An UPLC method for determination of structural analogues of DM1, the payload of trastuzumab emtansine (T-DM1)

An UPLC method for determination of structural analogues of DM1, the payload of trastuzumab emtansine (T-DM1) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An UPLC method for determination of structural analogues of DM1, the payload of trastuzumab emtansine (T-DM1) Junliang Yao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4628214/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Nov, 2024 Read the published version in Chromatographia → Version 1 posted 9 You are reading this latest preprint version Abstract DM1, a derivative of maytansine, is the payload of trastuzumab emtansine (T-DM1). In this work, a new gradient reverse-phase ultra-performance chromatographic (RP-UPLC) method was proposed for analysis of five structural analogues (DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L) of DM1. The chromatographic separation was accomplished by using a Waters BEH Phenyl column (50 × 2.1 mm, 1.7 µm), at the wavelength of 252 nm. Validation of the method was carried out according the ICH guidelines in terms of specificity, accuracy, precision, linearity and robustness. The developed method was proved to be convenient and reliable for quantitative determination of the DM1 analogue impurities. It can also be used for the related substances determination in DM1 bulk samples. DM1 UPLC Method development Validation maytansine Figures Figure 1 Figure 2 Figure 3 Figure 4 1、Introduction Ado-trastuzumab emtansine (T-DM1) is a famous antibody drug conjugate. It mainly consists of the HER2 monoclonal antibody trastuzumab and the potent microtubule-disturbing agent DM1 [ 1 ][ 2 ][ 3 ] . It has been approved by FDA and EMA for treatment of HER2-positive breast cancer in America and Europe with the brand name Kayla® [ 4 ][ 5 ][ 6 ] . DM1, payload of this ADC, is a maytansinoid derivative, which is modified by incorporation of new thiol containing ester side chains at the C3 position of maytansine to facilitate conjugation to the antibody [ 7 ][ 8 ] . Though synthesis of DM1 has been extensively studied [ 1 ][ 7 ][ 9 ] , the quality control of it has been seldom reported so far. Actually, the impurities of DM1 usually share the same basic parent structure of maytansine, some of them are even optical isomers, making it more difficult to separate and analyze. Therefore, it is very necessary to establish a novel method for the quality control of DM1 in bulk samples and manufacturing process. As is known to all, high-performance liquid chromatography (HPLC) is a well-established reliable analytical technique. It has been world-widely applied in quality control of pharmaceuticals as well as many other fields since decades ago [ 10 ][ 11 ] . However, it has been greatly challenged in recent years by ultra-performance liquid chromatography (UPLC), which is a new category of separation technique derived from the conventional HPLC [ 12 ][ 13 ] . The UPLC has attracted extensive attention because of its efficiency and sensitivity, resulted from the introduction of sub-2 µm particles for stationary phase [ 14 ][ 15 ][ 16 ] . This technique has been successfully applied to pharmaceutical and biomedical analysis of numerous drugs [ 17 ][ 18 ][ 19 ][ 20 ] . In the present study, we developed and validated an UPLC method for determination of the structural similar related substances in DM1. 2、Experimental 2.1. Chemicals and Samples The DM1 (DM1-4L) samples and related impurities were provided by Synthesis Department of Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd. Shanghai, China. The chemical structures of DM1 and impurities are shown in Fig. 1 . HPLC grade reagent acetonitrile and methanol were purchased from Fisher Scientific and phosphoric acid was purchased from ANPEL Laboratory Technologies (Shanghai) Inc. Ultrapure water was obtained by Millipore Milli-Q Advantage A10 water purification system. 2.2. Ultra-performance liquid chromatography Method development and validation was carried out on a Waters Acquity UPLC H-CLASS system equipped with a quaternary solvent delivery pump and a PDA detector. The chromatography data system Empower3 software was used to monitor the signal and process the results. A Waters BEH phenyl column (2.1×50 mm, 1.7 µm) was chosen to carry out the chromatographic separation. The mobile phase is composed of solvent A (0.05% H 3 PO 4 in water) and solvent B (Methanol). The flow rate was kept at 250 µL/min during elution. The UPLC gradient is shown in Table 1 . The monitoring wavelength of PDA was set as 252 nm and the temperature of the column heater was maintained at 40 °C. The injection volume was 2 µL. Both the standard and sample solutions were prepared using acetonitrile. Table 1 Gradient table of the UPLC method Time (min) Flow (µl/min) A (%) B (%) Curve * 0.0 250 60 40 4 7.5 250 10 90 6 8.0 250 10 90 6 9.0 250 60 40 6 15.0 250 60 40 6 * The gradient curve is available only in the Empower software. 2.3. Sample Preparation The standard stock solutions of AP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D (500 µg/mL of each) were prepared in acetonitrile and stored at 4 °C before and after every use. Working standard solution of each impurity was freshly prepared by diluting the stock solutions to about 2.5 µg/mL with acetonitrile before use. This standard solution was also used as system suitability solution. The sample solution was prepared by dissolving appropriate amount of DM1 in acetonitrile to give a final concentration of 500 µg/ml. 2.4. Method validation The UPLC method validation was carried out according to ICH guidelines in terms of precision, accuracy and linearity [ 21 ] . The specificity of the method was performed in the presence of DM1 and its related substances namely AP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D. The precision of the method was evaluated using six independent test solutions of DM1 spiked with 0.5% of each impurity relative to analyte concentration. The relative standard deviation (R.S.D %) of obtained recovery of each investigated component was calculated. The intermediate precision of the method was investigated by different analyst on different day with different instrument. The accuracy of the UPLC method was determined through the recovery experiments upon standard addition in triplicate at three concentration levels (low, medium and high) in bulk DM1 samples. The linearity was investigated using the test solutions prepared by diluting the standard stock solutions to 9 concentration levels from LOQ to 200% with reference to the specification limit (0.5%) of each impurity. The calibration curve was drawn by plotting the peak area versus concentration data of corresponding impurity and the coefficient of determination (R 2 ) was calculated by least-squares linear regression method. The limit of quantification (LOQ) and limit of detection (LOD) for the impurities were evaluated at a signal-to-noise ratio of 10/1 and 3/1, respectively, by monitoring a number of standard solutions diluted to designed concentrations. Precision research of each impurity at the LOQ level was also performed. The robustness of this method was investigated by evaluating the resolution variation between DM1 and its impurities upon altering the chromatographic parameters purposely. The column temperature was changed by (±) 5 °C. The flow rate was varied by 50 unit, i.e. from 200 to 300 µL/min. All the other chromatographic conditions were kept the same as described in Section 2.2 . 3、Results and discussion 3.1. Method Development AP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D are the potential related substances existing in DM1 bulk samples. AP-3 is the started material, DM1-2 and DM1-3L are the intermediates, DM1-3D and DM1-4L are the potential optical isomers. As shown in Fig. 1 , the only difference between DM1 and each impurity is the side chain located at C3 of maytansine molecule. The structural similarity makes it difficult to separate these impurities from DM1 with satisfactory resolution. Several UPLC columns with different stationary phases (C18 and phenyl) and from different manufacturers (Agilent and Waters) were used to observe the separation effect between each of the impurities. The influence of organic modifiers (acetonitrile and methanol) in the mobile phase were also examined subsequently. It was found that while using methanol in mobile phase and phenyl as stationary phase, the separation of DM1 and its impurities exhibits excellent selectivity. Considering the poor stability of these maytansine derivatives in alkaline condition, aqueous phase with low pH (0.05% H 3 PO 4 , v/v) was used during elution. We also tried to improve the UPLC performance by altering the column heater temperature, flow rate, and gradient curve. The results are displayed in Fig. 2 . To accomplish perfect resolution of all the six components (DM1 and its five impurities) and fast analysis speed, the final chromatographic conditions were employed as described in Section 2.2 . At the column temperature of 40 °C, flow rate of 250 µl/min, and gradient curve 4, a fast and selective separation was achieved in 6 min with a backpressure of less than 6000 psi. The system suitability results of the developed UPLC method are given in Table 2 . The representative UPLC chromatograms under the final optimized conditions are shown in Fig. 3 . Table 2 System suitability report Compound RT (min) Resolution Tailing factor Plate count DM1-2 4.5 -- 1.1 59899 AP-3 4.7 2.4 1.1 75413 DM1-4D 4.9 3.0 1.0 54718 DM1-4L 5.1 1.7 1.1 68244 DM1-3D 5.4 4.4 1.0 56671 DM1-3L 5.6 2.1 1.1 84240 3.2. Results of validation 3.2.1. Specificity The specificity of the UPLC method was ensured by determining the retention time of DM1 and every involved impurity standards separately, and no overlap of peak was observed in the chromatogram. The results are shown in Fig. 4 . 3.2.2. Precision The precision of the method was investigated using six independent injections of spiked DM1 samples. As shown in Table 3 , the R.S.D. of recovery for each of the impurities was below 5%. The intermediate precision was also found to meet the criteria (≤5%), indicating that the UPLC method is of good precision. Table 3 Precision of the investigated impurities Components Amount added (µg/ml) Mean recovery (%) R.S.D. (%) Precision DM1-2 2.00 93.1 0.9 AP-3 2.36 94.5 0.9 DM1-4D 2.91 108.4 1.2 DM1-3D 2.46 90.5 1.3 DM1-3L 2.61 94.0 1.2 Intermediate precision DM1-2 2.00 93.5 0.9 AP-3 2.36 93.5 1.3 DM1-4D 2.91 106.6 2.3 DM1-3D 2.46 89.4 3.6 DM1-3L 2.61 95.0 1.6 3.2.3. Limit of detection (LOD) and limit of quantification (LOQ) The limit of detection (LOD) of DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L were found to be 0.01%, 0.01%, 0.02%, 0.01%, and 0.01% of analyte concentration (500 µg/mL), respectively. The limit of quantification (LOQ) of DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L were 0.02%, 0.02%, 0.05%, 0.03%, and 0.03% of analyte concentration (500 µg/mL), with R.S.D below 10%, confirming good precision of the method at LOQ level. 3.2.4. Accuracy The accuracy of the method was investigated by utilizing a group of spiked DM1 bulk sample solutions containing certain amount of impurities. The experiment was performed in triplicate at concentration levels 80%, 100% and 120% of the specification limit. The percentage recovery of each impurity was calculated and given in Table 4 . It is shown that the recovery of each component ranged from 89.4–104.7%, within acceptable limits (85.0%~120.0%), and the R.S.D values were all less than 5.0%, indicating that the method has good reliability and accuracy. Table 4 Accuracy of the investigated impurities Level DM1-2 AP-3 DM1-4D DM1-3D DM1-3L At 80% (n = 3) Added (µg/mL) 1.60 1.89 2.33 1.97 2.09 Recovery (%) 93.1 94.0 104.7 93.6 97.7 R.S.D. (%) 2.2 2.3 2.2 2.2 2.5 At 100% (n = 3) Added (µg/mL) 2.00 2.36 2.91 2.46 2.61 Recovery (%) 92.9 92.0 103.8 89.4 95.2 R.S.D. (%) 0.9 0.7 1.5 0.7 1.5 At 120% (n = 3) Added (µg/mL) 2.40 2.83 3.50 2.95 3.13 Recovery (%) 94.2 93.0 101.2 93.4 95.9 R.S.D. (%) 2.3 2.7 1.0 2.2 2.3 3.2.5. Linearity Calibration curves were obtained over the range from LOQ to 200% of specification limit at nine concentration levels, and the correlation coefficient for each impurity obtained was greater than 0.990 (Table 5 ). The results indicated that the peak area of each impurity was linearly associated with the concentration. Table 5 Calibration curves of the investigated components Components Regression equation R 2 Linear range (µg/ml) DM1-2 y = 15697x-894.47 0.9955 0.1-4.0 AP-3 y = 14501x-1357.3 0.9935 0.1–4.7 DM1-4D y = 6963x + 363.8 0.9962 0.3–5.8 DM1-3D y = 11228x-1095.6 0.9960 0.2–4.9 DM1-3L y = 12070x-907.94 0.9959 0.1–5.2 3.2.6. Robustness The recoveries of all impurities were still within acceptable limits (85.0%~120.0%), even when chromatographic conditions were slightly changed deliberately suggesting that the UPLC method is of good robustness. 4、Conclusion In this paper, we have developed a novel UPLC method for the rapid separation and quantification of DM1-2, AP3, DM1-4D, DM1-3D and DM1-3L in the presence of DM1 bulk samples. The satisfactory data obtained from validation indicates that the method has good performance in terms of resolution, efficiency and sensitivity. Low consumption of organic solvent resulting from the reduced flow rate also indicate that it is environmentally friendly. In addition, the preparation of aqueous phase is much simpler, thus directly reduce the workload significantly. In conclusion, the proposed method is suitable for fast analysis of the five structural analogues of DM1. It can also be used for the related substances determination in DM1 bulk samples, or for the quality control during manufacturing process. Declarations Conflict of Interest The authors declare that they have no competing financial or personal interests, which could have influenced this work. Funding This work was funded by the project No.B003 in Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd., Shanghai, PR China. Author Contribution The Methodology, Validation, Investigation, Writing of manuscript, are all completed by Junliang Yao. Acknowledgement The author would like to thank Mr. Zhu of Synthesis Department for the materials support. Data Availability The data from this work will be provided upon reasonable request. References Lambert J M, Chari R V. Ado-trastuzumab Emtansine (T-DM1): an antibody-drug conjugate (ADC) for HER2-positive breast cancer[J]. J Med Chem, 2014, 57(16): 6949–6964. Sliwkowski M X, Mellman I. Antibody Therapeutics in Cancer[J]. 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Cite Share Download PDF Status: Published Journal Publication published 07 Nov, 2024 Read the published version in Chromatographia → Version 1 posted Editorial decision: Revision requested 23 Aug, 2024 Reviews received at journal 08 Aug, 2024 Reviewers agreed at journal 29 Jul, 2024 Reviews received at journal 19 Jul, 2024 Reviewers agreed at journal 05 Jul, 2024 Reviewers invited by journal 03 Jul, 2024 Editor assigned by journal 27 Jun, 2024 Submission checks completed at journal 26 Jun, 2024 First submitted to journal 24 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4628214","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328241115,"identity":"1eb9db72-43f4-4d76-97b7-092841631e77","order_by":0,"name":"Junliang Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYJACgwcVQIKBh+EA8VoSzpCqhSGxDaKFSBuOHz5QkDivLs+c/ezBwwU1DPL8YgQsMziTlmCQuO1wsWVPXsLhGccYDGfOTiCg5UCOAVDLgcQNN3gMDvOwMSQY3Cak5fwboJY5dVAt/4jRcgNkSwMzRAtvGxFaJG88SzBIOHa42OBMDlBLnwRhv/CdTz5m8KGmLs/g+BnjzzzfbOT5pQloUTjAwGYApGHKJPArBwH5BgbmB0haRsEoGAWjYBRgAgDGNUlJbtMFWAAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai Pharmaceuticals Holding Co., Ltd","correspondingAuthor":true,"prefix":"","firstName":"Junliang","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2024-06-24 07:21:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4628214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4628214/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10337-024-04377-1","type":"published","date":"2024-11-07T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60556015,"identity":"40ea9a55-5cbf-41a7-aeae-35cf3b8d03a4","added_by":"auto","created_at":"2024-07-18 06:29:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149994,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of DM1 and its analogue impurities.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4628214/v1/52cb87154ae3c0f051c1cadb.jpg"},{"id":60556530,"identity":"c2bc7c15-5c95-4157-a4ec-51c382d8e1cf","added_by":"auto","created_at":"2024-07-18 06:37:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":595198,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of flow rate, column temperature and gradient curve on UPLC performance.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4628214/v1/0a2c1f85788570ca71eec4fd.jpg"},{"id":60556014,"identity":"c1706987-4ef7-4f17-ae47-332bf2e9e762","added_by":"auto","created_at":"2024-07-18 06:29:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":930441,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms of DM1 and its analogues obtained from UPLC.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4628214/v1/ee8828757cddd1491c1039b5.jpg"},{"id":60556016,"identity":"aec47ef2-ddd2-410a-923e-282eb1e5a69f","added_by":"auto","created_at":"2024-07-18 06:29:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":399413,"visible":true,"origin":"","legend":"\u003cp\u003eUPLC chromatograms of individual impurity samples and DM1.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4628214/v1/a98d1765fca4edef1ee0fd80.jpg"},{"id":68750150,"identity":"02e490c7-f09f-414c-8ff7-e8c51e78b122","added_by":"auto","created_at":"2024-11-11 16:11:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2573232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4628214/v1/f1b3c8d1-99c5-4e1e-be0d-5f6bf67fda44.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An UPLC method for determination of structural analogues of DM1, the payload of trastuzumab emtansine (T-DM1)","fulltext":[{"header":"1、Introduction","content":"\u003cp\u003eAdo-trastuzumab emtansine (T-DM1) is a famous antibody drug conjugate. It mainly consists of the HER2 monoclonal antibody trastuzumab and the potent microtubule-disturbing agent DM1 \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. It has been approved by FDA and EMA for treatment of HER2-positive breast cancer in America and Europe with the brand name Kayla\u0026reg; \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. DM1, payload of this ADC, is a maytansinoid derivative, which is modified by incorporation of new thiol containing ester side chains at the C3 position of maytansine to facilitate conjugation to the antibody \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Though synthesis of DM1 has been extensively studied \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, the quality control of it has been seldom reported so far. Actually, the impurities of DM1 usually share the same basic parent structure of maytansine, some of them are even optical isomers, making it more difficult to separate and analyze. Therefore, it is very necessary to establish a novel method for the quality control of DM1 in bulk samples and manufacturing process.\u003c/p\u003e \u003cp\u003eAs is known to all, high-performance liquid chromatography (HPLC) is a well-established reliable analytical technique. It has been world-widely applied in quality control of pharmaceuticals as well as many other fields since decades ago \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, it has been greatly challenged in recent years by ultra-performance liquid chromatography (UPLC), which is a new category of separation technique derived from the conventional HPLC \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The UPLC has attracted extensive attention because of its efficiency and sensitivity, resulted from the introduction of sub-2 \u0026micro;m particles for stationary phase \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. This technique has been successfully applied to pharmaceutical and biomedical analysis of numerous drugs \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e][\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In the present study, we developed and validated an UPLC method for determination of the structural similar related substances in DM1.\u003c/p\u003e"},{"header":"2、Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Samples\u003c/h2\u003e \u003cp\u003eThe DM1 (DM1-4L) samples and related impurities were provided by Synthesis Department of Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd. Shanghai, China. The chemical structures of DM1 and impurities are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. HPLC grade reagent acetonitrile and methanol were purchased from Fisher Scientific and phosphoric acid was purchased from ANPEL Laboratory Technologies (Shanghai) Inc. Ultrapure water was obtained by Millipore Milli-Q Advantage A10 water purification system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Ultra-performance liquid chromatography\u003c/h2\u003e \u003cp\u003eMethod development and validation was carried out on a Waters Acquity UPLC H-CLASS system equipped with a quaternary solvent delivery pump and a PDA detector. The chromatography data system Empower3 software was used to monitor the signal and process the results. A Waters BEH phenyl column (2.1\u0026times;50 mm, 1.7 \u0026micro;m) was chosen to carry out the chromatographic separation. The mobile phase is composed of solvent A (0.05% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in water) and solvent B (Methanol). The flow rate was kept at 250 \u0026micro;L/min during elution. The UPLC gradient is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The monitoring wavelength of PDA was set as 252 nm and the temperature of the column heater was maintained at 40 \u0026deg;C. The injection volume was 2 \u0026micro;L. Both the standard and sample solutions were prepared using acetonitrile.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGradient table of the UPLC method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlow (\u0026micro;l/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCurve\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003e The gradient curve is available only in the Empower software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sample Preparation\u003c/h2\u003e \u003cp\u003eThe standard stock solutions of AP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D (500 \u0026micro;g/mL of each) were prepared in acetonitrile and stored at 4 \u0026deg;C before and after every use. Working standard solution of each impurity was freshly prepared by diluting the stock solutions to about 2.5 \u0026micro;g/mL with acetonitrile before use. This standard solution was also used as system suitability solution. The sample solution was prepared by dissolving appropriate amount of DM1 in acetonitrile to give a final concentration of 500 \u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Method validation\u003c/h2\u003e \u003cp\u003eThe UPLC method validation was carried out according to ICH guidelines in terms of precision, accuracy and linearity \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The specificity of the method was performed in the presence of DM1 and its related substances namely AP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D. The precision of the method was evaluated using six independent test solutions of DM1 spiked with 0.5% of each impurity relative to analyte concentration. The relative standard deviation (R.S.D %) of obtained recovery of each investigated component was calculated. The intermediate precision of the method was investigated by different analyst on different day with different instrument. The accuracy of the UPLC method was determined through the recovery experiments upon standard addition in triplicate at three concentration levels (low, medium and high) in bulk DM1 samples. The linearity was investigated using the test solutions prepared by diluting the standard stock solutions to 9 concentration levels from LOQ to 200% with reference to the specification limit (0.5%) of each impurity. The calibration curve was drawn by plotting the peak area versus concentration data of corresponding impurity and the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) was calculated by least-squares linear regression method. The limit of quantification (LOQ) and limit of detection (LOD) for the impurities were evaluated at a signal-to-noise ratio of 10/1 and 3/1, respectively, by monitoring a number of standard solutions diluted to designed concentrations. Precision research of each impurity at the LOQ level was also performed. The robustness of this method was investigated by evaluating the resolution variation between DM1 and its impurities upon altering the chromatographic parameters purposely. The column temperature was changed by (\u0026plusmn;) 5 \u0026deg;C. The flow rate was varied by 50 unit, i.e. from 200 to 300 \u0026micro;L/min. All the other chromatographic conditions were kept the same as described in Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3、Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Method Development\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAP-3, DM1-2, DM1-3D, DM1-3L, and DM1-4D are the potential related substances existing in DM1 bulk samples. AP-3 is the started material, DM1-2 and DM1-3L are the intermediates, DM1-3D and DM1-4L are the potential optical isomers. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the only difference between DM1 and each impurity is the side chain located at C3 of maytansine molecule. The structural similarity makes it difficult to separate these impurities from DM1 with satisfactory resolution. Several UPLC columns with different stationary phases (C18 and phenyl) and from different manufacturers (Agilent and Waters) were used to observe the separation effect between each of the impurities. The influence of organic modifiers (acetonitrile and methanol) in the mobile phase were also examined subsequently. It was found that while using methanol in mobile phase and phenyl as stationary phase, the separation of DM1 and its impurities exhibits excellent selectivity. Considering the poor stability of these maytansine derivatives in alkaline condition, aqueous phase with low pH (0.05% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, v/v) was used during elution. We also tried to improve the UPLC performance by altering the column heater temperature, flow rate, and gradient curve. The results are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. To accomplish perfect resolution of all the six components (DM1 and its five impurities) and fast analysis speed, the final chromatographic conditions were employed as described in Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. At the column temperature of 40 \u0026deg;C, flow rate of 250 \u0026micro;l/min, and gradient curve 4, a fast and selective separation was achieved in 6 min with a backpressure of less than 6000 psi. The system suitability results of the developed UPLC method are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The representative UPLC chromatograms under the final optimized conditions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSystem suitability report\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResolution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTailing factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlate count\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59899\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAP-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75413\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-4D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54718\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-4L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68244\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-3D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56671\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-3L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e84240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Results of validation\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Specificity\u003c/h2\u003e \u003cp\u003eThe specificity of the UPLC method was ensured by determining the retention time of DM1 and every involved impurity standards separately, and no overlap of peak was observed in the chromatogram. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Precision\u003c/h2\u003e \u003cp\u003eThe precision of the method was investigated using six independent injections of spiked DM1 samples. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the R.S.D. of recovery for each of the impurities was below 5%. The intermediate precision was also found to meet the criteria (\u0026le;5%), indicating that the UPLC method is of good precision.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrecision of the investigated impurities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmount added\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean recovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR.S.D. (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003ePrecision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAP-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-4D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e108.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-3D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-3L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eIntermediate precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAP-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-4D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e106.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-3D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDM1-3L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Limit of detection (LOD) and limit of quantification (LOQ)\u003c/h2\u003e \u003cp\u003eThe limit of detection (LOD) of DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L were found to be 0.01%, 0.01%, 0.02%, 0.01%, and 0.01% of analyte concentration (500 \u0026micro;g/mL), respectively. The limit of quantification (LOQ) of DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L were 0.02%, 0.02%, 0.05%, 0.03%, and 0.03% of analyte concentration (500 \u0026micro;g/mL), with R.S.D below 10%, confirming good precision of the method at LOQ level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Accuracy\u003c/h2\u003e \u003cp\u003eThe accuracy of the method was investigated by utilizing a group of spiked DM1 bulk sample solutions containing certain amount of impurities. The experiment was performed in triplicate at concentration levels 80%, 100% and 120% of the specification limit. The percentage recovery of each impurity was calculated and given in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It is shown that the recovery of each component ranged from 89.4\u0026ndash;104.7%, within acceptable limits (85.0%~120.0%), and the R.S.D values were all less than 5.0%, indicating that the method has good reliability and accuracy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAccuracy of the investigated impurities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDM1-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAP-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDM1-4D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDM1-3D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDM1-3L\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAt 80% (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e93.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e104.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e97.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR.S.D. (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAt 100% (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e92.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e103.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e89.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR.S.D. (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAt 120% (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e94.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e101.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR.S.D. (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5. Linearity\u003c/h2\u003e \u003cp\u003eCalibration curves were obtained over the range from LOQ to 200% of specification limit at nine concentration levels, and the correlation coefficient for each impurity obtained was greater than 0.990 (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results indicated that the peak area of each impurity was linearly associated with the concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalibration curves of the investigated components\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegression equation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinear range (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;15697x-894.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1-4.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAP-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;14501x-1357.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026ndash;4.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-4D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;6963x\u0026thinsp;+\u0026thinsp;363.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u0026ndash;5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-3D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;11228x-1095.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026ndash;4.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM1-3L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u0026thinsp;=\u0026thinsp;12070x-907.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026ndash;5.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6. Robustness\u003c/h2\u003e \u003cp\u003eThe recoveries of all impurities were still within acceptable limits (85.0%~120.0%), even when chromatographic conditions were slightly changed deliberately suggesting that the UPLC method is of good robustness.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4、Conclusion","content":"\u003cp\u003eIn this paper, we have developed a novel UPLC method for the rapid separation and quantification of DM1-2, AP3, DM1-4D, DM1-3D and DM1-3L in the presence of DM1 bulk samples. The satisfactory data obtained from validation indicates that the method has good performance in terms of resolution, efficiency and sensitivity. Low consumption of organic solvent resulting from the reduced flow rate also indicate that it is environmentally friendly. In addition, the preparation of aqueous phase is much simpler, thus directly reduce the workload significantly. In conclusion, the proposed method is suitable for fast analysis of the five structural analogues of DM1. It can also be used for the related substances determination in DM1 bulk samples, or for the quality control during manufacturing process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing financial or personal interests, which could have influenced this work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the project No.B003 in Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd., Shanghai, PR China.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe Methodology, Validation, Investigation, Writing of manuscript, are all completed by Junliang Yao.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe author would like to thank Mr. Zhu of Synthesis Department for the materials support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data from this work will be provided upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLambert J M, Chari R V. Ado-trastuzumab Emtansine (T-DM1): an antibody-drug conjugate (ADC) for HER2-positive breast cancer[J]. J Med Chem, 2014, 57(16): 6949\u0026ndash;6964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSliwkowski M X, Mellman I. Antibody Therapeutics in Cancer[J]. Science (Washington, DC, U. S.), 2013, 341(Copyright (C) 2017 American Chemical Society (ACS). All Rights Reserved.): 1192\u0026ndash;1198.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGebleux R, Casi G. Antibody-drug conjugates: Current status and future perspectives[J]. Pharmacol Ther, 2016, 167: 48\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrop I E, Lorusso P, Miller K D, et al. A Phase II Study of Trastuzumab Emtansine in Patients With Human Epidermal Growth Factor Receptor 2\u0026ndash;Positive Metastatic Breast Cancer Who Were Previously Treated With Trastuzumab, Lapatinib, an Anthracycline, a Taxane, and Capecitabine[J]. Journal of Clinical Oncology, 2012, 30(26): 3234\u0026ndash;3241.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallantyne A, Dhillon S. Trastuzumab emtansine: first global approval[J]. Drugs, 2013, 73(7): 755\u0026ndash;765.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapachristos A, Pippa N, Demetzos C, et al. Antibody-drug conjugates: a mini-review. The synopsis of two approved medicines[J]. Drug Deliv, 2016, 23(5): 1662\u0026ndash;1666.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiddison W C, Wilhelm S D, Cavanagh E E, et al. Semisynthetic maytansine analogues for the targeted treatment of cancer[J]. J Med Chem, 2006, 49(14): 4392\u0026ndash;4408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassady J M, Chan K K, Floss H G, et al. Recent developments in the maytansinoid antitumor agents[J]. Chemical \u0026amp; Pharmaceutical Bulletin, 2004, 52(1): 1\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFishkin N, Maloney E K, Chari R V J, et al. A novel pathway for maytansinoid release from thioether linked antibody-drug conjugates (ADCs) under oxidative conditions[J]. Chem. Commun. (Cambridge, U. K.), 2011, 47(38): 10752\u0026ndash;10754.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMallikarjuna Rao B, Sangaraju S, Srinivasu M K, et al. Development and validation of a specific stability indicating high performance liquid chromatographic method for rizatriptan benzoate[J]. J Pharm Biomed Anal, 2006, 41(4): 1146\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao B M, Srinivasu M K, Kumar K P, et al. A stability indicating LC method for rivastigmine hydrogen tartrate[J]. J Pharm Biomed Anal, 2005, 37(1): 57\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Saini G, Nair A, et al. UPLC: a preeminent technique in pharmaceutical analysis[J]. Acta Poloniae Pharmaceutica, 2012, 69(3): 371.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Li H, Jin C, et al. Development and validation of a UPLC method for quality control of rhubarb-based medicine: fast simultaneous determination of five anthraquinone derivatives[J]. J Pharm Biomed Anal, 2008, 47(4\u0026ndash;5): 765\u0026ndash;770.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDongre V G, Karmuse P P, Rao P P, et al. Development and validation of UPLC method for determination of primaquine phosphate and its impurities[J]. J Pharm Biomed Anal, 2008, 46(2): 236\u0026ndash;242.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRivera Velez S M, Morassi A, Court M H, et al. Development and validation of an ultrafast chromatographic method for quantification of the immunosuppressant mycophenolic acid in canine, feline and human plasma[J]. J Pharm Biomed Anal, 2016, 131: 94\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagiera S, Baranowska I, Lautenszleger A. UHPLC-UV method for the determination of flavonoids in dietary supplements and for evaluation of their antioxidant activities[J]. J Pharm Biomed Anal, 2015, 102(5): 468\u0026ndash;475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWren S A, Tchelitcheff P. Use of ultra-performance liquid chromatography in pharmaceutical development[J]. Journal of Chromatography A, 2006, 1119(1\u0026ndash;2): 140\u0026ndash;146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, Dong L, Huang J. Ultra performance liquid chromatography\u0026ndash;tandem mass spectrometry for the determination of epirubicin in human plasma[J]. Analytica Chimica Acta, 2005, 546(2): 167\u0026ndash;173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeshadri R K, Desai M M, Raghavaraju T V, et al. Simultaneous Quantitative Determination of Metoprolol, Atorvastatin and Ramipril in Capsules by a Validated Stability-Indicating RP-UPLC Method[J]. Scientia Pharmaceutica, 2010, 78(4): 821\u0026ndash;834.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFekete S, Fekete J, Ganzler K. Validated UPLC method for the fast and sensitive determination of steroid residues in support of cleaning validation in formulation area[J]. Journal of Pharmaceutical \u0026amp; Biomedical Analysis, 2009, 49(3): 833\u0026ndash;838.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eICH topic Q2(R1),Validation of analytical procedures: text and methodology. Step 4 version. 2005.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chromatographia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chro","sideBox":"Learn more about [Chromatographia](https://www.springer.com/journal/10337)","snPcode":"10337","submissionUrl":"https://submission.nature.com/new-submission/10337/3","title":"Chromatographia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DM1, UPLC, Method development, Validation, maytansine","lastPublishedDoi":"10.21203/rs.3.rs-4628214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4628214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDM1, a derivative of maytansine, is the payload of trastuzumab emtansine (T-DM1). In this work, a new gradient reverse-phase ultra-performance chromatographic (RP-UPLC) method was proposed for analysis of five structural analogues (DM1-2, AP-3, DM1-4D, DM1-3D, DM1-3L) of DM1. The chromatographic separation was accomplished by using a Waters BEH Phenyl column (50 \u0026times; 2.1 mm, 1.7 \u0026micro;m), at the wavelength of 252 nm. Validation of the method was carried out according the ICH guidelines in terms of specificity, accuracy, precision, linearity and robustness. The developed method was proved to be convenient and reliable for quantitative determination of the DM1 analogue impurities. It can also be used for the related substances determination in DM1 bulk samples.\u003c/p\u003e","manuscriptTitle":"An UPLC method for determination of structural analogues of DM1, the payload of trastuzumab emtansine (T-DM1)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 06:29:39","doi":"10.21203/rs.3.rs-4628214/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-23T13:11:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-08T06:21:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169550597047255765295770991192847734743","date":"2024-07-29T06:30:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T10:02:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204301264402506554358290234224871351754","date":"2024-07-05T13:22:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T14:22:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-27T14:19:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-26T12:45:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chromatographia","date":"2024-06-24T07:20:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chromatographia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chro","sideBox":"Learn more about [Chromatographia](https://www.springer.com/journal/10337)","snPcode":"10337","submissionUrl":"https://submission.nature.com/new-submission/10337/3","title":"Chromatographia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"31e99d15-1d92-4750-9b25-9fcb26a92bd2","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-11T16:06:01+00:00","versionOfRecord":{"articleIdentity":"rs-4628214","link":"https://doi.org/10.1007/s10337-024-04377-1","journal":{"identity":"chromatographia","isVorOnly":false,"title":"Chromatographia"},"publishedOn":"2024-11-07 15:57:55","publishedOnDateReadable":"November 7th, 2024"},"versionCreatedAt":"2024-07-18 06:29:39","video":"","vorDoi":"10.1007/s10337-024-04377-1","vorDoiUrl":"https://doi.org/10.1007/s10337-024-04377-1","workflowStages":[]},"version":"v1","identity":"rs-4628214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4628214","identity":"rs-4628214","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}