The aim of this study was to find lipid metabolite concentrations differences between healthy subjects and colorectal cancer patients. A novel high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) analytical method was developed for the analysis of nine lipid series (two sphingolipids, seven phospholipids) using the multiple reaction monitoring mode (MRM) on dried plasma spots. The extracted lipids were separated by HPLC using an Imtakt Unison UK-C8. The developed method was applied to human plasma samples obtained from healthy subjects (n = 40) and colorectal cancer patients (n = 40). A partial least squares discriminant analysis (PLS-DA) model, which is a multivariate statistical analysis method, was employed to analyse the quantitative results. The VIP score of the PLS-DA model was employed to effectively discriminate colorectal cancer patients from healthy subjects. Furthermore, a random forest classification method was employed. Through statistical processing, the lipid 18:1 Lyso PC in accordance with VIP score > 1 was identified, and four lipids in accordance with p-value < 0.05, 15:0–18:1 PC, 18:1 Lyso PC, 18:1 Lyso PE, and C15 Ceramide (d18:1/15:0) were identified. The results of this study corresponded to study of Zhao et al.(2007) that 18:1 LPC can be potential biomarker between normal and colorectal cancer patients. This method is expected to be practically useful due to its simple dried plasma spot use and excellent sensitivity for lipid screening when applied to various diseases, including colorectal cancer.

Adverse drug events are significant causes of emergency department visits. Systematic evaluation of adverse drug events leading to emergency department visits by age is lacking. This multicenter retrospective observational study evaluated the prevalence and features of adverse drug event-related emergency department visits across ages. We reviewed emergency department medical records obtained from three university hospitals between July 2014 and December 2014. The proportion of adverse drug events among total emergency department visits was calculated. The cause, severity, preventability, and causative drug(s) of each adverse drug event were analyzed and compared between age groups (children/adolescents [<18 years], adults [18–64 years], and the elderly [≥65 years]). Of 59,428 emergency department visits, 2,104 (3.5%) were adverse drug event-related. Adverse drug event-related emergency department visits were more likely to be female and older. Multivariate logistic regression analysis revealed that compared to non- adverse drug event-related cases, adverse drug event-related emergency department visitors were more likely to be female (60.6% vs. 53.6%, p<0.001, OR 1.285, 95% CI 1.025–1.603) and older (50.8 ± 24.6 years vs. 37.7 ± 24.4 years, p<0.001, OR 1.892, 95% CI: 1.397–2.297). Comorbidities such as diabetes, chronic kidney disease, chronic liver disease, and malignancies were also significantly associated with adverse drug event-related emergency department visits. Side effects were the most common type of adverse drug events across age groups, although main types differed substantially depending on age. Serious adverse drug events, hospitalizations, and adverse drug event-related deaths occurred more frequently in the elderly than in adults or children/adolescents. The proportion of adverse drug event-related emergency department visits that were preventable was 15.3%. Causative drugs of adverse drug events varied considerably depending on age group. Adverse drug event features differ substantially according to age group. The findings suggest that an age-specific approach should be adopted in the preventive strategies to reduce adverse drug events.

Chiral discrimination of thyroxine (T4) enantiomers was performed using ultra high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) on a chiral crown ether-derived ChiroSil SCA (-) column. The different composition of mobile phases and the effect of column oven temperatures were investigated and the optimum chromatographic separation with respect to resolution and analysis time was achieved using a mixture of 60

Liver cancer metastasis is known to be a poor prognosis and a leading cause of mortality. To overcome low therapeutic efficacy, understanding the physiological properties of liver cancer metastasis is required. However, the metastatic lesion is heterogeneous and complex. We investigate the distribution of lipids using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in an experimental metastasis model. We obtained the differentially expressed mass peaks in comparison between normal sites and metastatic lesions. The relationship of mass to charge ratio (m/z) and intensity were measured, m/z-indicated species were analyzed by MALDI-MS/MS analysis, and identification of these mass species was confirmed using the METASPACEannotation platform and Lipid Maps®. MALDI-MSI at m/z 725.6, 734.6, 735.6, 741.6, 742.6, 744.6, 756.6, and 772.6 showed significantly higher intensity, consistent with the metastatic lesions in hematoxylin-stained tissues. Sphingomyelin SM [d18:0/16:1], phosphatidylcholine (PC) [32:0], PC [31:0], PC [31:1], and PE [36:2] were highly expressed in metastatic lesions. Our results could provide information for understanding metastatic lesions. It suggests that the found lipids could be a biomarker for the diagnosis of metastatic lesions.

We developed and validated a fast, sensitive, and accurate analytical method using ultra high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI–MS/MS) for simultaneous extraction and quantification of 5 targeted omega fatty acids on human dried serum spots. MS/MS was performed with multiple reaction monitoring (MRM) mode under negative electrospray ionization. We targeted omega fatty acids which are likely to have a positive effect on various inflammatory reactions such as asthma, chronic obstructive pulmonary diseases and rheumatoid arthritis. If applicable, it is necessary to determine them as asthma biomarkers for intervention and public health. The coefficient of determination ( r 2 ) of calibration curve exhibited a good linear relationship between 0.9942 and 0.9963 in a range of 0.05–5 µg mL −1 . The analytical method was validated with excellent sensitivity (limit of detection; 0.005–0.01 µg mL −1 , limit of quantification; 0.03–0.1 µg mL) that allows targeted analyses of omega fatty acids. We obtained the recoveries of 92.6 to 110.4% (RSD, 0.2–9.6%) for intraday and 92.6 to 107.6% (RSD, 1.4–7.0%) for interday. Clinical correlation between the healthy subjects and asthmatic patients’ sera was evaluated with the ratio of omega-3/omega-6 fatty acids. The developed method combines the advantages of easy handling dried filter paper and UHPLC-MS/MS for effectiveness of extraction and analyses step. We evaluated the validation for the quantification and applicability of human serum as a possible role of asthma biomarkers.

We developed and validated a simple, sensitive, and reproducible analytical method using ultra high-pressure liquid chromatography (UHPLC)-QOrbitrap mass spectrometry to screen human urine samples for amphetamines. Amphetamines are classified under the group of abused drugs and are frequently used by jockeys. A rapid analysis method is necessary to ascertain the presence of minimal amounts of prohibited amphetamines in the urine samples of jockeys on a race day. For the method described herein, UHPLC conditions were set with a Luna Omega？？ C18 column using a gradient mobile phase. Six amphetamines were separated by gradient elution and detected by QOrbitrap MS in the positive ion mode. Validation parameters, including selectivity and limit of detection, were evaluated for the purpose of screening for amphetamines in human urine. Human urine samples were prepared for analysis using enzymatic hydrolysis and solidphase extraction. The limit of detection for screening the six amphetamines was between 0.1-10 ng/mL. We obtained recoveries between 31% and 76%, which is suitable for screening the amphetamines. This method combines the advantages of SPE and UHPLC-QOrbitrap MS, such as an effective extraction and simple analysis steps, and validated the amphetamine screening method for drug abuse control of jockies and improving public health.

Omega-3 and omega-6 fatty acids are reported to alleviate various inflammatory reactions such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, and inflammatory bowel disease. We have developed a new method for their quantitation in serum, utilizing ultra-high-performance liquid chromatography-electrospray ionizationtandem mass spectrometry (UHPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode, under negative ionization. MRM transitions were detected for five omega fatty acids, as follows: α-linolenic acid, m/z=277.20→277.20; eicosapentaenoic acid, m/z=301.10→257.30; docosahexaenoic acid, m/z=327.00→283.15; arachidonic acid, m/z=303.20→259.25; docosapentaenoic acid, m/z=329.10→285.25. After comparing several different solvents, we selected butanol:methanol (1:1, v/v) as the optimal extraction solvent. Chromatographic separations were carried out using an Acclaim RSLC 120 C18 column (150×2.1 mm, 2.2 μm) at 40oC. For the mobile phase, solvent A consisted of water containing 5% acetonitrile and 5% methanol, and solvent B consisted of methyl tert-butyl ether:acetonitrile:methanol (10:20:70, v/v/v) containing 2.5 mM ammonium acetate. The calibration curve showed excellent linearity within the range of 0.01-20 μg/mL (R2=0.9996-0.9999). The analytical method was validated and shown to have excellent sensitivity (LOD; 0.001-0.005 μg/mL, LOQ; 0.01-0.2 μg/mL), making it suitable for targeted analyses of omega fatty acids. Recovery ranged from 78.0 to 103.9% (RSD 0.5-5.6%). This new method uses relatively small amounts of serum and extraction solvent and is expected to be suitable for analysis of various clinical specimens, such as plasma, urine, and cerebrospinal fluid.

Sphingosine (SPH) and sphingosine-1-phosphate (S1P) are emerging as key players in asthma metabolism and in numerous cellular inflammation processes. To identify potential biomarkers of asthma and inflammatory therapeutics, it is essential to determine their levels. Herein, we developed a rapid and sensitive UHPLC-MS/MS method to simultaneously quantify SPH and S1P in human serum using C17-SPH and C17-S1P as internal standards. After methanol precipitation of serum proteins, the supernatants were analyzed by MS/MS performed in the positive ion mode by multiple reaction monitoring. UHPLC analysis (C18 column) was performed using two mobile phase systems (water containing 0.1% formic acid, and 85% acetonitrile containing 0.1% formic acid) within 5 min of the short run. The calibration curves were linear in the range of 0.002-1.5 µg/mL for S1P and SPH with an R 2 greater than 0.9999. The LOD and LOQ were 0.0002 and 0.0004 µg/mL for S1P, and 0.0005 and 0.001 µg/mL for SPH, respectively. The accuracy and precision of the method were in the range of 89.8-100.7% (RSD, 1.5-2.8%) for both SPH and S1P species. We were able to quantify both molecules in serum from healthy and asthmatic patients. These results suggest that SPH and S1P are promising potential biomarkers, and also contribute to the basic data for the construction of an omics-based platform for preventive index prior to asthma diagnosis.

Omega fatty acids play an important role as biomarkers of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, and inflammatory bowel disease. The purpose of our study was to develop a new analytical method for the detection of omega fatty acids from dried serum spots. We developed an ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS)-based method in the multiple reaction monitoring (MRM) mode with negative ionization. The detected MRM transitions for five omega fatty acids are as follow: α-linolenic acid (m/z=277.20→277.20), eicosapentaenoic acid (m/z=301.10→257.30), docosahexaenoic acid (m/z=327.00→283.15), arachidonic acid (m/z=303.20→259.25), and docosapentaenoic acid (m/z= 329.10→285.25). The calibration curve showed an excellent linearity within 0.1-2.5 μg/mL range (R2=0.9947~0.9994). The analytical methods howed excellent sensitivity (LOD: 0.005-0.01 μg/mL, LOQ: 0.03-0.1 μg/mL), which would allow for the targeted analyses of omega fatty acids. The recoveries of the omega fatty acid from the dried serum spots were 92.6%- 110.4% (RSD: ±0.2%-9.6%) and the retention time was within 5 min. This improved method offers rapid and sensitive quantification of omega fatty acids on dried serum spots using a small volume of serum and is expected to be applied to various biological specimens.

Enantioseparation of thyroxine using chiral crown ether type column.

While it has been recognized that airway microbiome in asthma is different from healthy subjects, much is not known well regarding the mechanisms how this microbiota affects airway inflammation in asthma. Short-chain fatty acids (SCFA), such as sodium acetate (SA), sodium propionate (SP) and sodium butyrate (SB), produced by bacteria, exert various immune modulating effects. In this experimental study, we examined the effects of locally administered SCFA into airway on the allergic inflammation using in vivo and in vitro model of asthma. Ovalbumin (OVA) induced asthma was induced by sensitizing mice using an intraperitoneal injection of OVA and intranasal administration of OVA afterwards. Each of three kinds of SCFA was administered into the airway via intranasal route at the time of OVA challenge. Airway inflammation was compared between each SCFA group and controls in terms of inflammatory cell counts in bronchoalveolar lavage (BAL) fluids and lung histology and cytokine expression. In addition, the effects of SCFA was also examined in house dust mite (HDM) treated BEAS-2B cells. SCFA (SA, SP, and SB)-treated mice showed a significant decrease in total inflammatory cell counts and eosinophil counts in BAL fluid and lung histology. Airway hyperresponsiveness was significantly attenuated in the SP administrated group. OVA-specific IgE level and the expression of IL-1 beta, inflammasome cytokine, were also significantly decreased with administration of SCFA. Moreover, SCFA attenuated the synthesis of IL-1 beta in HDM treated BEAS-2B cells. These findings suggest that airway SCFA have anti-inflammatory effects in asthma by downregulating allergic immune response and inflammasome pathway.

Bile acids are increasingly appreciated as bioactive molecules and important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties. bile acids have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To investigate bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein, we developed the analytical method of cholic acids following simple one step protein precipitation from biological sample by UPLC-MS/MS. MRM ions was m/z=407.2 for cholic acid. The R2 of calibration curves provided 0.9995 in the calibration range of 0.005~5 μg/mL. The quantification system was validated with excellent sensitivity that allows quantitative targeted analyses of bile acids. The LOD (0.001 μg/mL), LOQ (0.005 μg/mL), recoveries (96.8~101.3%± 2.7~4.0%) on intra-day assay, and (98.4~111.0%±2.3~3.4%) on inter-day assay achieved resonable validated data for bile acids analyses. The developed method was applied for drawing plasma and bile acid profile in both normal and disease status. Our study is characterized by rapid and simple sample preparation as well as successful application to plasma and bile. These results could be usable for routine diagnostic monitoring of bile acids on human biofluids.

A rapid and simple screening method was developed for the simultaneous analysis of 6 sulfur-containing amino acids from human plasma at ng level. The procedure involves simple protein precipitation followed by direct analysis using UPLC-MS/MS. Calibration curves showed an excellent linear relationship with coefficients of determination (r 2 ) of 0.9989-0.9998. LOD and LOQ were between 1-50 ng/mL and 10-100 ng/mL, respectively. When sulfur amino acids spiked into plasma sample, excellent linearity of detection response was demonstrated over the range between 10-10,000 ng/mL. This new analytical method is suitable for simultaneous screening of large number of samples in clinical application and metabolomics research.

Globotriaosylsphingosine (lyso Gb3) is considered as one of the biomarkers for Fabry disease. A rapid and simple UPLC-MS/MS method was developed for the determination of reliable biomarker, lyso Gb3. Total analytical procedure takes only 15 min including sample preparation and MS/MS analysis. Limit of detection was 0.85 ng/ml (S/N=3). The calibration curve was linear over the range of 2.0~400.0 ng/ml (R2=0.9999). Inter-day and intra-day assay accuracy were 93.4~100.6% (RSD, 0.6~6.0%) and 97.5~100.7% (RSD, 3.6~5.2%). Absolute recoveries of 97.6~98.6 showed excellence of a new analytical method. The method was applied to human and mice urines, proved the suitability for the quantification of lyso-Gb3 for screening, diagnosis and therapeutic monitoring of Fabry disease patients.

A rapid and sensitive ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometric (UHPLC-Q-TOF-MS) method was developed for quantification of imipramine, one of the most widely used tricyclic antidepressants, and desipramine, an active metabolite of imipramine, in mouse serum. The developed method included a simple protein precipitation with acetonitrile in 50 μL of serum and analyte separation on an Acquity UPLC BEH C18 column using a gradient elution of acetonitrile with 0.1% formic acid and 20 mM ammonium formate. As a result, the entire analysis time was <20 min including the sample preparation and the LC-MS analysis. The limit of quantification was 5.0 ng mL(-1) for both imipramine and desipramine, and calibration curves were linear over the concentration range of 5.0-1,000.0 and 5.0-250.0 ng mL(-1) for imipramine and desipramine, respectively. Intraday precisions at three levels were 2.2-3.6 and 1.7-4.2% for imipramine and desipramine, respectively, whereas interday precisions were 2.6-5.0 and 2.0-8.4% for imipramine and desipramine, respectively. Accuracy ranged between 93.6 and 106.6% for imipramine and 94.1 and 106.4% for desipramine. Absolute recovery was 96.0-97.6% for imipramine and 87.0-99.5% for desipramine. Finally, the described method was applied to mice administered with imipramine, demonstrating the suitability for quantification of imipramine and desipramine for therapeutic drug monitoring or bioequivalence studies.

A ketone body (acetoacetic acid, beta-hydroxybutyric acid, and acetone) increases from blood or urine when bio-energy dependence pays more fatty acid than glucose. However, in case oxidation of fat is greater than the capacity of the citric acid cycle the fatty acid oxidation is made from acetoacetyl CoA to acetoacetate then, again form beta-hydroxyburytic acid to acetone, the diffusion take place into the blood. Enzymes that oxidize ketone body in the brain and nerve tissue blood ketone dody is increased during prolonged fasting, brain used it as energy. In this study, we developed the rapid two step derivatization method for sensitive detection of the ketone body by GC-MS/SIM. The plasma was deproteinized and then the hydroxy and carboxyl groups of ketone body are subjected to extraction and drying then, keto-group were derivatized with hydoxylamine at 60 degrees C for 30 min for oximation. Then it was trimetyl-silylated with BSTFA at 80 degrees C for 30 min and analyzed using a GC-MS. The linear ranges were in between 0.001 mu g/mL and 250 mu g/mL for beta-hydroxy butyrate, and acetoacetate. The method detection limits were below 0.1 pg over each target compound determined. The mean recoveries (%) of target compounds were ranged from 88.2 % to 92.3 % at 1 mu g/mL, from 89.5 % to 94.8 % at 10 mu g/mL, with RSD of 6.3-9.4 %. This method could be applied to quantification of ketone bodies which are seen in the keto-acidosis in children and adults from a variety of diseases that cause ketones in the blood and urine.

The main purpose of newborn screening is to diagnose genetic, metabolic, and other inherited disorders, at their earliest to start treatment before the clinical manifestations become evident. Understanding and tracing the biochemical data obtained from tandem mass spectrometry is vital for early diagnosis of metabolic diseases associated with such disorders. Accordingly, it is important to focus on the entire diagnostic process, including differential and confirmatory diagnostic options, and the major factors that influence the results of biochemical analysis. Compared to regular biochemical testing, this is a complex process carried out by a medical physician specialist. It is comprised of an integrated program requiring multidisciplinary approach such as, pediatric specialist, expert scientist, clinical laboratory technician, and nutritionist. Tandem mass spectrometry is a powerful tool to improve screening of newborns for diverse metabolic diseases. It is likely to be used to analyze other treatable disorders or significantly improve existing newborn tests to allow broad scale and precise testing. This new era of various screening programs, new treatments, and the availability of detection technology will prove to be beneficial for the future generations.

Lactate and ketone bodies are considered biological markers for ketosis and several inherited metabolic disorders. In the current study, the specific ratios of lactate and ketone bodies as analytical tools for differential diagnosis of various lactic acidosis were devised. The study included a protein precipitation step following tert-butyldimethylsilyl derivatisation. Total run time was approximately 30 min including sample preparation and GS/MS analysis. The limits of detection were below 0.1 pg/mL over the targeted 4 analytes. The calibration curve was linear over the concentration range of 0.001~ 250 μg/mL for pyruvate, beta-hydroxybutyrate, and acetoacetate (R2 > 0.99). Inter-day accuracy and precision were 87.7~94.8% with RSD of 2.5~5.7% at 2 levels. Absolute recoveries (%) of target analytes were 87.0~98.4%. The method was validated for the quantification of lactate and ketone bodies for differentiation of lactic acidosis.

Alkaptonuria, a rare inherited metabolic disease, is characterized by a lack of homogentisate dioxygenase and accumulation of homogentisic acid (HGA), leading to homogentisic aciduria, arthritis, and ochronosis. In this study, a rapid analytical method, without an expensive and tedious solid phase extraction step, was developed to quantify HGA in plasma using GC-MS. HGA-spiked pooled plasma samples were subjected to liquid-liquid extraction (LLE) with ethyl acetate, followed by trimethylsilyl derivatization (TMS) and GC-MS quantification using selected ion monitoring. The formation of TMS derivative of the 1 carboxylic and 2 hydroxyl functional groups was performed by reacting BSTFA (with 10% TMCS) for 5 min at 80 degrees C. For selected ion monitoring, quantification and confirmation ions were determined based on specific ions (m/z 384, m/z 341 and m/z 252) of the TMS derivative of HGA. Calibration curves of pooled normal plasma specimens showed a linear relationship in the range of 1-100 ng/mu L. The precision and accuracy were within a relative standard deviation (RSD) of 1 to 15% and a bias of -5 to 25%. Recoveries were obtained in the range of 99-125% and 95-115% for intra-day and inter-day assay, respectively, at 2, 20 and 80 ng/mu L. The limit of detection (LOD) and limit of quantification (LOQ) were 0.4 ng/mu L and 4 ng/mu L, respectively. No homogentisic acid was excreted from normal Korean plasma samples. Collectively, the results from the present study suggest that this method could be useful for routine diagnosis and therapeutic monitoring of alkaptonuria patients with excellent sensitivity and rapidity.