Accurate LC-MS/MS Analysis of Diacylglycerols in Human Plasma with Eliminating Matrix Effect by Phospholipids Using Fluorous Biphasic Extraction.

We developed an accurate method for determining diacylglycerols (DAGs) in human plasma using a fluorous biphasic liquid-liquid extraction method, followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The lipid mixture in the plasma was first extracted with chloroform by using the Bligh-Dyer method. The resulting solution was subjected to fluorous biphasic liquid-liquid extraction to remove phospholipids, which are known to cause matrix effects during the LC-MS/MS analysis. In this method, phospholipids in a lipid mixture solution (nonfluorous solvent) were selectively extracted to tetradecafluorohexane (fluorous solvent) via the specificity of fluorous affinity by forming a complex with a perfluoropolyethercarboxylic acid-lanthanum(III) salt. The remaining DAGs in the nonfluorous solvent could be directly injected into the LC system through the positive electrospray ionization-MS/MS mode. The removal rate of the phospholipids through the fluorous biphasic extraction was more than 99.9%; thus, the matrix-effect-eliminating analysis of DAGs in human plasma with LC-MS/MS was enabled. Furthermore, the applicability of this method and the possibility of using DAGs as biomarkers were evaluated by applying this method to human plasma samples obtained from major depressive disorder as a related disease.

Liquid Chromatographic Determination of o-Phosphoethanolamine in Human Plasma Using Fluorescent Derivatization.

In this study, an HPLC analysis method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was developed for the determination of o-phosphoethanolamine (PEA), which is a potential biomarker for the diagnosis of major depressive disorder, in human plasma sample. After PEA was derivatized with AQC under mild conditions, the obtained derivative was subjected to purification with a titanium dioxide-modified monolithic silica spin column (MonoSpin® TiO). The eluate from the MonoSpin® TiO was directly injected into an amide-type hydrophilic interaction liquid chromatography (HILIC) column-equipped HPLC system, and the resulting derivative could be separated on the column under alkaline mobile phase conditions and subsequently detected fluorometrically at excitation and emission wavelengths of 250 and 395 nm, respectively. The limit of detection and limit of quantification for a 10 µL injection volume of PEA were 0.052 and 0.17 µM, respectively. The method was validated at 0.2, 1.0, and 5.0 nmol/mL levels in plasma sample, and the precision values were 2.0-6.6% as relative standard deviation and the correlation coefficient (r) of the calibration curve was 0.9995. Furthermore, applicability of this method was demonstrated by analyzing PEA levels in plasma samples from mental illness patients.

Discrimination of Malignant Pleural Mesothelioma Cell Lines Using Amino Acid Metabolomics with HPLC.

Malignant pleural mesothelioma (MPM) is a malignancy closely associated with asbestos exposure. Although early diagnosis provides a chance of effective treatment and better prognosis, invasive biopsy and cytological procedure are required for definitive diagnosis. In this study, we developed a method to differentiate between MPM and control cell lines, named "amino acid metabolomics," consisting in the assessment of the balance of their amino acid levels in the cell culture medium. Culture media of MESO-1 (MPM cell line) and Met-5A (control) cells were used in this study to evaluate amino acid levels using HPLC, following the fluorescence derivatization method. The time-dependent changes in amino acid levels were visualized on the score plot following principal component analysis, and the results revealed differential changes in amino acid levels between the two cell culture supernatants. A discriminative model based on linear discriminant analysis could distinguish MPM and control cells.

Deep eutectic solvent extraction of cortisol and cortisone from human saliva followed by LC-UV analysis.

A novel extraction method was developed for the determination of cortisol and cortisone. In this study, we prepared a hydrophobic deep eutectic solvent (DES) by mixing trioctylmethylammonium chloride and pentafluorophenol as a hydrogen bond acceptor and a hydrogen bond donor, respectively, for use as the extraction solvent. The extraction of cortisol and cortisone was performed by adding a small volume of the DES to the aqueous sample. After centrifugation, the resulting sedimented DES phase was injected into a reversed-phase liquid chroamtography column, and the analytes were detected with an ultraviolet detector at 254 nm. Under the optimized extraction conditions, the enrichment factors of cortisol and cortisone were 9.3 and 8.5, respectively. Furthermore, the linear dynamic ranges were established over a concentration range of 10-200 pmol mL-1 (r2 > 0.9992), and the limits of detection of cortisol and cortisone were found to be 2.1 and 1.8 pmol mL-1, respectively. The applicability of this method was evaluated by analyzing the cortisol and cortisone contents of human saliva samples.

Delayed mesh infection and mesh penetrating the transverse colon and small intestine after abdominal incisional hernia repair.

The occurrence of late-onset mesh infection and mesh invasion into the intestine after abdominal incisional hernia repair is extremely rare. Herein, we describe the first case of late-onset mesh infection and mesh penetrating the transverse colon and small intestine 5 years after incisional hernia repair using an expanded polytetrafluoroethylene mesh. The symptom was drainage from the reddish wound, and computed tomography scan revealed intestinal prolapse with local wall thickening. The mesh removal and small intestine and colon resection were conducted because the small intestine and transverse colon formed a mass containing the mesh inside. The events were caused by the lack of mesh fixation, and the dislodged mesh penetrating the intestinal tract caused the infection. For mesh infections in which conservative treatment is not effective, mesh removal and organ excision should not be delayed regardless whether there is a strong adhesion of the abdominal cavity.

Fluorous and Fluorogenic Derivatization for Selective Liquid Chromatographic Analysis of Cyanide in Human Plasma.

A liquid chromatographic (LC) method with fluorous derivatization for the determination of cyanide in human plasma is described. In this method, the cyanide was transformed to a fluorous and fluorogenic compound by derivatizing with 2,3-naphthalenedialdehyde and perfluoroalkylamine reagent under mild reaction conditions (a reaction time of 5 min at room temperature). The obtained derivative was successfully retained on the perfluoroalkyl-modified LC column with the use of a high concentration of organic solvent in the mobile phase, whereas non-fluorous derivative was hardly retained, followed by fluorometric detection at excitation and emission wavelengths of 420 and 490 nm, respectively. Under the optimized conditions, the limit of detection and the limit of quantification for cyanide in a 5-µL injection volume were 1.3 µg/L (S/N = 3) and 4.4 µg/L (S/N = 10), respectively. The recovery from spiked human plasma was achieved in the range of 54 - 90% within a relative standard deviation of 3.5%. The feasibility of this method was further evaluated by applying it to the analysis of human plasma samples.

Application of a fluorous derivatization method for characterization of glutathione-trapped reactive metabolites with liquid chromatography-tandem mass spectrometry analysis.

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.

Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics.

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drug-treated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro.

Amino Acid Metabolomics Using LC-MS/MS: Assessment of Cancer-Cell Resistance in a Simulated Tumor Microenvironment.

We performed a comprehensive quantification of 20 amino acids in RPMI 1640 medium-cultured human colorectal adenocarcinoma cells to evaluate the efficacy of 5-fluorouracil treatment under hypoxic and hypoglycemic conditions, which mimic the tumor microenvironment. In this study, we developed a simple and comprehensive analytical method by using LC-MS/MS connected to the Intrada amino acid column, which eluted amino acids within 9 min. The present method covered a linearity range of 3.6 - 1818 µM, except for Gly (227 - 1818 µM), Ala, Asp, His (7.1 - 1818 µM each), and Trp (3.6 - 909 µM). The limits of detection were in the range of 0.02 - 38.0 pmol per injection in a standard solution. Amino acid concentration data were analyzed using principal-component analysis to represent samples on two-dimensional graphs. Linear discriminant analysis was used to classify samples on the score plots. Using this approach, the effect of 5-fluorouracil treatment could be successfully discriminated at high discrimination rates. Moreover, several amino acids were extracted from corresponding loading plots as candidate markers for distinguishing the effects of the 5-fluorouracil treatment or tumor microenvironmental conditions. These results suggest that our proposed method might be a useful tool for evaluating the efficacy of anticancer drugs in the tumor microenvironment.

Screening of promising chemotherapeutic candidates from plants extracts.

Over the course of our studies investigating anti-proliferative properties of compounds originating from plants against human gastric adenocarcinoma (MK-1), human uterine carcinoma (HeLa), murine melanoma (B16F10), and two human T cell lymphotropic virus type 1 (HTLV-1)-infected T-cell lines (MT-1 and MT-2), we have screened 582 extracted samples obtained from a variety of parts from 370 plants. A few extracts showed anti-proliferative activity against all cell lines, but upon further investigation, toxicity toward selected cell lines was recognized. After activity-guided fractionation, isolation of the active principles was achieved. Structure-activity relationship studies identified the components and functionalities responsible for the specific selectivity against each cancer cell line. The effect of polyacetylenes against MK-1 cells was more potent than against HeLa and B16F10 cells. The compound having a 3,4-dihydroxyphenethyl group also showed an anti-proliferative effect against B16F10 cells. Some 6-methoxyflavone derivatives and 8-hydroxy furanocoumarins were good inhibitors of HeLa cell growth. The 17 compounds whose EC50 values were less than 1 nM did not show specific cellular selectivity. Because the cytotoxic effect of 24, 25-dihydrowithanolide D toward control cells was observed at a concentration about 100 times higher than those for the cancer cell lines, withanolide was identified as the most promising chemotherapeutic candidate in our experiments.

Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells.

Assessment of the efficacy of anticancer drugs by amino acid metabolomics using fluorescence derivatization-HPLC.

Metabolomic studies conducted for evaluating cancer pathogenesis and progression by monitoring the amino acids metabolic balance hold great promise for assessing current and future anticancer treatments. We performed a comprehensive quantification of 21 amino acids concentrations in cultured human colorectal adenocarcinoma cells treated with the anticancer drugs 5-fluorouracil, irinotecan, and cisplatin. A precolumn fluorescence derivatization-HPLC method involving 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was used. Amino acid concentration data were analyzed by principal-component analysis and partial least-squares multivariate statistical methods to represent samples on two-dimensional graphs. The hierarchical cluster analysis and linear discriminant analysis were used to classify the samples on the score plots. Unlike the cluster analysis approach, the linear discrimination analysis classification successfully distinguished anticancer drug-treated samples from the untreated controls. Moreover, three candidate amino acids (serine, aspartic acid, and methionine) were identified from the loading plots as potential biomarkers. Our proposed method might be able to evaluate the effectiveness of anticancer therapy even in small laboratories or medical institutions.

New α-glucosides of caffeoyl quinic acid from the leaves of Moringa oleifera Lam.

Two new caffeoyl quinic acid α-glucosides, together with three known caffeoyl quinic acids and five known flavonoid glucosides, were isolated from the leaves of Moringa oleifera Lam. The structures of the new compounds were elucidated as 4-O-(4'-O-α-D-glucopyranosyl)-caffeoyl quinic acid (1) and 4-O-(3'-O-α-D-glucopyranosyl)-caffeoyl quinic acid (2) by spectroscopic analyses.

New iridoid diesters of glucopyranose from Linaria canadensis (L.) Dum.

Two new iridoid diesters of glucopyranose were isolated from the aerial part of Linaria canadensis (L.) Dum. Eight known flavones, apigenin, diosmetin, genkwanin, luteolin, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, genkwanin 4'-O-rutinoside, and quercetin 7-O-rutinoside were also isolated. The chemical structures of the isolated compounds were elucidated based on the analyses of the spectroscopic data.

A new acylphloroglucinol glycoside from Solidago altissima L.

A new acylphloroglucinol glycoside was isolated from the leaves of Solidago altissima L. The chemical structure of the glycoside, which has a phloroglucinol moiety with a butyryl side chain, was elucidated based on the analysis of spectroscopic data.

Preparation and in vivo evaluation of a water-soluble prodrug for 2R-gamma-tocotrienol and as a two-step prodrug for 2,7,8-trimethyl-2S-(beta-carboxyethyl)-6-hydroxychroman (S-gamma-CEHC) in rat.

2R-gamma-Tocotrienol (gamma-T3) is currently receiving attention because it has beneficial effects not observed with alpha-tocopherol. To achieve the effective delivery of gamma-T3, we synthesized three kinds of ester derivatives of gamma-T3 and evaluated their use as hydrophilic prodrugs for gamma-T3 in vitro and in vivo. 2R-gamma-Tocotrienyl N,N-dimethylamino-acetate hydrochloride (compound 3) was a solid compound, with high solubility and stability in water, and was converted to gamma-T3 by esterases in rat and human liver. Intravenous administration of 3 in rats led to a rapid increase in the plasma, liver, heart, and kidney levels of gamma-T3. The bioavailability (plasma level) after intravenous administration was 82.5 +/- 13.4% and 100 +/- 11.3% for 3 and gamma-T3 in surfactant, respectively, and the availability in liver was 213 +/- 47.6% and 100 +/- 4.8% for 3 and gamma-T3 in surfactant, respectively. Furthermore, the systemic availability of 2,7,8-trimethyl-2S-(beta-carboxyethyl)-6-hydroxychroman (S-gamma-CEHC), a metabolite of gamma-T3, was 78.6% for compound 3, 47.1% for gamma-T3 in surfactant, and 100% for racemic gamma-CEHC. Based on these results, we identified compound 3 as the most promising water-soluble prodrug of gamma-T3 and two-step prodrug of S-gamma-CEHC.

Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells.

Four new triterpenes, together with 16 known triterpenes, were isolated from the floral spikes of Betula platyphylla var. japonica in a search for compounds capable of reversing multidrug resistance in cancer cells. The structures of the new triterpenes were elucidated as 3,4-seco-olean-4(23),13(18)-dien-3-oic acid (1), 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (2), 3-O-methylmalonylepiocotillol II (6), and 3-O-methylmalonylcabraleahydroxylactone (16) by spectroscopic examination. The cytotoxicity of the isolated triterpenes against human cancer cell lines as well as multidrug-resistant cancer cell lines was evaluated. Most of the isolated triterpenes showed very weak cellular toxicities. Although no discernible differences were found in the cytotoxicities for the tested compounds against sensitive and resistant cell lines, the cytotoxicities for several triterpenes against multidrug-resistant cancer cell lines (KB-C2 or K562/Adr) were enhanced in the presence of nontoxic concentrations of colchicine or doxorubicin. Compound 10 reversed the cytotoxicity of colchicine against KB-C2 cells at 8.1 microM and showed comparable potency to 5 microM verapamil.

The first topologically controlled synthesis of doubly bridged beta-cyclodextrin dimers.

Reaction 6(A),6(B)-di(O-tosyl)-beta-cyclodextrin with Na(2)S in DMF gave the cis-dimer of beta-cyclodextrin in 21% isolated yield while the trans-dimer was not detected.

Antiproliferative constituents from umbelliferae plants. IX. New triterpenoid glycosides from the fruits of Bupleurum rotundifolium.

The MeOH extract of the fruits of Bupleurum rotundifolium showed inhibitory activity against human gastric adenocarcinoma (MK-1) cell growth. Bioactivity-guided fractionation of the MeOH extract led to the isolation of four new triglycosides of 13beta,28-epoxy oleanane-type triterpenes, named rotundiosides O, Q, S and T; 12 new glycosides of oleanane-type triterpenes, named rotundiosides J-N, P, R, U-Y, and others; echinocystic acid 3-O-sulfate; and three known oleanane-type triterpene glycosides, rotundiosides A, F and G. The structures of the new isolates were determined based on chemical and spectroscopic evidence. The GI(50) of isolates against MK-1, HeLa and B16F10 cell lines are reported.

Glucocorticoid is involved in food-entrainable rhythm of mu-opioid receptor expression in mouse brainstem and analgesic effect of morphine.

The repeated manipulation of feeding schedule has a marked influence on the chronopharmaological aspects of many drugs. In this study, we investigated the role of endogenous glucocorticoid in the mechanism by which restricting the feeding time modulates the analgesic effect of morphine. Male ICR mice were housed under a light-dark cycle (light on from 07:00 to 19:00) with food and water ad libitum or under repeated time-restricted feeding (feeding time from 09:00 to 17:00) for 2 weeks before the experiment. Under the ad libitum feeding, mRNA levels of mu-opioid receptor and its binding capacity in mouse brainstem increased around the early dark phase, following the 24-h variation in circulating glucocorticoid levels. As a consequence, potent analgesic effects of morphine were observed in mice injected with the drug during the dark phase. Daily restricted feeding modulated the time-dependency of mu-opioid receptor function, accompanied by the alteration of the rhythm in circulating glucocorticoid levels. Under the time-restricted feeding, potent analgesic effects of morphine were found in mice injected with the drug during the light phase. Because the manipulation of feeding schedule was unable to produce the food-entrainable rhythm in the expression of mu-opioid receptor in the brainstem of adrenalectomized mice, endogenous rhythm of glucocorticoid secretion seems to be involved in the mechanism by which the time-restricted feeding modulates the analgesic effects of morphine.