Method for detecting morpholine ethanesulfonic acid and application thereof

Abstract

The invention discloses a method for detecting morpholine ethanesulfonic acid and application thereof. The method comprises the step of detecting a sample to be detected by using ion chromatography. According to the method, the residual quantity of morpholine ethanesulfonic acid is detected by adopting a high performance ion chromatograph, the lower detection limit can reach 0.04 mu g / ml, the quantitation limit can reach 0.13 mu g / ml, the method is better than the existing high performance liquid chromatography for detecting morpholine ethanesulfonic acid, and meanwhile, the method also has the characteristics of rapidness, simplicity, high sensitivity, strong interference resistance, simultaneous detection of multiple groups of data and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical technology, specifically relating to a method for detecting morpholine ethanesulfonic acid using ion chromatography. Background Technology

[0002] Morpholine ethanesulfonic acid (MES), also known as sodium 2-(N-methyl-4-pyrrolyl)ethanesulfonate, is a zwitterionic buffer that can replace highly toxic dimethylarsine, as well as ion buffers citrate and malate. It is commonly used in buffered media for bacteria, yeast, and mammalian cells. MES buffers play a crucial role in biochemical and biological research.

[0003] Carbodiimide crosslinking is widely used in biology, biomedicine, and biochemistry due to its simplicity and effectiveness. MES can provide a relatively stable pH range in the carbodiimide crosslinking system, providing a stable acid-base environment for the crosslinking reaction of carbodiimide.

[0004] Impurities in pharmaceutical quality standards refer to impurities introduced into drugs produced according to processes and raw materials approved by the National Medical Products Administration, either due to the manufacturing process or raw materials, or impurities generated during storage. The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (Q3A) specifies a confirmatory threshold of 0.1% or 1 mg for drug impurities in new active pharmaceutical ingredients. However, existing methods for detecting MES (Medium-terminated Excipients) suffer from drawbacks such as limited detection methods and low sensitivity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a novel method for detecting residual morpholine ethanesulfonic acid (MES) for impurity removal in manufacturing processes. This invention employs high-performance ion chromatography (HPLC) to detect MES residues, achieving a detection limit of 0.04 μg / ml and a quantitation limit of 0.13 μg / ml, which is superior to existing high-performance liquid chromatography (HPLC) methods for MES detection, providing data support for impurity removal checks in manufacturing processes.

[0006] One aspect of the present invention provides a method for detecting morpholine ethanesulfonic acid, comprising detecting the sample to be tested using ion chromatography.

[0007] In some embodiments, the method further includes: before injection, mixing and incubating the sample to be tested with a hydrogen peroxide solution, and then cooling to obtain an injection solution.

[0008] In the method of this invention, morpholine ethanesulfonic acid dissociates into ethanesulfonic acid anion in the presence of hydrogen peroxide. The ethanesulfonic acid anion can be detected using a highly efficient anion exchange-conductivity detection method, thereby realizing the detection of morpholine ethanesulfonic acid.

[0009] In some embodiments, the sample to be tested is diluted with a diluent. In some embodiments, the diluent is water, such as ultrapure water or deionized water.

[0010] In the method of this invention, the solution system of the sample to be tested significantly affects the peak elution in ion chromatography. Compared with solution systems such as PBS, using an ultrapure aqueous solution system is more advantageous for the ion chromatographic detection of morpholine ethanesulfonic acid.

[0011] In some embodiments, the mass concentration of the hydrogen peroxide solution is 20-40%. In some specific embodiments, the mass concentration of the hydrogen peroxide solution is 20%, 25%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, or any value between them. In some embodiments, the mass concentration of the hydrogen peroxide solution is 25-35%. In some specific embodiments, the mass concentration of the hydrogen peroxide solution is 29-32%.

[0012] In some embodiments, the volume of the hydrogen peroxide solution is 10 μl to 30 μl. In some embodiments, the volume of the hydrogen peroxide solution is 10 μl, 12.5 μl, 15 μl, 17.5 μl, 20 μl, 30 μl, or any value between them. In some preferred embodiments, the volume of the hydrogen peroxide solution is 10 μl to 20 μl. In some preferred embodiments, the volume of the hydrogen peroxide solution is 12 μl to 15 μl.

[0013] In some embodiments, the volume ratio of the test sample to the hydrogen peroxide solution is 500:(10-30). In some embodiments, the volume ratio of the test sample to the hydrogen peroxide solution is 500:10, 500:12.5, 500:15, 500:17.5, 500:20, 500:30, or any value between them. In some embodiments, the volume ratio of the test sample to the hydrogen peroxide solution is 500:(10-20). In some embodiments, the volume ratio of the test sample to the hydrogen peroxide solution is 500:(12-15).

[0014] In some embodiments, the incubation temperature is 90°C-110°C, and the incubation time is 6-10 hours. In some embodiments, the incubation temperature is 90°C, 95°C, 100°C, 105°C, 110°C, or any value between them. In some embodiments, the incubation time is 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any value between them.

[0015] In some embodiments, the injection temperature is 12-20°C, for example, 12°C, 14°C, 15°C, 16°C, 18°C, 20°C, or any value between them. In some preferred embodiments, the injection temperature is 14-16°C.

[0016] In some embodiments, the chromatographic column of the ion chromatography is an anion exchange column.

[0017] In some embodiments, the chromatographic column of the ion chromatography comprises an analytical column packed with a latex-polymerized anion exchanger.

[0018] In some embodiments, the ion chromatography column includes an IonPac AG 18 guard column and an IonPac AS18 analytical column. In some specific embodiments, the ion chromatography column includes Thermo Scientific. TM Dionex TM IonPac TM AG18 protective pillars and Thermo Scientific TM Dionex TM IonPac TM AS18 analytical column.

[0019] In some embodiments, the column temperature of the chromatographic column is 28-32°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C or any value between them.

[0020] In some embodiments, the method further includes gradient elution with a 2-35 mmol / L aqueous sodium hydroxide solution. In some embodiments, the concentration of the aqueous sodium hydroxide solution is 2 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, or any value between therewith.

[0021] In some embodiments, the gradient elution is performed using mobile phase A and mobile phase B, wherein mobile phase A is a 100 mmol / L sodium hydroxide aqueous solution and mobile phase B is ultrapure water.

[0022] Preferably, the gradient elution procedure includes:

[0023] (1) 0-2 min, 2% mobile phase A and 98% mobile phase B;

[0024] (2) 2-25 min, 2%-35% mobile phase A and 65%-98% mobile phase B;

[0025] (3) 25-35 min, 2% mobile phase A and 98% mobile phase B.

[0026] In some embodiments, the injection volume of the ion chromatograph is 40-60 μl, for example, 50 μl.

[0027] In some embodiments, the injection flow rate of the ion chromatography is 1-2 ml / min, for example, 1.50 ml / min.

[0028] In some embodiments, the elution flow rate of the ion chromatography is 1-2 ml / min, for example, 1.50 ml / min.

[0029] In some embodiments, the conductivity detector current of the ion chromatograph is 130-220 mA, for example, 130 mA, 150 mA, 170 mA, 190 mA, 200 mA, 210 mA, 211 mA, 220 mA or any value between them.

[0030] In some embodiments, the column oven temperature of the ion chromatograph is 28-32°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C or any value between them.

[0031] In some embodiments, the detection chamber temperature of the ion chromatograph is 32-38°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C or any value between them.

[0032] In some embodiments, the method further includes: obtaining the content of morpholine ethanesulfonic acid in the sample to be tested based on the chromatographic peak area of ​​morpholine ethanesulfonic acid obtained by detection.

[0033] In some embodiments, the method further includes preparing a standard curve using working solutions of morpholine ethanesulfonic acid reference standard at gradient concentrations.

[0034] In some embodiments, the concentration of the morpholine ethanesulfonic acid reference working solution is 2.0 μg / ml to 20.0 μg / ml. In some specific embodiments, the concentration of the morpholine ethanesulfonic acid reference working solution includes 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml.

[0035] Another aspect of the present invention provides an ion chromatography apparatus for the method described in the first aspect, the ion chromatography apparatus comprising a mobile phase delivery system, an injection system, a separation system, a detection system, and a data acquisition and analysis system connected in sequence; the mobile phase delivery system includes a mobile phase and a delivery pump; the injection system includes an autosampler; the separation system includes a guard column and an analytical column; the detection system includes a suppressor and a conductivity detector; and the data acquisition and analysis system includes a data acquisition and analysis unit.

[0036] In some embodiments, the device further includes a regenerator and a waste liquid pipe connected in sequence to the conductivity detector.

[0037] In some embodiments, the packing material of the analytical column contains a latex-aggregated anion exchanger.

[0038] In some embodiments, the protective post is an IonPac AG 18 protective post. In some specific embodiments, the protective post is a Thermo Scientific one. TM Dionex TM IonPac TM AG18 protective pillar.

[0039] In some embodiments, the analytical column is an IonPac AS18 analytical column. In some specific embodiments, the analytical column is a Thermo Scientific column. TM Dionex TM IonPac TM AS18 analytical column.

[0040] In some embodiments, the conductivity detector is an ASRS / AERS–4mm.

[0041] Another aspect of the present invention provides the application of the method described in the first aspect or the ion chromatography apparatus described in the second aspect in the detection of morpholine ethanesulfonic acid in pharmaceutical impurities.

[0042] This invention employs ion chromatography for the detection of morpholine ethanesulfonic acid, featuring rapid, simple, highly sensitive, and interference-resistant methods, as well as the ability to simultaneously detect multiple data sets. The detection limit reaches 0.04 μg / ml, and the quantitation limit reaches 0.13 μg / ml. The conductivity detector of this invention is primarily used for detecting inorganic anions and cations, and some polar organic compounds, such as certain carboxylic acids. Various strong acid and strong base anions and cations, such as chloride, sulfate, sodium, and potassium ions, exhibit excellent sensitivity on the conductivity detector. Furthermore, this invention utilizes a specific Dionex IonPac AS18 column, a high-capacity, hydroxide-selective anion exchange column. It can inject high-concentration samples without column overload or peak broadening, and is compatible with organic solvents, thereby improving analyte solubility, altering column selectivity, or effectively purifying the column. Attached Figure Description

[0043] Figure 1 The chromatogram of the 10.0 μg / ml MES reference standard from Example 1 is shown.

[0044] Figure 2 The standard curve of MES detection in Example 1 is shown.

[0045] Figure 3 Chromatograms of the MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml in Comparative Example 1 are shown.

[0046] Figure 4 Chromatograms of the MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml in Comparative Example 2 are shown.

[0047] Figure 5 Chromatograms of the MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml in Comparative Example 3 are shown. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0049] Example 1: Detection method for morpholine ethanesulfonic acid

[0050] Ion chromatography apparatus: Connect the eluent bottle containing 100 mmol / L sodium hydroxide solution (mobile phase A) and the eluent bottle containing ultrapure water (mobile phase B) to the channel respectively. Maintain a nitrogen protective pressure of 3-6 psi in both eluent bottles to prevent carbonate formation. Flush the tubing to remove air bubbles. Connect the IonPac AG 18 guard column (Thermo Scientific). TM Dionex TM IonPac TM AG18 and IonPac AS18 analytical columns (Thermo Scientific) TM Dionex TM IonPac TM The analysis column (AS18) is connected to the suppressor input, and the suppressor output is connected to the conductivity detector input. The conductivity detector output is connected to the regenerator input, and the regenerator output is connected to the waste liquid line.

[0051] Dilute the 1 mg / ml MES (SIGMA product code M3671-50G) reference stock solution 20-fold with ultrapure water to prepare a 0.05 mg / ml MES working solution. Prepare fresh before use. Using the 0.05 mg / ml MES working solution, prepare MES reference solutions at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml in cryopreserved tubes. Use ultrapure water as a blank. Dilute the test sample (ACYW135X group meningococcal polysaccharide conjugate vaccine stock solution developed and produced by Beijing Sinovac Biotech Co., Ltd.) to the MES concentration range and set aside.

[0052] Take 500 μl of blank, reference standard, and test sample into cryovials, and add 12.5 μl of 29-32% hydrogen peroxide solution (ThermoFisher product code 033323). Incubate in a metal bath at 100℃ for 8 h, remove and allow to room temperature, then transfer to sample vials. Place the sample vials into the injection tray of the ion chromatograph in the order listed in the sequence list, and inject them sequentially for analysis. Plot a standard curve with peak area (y) as the ordinate and MES solution concentration (x) as the abscissa to obtain the regression equation. Analyze the chromatogram of the test sample using instrument software, integrate the characteristic peaks of MES, and substitute them into the regression equation to calculate the MES residue. The parameters of ion chromatography are shown in Table 1 below.

[0053] Table 1 Chromatographic parameters detected by MES

[0054]

[0055] The above method was used to detect the residual amount of MES in the ACYW135X group meningococcal polysaccharide conjugate vaccine stock solution developed and produced by Beijing Sinovac Biotech Co., Ltd., and the linear range, specificity, accuracy, precision, robustness, limit of detection and limit of quantitation were analyzed.

[0056] Experiment 1. Linear Range

[0057] The peak areas of the reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml were determined three times. A standard curve was plotted with the concentration of the MES reference solution as the x-axis and the peak area as the y-axis, and the regression equation was calculated (see Table 2). For example, the retention time of the peak of the 10.0 μg / ml MES reference standard 1 was 5.097 min, and the coefficient of determination (R²) of the regression equation for the standard curve was >0.99. Figure 1 and Figure 2 As shown.

[0058] Table 2. Linearity and Range Validation Results

[0059]

[0060] The validation results in Table 2 show that when the concentration of MES reference standard is between 2.0 μg / ml and 20.0 μg / ml, the concentration of MES is linearly related to the peak area, with R2 ≥ 0.99, indicating that the method has good linearity.

[0061] Experiment 2. Specificity

[0062] This experiment investigates the interference of exogenous additive carbodiimide (hereinafter referred to as EDAC) in the MES residual detection during the production process.

[0063] Prepare a 1000 μg / ml EDAC solution and dilute it with purified water to 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml. Mix the 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml EDAC solutions with equal volumes of 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml MES reference standards, respectively, to achieve final MES and EDAC concentrations of 5.0 μg / ml, 10.0 μg / ml, and 15.0 μg / ml, respectively. Separately, mix the 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml EDAC solutions and the MES reference standard with equal volumes of ultrapure water to serve as blank controls.

[0064] The specific results are shown in Table 3. The recovery rate is calculated as: (MES content in the mixed MES and EDAC sample / theoretically added MES standard content) × 100%.

[0065] Table 3 Specificity Validation Results

[0066]

[0067]

[0068] The results in Table 3 show that EDAC did not elute under these ion chromatography conditions, and MES and EDAC did not appear at the same position. The theoretical recovery rate of the MES reference standard was 82.40%–100.13%, which meets the validation standard that the recovery rate should be between 80% and 120%, indicating that the method has good specificity.

[0069] Experiment 3. Accuracy

[0070] Three batches of group A and group X meningococcal conjugate stock solutions were each added in equal volumes to MES reference standards at concentrations of 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml, respectively, to achieve final MES concentrations of 5.0 μg / ml, 10.0 μg / ml, and 15.0 μg / ml, respectively, as spiked samples. Three separate batches of group A and group X meningococcal conjugate stock solutions were added in equal volumes to ultrapure water as blank controls. Equal volumes of 10.0 μg / ml, 20.0 μg / ml, and 30.0 μg / ml MES reference standards were mixed with ultrapure water to prepare spiked controls. The MES content in the spiked samples, blank controls, and spiked controls was then determined.

[0071] The results are shown in Tables 4 and 5. The spiked recovery rate was calculated as: (MES content in the spiked sample - MES content in the blank control) / MES content in the spiked control standard × 100%.

[0072] Table 4. Accuracy verification results for Group A

[0073]

[0074] Table 5. Accuracy verification results of the X group

[0075]

[0076]

[0077] The results in Tables 4 and 5 show that the MES spiked recovery rate is between 81% and 102%, which meets the validation criteria of a recovery rate between 80% and 120%.

[0078] Experiment 4. Precision

[0079] (1) Repeatability

[0080] In the same laboratory, the same operator injected 5 μg / ml MES reference standard 6 times consecutively. The relative standard deviation (RSD) of the 6 measurements was 3.64%, and the verification results met the standard of repeatability RSD (%) ≤ 4%.

[0081] (2) Intermediate precision

[0082] In the same laboratory, the same operator tested 2.0 μg / ml, 10.0 μg / ml, and 20.0 μg / ml MES reference standards at different time points (1, 2, and 3 days). The RSD% of the three batches of MES reference standards at different time points ranged from 2.22% to 7.39%, and the validation results met the validation standard requirement of RSD% ≤ 8%.

[0083] Experiment 5. Durability

[0084] The effect of different incubation times on the detection of MES residues was investigated. 10 μg / ml MES was incubated for 7 h 50 min, 8 h, and 8 h 10 min, respectively. The recovery rate of 10 μg / ml MES was between 100.90% and 103.27%, which met the validation criteria.

[0085] Experiment 6. Limit of Detection and Limit of Quantification

[0086] The signal-to-noise ratio (S / N) of the 2 μg / ml reference standard was determined by six different injections into the MES. The limit of detection (S / N = 3) was calculated to be 0.04 μg / ml, and the limit of quantitation (S / N = 10) was calculated to be 0.13 μg / ml. The results are shown in Table 6.

[0087] Table 6 Limit of Detection and Limit of Quantification

[0088]

[0089]

[0090] As can be seen from the above analysis, the linear range, specificity, accuracy, precision, robustness, detection limit and quantitation limit of the method of the present invention all meet the verification requirements of the "9101 Analytical Method Validation Guidelines" in Part III of the Chinese Pharmacopoeia for detection methods, and have guiding significance for the production process.

[0091] Comparative Example 1

[0092] The ion chromatography apparatus is the same as in Example 1.

[0093] Dilute the 1 mg / ml MES standard stock solution 20 times with ultrapure water to prepare a 0.05 mg / ml MES standard working solution. Prepare fresh before use. Take the cryovials and prepare standard solutions of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml using the 0.05 mg / ml MES standard. Use ultrapure water as a blank.

[0094] Take 500 μl of the reference standard into a cryovial and add 50.0 μl of 50% hydrogen peroxide solution. Incubate in a metal bath at 100 °C for 8 h, then remove and allow to cool to room temperature before transferring to a sample vial. Place the vial into the injection tray of an ion chromatograph and inject the vial sequentially for analysis.

[0095] The MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml all showed no peak at the characteristic peak of MES. Figure 3 As shown.

[0096] Conclusion: When the concentration and volume of the hydrogen peroxide solution were changed, no peaks were observed under these chromatographic conditions compared to the conditions in Example 1 (12.5 μl of 29-32% hydrogen peroxide solution), indicating that MES could not be effectively detected.

[0097] Comparative Example 2

[0098] The ion chromatography apparatus is the same as in Example 1, except that it is connected with an IonPac AG 15 guard column (Thermo Scientific). TM Dionex TM IonPac TM AG15 and IonPac AS15 analytical columns (Thermo Scientific) TM Dionex TM IonPac TM AS15). Among them, the filler of IonPac AS15 is surface-ammonized ethylvinylbenzene-divinylbenzene (EVB-DVB), and the filler of IonPac AS18 is latex-polymerized anion exchanger.

[0099] Dilute the 1 mg / ml MES standard stock solution 20 times with ultrapure water to prepare a 0.05 mg / ml MES standard working solution. Prepare fresh before use. Take the cryovials and prepare standard solutions of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml using the 0.05 mg / ml MES standard. Use ultrapure water as a blank.

[0100] Take 500 μl of the reference standard into a cryovial and add 12.5 μl of 29-32% hydrogen peroxide solution. Incubate in a metal bath at 100℃ for 8 h, then remove and allow to cool to room temperature before transferring to a sample vial. Place the vial into the injection tray of the ion chromatograph and inject the sample sequentially for analysis.

[0101] The MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml all showed no peak at the characteristic peak of MES. Figure 4 As shown.

[0102] Conclusion: When the types of analytical columns and guard columns are changed, compared to the conditions in Example 1 (IonPac AG 18 guard column (Thermo Scientific)... TM Dionex TM IonPac TM AG18 and IonPac AS18 analytical columns (Thermo Scientific) TM Dionex TM IonPac TM Under these chromatographic conditions, no peaks were observed in AS18, indicating that MES could not be effectively detected.

[0103] Comparative Example 3

[0104] The ion chromatography apparatus is the same as in Example 1.

[0105] Dilute the 1 mg / ml MES standard stock solution 20-fold with 10 mmol / L PBS buffer to prepare a 0.05 mg / ml MES standard working solution. Prepare fresh before use. Take the cryovials and prepare 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml solutions of the 0.05 mg / ml MES standard, respectively. Prepare a 10 mmol / L PBS buffer as a blank.

[0106] Take 500 μl of the reference standard into a cryovial and add 12.5 μl of 29-32% hydrogen peroxide solution (SIGMA product code 033323). Incubate in a metal bath at 100°C for 8 h, then remove and allow to cool to room temperature before transferring to a sample vial. Place the vial in the injection tray of the ion chromatograph and inject the vial sequentially for analysis.

[0107] The MES reference standards at concentrations of 2.0 μg / ml, 5.0 μg / ml, 10.0 μg / ml, 15.0 μg / ml, and 20.0 μg / ml all showed no peak at the characteristic peak of MES. Figure 5 As shown.

[0108] Conclusion: When the buffer dilution was changed, no peaks were observed under these chromatographic conditions compared to the conditions of Example 1 (where the diluent was water), indicating that MES could not be effectively detected. A comparison of Example 1 and Comparative Examples 1-3 shows that the chromatographic conditions of Comparative Examples 1-3 were ineffective for the qualitative and quantitative analysis of MES.

[0109] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for detecting morpholine ethanesulfonic acid, comprising detecting the sample to be tested using ion chromatography.

2. The method according to claim 1, characterized in that, The method further includes: before injection, mixing and incubating the sample to be tested with hydrogen peroxide solution, and cooling to obtain the injection solution; Preferably, the sample to be tested is diluted with a diluent; more preferably, the diluent is water. Preferably, the mass concentration of the hydrogen peroxide solution is 20-40%, more preferably 25-35%; Preferably, the volume of the hydrogen peroxide solution is 10 μl-30 μl, more preferably 10-20 μl; Preferably, the volume ratio of the sample to be tested to the hydrogen peroxide solution is 500:(10-30), more preferably 500:(10-20).

3. The method according to claim 2, characterized in that, The mixed incubation temperature is 90℃-110℃, and the time is 6-10 hours; and / or The injection temperature is 12-20℃, preferably 14-16℃.

4. The method according to claim 1, characterized in that, The chromatographic column used in the ion chromatography is an anion exchange column. Preferably, the chromatographic column of the ion chromatography comprises an analytical column containing a latex-aggregated anion exchanger as packing material; Preferably, the chromatographic column of the ion chromatography includes an IonPac AG 18 guard column and an IonPac AS18 analytical column; Preferably, the column temperature of the chromatographic column is 28-32℃.

5. The method according to claim 1, characterized in that, The method further includes gradient elution using a 2-35 mmol / L sodium hydroxide aqueous solution as the mobile phase; Preferably, the gradient elution is performed using mobile phase A and mobile phase B, wherein mobile phase A is a 100 mmol / L sodium hydroxide aqueous solution and mobile phase B is ultrapure water; Preferably, the gradient elution procedure includes: (1) 0-2 min, 2% mobile phase A and 98% mobile phase B; (2) 2-25 min, 2%-35% mobile phase A and 65%-98% mobile phase B; (3) 25-35 min, 2% mobile phase A and 98% mobile phase B.

6. The method according to claim 1, characterized in that, The injection volume for the ion chromatography is 40-60 μl; and / or The injection flow rate for the ion chromatography is 1-2 ml / min; and / or The elution flow rate for the ion chromatography is 1-2 ml / min; and / or The conductivity detector current of the ion chromatograph is 130-220 mA; and / or The column oven temperature for the ion chromatography is 28-32℃; and / or The detection chamber temperature of the ion chromatograph is 32-38℃.

7. The method according to claim 1, characterized in that, The method further includes: obtaining the content of morpholine ethanesulfonic acid in the sample to be tested based on the chromatographic peak area of ​​morpholine ethanesulfonic acid obtained by detection; Preferably, the method further includes preparing a standard curve using working solutions of morpholine ethanesulfonic acid reference standard with gradient concentrations. More preferably, the concentration of the morpholine ethanesulfonic acid reference working solution is 2.0 μg / ml to 20.0 μg / ml.

8. An ion chromatography apparatus for use in the method of any one of claims 1-7, the ion chromatography apparatus comprising a mobile phase delivery system, an injection system, a separation system, a detection system, and a data acquisition and analysis system connected in sequence; The mobile phase delivery system includes a mobile phase and a delivery pump; The sample introduction system includes an autosampler; The separation system includes a guard column and an analytical column; The detection system includes a suppressor and a conductivity detector; The data acquisition and analysis system includes a data acquisition and analysis unit; Preferably, the device further includes a regenerator and a waste liquid pipe connected in sequence to the conductivity detector.

9. The ion chromatography apparatus according to claim 8, characterized in that, The protective post is an IonPac AG 18 protective post, and / or The analytical column is an IonPac AS18 analytical column, and / or The conductivity detector is an ASRS / AERS–4mm.

10. The application of the method of any one of claims 1-7 or the ion chromatography apparatus of claim 8 or 9 in the detection of morpholine ethanesulfonic acid in pharmaceutical impurities.

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