Method for simultaneously determining N-sodium laureth sarcosinate and phenol red residues in injection based on UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method

Abstract

The invention discloses a method for simultaneously determining N-sodium laureth sarcosinate and phenol red residues in an injection based on a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method. According to the method, on the basis of an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, a 5mM ammonium acetate solution (containing 10% acetonitrile) is taken as a phase A, a 0.2% acetic acid acetonitrile solution is taken as a phase B, a Waters Acquity UPLCBEH C18 column is selected, ESI is taken as a mass spectrometry detection ion source, and the two process residues in the injection are accurately quantified at the same time in a negative ion scanning and multi-reaction monitoring mode. The method is simple in pretreatment, high in detection sensitivity and good in reproducibility, and can be used for corresponding injection process residue identification and quality control.

Description

Technical Field

[0001] The present invention relates to the fields of pharmacy and mass spectrometry analysis and detection, and in particular to a method for simultaneously determining the content of N -Analytical method for sodium lauryl sarcosinate and phenol red residues. Background Art

[0002] The Pharmacopoeia of the People's Republic of China (2020 edition) defines injections as sterile preparations for injection into the body, made from raw drug substances or suitable excipients. Injections can be categorized as injection solutions, sterile powders for injection, and concentrated solutions for injection. Injections refer to sterile liquid preparations for injection into the body, made from raw drug substances or suitable excipients, including solutions, emulsions, and suspensions. They can be administered subcutaneously, intradermally, intramuscularly, intravenously, intravenously, intravenously, intrathecally, and intrathecally.

[0003] Injectables can be categorized by physical state into injection solutions (water injections), powders for injection (powder injections), and tablets for injection. This is primarily determined by the drug's properties and medical requirements. Generally, water-soluble drugs are formulated as aqueous solutions or water-complex solutions (e.g., aqueous solutions supplemented with ethanol, propylene glycol, or glycerol). Some drugs are not suitable for aqueous solutions, such as those that are poorly soluble in water or require prolonged efficacy after injection. Instead, they can be formulated as oil solutions, water- or oil-based suspensions, or emulsions. However, these injections are generally intended only for intramuscular injection.

[0004] The Pharmacopoeia of the People's Republic of China stipulates that in addition to the main drug, other substances may be added to injections according to the needs of preparation and medical treatment to increase the effectiveness, safety and stability of the injection. Such substances are collectively referred to as additives for injections. The selected varieties and concentrations used are non-toxic to the body, have no incompatibility with the main drugs, and do not affect the main efficacy and content determination. The different uses of additives should also be indicated in the instructions for the finished preparation. Indicators such as phenol red and surfactants such as sodium N-lauryl sarcosinate may need to be added during the production process. At the finished product stage, such process residues need to be strictly controlled.

[0005] Currently, due to N - Sodium lauryl sarcosinate and phenol red compounds have different structural characteristics and their chromatographic behaviors are also different. N -Structural characteristics of sodium lauryl sarcosinate and phenol red. This method uses a C18 column for separation. However, there is currently no method that can simultaneously detect N - Sodium lauryl sarcosinate and phenol red, and considering the low sensitivity of HPLC detection, the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) method combines the high separation capability of ultra-high performance liquid chromatography with the high specificity and high sensitivity of mass spectrometry, which can achieveN -Simultaneous detection and accurate quantification of sodium lauryl sarcosinate and phenol red, with shorter detection time and less sample required. Summary of the Invention

[0006] Purpose of the invention: The present invention aims to provide a method based on UPLC-MS / MS for the simultaneous determination of N - The analytical method for the residual sodium lauryl sarcosinate and phenol red is to dilute the injection solution with 50% methanol solution, and then perform pre-treatment steps such as vortexing, shaking, and high-speed centrifugation, and finally take the supernatant for detection. This method can accurately and reliably determine the above-mentioned residues in the injection solution. N -Residues of sodium lauryl sarcosinate and phenol red are used for process residue identification and quality control.

[0007] The present invention provides the following solutions: First, accurately weigh appropriate amounts of sodium N-lauryl sarcosinate and phenol red reference substance, and use methanol or 50% methanol solution as the diluent to prepare a working stock solution of the reference substance at an appropriate concentration; Then, accurately measure an appropriate amount of the injection solution, dilute and extract it with 50% methanol solution, vortex to fully dissolve it, and then obtain the test solution after high-speed centrifugation; Next, accurately pipette an appropriate amount of each reference substance working stock solution and dilute it with a 50% methanol solution in a gradient to prepare a mixed standard working solution. Detect the solution using UPLC-MS / MS. Based on the peak area of ​​the obtained chromatogram, draw a linear fitting standard curve representing the relationship between the reference substance concentration and peak area, and calculate the corresponding linear fitting equation. Finally, accurately pipette an appropriate amount of the test solution and use the UPLC-MS / MS method for detection. Based on the measured chromatogram peak area, substitute it into the standard curve fitting equation to calculate the above two process residues in the test solution.

[0008] The UPLC-MS / MS method used in the present invention employed the following chromatographic conditions: an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm) was used as the chromatographic column, mobile phase A consisted of 5 mM ammonium acetate solution (containing 10% acetonitrile), mobile phase B consisted of 0.2% acetic acid in acetonitrile, and gradient elution conditions were: 0% B (0-0.2 min); 0-30% B (0.2-3 min); 30% B (30-5 min); 30-90% B (5-7 min); 90-98% B (7-10 min); 98% B (10-11 min); 98-0% B (11-11.1 min); and 0% B (11.1-12 min). The total LC run time was 12 min, the flow rate was 0.3 ml / min, the column temperature was 35°C, and the injection volume was 10 μl.

[0009] The mass spectrometry conditions for the UPLC-MS / MS method used in this invention are: an ESI ion source, negative ion simultaneous scanning, and MRM acquisition mode for targeted detection of two targets. The target MRM ion pairs and corresponding mass spectrometry parameters are listed in the table below. Furthermore, the ion source parameters for the positive ESI method were: curtain gas at 35.0 psi, collision gas at medium, ionization voltage at -4500 V, temperature at 450°C, and auxiliary heating gas at 50 and 50 psi, respectively.

[0010] *One ion pair is used as a quantitative ion pair for calibration and sample concentration quantification; the other is used as a qualitative ion pair for determining the elution time of the chromatographic peak.

[0011] The injection pretreatment process of the present invention is as follows: after the injection stock solution sample is taken out from the -80°C refrigerator, it is repeatedly frozen and thawed; 15 μl is accurately pipetted into a clean EP tube, 985 μl of 50% methanol aqueous solution is added, and the mixture is mixed at 2000 rpm on a mixer for 5 minutes, centrifuged at 12000 rpm for 5 minutes, and 100 μl of the supernatant is pipetted into a new EP tube for sample injection and analysis.

[0012] More preferably, the UPLC-MS / MS method of the present invention uses a QTRAP 6500 plus triple quadrupole mass spectrometer.

[0013] Furthermore, the UPLC-MS / MS method of the present invention N -The lowest limit of quantification of the process residue of sodium laurel sarcosinate is only 0.4 ng / ml, and the method has high sensitivity.

[0014] Beneficial effect: The present invention provides a method for simultaneously determining the N - The analytical method for sodium lauryl sarcosinate and phenol red residues has the following advantages compared with the existing technology: High sample detection efficiency: The method of the present invention adopts ESI negative ion synchronous scanning mode, which can achieve N -Simultaneous detection of sodium lauryl sarcosinate and phenol red effectively improves sample detection efficiency.

[0015] (2) The method is highly sensitive: the limits of quantification of the two process residues under the method described in the present invention are 0.4 ng / ml for sodium N-lauroylsarcosinate and 4 ng / ml for phenol red.

[0016] (3) Simple and convenient sample pretreatment: The present invention adopts vortex extraction method for sample pretreatment, which is relatively simple, convenient and fast to operate and can shorten the sample detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attachment Figure 1 The standard curves of the two process residual mixed reference solutions are shown in Figure 2. Attachment Figure 2 Mass spectra of two process residual mixed reference solution Attachment Figure 3 Mass spectra of the residual test sample solutions from two processes Attachment Figure 4 Mass spectra of the spiked solutions of two process residual blank samples DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to specific implementation cases: 1. Experimental equipment: The instruments mainly include AB Sciex 6500 plus UPLC-MS / MS (composed of Sciex Exion LC AD ultra-high performance liquid chromatograph and QTRAP 6500 plus mass spectrometer); Eppendorf Peb-11 multi-well mixer and Eppendorf5427R ultra-high-speed constant temperature centrifuge.

[0019] 2. Liquid chromatography conditions: Waters Acquity UPLC was used for liquid chromatography ® A BEH C18 column (100 × 2.1 mm, 1.7 μm) was used as the chromatographic column. The mobile phase A consisted of 5 mM ammonium acetate solution (containing 10% acetonitrile) and the mobile phase B consisted of 0.2% acetic acid in acetonitrile. The mobile phase gradient shown in Table 1 was used for 12 min at a flow rate of 0.3 ml / min. The column temperature was 35 °C and the injection volume was 10 μl.

[0020]

[0021] 3. Mass spectrometry conditions: Mass spectrometry detection was performed using an ESI ion source in negative ion scan mode and MRM acquisition mode to achieve targeted detection of five targets. The target MRM ion transitions and corresponding mass spectrometry parameters are listed in the table below. Furthermore, the ion source parameters for the ESI negative ion method were: curtain gas at 35.0 psi, collision gas at medium, ionization voltage at -4500 V, temperature at 450°C, and auxiliary heating gas at 50 and 50 psi, respectively. The target MRM ion transitions and corresponding mass spectrometry parameters are listed in Table 2.

[0022] *One ion pair is used as a quantitative ion pair for calibration and sample concentration quantification; the other is used as a qualitative ion pair for determining the elution time of the chromatographic peak.

[0023] 4. Preparation of mixed reference standard curve working solution: Preparation of reference substance working stock solution: weigh accurately N - Take appropriate amount of sodium lauric sarcosinate and phenol red reference substances, first dilute to a concentration of 1 mg / ml of each reference substance stock solution with methanol as the diluent, then pipette appropriate amount of each reference substance stock solution, add 50% methanol solution to mix and dilute, and prepare 10 μg / ml of each reference substance working stock solution 1; then pipette appropriate amount of each reference substance working stock solution 1, add 50% methanol solution to mix and dilute, and prepare 200 ng / ml of each reference substance working stock solution 2, which will be used for the subsequent preparation of mixed standard curve working solution.

[0024] Preparation of mixed standard curve working solution: Accurately pipette an appropriate amount of each reference substance working stock solution, and then dilute it with 50% methanol solution to prepare a mixed standard curve working solution with a concentration of 1.5-150 ng / ml. The specific preparation concentration and process are as follows: Linearity solution 1: Precisely pipette 7.5 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 992.5 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (1.5 ng / ml).

[0025] Linearization solution 2: Precisely pipette 15 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 985 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (3 ng / ml).

[0026] Linearity solution 3: Precisely pipette 30 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 970 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (6 ng / ml).

[0027] Linearity solution 4: Precisely pipette 75 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 925 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (15 ng / ml).

[0028] Linearization solution 5: Precisely pipette 150 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 850 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (30 ng / ml).

[0029] Linearity solution 6: Precisely pipette 300 μl N- Sodium lauroyl sarcosinate and phenol red reference working stock solution 2 were placed in a clean centrifuge tube, 700 μl of 50% methanol aqueous solution was added to 1 ml, and vortexed to mix (60 ng / ml).

[0030] Linearity solution 7: Precisely pipette 750 μl N - Prepare the working stock solution 2 of sodium lauroyl sarcosinate and phenol red reference substance in a clean centrifuge tube, add 250 μl of 50% methanol aqueous solution to 1 ml, and vortex to mix (150 ng / ml).

[0031] 5. Sample processing method: After the injection stock sample was taken out of the -80°C refrigerator, it was repeatedly frozen and thawed; 15 μl was accurately pipetted into a clean EP tube, 985 μl of 50% methanol aqueous solution was added, and the mixture was mixed at 2000 rpm on a mixer for 5 min. The mixture was centrifuged at 12000 rpm for 5 min, and 100 μl of the supernatant was transferred to a new EP tube for injection analysis.

[0032] 6. Experimental Results System Suitability: Results showed that the blank solution chromatogram was free of interfering peaks. The RSD for the peak area of ​​the sodium N-lauroylsarcosinate system suitability solution was 2.76%, and the RSD for the retention time shift was 0.05%. The RSD for the peak area of ​​the phenol red system suitability solution was 4.48%, and the RSD for the retention time shift was 0.43%, meeting the requirements.

[0033]

[0034] Limits of Detection and Quantification: The results showed that when the S / N ratio was ≥ 3, the limit of detection for sodium N-lauroylsarcosinate was 0.111 ng / ml, with a signal-to-noise ratio range of 35.50-200.61. The limit of detection for phenol red was 2.723 ng / ml, with a signal-to-noise ratio range of 58.93-89.65. When the S / N ratio was ≥ 10, the limit of quantification for sodium N-lauroylsarcosinate was 0.456 ng / ml, with a signal-to-noise ratio range of 189.70-246.98, an RSD of 4.92% for peak area, and an RSD of 0.13% for retention time. The limit of quantification for phenol red was 4.324 ng / ml, with a signal-to-noise ratio range of 129.70-169.87, an RSD of 5.12% for peak area, and an RSD of 0.42% for retention time, meeting the requirements.

[0035]

[0036]

[0037] Linearity: The results showed that the concentration of sodium N-lauroylsarcosinate showed a good linear relationship with the peak area in the concentration range of 1.525-152.518 ng / ml, and the linear relationship was good. The linear regression equation was: y =11347x -5691 (r= 0.9999, n=7); the concentration of phenol red showed a good linear relationship with the peak area in the concentration range of 1.479-147.900 ng / ml, and the linear regression equation was: y =441.94x +81.289 (r= 1.0000, n=7); both met the requirements.

[0038]

[0039]

[0040] Repeatability: The results show that in the repeatability test, N -The RSD of sodium lauroyl sarcosinate content was 2.51%, and the RSD of phenol red content was 3.73%, both meeting the requirements.

[0041]

[0042]

[0043] Intermediate precision: The results showed that in the test of person B, the RSD of the sodium N-lauryl sarcosinate content was 2.84%, and in the intermediate precision test, the RSD of the sodium N-lauryl sarcosinate content was 3.18%; the RSD of the phenol red content was 4.28%, and in the intermediate precision test, the RSD of the phenol red content was 10.53%, which met the requirements.

[0044]

[0045]

[0046] Accuracy: The results showed that the recoveries of sodium N-lauroylsarcosinate were in the range of 82.54-95.23%, with an RSD of 4.05%; the recoveries of phenol red were in the range of 78.05-97.29%, with an RSD of 7.01%, indicating that the method had good accuracy and met the requirements.

[0047]

[0048]

[0049] Stability: The results showed that the reference solution was injected at 7 time points within 24 hours. The peak area of ​​sodium lauroyl sarcosinate solution at each time point was compared with the peak area at 0 hour, and the ratio was in the range of 92.38%~102.56%. The peak area of ​​phenol red solution at each time point was compared with the peak area at 0 hour, and the ratio was in the range of 93.57%~100.12%. The test solution was injected at 7 time points within 24 hours. The peak area of ​​sodium lauroyl sarcosinate solution at each time point was compared with the peak area at 0 hour, and the ratio was in the range of 95. The peak area of ​​phenol red solution at each time point was compared with that at 0 hour, and the ratio was in the range of 91.47%~104.33%. Within 24 hours, the peak area of ​​sodium N-lauroylsarcosinate solution at each time point was compared with that at 0 hour, and the ratio was in the range of 95.90%~102.95%. The peak area of ​​phenol red solution at each time point was compared with that at 0 hour, and the ratio was in the range of 81.93%~101.91%, which met the requirements.

[0050]

[0051]

[0052]

[0053] Durability: The results showed that under various conditions, the ratio of sodium N-lauroylsarcosinate content to that under normal conditions was in the range of 95.59% to 101.05%, and the ratio of phenol red content to that under normal conditions was in the range of 91.83% to 103.07%, which met the requirements.

[0054]

[0055] In summary, the method of the present invention can achieve N -Simultaneous determination of sodium lauryl sarcosinate and phenol red residues. The method requires simple sample pretreatment, has high detection sensitivity and good reproducibility, and can be applied to the identification and quality control of injection process residues.

Claims

1. A UPLC-MS / MS method for simultaneous determination of N - A method for determining the residual content of sodium lauryl sarcosinate and phenol red, characterized in that The steps include: S1: Precision weighing N - Use appropriate amounts of sodium lauryl sarcosinate and phenol red reference substance, and use methanol or 50% methanol solution as diluent to prepare reference substance working stock solutions of appropriate concentrations; S2: Accurately measure an appropriate amount of injection solution, dilute and extract with 50% methanol solution, fully dissolve it with ultrasound, and then obtain the test solution after high-speed centrifugation; S3: Accurately pipette an appropriate amount of each reference substance working stock solution and dilute it with a 50% methanol solution to prepare a mixed standard working solution. Detect the solution using UPLC-MS / MS. Based on the obtained chromatogram peak area, draw a linear fitting standard curve representing the relationship between the reference substance concentration and peak area, and calculate the corresponding linear fitting equation. S4: Accurately pipette an appropriate amount of the test solution and use the UPLC-MS / MS method to detect it on the machine. Based on the measured chromatogram peak area, substitute it into the standard curve fitting equation to calculate the above two process residues in the test solution.

2. A method for simultaneous determination of UPLC-MS / MS as described in claim 1 N - A method for sodium lauryl sarcosinate and phenol red residues, characterized in that: In step S1, N - Sodium lauryl sarcosinate and phenol red reference substances were first diluted to a concentration of 1 mg / ml of each reference substance stock solution using methanol as the diluent. Then, an appropriate amount of each reference substance stock solution was pipetted and added to 50% methanol solution, mixed and diluted to prepare 10 μg / ml of each reference substance working stock solution 1; then, an appropriate amount of each reference substance working stock solution 1 was pipetted and added to 50% methanol solution, mixed and diluted to prepare 200 ng / ml of each reference substance working stock solution 2, which was used for the subsequent preparation of mixed standard curve working solution.

3. The method of simultaneous determination based on UPLC-MS / MS as described in claim 1 N - A method for sodium lauryl sarcosinate and phenol red residues, characterized in that: In step S2, an appropriate amount of the injection solution was diluted with 50% methanol for extraction, mixed at 2000 rpm on a mixer for 5 min, centrifuged at 12000 rpm for 5 min, and 100 μl of the supernatant was transferred to a new EP tube for sample injection and analysis.

4. The method of simultaneous determination based on UPLC-MS / MS as described in claim 1 N - A method for sodium lauryl sarcosinate and phenol red residues, characterized in that: In steps S3 and S4, the UPLC ultra-high performance liquid chromatography conditions are: Waters AcquityUPLC ® A BEH C18 column (100 × 2.1 mm, 1.7 μm) was used as the chromatographic column. The mobile phase A consisted of 5 mM ammonium acetate solution (containing 10% acetonitrile) and the mobile phase B consisted of 0.2% acetic acid in acetonitrile. The mobile phase gradient listed in the table below was run for 12 min at a flow rate of 0.3 ml / min. The column temperature was 35 °C, and the injection volume was 10 μl.

5. The method of simultaneous determination based on UPLC-MS / MS as described in claim 1 N - A method for sodium lauryl sarcosinate and phenol red residues, characterized in that: In steps S3 and S4, the mass spectrometry conditions are as follows: an ESI ion source is used, and targeted detection of two targets is performed in negative ion scanning and MRM acquisition mode. The target MRM ion pairs and corresponding mass spectrometry parameters are listed in the table below. In addition, the ion source parameters for the ESI negative ion method are: curtain gas of 35.0 psi, collision gas of medium, ionization voltage of -4500 V, temperature setting of 450°C, and auxiliary heating gas of 50 and 50 psi, respectively.

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