Characterization of silvestrol pharmacokinetics in mice using liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the plant natural product silvestrol in mice, using ansamitocin P-3 as the internal standard. The method was validated in plasma with a lower limit of quantification of 1 ng/mL, accuracy ranging from 87 to 114%, and precision (coefficient of variation) below 15%. The validated method was used to characterize pharmacokinetics in C57BL/6 mice and metabolism in mouse, human and rat plasma, and liver microsomes. Mice were dosed with silvestrol formulated in hydroxypropyl-ß-cyclodextrin via intravenous, intraperitoneal, and oral routes followed by blood sampling up to 24 h. Intraperitoneal systemic availability was 100%, but oral administration resulted in only 1.7% bioavailability. Gradual degradation of silvestrol was observed in mouse and human plasma, with approximately 60% of the parent drug remaining after 6 h. In rat plasma, however, silvestrol was completely converted to silvestric acid (SA) within 10 min. Evaluation in microsomes provided further evidence that the main metabolite formed was SA, which subsequently showed no cytotoxic or cytostatic activity in a silvestrol-sensitive lymphoblastic cell line. The ability of the analytical assay to measure tissue levels of silvestrol was evaluated in liver, brain, kidney, and spleen. Results indicated the method was capable of accurately measuring tissue levels of silvestrol and suggested it has a relatively low distribution to brain. Together, these data suggest an overall favorable pharmacokinetic profile of silvestrol in mice and provide crucial information for its continued development toward potential clinical testing.

A rapid and sensitive LC-MS/MS method for quantification of four anthocyanins and its application in a clinical pharmacology study of a bioadhesive black raspberry gel.

Cyanidin 3-glucoside (C3GLU), cyanidin 3-rutinoside (C3RUT), cyanidin 3-sambubioside (C3SAM) and cyanidin 3-(2(G)-xylosyl) rutinoside (C3XRUT) are the four constituent black raspberry anthocyanins that contribute significantly to the chemopreventive effects of freeze-dried black raspberries (FBR). A highly sensitive and specific LC-MS/MS assay was developed and validated to simultaneously quantify these four FBR anthocyanins in human saliva, plasma and oral tissue homogenates. In saliva, the lower limit of quantification (LLOQ) for these anthocyanins was 1.0 ng/mL. The within-run and between-run coefficients of variations (CVs) at the quality control concentrations (1.0, 5.0, 50 and 500 ng/mL) were all <12%, except for C3SAM and C3RUT at the LLOQ, which showed a within-run CV of 18.3% and between-run CV of 16.0%, respectively. The accuracy values ranged from 87.5 to 110%. In plasma, the LLOQ for C3GLU and C3RUT was 1.0 ng/mL and for C3SAM 5.0 ng/mL. The CVs at the above concentrations were <15%, except for C3GLU at the LLOQ, which showed the between-run CV of 16.9%. The accuracy values ranged from 90.7% to 112.7% except for C3GLU at the LLOQ, which showed 119.3%. In tissue homogenates, the LLOQ for C3GLU and C3RUT was 2.0 ng/mL, and C3SAM 5.0 ng/mL. The CVs and accuracy values at concentrations (2.0, 5.0, 50 and 500 ng/mL) were similar to those in human plasma. This assay was subsequently used in a pilot pharmacology study to evaluate the effects of topical application of a 10% (w/w) FBR bioadhesive gel to selected mucosal sites in the posterior mandibular gingiva. Measurable saliva and tissue levels of the FBR anthocyanins confirmed that gel-delivered anthocyanins are readily distributed to saliva and easily penetrate human oral mucosa.

Ion-pair solid-phase extraction and gas chromatography-mass spectrometric determination of acidic hydrolysis products of chemical warfare agents from aqueous samples.

The chemical warfare agents (CWA) degrade rapidly in aqueous samples and convert to acidic degradation products. Extraction and identification of the degradation products from complex matrices using simple sample preparation and sensitive detection and identification is the most important step in the off-site analysis of samples. In this present study, we report a simple sample preparation step based on ion-pair (IP) solid-phase extraction (SPE) for the extraction of acidic degradation products of CWA namely methyl, ethyl, propyl phosphonic acids, thiodiglycolic acid and benzilic acid. The analysis was performed on GC-MS in electron impact ionization mode. Three IP reagents triethylamine (TEA), tetrabutylammonium bromide (TBAB) and cetyltrimethyl ammonium bromide (CTAB) were used. The recoveries were estimated using the internal and external standard methods. The recovery of the compounds was almost negligible when TEA was used as IP reagent. The recoveries obtained when TBAB and CTAB were used as IP reagents were high and reproducible. The recovery of test chemicals is above 90%, except for methyl phosphonic acid and ethylphosphonic acid (20.6 +/- 3.2% and 35.8 +/- 2.5%, respectively). The minimum detection limits of the method were calculated for all chemicals in both full scan and selected ion monitoring modes. The test chemicals could be detected in microgram per litre quantities by the IP-SPE method.

Mass spectral studies of a series of N,N-dialkyl aminoethyl-2-chlorides and trimethyl silyl ethers of N,N-dialkyl aminoethane-2-ols under electron impact conditions.

The electron impact (EI) mass spectra of a series of N,N-dialkyl-aminoethyl-2-chlorides, N(R(1))(R(2))-CH(2)-CH(2)Cl and trimethylsilyl ethers of N,N-dialkyl aminoethane-2-ols, N(R(1))(R(2))-CH(2)-CH(2)-O-Si(CH(3))(3), where R(1) and R(2) = methyl, ethyl, propyl and isopropyl, which are precursors of VX type of compounds, are studied. All the compounds (1-20) show abundant molecular ions, in addition to a weak [M - H](+) ion, except the N,N-diisopropyl group containing compounds (8 and 18). A general EI fragmentation pattern for the above two series of compounds is discussed. The observed fragment ions are due to simple homolytic cleavages, and they are distinct to allow the identification of the compounds unequivocally including those of isomeric compounds. The primary fragmentation of compounds 1-20 is beta-cleavage, i.e. homolytic cleavage of C-C bond, which is linked to the nitrogen atom. Three types of beta-cleavages are possible for these compounds, in which the abundance of beta-cleavage product ions is found to depend on the size and structure of the alkyl group attached to nitrogen. The alpha-cleavage fragment ions are found only for N,N-dialkyl aminoethyl-2-chlorides but are absent in the corresponding trimethylsilyl ethers of N,N-dialkyl aminoethane-2-ols. The retention indices are calculated for all the studied compounds (1-20) and are in the ranges of 750.38-1079.24 for 1-10 and 905.23-1190.25 for 11-20.

Mass spectral analysis of chloropicrin under negative ion chemical ionization conditions.

A chemical ionization (CI) method is developed for the first time to obtain molecular weight information for chloropicrin (CP), which is used as a chemical warfare agent and as an insecticide. The study includes a detailed investigation on the behavior of CP under electron impact (EI) and CI. Reagent gases of different nature, i.e., methane, isobutane, ammonia, hydrogen, and carbon dioxide, were used for CI analysis. Negative ion mode is found more sensitive than positive ion mode for the EI/CI mass spectrometric analysis of CP, but none of the methods provided molecular weight information, except negative ion CI using ammonia as the reagent gas (NICI (NH3)). The NICI (NH3) showed formation of the quasi-molecular ion, [M + H]-, in addition to other adduct ions. The [M + H]- abundance critically depends on the source temperature, reagent gas pressure, and concentration of the analyte, and it can be 13% under optimized conditions by which CP can be confirmed unambiguously. This method meets the criteria used in official proficiency tests conducted by OPCW for confirming the molecular weight of the unknowns.

Mass spectral studies of N,N-dialkylaminoethanols.

Some dialkylaminoethanols, precursors of chemical warfare agents such as V-agents and nitrogen mustards, were analyzed by electron impact (EI) and electrospray ionization (ESI) mass spectrometry. The fragmentation pathways in EI and ESI-MS/MS methods are rationalized. The collision-induced dissociation (CID) spectra of [M+H](+) ions of aminoethanols in ESI mode are clearly distinguishable from one another, including those of isomeric normal and branched chain dialkylaminoethanols. Structures can be proposed based on the general fragmentation pathways of these molecules.