Clinical application of LC-MS/MS to detect fat-soluble vitamins in gastric cancer

Clinical application of LC-MS/MS to detect fat-soluble vitamins in gastric cancer | 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 Clinical application of LC-MS/MS to detect fat-soluble vitamins in gastric cancer Li Sun, Qianqian Gao, Yan Shen, Wangwei Sun, Yongliang Yao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3895853/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Gastric cancer is one of the most common malignant tumors, with the incidence and mortality ranking among the top four malignant tumors in the world. In recent years, more and more studies have reported that fat-soluble vitamins are involved in tumor metabolism. However, the sensitivity and specificity of conventional detection methods are low, while liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has the advantages of high sensitivity, high specificity, high stability and wide linear range, which makes up for the shortcomings of immunological methods. The purpose of this study was to accurately detect the content of fat-soluble vitamins in GC patients by LC-MS/MS, and to clarify the necessity of preoperative determination and regular postoperative monitoring. Methods Serum 25(OH)D 3 , 25(OH)D 2 , VA, VE and VK1 levels in gastric cancer patients and healthy volunteers were detected by LC-MS/MS. Results The results showed that the levels of serum 25(OH)D 3 , 25(OH)D 2 , VA and VK1 in gastric cancer patients were significantly lower than those in healthy volunteers, and there was no significant difference in VE content. Conclusion This study suggests that patients with gastric cancer should timely detect and supplement fat-soluble vitamins and LC-MS/MS will provide a more accurate detection protocol for the clinic. Gastric cancer Fat-soluble vitamins LC-MS/MS 25(OH)D3 25(OH)D2 Figures Figure 1 Figure 2 Introduction Gastric cancer is one of the most common malignant tumors, and according to the Global Cancer Statistics Report, gastric cancer ranked among the top four in terms of new incidence and mortality rate in 2020, with 1089103 new cases of gastric cancer globally in 2020, accounting for 5.6% of all new cases of malignant tumors; and 768793 deaths, accounting for 7.7% of all deaths from malignant tumors. Among them, the number of new cases and death cases in Asia accounted for 75.3% and 74.8% of the total, respectively [1]. In recent years, with the development of medical technology, the diagnosis rate, survival time and quality of life of early gastric cancer have been significantly improved. However, 2/3 of the patients are found in advanced stages, and even with chemotherapy, radiotherapy or immunotherapy based on surgery, the five-year survival rate is still less than 30% [2]. Most patients suffer from nutrient deficiencies, including fat-soluble vitamins, due to various dietary problems caused by surgical removal of gastric tissue. Cancer may alter the metabolism of fat-soluble vitamins, and alterations in the metabolism of fat-soluble vitamins may also be involved in the carcinogenesis process to some extent. Vitamin D receptors are present in most tissues and play a wide range of biological roles. In China, vitamin D deficiency is prevalent in the population involved in clinical vitamin D testing [3]. In addition to affecting calcium and phosphorus metabolism, an increasing number of studies have reported that vitamin D is involved in biological processes associated with antitumor effects, including cell proliferation, differentiation, survival, maturation, activation, and immune responses [4,5]. Kevin et al . reported that vitamin D deficiency was associated with the development of gastric cancer and correlated with the severity of gastric cancer [6]. Ren et al . reported that in gastric cancer patients, the vitamin D deficient patients had a higher overall mortality rate than those with adequate vitamin D [7]. Li et al . reported that vitamin D inhibited the growth of gastric cancer cells in vitro and in vivo by down-regulating the expression of CD44 [8]. Other fat-soluble vitamins, such as vitamin A, whose intake is negatively correlated with the risk of gastric cancer [9]. Vitamin E can effectively inhibit the growth of gastric cancer cells and sensitize cancer cells to chemotherapeutic drugs, such as celecoxib, adriamycin, erlotinib, gefitinib, gemcitabine, paclitaxel, statin [10]. Vitamin K1 can activate the ERK1/2 signaling pathway to inhibit the proliferation of gastric cancer cells and induces apoptosis in gastric cancer cells [11]. It has been reported that fat-soluble vitamin levels in gastric cancer patients decrease due to surgery [12]. Oh et al . reported that more than half of gastric cancer patients had preoperative vitamin D deficiency, and that patients treated with postoperative chemotherapy were at risk of vitamin D deficiency within a year after surgery [13]. Therefore, accurate preoperative detection of fat-soluble vitamins and timely targeted supplementation in gastric cancer patients are very important to improve patient prognosis. Fat-soluble vitamins are usually determined by immunological methods, but immunoassays have some limitations, with low sensitivity and specificity, such as the levels of 25(OH)D 3 and 25(OH)D 2 cannot be detected individually. And the immune antibody may also react with other metabolites, such as 24,25(OH) 2 D and 3-epi-25(OH)D, thus leading to inaccurate test results accuracy [14]. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method is a newly developed laboratory detection system that combines the high separation ability of chromatography and the analysis ability of mass spectrometry on the structure and quality of substances. Compared with immunological methods such as chemiluminescent enzyme immunoassay and electrochemical luminescence immunoassay, it has high sensitivity, specificity and accuracy. It requires a small sample volume and it can detect several kinds of fat-soluble vitamins together. LC-MS/MS has been recommended as the gold standard for vitamin D determination [15,16]. In this study, we measured the serum fat-soluble vitamins VA, 25(OH)D 3 , 25(OH)D 2 , VE, and VK1 levels in gastric cancer patients and healthy volunteers using LC-MS/MS. The results showed that the serum levels of VA, 25(OH)D 3 , 25(OH)D 2 and VK1 were lower in gastric cancer patients than in healthy volunteers, and there was no significant difference in VE. The study suggests that the detection of fat-soluble vitamin levels is important for improving prognosis, and LC-MS/MS will provide more accurate results and treatment options for clinical fat-soluble vitamin detection. Methods Human serum samples Serum was collected from 36 healthy volunteers and 33 patients diagnosed with gastric cancer by gastroscopy and postoperative histopathology and frozen in -80℃ ultra-low temperature refrigerator. Clinical characteristics: 33 cases of gastric cancer patients, including 22 (66.7%) males and 11 (33.3%) females, 25 (75.8%) above 60 years old, and 8 (24.2%) 60 and below; 36 cases of healthy volunteers, including 23 (63.9%) males and 13 (36.1%) females, 28 (77.8%) above 60 years old, and 28 (77.8%) and 8 (22.2%) were 60 and under. All study participants provided written consent prior to the study in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of the First People's Hospital of Kunshan. LC-MS/MS assay Sample preparation was performed according to the instruction manual of Fat-Soluble Vitamins Assay Kit (Liquid Chromatography-Tandem Mass Spectrometry) (hmibiotech, Shenzhen, China): 100 µL of calibrator, QC and serum samples were added to the 96-well plate, 100 µL of internal standard working solution was added to each well, and then the sample was shocked for 5 minutes at 12,000 rpm. 500 µL of extraction solution was added and shake at 12000 rpm for 15 min, centrifuge at 10 ℃ 4000 rpm for 20 min, aspirate 420 µL of supernatant into a new 96-well plate, nitrogen blow at room temperature for 10 min, add 80 µL of the reconstitution solution to each well, shake at 12000 rpm for 1 min and then centrifuge at 10 ℃ 4000 rpm for 20 min. The 96-well plate was put into the ACQUITY UPLC I -Class autosampler and waited for injection. The mass spectrometry analysis was performed on a TQ-XS mass spectrometer (Waters, Milford, USA), and the measurements were carried out using an atmospheric pressure chemical ionization source (APCI + source) in multiple reaction monitoring (MRM) scanning mode, and an ACQUITY UPLC BEH C18 column with a column temperature of 45 ℃. Other parameters: ion source temperature 150 ℃, probe temperature 450 ℃, desolvation gas flow rate 300 L/Hr, cone pore gas flow rate 150 L/Hr. VA parent ion 269.10 m/z, daughter ion 119.00 m/z, collision energy 20V, cone pore voltage 20V; 25(OH)D 3 parent ion 401.30 m/z, daughter ion 365.20 m/z, collision energy 10V, cone pore voltage 10V; 25(OH)D 2 parent ion 413.20 m/z, daughter ion 355.20 m/z, collision energy 10V, cone pore voltage 10V; VE parent ion 431.40 m/z, daughter ion 137.30 m/z, collision energy 60V, cone pore voltage 10V; VK1 parent ion 451.40 m/z, daughter ion 187.10 m/z, collision energy 25V, cone pore voltage 10V; VA-d6 parent ion 274.10 m/z, daughter ion 217.00 m/z, collision energy 20V, cone pore voltage 40V; 25(OH)D 3 -d6 parent ion 407.20 m/z, daughter ion 371.10 m/z, collision energy 10V, cone pore voltage 10V; 25(OH)D 2 -d6 parent ion 419.20 m/z, daughter ion 401.20 m/z, collision energy 5V, cone pore voltage 10V; VE-d6 parent ion 437.70 m/z, daughter ion 143.20 m/z, collision energy 50V, cone pore voltage 10V; VK1-d7 parent ion 458.30 m/z, daughter ion 194.20 m/z, collision energy 25V, cone pore voltage 10V. Data analysis LC-MS/MS data were analyzed using the TargetLynx quantitative program of MassLynxV4.2 (Waters) software, and the standard curve equations were plotted based on the peak area ratios of the detected substances to their intra-isotopic standards to obtain the mass spectral response values, and the sample concentrations were calculated. Unpaired t-tests were performed for the differences in serum fat-soluble vitamin levels between gastric cancer patients and healthy volunteers using GraphPad Prism 7.0 software, and all data were expressed as Mean ± SEM with a test level of α = 0.05. The reference ranges were VA 0.56–4.2 µmol/L; 25(OH)D > = 20 ng/mL; VE 11.6–58.1 µmol/L; VK1 0.22–4.88 µmol/L. Results LC-MS/MS detection of serum 25(OH)D 3 and 25(OH)D 2 levels in gastric cancer patients and healthy volunteers The results of LC-MS/MS assay showed that serum 25(OH)D 3 (21.90 ± 2.324 vs. 28.43 ± 2.189, P = 0.0446), 25(OH)D 2 (0.2970 ± 0.06284 vs. 1.183 ± 0.3872, P = 0.0338), and 25(OH)D (22.20 ± 2.346 vs 29.61 ± 2.279, P = 0.0267) levels were lower than serum levels in healthy volunteers (Fig. 1 A-C). LC-MS/MS detected serum VA, VE and VK1 levels in gastric cancer patients and healthy volunteers The results of LC-MS/MS assay showed that serum VA (2159 ± 176.4 vs 2932 ± 166.8, P = 0.0022) and VK1 (2.512 ± 0.4823 vs 4.550 ± 0.4622, P = 0.0033) levels in gastric cancer patients were lower than the serum levels in healthy volunteers, while there was no significant difference in VE (41910 ± 2813 vs 48110 ± 2722, ns.) (Fig. 2 A-C). Discussion LC-MS/MS is the method of choice for the detection of small molecules in clinical biochemistry, which has the advantages of high sensitivity, high specificity, high stability, and wide linear range, and has been widely used in the fields of chemical industry, biology, pharmaceuticals, food, clinical medicine, and environment [17]. In the field of clinical testing and diagnosis, LC-MS/MS, as a supplement to traditional diagnostic techniques, can provide a more reliable basis for more accurate diagnosis of many diseases [18]. The TQ-XS mass spectrometer used in this study is a new generation of revolutionary products in the mass spectrometry community, capable of analyzing a wider variety of compounds with a completely new level of ultra-high sensitivity, which has improved the reliability, reproducibility, and analytical performance of the quantitative analysis to a higher level through innovative research. 25(OH)D 3 and 25(OH)D 2 have small structural differences, and the activity of 25(OH)D 2 is lower than that of 25(OH)D 3 , and elevated 25(OH)D 2 may be accompanied by a decrease in 25(OH)D 3 , so testing the activity and function of both separately is a necessity to properly assess vitamin levels and the efficacy of supplementation therapy. Usually, immunological methods measure total 25(OH)D, while LC-MS/MS can detect 25(OH)D 3 and 25(OH)D 2 separately, which can be used for targeted supplementation according to the test results, avoiding the harms of under- and over-supplementation. LC-MS/MS can also be used to detect the blood concentration of different types of vitamin D drugs, preventing vitamin D toxicity, and providing a more precise and precise guidance to the clinical treatment [19,20]. In this study, serum 25(OH)D 3 , 25(OH)D 2 and 25(OH)D were found to be lower than the serum levels of healthy volunteers in gastric cancer patients by LC-MS/MS. Other fat-soluble vitamins, including vitamin A and vitamin K1, were also lower in serum levels of gastric cancer patients than in serum levels of healthy volunteers, and there was no significant difference in vitamin E. The sample size was limited in this study, and we will continue to expand the sample size and follow up the gastric cancer patients to further clarify the effects of various fat-soluble vitamins on the prognosis of gastric cancer patients. Although LC-MS/MS provides more accurate detection solutions for clinical small molecule detection, LC-MS/MS still faces serious challenges. Most of the current LC-MS/MS assays are Laboratory developde tests (LDT), and although a variety of mass spectrometry detection kits have been marketed, the lack of standardization of the methods makes it impossible to avoid deviations in results between different laboratories or even the same laboratory due to factors such as pretreatment methods, reagents, instrumentation, ion sources, columns, calibrators, methodology and performance validation. In addition, inappropriate interpretation and application of method validation guidelines, lack or inappropriate use of standard substances, and incomplete quality assurance measures can lead to inaccurate test results [21–23]. Relatively high operator requirements and the cost of the assay also hinder the use of LC-MS/MS in large-scale screening [24]. Conclusions In this study, we measured the serum fat-soluble vitamins VA, 25(OH)D 3 , 25(OH)D 2 , VE, and VK1 levels in gastric cancer patients and healthy volunteers using LC-MS/MS. The results showed that the serum levels of VA, 25(OH)D 3 , 25(OH)D 2 and VK1 were lower in gastric cancer patients than in healthy volunteers, and there was no significant difference in VE. This study suggests that the detection of fat-soluble vitamin levels is important for improving prognosis, and LC-MS/MS will provide more accurate results for clinical fat-soluble vitamin detection. Declarations Author contribution Sun L wrote the manuscript. Gao Q and Shen Y completed the experiment and verified the underlying data. Sun W was responsible for the graph draft. Yao Y designed and revised the manuscript. All authors contributed to the article and approved the submitted version. Funding This work was supported by National Natural Science Foundation of China (82203547), Natural Science Foundation of Jiangsu Province (BK20210136) and China Postdoctoral Science Foundation (2022M721433). Data availability Authors confrm that they have full control over all primary data and agree to allow the journal to review their data if requested. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the First People's Hospital of Kunshan. Competing interests The authors declare no competing interests. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. References International Agency for Research on Cancer, Data visualization tools for exploring the global cancer burden in 2020. Http://gco.iarc.fr/today/home. (Accessed 13 May 2022). Li D, Wang Y, Shi C, et al (2023) Targeting GPC3 high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in gastric cancer. Ann Med 55: 2189295. doi: 10.1080/07853890.2023.2189295. 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Wise SA, Camara JE, Burdette CQ, et al (2022) Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance. Anal Bioanal Chem 414:351–366. doi: 10.1007/s00216-021-03577-0. Binkley N, Dawson-Hughes B, Durazo-Arvizu R, et al (2017) Vitamin D measurement standardization: The way out of the chaos. J Steroid Biochem Mol Biol 173:117–121. doi: 10.1016/j.jsbmb.2016.12.002. Sempos CT, Heijboer AC, Bikle DD, et al (2018) Vitamin D assays and the definition of hypovitaminosis D: results from the First International Conference on Controversies in Vitamin D. Br J Clin Pharmacol 84:2194–2207. doi: 10.1111/bcp.13652. Chinese Association of Geriatric Research of Laboratory Medicine, Chinese Geriatrics Society of Laboratory Medicine (2021) Expert consensus on the standardization of liquid chromatography-tandem mass spectrometry method for the measurement of 25‐hydroxyvitamin D. Chin J Lab Med 44:587–595. doi: 10.3760/cma.j.cn114452-20201120-00846. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-3895853","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269329361,"identity":"7b480f66-d688-421e-b70d-3ded25343a4d","order_by":0,"name":"Li Sun","email":"","orcid":"","institution":"Affiliated Kunshan Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Sun","suffix":""},{"id":269329362,"identity":"ac5b12f8-91de-4779-b5fc-95d2a23afdcf","order_by":1,"name":"Qianqian Gao","email":"","orcid":"","institution":"Affiliated Kunshan Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Qianqian","middleName":"","lastName":"Gao","suffix":""},{"id":269329363,"identity":"f62d6b34-892f-4a16-ad4a-110890f3817a","order_by":2,"name":"Yan Shen","email":"","orcid":"","institution":"Affiliated Kunshan Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Shen","suffix":""},{"id":269329364,"identity":"d8d81155-e7fd-42d8-a2b1-e7bf43d28548","order_by":3,"name":"Wangwei Sun","email":"","orcid":"","institution":"Affiliated Kunshan Hospital of Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Wangwei","middleName":"","lastName":"Sun","suffix":""},{"id":269329365,"identity":"9abd2d73-354b-43dc-bd67-f231ead6cd61","order_by":4,"name":"Yongliang Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACgxs5jJ8//JCw42dmPviAWC3M0pI9FsmS7WzJBkRpkew5w8bMw1bBuOE8j5kAUVr42XuAWngkmI0PM5gxMNTYRBPUArKCmcdCgs/sMEPaA4ZjabkNxGixBtkC1HLcgLHhMHFapHnYJBg3NzO2SZCmZQMzMxvRWkCBLJEscZiN2SCBOL/wPwRGZZ0df//5jw8+1NgQ1oIKEkhTPgpGwSgYBaMAFwAA1tYyVfxwWBIAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Kunshan Hospital of Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Yongliang","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2024-01-25 03:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3895853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3895853/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50389368,"identity":"6a4b3e02-9212-4cd6-b445-40f527d22089","added_by":"auto","created_at":"2024-01-30 18:27:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40258,"visible":true,"origin":"","legend":"\u003cp\u003eLC-MS/MS detection of serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e (A), 25(OH)D\u003csub\u003e2\u003c/sub\u003e (B) and 25(OH)D (C) levels in 33 gastric cancer patients (GC) and 36 healthy volunteers (HC). \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, *.\u003c/p\u003e","description":"","filename":"Onlinefig101.png","url":"https://assets-eu.researchsquare.com/files/rs-3895853/v1/36ca973ff6c9fbe4b0448353.png"},{"id":50389370,"identity":"42085efd-13f1-4893-84cc-e3bcd6879103","added_by":"auto","created_at":"2024-01-30 18:27:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42675,"visible":true,"origin":"","legend":"\u003cp\u003eSerum VA (A), VE (B) and VK1 (C) levels were detected by LC-MS/MS in 33 gastric cancer patients (GC) and 36 healthy volunteers (HC). \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, **; \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, *; \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, ns.\u003c/p\u003e","description":"","filename":"Onlinefig201.png","url":"https://assets-eu.researchsquare.com/files/rs-3895853/v1/baa685bd0504f3a5c98297b5.png"},{"id":51399750,"identity":"f3e1be4f-c3e3-49ef-b266-259cbcacd16a","added_by":"auto","created_at":"2024-02-20 21:53:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":421891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3895853/v1/91bff298-4b29-4036-ab1d-f324cd0d7608.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical application of LC-MS/MS to detect fat-soluble vitamins in gastric cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer is one of the most common malignant tumors, and according to the Global Cancer Statistics Report, gastric cancer ranked among the top four in terms of new incidence and mortality rate in 2020, with 1089103 new cases of gastric cancer globally in 2020, accounting for 5.6% of all new cases of malignant tumors; and 768793 deaths, accounting for 7.7% of all deaths from malignant tumors. Among them, the number of new cases and death cases in Asia accounted for 75.3% and 74.8% of the total, respectively [1]. In recent years, with the development of medical technology, the diagnosis rate, survival time and quality of life of early gastric cancer have been significantly improved. However, 2/3 of the patients are found in advanced stages, and even with chemotherapy, radiotherapy or immunotherapy based on surgery, the five-year survival rate is still less than 30% [2].\u003c/p\u003e \u003cp\u003eMost patients suffer from nutrient deficiencies, including fat-soluble vitamins, due to various dietary problems caused by surgical removal of gastric tissue. Cancer may alter the metabolism of fat-soluble vitamins, and alterations in the metabolism of fat-soluble vitamins may also be involved in the carcinogenesis process to some extent. Vitamin D receptors are present in most tissues and play a wide range of biological roles. In China, vitamin D deficiency is prevalent in the population involved in clinical vitamin D testing [3]. In addition to affecting calcium and phosphorus metabolism, an increasing number of studies have reported that vitamin D is involved in biological processes associated with antitumor effects, including cell proliferation, differentiation, survival, maturation, activation, and immune responses [4,5]. Kevin \u003cem\u003eet al\u003c/em\u003e. reported that vitamin D deficiency was associated with the development of gastric cancer and correlated with the severity of gastric cancer [6]. Ren \u003cem\u003eet al\u003c/em\u003e. reported that in gastric cancer patients, the vitamin D deficient patients had a higher overall mortality rate than those with adequate vitamin D [7]. Li \u003cem\u003eet al\u003c/em\u003e. reported that vitamin D inhibited the growth of gastric cancer cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by down-regulating the expression of CD44 [8]. Other fat-soluble vitamins, such as vitamin A, whose intake is negatively correlated with the risk of gastric cancer [9]. Vitamin E can effectively inhibit the growth of gastric cancer cells and sensitize cancer cells to chemotherapeutic drugs, such as celecoxib, adriamycin, erlotinib, gefitinib, gemcitabine, paclitaxel, statin [10]. Vitamin K1 can activate the ERK1/2 signaling pathway to inhibit the proliferation of gastric cancer cells and induces apoptosis in gastric cancer cells [11]. It has been reported that fat-soluble vitamin levels in gastric cancer patients decrease due to surgery [12]. Oh \u003cem\u003eet al\u003c/em\u003e. reported that more than half of gastric cancer patients had preoperative vitamin D deficiency, and that patients treated with postoperative chemotherapy were at risk of vitamin D deficiency within a year after surgery [13]. Therefore, accurate preoperative detection of fat-soluble vitamins and timely targeted supplementation in gastric cancer patients are very important to improve patient prognosis.\u003c/p\u003e \u003cp\u003eFat-soluble vitamins are usually determined by immunological methods, but immunoassays have some limitations, with low sensitivity and specificity, such as the levels of 25(OH)D\u003csub\u003e3\u003c/sub\u003e and 25(OH)D\u003csub\u003e2\u003c/sub\u003e cannot be detected individually. And the immune antibody may also react with other metabolites, such as 24,25(OH)\u003csub\u003e2\u003c/sub\u003eD and 3-epi-25(OH)D, thus leading to inaccurate test results accuracy [14]. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method is a newly developed laboratory detection system that combines the high separation ability of chromatography and the analysis ability of mass spectrometry on the structure and quality of substances. Compared with immunological methods such as chemiluminescent enzyme immunoassay and electrochemical luminescence immunoassay, it has high sensitivity, specificity and accuracy. It requires a small sample volume and it can detect several kinds of fat-soluble vitamins together. LC-MS/MS has been recommended as the gold standard for vitamin D determination [15,16].\u003c/p\u003e \u003cp\u003eIn this study, we measured the serum fat-soluble vitamins VA, 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e, VE, and VK1 levels in gastric cancer patients and healthy volunteers using LC-MS/MS. The results showed that the serum levels of VA, 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e and VK1 were lower in gastric cancer patients than in healthy volunteers, and there was no significant difference in VE. The study suggests that the detection of fat-soluble vitamin levels is important for improving prognosis, and LC-MS/MS will provide more accurate results and treatment options for clinical fat-soluble vitamin detection.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman serum samples\u003c/h2\u003e \u003cp\u003eSerum was collected from 36 healthy volunteers and 33 patients diagnosed with gastric cancer by gastroscopy and postoperative histopathology and frozen in -80℃ ultra-low temperature refrigerator. Clinical characteristics: 33 cases of gastric cancer patients, including 22 (66.7%) males and 11 (33.3%) females, 25 (75.8%) above 60 years old, and 8 (24.2%) 60 and below; 36 cases of healthy volunteers, including 23 (63.9%) males and 13 (36.1%) females, 28 (77.8%) above 60 years old, and 28 (77.8%) and 8 (22.2%) were 60 and under. All study participants provided written consent prior to the study in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of the First People's Hospital of Kunshan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS assay\u003c/h2\u003e \u003cp\u003eSample preparation was performed according to the instruction manual of Fat-Soluble Vitamins Assay Kit (Liquid Chromatography-Tandem Mass Spectrometry) (hmibiotech, Shenzhen, China): 100 \u0026micro;L of calibrator, QC and serum samples were added to the 96-well plate, 100 \u0026micro;L of internal standard working solution was added to each well, and then the sample was shocked for 5 minutes at 12,000 rpm. 500 \u0026micro;L of extraction solution was added and shake at 12000 rpm for 15 min, centrifuge at 10 ℃ 4000 rpm for 20 min, aspirate 420 \u0026micro;L of supernatant into a new 96-well plate, nitrogen blow at room temperature for 10 min, add 80 \u0026micro;L of the reconstitution solution to each well, shake at 12000 rpm for 1 min and then centrifuge at 10 ℃ 4000 rpm for 20 min. The 96-well plate was put into the ACQUITY UPLC \u003cem\u003eI\u003c/em\u003e-Class autosampler and waited for injection.\u003c/p\u003e \u003cp\u003eThe mass spectrometry analysis was performed on a TQ-XS mass spectrometer (Waters, Milford, USA), and the measurements were carried out using an atmospheric pressure chemical ionization source (APCI\u0026thinsp;+\u0026thinsp;source) in multiple reaction monitoring (MRM) scanning mode, and an ACQUITY UPLC BEH C18 column with a column temperature of 45 ℃. Other parameters: ion source temperature 150 ℃, probe temperature 450 ℃, desolvation gas flow rate 300 L/Hr, cone pore gas flow rate 150 L/Hr. VA parent ion 269.10 m/z, daughter ion 119.00 m/z, collision energy 20V, cone pore voltage 20V; 25(OH)D\u003csub\u003e3\u003c/sub\u003e parent ion 401.30 m/z, daughter ion 365.20 m/z, collision energy 10V, cone pore voltage 10V; 25(OH)D\u003csub\u003e2\u003c/sub\u003e parent ion 413.20 m/z, daughter ion 355.20 m/z, collision energy 10V, cone pore voltage 10V; VE parent ion 431.40 m/z, daughter ion 137.30 m/z, collision energy 60V, cone pore voltage 10V; VK1 parent ion 451.40 m/z, daughter ion 187.10 m/z, collision energy 25V, cone pore voltage 10V; VA-d6 parent ion 274.10 m/z, daughter ion 217.00 m/z, collision energy 20V, cone pore voltage 40V; 25(OH)D\u003csub\u003e3\u003c/sub\u003e-d6 parent ion 407.20 m/z, daughter ion 371.10 m/z, collision energy 10V, cone pore voltage 10V; 25(OH)D\u003csub\u003e2\u003c/sub\u003e-d6 parent ion 419.20 m/z, daughter ion 401.20 m/z, collision energy 5V, cone pore voltage 10V; VE-d6 parent ion 437.70 m/z, daughter ion 143.20 m/z, collision energy 50V, cone pore voltage 10V; VK1-d7 parent ion 458.30 m/z, daughter ion 194.20 m/z, collision energy 25V, cone pore voltage 10V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eLC-MS/MS data were analyzed using the TargetLynx quantitative program of MassLynxV4.2 (Waters) software, and the standard curve equations were plotted based on the peak area ratios of the detected substances to their intra-isotopic standards to obtain the mass spectral response values, and the sample concentrations were calculated. Unpaired t-tests were performed for the differences in serum fat-soluble vitamin levels between gastric cancer patients and healthy volunteers using GraphPad Prism 7.0 software, and all data were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM with a test level of α\u0026thinsp;=\u0026thinsp;0.05. The reference ranges were VA 0.56\u0026ndash;4.2 \u0026micro;mol/L; 25(OH)D\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;20 ng/mL; VE 11.6\u0026ndash;58.1 \u0026micro;mol/L; VK1 0.22\u0026ndash;4.88 \u0026micro;mol/L.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS detection of serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e and 25(OH)D\u003csub\u003e2\u003c/sub\u003e levels in gastric cancer patients and healthy volunteers\u003c/h2\u003e \u003cp\u003eThe results of LC-MS/MS assay showed that serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e (21.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.324 vs. 28.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.189, P\u0026thinsp;=\u0026thinsp;0.0446), 25(OH)D\u003csub\u003e2\u003c/sub\u003e (0.2970\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06284 vs. 1.183\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3872, P\u0026thinsp;=\u0026thinsp;0.0338), and 25(OH)D (22.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.346 vs 29.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.279, P\u0026thinsp;=\u0026thinsp;0.0267) levels were lower than serum levels in healthy volunteers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS detected serum VA, VE and VK1 levels in gastric cancer patients and healthy volunteers\u003c/h2\u003e \u003cp\u003eThe results of LC-MS/MS assay showed that serum VA (2159\u0026thinsp;\u0026plusmn;\u0026thinsp;176.4 vs 2932\u0026thinsp;\u0026plusmn;\u0026thinsp;166.8, P\u0026thinsp;=\u0026thinsp;0.0022) and VK1 (2.512\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4823 vs 4.550\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4622, P\u0026thinsp;=\u0026thinsp;0.0033) levels in gastric cancer patients were lower than the serum levels in healthy volunteers, while there was no significant difference in VE (41910\u0026thinsp;\u0026plusmn;\u0026thinsp;2813 vs 48110\u0026thinsp;\u0026plusmn;\u0026thinsp;2722, ns.) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLC-MS/MS is the method of choice for the detection of small molecules in clinical biochemistry, which has the advantages of high sensitivity, high specificity, high stability, and wide linear range, and has been widely used in the fields of chemical industry, biology, pharmaceuticals, food, clinical medicine, and environment [17]. In the field of clinical testing and diagnosis, LC-MS/MS, as a supplement to traditional diagnostic techniques, can provide a more reliable basis for more accurate diagnosis of many diseases [18]. The TQ-XS mass spectrometer used in this study is a new generation of revolutionary products in the mass spectrometry community, capable of analyzing a wider variety of compounds with a completely new level of ultra-high sensitivity, which has improved the reliability, reproducibility, and analytical performance of the quantitative analysis to a higher level through innovative research. 25(OH)D\u003csub\u003e3\u003c/sub\u003e and 25(OH)D\u003csub\u003e2\u003c/sub\u003e have small structural differences, and the activity of 25(OH)D\u003csub\u003e2\u003c/sub\u003e is lower than that of 25(OH)D\u003csub\u003e3\u003c/sub\u003e, and elevated 25(OH)D\u003csub\u003e2\u003c/sub\u003e may be accompanied by a decrease in 25(OH)D\u003csub\u003e3\u003c/sub\u003e, so testing the activity and function of both separately is a necessity to properly assess vitamin levels and the efficacy of supplementation therapy. Usually, immunological methods measure total 25(OH)D, while LC-MS/MS can detect 25(OH)D\u003csub\u003e3\u003c/sub\u003e and 25(OH)D\u003csub\u003e2\u003c/sub\u003e separately, which can be used for targeted supplementation according to the test results, avoiding the harms of under- and over-supplementation. LC-MS/MS can also be used to detect the blood concentration of different types of vitamin D drugs, preventing vitamin D toxicity, and providing a more precise and precise guidance to the clinical treatment [19,20]. In this study, serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e and 25(OH)D were found to be lower than the serum levels of healthy volunteers in gastric cancer patients by LC-MS/MS. Other fat-soluble vitamins, including vitamin A and vitamin K1, were also lower in serum levels of gastric cancer patients than in serum levels of healthy volunteers, and there was no significant difference in vitamin E. The sample size was limited in this study, and we will continue to expand the sample size and follow up the gastric cancer patients to further clarify the effects of various fat-soluble vitamins on the prognosis of gastric cancer patients.\u003c/p\u003e \u003cp\u003eAlthough LC-MS/MS provides more accurate detection solutions for clinical small molecule detection, LC-MS/MS still faces serious challenges. Most of the current LC-MS/MS assays are Laboratory developde tests (LDT), and although a variety of mass spectrometry detection kits have been marketed, the lack of standardization of the methods makes it impossible to avoid deviations in results between different laboratories or even the same laboratory due to factors such as pretreatment methods, reagents, instrumentation, ion sources, columns, calibrators, methodology and performance validation. In addition, inappropriate interpretation and application of method validation guidelines, lack or inappropriate use of standard substances, and incomplete quality assurance measures can lead to inaccurate test results [21\u0026ndash;23]. Relatively high operator requirements and the cost of the assay also hinder the use of LC-MS/MS in large-scale screening [24].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we measured the serum fat-soluble vitamins VA, 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e, VE, and VK1 levels in gastric cancer patients and healthy volunteers using LC-MS/MS. The results showed that the serum levels of VA, 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e and VK1 were lower in gastric cancer patients than in healthy volunteers, and there was no significant difference in VE. This study suggests that the detection of fat-soluble vitamin levels is important for improving prognosis, and LC-MS/MS will provide more accurate results for clinical fat-soluble vitamin detection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eSun L wrote the manuscript. Gao Q and Shen Y completed the experiment and verified the underlying data. Sun W was responsible for the graph draft. Yao Y designed and revised the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by National Natural Science Foundation of China (82203547), Natural Science Foundation of Jiangsu Province (BK20210136) and China Postdoctoral Science Foundation (2022M721433).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eAuthors confrm that they have full control over all primary data and agree to allow the journal to review their data if requested.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the First People\u0026apos;s Hospital of Kunshan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eInternational Agency for Research on Cancer, Data visualization tools for exploring the global cancer burden in 2020. Http://gco.iarc.fr/today/home. (Accessed 13 May 2022).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi D, Wang Y, Shi C, et al (2023) Targeting GPC3\u003csup\u003ehigh\u003c/sup\u003e cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in gastric cancer. 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J Mol Diagn Ther 12:1591\u0026ndash;1594.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWise SA, Camara JE, Burdette CQ, et al (2022) Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance. Anal Bioanal Chem 414:351\u0026ndash;366. doi: 10.1007/s00216-021-03577-0.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBinkley N, Dawson-Hughes B, Durazo-Arvizu R, et al (2017) Vitamin D measurement standardization: The way out of the chaos. J Steroid Biochem Mol Biol 173:117\u0026ndash;121. doi: 10.1016/j.jsbmb.2016.12.002.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSempos CT, Heijboer AC, Bikle DD, et al (2018) Vitamin D assays and the definition of hypovitaminosis D: results from the First International Conference on Controversies in Vitamin D. Br J Clin Pharmacol 84:2194\u0026ndash;2207. doi: 10.1111/bcp.13652.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChinese Association of Geriatric Research of Laboratory Medicine, Chinese Geriatrics Society of Laboratory Medicine (2021) Expert consensus on the standardization of liquid chromatography-tandem mass spectrometry method for the measurement of 25‐hydroxyvitamin D. Chin J Lab Med 44:587\u0026ndash;595. doi: 10.3760/cma.j.cn114452-20201120-00846.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, Fat-soluble vitamins, LC-MS/MS, 25(OH)D3, 25(OH)D2","lastPublishedDoi":"10.21203/rs.3.rs-3895853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3895853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eGastric cancer is one of the most common malignant tumors, with the incidence and mortality ranking among the top four malignant tumors in the world. In recent years, more and more studies have reported that fat-soluble vitamins are involved in tumor metabolism. However, the sensitivity and specificity of conventional detection methods are low, while liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has the advantages of high sensitivity, high specificity, high stability and wide linear range, which makes up for the shortcomings of immunological methods. The purpose of this study was to accurately detect the content of fat-soluble vitamins in GC patients by LC-MS/MS, and to clarify the necessity of preoperative determination and regular postoperative monitoring.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSerum 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e, VA, VE and VK1 levels in gastric cancer patients and healthy volunteers were detected by LC-MS/MS.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that the levels of serum 25(OH)D\u003csub\u003e3\u003c/sub\u003e, 25(OH)D\u003csub\u003e2\u003c/sub\u003e, VA and VK1 in gastric cancer patients were significantly lower than those in healthy volunteers, and there was no significant difference in VE content.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study suggests that patients with gastric cancer should timely detect and supplement fat-soluble vitamins and LC-MS/MS will provide a more accurate detection protocol for the clinic.\u003c/p\u003e","manuscriptTitle":"Clinical application of LC-MS/MS to detect fat-soluble vitamins in gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 18:27:09","doi":"10.21203/rs.3.rs-3895853/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4fa7b582-64fc-4db6-8273-ff5a4ebbb385","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-20T21:45:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-30 18:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3895853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3895853","identity":"rs-3895853","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}