Method for simultaneously detecting residual quantity of EDC and EDU in rotator cuff patch

Abstract

The invention discloses a method for simultaneously detecting residual quantity of EDC and EDU in a rotator cuff patch. According to the present invention, acetonitrile and methanol are respectively added to perform extraction treatment on the rotator cuff patch, an ultra-high performance liquid chromatograph-mass spectrometer is adopted to detect the extraction liquid, and the residual amounts of EDC and EDU in the rotator cuff patch are calculated by using an external standard method according to a linear regression equation. The detection method is good in repeatability, high in sensitivity, simple, rapid, accurate in result and suitable for detecting the residual quantity of EDC and EDU in the rotator cuff patch, and the appearance time, parent ions and corresponding qualitative and quantitative ion information of EDC and EDU can be rapidly obtained by directly analyzing the standard solution through an ultra-high performance liquid chromatograph-mass spectrometer. By defining the key parameters, a targeted quantitative detection method can be directly established without a complicated pre-experiment or parameter exploration process, so that the development period of the method is effectively shortened, and the establishment efficiency and convenience of the detection method are improved.

Description

Technical Field

[0001] This invention belongs to the field of medical device testing technology, specifically a method for simultaneously detecting the residual amounts of EDC and EDU in rotator cuff patches. Background Technology

[0002] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, CAS No. 25952-53-8) is widely used as a condensing agent and crosslinking agent in pharmaceuticals, biology, and industrial auxiliaries. 1-[3-(dimethylamino)propyl]-3-ethylurea (EDU, CAS No. 32897-26-0) has wide applications in chemistry. It can be used as a reagent in organic synthesis to promote reactions and is also frequently used as a ligand for catalysts. Since rotator cuff patches are implanted, persistent contact medical devices that may come into contact with blood and tissue fluid, subsequently entering the systemic circulation, their toxicological risks are noteworthy. Therefore, it is necessary to detect EDC and EDU residues.

[0003] Currently, there are no relevant national or industry standards for testing methods for EDC and EDU. The commonly used testing methods are mostly liquid chromatography, which analyzes EDC and EDU separately, and the analysis time is long and the detection concentration is high.

[0004] Therefore, a simple and efficient ultra-high performance liquid chromatography-mass spectrometry method is needed to simultaneously detect EDC and EDU. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneously detecting the residual amounts of EDC and EDU in a shoulder and cuff patch in order to solve the problems mentioned above.

[0006] The technical solution adopted in this invention is as follows: A method for simultaneously detecting the residual amounts of EDC and EDU in a rotator cuff patch, the method comprising the following steps:

[0007] S1. Sample pretreatment: Place the samples in headspace vials, add acetonitrile and methanol extraction solutions respectively, seal and extract in a constant temperature shaking incubator to obtain sample extract; similarly, extract the sample blank in the same way to obtain the extract solution as sample blank extract.

[0008] S2. Solution preparation: Prepare mixed standard working solution, limit of detection solution, limit of quantitation solution, and mixed standard curve solution of EDC and EDU using methanol solution. Prepare accuracy solution and repeatability solution of EDC and EDU according to the sample extraction conditions.

[0009] S3. Qualitative analysis: The series of mixed standard working solutions were analyzed using ultra-high performance liquid chromatography-mass spectrometry. The precursor ions of EDC and EDU were obtained by Scan, and the quantitative and qualitative ions were obtained by Product Ion mode. The retention time was obtained by optimizing the liquid chromatography conditions.

[0010] S4. Establish test methods: Based on the peak times, qualitative ions, and quantitative ions of EDC and EDU standard substances, establish quantitative methods, and use ultra-high performance liquid chromatography-mass spectrometry to analyze the repeatability solution, detection limit solution, quantitation limit solution, mixed standard curve solution, and accuracy solution;

[0011] S5. Method Validation Data Analysis: Six injections of the repeatability solution were performed to obtain repeatability results; three injections of the detection limit solution were performed for analysis, and the signal-to-noise ratio was calculated; three injections of the quantitation limit solution were performed for analysis, and the signal-to-noise ratio was calculated; linear regression analysis was performed on the mixed standard curve solution to obtain the linear regression equation and establish a standard curve; the three concentrations of the standard working solutions (low, medium, and high) were analyzed to obtain accuracy results.

[0012] S6. Quantitative analysis: The sample solution was analyzed using ultra-high performance liquid chromatography-mass spectrometry to quantitatively analyze the residual amounts of EDC and EDU in the sample solution. This method has good repeatability, high sensitivity, high efficiency and speed, accurate results and convenient operation.

[0013] In a preferred embodiment, in step S1, the sample is placed in a headspace vial, and the extraction solutions are acetonitrile and methanol, respectively.

[0014] In a preferred embodiment, in step S1, after the bottle cap is sealed, the sample is extracted by shaking at a rate of 60 r / min in a constant temperature shaking incubator at 37°C for 24 h.

[0015] In a preferred embodiment, in step S2, the concentration of the mixed standard working solution is 10 μg / mL; the detection limit solution is 0.05 μg / mL of the mixed standard working solution; the quantitation limit solution is 0.10 μg / mL of the mixed standard working solution; and the concentration range of the mixed standard curve solution is set to 0.10 μg / mL, 0.20 μg / mL, 0.40 μg / mL, 0.60 μg / mL, 0.8 μg / mL, and 1.0 μg / mL.

[0016] In a preferred embodiment, in step S2, the repeatability solution and the accuracy solution are blank solutions respectively added to the standard solution, and extracted according to the sample extraction conditions to prepare standard working solutions with repeatability solution concentration of 0.50 μg / mL (n=6) and accuracy solution concentrations of 0.20 μg / mL, 0.50 μg / mL, and 0.80 μg / mL.

[0017] In a preferred embodiment, in step S3, the mixed standard working solution is analyzed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) system. The precursor ions of EDC and EDU are obtained by Scan, and the quantitative and qualitative ions are obtained by Product Ion mode. The retention times are optimized using liquid chromatography conditions. The peak elution time of the EDC standard is determined to be 1.53 min, the precursor ion to be 156.2 m / z, the qualitative ion to be 86.0 m / z, and the quantitative ion to be 58.0 m / z. The peak elution time of the EDU standard is determined to be 1.20 min, the precursor ion to be 174.2 m / z, the qualitative ion to be 103.0 m / z, and the quantitative ion to be 129.1 m / z.

[0018] In a preferred embodiment, in step S4, the repeatability solution is analyzed to obtain repeatability results; the detection limit solution is analyzed and the signal-to-noise ratio is calculated by repeated injections for 3 injections; the quantitation limit solution is analyzed and the signal-to-noise ratio is calculated by repeated injections for 3 injections.

[0019] In a preferred embodiment, in step S4, linear regression analysis is performed on the series of mixed standard curve solutions to obtain a linear regression equation and establish a standard curve; the accuracy solutions with concentrations of 0.20 μg / mL, 0.50 μg / mL, and 0.80 μg / mL are analyzed to obtain accuracy results.

[0020] In a preferred embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument in step S5 are as follows:

[0021] Flow rate: 0.3 mL / min; Column: ACQUITY UPLC BEH C18, 2.1×100 mm × 1.7 μm; Column temperature: 40℃; Injection volume: 0.2 μL; Ionization mode: positive mode; Mobile phase: Mobile phase A: 10 mmol / L ammonium acetate aqueous solution, Mobile phase B: methanol; Mobile phase ratio: A:B=95:5.

[0022] In a preferred embodiment, in step S6, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) are as follows:

[0023] Ion source: AJS ESI; Drying gas temperature: 300℃; Drying gas flow rate: 8 L / min; Sheath gas temperature: 250℃; Sheath gas flow rate: 11 L / min; Spray pressure: 35 psi; Capillary voltage: 4000 V(+); Nozzle voltage: 500 V(+); Scan mode: MRM; Ionization mode: Positive mode.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] 1. In this invention, by directly analyzing standard solutions using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS), the peak times, precursor ions, and corresponding qualitative and quantitative ion information of EDC and EDU can be rapidly obtained. By clarifying these key parameters, a targeted quantitative detection method can be directly established without complex pre-experiments or parameter exploration processes, effectively shortening the method development cycle and improving the efficiency and convenience of establishing the detection method.

[0026] 2. This invention employs a multi-dimensional validation process, including multiple injection tests of repeatable solutions, calculation of the signal-to-noise ratio of limit of detection and limit of quantitation solutions, linear regression analysis of the standard curve, and recovery rate tests of solutions with different concentrations and accuracy levels. This comprehensively covers the core indicators of the detection method, such as precision, sensitivity, linear range, and accuracy. These rigorous validation steps directly enhance the reliability of the detection results, ensuring the stability and credibility of the EDC and EDU residue detection data in rotator cuff patches.

[0027] 3. This invention enables the simultaneous detection of both EDC and EDU substances, avoiding the repetitive operations required for traditional separate detection. After extraction with acetonitrile and methanol, the extract can be directly tested, eliminating cumbersome pretreatment steps. Furthermore, the single-needle run time is only 4 minutes, significantly reducing the time required for a single test. These designs directly improve detection efficiency, simplify the operation process, and make the detection of residual substances in rotator cuff patches more efficient, faster, and easier to operate. Attached Figure Description

[0028] Figure 1 The images show the chromatograms and mass spectra of the EDC standard solutions in this embodiment of the invention.

[0029] Figure 2 The images show the chromatogram and mass spectrum of the EDC sample solution in this embodiment of the invention.

[0030] Figure 3 This is a standard curve diagram of EDC in an embodiment of the present invention;

[0031] Figure 4 The images show the chromatograms and mass spectra of the EDU standard solutions in this embodiment of the invention.

[0032] Figure 5 The images show the chromatogram and mass spectrum of the EDU sample solution in this embodiment of the invention.

[0033] Figure 6 This is a standard curve diagram of EDU in an embodiment of the present invention;

[0034] Figure 7 This is a schematic diagram of the process principle in this invention. Detailed Implementation

[0035] 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. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Example: Refer to Figure 1-7 A method for simultaneously detecting EDC and EDU residues in rotator cuff patches

[0037] A rotator cuff patch for implantation was selected, and the residual amounts of EDC and EDU in the extract of the rotator cuff patch were detected. The steps are as follows:

[0038] 1. Sample pretreatment: Take 3 sample pieces, place them in a headspace vial, add 12 mL of extraction solvent, seal the vial, and incubate at 37℃ with shaking at 60 r / min for 24 h. Prepare two parallel extracts. After the first extraction, separate the sample and extract, and perform a second extraction until the extraction limit is reached. Take a blank sample that does not contain the target analyte and prepare a blank extract using the same method.

[0039] Table 1 Extraction conditions

[0040]

[0041] 2. Solution preparation: Prepare a 10 μg / mL mixed standard working solution, a 0.50 μg / mL repeatability solution (n=6), mixed standard curve solutions of 0.10 μg / mL, 0.20 μg / mL, 0.40 μg / mL, 0.60 μg / mL, 0.8 μg / mL, and 1.0 μg / mL using methanol solution, a 0.05 μg / mL limit of detection solution, a 0.1 μg / mL limit of quantitation solution, and 0.20 μg / mL, 0.50 μg / mL, and 0.80 μg / mL accuracy solutions.

[0042] Table 2 Solution Preparation Information

[0043]

[0044] 3. Qualitative Analysis: A 10 μg / mL mixed standard working solution was analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to obtain chromatograms and mass spectra of EDC and EDU. The precursor ions of EDC and EDU were obtained by scanning, and the quantitative and qualitative ions were obtained by Product Ion mode. The retention times were optimized using HPLC conditions, and the peak elution time of the EDC standard was determined to be 1.53 min, the precursor ion to be 156.2 m / z, the qualitative ion to be 86.0 m / z, and the quantitative ion to be 58.0 m / z. The chromatograms and mass spectra are shown below. Figure 1 The peak elution time of the EDU standard was determined to be 1.20 min, the precursor ion to be 174.2 m / z, the qualitative ion to be 103.0 m / z, and the quantitative ion to be 129.1 m / z. The chromatogram and mass spectrum results are shown below. Figure 4 .

[0045] 4. Establishment of testing methods: Based on the peak times, qualitative ions, and quantitative ions of EDC and EDU standard substances, quantitative methods were established. The repeatability solution, limit of detection solution, limit of quantitation solution, mixed standard curve solution, and accuracy solution were analyzed using ultra-high performance liquid chromatography-mass spectrometry. The testing methods are shown in Table 3.

[0046] Table 3 LC-MS Instrument Conditions

[0047]

[0048] 5. Method Validation Data Analysis: Six injections of the repeatability solution were performed to obtain repeatability results; three injections of the detection limit solution were performed for analysis, and the signal-to-noise ratio (SNR) was calculated; three injections of the quantitation limit solution were performed for analysis, and the SNR was calculated; linear regression analysis was performed on the mixed standard curve solution to obtain the linear regression equation and establish the standard curve; three working standard solutions of low, medium, and high concentrations were analyzed to obtain accuracy results. The results of the method validation data analysis are shown in Table 7, and the standard curve is shown in [Table data missing]. Figure 3 , Figure 6 The validation data demonstrate that the method exhibits good repeatability, linearity, and accuracy, while also achieving a low limit of detection.

[0049] Table 4 Results of Limit of Detection and Limit of Quantification

[0050]

[0051] Table 5 Accuracy Results

[0052]

[0053] Table 6 Linear Results

[0054]

[0055] Table 7 Repeatability Results

[0056]

[0057] 5. Quantitative Analysis: The sample solution was analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to quantitatively determine the residual amounts of EDC and EDU in the sample solution; the detection results are shown in Table 8, and the chromatogram of the sample solution is shown in [Table 8]. Figure 2 and Figure 5 .

[0058] Table 8. Results of Extraction Tests

[0059]

[0060] As can be seen from the above, this invention can detect the residual amounts of EDC and EDU in rotator cuff patches, and employs a rigorous experimental design and verification process to ensure the accuracy and reliability of EDC and EDU detection in rotator cuff patches. In this invention, the rotator cuff patch is extracted with acetonitrile and methanol, and then the extract is detected using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The residual amounts of EDC and EDU in the rotator cuff patch are calculated based on a linear regression equation. The detection method of this invention has good repeatability, high sensitivity, is simple and rapid, and provides accurate results, making it suitable for detecting the residual amounts of EDC and EDU in rotator cuff patches.

[0061] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously detecting the residual amounts of EDC and EDU in a rotator cuff patch, characterized in that: The method includes the following steps: S1. Sample pretreatment: Place the samples in headspace vials, add acetonitrile and methanol extraction solutions respectively, seal and extract in a constant temperature shaking incubator to obtain sample extract; similarly, extract the sample blank in the same way to obtain the extract solution as sample blank extract. S2. Solution preparation: Prepare mixed standard working solution, limit of detection solution, limit of quantitation solution, and mixed standard curve solution of EDC and EDU using methanol solution. Prepare accuracy solution and repeatability solution of EDC and EDU according to the sample extraction conditions. S3. Qualitative analysis: The series of mixed standard working solutions were analyzed using ultra-high performance liquid chromatography-mass spectrometry. The precursor ions of EDC and EDU were obtained by Scan, and the quantitative and qualitative ions were obtained by Product Ion mode. The retention time was obtained by optimizing the liquid chromatography conditions. S4. Establish test methods: Based on the peak times, qualitative ions, and quantitative ions of EDC and EDU standard substances, establish quantitative methods, and use ultra-high performance liquid chromatography-mass spectrometry to analyze the repeatability solution, detection limit solution, quantitation limit solution, mixed standard curve solution, and accuracy solution; S5. Method Validation Data Analysis: Six injections of the repeatability solution were performed to obtain repeatability results; three injections of the detection limit solution were performed for analysis, and the signal-to-noise ratio was calculated; three injections of the quantitation limit solution were performed for analysis, and the signal-to-noise ratio was calculated; linear regression analysis was performed on the mixed standard curve solution to obtain the linear regression equation and establish a standard curve; the three concentrations of the standard working solutions (low, medium, and high) were analyzed to obtain accuracy results. S6. Quantitative analysis: The sample solution was analyzed using ultra-high performance liquid chromatography-mass spectrometry to quantitatively analyze the residual amounts of EDC and EDU in the sample solution. This method has good repeatability, high sensitivity, high efficiency and speed, accurate results and convenient operation.

2. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S1, the sample is placed in a headspace vial, and the extraction solutions are acetonitrile and methanol, respectively.

3. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S1, after the bottle cap is sealed, the sample is extracted by shaking at a rate of 60 r / min in a constant temperature shaking incubator at 37°C for 24 h.

4. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S2, the concentration of the mixed standard working solution is 10 μg / mL; the detection limit solution is 0.05 μg / mL of the mixed standard working solution; the quantitation limit solution is 0.10 μg / mL of the mixed standard working solution; and the concentration range of the mixed standard curve solution is set to 0.10 μg / mL, 0.20 μg / mL, 0.40 μg / mL, 0.60 μg / mL, 0.8 μg / mL, and 1.0 μg / mL.

5. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S2, the repeatability solution and the accuracy solution are blank solutions, respectively, to be added to the standard solution and extracted according to the sample extraction conditions to prepare standard working solutions with a repeatability solution concentration of 0.50 μg / mL (n=6) and accuracy solutions concentrations of 0.20 μg / mL, 0.50 μg / mL, and 0.80 μg / mL.

6. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S3, the mixed standard working solution is analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The precursor ions of EDC and EDU are obtained by Scan, and the quantitative and qualitative ions are obtained by Product Ion mode. The retention times are optimized using liquid chromatography conditions. The peak elution time of the EDC standard is determined to be 1.53 min, the precursor ion to be 156.2 m / z, the qualitative ion to be 86.0 m / z, and the quantitative ion to be 58.0 m / z. The peak elution time of the EDU standard is determined to be 1.20 min, the precursor ion to be 174.2 m / z, the qualitative ion to be 103.0 m / z, and the quantitative ion to be 129.1 m / z.

7. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S4, the repeatability solution is analyzed to obtain repeatability results; the detection limit solution is repeatedly injected three times and analyzed to calculate the signal-to-noise ratio; the quantitation limit solution is repeatedly injected three times and analyzed to calculate the signal-to-noise ratio.

8. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S4, linear regression analysis is performed on the series of mixed standard curve solutions to obtain the linear regression equation and establish the standard curve; the accuracy solutions with concentrations of 0.20 μg / mL, 0.50 μg / mL, and 0.80 μg / mL are analyzed to obtain the accuracy results.

9. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S5, the chromatographic conditions for the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) are as follows: Flow rate: 0.3 mL / min; Column: ACQUITY UPLC BEH C18, 2.1 × 100 mm × 1.7 μm; Column temperature: 40℃; Injection volume: 0.2 μL; Ionization mode: Positive mode; Mobile phase: Mobile phase A: 10 mmol / L ammonium acetate aqueous solution, Mobile phase B: methanol; Mobile phase ratio: A:B = 95:

5.

10. The method for simultaneously detecting EDC and EDU residues in a rotator cuff patch as described in claim 1, characterized in that: In step S6, the mass spectrometry conditions for the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument are as follows: Ion source: AJS ESI; Drying gas temperature: 300℃; Drying gas flow rate: 8 L / min; Sheath gas temperature: 250℃; Sheath gas flow rate: 11 L / min; Spray pressure: 35 psi; Capillary voltage: 4000 V(+); Nozzle voltage: 500 V(+); Scan mode: MRM; Ionization mode: Positive mode.

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