sample %.

[0075] The detection method for the loss on drying content of the sample follows USP<731>.

[0076] (4) Chromatographic conditions adopted in the following examples

[0077] Chromatographic column: DAICEL CROWNPAK CR(+) 4.0 mm × 150 mm, 5 μm

[0078] Mobile phase: 0.95% perchloric acid aqueous solution (pH about 1.0 - 1.3)

[0079] Flow rate: 1.0 mL / min

[0080] Column temperature: 35 °C

[0081] Wavelength: 200 nm

[0082] Sample injection volume: 1 μL

[0083] Running time: 45 min

[0084] Diluent: ultrapure water

[0085] Blank solution: ultrapure water

[0086] (5) Solution preparation:

[0087] System suitability solution: Weigh 0.025 g ± 0.001 g of D - tryptophan reference standard (accurate to 0.0001 g) and 0.025 g ± 0.001 g of D - tryptophan enantiomer reference (accurate to 0.0001 g) into a 25 - mL volumetric flask, dissolve with ultrapure water, dilute to volume, and filter through a 0.22 - μm aqueous needle filter for standby.

[0088] Sample solution preparation: Weigh 0.25 g ± 0.01 g of the sample (accurate to 0.0001 g) into a 50 - mL volumetric flask, dissolve with ultrapure water, dilute to volume, and filter through a 0.22 - μm aqueous needle filter for standby. Conduct parallel tests simultaneously.

[0089] Example 1: System suitability experiment

[0090] According to the above chromatographic conditions, conduct system suitability experiments on D - tryptophan and D - tryptophan enantiomer reference standards respectively. The liquid chromatograms of the two are as shown in Figure 1 and Figure 2 shown below. The test data results of the two are as follows:

[0091] Table 1 System suitability experiment test

[0092]

[0093]

[0094] As can be seen from the test results in Table 1, the relative standard deviations (RSD) of the retention times and peak areas of the 6 - needle D - tryptophan standard solution and the D - tryptophan enantiomer standard solution are both less than <0.1%, indicating that the same bottle of mobile phase is well configured and the system suitability is good. The retention times Rt of the two standards: D - tryptophan is about 31.166 min, and D - tryptophan enantiomer is about 38.823 min.

[0095] In addition, the tailing factor of D - tryptophan is 1.0 - 1.5, and the tailing factor of D - tryptophan enantiomer is 0.8 - 1.3; the resolution between D - tryptophan enantiomer and D - tryptophan is ≥2, indicating that this method can well distinguish D - tryptophan and D - tryptophan enantiomer, has good specificity, and can be used to detect the purity of D - tryptophan enantiomer.

[0096] Example 2: Detection limit and quantification limit of D - tryptophan enantiomer

[0097] Run the sample according to the chromatographic conditions in the example to obtain the peak area, view the S / N data according to the instrument's built - in software, and calculate the detection limit and quantification limit according to the following formula:

[0098] Calculation formula for detection limit:

[0099]

[0100] Calculation formula for quantification limit:

[0101]

[0102] For the first test result of the D - tryptophan enantiomer standard in Table 1 of Example 1, the peak area of the D - tryptophan enantiomer is 24831081, and the S / N of the D - tryptophan enantiomer in the instrument's built - in software is 33333.3. Through the above formula calculation, the detection limit of D - tryptophan enantiomer is 0.009%, and the quantification limit is 0.03%.

[0103] Example 3: Detection of the purity of D - tryptophan enantiomer in sample #1

[0104] According to the above chromatographic conditions, perform chromatographic detection in the following injection order: 1 needle of blank, 1 needle of system suitability, 1 needle of sample #1 solution and its parallel test, and record the chromatogram.

[0105] The chromatogram of sample #1 is as Figure 3As shown, the retention times of D-tryptophan and the D-tryptophan enantiomer are 31.588 min and 40.292 min respectively, which are basically the same as those of the standard product. The content of the D-tryptophan enantiomer in the sample was calculated by the area percentage method = peak area of D-tryptophan enantiomer 1838 / (peak area of D-tryptophan 19994985 + peak area of D-tryptophan enantiomer 1838) = 0.009%, which is not much different from the result of 1 - 99.98%, indicating that the detection method of the present invention is accurate and reliable.

[0106] Example 4: Detect the purity of the D-tryptophan enantiomer in sample #2

[0107] According to the above chromatographic conditions, chromatographic detection was carried out in the following injection sequence: 1 blank injection, 1 system suitability injection, 1 injection of sample #2 solution and parallel test respectively, and the chromatogram was recorded.

[0108] The chromatogram of sample #2 is as Figure 4 shown. The retention times of D-tryptophan and the D-tryptophan enantiomer are 30.412 min and 38.087 min respectively, which are basically the same as those of the standard product. The content of the D-tryptophan enantiomer in the sample was calculated by the area percentage method = peak area of D-tryptophan enantiomer 11348 / (peak area of D-tryptophan 19313130 + peak area of D-tryptophan enantiomer 11348) = 0.05872%, which is basically the same as the result of 1 - 99.94%, indicating that the detection method of the present invention is accurate and reliable.

[0109] In the above Examples 3 and 4, there are slight differences between the retention times of D-tryptophan and the D-tryptophan enantiomer during sample detection and those of the standard product, but they are acceptable in the art. During the actual detection process, the retention times of D-tryptophan and the D-tryptophan enantiomer will change. The specific reason is that the chromatographic column used is a chiral column and is very sensitive to slight changes in pH. Although the pH value is detected with a pH meter each time the mobile phase is prepared to make the pH value basically the same, there are still slight differences; moreover, different mobile phase temperatures will also cause this situation to occur. For example, there will be slight differences in the peak emergence when preparing samples in spring and summer. Therefore, when using the detection method of the present invention, the isomer standard will be run together each time, and the position of the isomer peak can be found. This situation is acceptable for the isomer chromatogram using the peak area percentage method.

[0110] According to the above examples, the method described in the present invention has a simple pretreatment method, a short analysis time, good system suitability, accurate and reliable results, and good repeatability. It can effectively detect the purity of the D-tryptophan enantiomer and has very good application prospects.

[0111] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

[0112] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting the enantiomeric purity of D-tryptophan, characterized in that, It includes the following steps: S1. Take equal masses of D-tryptophan and D-tryptophan enantiomer standard products, dissolve them in water to prepare a system suitability solution; S2. Use the system suitability solution to adjust the chromatographic conditions until the resolution between D-tryptophan enantiomer and D-tryptophan is ≥2 to obtain the suitable chromatographic conditions; S3. Perform high performance liquid chromatography analysis on the sample solution according to the suitable chromatographic conditions, and calculate according to the percentage of peak area to obtain the purity of D-tryptophan enantiomer.

2. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, When performing high performance liquid chromatography analysis, the injection sequence is ultrapure water blank control, system suitability solution, and sample solution.

3. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, The concentrations of D-tryptophan and D-tryptophan enantiomer in the system suitability solution are both 1 mg / mL.

4. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, The concentration of the sample in the sample solution is 5 mg / mL.

5. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, wherein The suitable chromatographic conditions are as follows: Chromatographic column: DAICEL CROWNPAK CR(+) 4.0 mm×150 mm, 5 μm; Mobile phase: 0.95% perchloric acid aqueous solution with a pH of 1.0 - 1.3; Flow rate: 1.0 mL / min; Wavelength: 200 nm; Column temperature: 35°C; Injection volume: 1 μL; Running time: 45 min.

6. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, wherein Under the suitable chromatographic conditions, the theoretical plate number of D-tryptophan is ≥4000; the theoretical plate number of D-tryptophan enantiomer is ≥5000.

7. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, Under the suitable chromatographic conditions, the tailing factor of D-tryptophan is 1.0 - 1.5; the tailing factor of D-tryptophan enantiomer is 0.8 - 1.

3.

8. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, Under the suitable chromatographic conditions, the detection limit of D-tryptophan enantiomer is 0.009%, and the quantitation limit is 0.03%.

9. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, Under the suitable chromatographic conditions, the retention time of D-tryptophan is about 31.166 min, and the retention time of D-tryptophan enantiomer is about 38.823 min.

10. The method for detecting the enantiomeric purity of D-tryptophan according to claim 1, characterized in that, When performing high performance liquid chromatography analysis on the sample solution according to the suitable chromatographic conditions, parallel samples are also set, and the absolute value of the error of the parallel determination calculation result is not greater than 0.03%.

Images