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.

Simultaneous quantification of TB-500 and its metabolites in in-vitro experiments and rats by UHPLC-Q-Exactive orbitrap MS/MS and their screening by wound healing activities in-vitro.

BACKGROUND: TB-500 (Ac-LKKTETQ), derived from the active site of thymosin ß4 (Tß4), has various biological functions in its unacetylated form, LKKTETQ. These functions include actin binding, dermal wound healing, angiogenesis, and skin repair. The biological effects of TB-500, however, have not been documented. And the analysis of TB-500 and its metabolites have been neither simultaneously quantified nor structurally identified using synthesized authentic standards. METHODS: This study was aimed to investigating simultaneous analytical methods of TB-500 and its metabolites in in-vitro and urine samples by using UHPLC-Q-Exactive orbitrap MS, and to comparing the biological activity of its metabolites with the parent TB-500. The metabolism of TB-500 was investigated in human serum, various in-vitro enzyme systems, and urine samples from rats treated with TB-500, and their biological activities measured by cytotoxicity and wound healing experiments were also evaluated in fibroblasts. RESULTS: The simultaneous analytical method for TB-500 and its metabolites was developed and validated. The study found that Ac-LK was the primary metabolite with the highest concentration in rats at 0-6 h intervals. Also, the metabolite Ac-LKK was a long-term metabolite of TB-500 detected up to 72 hr. No cytotoxicity of the parent and its metabolites was found. Ac-LKKTE only showed a significant wound healing activity compared to the control. CONCLUSION: The study provides a valuable tool for quantifying TB-500 and its metabolites, contributing to the understanding of metabolism and potential therapeutic applications. Our results also suggest that the previously reported wound-healing activity of TB-500 in literature may be due to its metabolite Ac-LKKTE rather than the parent form.

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.

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.

Detection and quantification of the metabolite Ac-Tß1-14 in in vitro experiments and urine of rats treated with Ac-Tß4: A potential biomarker of Ac-Tß4 for doping tests.

Thymosin ß4 (Tß4) was reported to exert various beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti-Doping Agency. However, no metabolism studies of Tß4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tß31-43 , Ac-Tß17-43 , Ac-Tß1-11 , Ac-Tß1-14 , Ac-Tß1-15 , and Ac-Tß1-17 ) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tß4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tß4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac-Tß1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac-Tß1-14 were 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r2 = 0.9998). As a result, among the six metabolites above, Ac-Tß1-14 , as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tß4, and the urine of rats intraperitoneally treated with 20-mg/kg Tß4. And the metabolite Ac-Tß1-14 was quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac-Tß1-14 was not detected in non-treated control groups, including human blank urine. These results suggest that Ac-Tß1-14 in urine is a potential biomarker for screening the parent Tß4 in doping tests.

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.

Enhancing adoptive T-cell therapy with fucoidan-based IL-2 delivery microcapsules.

Adoptive cell therapy (ACT) with antigen-specific T cells is a promising treatment approach for solid cancers. Interleukin-2 (IL-2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL-2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL-2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL-2 binding glycosaminoglycan, and poly-l-lysine, a cationic counterpart (FPC2). IL-2-laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2-IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2-IG-IL-2 increased expansion of tumor-infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor-reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2-IG-IL-2. The immune-favorable tumor microenvironment induced by FPC2-IG-IL-2 enabled adoptively transferred TCR-engineered T cells to effectively eradicate tumors. FPC2-IG delivery system is a promising strategy for T-cell-based immunotherapies.

Influence of mobile phase composition on the analytical sensitivity of LC-ESI-MS/MS for the concurrent analysis of bisphenols, parabens, chlorophenols, benzophenones, and alkylphenols.

Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a "dilute and shoot" approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.

Simple visualization method for the c.577del of erythropoietin variant: CRISPR/dCas9-based single nucleotide polymorphism diagnosis.

One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild-type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti-Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti-doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant-specific single-guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native-PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild-type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3 h using only 3 µl of the whole blood.

Comprehensive LC-MS/MS method combined with tandem hybrid hydrolysis for multiple exposure assessment of multiclass environmental pollutants.

Environmental pollutants (EPOLs), such as phthalates, volatile organic compounds, phenols, parabens, polycyclic aromatic hydrocarbons, pyrethroids, and environmental tobacco smoke, are highly heterogeneous compounds. Recently, attention has been drawn to the assessment of the combinatory effects of multiple EPs. To correlate multiple exposures with potential health implications, advanced comprehensive analytical methods covering multiclass EPOLs are essential. However, because of several technical problems associated with enzyme hydrolysis, simultaneous extraction, and multiresidue liquid chromatography-tandem mass spectrometry analysis, it is difficult to establish a comprehensive method covering a number of EPOLs in a single sample preparation and analytical run. We developed tandem hybrid hydrolysis, modified direct injection, and a comprehensive mobile phase to overcome these technical problems and established a comprehensive analytical method for simultaneous biomonitoring of multiclass EPOLs. Tandem hybrid hydrolysis using ß-glucuronidase and consecutive acid hydrolysis allowed selective hydrolysis of glucuronide- and sulfate-conjugated metabolites without phthalate degradation. The comprehensive mobile phase composed of 0.01% acetic acid and acetonitrile enabled us to simultaneously analyze 86 EPOLs, with good chromatographic behavior and ionization efficiency. Modified direct injection allowed a small amount of sample and simultaneous urinary extraction. The method was validated and applied to 39 urine samples from 19 mother-newborn pairs for multiple exposure assessment. Results showed that BP-3, a general component in sunblock products, and monoethyl phthalate, a metabolite of diethyl phthalate, exhibit a clear positive correlation between mothers and newborns. Therefore, the developed method has potential as a novel analytical tool for long-term, large-scale, and data-rich human biomonitoring of EPOLs.

Fluoride-assisted liquid chromatography-tandem mass spectrometry method for simultaneous analysis of propofol and its metabolites without derivatization in urine.

The misuse of propofol for recreational purposes has become a serious social issue. Accordingly, practical and sensitive analytical methods to investigate the chronic abuse and toxicity of propofol are required. However, current propofol determination methods using liquid chromatography-mass spectrometry (LC-MS/MS) suffer from problems associated with loss in sample preparation due to its volatility and its poor ionization efficiency and collision-induced dissociation in mass spectrometry. Herein, we have developed a sensitive and accurate fluoride-assisted LC-MS/MS method combined with direct-injection for propofol determination. Ionization via fluoride-ion attachment/induced deprotonation, effected by ammonium fluoride in the mobile phase, was found to dramatically improve the sensitivity of propofol without derivatization. Furthermore, direct injection without derivatization enables the simultaneous analysis of propofol and its phase II metabolites without analyte loss. The optimal concentration of ammonium fluoride in the mobile phase was found to be 1 mM under methanol conditions. The linearity is good (R2 ≥ 0.999) and the intra- and inter-day precisions for propofol determination are between 1.9 and 8.7%. The accuracies range from 87.5% to 105.4% and the limits of detection and quantitation for propofol in urine are 0.15 and 0.44 ng mL-1, respectively. The present method was successfully applied to human urine and showed a sufficient sensitivity to determine propofol and five phase II metabolites over 48 h in human urine after administration. Consequently, the fluoride-assisted LC-MS/MS method was demonstrated to be sensitive, accurate, and practical for the determination of propofol and its metabolites.

Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

Optimization, validation, and comparison of a rapid method for the quantification of insulin-like growth factor 1 in serum using liquid chromatography-high-resolution mass spectrometry.

Human insulin-like growth factor 1 (IGF-I) is the primary mediator of the effects of the growth hormone (GH). Therefore, it has been used as a biomarker to detect the abuse of GH in sports. The measurement of IGF-I relies on mass-based and immunological approaches to analysis. Among the mass-based analysis methods, liquid chromatography-mass spectrometry (LC-MS) has a number of functional advantages. LC-MS measurements based on the quantification of IGF-I, according to trypsin digestion, are used in the most common method of analyzing doping. However, this method is time-consuming and subject to experimental variability. In this study, we optimized a rapid method for detecting IGF-I without the trypsin digestion step. This method of analysis uses an ultra-centrifugal filter and an LC-HRMS through narrow-range mass scan method. To verify the validity of this method, eight categories of validation testing were applied with the following results: linearity, R2 > 0.99; limit of detection, 15 ng/ml; limit of quantification, 20 ng/ml; accuracy, >99%; recovery rate, >95%; carryover, <0.03; and inter- and intra-day precision values, %CV < 2% and %CV < 6%, respectively. Furthermore, we discussed the correlation of the quantified concentration from two other methods, immunoradiometric assay (IRMA) and parallel reaction monitoring method, using 209 serum samples. In conclusion, although both mass spectrometry-based methods worked equally well in terms of analytical performance and correlation with IRMA results, narrow-range mass scan method had several advantages, such as time and cost savings and reliable reproducibility, over the existing methods.

New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis.

The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping.

An Exploratory Pilot Study with Plasma Protein Signatures Associated with Response of Patients with Depression to Antidepressant Treatment for 10 Weeks.

Major depressive disorder (MDD) is a leading cause of global disability with a chronic and recurrent course. Recognition of biological markers that could predict and monitor response to drug treatment could personalize clinical decision-making, minimize unnecessary drug exposure, and achieve better outcomes. Four longitudinal plasma samples were collected from each of ten patients with MDD treated with antidepressants for 10 weeks. Plasma proteins were analyzed qualitatively and quantitatively with a nanoflow LC-MS/MS technique. Of 1153 proteins identified in the 40 longitudinal plasma samples, 37 proteins were significantly associated with response/time and clustered into six according to time and response by the linear mixed model. Among them, three early-drug response markers (PHOX2B, SH3BGRL3, and YWHAE) detectable within one week were verified by liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) in the well-controlled 24 patients. In addition, 11 proteins correlated significantly with two or more psychiatric measurement indices. This pilot study might be useful in finding protein marker candidates that can monitor response to antidepressant treatment during follow-up visits within 10 weeks after the baseline visit.

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT-Based Mass Spectrometry.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites-the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high-resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region-specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell-cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.

A rapid non-target screening method for determining prohibited substances in human urine using liquid chromatography/high-resolution tandem mass spectrometry.

Target analysis using liquid chromatography-tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non-target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid-phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high-resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%-131%), matrix effects (35%-237%), limites of detection (0.0002-100 ng/mL), and intra- and inter-day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non-target screening method is efficient for conducting anti-doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.

Development of a multi-functional concurrent assay using weak cation-exchange solid-phase extraction (WCX-SPE) and reconstitution with a diluted sample aliquot for anti-doping analysis.

RATIONALE: In addition to the development of adequate screening methods for multiple compounds, the World Anti-Doping Agency (WADA) requires anti-doping laboratories to analyze prohibited substances and their metabolites from various classes. This task presents a difficult challenge for all agencies and interests involved in the field of doping control. METHODS: A screening method is reported in which hybrid sample preparation was performed using a combination of weak cation-exchange solid-phase extraction (WCX-SPE) and the 'Dilute and Shoot' strategy in order to take advantage of both the methodologies. Target substances were extracted using a WCX cartridge and reconstituted with a diluted sample aliquot that included 20% of an untreated urine sample. The target substances were further analyzed by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS). RESULTS: The SPE procedure was optimized using a cartridge-washing step, elution conditions, and elution volume. The cartridge-washing step, which was performed using 10% methanol, improved the overall recovery of target substances. Since the recovery was observed to vary according to the pH of the eluting solution, we applied an elution step using both an acid and a basic organic solvent to achieve complementary recovery. Reconstitution of the diluted aliquot sample was performed to recover the polar substances. CONCLUSIONS: The method was validated and applied to real samples in accordance with the external quality assessment scheme of WADA and to the previously reported samples that had provided positive test results. This novel method using hybrid sample preparation and LC/MS could be useful to screen multiple classes of the 264 targeted substances in anti-doping analysis.