Development and validation of a liquid chromatography-tandem mass spectrometry assay for the quantification of lurbinectedin in human plasma and urine.

Lurbinectedin is a novel highly selective inhibitor of RNA polymerase II triggering caspase-dependent apoptosis of cancerous cells. This article describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify lurbinectedin in human plasma and urine. Plasma samples were pre-treated with 1 M aqueous ammonia after which they were brought onto supported liquid extraction (SLE) columns. Lurbinectedin was eluted from the columns using tert-butyl methyl ether (TBME). Urine was first diluted in plasma and lurbinectedin was extracted from this matrix by liquid-liquid extraction using TBME. Samples were measured by LC-MS/MS in the positive electron ion spray mode. The method was linear over 0.1-100 ng/mL and 1-1000 ng/mL in plasma and urine, respectively, with accuracies and precisions within ±15% (20% for LLOQ) and below 15% (20% for LLOQ), respectively. The method was developed to support a mass balance study in which patients received a dose of 5 mg lurbinectedin.

Developing an activity and absorption-based quality control platform for Chinese traditional medicine: Application to Zeng-Sheng-Ping(Antitumor B).

ETHNOPHARMACOLOGICAL RELEVANCE: Zeng-Sheng-Ping (ZSP), also called antitumor B, is a marketed Chinese traditional medicine used for cancer prevention. AIM OF THE STUDY: Currently, for the quality control of Chinese traditional medicines, marker compounds are not selected based on bioactivities and pharmaceutical behaviors in most of the cases. Therefore, even if the "quality" of the medicine is controlled, the pharmacological effect could still be inconsistent. The aim of this study is to establish an activity and absorption-based platform to select marker compound(s) for the quality control of Chinese traditional medicines. MATERIALS AND METHODS: We used ZSP as a reference Chinese traditional medicine to establish the platform. Activity guided fractionation approach was used to purify the major components from ZSP. NMR and MS spectra were used to elucidate the structure of the isolated compounds. MTT assay against oral carcinoma cell line (SCC2095) was performed to evaluate the activities. UPLC-MS/MS was used to quantify the pure compounds in ZSP and the active fraction. The permeabilities of the identified compounds were evaluated in the Caco-2 cell culture model. The intracellular accumulation of the isolated compounds was evaluated in the SCC2095 cells. RESULTS: The major compounds were identified from ZSP. The contents, anti-proliferation activities, permeabilities, and intracellular accumulations of these compounds were also evaluated. The structure of these purified compounds were identified by comparing the NMR and MS data with those of references as rutaevine (1), limonin (2), evodol (3), obacunone (4), fraxinellone (5), dictamnine (6), maackiain (7), trifolirhizin (8), and matrine (9). The IC50 of compounds 5, 6, and 7 against SCC2095 cells were significantly lower than that of ZSP. The uptake permeability of compounds 5, 6, and 7 were 2.58 ± 0.3 × 10(-5), 4.33 ± 0.5 × 10(-5), and 4.27 ± 0.8 × 10(-5) respectively in the Caco-2 cell culture model. The intracellular concentrations of these compounds showed that compounds 5, 6, and 7 were significantly accumulated inside the cells. CONCLUSION: Based on the activity against oral carcinoma cell line as well as the absorption permeability, compound 5, 6, and 7 are selected as quality control markers for ZSP. An activity and absorption-based platform was established and successfully used for the quality control of ZSP.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

Simultaneous determination of evodiamine and evodine in Beagle dog plasma using liquid chromatography tandem mass spectrometry.

A sensitive, rapid, and specific liquid chromatography/tandem mass spectrometry assay has been established and validated for the quantitation of evodiamine and evodine in Beagle dog plasma. Plasma samples of 0.2 ml were processed by liquid-liquid extraction with n-hexane/ethyl acetate (2:1, v/v). Chromatographic separations were done on a Symmetry C18 column (100 mm × 4.6 mm, ID, 5 µm) at 35°C with a linear gradient of methanol and 20 mM ammonium formate containing 0.2% formic acid. Evodiamine, evodine, and glibenclamide [internal standard (IS)] were ionized with an electrospray ionization source operated in positive ion mode. The MS/MS transitions were m/z 304.1 â†’ 161.1 for evodiamine, m/z 471.2 â†’ 425.1 for evodine, and m/z 494.1 â†’ 369.1 for IS. Calibration curves were linear over the concentration range of 0.1-100 ng/ml for evodiamine and 0.5-500 ng/ml for evodine. The mean extraction recoveries were 88.10 ± 3.21% for evodiamine and 81.24 ± 4.07% for evodine. The intra- and inter-day precisions were less than 11.10% and 12.81%, and the accuracy was within ± 11.76% for both analytes. Evodiamine and evodine were stable during storage and analytical periods. The validated method has been successfully applied to a pharmacokinetic study of evodiamine and evodine in beagle dogs after oral administration.

Ohioensin F suppresses TNF-α-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-κB pathways in vascular smooth muscle cells.

AIMS: The expression of cell adhesion molecules on vascular smooth muscle cells is central to leukocyte recruitment and progression of atherosclerotic disease. Ohioensin F, a chemical compound of the Antarctic moss Polyerichastrum alpinum, exhibited inhibitory activity against protein tyrosine phosphatase 1B and antioxidant activity. However, published scientific information regarding other biological activities and pharmacological function of ohioensin F is scarce. In the present study, we aimed to examine the in vitro effects of ohioensin F on the ability to suppress TNF-α-induced adhesion molecule expression in vascular smooth muscle cells (VSMCs). MAIN METHODS: The inhibitory effect of ohioensin F on TNF-α-induced upregulation in expression of adhesion molecules was investigated by enzyme-linked immunosorbent assay, cell adhesion assay, RT-PCR, western blot analysis, immunofluorescence, and transfection and reporter assay, respectively. KEY FINDINGS: Pretreatment of VSMCs with ohioensin F at nontoxic concentrations of 0.1-10 µg/ml dose-dependently inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In addition, ohioensin F suppressed adhesion of THP-1 monocytes to TNF-α-stimulated VSMCs. Ohioensin F reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Finally, ohioensin F inhibited TNF-α-induced CAM mRNA expression and NK-κB translocation. SIGNIFICANCE: These results suggest a new mechanism of ohioensin F's anti-inflammatory action, owing to the negative regulation of TNF-α-induced adhesion molecule expression, monocyte adhesion and ROS production in vascular smooth muscle cells. Our finding also supports ohioensin F as a potential pharmacological, anti-inflammatory molecule that has a protective effect on the atherosclerotic lesion.

Structure of the lycorinine alkaloid nobilisitine A.

The structure 3 recently proposed, on the basis of computed NMR chemical shifts, for the natural product nobilisitine A has been synthesized from its C5-epimer (+)-clividine (4). The spectral data derived from compound 3 match those reported for the natural product.

RP-HPLC-DAD for simultaneous estimation of mahanine and mahanimbine in Murraya koenigii.

Murraya koenigii leaves (Rutaceae) are widely used as food condiments in various food preparations in India. They possess a wide range of biological activities including antioxidant, antibacterial, anticancer, hypoglycemic and hypolipidemic activity. A rapid reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed for quantitative estimation of mahanine and mahanimbine, two major bioactive alkaloids in this plant. The amounts of mahanine and mahanimbine were detected as 9.56 ± 1.04 and 4.32 ± 0.81% w/w in the extract, with the retention times of 6.26 ± 0.66 and 10.40 ± 0.95 minutes. The limits of detection and quantification were estimated to be 29.30 and 81.12 µg/mL and 1.67 and 6.31 µg/mL, respectively. This specific and precise validated method can be useful for the routine analysis and quantitative determination of mahanine and mahanimbine in this therapeutically potent medicinal plant.

HPLC isolation of antioxidant constituents from Xanthoparmelia spp.

A chromatographic method is described for the purification and characterization of secondary lichen substances with biological activity. A simple reversed-phase high-performance liquid chromatography method with gradient elution has been developed that allows the determination and isolation of salazinic, usnic and stictic acids from lichen samples in a single run and the quantification of every acid in the tested extracts. The antioxidant activity of both the isolated compounds and the respective lichen belonging to Xanthoparmelia genus was determined by the Oxygen Radical Absorbance Capacity (ORAC) assay; their effect as free radical scavengers, effect on cell survival by the 3(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium reduction assay and 2',7'-dichlorofluorescin diacetate method were tested on U373 MG human astrocytome cell line. Both lichens extracts and all isolated compounds protected U373 MG cells from hydrogen peroxide-induced damage, suggesting that they could act as antioxidant agents in those neurodegenerative disorders associated with oxidative damage, such as Alzheimer's disease and Parkinson's disease.

Polyphenolic profiles and antioxidant activities of heartnut (Juglans ailanthifolia Var. cordiformis) and Persian walnut (Juglans regia L.).

The polyphenolic compositions of three heartnut (Juglans ailanthifolia var. cordiformis) varieties (Imshu, Campbell CW1, and Campbell CW3) were examined and compared with those of two Persian walnut (Juglans regia L.) varieties (Combe and Lake). The nuts were defatted, extracted, and separated into three different fractions, the free phenolic acid (FPA), acid-hydrolyzable phenolic acid (AHPA), and bound phenolic acid (BPA) fractions. The total phenolic contents (TPCs) in both FPA and AHPA of the Persian walnuts were significantly higher (P < 0.001) than those of the heartnuts, but not in the BPA (P = 0.20). LC-ESI-MS(n)() studies revealed that except for the FPA fraction, the major polyphenolics in both heartnut and Persian walnut were ellagic acid and valoneic acid dilactone. Persian walnuts contained an average of 0.29 and 1.31 mg of ellagic acid/g nut in the 80% methanol extractable fractions FPA and AHPA, respectively. Heartnuts contained an average of 0.16 and 0.60 mg of ellagic acid/g nut in the respective fractions. Bound ellagic acid in the residue was 0.93 and 0.70 mg/g of nut in the Persian walnut and in the heartnut, respectively. Valoneic acid dilactone was tentatively identified and quantified as milligrams of ellagic acid equivalent per gram of nut. These components were found to contribute to the strong total antioxidant activities measured using ferric reducing antioxidant power and photochemiluminescence methods.

Iron-catalyzed homo-coupling of simple and functionalized arylmagnesium reagents.

Iron-catalyzed homo-coupling of simple and functionalized arylmagnesium reagents is described. The reaction is highly chemoselective (CN, COOEt and NO(2) groups are tolerated). The procedure was used to perform intramolecular couplings. This cyclization reaction is the key step of the total synthesis of the N-methylcrinasiadine.

Hydration, stability, and phase transformations of a new antitumor drug.

We studied hydration equilibria and phase transformations in a cytotoxic drug (BBR3576). The original sample is a hydrated compound that undergoes a structural phase transition when it looses about half of its structural water. Such a structural transition is completely reversible: the partially dehydrated form is stable up to 130 degrees C (or up to 140 degrees C for several minutes) and reverts to the original form upon rehydration. Completely different phase relationships are observed if an original sample is fully dehydrated: when all water has been released, a metastable anhydrous phase forms, which undergoes an irreversible exothermic conversion to a stable phase. Upon rehydration at room temperature of such an anhydrous phase, a new hydrated form is obtained, which is definitely different from the original one.

[Isolation and quantitative analysis of the alpha-amylase inhibitor in Lagerstroemia speciosa (L.) Pers. (Banaba)].

Banaba [Lagerstroemia speciosa (L.) Pers.] has been used as a folk medicine for diabetes in the Philippines. Using bioassay-guided separation, valoneaic acid dilactone (1) was isolated from the leaves as a potent alpha-amylase inhibitor. A simple and efficient method for the quantitative determination of valoneaic acid and its derivatives in Banaba extract was established. Valoneaic acid exists as the structural part of the polyphenols, which like flosin A, reginin A, and lagerstroemin, are characteristic constituents of Banaba. These derivatives were hydrolyzed to valoneaic acid by HCl and extracted with 2-butanone. This extract was subjected to HPLC analysis, and the contents of valoneaic acid determined as the whole valoneaic acid contents. Using this method, the whole valoneaic acid contents were measured in eight Banaba leaf decoctions. The alpha-amylase-inhibiting activities of the decoctions were dependent on the whole valoneaic acid contents. In addition, a strong linear correlation was observed between the whole valoneaic acid contents and total polyphenol contents. This analytical procedure is applicable to the chemical evaluation of Banaba.