Method for detecting related substances in lipoic acid preparation

Abstract

The invention provides a method for detecting related substances in a lipoic acid preparation, and relates to the technical field of medicine detection. The detection method is a high performance liquid chromatography method, a mobile phase A is 0.001-0.008 mol / L disodium hydrogen phosphate solution, and the pH value is adjusted to 2.8-3.2 by phosphoric acid; a mobile phase B is a gradient elution mode by using a mixture of acetonitrile, water and trifluoroacetic acid; according to the method, six specific impurities including an impurity A, an impurity C, an impurity D, an impurity E, an impurity F and an impurity H can be detected by selecting a specific diluent, an extraction method, a mobile phase, a chromatographic column, elution conditions and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a detection method of related substances in lipoic acid preparation. BACKGROUND

[0002] Lipoic acid (chemical name: 1, 2-dithiolane-3-pentanoic acid, molecular formula: C8H 14 O2S2) is a biological antioxidant, which can reduce the lipid oxidation of nerve tissue, prevent the glycosylation of protein, and inhibit aldose reductase to prevent glucose or hemiacetal from being converted into sorbitol, and enhance the metabolism of glucose, so that lipoic acid can prevent and treat diabetes, control blood sugar, and prevent neuropathy caused by hyperglycemia. Animal experiments have proved that lipoic acid has the same free radical scavenging function as vitamin C, vitamin E and glutathione. Foreign clinical trials have proved that lipoic acid can significantly relieve the numbness, pain, burning sensation and other nervous system symptoms of patients, and can improve the autonomic nervous system disease of the heart of diabetes, so that the heart rate variability is restored to a certain extent. At present, lipoic acid is used in clinical treatment of diabetes, senile dementia and related diseases caused by alcoholism.

[0003] Due to the chemical structure of lipoic acid as shown in formula I, due to the particularity of the structure, lipoic acid raw materials and preparations are unstable and prone to impurities, mainly including impurity A, reduction impurities, mono-oxidized impurities, di-oxidized impurities 1, di-oxidized impurities 2, impurity H and the like. At present, the dosage forms of lipoic acid on the market mainly include injection, capsule or tablet. Due to the thermal instability of lipoic acid, there is a safety risk in the production of tablets, capsules or injections. It is of great significance to explore the liquid chromatography conditions suitable for the determination of lipoic acid content and degradation products of lipoic acid in different dosage forms, and to establish a method for determining the related substances of lipoic acid, for controlling the quality of lipoic acid.

[0004]

[0005] Formula I There are reports on the detection of lipoic acid content in the prior art. For example, “Study on High Performance Liquid Chromatography Determination of Lipoic Acid Tablets Content and Degradation Products” (Ye Ruhan, Zhang Yi, Peng Lei, et al., Journal of Analysis and Testing, 1004-4957 (2013) 07-0894-04) discloses a high performance liquid chromatography method for rapid determination of lipoic acid and its degradation products in lipoic acid tablets. The method is simple, rapid, accurate, selective, sensitive, and can be used for the determination of lipoic acid tablet content and degradation products.

[0006] Chinese patent application CN110118835A discloses a method for determining related substances in lipoic acid injection by high performance liquid chromatography. The mobile phase of the method includes mobile phase A and mobile phase B, the mobile phase A is potassium dihydrogen phosphate A solution, and the mobile phase B is a mixed solution of methanol and acetonitrile. By adopting a specific gradient elution mode, the related substances (including lipoic acid and at least 11 common impurities) in lipoic acid injection can be separated in the high performance liquid chromatogram. Through optimization of the conditions, the sensitivity of the detection of each component and the accuracy of the content are further improved.

[0007] Developing a new detection method for related substances in lipoic acid preparation can detect different impurities, has good resolution and high stability, which is a problem to be solved by researchers in the field. SUMMARY

[0008] The present application provides a detection method for related substances in lipoic acid preparation, which can detect different impurities, has good resolution and high stability. The research idea of the present application can provide a reference for the analysis of related substance methods of other lipoic acid products.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows: On the one hand, the present application provides a detection method for related substances in lipoic acid preparation, which is high performance liquid chromatography, and the chromatographic conditions are as follows: Chromatographic column: the filler is octadecylsilane bonded silica gel; Mobile phase: The mobile phase A is 0.001-0.008 mol / L disodium hydrogen phosphate solution, and the pH is adjusted to 2.8-3.2 by phosphoric acid; The mobile phase B is a mixture of acetonitrile, water and trifluoroacetic acid, and the mass ratio is 480-550:45-55:0.05-0.1; Elution gradient: 0-35min, the volume of mobile phase A decreases from 95-90% to 88-80%, and the volume of mobile phase B increases from 5-10% to 12-20%; 35-80min, the volume of mobile phase A decreases from 88-80% to 30-20%, and the volume of mobile phase B increases from 12-20% to 70-80%; 80-90min, the volume of mobile phase A is 30-20%, and the volume of mobile phase B is 70-80%; The lipoic acid preparation includes lipoic acid injection and / or lipoic acid solid preparation; The related substances include the following impurities:

[0010] Preferably, the chromatographic column is a Phenomenex Kinetex XB-C18 column, 4.6 mm × 250 mm, 5 μm.

[0011] Preferably, the mobile phase is: Mobile phase A is a 0.002-0.006 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 2.9-3.1 by phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 480-520:48-55:0.06-0.1.

[0012] More preferably, the mobile phase is: Mobile phase A is a 0.004 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 3.0 using phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 500:50:0.08.

[0013] Preferably, the gradient elution is as follows: From 0 to 35 minutes, the volume of mobile phase A decreased from 95-93% to 85-80%, while the volume of mobile phase B increased from 5-7% to 15-20%. Over 35-80 minutes, the volume of mobile phase A decreased from 85-80% to 30-25%, while the volume of mobile phase B increased from 15-20% to 75-80%. The process takes 80-90 minutes, with the volume of mobile phase A at 30-25% and the volume of mobile phase B at 75-80%.

[0014] More preferably, the gradient elution is as follows: From 0 to 5 minutes, the volume of mobile phase A was 93% and the volume of mobile phase B was 7%. Over 5-20 minutes, the volume of mobile phase A decreased from 93% to 85%, while the volume of mobile phase B increased from 7% to 15%. After 20-35 minutes, the volume of mobile phase A is 85% and the volume of mobile phase B is 15%. Over 35-50 minutes, the volume of mobile phase A decreased from 85% to 70%, while the volume of mobile phase B increased from 15% to 30%. Over 50-80 minutes, the volume of mobile phase A decreased from 70% to 25%, while the volume of mobile phase B increased from 30% to 75%. The time is 80-95 min, the volume of mobile phase A is 25%, and the volume of mobile phase B is 75%.

[0015] Preferably, the injection volume of the high-performance liquid chromatography (HPLC) is 10-30 μL. More preferably, the injection volume of the HPLC is 10-20 μL. More preferably, the injection volume of the HPLC is 10 μL.

[0016] Preferably, the detection wavelength of the high performance liquid chromatography is 210-230 nm.

[0017] Preferably, the column temperature of the high-performance liquid chromatography is 34-38℃.

[0018] Preferably, the flow rate of the high-performance liquid chromatography (HPLC) is 1.8-2.2 mL / min. More preferably, the flow rate of the HPLC is 2.0 mL / min.

[0019] Preferably, the detection method includes the following steps: S1: Solution preparation: (1) Test solution: Zinc sulfate solid preparations: Take the sample to be tested, extract it with diluent, and use it as the test solution; (2) Mixed reference solution: Take at least one of impurities A, C, D, E, F, and H and mix it with lipoic acid, dissolve it with diluent, and the concentrations of impurities A, C, D, E, F, and H are 0.001-0.008 mg / mL, respectively; the concentration of lipoic acid is 1.2-1.8 mg / mL; (3) Control solution: Take the test solution and dilute it quantitatively with diluent to prepare a solution containing 5-7 μg / mL of thioctic acid per 1 mL; S2: Inject the test solution, mixed reference solution, and reference solution into a high-performance liquid chromatograph for detection.

[0020] Preferably, in step S1, the diluent is a mixture of methanol, acetonitrile and 5mM ammonium formate, and the mass ratio of methanol, acetonitrile and 5mM ammonium formate is 20-30:60-70:5-10. More preferably, the mass ratio of methanol, acetonitrile, and 5mM ammonium formate is 25:65:10.

[0021] Preferably, in step S1, the extraction is ultrasonic extraction, with an ultrasonic power of 200-300 kW, a temperature of 20-30°C, and a time of 15-30 min.

[0022] More preferably, in step S1, the extraction is ultrasonic extraction, with an ultrasonic power of 250 kW, a temperature of 25 °C, and a time of 20 min.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention, by selecting specific mixed solvents, extraction methods, mobile phases, chromatographic columns, elution conditions, etc., can detect six specific impurities. Experimental results show that this method is simple, rapid, accurate, selective, and highly sensitive, which can greatly save analysis time and is suitable for the determination of related substances in the stability study of lipoic acid. Attached Figure Description

[0024] Figure 1 This is a chromatogram of relevant substances in the thioctic acid preparation of Example 1 of the present invention. Detailed Implementation

[0025] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0026] Instruments and reagents Example 1 A method for detecting related substances in thioctic acid preparations, comprising the following steps: Preparation of diluent: a mixture of methanol, acetonitrile and 5mM ammonium formate, wherein the mass ratio of methanol, acetonitrile and 5mM ammonium formate is 25:65:10.

[0027] (1) Test solution: Zinc sulfate solid preparation: Take this product, dissolve it in diluent, and extract it by ultrasonic extraction at a power of 250 kW, a temperature of 25 °C, and a time of 20 min to obtain the test solution. (2) Mixed reference solution: Take the reference standards of impurities A, C, D, E, F, H and lipoic acid, mix them together, and dissolve them with diluent. The concentrations of impurities A, C, D, E, F, and H are 0.005 mg / mL, 0.003 mg / mL, 0.002 mg / mL, 0.005 mg / mL, 0.008 mg / mL, and 0.006 mg / mL, respectively. The concentration of lipoic acid is 1.6 mg / mL. (3) Control solution: Take the test solution and dilute it quantitatively with diluent to prepare a solution containing 6 μg / mL of thioctic acid per 1 mL; S2: Inject the test solution, mixed reference solution, and reference solution into a high-performance liquid chromatograph for detection. The chromatographic conditions are as follows: The chromatographic column was a Phenomenex Kinetex XB-C18 column, 4.6 mm × 250 mm, 5 μm.

[0028] The mobile phase is: Mobile phase A is a 0.004 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 3.0 using phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 500:50:0.08.

[0029] The injection volume was 10 μL; The detection wavelength is 220nm; The column temperature is 35℃; The flow rate was 2.0 mL / min; Gradient elution is as follows: From 0 to 5 minutes, the volume of mobile phase A was 93% and the volume of mobile phase B was 7%. Over 5-20 minutes, the volume of mobile phase A decreased from 93% to 85%, while the volume of mobile phase B increased from 7% to 15%. After 20-35 minutes, the volume of mobile phase A is 85% and the volume of mobile phase B is 15%. Over 35-50 minutes, the volume of mobile phase A decreased from 85% to 70%, while the volume of mobile phase B increased from 15% to 30%. Over 50-80 minutes, the volume of mobile phase A decreased from 70% to 25%, while the volume of mobile phase B increased from 30% to 75%. The time is 80-95 min, the volume of mobile phase A is 25%, and the volume of mobile phase B is 75%.

[0030] The obtained spectrum is as follows Figure 1 As shown, the spectral information is shown in Table 1.

[0031] Table 1. Spectral Information

[0032] Example 2 A method for detecting related substances in thioctic acid preparations, which differs from Example 1 in that the diluent and mobile phase are changed, as shown in Table 2 below: Table 2. Diluents and Mobile Phase

[0033] The rest is the same as in Example 1.

[0034] The results showed that the characteristic peaks of zinc sulfate solid preparations could also be separated under the diluent and mobile phase conditions of 2-1 and 2-2 in Example 2, but the separation degree and peak shape of each major chromatographic peak were the best under the conditions of Example 1.

[0035] Example 3 A method for detecting related substances in thioctic acid preparations differs from Example 1 in that the detection wavelengths are 210 nm, 220 nm, and 230 nm, while all other wavelengths remain the same. Results show that the characteristic peaks of zinc sulfate solid preparations can be separated at detection wavelengths of 210 nm, 220 nm, and 230 nm, but the peaks are more complete and the characteristic peaks are more pronounced at the detection wavelength of 220 nm used in Example 1.

[0036] Example 4 A method for detecting related substances in thioctic acid preparations, differing from Example 1 in that column temperatures of 34℃ and 38℃ were used, while all other parameters remained the same. Results showed that the main chromatographic peaks of the zinc sulfate solid preparation exhibited good resolution and peak shape under column temperatures of 34℃ and 38℃, with no significant difference from Example 1.

[0037] Example 5 A method for detecting related substances in thioctic acid preparations, which differs from Example 1 in that it uses 0.8 mL·min -1 0.9 mL·min -1 1.1 mL·min -1 1.2 mL·min -1 The experiment was conducted at a flow rate of 1.0 mL / min, with all other parameters remaining the same. The results showed that a flow rate of 1.0 mL / min was optimal. -1 At this time, the resolution of each major chromatographic peak is optimal, and the peak shape is best.

[0038] Comparative Example 1 A method for detecting related substances in lipoic acid preparations, differing from Example 1 in that the diluent is changed, while the rest is the same as in Example 1. The characteristic peak resolution results are shown in Table 3 below: Table 3. Resolution

[0039] As shown in Table 3, compared with Example 1, the resolution of the major chromatographic peaks of the thioctic acid preparation deteriorated significantly after changing the diluent, especially the impurities E, F, and H, which could not be separated.

[0040] Comparative Example 2 A method for detecting related substances in a thioctic acid preparation, which differs from Example 1 in that the elution gradient is changed to the elution gradient described in Example 1 of Chinese Patent CN114397376A, while the rest is the same as Example 1. The results showed that there were no characteristic peaks of impurities E and F, and the resolution was significantly worse and the peak shape was poor.

[0041] Comparative Example 3 A method for detecting related substances in thioctic acid preparations, differing from Example 1 in that the chromatographic column was changed to a Phenomen ACE Excel 5C18-Amide column (4.6 mm × 250 mm, 5 μm), while the rest remained the same as in Example 1. The characteristic peak resolution results are shown in Table 4 below: Table 4. Resolution

[0042] As shown in Table 4, compared with Example 1, the resolution of the major chromatographic peaks of the thioctic acid preparation deteriorated significantly after changing the chromatographic column, especially impurities D and E could not be separated.

[0043] Detection example The specificity, linearity, stability, and repeatability were examined according to the detection method in Example 1.

[0044] 1. Exclusivity Accurately measure 10 µL each of blank solvent (a mixture of methanol, acetonitrile, and 5 mM ammonium formate in a mass ratio of 25:65:10) and test solution, inject them separately into the liquid chromatograph, and determine the result.

[0045] Result: The blank solvent did not interfere with the test sample.

[0046] 2. Linear relationship Accurately weigh and mix the reference standards of impurities A, C, D, E, F, and H with lipoic acid. Dilute with the diluent described in Example 1 to prepare a reference standard mixed solution with a specific concentration gradient. Inject 10 µL of the reference standard mixed solution from low to high concentration and determine the linear relationship between the concentration (μg / mL) of each component and the corresponding peak area. The results showed that the reference standard concentrations of impurities A, C, D, E, F, H, and zinc sulfate had good linear relationships with the corresponding peak areas, and the linear range of the injection amount for each component met the detection requirements. Correlation coefficient: R 2 =0.9999.

[0047] 3. Stability Following the preparation method of the test solution in Example 1, the retention time of the same sample was measured at 0, 4, 8, 12, 36, and 48 hours. The RSD% was 0.15%-0.80%, indicating that the sample solution had good stability within 48 hours.

[0048] 4. Repeatability Following the test solution preparation method in Example 1, six test solutions were prepared in parallel for the same batch of samples, and the content was determined. The RSD% results were 0.47%-0.90%, indicating that the method has good repeatability.

[0049] This invention, by selecting specific diluents, extraction methods, mobile phases, chromatographic columns, elution conditions, etc., can detect six specific impurities: impurity A, impurity C, impurity D, impurity E, impurity F, and impurity H. Experimental results show that this method is simple, rapid, accurate, selective, and highly sensitive, which can greatly save analysis time and is suitable for the determination of related substances in the stability study of lipoic acid.

[0050] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting related substances in lipoic acid preparations, characterized in that, The method used is high-performance liquid chromatography (HPLC), and the chromatographic conditions are as follows: Column: The packing material is octadecylsilane-bonded silica gel; Mobile phase: Mobile phase A is a 0.001-0.008 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 2.8-3.2 by phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 480-550:45-55:0.05-0.

1. Elution gradient: From 0 to 35 minutes, the volume of mobile phase A decreased from 95-90% to 88-80%, while the volume of mobile phase B increased from 5-10% to 12-20%. Over 35-80 minutes, the volume of mobile phase A decreased from 88-80% to 30-20%, while the volume of mobile phase B increased from 12-20% to 70-80%. The process takes 80-90 minutes, with mobile phase A at 30-20% volume and mobile phase B at 70-80% volume. The lipoic acid preparation is a solid lipoic acid preparation. The relevant substances include the following impurities: Impurity A: Impurity C: Impurity D: Impurity E: Impurity F: Impurity H: .

2. The detection method according to claim 1, characterized in that, The chromatographic column was a Phenomenex Kinetex XB-C18 column, 4.6 mm × 250 mm, 5 μm.

3. The detection method according to claim 1, characterized in that, The mobile phase is: Mobile phase A is a 0.002-0.006 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 2.9-3.1 by phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 480-520:48-55:0.06-0.

1.

4. The detection method according to claim 3, characterized in that, The mobile phase is: Mobile phase A is a 0.004 mol / L disodium hydrogen phosphate solution, with the pH adjusted to 3.0 using phosphoric acid; Mobile phase B is a mixture of acetonitrile, water, and trifluoroacetic acid in a mass ratio of 500:50:0.

08.

5. The detection method according to claim 1, characterized in that, The gradient elution is as follows: From 0 to 35 minutes, the volume of mobile phase A decreased from 95-93% to 85-80%, while the volume of mobile phase B increased from 5-7% to 15-20%. Over 35-80 minutes, the volume of mobile phase A decreased from 85-80% to 30-25%, while the volume of mobile phase B increased from 15-20% to 75-80%. The process takes 80-90 minutes, with the volume of mobile phase A at 30-25% and the volume of mobile phase B at 75-80%.

6. The detection method according to claim 5, characterized in that, The gradient elution is as follows: From 0 to 5 minutes, the volume of mobile phase A was 93% and the volume of mobile phase B was 7%. Over 5-20 minutes, the volume of mobile phase A decreased from 93% to 85%, while the volume of mobile phase B increased from 7% to 15%. After 20-35 minutes, the volume of mobile phase A is 85% and the volume of mobile phase B is 15%. Over 35-50 minutes, the volume of mobile phase A decreased from 85% to 70%, while the volume of mobile phase B increased from 15% to 30%. Over 50-80 minutes, the volume of mobile phase A decreased from 70% to 25%, while the volume of mobile phase B increased from 30% to 75%. The time is 80-95 min, the volume of mobile phase A is 25%, and the volume of mobile phase B is 75%.

7. The detection method according to claim 1, characterized in that, The injection volume for the high-performance liquid chromatography method is 10-30 μL, and the column temperature is 34-38℃.

8. The detection method according to claim 1, characterized in that, The detection wavelength of the high-performance liquid chromatography method is 210-230 nm.

9. The detection method according to claim 1, characterized in that, The flow rate for the high-performance liquid chromatography method is 1.8-2.2 mL / min.

10. The detection method according to claim 1, characterized in that, Includes the following steps: S1: Solution preparation: (1) Test solution: Zinc sulfate solid preparations: Take the sample to be tested, extract it with diluent, and use it as the test solution; (2) Mixed reference solution: Take at least one of impurities A, C, D, E, F, and H and mix it with lipoic acid, dissolve it with diluent, and the concentrations of impurities A, C, D, E, F, and H are 0.001-0.008 mg / mL, respectively; the concentration of lipoic acid is 1.2-1.8 mg / mL; (3) Control solution: Take the test solution and dilute it quantitatively with diluent to prepare a solution containing 5-7 μg / mL of thioctic acid per 1 mL; S2: Inject the test solution, mixed reference solution, and reference solution into a high-performance liquid chromatograph for detection.

11. The detection method according to claim 10, characterized in that, In step S1, the diluent is a mixture of methanol, acetonitrile and 5mM ammonium formate, and the mass ratio of methanol, acetonitrile and 5mM ammonium formate is 20-30:60-70:5-10.

12. The detection method according to claim 11, characterized in that, The mass ratio of methanol, acetonitrile, and 5mM ammonium formate is 25:65:

10.

13. The detection method according to claim 10, characterized in that, In step S1, the extraction is ultrasonic extraction, with an ultrasonic power of 200-300 kW, a temperature of 20-30°C, and a time of 15-30 min.

14. The detection method according to claim 13, characterized in that, In step S1, the extraction is ultrasonic extraction, with an ultrasonic power of 250 kW, a temperature of 25 °C, and a time of 20 min.

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