Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometry

Analysis of caffeine and its metabolites is of interest with respect to caffeine exposure, for kinetic and metabolism studies and for opportunistic in vivo estimation of drug metabolizing enzyme activity in humans and animals. For the latter, analysis is usually done by high-performance liquid chromatography (HPLC) with UV detection. However, this method is close to the detection limit for certain of the metabolites and requires very long chromatography, 30-60 min. We have developed a fast method for the quantification of caffeine and its metabolites 1-methylxanthine, 1-methyluric acid, 1,7-dimethyluric acid, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by HPLC tandem mass spectrometry (MS/MS) in urine that requires only its dilution with buffer and centrifugation before injection into the HPLC/MS/MS system. The chromatography lasts 7 min and is followed by 4.5 min for re-equilibration of the HPLC column, giving a total analysis time of 11.5 min. The method provides a great sensitivity improvement with detection limits for all analytes 相似文献   

Temperature dependence of near-resonant vibration ↔ rotation energy transfer

V ? R,T deactivation of the ν₄ (243 cm^?1) level in BCl₃ by HCl has been measured over the range 220–340 K, with an infrared double resonance technique. The deactivation probabilities show an inverse temperature dependence below room temperature, indicating that long-range attractive forces are important in the resonant transfer process. The results are compared with the theory of Sharma and Brau.     

Direct optical detection of singlet oxygen from a single cell

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1deltag --> X3sigmag-) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.     

Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem

Diagnostic PCR has been used to analyse a wide range of biological materials. Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification. PCR is often inhibited by contamination of DNA templates. To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published. The methods are either chemical or based on filtering. Conventional ways of filtering include mechanical filters or washing e.g. by centrifugation. Another way of filtering is the use of electric fields. It has been shown that a cell will experience a force when an inhomogeneous electric field is applied. The effect is called dielectrophoresis (DEP). The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid. DEP has been applied to manipulate cells in many microstructures. In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system. Haemoglobin and heparin - natural components of blood - were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented. The usefulness of DEP in a microsystem to withhold baker's yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed. This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem.     

Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity

The development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1-0.5; rocket immunoelectrophoresis, 0.5-0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico-chemical differences between different molecular forms of the lipase, e.g. forms with different pI.     

Understanding the factors controlling the photo-oxidation of natural DNA by enantiomerically pure intercalating ruthenium polypyridyl complexes through TA/TRIR studies with polydeoxynucleotides and mixed sequence oligodeoxynucleotides

Ruthenium polypyridyl complexes which can sensitise the photo-oxidation of nucleic acids and other biological molecules show potential for photo-therapeutic applications. In this article a combination of transient visible absorption (TrA) and time-resolved infra-red (TRIR) spectroscopy are used to compare the photo-oxidation of guanine by the enantiomers of [Ru(TAP)₂(dppz)]²⁺ in both polymeric {poly(dG-dC), poly(dA-dT) and natural DNA} and small mixed-sequence duplex-forming oligodeoxynucleotides. The products of electron transfer are readily monitored by the appearance of a characteristic TRIR band centred at ca. 1700 cm⁻¹ for the guanine radical cation and a band centered at ca. 515 nm in the TrA for the reduced ruthenium complex. It is found that efficient electron transfer requires that the complex be intercalated at a G-C base-pair containing site. Significantly, changes in the nucleobase vibrations of the TRIR spectra induced by the bound excited state before electron transfer takes place are used to identify preferred intercalation sites in mixed-sequence oligodeoxynucleotides and natural DNA. Interestingly, with natural DNA, while it is found that quenching is inefficient in the picosecond range, a slower electron transfer process occurs, which is not found with the mixed-sequence duplex-forming oligodeoxynucleotides studied.

Efficient electron transfer requires the complex to be intercalated at a G-C base-pair. Identification of preferred intercalation sites is achieved by TRIR monitoring of the nucleobase vibrations before electron transfer.     

Nonadiabatic alignment of asymmetric top molecules: rotational revivals

The rotational revival structure of asymmetric top molecules, following irradiation by an intense picosecond laser pulse, is explored theoretically and experimentally. Numerically we solve nonperturbatively for the rotational dynamics of a general asymmetric top subject to a linearly polarized intense pulse, and analyze the dependence of the dynamical alignment on the field and system parameters. Experimentally we use time-resolved photofragment imaging to measure the alignment of two molecules with different asymmetry, iodobenzene, and iodopentafluorobenzene. Our numerical results explain the experimental observations and generalize them to other molecules. The rotational revival structure of asymmetric tops differs qualitatively from the intensively studied linear top case. Potentially it provides valuable structural information about molecules.     

Influence of epidermal thickness,pigmentation and redness on skin autofluorescence

Detection of autofluorescence at the skin surface is highly influenced by melanin and hemoglobin. Epidermal absorption and scattering may also be an influencing factor and is represented in this article as a quantitative parameter, epidermal thickness. To examine this parameter we measured the 370 nm fluorescence in vivo after excitation with 330 nm and the 455 nm fluorescence after excitation with 330 and 370 nm. Measurements were performed on sun-exposed skin at the dorsal aspect of the forearm and shoulder and on nonexposed buttock skin. Skin pigmentation and redness of the same body sites were measured by reflectance spectroscopy. The thickness of the stratum corneum and the cellular part of epidermis was quantified by light microscopy of skin biopsies. Multiple regression analysis was used to find correlations between autofluorescence and the potential influencing factors. We found a highly significant correlation of skin autofluorescence with pigmentation and redness for both emission wavelengths (Em). A small but significant correlation to epidermal thickness was found only for excitation wavelength (Ex) 370 nm and Em455 nm if body site was included in the analysis. No correlation between Ex330:Em370 and Ex330:Em455 and thickness of epidermis was found. For practical use, correction of skin autofluorescence for pigmentation is essential, correction for redness is of less importance and correction for epidermal thickness is unnecessary.