High performance liquid chromatography detection method of glucosamine sulfate capsule

Abstract

The invention discloses a high performance liquid chromatography detection method for glucosamine sulfate capsules. On-line derivatization and detection are carried out in a high performance liquid chromatography by using a derivatization reagent. The method realizes rapid detection of the dissolution curve of the glucosamine sulfate capsule, is low in detection cost, has good sensitivity, specificity, accuracy and stability, is an effective detection method for the dissolution curve of the glucosamine sulfate capsule, and further finally ensures the product quality and curative effect of the glucosamine sulfate capsule.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to a high-performance liquid chromatography method for the detection of glucosamine sulfate capsules. Background Technology

[0002] Glucosamine sulfate is a slow-acting drug for improving osteoarthritis (OA). It is a natural amino monosaccharide and a precursor in the synthesis of glucosamine in cartilage. It also stimulates chondrocytes to produce glucosamine with a normal polymeric structure, promoting the synthesis and repair of the cartilage matrix. Furthermore, glucosamine sulfate can inhibit the production of inflammatory factors, thereby relieving joint pain, improving joint function, and slowing disease progression. Clinical trial results show that it simultaneously improves OA symptoms and protects articular cartilage, making it an ideal drug for treating OA.

[0003] Glucosamine sulfate capsules were first approved for marketing in Italy in 1982 by ROTTAPHARMS.PA. They were launched in China in 2007 under the brand name Isogene / Vigol, with a strength of 0.25g (calculated as glucosamine sulfate), for the treatment of primary or secondary osteoarthritis.

[0004] After oral administration of solid dosage forms, drug absorption depends on factors such as drug dissolution or release from the formulation, drug dissolution under physiological conditions, and penetration into the gastrointestinal tract. Therefore, in vivo dissolution and rehydration have a significant impact on absorption. In vitro dissolution tests can evaluate the consistency of intra-batch and inter-batch quality of the formulation and assess the similarity between generic and reference formulations by detecting multiple dissolution curves, thereby ensuring drug quality and efficacy.

[0005] For the detection of dissolution curves of glucosamine sulfate capsules, the methods for determining the content of glucosamine sulfate can be referenced. Currently, the three commonly used methods are: direct injection HPLC, derivatization, and titration. Direct injection HPLC uses an amino column, which is not durable, has high detection costs, suffers from significant interference at low wavelengths, and has low sensitivity. Derivatization can improve detection sensitivity, but the derivatized products have poor stability, are complex to operate, and require immediate preparation. Titration has poor specificity and low sensitivity. All three methods have limitations when applied to the detection of dissolution curves for this product. Summary of the Invention

[0006] The purpose of this invention is to provide a method for online derivatization and detection of the dissolution curve of glucosamine sulfate capsules using high performance liquid chromatography, thereby ensuring drug safety and quality reliability.

[0007] The specific technical solution of this invention is as follows:

[0008] A high-performance liquid chromatography (HPLC) method for the detection of glucosamine sulfate capsules involves online derivatization and detection using a derivatization reagent on an HPLC system. The chromatographic conditions include an octadecylsilane-bonded silica gel column, isocratic elution, mobile phase A being acetate buffer, and mobile phase B being methanol or a mixture of methanol and acetonitrile. An ultraviolet (UV) detector is used. The derivatization reagent is a borate buffer containing phthalaldehyde and 3-mercaptopropionic acid.

[0009] Each 1 ml of the derivatization reagent contains 4 mg of o-phthalaldehyde and 4 μl of 3-mercaptopropionic acid.

[0010] The online derivatization conditions are as follows: accurately pipette 50-100 μl of derivatization reagent and test solution, add them to a vial pre-filled with 400-800 μl of borate buffer, mix well, derivatize for 1-5 min, and then inject into the high performance liquid chromatograph.

[0011] The mobile phase A is an acetate buffer solution with a pH of 5–7; the mobile phase B is methanol; the volume ratio of mobile phase A to mobile phase B is 9:1–6:4; the flow rate of the mobile phase is 1.0–2.0 ml / min; the column temperature is 20–40℃; and the detection wavelength is 300–350 nm.

[0012] Preferably, the pH value of mobile phase A is 5.9; mobile phase B is methanol; the volume ratio of mobile phase A to mobile phase B is 8:2; and the detection wavelength is 344 nm.

[0013] The specific implementation method of the detection method described in this invention is as follows: it includes the following steps:

[0014] (1) Preparation of borate buffer: Take sodium tetraborate, add water, heat to dissolve, let cool at room temperature, quickly adjust the pH to 9.5 with 10% sodium hydroxide solution, dilute with water, store at room temperature, if crystals precipitate, heat to dissolve or take the supernatant; 3.82g sodium tetraborate, add 80ml water, dilute with water to 100ml.

[0015] (2) Preparation of derivatization reagent: Take o-phthalaldehyde, dissolve it in anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and borate buffer, mix well, and let stand in the dark for 30 minutes before use; take 50 mg of o-phthalaldehyde, 1.25 ml of anhydrous methanol, 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer.

[0016] (3) Preparation of reference solution: Take an appropriate amount of glucosamine sulfate reference standard, dissolve it in water and dilute it quantitatively to prepare a solution containing 0.35 mg of glucosamine sulfate per 1 ml;

[0017] (4) Preparation of dissolution test solution: Take 1 glucosamine sulfate capsule, add 900ml of water, add a sedimentation basket, and follow the dissolution and release determination method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Method II), using 900ml of water as the dissolution medium and a rotation speed of 50 rpm. After 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes, take 10ml of the solution at each time, and promptly add 10ml of the solution to the dissolution cup to dissolve and evenly disperse the capsule. Filter the solution and take the filtrate to obtain the test solution.

[0018] (5) Use derivatization reagents to perform online derivatization and detection on a high performance liquid chromatograph; accurately pipette 50-100 μl of derivatization reagent and test solution, add to a vial pre-filled with 400-800 μl of borate buffer, mix well, derivatize for 1-5 min, and then inject into the high performance liquid chromatograph.

[0019] (6) The elution method is isocratic elution. Mobile phase A is acetate buffer, and mobile phase B is methanol or a mixture of methanol and acetonitrile. The detector is a UV detector. The pH of the acetate buffer solution is 5-7. Mobile phase B is methanol. The volume ratio of mobile phase A to mobile phase B is 9:1-6:4. The flow rate of the mobile phase is 1.0-2.0 ml / min. The column temperature is 20-40℃. The detection wavelength is 300-350 nm.

[0020] More preferably, mobile phase A is acetate buffer with a solution pH of 5.9; mobile phase B is methanol; the volume ratio of mobile phase A to mobile phase B is 8:2; the detection wavelength is 344 nm; take 100 μl of derivatization reagent and 100 μl of reference solution (or test solution), 400 μl of borate buffer, and derivatize for 1 min.

[0021] This invention employs online HPLC derivatization followed by immediate sample injection, which solves the problem of poor stability. At the same time, it is simple to operate, highly sensitive, and has good repeatability. Attached Figure Description

[0022] Figure 1 HPLC chromatogram of the dissolution test solution detected by direct HPLC injection in Example 1.

[0023] Figure 2 HPLC chromatogram of the dissolution test solution detected by derivatization method (derivatization for 1 minute) in Example 2.

[0024] Figure 3 HPLC chromatogram of the dissolution test solution detected by online derivatization in Example 3.

[0025] Figure 4 HPLC chromatogram for specificity confirmation by online derivatization in Example 4. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0027] Example 1: Detection using HPLC direct injection method

[0028] Chromatographic conditions: The column was HEXI-TECH Morphling WD-NH2 (4.6×150mm, 5μm); the mobile phase was phosphate buffer (0.7g potassium dihydrogen phosphate dissolved in 1000ml of water, and the pH was adjusted to 7.5 with ammonia)-acetonitrile (35:65); the flow rate was 1.5ml / min; the column temperature was 35℃; a UV detector was used with a detection wavelength of 195nm; and the injection volume was 50μl.

[0029] Experimental steps:

[0030] Solvent: Water

[0031] Dissolve the test sample solution: Take one glucosamine sulfate capsule, add 900 ml of water, sonicate for 10 minutes to dissolve and evenly disperse the capsule, filter, and collect the filtrate to obtain the test sample solution.

[0032] Sensitivity solution: Take 1 ml of the dissolved test sample solution, dilute with water to 10 ml, and shake well to obtain the solution.

[0033] Take 50 μl each of the dissolved test solution and the sensitivity solution, inject them into the liquid chromatograph, and detect them under the chromatographic conditions described above. Record the chromatograms.

[0034] The results are as follows Figure 1 As shown, glucosamine eluted at around 4.8 min. The signal-to-noise ratio of the glucosamine sulfate peak in the chromatogram of the sensitive solution (equivalent to 10% sample dissolution) was about 4, which was lower than the limit of quantitation. It can be seen that under these chromatographic conditions, the baseline fluctuation was large, the peak response was weak, and the detection sensitivity was low.

[0035] Example 2: Derivatization method for detection

[0036] Chromatographic conditions: The column was a GL Sciences Inert Sustain C18 (4.6 × 150 mm, 5 μm); the mobile phase was acetate buffer (6.8 g sodium acetate dissolved in 1000 ml of water, and the pH was adjusted to 5.9 with acetic acid) - methanol (80:20); the flow rate was 1.5 ml / min; the column temperature was 30 ℃; a UV detector was used with a detection wavelength of 344 nm; and the injection volume was 20 μl.

[0037] Experimental steps:

[0038] Borate buffer: Dissolve 3.82 g of sodium tetraborate in approximately 80 ml of water by slow heating. Allow to cool to lukewarm temperature at room temperature. Quickly adjust the pH to 9.5 with 10% sodium hydroxide solution and dilute to 100 ml with water. Store at room temperature. If crystals precipitate, dissolve by heating or collect the supernatant.

[0039] Derivatization reagent: Dissolve 50 mg of o-phthalaldehyde in 1.25 ml of anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer, and mix well (let stand in the dark for 30 minutes before use).

[0040] Dissolve the test sample solution: Take one glucosamine sulfate capsule, add 900 ml of water, sonicate for 10 minutes to dissolve and evenly disperse the capsule, filter, and collect the filtrate to obtain the test sample solution.

[0041] Take 1 ml of the dissolved test sample solution, 1 ml of derivatization reagent, and 4 ml of borate buffer, mix well, and derivatize for 1 minute, 5 minutes, 10 minutes, and 20 minutes respectively. Immediately inject the mixture into the liquid chromatograph and detect it under the above chromatographic conditions. Record the chromatogram. The results are as follows: Figure 2 As shown in the chromatographic diagram (derivatives for 1 minute), under these chromatographic conditions, glucosamine produces two derivative peaks: α-glucosamine derivative at approximately 5.6 min and β-glucosamine derivative at approximately 8.8 min. The signal-to-noise ratio of the glucosamine sulfate derivative peak in the chromatogram of the sensitive solution (equivalent to 10% sample dissolution) is approximately 100, which meets the detection requirements. However, after derivatization of the sample solution for 1, 5, 10, and 20 minutes, the peak areas of the glucosamine sulfate derivative in each chromatogram are as follows:

[0042]

[0043] The results showed that the peak area at 20 minutes of derivatization was equivalent to 90.8% of that at 1 minute. This indicates that as the storage time increases, the glucosamine sulfate derivative degrades, resulting in poor solution stability. It is necessary to prepare and process the solution immediately. However, since a single dissolution curve contains 72 dissolution samples, preparing and processing the solution immediately would undoubtedly be time-consuming and labor-intensive, making the detection process cumbersome and costly.

[0044] Example 3: Online derivatization detection according to the present invention

[0045] Chromatographic conditions: The column was a GL Sciences Inert Sustain C18 (4.6 × 150 mm, 5 μm); the mobile phase was acetate buffer (6.8 g sodium acetate dissolved in 1000 ml of water, and the pH was adjusted to 5.9 with acetic acid) - methanol (80:20); the flow rate was 1.5 ml / min; the column temperature was 30 ℃; a UV detector was used with a detection wavelength of 344 nm; and the injection volume was 20 μl.

[0046] Experimental steps:

[0047] Borate buffer: Dissolve 3.82 g of sodium tetraborate in approximately 80 ml of water by slow heating. Allow to cool to lukewarm temperature at room temperature. Quickly adjust the pH to 9.5 with 10% sodium hydroxide solution and dilute to 100 ml with water. Store at room temperature. If crystals precipitate, dissolve by heating or collect the supernatant.

[0048] Derivatization reagent: Dissolve 50 mg of o-phthalaldehyde in 1.25 ml of anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer, and mix well (let stand in the dark for 30 minutes before use).

[0049] Dissolve the test sample solution: Take one glucosamine sulfate capsule, add 900 ml of water, sonicate for 10 minutes to dissolve and evenly disperse the capsule, filter, and collect the filtrate to obtain the test sample solution.

[0050] Sensitivity solution: Take 1 ml of the dissolved test sample solution, dilute with water to 10 ml, and shake well to obtain the solution.

[0051] Accurately pipette 100 μl of the sensitivity solution and the dissolution test solution, add 100 μl of derivatization reagent, and then add to a vial pre-filled with 400 μl of borate buffer. Mix well, derivatize for 1 minute, and then inject into the liquid chromatograph. Detect under the above chromatographic conditions and record the chromatogram.

[0052] The results are as follows Figure 3 As shown, under these chromatographic conditions, glucosamine eluted two derivative peaks: the α-glucosamine derivative at approximately 6.1 min and the β-glucosamine derivative at approximately 9.7 min. The signal-to-noise ratio of the glucosamine sulfate derivative peak in the chromatogram of the sensitive solution (equivalent to 10% sample dissolution) was approximately 100. Except for a slight difference in elution time (possibly related to the instrument's dead volume), the results were basically consistent with those of Example 2. The sensitivity met the detection requirements. Using the instrument for online derivatization before injection solved the problem of poor solution stability and greatly improved the detection efficiency.

[0053] Example 4: Specificity Confirmation of the Online Derivatization Method of the Present Invention

[0054] Chromatographic conditions: The column was a GL Sciences Inert Sustain C18 (4.6 × 150 mm, 5 μm); the mobile phase was acetate buffer (6.8 g sodium acetate dissolved in 1000 ml of water, and the pH was adjusted to 5.9 with acetic acid) - methanol (80:20); the flow rate was 1.5 ml / min; the column temperature was 30 ℃; a UV detector was used with a detection wavelength of 344 nm; and the injection volume was 20 μl.

[0055] Experimental steps:

[0056] Borate buffer: Dissolve 3.82 g of sodium tetraborate in approximately 80 ml of water by slow heating. Allow to cool to lukewarm temperature at room temperature. Quickly adjust the pH to 9.5 with 10% sodium hydroxide solution and dilute to 100 ml with water. Store at room temperature. If crystals precipitate, dissolve by heating or collect the supernatant.

[0057] Derivatization reagent: Dissolve 50 mg of o-phthalaldehyde in 1.25 ml of anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer, and mix well (let stand in the dark for 30 minutes before use).

[0058] Blank excipient solution: Take 600mg corn starch, 285mg lactose, 50mg magnesium stearate and 25mg talc, mix them evenly, accurately weigh 96mg, add 900ml water, sonicate for 10 minutes to dissolve the sample and disperse it evenly, filter, and take the filtrate to obtain the blank excipient solution.

[0059] Dissolve the test sample solution: Take one glucosamine sulfate capsule, add 900 ml of water, sonicate for 10 minutes to dissolve and evenly disperse the capsule, filter, and collect the filtrate to obtain the test sample solution.

[0060] Accurately pipette 100 μl of the blank excipient solution and the test solution, add 100 μl of derivatization reagent, and then add to a vial pre-filled with 400 μl of borate buffer. Mix well, derivatize for 1 minute, and then inject into the liquid chromatograph. Detect under the above chromatographic conditions and record the chromatogram.

[0061] The results are as follows Figure 4 As shown, the blank excipient solution did not produce peaks at the positions of the α-glucosamine derivative peak and the β-glucosamine derivative peak, and did not interfere with the detection of α-glucosamine and β-glucosamine.

[0062] Example 5: Linearity Examination of the Online Derivatization Method of the Present Invention

[0063] Chromatographic conditions: The column was a GL Sciences Inert Sustain C18 (4.6 × 150 mm, 5 μm); the mobile phase was acetate buffer (6.8 g sodium acetate dissolved in 1000 ml of water, and the pH was adjusted to 5.9 with acetic acid) - methanol (80:20); the flow rate was 1.5 ml / min; the column temperature was 30 ℃; a UV detector was used with a detection wavelength of 344 nm; and the injection volume was 20 μl.

[0064] Experimental steps:

[0065] Borate buffer: Dissolve 3.82 g of sodium tetraborate in approximately 80 ml of water by slow heating. Allow to cool to lukewarm temperature at room temperature. Quickly adjust the pH to 9.5 with 10% sodium hydroxide solution and dilute to 100 ml with water. Store at room temperature. If crystals precipitate, dissolve by heating or collect the supernatant.

[0066] Derivatization reagent: Dissolve 50 mg of o-phthalaldehyde in 1.25 ml of anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer, and mix well (let stand in the dark for 30 minutes before use).

[0067] Serial linearity test solutions: Take an appropriate amount of glucosamine sulfate reference standard, dissolve it in water and dilute it quantitatively to prepare solutions containing approximately 0.035 mg, 0.07 mg, 0.14 mg, 0.28 mg, 0.35 mg and 0.42 mg per ml, respectively, and shake well to obtain the solution.

[0068] Accurately pipette 100 μl of each of the series of linear test solutions, add 100 μl of derivatization reagent, and then add to a vial pre-filled with 400 μl of borate buffer. Mix well, derivatize for 1 minute, and then inject into the liquid chromatograph. Detect under the above chromatographic conditions and record the chromatogram.

[0069] Linear regression analysis using the least squares method on concentration and peak area showed that glucosamine exhibited a good linear relationship between concentration and peak area in the range of 0.035 mg / ml to 0.42 mg / ml (equivalent to 10% to 120% of the dissolution amount). The linear equation was y = 7415540.17x + 15121.08, with a correlation coefficient r of 1.000 and an RSD of 3.8% for the response factor.

[0070] Example 6: Repeatability Confirmation of the Online Derivatization Method of the Present Invention

[0071] Chromatographic conditions: The column was a GL Sciences Inert Sustain C18 (4.6 × 150 mm, 5 μm); the mobile phase was acetate buffer (6.8 g sodium acetate dissolved in 1000 ml of water, and the pH was adjusted to 5.9 with acetic acid) - methanol (80:20); the flow rate was 1.5 ml / min; the column temperature was 30 ℃; a UV detector was used with a detection wavelength of 344 nm; and the injection volume was 20 μl.

[0072] Experimental steps:

[0073] Borate buffer: Dissolve 3.82 g of sodium tetraborate in approximately 80 ml of water by slow heating. Allow to cool to lukewarm temperature at room temperature. Quickly adjust the pH to 9.5 with 10% sodium hydroxide solution and dilute to 100 ml with water. Store at room temperature. If crystals precipitate, dissolve by heating or collect the supernatant.

[0074] Derivatization reagent: Dissolve 50 mg of o-phthalaldehyde in 1.25 ml of anhydrous methanol, add 50 μl of 3-mercaptopropionic acid and 11.2 ml of borate buffer, and mix well (let stand in the dark for 30 minutes before use).

[0075] Dissolve the test sample solution: Take one glucosamine sulfate capsule, add 900 ml of water, sonicate for 10 minutes to dissolve and evenly disperse the capsule, filter, and collect the filtrate to obtain the test sample solution.

[0076] Accurately pipette 100 μl of the dissolution test solution and 100 μl of the derivatization reagent into a vial pre-filled with 400 μl of borate buffer, mix well, derivatize for 1 minute, and then inject into the liquid chromatograph. Detect under the above chromatographic conditions, record the chromatogram, and perform parallel derivatization and injection 6 times.

[0077] The results of the injection of the dissolved sample solution after six derivatizations using the instrument are as follows:

[0078]

[0079] The results showed that the six leaching test solutions were basically consistent, with the RSD of the total peak area being less than 1.0%, indicating good repeatability of the online derivatization method.

[0080] Summarize

[0081] As shown in the results of Examples 1-3, the online derivatization method for detecting the dissolution curve of glucosamine sulfate capsules is highly sensitive, easy to operate, and solves problems such as the instability of chromatographic columns and sample solutions, greatly reducing various detection costs. In addition, Examples 4-6 demonstrate that the online derivatization method described in this invention has strong specificity and good repeatability, and can meet the detection requirements of different dissolution amounts of glucosamine sulfate capsules.

Claims

1. A high-performance liquid chromatography method for the detection of glucosamine sulfate capsules, characterized in that, Online derivatization and detection were performed using a high-performance liquid chromatograph (HPLC) with a derivatizing reagent. The chromatographic conditions were: an octadecylsilane-bonded silica column, isocratic elution, mobile phase A being acetate buffer, and mobile phase B being methanol or a mixture of methanol and acetonitrile. The detector was an ultraviolet detector. The derivatizing reagent was a borate buffer containing o-phthalaldehyde and 3-mercaptopropionic acid.

2. The detection method as described in claim 1, characterized in that, Each 1 ml of the derivatization reagent contains 4 mg of o-phthalaldehyde and 4 μl of 3-mercaptopropionic acid.

3. The detection method as described in claim 1, characterized in that, The online derivatization conditions are as follows: the derivatization reagent and the test solution are taken and added to a vial pre-filled with borate buffer, mixed well, derivatized for 1 to 5 minutes, and then injected into the high-performance liquid chromatograph.

4. The detection method as described in claim 3, characterized in that, The volume ratio of the biochemical reagent, the test solution, and the borate buffer is 1:1:

4.

5. The detection method as described in claim 1, characterized in that, The acetate buffer solution has a pH of 5–7; mobile phase B is methanol; the volume ratio of mobile phase A to mobile phase B is 9:1–6:4; the flow rate of the mobile phase is 1.0–2.0 ml / min; the column temperature is 20–40 °C; and the detection wavelength is 300–350 nm.

6. The detection method as described in claim 5, characterized in that, The mobile phase A is an acetate buffer solution with a pH of 5.9; the mobile phase B is methanol; the volume ratio of mobile phase A to mobile phase B is 8:2; and the detection wavelength is 344 nm.

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