Development of an Ephedrine In-House Matrix Reference Material and Its Application to Doping Analysis.

Biomatrix-based reference materials (RMs) improve the quality of laboratory test results by better representing actual samples. However, a matrix RM of ephedrine (EP) for threshold substances that require accurate analysis results has not yet been developed. Therefore, this study aimed to develop an in-house matrix RM for EP and subsequently apply it to analytical procedures. During the development of the in-house matrix EP RM, the system underwent homogeneity and stability studies. Additionally, it was subjected to interlaboratory comparison study in 11 laboratories, including 10 World Anti-Doping Agency (WADA)-accredited laboratories and our laboratory. Stability testing revealed no significant changes in the RM characteristics. For homogeneity, 10 random batches out of 200 were analyzed to confirm the uniformity within and between bottles. These results, combined with data from 11 laboratories, ensured retroactive validation. The traceability value of the in-house matrix EP RM was assigned as 9.83 ± 0.57 µg/mL (k = 2) by interlaboratory comparison studies and traceable uncertain evaluation. The feasibility of this method as a single calibration standard was confirmed in two laboratories. This substance is reliable and consistent for quality control during EP quantification, ensuring accurate and trustworthy outcomes. Consequently, this study establishes a framework and guidelines for producing in-house matrix RMs and serves as a reference for generating similar matrix RMs in other contexts.

Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests.

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.

Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development.

Lipids are indispensable for energy storage, membrane structure and cell signalling. However, dynamic changes in various categories of endogenous lipids in mammalian early embryonic development have not been systematically characterized. Here we comprehensively investigated the dynamic lipid landscape during mouse and human early embryo development. Lipid signatures of different developmental stages are distinct, particularly for the phospholipid classes. We highlight that the high degree of phospholipid unsaturation is a conserved feature as embryos develop to the blastocyst stage. Moreover, we show that lipid desaturases such as SCD1 are required for in vitro blastocyst development and blastocyst implantation. One of the mechanisms is through the regulation of unsaturated fatty-acid-mediated fluidity of the plasma membrane and apical proteins and the establishment of apical-basal polarity during development of the eight-cell embryo to the blastocyst. Overall, our study provides an invaluable resource about the remodelling of the endogenous lipidome in mammalian preimplantation embryo development and mechanistic insights into the regulation of embryogenesis and implantation by lipid unsaturation.

Impaired Capillary Recruitment Capacity in Obesity: A Subgroup Analysis of Prospective Observational Study on Anesthesia Effects.

BACKGROUND This subgroup analysis of prospective observational research, involving 71 participants, compared the effects of anesthesia on microvascular reactivity in obese vs lean individuals using near-infrared spectroscopy and vascular occlusion tests. The correlation between the body mass index (BMI) and microvascular reactivity under general anesthesia was also investigated. MATERIAL AND METHODS This study enrolled adult patients classified as American Society of Anesthesiologists physical status I or II, undergoing elective surgery under general anesthesia. The microcirculatory variables measured before (Tpre) and 30 min following the induction of anesthesia (Tpost) were as follows: baseline tissue oxygen saturation (StO2), occlusion slope (∇occl), and recovery slope (∇recov). The patients were grouped according to their BMI (lean [BMI <25 kg/m²] vs obese [BMI ≥25 kg/m²]). Data are presented as medians and interquartile ranges. RESULTS There were 43 patients in the lean group and 28 in the obese group. At Tpre, baseline StO2, ∇occl, and ∇recov were not different between the 2 groups (P=0.860, 0.659, and 0.518, respectively). At Tpost, the baseline StO2 and ∇occl were not different between the 2 groups (P=0.343 and 0.791); however, the ∇recov was lower in the obese group than in the lean group (3.245 [2.737, 3.977] vs 4.131 [3.491, 4.843], P=0.003). At Tpost, BMI showed a moderate correlation with ∇recov (correlation coefficient: -0.319, P=0.007). CONCLUSIONS In obese patients, capillary recruitment capacity during general anesthesia is compromised compared to lean patients.

Sedation with propofol and isoflurane differs in terms of microcirculatory parameters: A randomized animal study using dorsal skinfold chamber mouse model.

OBJECTIVE: This study aimed to explore the effects of sedative doses of propofol and isoflurane on microcirculation in septic mice compared to controls. Isoflurane, known for its potential as a sedation drug in bedside applications, lacks clarity regarding its impact on the microcirculation system. The hypothesis was that propofol would exert a more pronounced influence on the microvascular flow index, particularly amplified in septic conditions. MATERIAL AND METHODS: Randomized study was conducted from December 2020 to October 2021 involved 60 BALB/c mice, with 52 mice analyzed. Dorsal skinfold chambers were implanted, followed by intraperitoneal injections of either sterile 0.9 % saline or lipopolysaccharide for the control and sepsis groups, respectively. Both groups received propofol or isoflurane treatment for 120 min. Microcirculatory parameters were obtained via incident dark-field microscopy videos, along with the mean blood pressure and heart rate at three time points: before sedation (T0), 30 min after sedation (T30), and 120 min after sedation (T120). Endothelial glycocalyx thickness and syndecan-1 concentration were also analyzed. RESULTS: In healthy controls, both anesthetics reduced blood pressure. However, propofol maintained microvascular flow, differing significantly from isoflurane at T120 (propofol, 2.8 ± 0.3 vs. isoflurane, 1.6 ± 0.9; P < 0.001). In the sepsis group, a similar pattern occurred at T120 without statistical significance (propofol, 1.8 ± 1.1 vs. isoflurane, 1.2 ± 0.7; P = 0.023). Syndecan-1 levels did not differ between agents, but glycocalyx thickness index was significantly lower in the isoflurane-sepsis group than propofol (P = 0.001). CONCLUSIONS: Propofol potentially offers protective action against microvascular flow deterioration compared to isoflurane, observed in control mice. Furthermore, a lower degree of sepsis-induced glycocalyx degradation was evident with propofol compared to isoflurane.

Effect of the Interaction between Transition Metal Redox Center and Cyanide Ligand on Structural Evolution in Prussian White Cathodes.

Transition metal (TM) based Prussian whites, comprising a cyanide anion ((C≡N)-) and TM cations in an alternative manner, have been widely adopted as cathode materials for rechargeable batteries. Prussian whites are characterized by the TM electronic states that exclusively adopt low spin (LS) toward the C atom and high spin (HS) toward the N atom through the hybridized covalent bonding in the TM─C≡N─TM unit with the average oxidation states of the TM ions being 2+, considerably affecting the phase transition behavior upon the release and storage of carrier ions; however, there have been only a few studies on their associated features. Herein, Prussian whites with different HS TM ions were synthesized via coprecipitation and the phase transition behavior controlled by the π electron interaction between the cyanide anions and TM ions during battery operations was investigated. In situ X-ray characterizations reveal that the combined effect of π backdonation in the LS Fe-C unit and π donation in the HS TM-N unit effectively controls the bond length of the TM─C≡N─TM building unit, thus markedly influencing the lattice volume of a series of Prussian white cathodes during the charge/discharge process. This study presents a comprehensive understanding of the structure-property relationship of the Prussian white cathodes involving π electron interactions during battery operations.

Application of Skyline software for detecting prohibited substances in doping control analysis.

As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.

Cost-effectiveness of the anti-vascular endothelial growth factor intravitreal injection and panretinal photocoagulation for patients with proliferative diabetic retinopathy in South Korea.

BACKGROUND: We determined the cost-effectiveness of the anti-vascular endothelial growth factor (VEGF) intravitreal injection versus panretinal photocoagulation (PRP) for patients with proliferative diabetic retinopathy (PDR) in South Korea. METHODS: We simulated four treatment strategies using PRP and the anti-VEGF injection by constructing a Markov model for a hypothetical cohort of 50-year-old PDR patients: (1) PRP only; (2) anti-VEGF injection only; (3) PRP first; and (4) anti-VEGF injection first. RESULTS: In this cost-effectiveness analysis, compared with only-PRP, the incremental cost-effectiveness ratio was $95,456 per quality-adjusted life-year (QALY) for PRP first, $34,375 per QALY for anti-VEGF injection first, and $33,405 per QALY for anti-VEGF injection only from a healthcare perspective. From the societal and payer perspective, strategy (2) was more cost-saving and effective than (1). In the probabilistic sensitivity analysis, only-PRP was cost-effective up to the willingness-to-pay (WTP) of about $42,000, while anti-VEGF injection only was cost-effective from a healthcare perspective. From the societal and payer perspectives, regardless of the value of WTP, anti-VEGF injection only was the most cost-effective strategy. CONCLUSION: In our study, the anti-VEGF injection for PDR was cost-effective from the payer and societal perspectives.

Rapid azoospermia classification by stimulated Raman scattering and second harmonic generation microscopy.

Disease diagnosis and classification pose significant challenges due to the limited capabilities of traditional methods to obtain molecular information with spatial distribution. Optical imaging techniques, utilizing (auto)fluorescence and nonlinear optical signals, introduce new dimensions for biomarkers exploration that can improve diagnosis and classification. Nevertheless, these signals often cover only a limited number of species, impeding a comprehensive assessment of the tissue microenvironment, which is crucial for effective disease diagnosis and therapy. To address this challenge, we developed a multimodal platform, termed stimulated Raman scattering and second harmonic generation microscopy (SRASH), capable of simultaneously providing both chemical bonds and structural information of tissues. Applying SRASH imaging to azoospermia patient samples, we successfully identified lipids, protein, and collagen contrasts, unveiling molecular and structural signatures for non-obstructive azoospermia. This achievement is facilitated by LiteBlendNet-Dx (LBNet-Dx), our diagnostic algorithm, which achieved an outstanding 100% sample-level accuracy in classifying azoospermia, surpassing conventional imaging modalities. As a label-free technique, SRASH imaging eliminates the requirement for sample pre-treatment, demonstrating great potential for clinical translation and enabling molecular imaging-based diagnosis and therapy.

Glial cell proteome using targeted quantitative methods for potential multi-diagnostic biomarkers.

Glioblastoma is one of the most malignant primary brain cancer. Despite surgical resection with modern technology followed by chemo-radiation therapy with temozolomide, resistance to the treatment and recurrence is common due to its aggressive and infiltrating nature of the tumor with high proliferation index. The median survival time of the patients with glioblastomas is less than 15 months. Till now there has been no report of molecular target specific for glioblastomas. Early diagnosis and development of molecular target specific for glioblastomas are essential for longer survival of the patients with glioblastomas. Development of biomarkers specific for glioblastomas is most important for early diagnosis, estimation of the prognosis, and molecular target therapy of glioblastomas. To that end, in this study, we have conducted a comprehensive proteome study using primary cells and tissues from patients with glioblastoma. In the discovery stage, we have identified 7429 glioblastoma-specific proteins, where 476 proteins were quantitated using Tandem Mass Tag (TMT) method; 228 and 248 proteins showed up and down-regulated pattern, respectively. In the validation stage (20 selected target proteins), we developed quantitative targeted method (MRM: Multiple reaction monitoring) using stable isotope standards (SIS) peptide. In this study, five proteins (CCT3, PCMT1, TKT, TOMM34, UBA1) showed the significantly different protein levels (t-test: p value ≤ 0.05, AUC ≥ 0.7) between control and cancer groups and the result of multiplex assay using logistic regression showed the 5-marker panel showed better sensitivity (0.80 and 0.90), specificity (0.92 and 1.00), error rate (10 and 2%), and AUC value (0.94 and 0.98) than the best single marker (TOMM34) in primary cells and tissues, respectively. Although we acknowledge that the model requires further validation in a large sample size, the 5 protein marker panel can be used as baseline data for the discovery of novel biomarkers of the glioblastoma.

Untargeted Metabolomics Analysis Reveals Toxicity Based on the Sex and Sexual Maturity of Single Low-Dose DEHP Exposure.

Di-(2-Ethylhexyl) phthalate (DEHP) is a prevalent environmental endocrine disruptor that affects homeostasis, reproduction, and developmental processes. The effects of DEHP have been shown to differ based on sex and sexual maturity. This study examines the metabolic profiles of mature adult rats from both sexes, aged 10 weeks, and adolescent female rats, aged 6 weeks, following a single 5 mg/kg of body weight DEHP oral administration. An untargeted metabolomic analysis was conducted on urine samples collected at multiple times to discern potential sex- and maturity-specific DEHP toxicities. Various multivariate statistical analyses were employed to identify the relevant metabolites. The findings revealed disruptions to the steroid hormone and primary bile acid biosynthesis. Notably, DEHP exposure increased hyocholic, muricholic, and ketodeoxycholic acids in male rats. Moreover, DEHP exposure was linked to heart, liver, and kidney damage, as indicated by increased plasma GOT1 levels when compared to the levels before DEHP exposure. This study provides detailed insights into the unique mechanisms triggered by DEHP exposure concerning sex and sexual maturity, emphasizing significant distinctions in lipid metabolic profiles across the different groups. This study results deepens our understanding of the health risks linked to DEHP, informing future risk assessments and policy decisions.

Effects of sevoflurane on metalloproteinase and natural killer group 2, member D (NKG2D) ligand expression and natural killer cell-mediated cytotoxicity in breast cancer: an in vitro study.

BACKGROUND: We investigated the effects of sevoflurane exposure on the expression of matrix metalloproteinase (MMP), expression and ablation of natural killer group 2, member D (NKG2D) ligands (UL16-binding proteins 1-3 and major histocompatibility complex class I chain-related molecules A/B), and natural killer (NK) cell-mediated cytotoxicity in breast cancer cells. METHODS: Three human breast cancer cell lines (MCF-7, MDA-MB-453, and HCC-70) were incubated with 0 (control), 600 (S6), or 1200 µM (S12) sevoflurane for 4 h. The gene expression of NKG2D ligands and their protein expression on cancer cell surfaces were measured using multiplex polymerase chain reaction (PCR) and flow cytometry, respectively. Protein expression of MMP-1 and -2 and the concentration of soluble NKG2D ligands were analyzed using western blotting and enzyme-linked immunosorbent assays, respectively. RESULTS: Sevoflurane downregulated the mRNA and protein expression of the NKG2D ligand in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells but did not affect the expression of MMP-1 or -2 or the concentration of soluble NKG2D ligands in the MCF-7, MDA-MB-453, and HCC-70 cells. Sevoflurane attenuated NK cell-mediated cancer cell lysis in a dose-dependent manner in MCF-7, MDA-MB-453, and HCC-70 cells (P = 0.040, P = 0.040, and P = 0.040, respectively). CONCLUSIONS: Our results demonstrate that sevoflurane exposure attenuates NK cell-mediated cytotoxicity in breast cancer cells in a dose-dependent manner. This could be attributed to a sevoflurane-induced decrease in the transcription of NKG2D ligands rather than sevoflurane-induced changes in MMP expression and their proteolytic activity.

Perioperative considerations of pyruvate dehydrogenase complex deficiency: a case report of two consecutive anesthesia.

BACKGROUND: Pyruvate dehydrogenase complex (PDHC) deficiency is a rare mitochondrial disorder caused by a genetic mutation affecting the activity of the PDHC enzyme, which plays a major role in the tricarboxylic cycle. Few cases of surgery or anesthesia have been reported. Moreover, there is no recommended anesthetic method. CASE: A 24-month-old child with a PDHC deficiency presented to the emergency room with respiratory failure, mental decline, systemic cyanosis, and lactic acidosis. During hospitalization period, the patient presented with pneumothorax, pneumoperitoneum, and multiple air pockets in the heart. Two surgeries were performed under general anesthesia using an inhalational anesthetic agent. The patient was discharged with home ventilation. CONCLUSIONS: Anesthesiologists should be wary of multiple factors when administering anesthesia to patients with PDHC deficiency, including airway abnormalities, acid-base imbalance, intraoperative fluid management, selection of appropriate anesthetics, and monitoring of lactic acid levels.

Two Years of Experience and Methodology of Korean COVID-19 Living Clinical Practice Guideline Development.

BACKGROUND: In Korea, during the early phase of the coronavirus disease 2019 (COVID-19) pandemic, we responded to the uncertainty of treatments under various conditions, consistently playing catch up with the speed of evidence updates. Therefore, there was high demand for national-level evidence-based clinical practice guidelines for clinicians in a timely manner. We developed evidence-based and updated living recommendations for clinicians through a transparent development process and multidisciplinary expert collaboration. METHODS: The National Evidence-based Healthcare Collaborating Agency (NECA) and the Korean Academy of Medical Sciences (KAMS) collaborated to develop trustworthy Korean living guidelines. The NECA-supported methodological sections and 8 professional medical societies of the KAMS worked with clinical experts, and 31 clinicians were involved annually. We developed a total of 35 clinical questions, including medications, respiratory/critical care, pediatric care, emergency care, diagnostic tests, and radiological examinations. RESULTS: An evidence-based search for treatments began in March 2021 and monthly updates were performed. It was expanded to other areas, and the search interval was organized by a steering committee owing to priority changes. Evidence synthesis and recommendation review was performed by researchers, and living recommendations were updated within 3-4 months. CONCLUSION: We provided timely recommendations on living schemes and disseminated them to the public, policymakers and various stakeholders using webpages and social media. Although the output was successful, there were some limitations. The rigor of development issues, urgent timelines for public dissemination, education for new developers, and spread of several new COVID-19 variants have worked as barriers. Therefore, we must prepare systematic processes and funding for future pandemics.

Comparison of safety and efficacy between therapeutic or intermediate versus prophylactic anticoagulation for thrombosis in COVID-19 patients: a systematic review and meta-analysis.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) infections often have macrovascular or microvascular thrombosis and inflammation, which are known to be associated with a poor prognosis. Heparin has been hypothesized that administration of heparin with treatment dose rather than prophylactic dose for prevention of deep vein thrombosis in COVID-19 patients. METHODS: Studies comparing therapeutic or intermediate anticoagulation with prophylactic anticoagulation in COVID-19 patients were eligible. Mortality, thromboembolic events, and bleeding were the primary outcomes. PubMed, Embase, the Cochrane Library, and KMbase were searched up to July 2021. A meta-analysis was performed using random-effect model. Subgroup analysis was conducted according to disease severity. RESULTS: Six randomized controlled trials (RCTs) with 4,678 patients and four cohort studies with 1,080 patients were included in this review. In the RCTs, the therapeutic or intermediate anticoagulation was associated with significant reductions in the occurrence of thromboembolic events (5 studies, n=4,664; relative risk [RR], 0.72; P=0.01), and a significant increase in bleeding events (5 studies, n=4,667; RR, 1.88; P=0.004). In the moderate patients, therapeutic or intermediate anticoagulation was more beneficial than prophylactic anticoagulation in terms of thromboembolic events, but showed significantly higher bleeding events. In the severe patients, the incidence of thromboembolic and bleeding events in the therapeutic or intermediate. CONCLUSIONS: The study findings suggest that prophylactic anticoagulant treatment should be used in patients with moderate and severe COVID-19 infection groups. Further studies are needed to determine more individualized anticoagulation guidance for all COVID-19 patients.

Acetate-Mediated Odorant Receptor OR51E2 Activation Results in Calcitonin Secretion in Parafollicular C-Cells: A Novel Diagnostic Target of Human Medullary Thyroid Cancer.

Medullary thyroid cancer originates from parafollicular C-cells in the thyroid. Despite successful thyroidectomy, localizing remnant cancer cells in patients with elevated calcitonin and carcinoembryonic antigen levels remains a challenge. Extranasal odorant receptors are expressed in cells from non-olfactory tissues, including C-cells. This study evaluates the odorant receptor signals from parafollicular C-cells, specifically, the presence of olfactory marker protein, and further assesses the ability of the protein in localizing and treating medullary thyroid cancer. We used immunohistochemistry, immunofluorescent staining, Western blot, RNA sequencing, and real time-PCR to analyze the expression of odorant receptors in mice thyroids, thyroid cancer cell lines, and patient specimens. We used in vivo assays to analyze acetate binding, calcitonin secretion, and cAMP pathway. We also used positron emission tomography (PET) to assess C11-acetate uptake in medullary thyroid cancer patients. We investigated olfactory marker protein expression in C-cells in patients and found that it co-localizes with calcitonin in C-cells from both normal and cancer cell lines. Specifically, we found that OR51E2 and OR51E1 were expressed in thyroid cancer cell lines and human medullary thyroid cancer cells. Furthermore, we found that in the C-cells, the binding of acetate to OR51E2 activates its migration into the nucleus, subsequently resulting in calcitonin secretion via the cAMP pathway. Finally, we found that C11-acetate, a positron emission tomography radiotracer analog for acetate, binds competitively to OR51E2. We confirmed C11-acetate uptake in cancer cells and in human patients using PET. We demonstrated that acetate binds to OR51E2 in C-cells. Using C11-acetate PET, we identified recurrence sites in post-operative medullary thyroid cancer patients. Therefore, OR51E2 may be a novel diagnostic and therapeutic target for medullary thyroid cancer.

Spatial sterol metabolism unveiled by stimulated Raman imaging.

Spatiotemporal dynamics of small-molecule metabolites have gained increasing attention for their essential roles in deciphering the fundamental machinery of life. However, subcellular-level regulatory mechanisms remain less studied, particularly due to a lack of tools to track small-molecule metabolites. To address this challenge, we developed high-resolution stimulated Raman scattering (SRS) imaging of a genetically engineered model (GEM) to map metabolites in subcellular resolution. As a result, an unexpected regulatory mechanism of a critical metabolite, sterol, was discovered in yeast by amplifying the strength of vibrational imaging by genetic modulation. Specifically, isozymes of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were evident to promote ergosterol distribution to distinct subcellular locations, where ergosterol was enriched by a local HMGR-directed synthesis. The heterogeneity of this expression pattern thus provides new insights into sterol metabolism and related disease treatment strategies. These findings demonstrate SRS-GEM as a promising platform for new possibilities in investigating metabolic regulation, disease mechanisms, and biopharmaceutical research.

Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC-HRMS.

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood's capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.