Establishment of the quick method to measure plasma cefmetazole by high- performance liquid chromatography and its clinical application

Establishment of the quick method to measure plasma cefmetazole by high- performance liquid chromatography and its clinical application | 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 Establishment of the quick method to measure plasma cefmetazole by high- performance liquid chromatography and its clinical application Yuna Sadaka, Kokoro Nakajima, Yuki Sasaki, Mao Tsurugaya, Yasuhisa Oida, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6736302/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Journal of Pharmaceutical Health Care and Sciences → Version 1 posted You are reading this latest preprint version Abstract Background Cefmetazole (CMZ) is widely used in Japan as a treatment for urinary tract infections (UTI), intra-abdominal infections, and bacteremia and as an antimicrobial prophylaxis. For the safe use of CMZ, evidence related to pharmacokinetic/pharmacodynamic analyses of CMZ in UTI is necessary. Thus, we attempted to establish a method to quantify CMZ plasma concentration using high-performance liquid chromatography (HPLC) and to verify its clinical application using plasma samples collected from Japanese patients with UTI. Methods Plasma samples were deproteinized by adding acetonitrile (MeCN) containing an internal standard substance (IS), barbital sodium. After centrifugation, the supernatant was collected and evaporated to dryness under a stream of nitrogen gas. The residue was reconstituted with the mobile phase and injected into the HPLC system equipped with a COSMOSIL® 5C 18 -MS-II column. In the mobile phase, 5-mM sodium citrate buffer (pH 3.2)/MeCN (85/15, v/v %) was added at an isocratic flow rate of 1.2 mL/min. CMZ and IS were detected at 272 and 229 nm, respectively, and the total run time was 15 min. The method’s application in clinical samples was evaluated by measuring plasma samples from 23 patients with UTI treated with CMZ. Samples were collected 2–3 h after the initiation of infusion (post-peak level) and 6–24 h after the last dose (trough level). The relationship between plasma CMZ concentrations and creatinine clearance (Ccr) was evaluated. Results The established method was found to have good linearity (0.2–200 µg/mL), precision, and accuracy. Furthermore, our method was applicable to the measurement of plasma in patients with UTI. These results suggest that the established method is suitable for measuring plasma CMZ levels for research to promote the proper use of CMZ. Furthermore, a negative correlation was observed between Ccr and the post-peak level, but not trough levels, of plasma CMZ. Conclusions Our developed method is simple and precise for quantifying plasma CMZ levels and is applicable in clinical settings. cefmetazole HPLC urinary tract infection plasma concentration PK/PD analysis clinical trials Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (ESBL- E ) are resistant to third-generation cephalosporin antibiotics. The incidence of infections caused by ESBL- E has increased recently and is considered a global threat ( 1 , 2 ). Selecting therapeutic agents for ESBL- E in clinical practice is often challenging. Carbapenem-resistant Acinetobacter baumannii and Enterobacterales are categorized as “critical priority” in the World Health Organization (WHO) Bacterial Priority Pathogens List 2024 ( 3 ). Although carbapenems are frequently used to treat severe ESBL- E infections, excessive carbapenem use is associated with increased carbapenem resistance ( 4 , 5 ). Cefmetazole (CMZ), a second-generation cephamycin antibiotic, possesses antibacterial efficacy against ESBL-producing bacteria ( 6 ). Moreover, several reports have demonstrated the clinical effectiveness of CMZ against ESBL- E infections ( 7 – 9 ). Recently, CMZ has been widely used in Japan as a treatment for urinary tract infection (UTI), intra-abdominal infections, and bacteremia ( 10 ) and as an antimicrobial prophylaxis in patients undergoing lower gastrointestinal tract and uterine surgery for both ESBL- E and non-ESBL- E infections ( 11 , 12 ). In particular, considering the high urinary recovery rate of CMZ as an unchanged form ( 13 ), CMZ would be suitable to treat ESBL- E- induced UTI, although appropriate dosage adjustment would be necessary depending on the renal function of individual patients. There is no description related to CMZ in the SANFORD GUIDE ( 14 ), Infectious Diseases Society of America 2023 Guidance ( 15 ), Up To Date (online database), and Global Guidelines for the Prevention of Surgical Site Infection (WHO) ( 16 ), probably because of the lack of global evidence without prescribing CMZ in the USA and several EU countries. Thus, new evidence to prove the effectiveness and safety of CMZ, such as pharmacokinetic/pharmacodynamic (PK-PD) analyses, would be necessary to change this situation. Regarding the measurement of CMZ, several methods using high-performance liquid chromatography (HPLC) ( 17 , 18 ) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) ( 19 , 20 ) have been reported. However, few methods can measure concentrations over a wide range at a faster rate. Furthermore, although plasma concentration data after a single dose of CMZ have been reported ( 21 – 24 ), only a few studies have examined the plasma concentration of CMZ after repeated administration, including peak and trough levels. Furthermore, no previous study has demonstrated whether dose adjustment according to renal function assessed based on plasma CMZ concentration is appropriate. In this study, we established a method for measuring plasma CMZ concentrations using HPLC and validated it based on the Food and Drug Administration (FDA) guidelines for conducting clinical trials ( 25 ). We then focused on the PK/PD study of CMZ in patients with UTI, where no PD target has been established ( 26 , 27 ). Furthermore, we measured plasma CMZ concentration in patients with UTI treated with CMZ using this method and revealed the relationship between CMZ concentration and renal function. Materials and Methods Chemicals and Reagents Cefmetazole sodium (purity 98%) was purchased from SIGMA-Aldrich Co., LLC. (St. Louis, USA). Barbital sodium (purity > 98%), used as the internal standard (IS), was purchased from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Distilled water and acetonitrile (MeCN) for HPLC grading were purchased from Hikari-Pharm. Co., Ltd. (Osaka, Japan) and FUJIFILM Wako Co., Ltd. (Tokyo, Japan), respectively. The human plasma used for the calibration curves and validation was purchased from Cosmo Bio., Co., Ltd. (Tokyo, Japan). The citric acid and trisodium citrate dihydrate used for mobile phase preparation were purchased from FUJIFILM Wako Co., Ltd., and Millex®-LG (0.20 µm, Merck, Darmstadt, Germany) was used for sample filtration. Liquid chromatography HPLC analysis was performed using a SHIMAZU LC-20AD system (Shimadzu Co., Ltd., Kyoto, Japan). Samples were separated using a COSMOSIL® 5C 18 -MS-II column (4.6 ID × 250 mm, Nacalai Tesque Co., Ltd., Kyoto, Japan). The mobile phase consisted of 5-mM sodium citrate buffer (pH 3.2) and MeCN (85/15, v/v %). The flow rate was maintained at 1.2 mL/min, and the column temperature was set at 45°C. The total run time was 15 min. The injection volume was 50 µL, and the detection wavelengths were 272 (CMZ) and 229 (IS) nm, respectively. Preparation of stock solutions CMZ and IS were dissolved in 15% MeCN in water to 20 and 1 mg/mL, respectively, as stock solutions and then stored at − 30°C until analysis. Preparation of standard and quality control (QC) samples The CMZ-stock solution was further diluted with 15% MeCN in water to obtain working solutions at several concentrations. The IS-stock solution was further diluted with MeCN at 40 µg/mL. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. The final concentrations of calibration standards were 0.2, 0.5, 1, 10, 50, 100, and 200 µg/mL in plasma. In this method, the calibration curves were prepared in two ranges: high (10–200 µg/mL) and low (0.2–10 µg/mL) concentrations. Therefore, three QC samples were used for each calibration curve. For the low-concentration calibration curves, the final concentrations of low-quality control (LQC), medium-QC (MQC), and high-QC (HQC) samples were set at 0.5, 5.0, and 10.0 µg/mL, respectively. For the high-concentration calibration curves, the final concentrations of LQC, MQC, and HQC were 20, 100, and 200 µg/mL, respectively. Preparation of samples to measure plasma CMZ Standard samples for the calibration curves were prepared using the following method. A 200-µL plasma sample was mixed with 20-µL CMZ working solutions and 600-µL IS in MeCN. The mixtures were centrifuged at 12,500 rpm for 10 min at 4°C. The supernatant was then completely dried under a nitrogen stream. The residue was eluted with 200 µL of the mobile phase and then filtered. Finally, 50 µL of the sample was injected into the HPLC system. Determination of plasma CMZ concentration Calibration curves were constructed by plotting the peak ratio (standard to internal standard) versus the nominal concentration. Using the regression equation, the plasma CMZ concentration was calculated from the CMZ/IS ratio of each sample. Method validation The method was fully validated according to the FDA guidelines for biological method validation ( 25 ). Accuracy was considered acceptable when the relative error (RE) was within ± 15%, and precision was considered acceptable at a relative standard deviation (RSD) of ≤ 15%, except lower limit of quantification (LLOQ) (RE ≤ ± 20 and RSD ≤ 20%). Selectivity If plasma-derived components did not interfere with the peak regions of CMZ and IS, then selectivity was evaluated. Blank plasma without CMZ and IS and plasma containing 200 µg/mL (in plasma concentration) of CMZ were measured using the aforementioned method. Chromatograms were compared and validated. Intra-day accuracy and precision QC samples were prepared using the method described above, and the same QC samples were measured five times repeatedly within 1 day to assess reproducibility within each run; accuracy and precision were then calculated. Inter-day accuracy and precision QC samples were measured once a day for 5 days, and the reproducibility between each analytical run was assessed by calculating the accuracy and precision. Recovery As with the QC samples, the peak area ratio of samples in which CMZ working solution was spiked in human plasma, and pretreatment (a) was then compared with the peak area ratio of samples treated with human plasma, and the working solution of CMZ was then spiked (b). The recovery rate was calculated using the following formula: Recovery (%)= \(\:\:\frac{\left(\text{a}\right)}{\left(\text{b}\right)}\) ×100 Stability The stability of CMZ was evaluated in QC samples at each of the three concentrations (HQC, MQC, and LLOQ) for high- and low-concentration calibration curves, respectively. HPLC measurements were performed in three runs under the following conditions: Freeze–thaw stability The stability of the samples when repeatedly frozen and thawed, which can occur during sample storage, was assessed. QC samples of three concentrations of CMZ in plasma were frozen and stored at − 30°C for 24 h and then thawed at room temperature, for three cycles. The three freeze–thaw samples were prepared using the aforementioned method. Bench-top stability To assess stability under conditions that may occur during sample preparation, QC samples (CMZ-containing plasma) were left at room temperature for 24 h and then prepared using the aforementioned method. Short-term stability The short-term stability of CMZ in plasma was assessed by analyzing CMZ-containing plasma after storage at − 30°C for 1 week according to the aforementioned method. Long-term stability The long-term stability of CMZ was assessed by analyzing CMZ-containing plasma stored at − 30ºC for 2 months. CMZ stock solutions were assessed for stability under conditions that may occur during long-term sample storage. Autosampler stability QC plasma samples containing CMZ were prepared using the above-mentioned method and left in an autosampler (room temperature) for 24 h. The stability of the samples was then measured and assessed from the time of sample preparation until measurement. Measurement of plasma CMZ concentrations in patients with UTI Twenty-three patients with UTI treated with CMZ admitted to the Department of Urology at Gifu General Medical Center between October 2022 and January 2023 were included in this study. The exclusion criteria were as follows: (i) patients who did not provide informed consent, (ii) those who were younger than 15 years, (iii) those with urinary bacteria < 10 5 CFU/mL, (iv) those receiving renal replacement therapy (hemodialysis or peritoneal dialysis) or those with creatinine clearance (Ccr) < 10 mL/min, (v) those who received CMZ for perioperative antimicrobial prophylaxis, and (vi) those deemed unfit for inclusion by physicians. All patients received 1 g of CMZ for 1 h according to their renal function (Table 1). This study was approved by the Ethics Committee of Gifu Pharmaceutical University (no. 5–13) and the Ethics Review Committee of Gifu General Medical Center (no. 758-4). Blood samples were collected 2–3 h after infusion initiation (defined as the “post-peak level”) and 6–24 h after the last dose (defined as the “trough level”). Of the trough samples, three that were collected significantly later than the scheduled interval (> 2 h after the scheduled infusion time) were excluded from statistical analysis. Although peak plasma concentrations should be obtained at the end of infusion (i.e., in this study, 1 h after infusion initiation), blood samples were collected 2–3 h after infusion initiation. Ohkawa et al. reported a more significant difference in plasma concentration between patients with normal and impaired renal function ( 22 ) 2–4 h after the start of infusion than the maximum concentration in plasma (C max ), and Halstenson et al. reported that C max and volume of distribution at steady state (V ss ) were not altered in the presence of renal insufficiency ( 21 ). Therefore, blood samples were collected slightly after C max to confirm the differences in plasma CMZ concentration between patients with normal and impaired renal function. Blood samples were separated into plasma and stored at − 60°C until measurement. Twenty microliters of 15% MeCN and 600 µL of IS in MeCN were spiked into 200 µL of patient plasma; the mixture was then prepared as previously described and measured using HPLC. Statistical analysis The relationship between plasma CMZ concentration and Ccr in patients with UTI was evaluated using Pearson’s product-moment correlation coefficient, with values of p < 0.05 considered statistically significant. Statistical analyses were performed using JMP® Pro (ver. 17.2.0, North Carolina, US). Results and Discussion Method validation Selectivity and linearity Figure 1 illustrates typical chromatograms of plasma samples containing CMZ and IS and blank plasma samples without CMZ and IS. CMZ and IS were detected at 9.9 and 7.9 min, respectively. No significant interfering peaks were observed in the CMZ and IS peak regions. The proposed method can be performed in approximately 15 min to measure one sample, equivalent to that observed in a previous report ( 17 ). Furthermore, the calibration curve exhibited good linearity in the plasma concentration range of 0.2–200 µg /mL (R 2 > 0.99) (Fig. 2 ). The range of the calibration curve in the proposed method (0.2–200 µg/mL in plasma concentration) was slightly wider than that observed in previous reports ( 17 , 18 , 28 ). The calibration range of CMZ in the proposed method was selected to measure various plasma concentrations expected in patients. The average C max of 1 g of CMZ intravenous administration for 1 h in healthy adults is approximately 76.2 µg/mL, and T 1/2 is approximately 1.1 h ( 13 ). Moreover, the serum CMZ concentration is higher in patients with renal dysfunction with Ccr ≤ 70 mL/min than in healthy adults, and T 1/2 is prolonged ( 13 ). In another previous report, T 1/2 in patients with Ccr of 10–39 mL/min was reported to be approximately 6 h ( 21 ). The C max of the fifth dose (steady state) was estimated to increase to approximately 130 µg/mL when the patients were given 1 g twice daily. Therefore, the upper limit of the calibration curve was set to 200 µg/mL. In contrast, the lower limit of the calibration curve was set to 0.2 µg/mL because it was estimated to be approximately 0.3 µg/mL approximately 8–9 h after a single dose of 1 g of CMZ to healthy adults. Furthermore, in the proposed method, the calibration curve was used separately for high (10–200 µg/mL) and low (0.2–10 µg/mL) concentrations based on an IS ratio of 10 µg/mL, enabling the calculation of a more accurate concentration. In this study, six of the 45 points of the post-peak level and two of the 14 points of the trough level were > 100 µg/mL and < 0.5 µg/mL, respectively. Although several methods for measuring CMZ concentrations using HPLC have been reported, they can measure up to 0.4–0.5 µg/mL ( 17 , 18 , 28 ). In contrast, the LLOQ of the proposed method is 0.2 µg/mL, and using a calibration curve separately for high and low concentrations enables quantification at lower concentrations than previously reported ( 17 , 18 , 28 ). Accuracy and precision Intra- and inter-day accuracy and precision of CMZ plasma samples met the criteria of the FDA guidelines. The RE was within ± 12%, and RSD was < 15% for all QC samples (Table 2). The RSD (1.2–14.1%) for each concentration in the intra- and inter-day reproducibility was lower than the FDA guidelines ( 25 ) acceptance criteria of 15%, suggesting that the proposed method has high reproducibility (Table 2). Recovery The recovery rates of CMZ in plasma were > 93.2% for all seven concentrations (Table 2), and small variations were observed among concentrations. Stability Table 3 presents the results of the stability analysis under various conditions of CMZ in plasma. The RE and RSD of CMZ in plasma were all within 15% for bench-top stability (24 h at room temperature), autosampler stability (up to 24 h after sample preparation), and short- and long-term stability (1 week or 2 months at − 30°C). After three freeze-thaw cycles, the RE and RSD were within 7%, indicating that freeze-thaw had no effects. In all stability tests, the accuracy and precision met the acceptance criteria of the FDA guidelines (accuracy: ≤ ±15%; precision: ≤15%) ( 25 ). Furthermore, the CMZ stock solution was stable at − 30°C for 3 months. Application of the developed method to clinical samples Patient characteristics Table 4 presents the patient characteristics. KO et al. demonstrated that CMZ is partially eliminated by renal tubule secretion, and its serum concentration can be increased by pre-administration of probenecid ( 29 ). However, in our study, none of the patients received probenecid. Although many concomitant medications were taken, none affected the CMZ measurements. Chromatograms of patient samples and the relationship between plasma CMZ levels and renal function Figure 3 presents representative chromatograms of patient samples. No significant interfering peaks were observed in the CMZ and IS peak regions, confirming that both CMZ and IS peaks were detected. Therefore, the proposed method can be considered useful for clinical studies. A negative correlation was observed between Ccr and post-peak level of plasma CMZ ( R = − 0.67, p < 0.0001) (Fig. 4 a). That is, plasma CMZ concentrations tended to be higher early after administration in patients with impaired renal function. These results were consistent with those of previous studies ( 13 , 21 , 22 ), indicating that renal function should be considered when determining the optimal dosage of CMZ. In contrast, no significant correlation was observed between Ccr and the trough level of plasma CMZ ( R = − 0.53, p = 0.093) (Fig. 4 b). This study is the first to report CMZ concentrations including both post-peak and trough levels, after repeated administration rather than after a single dose. These results suggest that even in patients with impaired renal function, little CMZ accumulation can be observed with appropriate dosage adjustment (prolonging the dosing interval) according to renal function. This finding is consistent with that of a previous study: the dosing interval should be increased to avoid excessive drug accumulation, and the dose of CMZ may not need to be adjusted according to renal function because the V ss of CMZ does not vary with renal function ( 21 ). This study has limitations. A few patient samples were used to evaluate the correlation between Ccr and plasma CMZ concentrations. Other factors such as low body weight were not examined, and the cause of the deviation from the correlation could not be determined. Accordingly, further studies with large sample sizes are necessary. In the future, plasma CMZ concentration data will be collected by analyzing patient samples, and a population pharmacokinetic (PPK) model will be developed. This model will enable the simulation of plasma concentrations and the prediction of therapeutic efficacy by integrating data such as minimum inhibitory concentration distribution and clinical outcomes. Ultimately, this approach will enable us to propose an optimal dosage of CMZ for patients with UTI. Conclusion In this study, we established an HPLC-UV method for quantifying plasma CMZ concentrations to facilitate clinical studies. The selectivity, reproducibility, and stability were confirmed through validation based on the FDA guidelines ( 25 ). The proposed method enables the quantification of various CMZ concentrations for clinical PPK/PD studies. Abbreviations Ccr: creatinine clearance C max : maximum concentration in plasma CMZ: cefmetazole V ss: distribution at steady state ESBL: extended-spectrum β-lactamase ESBL- E : extended-spectrum β-lactamase-producing Enterobacterales FDA: Food and Drug Administration HPLC: high-performance liquid chromatography HQC: high-quality control IS: internal standard LC-MS/MS: liquid chromatography-tandem mass spectrometry LLOQ: lower limit of quantification LQC: low-quality control MeCN: acetonitrile MQC: medium-quality control PK/PD: pharmacokinetic/pharmacodynamic PPK: population pharmacokinetic QC: quality control RE: relative error RSD: relative standard deviation T 1/2 : half-life UTI: urinary tract infection WHO: World Health Organization Declarations Ethics approval and consent to participate The study was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of Gifu Pharmaceutical University (no. 5-13) and the Ethics Review Committee of Gifu General Medical Center (no. 758-4). Before implementing the study, written informed consent was obtained from all participants or substitute decision-makers. Consent for publication Not applicable. Availability of data and materials The data supporting the findings of this study are available. Competing interests The authors declare that they have no conflicts of interest. Funding This study did not receive funding from any grants. All research funds were provided by Gifu Pharmaceutical University. Authors’ contribution YS, YO, MS, TH, KI, YT, and KK participated in the research design. YS, KN, YS, and MT performed experiments and analyzed data. YS collected the clinical data. YS, YO, MS, and KK performed statistical analyses. YS, YO, MS, TY, and KK drafted the manuscript, which was revised and approved by all authors. Acknowledgments Thank you for the English editing by professional human editors at Enago (ZYDCFJ-5). References Woerther PL, Burdet C, Chachaty E, Andremont A. 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Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.CMZJPHCS01.docx Table2.CMZJPHCS01.docx Table3.CMZJPHCS01.docx Table4.CMZJPHCS01.docx Cite Share Download PDF Status: Published Journal Publication published 27 Dec, 2025 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. 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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-6736302","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464044367,"identity":"b16f9c8b-a593-47f9-8a95-4cd161787edd","order_by":0,"name":"Yuna Sadaka","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yuna","middleName":"","lastName":"Sadaka","suffix":""},{"id":464044368,"identity":"bae50e03-5c44-435f-82d8-eaf93a2ba3d8","order_by":1,"name":"Kokoro Nakajima","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Kokoro","middleName":"","lastName":"Nakajima","suffix":""},{"id":464044369,"identity":"10c64225-809b-4183-9e28-bf4b01c570e0","order_by":2,"name":"Yuki Sasaki","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Sasaki","suffix":""},{"id":464044370,"identity":"f99a621e-b267-45a6-91bd-6f956b167491","order_by":3,"name":"Mao Tsurugaya","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Mao","middleName":"","lastName":"Tsurugaya","suffix":""},{"id":464044371,"identity":"a51e70fa-05c1-4bda-a08d-2aa5bfa4892f","order_by":4,"name":"Yasuhisa Oida","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yasuhisa","middleName":"","lastName":"Oida","suffix":""},{"id":464044372,"identity":"0813d1f7-e603-4108-a047-1d7e0685a0aa","order_by":5,"name":"Midori Soda","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Midori","middleName":"","lastName":"Soda","suffix":""},{"id":464044373,"identity":"9065690a-cf95-47dd-8dc9-75e0b9303072","order_by":6,"name":"Tomoyuki Hirashita","email":"","orcid":"","institution":"Department of Pharmacy, Gifu Prefectural General Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Hirashita","suffix":""},{"id":464044374,"identity":"7ee6756e-a4bb-43fe-8d1a-86073ca3a77e","order_by":7,"name":"Kotoe Inoue","email":"","orcid":"","institution":"Department of Pharmacy, Gifu Prefectural General Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Kotoe","middleName":"","lastName":"Inoue","suffix":""},{"id":464044375,"identity":"5b0c2987-8edf-4504-af4b-d6f093bc00a0","order_by":8,"name":"Yoshito Takahashi","email":"","orcid":"","institution":"Department of Urology, Gifu Prefectural General Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yoshito","middleName":"","lastName":"Takahashi","suffix":""},{"id":464044376,"identity":"a0a379b7-0fd5-47ec-abce-70c026901c74","order_by":9,"name":"Tsuyoshi Yokoi","email":"","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Tsuyoshi","middleName":"","lastName":"Yokoi","suffix":""},{"id":464044377,"identity":"350ce3dd-95c5-42b0-bfd3-eb1584c224d6","order_by":10,"name":"Kiyoyuki Kitaichi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3QsarCMBSA4RMCuuSSNUG9z1AoiE6+Sovg5CM4JAg6unZw8yUcUwK6CK4KHSyFTg66OYmJXvAuxo6C+YczhH6cpAA+3wfGsBkRKEIBidPz2Enwg3CBZFKNgF0DCgIFKHF8+IxPaXo6jLJmuF3IMYEsFnV9gO7yNWlgjFm0Kkl7l1tSxoIMAuCb1+TXvIXFQhuSToufq44FDM3yiZPgiyVhktothtCjm5iL1e5bAir/CHuzhY9xuxOtNGE7JOUcynDCykC53sK2abG/jHSPzta5OELWmtF+XnDHH/uHIzsV1MzUXFQhVD2IDZ0rEZ/P5/uSbt55UoLhULlOAAAAAElFTkSuQmCC","orcid":"","institution":"Laboratory of Pharmaceutics, Department of Biomedical Pharmaceutics, Gifu Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Kiyoyuki","middleName":"","lastName":"Kitaichi","suffix":""}],"badges":[],"createdAt":"2025-05-24 02:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6736302/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6736302/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40780-025-00535-1","type":"published","date":"2025-12-27T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83814252,"identity":"dac74f30-02ab-4ca0-b936-bc1891e94a89","added_by":"auto","created_at":"2025-06-03 07:24:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":424812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypical chromatograms of CMZ (200 μg/mL) and IS (40 μg/mL) in plasma and blank plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) CMZ-spiked plasma (272 nm), (b) IS-spiked plasma (229 nm), (c) blank plasma (272 nm), (d) blank plasma (229 nm).\u003c/p\u003e","description":"","filename":"Fig.1CMZJPHCS01.png","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/1c4d43310a049f778335488a.png"},{"id":83815878,"identity":"9212e4e8-16a8-441a-ae2a-d930ee612ac1","added_by":"auto","created_at":"2025-06-03 07:40:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStandard calibration curves of CMZ in plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Low concentration, (b) high concentration.\u003c/p\u003e","description":"","filename":"Fig.2CMZJPHCS01.png","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/b41692b0438d3b92f8f5a78d.png"},{"id":83816210,"identity":"6a78325e-689d-4f70-8295-3de133c95d33","added_by":"auto","created_at":"2025-06-03 07:48:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative chromatograms of the clinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) CMZ (272 nm), (b) IS (229 nm).\u003c/p\u003e","description":"","filename":"Fig.3CMZJPHCS01.png","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/6566d54053a098f41a398fb8.png"},{"id":83814269,"identity":"8052d58d-20bc-4860-b9c8-a2ac1fa5ee43","added_by":"auto","created_at":"2025-06-03 07:24:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":282648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between Ccr (mL/min) and CMZ concentration (μg/mL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Post-peak (\u003cem\u003eR\u003c/em\u003e = −0.67, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), (b) trough (\u003cem\u003eR\u003c/em\u003e = −0.53, \u003cem\u003ep\u003c/em\u003e = 0.093).\u003c/p\u003e","description":"","filename":"Fig.4CMZJPHCS01.png","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/9e6d4b1dc57a59becfe4b2fd.png"},{"id":99172510,"identity":"7ca287bd-5d21-4bb3-968e-f4fb758931a2","added_by":"auto","created_at":"2025-12-29 16:10:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2281117,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/97bd2fab-fc75-4597-8f10-a588c9986f5d.pdf"},{"id":83814254,"identity":"c13edc97-865c-421a-9017-ab05d8e97760","added_by":"auto","created_at":"2025-06-03 07:24:53","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17642,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.CMZJPHCS01.docx","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/e6d3e4b4e812c073f084b732.docx"},{"id":83814268,"identity":"30c55c77-cec8-412a-9c7f-e73c9d2a64c9","added_by":"auto","created_at":"2025-06-03 07:24:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18644,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.CMZJPHCS01.docx","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/f20da9ada5a63933dbfd62f3.docx"},{"id":83814869,"identity":"2e829ea6-27ef-449c-9e80-58a56a294265","added_by":"auto","created_at":"2025-06-03 07:32:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19136,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.CMZJPHCS01.docx","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/5beb560b88a95fde7513ae7a.docx"},{"id":83815877,"identity":"68c9bc9f-9df2-4091-9e7d-fc85496090de","added_by":"auto","created_at":"2025-06-03 07:40:53","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20199,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.CMZJPHCS01.docx","url":"https://assets-eu.researchsquare.com/files/rs-6736302/v1/7d7844b1f83a339d5fc588a8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of the quick method to measure plasma cefmetazole by high- performance liquid chromatography and its clinical application","fulltext":[{"header":"Background","content":"\u003cp\u003eExtended-spectrum β-lactamase (ESBL)-producing \u003cem\u003eEnterobacterales\u003c/em\u003e (ESBL-\u003cem\u003eE\u003c/em\u003e) are resistant to third-generation cephalosporin antibiotics. The incidence of infections caused by ESBL-\u003cem\u003eE\u003c/em\u003e has increased recently and is considered a global threat (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Selecting therapeutic agents for ESBL-\u003cem\u003eE\u003c/em\u003e in clinical practice is often challenging. Carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eEnterobacterales\u003c/em\u003e are categorized as \u0026ldquo;critical priority\u0026rdquo; in the World Health Organization (WHO) Bacterial Priority Pathogens List 2024 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Although carbapenems are frequently used to treat severe ESBL-\u003cem\u003eE\u003c/em\u003e infections, excessive carbapenem use is associated with increased carbapenem resistance (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCefmetazole (CMZ), a second-generation cephamycin antibiotic, possesses antibacterial efficacy against ESBL-producing bacteria (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Moreover, several reports have demonstrated the clinical effectiveness of CMZ against ESBL-\u003cem\u003eE\u003c/em\u003e infections (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Recently, CMZ has been widely used in Japan as a treatment for urinary tract infection (UTI), intra-abdominal infections, and bacteremia (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) and as an antimicrobial prophylaxis in patients undergoing lower gastrointestinal tract and uterine surgery for both ESBL-\u003cem\u003eE\u003c/em\u003e and non-ESBL-\u003cem\u003eE\u003c/em\u003e infections (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In particular, considering the high urinary recovery rate of CMZ as an unchanged form (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), CMZ would be suitable to treat ESBL-\u003cem\u003eE-\u003c/em\u003einduced UTI, although appropriate dosage adjustment would be necessary depending on the renal function of individual patients.\u003c/p\u003e \u003cp\u003eThere is no description related to CMZ in the SANFORD GUIDE (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), Infectious Diseases Society of America 2023 Guidance (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), Up To Date (online database), and Global Guidelines for the Prevention of Surgical Site Infection (WHO) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), probably because of the lack of global evidence without prescribing CMZ in the USA and several EU countries. Thus, new evidence to prove the effectiveness and safety of CMZ, such as pharmacokinetic/pharmacodynamic (PK-PD) analyses, would be necessary to change this situation.\u003c/p\u003e \u003cp\u003eRegarding the measurement of CMZ, several methods using high-performance liquid chromatography (HPLC) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) have been reported. However, few methods can measure concentrations over a wide range at a faster rate. Furthermore, although plasma concentration data after a single dose of CMZ have been reported (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), only a few studies have examined the plasma concentration of CMZ after repeated administration, including peak and trough levels. Furthermore, no previous study has demonstrated whether dose adjustment according to renal function assessed based on plasma CMZ concentration is appropriate.\u003c/p\u003e \u003cp\u003eIn this study, we established a method for measuring plasma CMZ concentrations using HPLC and validated it based on the Food and Drug Administration (FDA) guidelines for conducting clinical trials (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). We then focused on the PK/PD study of CMZ in patients with UTI, where no PD target has been established (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Furthermore, we measured plasma CMZ concentration in patients with UTI treated with CMZ using this method and revealed the relationship between CMZ concentration and renal function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eCefmetazole sodium (purity 98%) was purchased from SIGMA-Aldrich Co., LLC. (St. Louis, USA). Barbital sodium (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%), used as the internal standard (IS), was purchased from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Distilled water and acetonitrile (MeCN) for HPLC grading were purchased from Hikari-Pharm. Co., Ltd. (Osaka, Japan) and FUJIFILM Wako Co., Ltd. (Tokyo, Japan), respectively. The human plasma used for the calibration curves and validation was purchased from Cosmo Bio., Co., Ltd. (Tokyo, Japan). The citric acid and trisodium citrate dihydrate used for mobile phase preparation were purchased from FUJIFILM Wako Co., Ltd., and Millex\u0026reg;-LG (0.20 \u0026micro;m, Merck, Darmstadt, Germany) was used for sample filtration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLiquid chromatography\u003c/h3\u003e\n\u003cp\u003eHPLC analysis was performed using a SHIMAZU LC-20AD system (Shimadzu Co., Ltd., Kyoto, Japan). Samples were separated using a COSMOSIL\u0026reg; 5C\u003csub\u003e18\u003c/sub\u003e-MS-II column (4.6 ID \u0026times; 250 mm, Nacalai Tesque Co., Ltd., Kyoto, Japan). The mobile phase consisted of 5-mM sodium citrate buffer (pH 3.2) and MeCN (85/15, v/v %). The flow rate was maintained at 1.2 mL/min, and the column temperature was set at 45\u0026deg;C. The total run time was 15 min. The injection volume was 50 \u0026micro;L, and the detection wavelengths were 272 (CMZ) and 229 (IS) nm, respectively.\u003c/p\u003e\n\u003ch3\u003ePreparation of stock solutions\u003c/h3\u003e\n\u003cp\u003eCMZ and IS were dissolved in 15% MeCN in water to 20 and 1 mg/mL, respectively, as stock solutions and then stored at \u0026minus;\u0026thinsp;30\u0026deg;C until analysis.\u003c/p\u003e\n\u003ch3\u003ePreparation of standard and quality control (QC) samples\u003c/h3\u003e\n\u003cp\u003eThe CMZ-stock solution was further diluted with 15% MeCN in water to obtain working solutions at several concentrations. The IS-stock solution was further diluted with MeCN at 40 \u0026micro;g/mL. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. The final concentrations of calibration standards were 0.2, 0.5, 1, 10, 50, 100, and 200 \u0026micro;g/mL in plasma. In this method, the calibration curves were prepared in two ranges: high (10\u0026ndash;200 \u0026micro;g/mL) and low (0.2\u0026ndash;10 \u0026micro;g/mL) concentrations. Therefore, three QC samples were used for each calibration curve. For the low-concentration calibration curves, the final concentrations of low-quality control (LQC), medium-QC (MQC), and high-QC (HQC) samples were set at 0.5, 5.0, and 10.0 \u0026micro;g/mL, respectively. For the high-concentration calibration curves, the final concentrations of LQC, MQC, and HQC were 20, 100, and 200 \u0026micro;g/mL, respectively.\u003c/p\u003e\n\u003ch3\u003ePreparation of samples to measure plasma CMZ\u003c/h3\u003e\n\u003cp\u003eStandard samples for the calibration curves were prepared using the following method. A 200-\u0026micro;L plasma sample was mixed with 20-\u0026micro;L CMZ working solutions and 600-\u0026micro;L IS in MeCN. The mixtures were centrifuged at 12,500 rpm for 10 min at 4\u0026deg;C. The supernatant was then completely dried under a nitrogen stream. The residue was eluted with 200 \u0026micro;L of the mobile phase and then filtered. Finally, 50 \u0026micro;L of the sample was injected into the HPLC system.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of plasma CMZ concentration\u003c/h2\u003e \u003cp\u003eCalibration curves were constructed by plotting the peak ratio (standard to internal standard) \u003cem\u003eversus\u003c/em\u003e the nominal concentration. Using the regression equation, the plasma CMZ concentration was calculated from the CMZ/IS ratio of each sample.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethod validation\u003c/h3\u003e\n\u003cp\u003eThe method was fully validated according to the FDA guidelines for biological method validation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Accuracy was considered acceptable when the relative error (RE) was within \u0026plusmn;\u0026thinsp;15%, and precision was considered acceptable at a relative standard deviation (RSD) of \u0026le;\u0026thinsp;15%, except lower limit of quantification (LLOQ) (RE\u0026thinsp;\u0026le;\u0026thinsp;\u0026plusmn;\u0026thinsp;20 and RSD\u0026thinsp;\u0026le;\u0026thinsp;20%).\u003c/p\u003e\n\u003ch3\u003eSelectivity\u003c/h3\u003e\n\u003cp\u003eIf plasma-derived components did not interfere with the peak regions of CMZ and IS, then selectivity was evaluated. Blank plasma without CMZ and IS and plasma containing 200 \u0026micro;g/mL (in plasma concentration) of CMZ were measured using the aforementioned method. Chromatograms were compared and validated.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntra-day accuracy and precision\u003c/h2\u003e \u003cp\u003eQC samples were prepared using the method described above, and the same QC samples were measured five times repeatedly within 1 day to assess reproducibility within each run; accuracy and precision were then calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInter-day accuracy and precision\u003c/h2\u003e \u003cp\u003eQC samples were measured once a day for 5 days, and the reproducibility between each analytical run was assessed by calculating the accuracy and precision.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRecovery\u003c/h2\u003e \u003cp\u003eAs with the QC samples, the peak area ratio of samples in which CMZ working solution was spiked in human plasma, and pretreatment (a) was then compared with the peak area ratio of samples treated with human plasma, and the working solution of CMZ was then spiked (b). The recovery rate was calculated using the following formula:\u003c/p\u003e \u003cp\u003eRecovery (%)=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{\\left(\\text{a}\\right)}{\\left(\\text{b}\\right)}\\)\u003c/span\u003e\u003c/span\u003e \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStability\u003c/h2\u003e \u003cp\u003eThe stability of CMZ was evaluated in QC samples at each of the three concentrations (HQC, MQC, and LLOQ) for high- and low-concentration calibration curves, respectively. HPLC measurements were performed in three runs under the following conditions:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFreeze\u0026ndash;thaw stability\u003c/h2\u003e \u003cp\u003eThe stability of the samples when repeatedly frozen and thawed, which can occur during sample storage, was assessed. QC samples of three concentrations of CMZ in plasma were frozen and stored at \u0026minus;\u0026thinsp;30\u0026deg;C for 24 h and then thawed at room temperature, for three cycles. The three freeze\u0026ndash;thaw samples were prepared using the aforementioned method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBench-top stability\u003c/h2\u003e \u003cp\u003eTo assess stability under conditions that may occur during sample preparation, QC samples (CMZ-containing plasma) were left at room temperature for 24 h and then prepared using the aforementioned method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eShort-term stability\u003c/h2\u003e \u003cp\u003eThe short-term stability of CMZ in plasma was assessed by analyzing CMZ-containing plasma after storage at \u0026minus;\u0026thinsp;30\u0026deg;C for 1 week according to the aforementioned method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLong-term stability\u003c/h2\u003e \u003cp\u003eThe long-term stability of CMZ was assessed by analyzing CMZ-containing plasma stored at \u0026minus;\u0026thinsp;30\u0026ordm;C for 2 months. CMZ stock solutions were assessed for stability under conditions that may occur during long-term sample storage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAutosampler stability\u003c/h2\u003e \u003cp\u003eQC plasma samples containing CMZ were prepared using the above-mentioned method and left in an autosampler (room temperature) for 24 h. The stability of the samples was then measured and assessed from the time of sample preparation until measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of plasma CMZ concentrations in patients with UTI\u003c/h2\u003e \u003cp\u003eTwenty-three patients with UTI treated with CMZ admitted to the Department of Urology at Gifu General Medical Center between October 2022 and January 2023 were included in this study. The exclusion criteria were as follows: (i) patients who did not provide informed consent, (ii) those who were younger than 15 years, (iii) those with urinary bacteria\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU/mL, (iv) those receiving renal replacement therapy (hemodialysis or peritoneal dialysis) or those with creatinine clearance (Ccr)\u0026thinsp;\u0026lt;\u0026thinsp;10 mL/min, (v) those who received CMZ for perioperative antimicrobial prophylaxis, and (vi) those deemed unfit for inclusion by physicians. All patients received 1 g of CMZ for 1 h according to their renal function (Table\u0026nbsp;1). This study was approved by the Ethics Committee of Gifu Pharmaceutical University (no. 5\u0026ndash;13) and the Ethics Review Committee of Gifu General Medical Center (no. 758-4).\u003c/p\u003e \u003cp\u003eBlood samples were collected 2\u0026ndash;3 h after infusion initiation (defined as the \u0026ldquo;post-peak level\u0026rdquo;) and 6\u0026ndash;24 h after the last dose (defined as the \u0026ldquo;trough level\u0026rdquo;). Of the trough samples, three that were collected significantly later than the scheduled interval (\u0026gt;\u0026thinsp;2 h after the scheduled infusion time) were excluded from statistical analysis. Although peak plasma concentrations should be obtained at the end of infusion (i.e., in this study, 1 h after infusion initiation), blood samples were collected 2\u0026ndash;3 h after infusion initiation. Ohkawa et al. reported a more significant difference in plasma concentration between patients with normal and impaired renal function (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) 2\u0026ndash;4 h after the start of infusion than the maximum concentration in plasma (C\u003csub\u003emax\u003c/sub\u003e), and Halstenson et al. reported that C\u003csub\u003emax\u003c/sub\u003e and volume of distribution at steady state (V\u003csub\u003ess\u003c/sub\u003e) were not altered in the presence of renal insufficiency (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Therefore, blood samples were collected slightly after C\u003csub\u003emax\u003c/sub\u003e to confirm the differences in plasma CMZ concentration between patients with normal and impaired renal function.\u003c/p\u003e \u003cp\u003eBlood samples were separated into plasma and stored at \u0026minus;\u0026thinsp;60\u0026deg;C until measurement. Twenty microliters of 15% MeCN and 600 \u0026micro;L of IS in MeCN were spiked into 200 \u0026micro;L of patient plasma; the mixture was then prepared as previously described and measured using HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe relationship between plasma CMZ concentration and Ccr in patients with UTI was evaluated using Pearson\u0026rsquo;s product-moment correlation coefficient, with values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Statistical analyses were performed using JMP\u0026reg; Pro (ver. 17.2.0, North Carolina, US).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003eSelectivity and linearity\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates typical chromatograms of plasma samples containing CMZ and IS and blank plasma samples without CMZ and IS. CMZ and IS were detected at 9.9 and 7.9 min, respectively. No significant interfering peaks were observed in the CMZ and IS peak regions. The proposed method can be performed in approximately 15 min to measure one sample, equivalent to that observed in a previous report (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Furthermore, the calibration curve exhibited good linearity in the plasma concentration range of 0.2\u0026ndash;200 \u0026micro;g /mL (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The range of the calibration curve in the proposed method (0.2\u0026ndash;200 \u0026micro;g/mL in plasma concentration) was slightly wider than that observed in previous reports (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The calibration range of CMZ in the proposed method was selected to measure various plasma concentrations expected in patients. The average C\u003csub\u003emax\u003c/sub\u003e of 1 g of CMZ intravenous administration for 1 h in healthy adults is approximately 76.2 \u0026micro;g/mL, and T\u003csub\u003e1/2\u003c/sub\u003e is approximately 1.1 h (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Moreover, the serum CMZ concentration is higher in patients with renal dysfunction with Ccr\u0026thinsp;\u0026le;\u0026thinsp;70 mL/min than in healthy adults, and T\u003csub\u003e1/2\u003c/sub\u003e is prolonged (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In another previous report, T\u003csub\u003e1/2\u003c/sub\u003e in patients with Ccr of 10\u0026ndash;39 mL/min was reported to be approximately 6 h (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The C\u003csub\u003emax\u003c/sub\u003e of the fifth dose (steady state) was estimated to increase to approximately 130 \u0026micro;g/mL when the patients were given 1 g twice daily. Therefore, the upper limit of the calibration curve was set to 200 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eIn contrast, the lower limit of the calibration curve was set to 0.2 \u0026micro;g/mL because it was estimated to be approximately 0.3 \u0026micro;g/mL approximately 8\u0026ndash;9 h after a single dose of 1 g of CMZ to healthy adults. Furthermore, in the proposed method, the calibration curve was used separately for high (10\u0026ndash;200 \u0026micro;g/mL) and low (0.2\u0026ndash;10 \u0026micro;g/mL) concentrations based on an IS ratio of 10 \u0026micro;g/mL, enabling the calculation of a more accurate concentration. In this study, six of the 45 points of the post-peak level and two of the 14 points of the trough level were \u0026gt;\u0026thinsp;100 \u0026micro;g/mL and \u0026lt;\u0026thinsp;0.5 \u0026micro;g/mL, respectively. Although several methods for measuring CMZ concentrations using HPLC have been reported, they can measure up to 0.4\u0026ndash;0.5 \u0026micro;g/mL (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In contrast, the LLOQ of the proposed method is 0.2 \u0026micro;g/mL, and using a calibration curve separately for high and low concentrations enables quantification at lower concentrations than previously reported (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAccuracy and precision\u003c/h2\u003e \u003cp\u003e Intra- and inter-day accuracy and precision of CMZ plasma samples met the criteria of the FDA guidelines. The RE was within \u0026plusmn;\u0026thinsp;12%, and RSD was \u0026lt;\u0026thinsp;15% for all QC samples (Table\u0026nbsp;2). The RSD (1.2\u0026ndash;14.1%) for each concentration in the intra- and inter-day reproducibility was lower than the FDA guidelines (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) acceptance criteria of 15%, suggesting that the proposed method has high reproducibility (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eRecovery\u003c/h2\u003e \u003cp\u003eThe recovery rates of CMZ in plasma were \u0026gt;\u0026thinsp;93.2% for all seven concentrations (Table\u0026nbsp;2), and small variations were observed among concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eStability\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;3 presents the results of the stability analysis under various conditions of CMZ in plasma. The RE and RSD of CMZ in plasma were all within 15% for bench-top stability (24 h at room temperature), autosampler stability (up to 24 h after sample preparation), and short- and long-term stability (1 week or 2 months at \u0026minus;\u0026thinsp;30\u0026deg;C). After three freeze-thaw cycles, the RE and RSD were within 7%, indicating that freeze-thaw had no effects. In all stability tests, the accuracy and precision met the acceptance criteria of the FDA guidelines (accuracy: \u0026le; \u0026plusmn;15%; precision: \u0026le;15%) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Furthermore, the CMZ stock solution was stable at \u0026minus;\u0026thinsp;30\u0026deg;C for 3 months.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eApplication of the developed method to clinical samples\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003ePatient characteristics\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;4 presents the patient characteristics. KO et al. demonstrated that CMZ is partially eliminated by renal tubule secretion, and its serum concentration can be increased by pre-administration of probenecid (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, in our study, none of the patients received probenecid. Although many concomitant medications were taken, none affected the CMZ measurements.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eChromatograms of patient samples and the relationship between plasma CMZ levels and renal function\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents representative chromatograms of patient samples. No significant interfering peaks were observed in the CMZ and IS peak regions, confirming that both CMZ and IS peaks were detected. Therefore, the proposed method can be considered useful for clinical studies.\u003c/p\u003e \u003cp\u003eA negative correlation was observed between Ccr and post-peak level of plasma CMZ (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.67, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). That is, plasma CMZ concentrations tended to be higher early after administration in patients with impaired renal function. These results were consistent with those of previous studies (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), indicating that renal function should be considered when determining the optimal dosage of CMZ. In contrast, no significant correlation was observed between Ccr and the trough level of plasma CMZ (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.53, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.093) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This study is the first to report CMZ concentrations including both post-peak and trough levels, after repeated administration rather than after a single dose. These results suggest that even in patients with impaired renal function, little CMZ accumulation can be observed with appropriate dosage adjustment (prolonging the dosing interval) according to renal function. This finding is consistent with that of a previous study: the dosing interval should be increased to avoid excessive drug accumulation, and the dose of CMZ may not need to be adjusted according to renal function because the V\u003csub\u003ess\u003c/sub\u003e of CMZ does not vary with renal function (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has limitations. A few patient samples were used to evaluate the correlation between Ccr and plasma CMZ concentrations. Other factors such as low body weight were not examined, and the cause of the deviation from the correlation could not be determined. Accordingly, further studies with large sample sizes are necessary.\u003c/p\u003e \u003cp\u003eIn the future, plasma CMZ concentration data will be collected by analyzing patient samples, and a population pharmacokinetic (PPK) model will be developed. This model will enable the simulation of plasma concentrations and the prediction of therapeutic efficacy by integrating data such as minimum inhibitory concentration distribution and clinical outcomes. Ultimately, this approach will enable us to propose an optimal dosage of CMZ for patients with UTI.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we established an HPLC-UV method for quantifying plasma CMZ concentrations to facilitate clinical studies. The selectivity, reproducibility, and stability were confirmed through validation based on the FDA guidelines (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The proposed method enables the quantification of various CMZ concentrations for clinical PPK/PD studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCcr: creatinine clearance\u003c/p\u003e\n\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e: maximum concentration in plasma\u003c/p\u003e\n\u003cp\u003eCMZ: cefmetazole\u003c/p\u003e\n\u003cp\u003eV\u003csub\u003ess:\u0026nbsp;\u003c/sub\u003edistribution at steady state\u003c/p\u003e\n\u003cp\u003eESBL:\u0026nbsp;extended-spectrum β-lactamase\u003c/p\u003e\n\u003cp\u003eESBL-\u003cem\u003eE\u003c/em\u003e:\u0026nbsp;extended-spectrum β-lactamase-producing \u003cem\u003eEnterobacterales\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFDA: Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eHPLC: high-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eHQC: high-quality control\u003c/p\u003e\n\u003cp\u003eIS: internal standard\u003c/p\u003e\n\u003cp\u003eLC-MS/MS: liquid chromatography-tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eLLOQ: lower limit of quantification\u003c/p\u003e\n\u003cp\u003eLQC: low-quality control\u003c/p\u003e\n\u003cp\u003eMeCN: acetonitrile\u003c/p\u003e\n\u003cp\u003eMQC: medium-quality control\u003c/p\u003e\n\u003cp\u003ePK/PD: pharmacokinetic/pharmacodynamic\u003c/p\u003e\n\u003cp\u003ePPK: population pharmacokinetic\u003c/p\u003e\n\u003cp\u003eQC: quality control\u003c/p\u003e\n\u003cp\u003eRE: relative error\u003c/p\u003e\n\u003cp\u003eRSD: relative standard deviation\u003c/p\u003e\n\u003cp\u003eT\u003csub\u003e1/2\u003c/sub\u003e: half-life\u003c/p\u003e\n\u003cp\u003eUTI: urinary tract infection\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of Gifu Pharmaceutical University (no. 5-13) and the Ethics Review Committee of Gifu General Medical Center (no. 758-4). Before implementing the study, written informed consent was obtained from all participants or substitute decision-makers.\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 materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not receive funding from any grants. All research funds were provided by Gifu Pharmaceutical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYS, YO, MS, TH, KI, YT, and KK participated in the research design. YS, KN, YS, and MT performed experiments and analyzed data. YS collected the clinical data. YS, YO, MS, and KK performed statistical analyses. YS, YO, MS, TY, and KK drafted the manuscript, which was revised and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank you for the English editing by professional human editors at Enago\u0026nbsp;(ZYDCFJ-5).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWoerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum β-lactamases in the community: toward the globalization of CTX-M. 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May 2018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry\u003c/span\u003e\u003cspan address=\"https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamada Y, Matsumura Y, Nagashima M, Akazawa T, Doi Y, Hayakawa K. Retrospective evaluation of appropriate dosing of cefmetazole for invasive urinary tract infection due to extended-spectrum β-lactamase-producing Escherichia coli. 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J Pharm Health Care Sci. 2017;3:1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s40780-016-0071-6\u003c/span\u003e\u003cspan address=\"10.1186/s40780-016-0071-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo H, Cathcart KS, Griffith DL, Peters GR, Adams WJ. Pharmacokinetics of intravenously administered cefmetazole and cefoxitin and effects of probenecid on cefmetazole elimination. Antimicrob Agents Chemother. 1989;33:356\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.33.3.356\u003c/span\u003e\u003cspan address=\"10.1128/aac.33.3.356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 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":"cefmetazole, HPLC, urinary tract infection, plasma concentration, PK/PD analysis, clinical trials","lastPublishedDoi":"10.21203/rs.3.rs-6736302/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6736302/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCefmetazole (CMZ) is widely used in Japan as a treatment for urinary tract infections (UTI), intra-abdominal infections, and bacteremia and as an antimicrobial prophylaxis. For the safe use of CMZ, evidence related to pharmacokinetic/pharmacodynamic analyses of CMZ in UTI is necessary. Thus, we attempted to establish a method to quantify CMZ plasma concentration using high-performance liquid chromatography (HPLC) and to verify its clinical application using plasma samples collected from Japanese patients with UTI.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePlasma samples were deproteinized by adding acetonitrile (MeCN) containing an internal standard substance (IS), barbital sodium. After centrifugation, the supernatant was collected and evaporated to dryness under a stream of nitrogen gas. The residue was reconstituted with the mobile phase and injected into the HPLC system equipped with a COSMOSIL\u0026reg; 5C\u003csub\u003e18\u003c/sub\u003e-MS-II column. In the mobile phase, 5-mM sodium citrate buffer (pH 3.2)/MeCN (85/15, v/v %) was added at an isocratic flow rate of 1.2 mL/min. CMZ and IS were detected at 272 and 229 nm, respectively, and the total run time was 15 min. The method\u0026rsquo;s application in clinical samples was evaluated by measuring plasma samples from 23 patients with UTI treated with CMZ. Samples were collected 2\u0026ndash;3 h after the initiation of infusion (post-peak level) and 6\u0026ndash;24 h after the last dose (trough level). The relationship between plasma CMZ concentrations and creatinine clearance (Ccr) was evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe established method was found to have good linearity (0.2\u0026ndash;200 \u0026micro;g/mL), precision, and accuracy. Furthermore, our method was applicable to the measurement of plasma in patients with UTI. These results suggest that the established method is suitable for measuring plasma CMZ levels for research to promote the proper use of CMZ. Furthermore, a negative correlation was observed between Ccr and the post-peak level, but not trough levels, of plasma CMZ.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur developed method is simple and precise for quantifying plasma CMZ levels and is applicable in clinical settings.\u003c/p\u003e","manuscriptTitle":"Establishment of the quick method to measure plasma cefmetazole by high- performance liquid chromatography and its clinical application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 07:24:48","doi":"10.21203/rs.3.rs-6736302/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":"6d469833-e32c-4194-adc1-7a0d97aaa45c","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:06:46+00:00","versionOfRecord":{"articleIdentity":"rs-6736302","link":"https://doi.org/10.1186/s40780-025-00535-1","journal":{"identity":"journal-of-pharmaceutical-health-care-and-sciences","isVorOnly":false,"title":"Journal of Pharmaceutical Health Care and Sciences"},"publishedOn":"2025-12-27 15:57:20","publishedOnDateReadable":"December 27th, 2025"},"versionCreatedAt":"2025-06-03 07:24:48","video":"","vorDoi":"10.1186/s40780-025-00535-1","vorDoiUrl":"https://doi.org/10.1186/s40780-025-00535-1","workflowStages":[]},"version":"v1","identity":"rs-6736302","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6736302","identity":"rs-6736302","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}