Comparison of an HPLC-UV system (LM1010) and UPLC-MS/MS for plasma voriconazole measurement in routine clinical practice

Comparison of an HPLC-UV system (LM1010) and UPLC-MS/MS for plasma voriconazole measurement in routine clinical practice | 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 Comparison of an HPLC-UV system (LM1010) and UPLC-MS/MS for plasma voriconazole measurement in routine clinical practice Junichi Nakagawa, Kayo Ueno, Katsuyoshi Osanai, Masahiro Ishiyama, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8825916/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Journal of Pharmaceutical Health Care and Sciences → Version 1 posted You are reading this latest preprint version Abstract Background We aimed to evaluate the clinical utility of the LM1010 high-performance liquid chromatography with ultraviolet-visible detection instrument for measuring plasma concentrations of voriconazole (VRCZ) under routine clinical conditions, including in patients with diverse clinical backgrounds. Methods In total, 213 samples were collected from two hospitals (groups A and B). The plasma samples were analyzed using LM1010 and ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS). Laboratory test values and information on concomitant medications were collected from electronic medical records. Results In total, 66 samples from group A and 135 samples from group B were included in the analysis. VRCZ plasma concentrations measured by LM1010 and UPLC-MS/MS were strongly correlated in both groups ( r = 0.983 and 0.996; both P < 0.001). The Bland-Altman analysis demonstrated proportional bias in both groups A and B, and the slopes of the regression lines were 0.045 (95% confidence interval [CI], 0.011–0.079) and 0.038 (95% CI, 0.025–0.052), respectively. Interfering peaks were observed in the chromatograms of eight samples in group B, but their effect on the intermethod difference was small. No clinical laboratory test values or concomitant medications were identified as factors affecting the intermethod difference. Conclusions The measurement of VRCZ plasma concentration by LM1010 was robust under heterogeneous clinical conditions and was useful for clinical therapeutic drug monitoring of VRCZ. Voriconazole LM1010 therapeutic drug monitoring high-performance liquid chromatography with ultraviolet-visible detection Figures Figure 1 Figure 2 Introduction Voriconazole (VRCZ), an azole antifungal agent, is widely used for the treatment and prophylaxis of invasive fungal infections. The plasma concentration of VRCZ is related to its therapeutic effect and the risk of side effects, such as liver dysfunction; therefore, therapeutic drug monitoring (TDM) is recommended [ 1 ]. The current gold standard for measurement of the plasma concentration of VRCZ is liquid chromatography-tandem mass spectrometry (LC-MS/MS) [ 2 ]. However, due to the high cost of equipment and the need for skilled technicians, only a limited number of institutions in Japan are able to perform in-house testing, and most rely on external laboratories [ 3 , 4 ]. VRCZ is primarily metabolized to voriconazole N -oxide (VNO) by cytochrome P450 (CYP) 2C19, with contributions from CYP2C9 and CYP3A4 [ 5 , 6 ]. The pharmacokinetics of VRCZ are highly variable among individuals due to CYP2C19 polymorphisms, and even within the same individual, they can vary significantly over several days in response to changes in the inflammatory response [ 7 ]. Therefore, optimal individualized VRCZ therapy requires frequent measurement of plasma concentrations and rapid feedback of the results; however, with outsourcing, it takes approximately 5 days from blood sampling to receive feedback from the results in Japan [ 4 ]. Thus, there is a clinical need for a cost-effective and user-friendly analytical platform that enables rapid in-hospital measurement of VRCZ plasma concentrations. In Japan, the LM1010 high-performance liquid chromatography with ultraviolet-visible detection (HPLC-UV) system, which has been approved for clinical use as a medical device, is available for the measurement of plasma VRCZ concentrations. However, the robustness of this system under heterogeneous clinical and analytical conditions encountered in routine practice remains insufficiently characterized. In particular, the influence of differences in clinical indications, sample handling procedures, measurement timing, and laboratory environments on intermethod agreement has not been fully examined. In real-world settings, VRCZ is used both for prophylaxis and treatment across diverse patient populations, and plasma samples may be processed under varying pre-analytical conditions, including delayed analysis after frozen storage or immediate same-day measurement. The purpose of this study was to evaluate the clinical utility and analytical accuracy of the LM1010 instrument for measuring plasma VRCZ concentrations under real-world clinical conditions. By comparing LM1010 with ultra performance LC (UPLC)-MS/MS across two independent clinical settings involving different therapeutic indications and sample handling conditions and by systematically assessing the impact of concomitant medications and laboratory test values on intermethod variability, we aimed to clarify the robustness of the LM1010 for routine TDM. Methods Chemical and reagents The VRCZ used in the UPLC-MS/MS analyses was purchased from LKT Laboratories (St. Paul, MN, USA). VRCZ-d3 was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used as an internal standard for UPLC-MS/MS. The 5 µg/mL VRCZ standard solution and mobile phases A and B used in the LM1010 analysis were purchased from Hitachi High-Tech Analysis (Tokyo, Japan). The solid-phase extraction spin column set used for sample preparation for LM1010 analysis was purchased from Hitachi High-Tech Analysis (Tokyo, Japan). Patients and blood sampling Blood samples were collected from Japanese patients undergoing treatment with VRCZ at Aomori Prefectural Central Hospital between July 2019 and August 2021 (group A) or at Hirosaki University Hospital between June 2023 and December 2024 (group B). In group A, VRCZ was primarily used for the prophylaxis of infections in patients undergoing bone marrow transplantation, whereas in group B, it was mainly used for the treatment of invasive fungal infections. In both groups, blood samples were collected immediately before the next dose in heparin sodium tubes and centrifuged at 3,500 rpm for 10 min at 4°C. To ensure consistency and minimize interoperator variability, all plasma VRCZ concentration measurements were performed at Hirosaki University Hospital by a single experienced analyst using two different analytical platforms: the LM1010 HPLC-UV system and the UPLC-MS/MS system. Laboratory test values ​​on the same day as blood sampling for both groups A and B and information on concomitant medications administered from the day before to the day of blood sampling for group B were collected from electronic medical records. Because this study included retrospectively collected samples, the availability of chromatographic data differed between the two groups, and chromatograms were not available for retrospective evaluation in group A. In addition, detailed information on concomitant medications administered from the day before to the day of blood sampling was available only for group B. The study protocol was approved by the Ethics Committee of Hirosaki University Graduate School of Medicine (project identification code: 2023-036-1). Measurement of VRCZ plasma concentrations by LM1010 HPLC Plasma VRCZ concentrations were measured using an automated HPLC-UV system (LM1010; Hitachi High-Tech Analysis, Tokyo, Japan). The plasma concentration of VRCZ was measured using LM1010 according to the operating instructions provided by Hitachi High-Tech. Separated plasma samples from group A were shipped frozen to Hirosaki University Hospital on the day of blood sampling and analyzed within 7 days of blood sampling. In group B, separated plasma samples were analyzed on the same day as blood collection. The plasma sample (150 µL) was loaded into a spin column preconditioned with 500 µL pretreatment solution A and 500 µL pretreatment solution B, and the column was centrifuged at 2,400 × g for 3 min at room temperature. After the column was washed with pretreatment solution B, the sample was eluted with 150 µL pretreatment solution C. The processed VRCZ sample was vortexed for 10 s and subjected to LM1010. HPLC conditions were the default settings, and peak selection for VRCZ as well as calculation of plasma concentrations were performed automatically by the analysis software of the LM1010 system. Measurement of VRCZ and VNO plasma concentrations by UPLC-MS/MS The remaining aliquots for UPLC-MS/MS were stored at − 80°C, and all measurements were performed within 6 months of sample collection to minimize potential degradation of analytes [ 8 ]. The UPLC-MS/MS system consisted of an ACQUITY UPLC System (Waters, MA, USA) and Xevo TQD (Waters). The conditions for plasma concentration analysis of VRCZ were described in our previous report [ 9 ]. The range of the VRCZ calibration curve was 0.25–10 µg/mL. Statistical procedures Comparisons of VRCZ concentrations measured by LM1010 and clinical laboratory values between groups A and B were performed using the Mann-Whitney U test. Pearson correlation analysis and Bland-Altman plots were performed to compare the plasma concentrations of VRCZ measured by LM1010 HPLC and UPLC-MS/MS. In the Bland-Altman plots, the limits of the agreement (LOAs) of intermethod differences were defined as the mean bias ± 1.96 × standard deviation. The fixed bias and proportional bias between LM1010 and UPLC-MS/MS were assessed using one-sample t-tests and linear regression analysis, respectively. Patient samples with measured VRCZ plasma concentrations less than 0.2 µg/mL by LM1010 or less than 0.25 µg/mL by UPLC-MS/MS were excluded from the analyses because of the lower limit of quantification. Relationships between the day of blood sampling, clinical test values, and differences in measurement results between the two methods were assessed using Spearman’s rank correlation. A P value less than 0.05 was considered to indicate statistical significance. Statistical analyses were performed with SPSS 28.0 for Windows (SPSS IBM Japan Inc., Tokyo, Japan). Results Patient characteristics In total, 73 samples were collected from 50 patients in group A, whereas in group B, 140 samples were collected from 32 patients. Blood sampling and LM1010 analysis were performed on 192 different days (69 days for group A and 123 days for group B). In group A, seven samples showed concentrations less than or equal to 0.2 µg/mL by LM1010; among these, six had values less than 0.25 µg/mL by UPLC-MS/MS, while one sample had a concentration of 0.4 µg/mL by UPLC-MS/MS. In group B, all five samples with VRCZ concentrations less than or equal to 0.2 µg/mL by LM1010 also had concentrations less than 0.25 µg/mL by UPLC-MS/MS in group B. In total, 12 samples (7 from group A and 5 from group B) with VRCZ concentrations below the lower limit of quantification were excluded from subsequent analyses. The laboratory test values for groups A and B are shown in Table 1. The median trough VRCZ concentration measured by LM1010 tended to be higher in group B than in group A (2.3 versus 1.9 µg/mL, respectively, P = 0.080). The median values of aspartate aminotransferase (AST), serum total bilirubin (T-Bil), and C-reactive protein (CRP) were significantly higher in group B than in group A (AST: 29 versus 24 U/L, respectively, P = 0.028; total bilirubin: 0.40 versus 0.36 mg/dL, respectively, P = 0.018; CRP: 1.22 versus 0.68 mg/dL, respectively, P = 0.026). The list of concomitant medications available for group B is shown in Supplementary Table 1. In total, 108 medications were administered between the day before and the day of blood sampling. Chromatograms by LM1010 HPLC In the LM1010 analysis, interfering peaks partially overlapping with the VRCZ peak were detected in eight patient samples from group B (Supplementary Fig. 1). There were no concomitant medications commonly administered to any of the patients who provided these samples. Although markedly elevated bilirubin levels were observed in two samples with interfering peaks, no consistent abnormalities in laboratory test values were identified across the eight samples (Supplementary Table 2). In addition, similar peak interference was not consistently observed in samples with elevated T-Bil levels across the entire study population, and no systematic association could be established. Comparison of LM1010 HPLC and UPLC-MS/MS The measured VRCZ plasma concentrations showed a strong correlation between LM1010 and UPLC-MS/MS in both groups A and B ( r = 0.983, n = 66 and 0.997, n = 135 respectively; both P < 0.001). The slopes of the linear regression equations were 0.947 (95% confidence interval [CI], 0.914–0.980) for group A and 0.959 (95% CI, 0.947–0.972) for group B (Fig. 1 ). Bland-Altman plots of the differences and ratios between the two methods are shown in Fig. 2 and Supplementary Fig. 2. In group A, the mean intermethod difference in VRCZ plasma concentration was 0.09 µg/mL (95% CI, 0.03–0.15), with fixed bias ( P = 0.003). For all but four (6.2%) samples, the differences were within the LOA (-0.39 to 0.58 µg/mL). Linear regression analysis showed that the slope of the regression line was 0.045 (95% CI, 0.011–0.079), and significant proportional bias was observed ( r 2 = 0.098, P = 0.010). In group B, the mean intermethod difference was 0.14 ug/mL (95% CI, 0.11–0.18), with fixed bias ( P < 0.001). For all but six (4.4%) samples, the differences were within the LOA (-0.23 to 0.52 µg/mL). Linear regression analysis showed that the slope of the regression line was 0.038 (95% CI, 0.025–0.052), and significant proportional bias was observed ( r 2 = 0.194, P < 0.001). The difference for all eight samples in which interference peaks were detected was within the LOA (Supplementary Table 2). In the ratio plot, the mean ratios of the measured VRCZ plasma concentration by LM1010 to UPLC-MS/MS were 1.055 (95% CI, 1.013–1.096, P < 0.001) in group A and 1.061 (95% CI, 1.048–1.074, P < 0.001) in group B, indicating a small, fixed bias toward higher concentrations measured by LM1010. No significant proportional bias was found between the ratio and means of measurements in both groups A and B ( P = 0.746 and 0.225, respectively). Table 2 shows the agreement between LM1010 and UPLC-MS/MS based on concentration category classifications according to the recommended therapeutic range for VRCZ in Japan [ 10 ]. Intermethod discordance classifications were observed in four samples (6.1%) in group A and 10 samples (7.4%) in group B. A list of samples showing discordant classifications is provided in Supplementary Table 3; in all but one of these samples, the VRCZ plasma concentrations measured by LM1010 were higher than those measured by UPLC-MS/MS. There was no significant association between intermethod differences in VRCZ concentrations measured by LM1010 and UPLC-MS/MS and the time elapsed since the initiation of TDM in either group A or group B ( P = 0.303 and 0.102, respectively). Discussion In this study, we compared VRCZ plasma concentrations measured by LM1010 and UPLC-MS/MS in patients with a variety of concomitant medications based on samples collected from two institutions. Despite heterogeneity in clinical indications, concomitant medications, and samples, plasma VRCZ concentrations measured by LM1010 showed strong agreement with those measured by UPLC-MS/MS. These findings indicated that LM1010 provided sufficiently robust and clinically reliable measurements for routine VRCZ TDM. Our results showed that the measured VRCZ plasma concentrations were approximately 6% higher by LM1010 than by UPLC-MS/MS in both groups, indicating a small but significant systematic bias between the two methods. This tendency toward higher values by LM1010 may be attributable to the inherent characteristics of UV-based detection, whereby at higher analyte concentrations, enlargement of the VRCZ peak increases the relative contribution of peak tailing and minor co-eluting components to the integrated peak area, resulting in slight overestimation. However, discordant concentration category classifications based on the recommended therapeutic range in Japan were observed in fewer than 8% of samples in each group, and only one sample per group showed both an intermethod difference outside the limits of agreement and a discordant classification. These results suggest that the observed inter-method differences are unlikely to meaningfully affect clinical decision-making in routine VRCZ TDM. In addition, no significant association was observed between the day of blood sampling and the intermethod difference over extended sampling periods, supporting the long-term robustness of LM1010 measurements. While recent clinical studies have highlighted the practical advantages of LM1010 in VRCZ TDM in terms of workflow efficiency and turnaround time [ 11 , 12 ], our current findings extend these observations by demonstrating the analytical robustness of LM1010 across heterogeneous clinical conditions. In this study, CRP, AST, and total bilirubin levels were significantly higher in group B than in group A, indicating distinct clinical backgrounds between the two cohorts (Table 1). In addition, although the difference did not reach statistical significance, the median trough VRCZ concentration measured by LM1010 tended to be higher in group B than in group A. Inflammatory conditions are known to suppress the expression of pregnane X receptor, thereby reducing CYP2C19 expression and increasing VRCZ exposure [ 9 , 13 ]. Moreover, impaired hepatic function has also been associated with decreased voriconazole metabolism and elevated plasma concentrations [ 14 ]. Therefore, the trend toward higher measured VRCZ concentrations in group B may be explained, at least in part, by increased inflammatory activity, as reflected by elevated CRP levels, and mildly impaired liver function. These findings reflect clinically relevant background differences rather than limitations in the analytical performance of the LM1010 system. Importantly, despite variations in patient backgrounds and VRCZ exposure levels between institutions, LM1010 consistently provided accurate measurements that reflected these clinical differences. There were several limitations to this study. First, the cause of the interfering peaks detected in the LM1010 analysis was not identified. Since all but one sample in which an interfering peak was detected had been administered trimethoprim-sulfamethoxazole concomitantly, we evaluated a mixed standard solution of trimethoprim and sulfamethoxazole using LM1010 analysis; however, no peaks corresponding to the interfering peaks were detected (data not shown). Second, the interval between administration of concomitant medications and blood sampling was not evaluated, and medication adherence on the day before blood sampling was also not assessed. Concomitant medications with short elimination half-lives may not have been present in the plasma at the time of blood collection. In conclusion, plasma VRCZ concentrations measured using the LM1010 instrument showed strong agreement with those measured by UPLC-MS/MS under routine clinical conditions. These findings support that measurement of VRCZ plasma concentration using LM1010 is robust in patients with diverse backgrounds and is useful for clinical TDM of VRCZ. Abbreviations AST, aspartate aminotransferase CRP, C-reactive protein HPLC-UV, high-performance liquid chromatography with ultraviolet-visible detection T-Bil, total bilirubin TDM, therapeutic drug monitoring UPLC-MS/MS, ultra performance liquid chromatography-tandem mass spectrometry VRCZ, voriconazole Declarations Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Hirosaki University Graduate School of Medicine (project identification code: 2023-036-1) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in this study. Consent for publication Not applicable. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests Dr. Niioka received speaker’s honoraria and collaborative research funding from Hitachi High-Tech Analysis. Two co-authors, Mr. Satoru Morikawa and Ms. Miyuki Matsushita, are employees of Hitachi High-Tech Analysis, the manufacturer of the LM1010 system evaluated in this study. The remaining authors declare no conflicts of interest. Funding Dr. Niioka received collaborative research funding from Hitachi High-Tech Analysis. Authors’ contributions All authors contributed to the study conception and design. Conceptualization: Takenori Niioka; investigation: Junichi Nakagawa; plasma concentration measurements: Kayo Ueno; blood sample collection: Katsuyoshi Osanai and Masahiro Ishiyama; formal analysis: Junichi Nakagawa; writing—original draft preparation: Junichi Nakagawa; writing—review and editing: Hirofumi Tomita and Takenori Niioka. All authors read and approved the final manuscript. Acknowledgments We would like to thank Mr. Shoji Yamamoto, Ms. Yuki Ono, and Ms. Kanae Wakasaya for their contributions to blood sample collection at Aomori Prefectural Central Hospital. References Takesue Y, Hanai Y, Oda K, Hamada Y, Ueda T, Mayumi T, et al. Japanese Antimicrobial Therapeutic Drug Monitoring Guideline Committee. Clinical Practice Guideline for the Therapeutic Drug Monitoring of Voriconazole in Non-Asian and Asian Adult Patients: Consensus Review by the Japanese Society of Chemotherapy and the Japanese Society of Therapeutic Drug Monitoring. Clin Ther. 2022;44(12):1604–23. https://doi.org/10.1016/j.clinthera.2022.10.005 Mak J, Sujishi KK, French D. Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify serum voriconazole. J Chromatogr B Analyt Technol Biomed Life Sci. 2015;986–7:94–9. https://doi.org/10.1016/j.jchromb.2015.02.011 Hamada Y, Tokimatsu I, Mikamo H, Kimura M, Seki M, Takakura S, et al. 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Therapeutic drug monitoring and safety of voriconazole in patients with liver dysfunction. Antimicrob Agents Chemother. 2024;68(11):e0112624. https://doi.org/10.1128/aac.01126-24 Tables Tables 1 and 2 are available in the supplementary files section Additional Declarations Competing interest reported. Dr. Niioka received speaker’s honoraria and collaborative research funding from Hitachi High-Tech Analysis. Two co-authors, Mr. Satoru Morikawa and Ms. Miyuki Matsushita, are employees of Hitachi High-Tech Analysis, the manufacturer of the LM1010 system evaluated in this study. The remaining authors declare no conflicts of interest. Supplementary Files ESM1cleancopy.docx Table1.xlsx Table2.xlsx Cite Share Download PDF Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Journal of Pharmaceutical Health Care and Sciences → 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8825916","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591854736,"identity":"04ab51b1-7876-4f22-9a1e-b82ca7c509bf","order_by":0,"name":"Junichi Nakagawa","email":"","orcid":"","institution":"Hirosaki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junichi","middleName":"","lastName":"Nakagawa","suffix":""},{"id":591854737,"identity":"64430310-04bb-46d8-8fb5-bb6a28c23619","order_by":1,"name":"Kayo Ueno","email":"","orcid":"","institution":"Hirosaki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kayo","middleName":"","lastName":"Ueno","suffix":""},{"id":591854738,"identity":"00e96dd8-09e8-4de0-8cf5-73d3bf2150f1","order_by":2,"name":"Katsuyoshi Osanai","email":"","orcid":"","institution":"Aomori Prefectural Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Katsuyoshi","middleName":"","lastName":"Osanai","suffix":""},{"id":591854739,"identity":"c4ebc52a-8601-4b61-b639-227c7b083bc8","order_by":3,"name":"Masahiro Ishiyama","email":"","orcid":"","institution":"Hirosaki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Ishiyama","suffix":""},{"id":591854740,"identity":"30b55d51-e8a1-4138-b04c-f871ad0e8f10","order_by":4,"name":"Miyuki Matsushita","email":"","orcid":"","institution":"Hitachi High-Tech Analysis Corporation","correspondingAuthor":false,"prefix":"","firstName":"Miyuki","middleName":"","lastName":"Matsushita","suffix":""},{"id":591854741,"identity":"3ca7eb78-fa13-41f8-8791-2bc26934538e","order_by":5,"name":"Satoru Morikawa","email":"","orcid":"","institution":"Hitachi High-Tech Analysis Corporation","correspondingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Morikawa","suffix":""},{"id":591854742,"identity":"ff8dca23-513d-4db4-89d6-56ce0b965c65","order_by":6,"name":"Hirofumi Tomita","email":"","orcid":"","institution":"Hirosaki University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hirofumi","middleName":"","lastName":"Tomita","suffix":""},{"id":591854743,"identity":"840b5cab-8cd4-467d-bb00-b18cc06e4b80","order_by":7,"name":"Takenori Niioka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYJCCAwkMCQz8EowNII4BG1SUGZdyHpgWyRmkaAGCBAaDGxCWAUFH2TPwHjzwoCJN3vh2c9uHD3/sjPkYeAwYftQwsJvjtIUv4UDCmRzDbXcONs+c2ZZsxgbUwthzjIHZsgGHFvk3BgcS2yoYt91IbGbmbWC2YQOKMPA2MDAbHMBlCw9Yi/3mGUAtPH/qbcC2/CWsJSdxgwRIC9thsMOY8dpyAKgl4Uxa8gygwxhnth03ZmNgKzgsc0wCp1/YG3iMP/6oSLbtn5H+mOHDn2rD+Q3MGx++qbFJxhVi2AHQSRLJhGMIHdiRrmUUjIJRMAqGKQAA31lRLxrcNU4AAAAASUVORK5CYII=","orcid":"","institution":"Hirosaki University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Takenori","middleName":"","lastName":"Niioka","suffix":""}],"badges":[],"createdAt":"2026-02-09 05:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8825916/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8825916/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40780-026-00573-3","type":"published","date":"2026-04-17T15:59:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102991459,"identity":"6e9cc416-b7fa-4960-a4a6-f265d694c9f5","added_by":"auto","created_at":"2026-02-19 11:32:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationships between voriconazole plasma concentrations measured by LM1010 and UPLC-MS/MS.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/831ebb149176a699fbb8da3b.png"},{"id":102991460,"identity":"a03e7d3f-f222-4289-b0a7-12a19b994037","added_by":"auto","created_at":"2026-02-19 11:32:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBland-Altman plots of differences between LM1010 and UPLC-MS/MS measurements of VRCZ.\u003c/strong\u003e Mean of measurements represents the mean voriconazole concentration measured by LM1010 and UPLC-MS/MS. The dashed lines indicate the mean difference ± 1.96 × standard deviation and zero, and the solid line represents the regression line.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/2a6f0c15d8fa8f3bf9517ad4.png"},{"id":107352662,"identity":"6d84bbdf-e23e-48c4-89e0-233b19a71a04","added_by":"auto","created_at":"2026-04-20 16:14:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":340267,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/4c8e3385-12ec-4af0-aea9-da420b2dc26c.pdf"},{"id":102991463,"identity":"543d02de-87b2-491a-9d2b-338ac9cc00b0","added_by":"auto","created_at":"2026-02-19 11:32:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":898269,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1cleancopy.docx","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/a094efc23a2a5e692ee4068a.docx"},{"id":102991462,"identity":"0b778d26-9800-4084-b163-be21e0b0b35c","added_by":"auto","created_at":"2026-02-19 11:32:02","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11256,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/7c5c28e24843e85644acf7b7.xlsx"},{"id":103049761,"identity":"0e37e559-0f0d-4b93-b47f-797f71dade65","added_by":"auto","created_at":"2026-02-20 07:45:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11562,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8825916/v1/4aeef2022fe04a31b5bfb316.xlsx"}],"financialInterests":"Competing interest reported. Dr. Niioka received speaker’s honoraria and collaborative research funding from Hitachi High-Tech Analysis. Two co-authors, Mr. Satoru Morikawa and Ms. Miyuki Matsushita, are employees of Hitachi High-Tech Analysis, the manufacturer of the LM1010 system evaluated in this study. The remaining authors declare no conflicts of interest.","formattedTitle":"Comparison of an HPLC-UV system (LM1010) and UPLC-MS/MS for plasma voriconazole measurement in routine clinical practice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVoriconazole (VRCZ), an azole antifungal agent, is widely used for the treatment and prophylaxis of invasive fungal infections. The plasma concentration of VRCZ is related to its therapeutic effect and the risk of side effects, such as liver dysfunction; therefore, therapeutic drug monitoring (TDM) is recommended [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The current gold standard for measurement of the plasma concentration of VRCZ is liquid chromatography-tandem mass spectrometry (LC-MS/MS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, due to the high cost of equipment and the need for skilled technicians, only a limited number of institutions in Japan are able to perform in-house testing, and most rely on external laboratories [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVRCZ is primarily metabolized to voriconazole \u003cem\u003eN\u003c/em\u003e-oxide (VNO) by cytochrome P450 (CYP) 2C19, with contributions from CYP2C9 and CYP3A4 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The pharmacokinetics of VRCZ are highly variable among individuals due to \u003cem\u003eCYP2C19\u003c/em\u003e polymorphisms, and even within the same individual, they can vary significantly over several days in response to changes in the inflammatory response [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, optimal individualized VRCZ therapy requires frequent measurement of plasma concentrations and rapid feedback of the results; however, with outsourcing, it takes approximately 5 days from blood sampling to receive feedback from the results in Japan [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, there is a clinical need for a cost-effective and user-friendly analytical platform that enables rapid in-hospital measurement of VRCZ plasma concentrations.\u003c/p\u003e \u003cp\u003eIn Japan, the LM1010 high-performance liquid chromatography with ultraviolet-visible detection (HPLC-UV) system, which has been approved for clinical use as a medical device, is available for the measurement of plasma VRCZ concentrations. However, the robustness of this system under heterogeneous clinical and analytical conditions encountered in routine practice remains insufficiently characterized. In particular, the influence of differences in clinical indications, sample handling procedures, measurement timing, and laboratory environments on intermethod agreement has not been fully examined. In real-world settings, VRCZ is used both for prophylaxis and treatment across diverse patient populations, and plasma samples may be processed under varying pre-analytical conditions, including delayed analysis after frozen storage or immediate same-day measurement.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to evaluate the clinical utility and analytical accuracy of the LM1010 instrument for measuring plasma VRCZ concentrations under real-world clinical conditions. By comparing LM1010 with ultra performance LC (UPLC)-MS/MS across two independent clinical settings involving different therapeutic indications and sample handling conditions and by systematically assessing the impact of concomitant medications and laboratory test values on intermethod variability, we aimed to clarify the robustness of the LM1010 for routine TDM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemical and reagents\u003c/h2\u003e \u003cp\u003eThe VRCZ used in the UPLC-MS/MS analyses was purchased from LKT Laboratories (St. Paul, MN, USA). VRCZ-d3 was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used as an internal standard for UPLC-MS/MS. The 5 \u0026micro;g/mL VRCZ standard solution and mobile phases A and B used in the LM1010 analysis were purchased from Hitachi High-Tech Analysis (Tokyo, Japan). The solid-phase extraction spin column set used for sample preparation for LM1010 analysis was purchased from Hitachi High-Tech Analysis (Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatients and blood sampling\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected from Japanese patients undergoing treatment with VRCZ at Aomori Prefectural Central Hospital between July 2019 and August 2021 (group A) or at Hirosaki University Hospital between June 2023 and December 2024 (group B). In group A, VRCZ was primarily used for the prophylaxis of infections in patients undergoing bone marrow transplantation, whereas in group B, it was mainly used for the treatment of invasive fungal infections. In both groups, blood samples were collected immediately before the next dose in heparin sodium tubes and centrifuged at 3,500 rpm for 10 min at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo ensure consistency and minimize interoperator variability, all plasma VRCZ concentration measurements were performed at Hirosaki University Hospital by a single experienced analyst using two different analytical platforms: the LM1010 HPLC-UV system and the UPLC-MS/MS system.\u003c/p\u003e \u003cp\u003eLaboratory test values ​​on the same day as blood sampling for both groups A and B and information on concomitant medications administered from the day before to the day of blood sampling for group B were collected from electronic medical records. Because this study included retrospectively collected samples, the availability of chromatographic data differed between the two groups, and chromatograms were not available for retrospective evaluation in group A. In addition, detailed information on concomitant medications administered from the day before to the day of blood sampling was available only for group B. The study protocol was approved by the Ethics Committee of Hirosaki University Graduate School of Medicine (project identification code: 2023-036-1).\u003c/p\u003e\n\u003ch3\u003eMeasurement of VRCZ plasma concentrations by LM1010 HPLC\u003c/h3\u003e\n\u003cp\u003ePlasma VRCZ concentrations were measured using an automated HPLC-UV system (LM1010; Hitachi High-Tech Analysis, Tokyo, Japan). The plasma concentration of VRCZ was measured using LM1010 according to the operating instructions provided by Hitachi High-Tech. Separated plasma samples from group A were shipped frozen to Hirosaki University Hospital on the day of blood sampling and analyzed within 7 days of blood sampling. In group B, separated plasma samples were analyzed on the same day as blood collection. The plasma sample (150 \u0026micro;L) was loaded into a spin column preconditioned with 500 \u0026micro;L pretreatment solution A and 500 \u0026micro;L pretreatment solution B, and the column was centrifuged at 2,400 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 min at room temperature. After the column was washed with pretreatment solution B, the sample was eluted with 150 \u0026micro;L pretreatment solution C. The processed VRCZ sample was vortexed for 10 s and subjected to LM1010. HPLC conditions were the default settings, and peak selection for VRCZ as well as calculation of plasma concentrations were performed automatically by the analysis software of the LM1010 system.\u003c/p\u003e\n\u003ch3\u003eMeasurement of VRCZ and VNO plasma concentrations by UPLC-MS/MS\u003c/h3\u003e\n\u003cp\u003eThe remaining aliquots for UPLC-MS/MS were stored at \u0026minus;\u0026thinsp;80\u0026deg;C, and all measurements were performed within 6 months of sample collection to minimize potential degradation of analytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The UPLC-MS/MS system consisted of an ACQUITY UPLC System (Waters, MA, USA) and Xevo TQD (Waters). The conditions for plasma concentration analysis of VRCZ were described in our previous report [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The range of the VRCZ calibration curve was 0.25\u0026ndash;10 \u0026micro;g/mL.\u003c/p\u003e\n\u003ch3\u003eStatistical procedures\u003c/h3\u003e\n\u003cp\u003eComparisons of VRCZ concentrations measured by LM1010 and clinical laboratory values between groups A and B were performed using the Mann-Whitney U test. Pearson correlation analysis and Bland-Altman plots were performed to compare the plasma concentrations of VRCZ measured by LM1010 HPLC and UPLC-MS/MS. In the Bland-Altman plots, the limits of the agreement (LOAs) of intermethod differences were defined as the mean bias\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 \u0026times; standard deviation. The fixed bias and proportional bias between LM1010 and UPLC-MS/MS were assessed using one-sample t-tests and linear regression analysis, respectively. Patient samples with measured VRCZ plasma concentrations less than 0.2 \u0026micro;g/mL by LM1010 or less than 0.25 \u0026micro;g/mL by UPLC-MS/MS were excluded from the analyses because of the lower limit of quantification. Relationships between the day of blood sampling, clinical test values, and differences in measurement results between the two methods were assessed using Spearman\u0026rsquo;s rank correlation.\u003c/p\u003e \u003cp\u003eA \u003cem\u003eP\u003c/em\u003e value less than 0.05 was considered to indicate statistical significance. Statistical analyses were performed with SPSS 28.0 for Windows (SPSS IBM Japan Inc., Tokyo, Japan).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePatient characteristics\u003c/h2\u003e \u003cp\u003eIn total, 73 samples were collected from 50 patients in group A, whereas in group B, 140 samples were collected from 32 patients. Blood sampling and LM1010 analysis were performed on 192 different days (69 days for group A and 123 days for group B). In group A, seven samples showed concentrations less than or equal to 0.2 \u0026micro;g/mL by LM1010; among these, six had values less than 0.25 \u0026micro;g/mL by UPLC-MS/MS, while one sample had a concentration of 0.4 \u0026micro;g/mL by UPLC-MS/MS. In group B, all five samples with VRCZ concentrations less than or equal to 0.2 \u0026micro;g/mL by LM1010 also had concentrations less than 0.25 \u0026micro;g/mL by UPLC-MS/MS in group B. In total, 12 samples (7 from group A and 5 from group B) with VRCZ concentrations below the lower limit of quantification were excluded from subsequent analyses. The laboratory test values for groups A and B are shown in Table 1. The median trough VRCZ concentration measured by LM1010 tended to be higher in group B than in group A (2.3 versus 1.9 \u0026micro;g/mL, respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.080). The median values of aspartate aminotransferase (AST), serum total bilirubin (T-Bil), and C-reactive protein (CRP) were significantly higher in group B than in group A (AST: 29 versus 24 U/L, respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028; total bilirubin: 0.40 versus 0.36 mg/dL, respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018; CRP: 1.22 versus 0.68 mg/dL, respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026). The list of concomitant medications available for group B is shown in Supplementary Table\u0026nbsp;1. In total, 108 medications were administered between the day before and the day of blood sampling.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromatograms by LM1010 HPLC\u003c/h3\u003e\n\u003cp\u003eIn the LM1010 analysis, interfering peaks partially overlapping with the VRCZ peak were detected in eight patient samples from group B (Supplementary Fig.\u0026nbsp;1). There were no concomitant medications commonly administered to any of the patients who provided these samples. Although markedly elevated bilirubin levels were observed in two samples with interfering peaks, no consistent abnormalities in laboratory test values were identified across the eight samples (Supplementary Table\u0026nbsp;2). In addition, similar peak interference was not consistently observed in samples with elevated T-Bil levels across the entire study population, and no systematic association could be established.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparison of LM1010 HPLC and UPLC-MS/MS\u003c/h2\u003e \u003cp\u003eThe measured VRCZ plasma concentrations showed a strong correlation between LM1010 and UPLC-MS/MS in both groups A and B (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.983, n\u0026thinsp;=\u0026thinsp;66 and 0.997, n\u0026thinsp;=\u0026thinsp;135 respectively; both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The slopes of the linear regression equations were 0.947 (95% confidence interval [CI], 0.914\u0026ndash;0.980) for group A and 0.959 (95% CI, 0.947\u0026ndash;0.972) for group B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBland-Altman plots of the differences and ratios between the two methods are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2. In group A, the mean intermethod difference in VRCZ plasma concentration was 0.09 \u0026micro;g/mL (95% CI, 0.03\u0026ndash;0.15), with fixed bias (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). For all but four (6.2%) samples, the differences were within the LOA (-0.39 to 0.58 \u0026micro;g/mL). Linear regression analysis showed that the slope of the regression line was 0.045 (95% CI, 0.011\u0026ndash;0.079), and significant proportional bias was observed (\u003cem\u003er\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.098, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010). In group B, the mean intermethod difference was 0.14 ug/mL (95% CI, 0.11\u0026ndash;0.18), with fixed bias (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). For all but six (4.4%) samples, the differences were within the LOA (-0.23 to 0.52 \u0026micro;g/mL). Linear regression analysis showed that the slope of the regression line was 0.038 (95% CI, 0.025\u0026ndash;0.052), and significant proportional bias was observed (\u003cem\u003er\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.194, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The difference for all eight samples in which interference peaks were detected was within the LOA (Supplementary Table\u0026nbsp;2). In the ratio plot, the mean ratios of the measured VRCZ plasma concentration by LM1010 to UPLC-MS/MS were 1.055 (95% CI, 1.013\u0026ndash;1.096, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in group A and 1.061 (95% CI, 1.048\u0026ndash;1.074, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in group B, indicating a small, fixed bias toward higher concentrations measured by LM1010. No significant proportional bias was found between the ratio and means of measurements in both groups A and B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.746 and 0.225, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;2 shows the agreement between LM1010 and UPLC-MS/MS based on concentration category classifications according to the recommended therapeutic range for VRCZ in Japan [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Intermethod discordance classifications were observed in four samples (6.1%) in group A and 10 samples (7.4%) in group B. A list of samples showing discordant classifications is provided in Supplementary Table\u0026nbsp;3; in all but one of these samples, the VRCZ plasma concentrations measured by LM1010 were higher than those measured by UPLC-MS/MS. There was no significant association between intermethod differences in VRCZ concentrations measured by LM1010 and UPLC-MS/MS and the time elapsed since the initiation of TDM in either group A or group B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.303 and 0.102, respectively).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we compared VRCZ plasma concentrations measured by LM1010 and UPLC-MS/MS in patients with a variety of concomitant medications based on samples collected from two institutions. Despite heterogeneity in clinical indications, concomitant medications, and samples, plasma VRCZ concentrations measured by LM1010 showed strong agreement with those measured by UPLC-MS/MS. These findings indicated that LM1010 provided sufficiently robust and clinically reliable measurements for routine VRCZ TDM.\u003c/p\u003e \u003cp\u003eOur results showed that the measured VRCZ plasma concentrations were approximately 6% higher by LM1010 than by UPLC-MS/MS in both groups, indicating a small but significant systematic bias between the two methods. This tendency toward higher values by LM1010 may be attributable to the inherent characteristics of UV-based detection, whereby at higher analyte concentrations, enlargement of the VRCZ peak increases the relative contribution of peak tailing and minor co-eluting components to the integrated peak area, resulting in slight overestimation. However, discordant concentration category classifications based on the recommended therapeutic range in Japan were observed in fewer than 8% of samples in each group, and only one sample per group showed both an intermethod difference outside the limits of agreement and a discordant classification. These results suggest that the observed inter-method differences are unlikely to meaningfully affect clinical decision-making in routine VRCZ TDM. In addition, no significant association was observed between the day of blood sampling and the intermethod difference over extended sampling periods, supporting the long-term robustness of LM1010 measurements. While recent clinical studies have highlighted the practical advantages of LM1010 in VRCZ TDM in terms of workflow efficiency and turnaround time [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], our current findings extend these observations by demonstrating the analytical robustness of LM1010 across heterogeneous clinical conditions.\u003c/p\u003e \u003cp\u003eIn this study, CRP, AST, and total bilirubin levels were significantly higher in group B than in group A, indicating distinct clinical backgrounds between the two cohorts (Table\u0026nbsp;1). In addition, although the difference did not reach statistical significance, the median trough VRCZ concentration measured by LM1010 tended to be higher in group B than in group A. Inflammatory conditions are known to suppress the expression of pregnane X receptor, thereby reducing CYP2C19 expression and increasing VRCZ exposure [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, impaired hepatic function has also been associated with decreased voriconazole metabolism and elevated plasma concentrations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the trend toward higher measured VRCZ concentrations in group B may be explained, at least in part, by increased inflammatory activity, as reflected by elevated CRP levels, and mildly impaired liver function. These findings reflect clinically relevant background differences rather than limitations in the analytical performance of the LM1010 system. Importantly, despite variations in patient backgrounds and VRCZ exposure levels between institutions, LM1010 consistently provided accurate measurements that reflected these clinical differences.\u003c/p\u003e \u003cp\u003eThere were several limitations to this study. First, the cause of the interfering peaks detected in the LM1010 analysis was not identified. Since all but one sample in which an interfering peak was detected had been administered trimethoprim-sulfamethoxazole concomitantly, we evaluated a mixed standard solution of trimethoprim and sulfamethoxazole using LM1010 analysis; however, no peaks corresponding to the interfering peaks were detected (data not shown). Second, the interval between administration of concomitant medications and blood sampling was not evaluated, and medication adherence on the day before blood sampling was also not assessed. Concomitant medications with short elimination half-lives may not have been present in the plasma at the time of blood collection.\u003c/p\u003e \u003cp\u003eIn conclusion, plasma VRCZ concentrations measured using the LM1010 instrument showed strong agreement with those measured by UPLC-MS/MS under routine clinical conditions. These findings support that measurement of VRCZ plasma concentration using LM1010 is robust in patients with diverse backgrounds and is useful for clinical TDM of VRCZ.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAST, aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eCRP, C-reactive protein\u003c/p\u003e\n\u003cp\u003eHPLC-UV, high-performance liquid chromatography with ultraviolet-visible detection\u003c/p\u003e\n\u003cp\u003eT-Bil, total bilirubin\u003c/p\u003e\n\u003cp\u003eTDM, therapeutic drug monitoring\u003c/p\u003e\n\u003cp\u003eUPLC-MS/MS, ultra performance liquid chromatography-tandem\u0026nbsp;mass spectrometry\u003c/p\u003e\n\u003cp\u003eVRCZ, voriconazole\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Hirosaki University Graduate School of Medicine (project identification code: 2023-036-1) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Niioka received speaker’s honoraria and collaborative research funding from Hitachi High-Tech Analysis. Two co-authors,\u0026nbsp;Mr.\u0026nbsp;Satoru Morikawa and Ms.\u0026nbsp;Miyuki\u0026nbsp;Matsushita, are employees of Hitachi High-Tech\u0026nbsp;Analysis, the manufacturer of\u0026nbsp;the\u0026nbsp;LM1010 system evaluated in this study. The remaining authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Niioka received collaborative research funding from Hitachi High-Tech Analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Conceptualization: Takenori Niioka; investigation: Junichi Nakagawa; plasma concentration measurements: Kayo Ueno; blood sample collection: Katsuyoshi Osanai and Masahiro Ishiyama; formal analysis: Junichi Nakagawa; writing—original draft preparation: Junichi Nakagawa; writing—review and editing: Hirofumi Tomita and Takenori Niioka. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Mr. Shoji Yamamoto, Ms. Yuki Ono, and Ms. Kanae Wakasaya for their contributions to blood sample collection at Aomori Prefectural Central Hospital.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTakesue Y, Hanai Y, Oda K, Hamada Y, Ueda T, Mayumi T, et al. Japanese Antimicrobial Therapeutic Drug Monitoring Guideline Committee. Clinical Practice Guideline for the Therapeutic Drug Monitoring of Voriconazole in Non-Asian and Asian Adult Patients: Consensus Review by the Japanese Society of Chemotherapy and the Japanese Society of Therapeutic Drug Monitoring. Clin Ther. 2022;44(12):1604\u0026ndash;23. https://doi.org/10.1016/j.clinthera.2022.10.005\u003c/li\u003e\n \u003cli\u003eMak J, Sujishi KK, French D. Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify serum voriconazole. J Chromatogr B Analyt Technol Biomed Life Sci. 2015;986\u0026ndash;7:94\u0026ndash;9. https://doi.org/10.1016/j.jchromb.2015.02.011\u003c/li\u003e\n \u003cli\u003eHamada Y, Tokimatsu I, Mikamo H, Kimura M, Seki M, Takakura S, et al. Practice guidelines for therapeutic drug monitoring of voriconazole: a consensus review of the Japanese Society of Chemotherapy and the Japanese Society of Therapeutic Drug Monitoring; J Infect Chemother. 2013;19(3):381\u0026ndash;92. https://doi.org/10.1007/s10156-013-0607-8\u003c/li\u003e\n \u003cli\u003eYasu T, Nomura Y, Gando Y, Matsumoto Y, Sugita T, Kosugi N, Kobayashi, M. High-performance liquid chromatography for ultra-simple determination of plasma voriconazole concentration. J Fungi (Basel). 2022;8(10):1035. https://doi.org/10.3390/jof8101035\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Theuretzbacher U, Ihle F, Derendorf H. Pharmacokinetic/pharmacodynamic profile of voriconazole. Clin Pharmacokinet. 2006;45(7):649\u0026ndash;63. https://doi.org/10.2165/00003088-200645070-00002\u003c/li\u003e\n \u003cli\u003eHyland R, Jones BC, Smith DA. Identification of the cytochrome P450 enzymes involved in the N-oxidation of voriconazole. Drug Metab Dispos. 2003;31(5):540\u0026ndash;7. https://doi.org/10.1124/dmd.31.5.540\u003c/li\u003e\n \u003cli\u003eKlomp SD, Veringa A, Alffenaar JC, de Boer MGJ, Span LFR, Guchelaar HJ, Swen, JJ. Inflammation altered correlation between CYP2C19 genotype and CYP2C19 activity in patients receiving voriconazole. Clin Transl Sci. 2024;17(7):e13887. https://doi.org/10.1111/cts.13887\u003c/li\u003e\n \u003cli\u003eGordien JB, Pigneux A, Vigouroux S, Tabrizi R, Accoceberry I, Bernadou JM, Rouault A, Saux MC, Breilh D. Simultaneous determination of five systemic azoles in plasma by high-performance liquid chromatography with ultraviolet detection. J Pharm Biomed Anal. 2009;50(5):932\u0026ndash;8. https://doi.org/10.1016/j.jpba.2009.06.030\u003c/li\u003e\n \u003cli\u003eAiuchi N, Nakagawa J, Sakuraba H, Takahata T, Kamata K, Saito N, et al. Impact of polymorphisms of pharmacokinetics-related genes and the inflammatory response on the metabolism of voriconazole. Pharmacol Res Perspect. 2022;10(2):e00935. https://doi.org/10.1002/prp2.935\u003c/li\u003e\n \u003cli\u003eKato H, Umemura T, Hagihara M, Shiota A, Asai N, Hamada Y, Mikamo H, Iwamoto T. Development of a therapeutic drug-monitoring algorithm for outpatients receiving voriconazole: a multicentre retrospective study. Br J Clin Pharmacol. 2024;90(5):1222\u0026ndash;30. https://doi.org/10.1111/bcp.1600\u003c/li\u003e\n \u003cli\u003eOda K, Uchino S, Kurogi K, Horikawa M, Matsumoto N, Yonemaru K. Clinical evaluation of an authorized medical equipment based on high performance liquid chromatography for measurement of serum voriconazole concentration. J Pharm Health Care Sci. 2021;7(1):42.\u0026nbsp;https://doi.org/10.1186/s40780-021-00225-8\u003c/li\u003e\n \u003cli\u003eMorikawa S, Yagi Y, Okazaki M, Yanagisawa N, Ishida T, Jobu K, et al. Rapid Therapeutic drug monitoring of voriconazole based on high-performance liquid chromatography: a single-center pilot study in outpatients. Antibiotics (Basel). 2025;14(5):474. https://doi.org/10.3390/antibiotics14050474\u003c/li\u003e\n \u003cli\u003evan Wanrooy MJ, Span LF, Rodgers MG, van den Heuvel ER, Uges DR, van der Werf TS, et al. Inflammation is associated with voriconazole trough concentrations. Antimicrob Agents Chemother. 2014;58(12):7098\u0026ndash;101. https://doi.org/10.1128/aac.03820-14\u003c/li\u003e\n \u003cli\u003eHu L, Su Y, Tang X, Li Y, Feng J, He G. Therapeutic drug monitoring and safety of voriconazole in patients with liver dysfunction. Antimicrob Agents Chemother. 2024;68(11):e0112624. https://doi.org/10.1128/aac.01126-24\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"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":"Voriconazole, LM1010, therapeutic drug monitoring, high-performance liquid chromatography with ultraviolet-visible detection","lastPublishedDoi":"10.21203/rs.3.rs-8825916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8825916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWe aimed to evaluate the clinical utility of the LM1010 high-performance liquid chromatography with ultraviolet-visible detection instrument for measuring plasma concentrations of voriconazole (VRCZ) under routine clinical conditions, including in patients with diverse clinical backgrounds.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn total, 213 samples were collected from two hospitals (groups A and B). The plasma samples were analyzed using LM1010 and ultra performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS). Laboratory test values and information on concomitant medications were collected from electronic medical records.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn total, 66 samples from group A and 135 samples from group B were included in the analysis. VRCZ plasma concentrations measured by LM1010 and UPLC-MS/MS were strongly correlated in both groups (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.983 and 0.996; both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The Bland-Altman analysis demonstrated proportional bias in both groups A and B, and the slopes of the regression lines were 0.045 (95% confidence interval [CI], 0.011\u0026ndash;0.079) and 0.038 (95% CI, 0.025\u0026ndash;0.052), respectively. Interfering peaks were observed in the chromatograms of eight samples in group B, but their effect on the intermethod difference was small. No clinical laboratory test values or concomitant medications were identified as factors affecting the intermethod difference.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe measurement of VRCZ plasma concentration by LM1010 was robust under heterogeneous clinical conditions and was useful for clinical therapeutic drug monitoring of VRCZ.\u003c/p\u003e","manuscriptTitle":"Comparison of an HPLC-UV system (LM1010) and UPLC-MS/MS for plasma voriconazole measurement in routine clinical practice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 11:31:55","doi":"10.21203/rs.3.rs-8825916/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":"679fed89-ee18-4535-911a-1ce0c3748bcc","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:13:05+00:00","versionOfRecord":{"articleIdentity":"rs-8825916","link":"https://doi.org/10.1186/s40780-026-00573-3","journal":{"identity":"journal-of-pharmaceutical-health-care-and-sciences","isVorOnly":false,"title":"Journal of Pharmaceutical Health Care and Sciences"},"publishedOn":"2026-04-17 15:59:19","publishedOnDateReadable":"April 17th, 2026"},"versionCreatedAt":"2026-02-19 11:31:55","video":"","vorDoi":"10.1186/s40780-026-00573-3","vorDoiUrl":"https://doi.org/10.1186/s40780-026-00573-3","workflowStages":[]},"version":"v1","identity":"rs-8825916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8825916","identity":"rs-8825916","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}