Early restricted oxygen therapy after resuscitation from cardiac arrest (ER-OXYTRAC): protocol for a stepped-wedge cluster randomised controlled trial.

INTRODUCTION: Cardiac arrest is a critical condition, and patients often experience postcardiac arrest syndrome (PCAS) even after the return of spontaneous circulation (ROSC). Administering a restricted amount of oxygen in the early phase after ROSC has been suggested as a potential therapy for PCAS; however, the optimal target for arterial partial pressure of oxygen or peripheral oxygen saturation (SpO2) to safely and effectively reduce oxygen remains unclear. Therefore, we aimed to validate the efficacy of restricted oxygen treatment with 94%-95% of the target SpO2 during the initial 12 hours after ROSC for patients with PCAS. METHODS AND ANALYSIS: ER-OXYTRAC (early restricted oxygen therapy after resuscitation from cardiac arrest) is a nationwide, multicentre, pragmatic, single-blind, stepped-wedge cluster randomised controlled trial targeting cases of non-traumatic cardiac arrest. This study includes adult patients with out-of-hospital or in-hospital cardiac arrest who achieved ROSC in 39 tertiary centres across Japan, with a target sample size of 1000. Patients whose circulation has returned before hospital arrival and those with cardiac arrest due to intracranial disease or intoxication are excluded. Study participants are assigned to either the restricted oxygen (titration of a fraction of inspired oxygen with 94%-95% of the target SpO2) or the control (98%-100% of the target SpO2) group based on cluster randomisation per institution. The trial intervention continues until 12 hours after ROSC. Other treatments for PCAS, including oxygen administration later than 12 hours, can be determined by the treating physicians. The primary outcome is favourable neurological function, defined as cerebral performance category 1-2 at 90 days after ROSC, to be compared using an intention-to-treat analysis. ETHICS AND DISSEMINATION: This study has been approved by the Institutional Review Board at Keio University School of Medicine (approval number: 20211106). Written informed consent will be obtained from all participants or their legal representatives. Results will be disseminated via publications and presentations. TRIAL REGISTRATION NUMBER: UMIN Clinical Trials Registry (UMIN000046914).

Reactivation of Anticancer Immunity by Resetting Interorgan Crosstalk in Immune-Suppressive Cells with a Nanoparticulated Anti-Inflammatory Drug.

The reactivation of anticancer immunity is a fundamental principle in cancer immunotherapy as evidenced by the use of immune checkpoint inhibitors (ICIs). While treatment with the ICIs is shown to have remarkable and durable therapeutic effects in the responders, the low objective response rate (<40%) continues to be a major problem. Since myeloid-derived suppressor cells (MDSCs), heterogenous cells with strong immunosuppressive activity that originate in the hematopoietic system, suppress the anticancer immunity via parallel immune checkpoint-dependent and independent pathways, these cells are potential targets for improving the efficacy of cancer immunotherapy. In this study, it is demonstrated that MDSCs can be depleted by delivering synthetic glucocorticoid dexamethasone to phagocytic cells in the spleen using a lipid nanoparticle. Since the interaction of nanoparticles with T cells is intrinsically poor, this strategy also enables the "detargeting" from T cells, thus avoiding the nonspecific suppression of cytotoxic immune responses against cancer cells. In addition to the direct anticancer effect of the nanoparticulated dexamethasone, their synergistic anticancer effect with ICIs is also reported.

Outcome of extracorporeal membrane oxygenation use in severe accidental hypothermia with cardiac arrest and circulatory instability: A multicentre, prospective, observational study in Japan (ICE-CRASH study).

AIM: To elucidate the effectiveness of extracorporeal membrane oxygenation (ECMO) in accidental hypothermia (AH) patients with and without cardiac arrest (CA), including details of complications. METHODS: This study was a multicentre, prospective, observational study of AH in Japan. All adult (aged ≥18 years) AH patients with body temperature ≤32 °C who presented to the emergency department between December 2019 and March 2022 were included. Among the patients, those with CA or circulatory instability, defined as severe AH, were selected and divided into the ECMO and non-ECMO groups. We compared 28-day survival and favourable neurological outcomes at discharge between the ECMO and non-ECMO groups by adjusting for the patients' background characteristics using multivariable logistic regression analysis. RESULTS: Among the 499 patients in this study, 242 patients with severe AH were included in the analysis: 41 in the ECMO group and 201 in the non-ECMO group. Multivariable analysis showed that the ECMO group was significantly associated with better 28-day survival and favourable neurological outcomes at discharge in patients with CA compared to the non-ECMO group (odds ratio [OR] 0.17, 95% confidence interval [CI]: 0.05-0.58, and OR 0.22, 95%CI: 0.06-0.81). However, in patients without CA, ECMO not only did not improve 28-day survival and neurological outcomes, but also decreased the number of event-free days (ICU-, ventilator-, and catecholamine administration-free days) and increased the frequency of bleeding complications. CONCLUSIONS: ECMO improved survival and neurological outcomes in AH patients with CA, but not in AH patients without CA.

Therapeutic strategies for pseudoaneurysm following blunt liver and spleen injuries: A multicenter cohort study in the pediatric population.

BACKGROUND: Little guidance exists for the treatment of pseudoaneurysm (PA) following pediatric blunt liver and/or spleen injuries (BLSIs). We aimed to describe the incidence of delayed PA development and the subsequent clinical course of PA in pediatric BLSIs. METHODS: This multicenter retrospective cohort study from Japan included pediatric patients (16 years and younger) who sustained BLSIs from 2008 to 2019. The cohort was divided into four groups based on hemostatic intervention within 48 hours of admission, namely, nonoperative management (NOM), NOM with interventional radiology (IR), operative management (OM), and combined IR/OM. Descriptive statistics were used to describe the incidence of delayed PA among the groups and to characterize the clinical course of any PAs. RESULTS: A total of 1,407 children (median age, 9 years) from 83 institutions were included. The overall number (incidence) of cases of delayed PA formation was 80 (5.7%), and the number with delayed PA rupture was 16 cases (1.1%) in the entire cohort. Patients treated with NOM (1,056), NOM with IR (276), OM (53), and combined IR/OM (22) developed 43 (4.1%), 32 (12%), 2 (3.8%), and 3 (14%) delayed PAs, respectively. Among patients who developed any PAs, 39% of patients underwent prophylactic IR for unruptured PA, while 13% required emergency angioembolization for delayed PA rupture, with one ruptured case requiring total splenectomy. At least 45% of patients experienced spontaneous resolution of PA without any interventions. CONCLUSION: Our results suggest that the risk of delayed PA still exists even after acute phase IR as an adjunct to NOM for BLSIs in children, indicating the necessity of a period of further observation. While endovascular interventions are usually successful for PA management, including rupture cases, given the high incidence of spontaneous resolution, the ideal management of PA remains to be investigated in future studies. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Multiacquisition Variable-Resonance Image Combination Selective Can Improve Image Quality and Reproducibility for Metallic Implants in the Lumbar Spine.

The aim of this study is to evaluate how metallic artifacts in the lumbar spine can affect images obtained from magnetic resonance (MR) sequences. We performed a phantom experiment by scanning an agar containing an orthopedic metallic implant using 64-channel multidetector row computed tomography (CT) and a 3-tesla MR unit. We compared the reproducibility in each measurement, enlargement or reduction ratio of the CT and MR measurements, and signal deviation in each voxel from the control. The reproducibility on CT and multiacquisition variable-resonance image combination selective (MAVRIC SL) was good, but that on the other MR sequences showed either fixed bias or proportional bias. The reduction ratios of the distance between the nails were significantly smaller in MAVRIC SL than in the other MR sequences after CT measurements (p<0.001, respectively). MAVRIC SL was able to reduce the metallic artifact, permitting observation of the tissue surrounding the metal with good reproducibility.

Development of Sentinel LN Imaging with a Combination of HAase Based on a Comprehensive Analysis of the Intra-lymphatic Kinetics of LPs.

The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.

Urinary glucose and ketone bodies as indicators of acute caffeine poisoning.

AIM: In various countries, many fatal health problems have been reported due to high intake of caffeine-rich energy drinks, tablets, and powders. In patients with acute caffeine poisoning, determination of blood caffeine concentration is an important yet difficult task. We aimed to assess whether the presence of glucose and ketone bodies in urine reflected the blood caffeine concentration in patients with acute caffeine poisoning. METHODS: From April 2010 to March 2018, 25 patients with an overdose of only caffeine-rich tablets were admitted to our hospital. Their clinical features were investigated. In addition, we investigated whether the glucose and ketone bodies in the urine reflected blood caffeine concentration in 23 patients who underwent the urine qualitative test at admission. RESULTS: The majority of the patients were young healthy women, whose average caffeine ingestion was 15.6 ± 8.1 g. Initial urine examinations showed glucose in 60% (14/23) of patients and ketone bodies in 57% (13/23) of patients. Ketone bodies or glucose were found in 78% (18/23) of the patients. The correlation between blood caffeine concentration and urinary glucose was R = 0.625, blood caffeine concentration and ketone bodies was R = 0.596, and blood caffeine and both was R = 0.76. CONCLUSION: Urine qualitative test is effective for differential diagnosis and severity assessment of acute caffeine poisoning in patients.

Vitamin E Scaffolds of pH-Responsive Lipid Nanoparticles as DNA Vaccines in Cancer and Protozoan Infection.

DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.

Development of a lipoplex-type mRNA carrier composed of an ionizable lipid with a vitamin E scaffold and the KALA peptide for use as an ex vivo dendritic cell-based cancer vaccine.

A dendritic cells (DCs)-based vaccine (DC-vaccine) system is an attractive technology for eliciting antigen-specific immune responses that can protect subjects from infectious diseases and for curing various types of cancers. For the insertion of a foreign antigen to DCs, the transfection of an antigen-coding mRNA to the cells is a promising approach. In order to introduce an antigen, a carrier for mRNA transfection is required, since the mRNA molecule per se is unstable in serum-containing medium. We previously reported on an ionizable lipid-like material with vitamin E-scaffolds (ssPalmE) as a material for a lipid nanoparticle (LNP)-based carrier for nucleic acids. In the present study, we report on the development of a lipoplex-type mRNA carrier for use as a DC-vaccine by using a combination of an ssPalmE-LNP and an α-helical cationic peptide "KALA" (ssPalmE-KALA). The transfection of mRNAs complexed with the ssPalmE-KALA achieved a significantly higher protein expression and the production of proinflammatory cytokines from murine bone marrow derived DCs (BMDCs) in comparison with a lipoplex that was prepared with an ssPalm with fatty acid-scaffolds (myristic acid; ssPalmM-KALA). A cellular uptake process and a pH-responsive membrane-destabilization activity cannot explain the preferred protein expression and immune-stimulation caused by the ssPalmE-KALA. Proteomic analyses suggest that transfection with the ssPalmM-KALA stimulates a down-regulatory pathway of translation, while the transfection with the ssPalmE-KALA does not stimulate it. In the vaccination with the BMDCs that were preliminarily transfected with an ovalbumin (OVA)-encoding mRNA elicited the induction OVA specific cytotoxic T-lymphocyte activity in vivo. In parallel, the vaccination induced significant prophylactic anti-tumor effects against a model tumor that stably expressed the OVA protein. Based on the above findings, the ssPalmE-KALA appears to be a potent ex vivo DCs-based RNA vaccine platform.

DNA-loaded nano-adjuvant formed with a vitamin E-scaffold intracellular environmentally-responsive lipid-like material for cancer immunotherapy.

Cytoplasmic DNA triggers cellular immunity via activating the stimulator of interferon genes pathway. Since DNA is degradable and membrane impermeable, delivery system would permit cytoplasmic delivery by destabilizing the endosomal membrane for the use as an adjuvant. Herein, we report on the development of a plasmid DNA (pDNA)-encapsulating lipid nanoparticle (LNP). The structural components include an SS-cleavable and pH-activated lipid-like material that mounts vitamin E as a hydrophobic scaffold, and dual sensing motifs that are responsive to the intracellular environment (ssPalmE). The pDNA-encapsulating LNP (ssPalmE-LNP) induced a high interferon-ß production in Raw 264.7 cells. The subcutaneous injection of ssPalmE-LNP strongly enhanced antigen-specific cytotoxic T cell activity. The ssPalmE-LNP treatment efficiently induced antitumor effects against E.G7-OVA tumor and B16-F10 melanoma metastasis. Furthermore, when combined with an anti-programmed death 1 antibody, an extensive therapeutic antitumor effect was observed. Therefore, the ssPalmE-LNP is a promising carrier of adjuvants for cancer immunotherapy.

Development of a mouse model for the visual and quantitative assessment of lymphatic trafficking and function by in vivo imaging.

Methods for quantitative analysis of long distance lymphatic transport of nanoparticles in live animals are yet to be established. We established a mouse model for analysis of time-dependent transport just beneath the abdominal skin to investigate lymph node-to-lymph node trafficking by in vivo imaging. For this purpose, popliteal lymph nodes (PLNs) as well as efferent and afferent lymphatic vessels, marginal veins, and feeding blood vessels were surgically resected to change the lymphatic flow from footpad injections. Using this model, we observed a novel lymphatic flow from the footpad to the proper axillary lymph node (ALN) via the inguinal lymph node (ILN). This drainage pathway was maintained over 12 weeks. Time-dependent transportation of 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide-labelled liposomes from the footpad to the ILN was successfully quantified by an in vivo imaging system. Moreover, congestion and development of a new collateral lymphatic route was visualised under a lymphedema status. Histological analysis of abdominal skin tissues of this model revealed that PLN resection had no effect on the abdominal lymphatic system between the ILN and ALN. These data indicate that this model might be useful to clarify the mechanisms of lymphedema and study direct transportation of lymph or other substances between lymph nodes.

Identification and Evaluation of the Minimum Unit of a KALA Peptide Required for Gene Delivery and Immune Activation.

The KALA peptide (WEAKLAKALAKALAKHLAKALAKALKA) is an amphiphilic peptide that forms an α-helical structure at physiological pH. We previously reported that, when a plasmid DNA-encapsulating liposomal membrane is modified with the KALA peptide, transgene expression and immune activation are facilitated in bone marrow-derived dendritic cells (BMDCs). However, the minimum unit of the KALA peptide and the importance of its secondary structure for these activities are not completely known at this time. We herein report on the identification of the minimum unit of the KALA peptide (short-KALA) required for activity, as determined by the stepwise removal of "K-A-L-A" units. We evaluated the activities of 4 types of short-KALAs by modifying plasmid DNA-encapsulating multi-functional envelop-type nano devices. Among the peptides tested, a short-KALA3 (WEAKLAKALAKALA) was the shortest KALA peptide that could form an α-helical structure, as well as to elicit transgene expression and immune activation in BMDCs. Furthermore, the function of the short-KALA3 as an inducer of cellular uptake was retained, while uptake was completely lost in more shortened versions of KALA (short KALA4), in that transgene expression and immunological activation were both completely lost. These collective data show that the KALA peptide must form an α-helical structure to induce cellular uptake in BMDCs.

Modifying Antigen-Encapsulating Liposomes with KALA Facilitates MHC Class I Antigen Presentation and Enhances Anti-tumor Effects.

For a successful anti-cancer vaccine, antigen presentation on the major histocompatibility complex (MHC) class I is a requirement. To accomplish this, an antigen must be delivered to the cytoplasm by overcoming the endosome/lysosome. We previously reported that a lipid nanoparticle modified with a KALA peptide (WEAKLAKALAKALAKHLAKALAKALKA), an α-helical cationic peptide, permits the encapsulated pDNA to be efficiently delivered to the cytoplasm in bone marrow-derived dendritic cells (BMDCs). Herein, we report on the use of KALA-modified liposomes as an antigen carrier, in an attempt to induce potent antigen-specific cellular immunity. The subcutaneous injection of KALA-modified ovalbumin (OVA)-encapsulating liposomes (KALA-OVA-LPs) elicited a much more potent OVA-specific cytotoxic T lymphocyte activity and anti-tumor effect in comparison with particles that were modified with octa-arginine (R8), a cell-penetrating peptide (R8-OVA-LPs). In addition, the numbers of OVA-specific CD8+ T cells were increased by immunization the KALA-OVA-LPs. The treatment of BMDCs with KALA-OVA-LPs induced a substantial MHC class I antigen presentation. Furthermore, the acidic pH-dependent membrane destabilization activity of KALA-OVA-LPs strongly suggests that they are able to escape from endosomes/lysosomes and thereby deliver their cargos to the cytoplasm. Collectively, the KALA-modified liposome is a potential antigen delivery platform for use as a protein vaccine.

Risk factors for ground-level falls differ by sex.

BACKGROUND: The populations of many developed countries have been aging in recent years, resulting in increasing numbers of elderly-related injuries. Conventionally regarded as minor, injuries from ground-level falls are now associated with a higher risk of death for elderly people. METHODS: The subjects of this study were 15662 adult patients with injuries from ground-level falls who were registered in the Japan Trauma Data Bank between 2007 and 2013. Logistic regression analysis was used to evaluate the effects of age, sex, Injury Severity Score, and Revised Trauma Score (RTS) on inhospital mortality. Patients aged 60 years or older were further categorized into 4 subgroups by age and sex, and the effect of the presence of injuries of Abbreviated Injury Scale greater than or equal to 3 in each region on inhospital mortality was analyzed. RESULTS: Logistic regression analysis for inhospital mortality showed significant interactions between sex and age and between sex and RTS, and subgroup analysis by sex was, therefore, performed. The odds ratio (95% confidence interval) for inhospital mortality compared with patients older than 60 years was 2.75 (1.90-3.96) for men aged 60 to 79 years and 5.44 (3.77-7.85) for men 80 years or older and 1.46 (0.83-2.58) for women aged 60 to 79 years and 2.32 (1.35-4.01) for women 80 years or older. The odds ratios (95% confidence interval) for RTS less than 7.840 was 6.89 (5.56-8.55) for men and 9.97 (7.59-13.10) for women. CONCLUSIONS: The effects of age and RTS on inhospital mortality of patients after ground-level falls differed by sex.

A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant.

Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms α-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-ß and IL-1ß production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.

Risk factors for QT prolongation associated with acute psychotropic drug overdose.

BACKGROUND: Antipsychotic/Antidepressant use is a risk factor for QT interval (QT) prolongation and sudden cardiac death. However, it is unclear which drugs are risk factors for QT prolongation and torsades de pointes in cases of psychotropic drug overdose. METHODS: After correction of QT data by Bazett formula (QTc), QTc was classified into 3 categories (QTc<440 milliseconds, 440 milliseconds≤QTc<500 milliseconds, and QTc≥500 milliseconds), and the blood concentration of each drug was classified as not detected, therapeutic range, or toxic range. The association of the blood concentration of each drug with QTc was analyzed using the ordinal logistic regression model. Drugs that induced QT-heart rate pairs higher than the at-risk line of Isbister's QT-heart rate nomogram (QT nomogram) were further analyzed using the binomial logistic regression model. RESULTS: A total of 649 patients were enrolled in the study. The independent risk factors for QTc prolongation were therapeutic and toxic range of phenotiazine antipsychotic drug (therapeutic range: odds ratio [OR], 1.56 [P=.039]; toxic range: OR, 3.85 [P<.001]), and toxic range of cyclic antidepressants (OR, 2.39; P=.018). In addition, toxic range of phenotiazine antipsychotic drug (OR, 3.87; P=.012) and tricyclic antidepressants (OR, 4.94; P<.001) were risk factors for QT higher than the at-risk line of the QT nomogram. CONCLUSIONS: The possibility of QT prolongation and torsades de pointes due to overdose of phenotiazine antipsychotic drug or tricyclic antidepressants requires particular consideration.

Serum concentration of eperisone hydrochloride correlates with QT interval.

BACKGROUND: Eperisone hydrochloride is a centrally acting muscle relaxant prescribed for muscle stiffness that acts by depressing the activities of α and γ efferent neurons in the spinal cord and supraspinal structures. Although a case of eperisone-induced severe QT prolongation had been reported, the relationship between serum eperisone concentration and QT interval remains obscure. OBJECTIVE: The aim of this study was to investigate the relationship between serum eperisone concentration and QT interval. METHODS: Four patients who overdosed on eperisone were admitted to our hospital between January 2010 and December 2011. We took simultaneous serial measurements of serum eperisone concentration and QT interval in the intensive care unit. In total, 22 measurement points were plotted for these patients. We analyzed the correlation between the serum eperisone concentration and corrected QT (QTc) interval. RESULTS: Three men and one woman (mean age, 50 years) overdosed on eperisone with an average dose of 3087.5 mg (therapeutic dose, 150 mg/day). The mean QTc interval at arrival was 592 ms (range, 444-825 ms), and the mean serum eperisone concentration at arrival was 1257.5 ng/mL (range, 14.5-4120.0 ng/mL). The correlation coefficient was 0.833 between serum eperisone concentration and QTc interval (P < .001). CONCLUSION: Serum eperisone concentration correlates with QTc interval in patients who overdose on eperisone.

Rapid simultaneous determination of eperisone, tolperisone, and tizanidine in human serum by using a MonoSpin® C18 extraction column and liquid chromatography/tandem mass spectrometry.

A method was developed for rapid toxicological analysis of eperisone, tolperisone, and tizanidine in human serum using a MonoSpin® C18 extraction column and LC/MS/MS. The method was validated for LOD, linearity, precision, and extraction recovery. This method was rapid with an LOD of 0.5 ng/mL, linearity range 1-500.0 ng/mL (r2 = 0.999), and RSD value below 14.6%. Extraction recovery from the sample was greater than 98.6, 98.8, and 88.5% for eperisone, tolperisone, and tizanidine, respectively. Results showed that combination of the MonoSpin C18 extraction column and LC/MS/MS is a simple and rapid method for the analysis of these three analytes, and a method is described for simultaneous quantitative determination of the analytes in human serum by LC/MSIMS. This method was used to determine the serum levels of eperisone in a patient with eperisone poisoning, and could be successfully applied for screening analyses in clinical cases other than poisoning.