A method for detecting enantiomers of halofuginone lactate

Abstract

The invention discloses a method for detecting the enantiomers of fufurin lactate, comprising the following steps: using high performance liquid chromatography to detect a test sample of fufurin lactate to obtain a high performance liquid chromatogram; calculating the peak area ratio of the enantiomers of fufurin lactate in the high performance liquid chromatogram; the chromatographic column is: Chiralcel IC (250mm×4.6mm, 5µm); the mobile phase of the high performance liquid chromatography is n-heptane / isopropanol / ethanol / triethylamine, and the volume ratio is 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5; the solvent of the solution to be tested is ethanol / isopropanol / triethylamine, and the volume ratio is 100 / 100 / 0.1. The method of the invention has good peak shape and separation of each peak, and can efficiently and sensitively detect the enantiomers in fufurin lactate. When impurities are mixed into halofuginone lactate, the method of the present invention can also effectively detect the ratio of halofuginone lactate enantiomer I and halofuginone lactate enantiomer II. The detection method of the present invention has the characteristics of simple operation, high sensitivity and high accuracy, and each chromatographic peak has high separation and does not interfere with each other, has good peak shape, and the accuracy of the result is guaranteed.

Description

Technical Field

[0001] The invention relates to a method for detecting the enantiomers of halofuginone lactate, and belongs to the technical field of detection of halofuginone lactate. Background Art

[0002] Halofuginonelactate is one of the active ingredients in the traditional Chinese medicine Changshan. The chemical name of Halofuginonelactate is 7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl(acetonyl)]-4(3H)-quinazolinone lactate, and its chemical structure is:

[0003]

[0004] Halofuginone lactate has anti-malarial, anti-parasitic and anti-tumor biological activities, and is often used to control common parasitic infections in poultry and livestock, such as avian coccidiosis and bovine coccidiosis. Halofuginone lactate can also inhibit microbial infection and inflammatory response in livestock and poultry, and is used for diarrhea caused by cryptosporidiosis in newborn cattle / sheep. It has the advantages of broad spectrum, high efficiency, irreversibility, no recurrence after discontinuation of drug use, no cross-resistance, low toxicity, and high safety.

[0005] Halofuginone lactate has a chiral center and exists as a pair of enantiomers. The isomers may exhibit different properties in the body, such as drug disintegration, dissolution, and the rate of absorption by the body. Therefore, the detection of the ratio of enantiomers in drug products is helpful to control and improve the quality of drugs. However, there is no detection method for the ratio of isomers of halofuginone lactate in various pharmacopoeias. Summary of the invention

[0006] The invention provides a method for detecting the enantiomers of halofuginone lactate, which can realize efficient and accurate detection of the enantiomers of halofuginone lactate.

[0007] To achieve the above object, the technical scheme adopted by the present invention is: a method for detecting the enantiomers of fufurin lactate, comprising the following steps: using high performance liquid chromatography to detect a test sample of fufurin lactate to obtain a high performance liquid chromatogram; calculating the peak area ratio of the enantiomers of fufurin lactate in the high performance liquid chromatogram; the chromatographic column is: Chiralcel IC (250mm×4.6mm, 5μm); the mobile phase of the high performance liquid chromatography is n-heptane / isopropanol / ethanol / triethylamine, and the volume ratio is 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5; the solvent of the test solution is ethanol / isopropanol / triethylamine, and the volume ratio is 100 / 100 / 0.1.

[0008] Furthermore, the mobile phase is n-heptane / isopropanol / ethanol / triethylamine

[0009] =720 / 200 / 80 / 4.5.

[0010] Furthermore, the peak area of ​​the enantiomer of halofuginone lactate is calculated by using an external standard method, an internal standard method or an area normalization method.

[0011] Furthermore, when the test sample of halofuginone lactate is tested by high performance liquid chromatography, the temperature of the chromatographic column is 24°C to 26°C.

[0012] Furthermore, the chromatographic column temperature is 25°C.

[0013] Furthermore, when the test sample of halofuginone lactate is detected by high performance liquid chromatography, the flow rate of the mobile phase is 0.68 mL / min to 0.72 mL / min.

[0014] Furthermore, the flow rate of the mobile phase is 0.7 mL / min.

[0015] Furthermore, when the test sample of halofuginone lactate is detected by high performance liquid chromatography, the detection wavelength is 254 nm; the injection volume is 20 μl; and the running time is 30 to 40 minutes.

[0016] Furthermore, it also includes drawing a quantitative standard curve of the enantiomers of halofuginone lactate, substituting the peak area of ​​the enantiomers of halofuginone lactate in the test sample into the quantitative standard curve for calculation, thereby quantitatively detecting the enantiomers in the halofuginone lactate test solution.

[0017] Furthermore, drawing the quantitative standard curve includes the following steps: preparing standard test solutions with different concentration gradients; using high performance liquid chromatography to detect the standard test solutions with different concentration gradients to obtain high performance liquid chromatograms corresponding to each standard test solution; calculating the peak area of ​​fusinone lactate enantiomer I and the peak area of ​​fusinone lactate enantiomer II in the high performance liquid chromatogram, and drawing corresponding quantitative standard curves according to the peak areas.

[0018] The method of the present invention has good peak shape and separation of each peak under the conditions of column temperature of 24°C to 26°C, mobile phase flow rate of 0.68mL / min to 0.72mL / min, and mobile phase volume ratio of 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5, and can efficiently and sensitively detect the enantiomers in halofuginone lactate. When impurities are mixed into halofuginone lactate, the method of the present invention can also effectively detect the ratio of halofuginone lactate enantiomer I and halofuginone lactate enantiomer II.

[0019] The detection method of the enantiomers of halofuginone lactate in the present invention has the characteristics of simple operation, high sensitivity and high accuracy, and each chromatographic peak has high separation degree and does not interfere with each other, has good peak shape, and the accuracy of the result is guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a line graph of enantiomer I of halofuginone lactate in Example 1 of the present invention;

[0021] Figure 2 is a line graph of enantiomer II of halofuginone lactate in Example 1 of the present invention;

[0022] Figure 3 is a high performance liquid chromatogram of halofuginone lactate test solution 2 in Example 1 of the present invention;

[0023] Figure 4 is a high performance liquid chromatogram of the qualitative solution 1 in Example 8 of the present invention;

[0024] Figure 5 is a high performance liquid chromatogram of the qualitative solution 2 in Example 8 of the present invention;

[0025] Figure 6 This is a high performance liquid chromatogram of the test sample in Example 8 of the present invention. DETAILED DESCRIPTION

[0026] In order to better understand the essence of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0027] The invention provides a method for detecting the enantiomers of halofuginone lactate, comprising the following steps:

[0028] The test solution of halofuginone lactate is subjected to high performance liquid chromatography to obtain a chromatogram; and the peak area ratio of halofuginone lactate enantiomers is obtained by calculation;

[0029] The solvent of the test solution: ethanol / isopropanol / triethylamine, the volume ratio is 100 / 100 / 0.1;

[0030] Chromatographic column: Chiralcel IC (250 mm × 4.6 mm, 5 μm);

[0031] The column temperature is 24°C to 26°C;

[0032] Mobile phase: n-heptane / isopropanol / ethanol / triethylamine = 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5 (V / V / V);

[0033] Mobile phase flow rate: 0.68 mL / min~0.72 mL / min;

[0034] Detection wavelength: 254nm;

[0035] Injection volume: 20 μl;

[0036] Running time: 30 to 40 minutes.

[0037] In the present invention, if it is necessary to quantitatively detect the enantiomers of halofuginone lactate, the detection method further includes the step of drawing a quantitative standard curve. Specifically:

[0038] S1. Prepare standard test solutions with different concentration gradients.

[0039] Take 25 mg of halofuginone lactate in a 25 mL volumetric bottle, add solvent to dissolve it completely by ultrasonication, dilute it to the scale with the solvent, and mix it well as a stock solution; the solvent is ethanol / isopropanol / triethylamine, and the volume ratio is 100 / 100 / 0.1.

[0040] Accurately measure 2.5mL, 3mL, 4mL, 5mL, 5.5mL and 6mL of the stock solution, place them in 10mL volumetric flasks respectively, dilute to the scale with solvent, mix well, and obtain solutions with different concentration gradients.

[0041] S2. Inject the standard sample solutions with different concentration gradients and the blank solution in step S1 into the chromatographic column, elute with the mobile phase and record the chromatogram; calculate the peak area of ​​fusinone lactate enantiomer I and the peak area of ​​fusinone lactate enantiomer II under different concentration gradients, and draw corresponding quantitative standard curves according to the peak areas.

[0042] Wherein, the blank solution is the solvent in step S1;

[0043] The chromatographic column was: Chiralcel IC (250 mm × 4.6 mm, 5 μm);

[0044] The column temperature is 24°C to 26°C;

[0045] Mobile phase: n-heptane / isopropanol / ethanol / triethylamine = 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5 (V / V / V);

[0046] Mobile phase flow rate: 0.68 mL / min~0.72 mL / min;

[0047] Detection wavelength: 254nm;

[0048] Injection volume: 20 μl;

[0049] Running time: 30 to 40 minutes.

[0050] In the detection method of the present invention, the peak area can be calculated by using an external standard method, an internal standard method, an area normalization method, etc.

[0051] S3. The fufurin lactate test solution is tested by high performance liquid chromatography, and the peak areas of the obtained fufurin lactate enantiomer I and the fufurin lactate enantiomer II are respectively introduced into the corresponding quantitative standard curves in step S2, thereby quantitatively detecting the enantiomers in the fufurin lactate test solution.

[0052] Example 1

[0053] S1. Prepare standard test solutions with different concentration gradients.

[0054] S1.1. Take 25 mg of halofuginone lactate in a 25 mL volumetric flask, add solvent and ultrasonically dissolve it completely, dilute it to the scale with the solvent, and mix it well as a stock solution; the solvent is ethanol / isopropanol / triethylamine, and the volume ratio is 100 / 100 / 0.1.

[0055] S1.2. Accurately measure 2.5mL, 3mL, 4mL, 5mL, 5.5mL and 6mL of the stock solution and place them in 10mL volumetric flasks respectively. Dilute to the scale with solvent and mix well to obtain solutions with different concentration gradients.

[0056] S2. Draw a quantitative standard curve.

[0057] S2.1. Inject the standard sample solutions with different concentration gradients and the blank solution in step S1.2 into the chromatographic column, elute with the mobile phase and record the chromatogram; wherein the blank solution is the solvent in step 1.

[0058] The chromatographic column is Chiralcel IC (250 mm × 4.6 mm, 5 μm); the chromatographic column temperature is 25°C.

[0059] Chromatograph: Shimadzu LC-20A.

[0060] Mobile phase: n-heptane / isopropanol / ethanol / triethylamine = 720 / 200 / 80 / 4.5 (V / V / V); mobile phase flow rate: 0.7 mL / min; detection wavelength: 254 nm; injection volume: 20 μl; running time: 30 min.

[0061] S2.2. The peak area calculation results are shown in Tables 1 and 2, and the quantitative standard curve is shown in Figure 1 and Figure 2 shown.

[0062] Table 1 Quantitative standard curve of halofuginone lactate enantiomer Ⅰ

[0063]

[0064] Table 2 Quantitative standard curve of halofuginone lactate enantiomer II

[0065]

[0066] It can be seen from Tables 1 and 2 that, within the measured concentration range of 50% to 120% (i.e., concentration of 2.5 mL to 6 mL), the peak area of ​​enantiomer I of fufurin lactate showed a good linear relationship with the concentration, and the correlation coefficient was r1 of 0.9995; the peak area of ​​enantiomer II of fufurin lactate showed a good linear relationship with the concentration, and the correlation coefficient was r2 of 0.9997; both r1 and r2 were greater than the reference value of 0.999 of the prescribed standard (Chinese Pharmacopoeia 2015 edition).

[0067] S3. The halofuginone lactate test solution is tested by high performance liquid chromatography to obtain the ratio of enantiomers in the halofuginone lactate test solution.

[0068] S3.1. Preparation of halofuginone lactate test solution 1: Weigh about 10.0 mg of halofuginone lactate test sample, accurately weigh and place in a 25 mL volumetric flask, dissolve and dilute to the scale with a solvent, and mix well.

[0069] Preparation of halofuginone lactate test solution 2: Weigh about 12.5 mg of halofuginone lactate test sample, accurately weigh and place in a 25 mL volumetric flask, dissolve and dilute to the scale with a solvent, and mix well.

[0070] Preparation of halofuginone lactate test solution 3: Weigh about 15.0 mg of halofuginone lactate test sample, accurately weigh and place in a 25 mL volumetric flask, dissolve and dilute to the scale with a solvent, and mix well.

[0071] S3.2. Take 3 portions of each of the test solution 1, test solution 2 and test solution 3 prepared in step S3.1, for a total of 9 portions, use the solvent as the blank solution, and perform HPLC detection according to step S2.1, and record the chromatograms.

[0072] S3.3. The peak areas of the enantiomers in test solution 1, test solution 2 and test solution 3 were calculated by the area normalization method, and the RSD of the enantiomer peak area ratio was calculated. The results are shown in Table 3.

[0073] Table 3

[0074]

[0075]

[0076] As can be seen from Table 3, the RSD of the peak area ratio of enantiomer I of fufurin lactate in test solutions 1 to 3 is 0.40%, and the RSD of the peak area ratio of enantiomer II of fufurin lactate is 0.47%, both of which meet the standard requirement of RSD≤2.0%. Therefore, the detection method of the present invention can accurately detect the enantiomers in fufurin lactate.

[0077] S3.4. Substitute the enantiomer peak area calculated in step S3.3 into the quantitative standard curve drawn in step S2.2 to quantitatively detect the enantiomer I and enantiomer II of fufurin lactate in test solution 1, test solution 2 and test solution 3.

[0078] Example 2

[0079] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0080] Among them, the chromatographic column temperature is 24°C.

[0081] Example 3

[0082] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0083] Among them, the chromatographic column temperature is 26°C.

[0084] Example 4

[0085] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0086] Among them, the flow rate of mobile phase is: 0.68mL / min.

[0087] Example 5

[0088] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0089] Among them, the flow rate of mobile phase is: 0.72mL / min.

[0090] Example 6

[0091] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0092] Wherein, the mobile phase: n-heptane / isopropanol / ethanol / triethylamine=710 / 205 / 85 / 4.5 (V / V / V).

[0093] Example 7

[0094] The method in Example 1 was used to perform HPLC detection on the halofuginone lactate test solution 2, and the detection was repeated three times.

[0095] Wherein, the mobile phase: n-heptane / isopropanol / ethanol / triethylamine=730 / 195 / 75 / 4.5 (V / V / V).

[0096] The areas and ratios of enantiomers I and II in the test solution 2 of hawthorn lactate in Examples 2 to 7 are shown in Table 4.

[0097] Table 4

[0098]

[0099]

[0100]

[0101] As shown in Table 4, under the conditions of column temperature of 24°C to 26°C, mobile phase flow rate of 0.68mL / min to 0.72mL / min, and mobile phase volume ratio of 710 / 205 / 85 / 4.5 to 730 / 195 / 75 / 4.5, each RSD meets the requirement of RSD≤2.0% of the 2015 edition of the Chinese Pharmacopoeia, that is, within the above conditions, the method of the present invention can effectively detect the enantiomers in halofuginone lactate.

[0102] Example 8

[0103] The cis isomer impurity is added to the test solution, and the separation degree between the two enantiomer main peaks and the cis impurity in the analysis result is analyzed by the method of the present invention to verify the specificity of the method of the present invention.

[0104] The chromatographic column is Chiralcel IC (250 mm × 4.6 mm, 5 μm); the chromatographic column temperature is 25°C.

[0105] Chromatograph: Shimadzu LC-20A.

[0106] Solvent: ethanol / isopropanol / triethylamine, volume ratio is 100 / 100 / 0.1.

[0107] Mobile phase: n-heptane / isopropanol / ethanol / triethylamine = 720 / 200 / 80 / 4.5 (V / V / V); mobile phase flow rate: 0.7 mL / min; detection wavelength: 254 nm; injection volume: 20 μl.

[0108] Blank solution: solvent.

[0109] Qualitative solution 1 (500 μg / mL): Take 25 mg of halofuginone lactate reference substance in a 50 mL volumetric flask, add solvent and ultrasonically dissolve it completely, dilute it to the scale with solvent, and mix well.

[0110] Reference solution (200 μg / mL): Take 10 mg of the cis-isomer impurity reference substance in a 50 mL volumetric flask, add solvent and ultrasonically dissolve it completely, dilute it to the scale with solvent, and mix well.

[0111] Qualitative solution 2: Accurately measure 6 mL of reference solution into a 50 mL volumetric flask, add solvent to dilute to the mark, and mix well.

[0112] Preparation of the test product: Take about 25 mg of halofuginone lactate in a 50 mL volumetric bottle, add 6 mL of reference solution, add a certain amount of solvent to dissolve it completely by ultrasonication, dilute it to the scale with solvent, and mix well.

[0113] According to the above chromatographic conditions, blank solution, qualitative solution 1, qualitative solution 2, and the sample to be tested were injected respectively, and the chromatograms were recorded. The results are shown in Table 5 and Table 6. Figures 4 to 6 shown.

[0114] Table 5 Specificity test results of the method of the present invention (retention time)

[0115]

[0116] Table 6 Specificity test results of the method of the present invention (separation degree)

[0117] name Separation Enantiomer I / Cis isomer Ⅰ 1.73 Enantiomer II 2.28 Cis isomer II 6.44

[0118] From Table 5, Table 6 and Figure 6 It can be seen that the minimum separation between the two enantiomer peaks of the test sample and the cis impurity peak is 1.73 (Chinese Pharmacopoeia 2015 version stipulates: minimum separation ≥ 1.5), which meets the requirements of the standard. When impurities are mixed into halofuginone lactate, the method of the present invention can also effectively detect the ratio of halofuginone lactate enantiomer I and halofuginone lactate enantiomer II.

[0119] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the scope of the claims of the present invention to be approved.

Claims

1. A method for detecting the enantiomers of halofuginone lactate, characterized in that: The following steps are involved: The test solution of halofuginone lactate is tested by high performance liquid chromatography to obtain a high performance liquid chromatogram; Calculating the peak area ratio of the enantiomers of halofuginone lactate in the high performance liquid chromatogram; The chromatographic columns were: Chiralcel IC, 250 mm × 4.6 mm, 5 μm; The mobile phase of the HPLC is n-heptane / isopropanol / ethanol / triethylamine, and the volume ratio is 720 / 200 / 80 / 4.5 or 710 / 205 / 85 / 4.5 or 730 / 195 / 75 / 4.5; the column temperature is 24° C. to 26° C.; the mobile phase flow rate is 0.68 mL / min to 0.72 mL / min ； Detection wavelength 254nm ； Preparation of halofuginone lactate test solution: weigh halofuginone lactate test sample, accurately weigh and place in a volumetric bottle, dissolve and dilute to scale with solvent, mix well to obtain; The solvent of the solution to be tested is ethanol / isopropanol / triethylamine, and the volume ratio thereof is 100 / 100 / 0.

1.

2. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: The mobile phase is n-heptane / isopropanol / ethanol / triethylamine=720 / 200 / 80 / 4.

5.

3. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: The peak area of ​​the enantiomer of halofuginone lactate is calculated by using an external standard method, an internal standard method or an area normalization method.

4. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: The chromatographic column temperature is 25°C.

5. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: The flow rate of the mobile phase is 0.7 mL / min.

6. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: When the test solution of halofuginone lactate is detected by high performance liquid chromatography, the injection volume is 20 μl and the running time is 30 to 40 minutes.

7. The method for detecting the enantiomers of halofuginone lactate according to claim 1, characterized in that: It also includes the preparation of a quantitative standard curve for the enantiomers of halofuginone lactate; The peak area of ​​the enantiomer of halofuginone lactate in the test solution is substituted into the quantitative standard curve for calculation, thereby quantitatively detecting the enantiomer in the test solution.

8. The method for detecting the enantiomers of halofuginone lactate according to claim 7, characterized in that: Drawing the quantitative standard curve comprises the following steps: Prepare standard test solutions with different concentration gradients; High performance liquid chromatography was used to detect standard test solutions with different concentration gradients. Obtaining the HPLC chromatogram corresponding to each standard test solution; Calculating the peak area of ​​the enantiomer of halofuginone lactate in the high performance liquid chromatogram, And according to the peak areas, corresponding quantitative standard curves are drawn respectively.

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