Polycyclic aromatic hydrocarbons (PAHs) are abundant pollutants, and many PAHs are carcinogenic, ... more Polycyclic aromatic hydrocarbons (PAHs) are abundant pollutants, and many PAHs are carcinogenic, but only after metabolic activation. Benzo[a]pyrene (BaP) is among the most carcinogenic PAHs. The dose and time response of two enzymes involved in BaP metabolism and the amounts of BaP metabolites excreted into the bile were evaluated in an experiment with dab (Limanda limanda). Ninety dab were exposed orally to one of five doses of BaP (0, 0.08, 0.4, 2, or 10 mg/kg) and sampled at 3, 6, or 12 d after exposure. None of the doses studied caused significant induction of either microsomal ethoxyresorufin-O-deethylase (EROD), which reflects cytochrome P450 1A (CYP1A) activity, or cytosolic glutathione-S-transferase activity (GST). Concentrations of biliary BaP metabolites significantly increased with dose and significantly decreased with time after exposure. It is concluded that biliary BaP metabolites provide a much more sensitive method than EROD (CYP1A) or GST activity to monitor recent exposure to PAHs in dab.
Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brai... more Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron co-culture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe2+ (Fenton reaction). Raman peaks related to nucleic acids (725 cm-1, 782 cm-1, 1092 cm-1, 1320 cm-1, 1340 cm-1, 1420 cm-1, 1576 cm-1) showed time-dependent changes over the experimental period (60 min), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when co-treated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra were observed for untreated control cells and for cells exposed to Fe2+ only, H2O2 only and ascorbate only. A live dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent non invasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.
Ultraviolet resonance Raman spectroscopy (RRS) is presented as a novel identification tool for co... more Ultraviolet resonance Raman spectroscopy (RRS) is presented as a novel identification tool for conventional-size column liquid chromatography (LC). The on-line coupling was made using a standard Z-shaped flow cell. A continuous-wave frequency-doubled argon ion laser operating at a wavelength of 244 nm was used for excitation. "On-thefly" resonance Raman spectra of four model compounds, fluorene, phenanthrene, fluoranthene, and pyrene, were recorded after a standard acetonitrile/water reversedphase LC separation. When applying a large-volumeinjection procedure (32 mL), detection limits were at the nanogram per milliliter level. The results indicate that UV-RRS gives detailed spectral information at an appropriate sensitivity level so that coupling with LC becomes feasible.
Hyphenation of thin layer chromatography (TLC) with surface-based spectral methods requires a hom... more Hyphenation of thin layer chromatography (TLC) with surface-based spectral methods requires a homogeneous surface for direct and quantitative analysis on the chromatographic plate after separation. Since most chromatographic materials do not produce strong background signals in Raman spectroscopy (RS) or surface-enhanced RS (SERS), we tested the suitability of two different chromatographic substrates and one interface for coupling SERS with TLC.
Tranexamic acid (TA) is a synthetic antifibrinolytic agent that is being considered as a candidat... more Tranexamic acid (TA) is a synthetic antifibrinolytic agent that is being considered as a candidate adjuvant drug for site-specific pharmaco-laser therapy of port wine stains. For drug utility studies, a high-performance liquid chromatography (HPLC)-fluorescence method was developed for the quantification of TA in blood. Platelet-poor plasma was prepared, size-separated using 3 kDa cut-off centrifuge filters, and derivatized with naphthalene-2-3-dicarboxaldehyde (NDA) and cyanide. The excess of NDA was quenched after 2 min by adding tryptophan. The derivatives were separated on a 2.1 mm C18 column using an acetate buffer/acetonitrile gradient. Excellent separation from plasma background was obtained at pH 5.5. Quantification was carried out at 440/520 nm. The limit of detection was 0.5 M and the mean ± SD recovery from whole blood was 81.7 ± 10.9%. Derivatized TA samples were stable for at least 36 h at 4 • C. The method was successfully applied to a heat-induced TA release study from thermosensitive liposomes.
In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid ch... more In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 3 10 À6 A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10 À4 A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 3 10 À5 A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.
In this preliminary study, we evaluated four different types of substrate for the at-line couplin... more In this preliminary study, we evaluated four different types of substrate for the at-line coupling of capillary electrophoresis and surface-enhanced (resonance) Raman spectroscopy, CE–SER(R)S, with emphasis on spectral repeatability. We tested Sub1: etched silver foil, Sub2: a vapour-deposited silver film, Sub3: a silver oxalate-precoated silica TLC plate and Sub4: a silica TLC plate on which colloid and poly(l-lysine) were manually
This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-ox... more This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.
Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous s... more Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous solutions are shown to exhibit relatively strong room temperature phosphorescence (RTP). Among the various CDs tested, ␣-CD showed the strongest RTP signals. Interestingly, these signals differed significantly for the two enantiomers of CQ; the phosphorescence lifetime of (+)-CQ was about four times longer than that of (−)-CQ, being 352 ± 16 and 89 ± 6 s, respectively. This enantiomeric selectivity is attributed to a difference in dissociation rates (competing with the radiative emission process) for the diastereoisomeric inclusion complexes dealt with, which have a 2:1 stoichiometry (␣-CD:CQ:␣-CD). Time-resolved RTP detection using different delay times enables the determination of the two enantiomers in a mixture without involving a separation technique. The minimum detectable fraction of (+)-CQ in a 2 mM sample was 13%.
Adsorption kinetics and molecular interactions on different surface interfaces are studied by mea... more Adsorption kinetics and molecular interactions on different surface interfaces are studied by means of evanescent-wave cavity ring-down spectroscopy, using total internal reflection surfaces onto which different self-assembled monolayers are covalently attached. The adsorption of cytochrome c (a positively charged, spherical heme protein) to a negatively charged bare silica surface, as well as to C 18 -coated (hydrophobic) and C 3 NH 2 -coated (positively charged) silica have been studied. It is experimentally verified that these surface layers do not interfere with the sensitive measurement of adsorbed cyt c monolayers using the evanescent wave in a ring-down scheme. Attaching monolayers covalently to the silica total internal reflection surface is a first step towards the development of a biosensor that makes use of immobilized biomolecules for specific detection of analytes in solution.
Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberell... more Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional ...
Journal of Photochemistry and Photobiology A: Chemistry, 2011
ABSTRACT A paper by Amat et al. [2] reported that the ATP-driven oxidation of luciferin to electr... more ABSTRACT A paper by Amat et al. [2] reported that the ATP-driven oxidation of luciferin to electronically excited oxyluciferin catalyzed by luciferase was accelerated when ATP was priorly irradiated at non-resonant optical frequencies (NROF) at 635 and 830nm. In another paper by Amat et al. [3], increased fluorescence intensities of ATP–Mg complexes, which showed lower fluorescence than ATP when excited at 260nm, were reported in consequence of concomitant NROF irradiation (i.e., 655 and 830nm). It was postulated that NROF-induced electric field changes may alter the charge distribution in ATP's phosphate chain, resulting in lowering of the activation energy of its terminal phosphate. Here we use spectrofluorometry to further investigate this hypothesis. The effect of NROF (at 632.8nm) on the intrinsic fluorescence of non-complexed and Mg-chelated ATP in aqueous solution and the influence of NROF (514.5nm and 632.8nm) on the rate of the luciferin–luciferase reaction was studied. We found that neither the intrinsic fluorescence of ATP nor its biochemical behavior during the firefly luciferin–luciferase reaction was affected by laser irradiation with NROF. Consequently, no evidence was found supporting the postulation that NROF-induced alternations on the charge distribution of the phosphate chain affect the reactivity of ATP.
The effect of disturbed root nodulation on the quantitative and qualitative composition of the ma... more The effect of disturbed root nodulation on the quantitative and qualitative composition of the main isoflavonoid glucosideYmalonates, glucosides, and aglycones in the leaves of Trifolium pratense L. grown under waterlogging conditions was investigated. Isoflavonoids are involved in the regulation of root nodule activity and the establishment of the mycorrhizal association. Isoflavonoid determination was performed using reversed-phase liquid chromatography coupled to mass spectrometric and UV absorbance detection. In response to waterlogging, the concentrations of biochanin A and biochanin AY7-O-glucosideYmalonate, biochanin AY7-O-glucoside, and gen-isteinY7-O-glucoside in the leaves increased two-to threefold after a lag period of 3 wk because of disturbed root nodulation. The other isoflavones detectedVformononetin, formononetinY7-O-glucosideYmalonate, and for-mononetinY7-O-glucosideVdid not show any significant changes related to waterlogging. After restoring normal soil water conditions, the concentrations of biochanin A and its glucoside and glucosideYmalonate rapidly returned to the initial values, whereas the concentration of genisteinY7-O-glucoside remained high.
... Correspondence: Gerard J. Stroomberg,, Vrije ... Compared to PAH metabolism studies on benzo[... more ... Correspondence: Gerard J. Stroomberg,, Vrije ... Compared to PAH metabolism studies on benzo[a]pyrene in other invertebrates, such as sea star (Asterias rubens) [17] or shore crab ... 5Van der Oost R, Van Schooten FJ, Ariese F, Heida H, Satumalay K, Vermeulen NPE: 1994. ...
Samples of sediment and eel taken from six sites in Amsterdam with different levels of water poll... more Samples of sediment and eel taken from six sites in Amsterdam with different levels of water pollution were ana lyzed for 16 parental PAHs In addition, biliary PAH metabolites and hepatic PAH-DNA adducts were determined in the eel to evaluate biomonitoring techniques for PAH exposure There was a clear difference between PAH profiles in sediments and eel Mainly two-and three-ring PAHs were detected in eel, whereas four-ring PAHs predominated in the sediments Because PAH bioaccumulation was highest in eel from the reference sites, tissue levels of the parental PAH are probably not the most accurate monitor of PAH exposure in fish An elevated excretion of 1 OH pyrene (determined by synchronous scan fluores cence) was observed in the bile of fish from three of the four polluted sites, indicating that this parameter may be used as a biomarker for PAH exposure A significant increase in PAH-DNA adduct levels (determined by 3zP postlabeling) was observed in the liver of eel from all polluted sites Therefore, this parameter seems to be a sensitive biomarker for exposure to mutagenic and carcinogenic PAHs
A new CE detection method was developed for the chiral drug bupropion (a second-generation antide... more A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.
The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum al... more The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom-induced room-temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern-Volmer plots based on the phosphorescence lifetimes indicate that (R)-FBP causes a stronger Trp quenching than (S)-FBP. For the methyl esters of FBP, the opposite is observed: (S)-(FBPMe) quenches more than (R)-FBPMe. The Stern-Volmer plots of (R)-FBP and (R)-FBPMe are similar although their high-affinity binding sites are different. The methylation of (S)-FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10(7) M(-1) s(-1) of the R-enantiomers and 2.5 × 10(7) M(-1) s(-1) for the S-enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs.
A Raman instrument was assembled and tested that rejects typically 98-99% of background fluoresce... more A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm À1 in the blue and 15 cm À1 in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (k exc ¼ 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (k exc ¼ 405 nm). Advantages and disadvantages of this approach for RRS are discussed.
In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid ch... more In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 3 10 À6 A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10 À4 A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 3 10 À5 A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.
Polycyclic aromatic hydrocarbons (PAHs) are abundant pollutants, and many PAHs are carcinogenic, ... more Polycyclic aromatic hydrocarbons (PAHs) are abundant pollutants, and many PAHs are carcinogenic, but only after metabolic activation. Benzo[a]pyrene (BaP) is among the most carcinogenic PAHs. The dose and time response of two enzymes involved in BaP metabolism and the amounts of BaP metabolites excreted into the bile were evaluated in an experiment with dab (Limanda limanda). Ninety dab were exposed orally to one of five doses of BaP (0, 0.08, 0.4, 2, or 10 mg/kg) and sampled at 3, 6, or 12 d after exposure. None of the doses studied caused significant induction of either microsomal ethoxyresorufin-O-deethylase (EROD), which reflects cytochrome P450 1A (CYP1A) activity, or cytosolic glutathione-S-transferase activity (GST). Concentrations of biliary BaP metabolites significantly increased with dose and significantly decreased with time after exposure. It is concluded that biliary BaP metabolites provide a much more sensitive method than EROD (CYP1A) or GST activity to monitor recent exposure to PAHs in dab.
Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brai... more Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron co-culture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe2+ (Fenton reaction). Raman peaks related to nucleic acids (725 cm-1, 782 cm-1, 1092 cm-1, 1320 cm-1, 1340 cm-1, 1420 cm-1, 1576 cm-1) showed time-dependent changes over the experimental period (60 min), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when co-treated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra were observed for untreated control cells and for cells exposed to Fe2+ only, H2O2 only and ascorbate only. A live dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent non invasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.
Ultraviolet resonance Raman spectroscopy (RRS) is presented as a novel identification tool for co... more Ultraviolet resonance Raman spectroscopy (RRS) is presented as a novel identification tool for conventional-size column liquid chromatography (LC). The on-line coupling was made using a standard Z-shaped flow cell. A continuous-wave frequency-doubled argon ion laser operating at a wavelength of 244 nm was used for excitation. "On-thefly" resonance Raman spectra of four model compounds, fluorene, phenanthrene, fluoranthene, and pyrene, were recorded after a standard acetonitrile/water reversedphase LC separation. When applying a large-volumeinjection procedure (32 mL), detection limits were at the nanogram per milliliter level. The results indicate that UV-RRS gives detailed spectral information at an appropriate sensitivity level so that coupling with LC becomes feasible.
Hyphenation of thin layer chromatography (TLC) with surface-based spectral methods requires a hom... more Hyphenation of thin layer chromatography (TLC) with surface-based spectral methods requires a homogeneous surface for direct and quantitative analysis on the chromatographic plate after separation. Since most chromatographic materials do not produce strong background signals in Raman spectroscopy (RS) or surface-enhanced RS (SERS), we tested the suitability of two different chromatographic substrates and one interface for coupling SERS with TLC.
Tranexamic acid (TA) is a synthetic antifibrinolytic agent that is being considered as a candidat... more Tranexamic acid (TA) is a synthetic antifibrinolytic agent that is being considered as a candidate adjuvant drug for site-specific pharmaco-laser therapy of port wine stains. For drug utility studies, a high-performance liquid chromatography (HPLC)-fluorescence method was developed for the quantification of TA in blood. Platelet-poor plasma was prepared, size-separated using 3 kDa cut-off centrifuge filters, and derivatized with naphthalene-2-3-dicarboxaldehyde (NDA) and cyanide. The excess of NDA was quenched after 2 min by adding tryptophan. The derivatives were separated on a 2.1 mm C18 column using an acetate buffer/acetonitrile gradient. Excellent separation from plasma background was obtained at pH 5.5. Quantification was carried out at 440/520 nm. The limit of detection was 0.5 M and the mean ± SD recovery from whole blood was 81.7 ± 10.9%. Derivatized TA samples were stable for at least 36 h at 4 • C. The method was successfully applied to a heat-induced TA release study from thermosensitive liposomes.
In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid ch... more In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 3 10 À6 A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10 À4 A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 3 10 À5 A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.
In this preliminary study, we evaluated four different types of substrate for the at-line couplin... more In this preliminary study, we evaluated four different types of substrate for the at-line coupling of capillary electrophoresis and surface-enhanced (resonance) Raman spectroscopy, CE–SER(R)S, with emphasis on spectral repeatability. We tested Sub1: etched silver foil, Sub2: a vapour-deposited silver film, Sub3: a silver oxalate-precoated silica TLC plate and Sub4: a silica TLC plate on which colloid and poly(l-lysine) were manually
This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-ox... more This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.
Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous s... more Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous solutions are shown to exhibit relatively strong room temperature phosphorescence (RTP). Among the various CDs tested, ␣-CD showed the strongest RTP signals. Interestingly, these signals differed significantly for the two enantiomers of CQ; the phosphorescence lifetime of (+)-CQ was about four times longer than that of (−)-CQ, being 352 ± 16 and 89 ± 6 s, respectively. This enantiomeric selectivity is attributed to a difference in dissociation rates (competing with the radiative emission process) for the diastereoisomeric inclusion complexes dealt with, which have a 2:1 stoichiometry (␣-CD:CQ:␣-CD). Time-resolved RTP detection using different delay times enables the determination of the two enantiomers in a mixture without involving a separation technique. The minimum detectable fraction of (+)-CQ in a 2 mM sample was 13%.
Adsorption kinetics and molecular interactions on different surface interfaces are studied by mea... more Adsorption kinetics and molecular interactions on different surface interfaces are studied by means of evanescent-wave cavity ring-down spectroscopy, using total internal reflection surfaces onto which different self-assembled monolayers are covalently attached. The adsorption of cytochrome c (a positively charged, spherical heme protein) to a negatively charged bare silica surface, as well as to C 18 -coated (hydrophobic) and C 3 NH 2 -coated (positively charged) silica have been studied. It is experimentally verified that these surface layers do not interfere with the sensitive measurement of adsorbed cyt c monolayers using the evanescent wave in a ring-down scheme. Attaching monolayers covalently to the silica total internal reflection surface is a first step towards the development of a biosensor that makes use of immobilized biomolecules for specific detection of analytes in solution.
Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberell... more Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional ...
Journal of Photochemistry and Photobiology A: Chemistry, 2011
ABSTRACT A paper by Amat et al. [2] reported that the ATP-driven oxidation of luciferin to electr... more ABSTRACT A paper by Amat et al. [2] reported that the ATP-driven oxidation of luciferin to electronically excited oxyluciferin catalyzed by luciferase was accelerated when ATP was priorly irradiated at non-resonant optical frequencies (NROF) at 635 and 830nm. In another paper by Amat et al. [3], increased fluorescence intensities of ATP–Mg complexes, which showed lower fluorescence than ATP when excited at 260nm, were reported in consequence of concomitant NROF irradiation (i.e., 655 and 830nm). It was postulated that NROF-induced electric field changes may alter the charge distribution in ATP's phosphate chain, resulting in lowering of the activation energy of its terminal phosphate. Here we use spectrofluorometry to further investigate this hypothesis. The effect of NROF (at 632.8nm) on the intrinsic fluorescence of non-complexed and Mg-chelated ATP in aqueous solution and the influence of NROF (514.5nm and 632.8nm) on the rate of the luciferin–luciferase reaction was studied. We found that neither the intrinsic fluorescence of ATP nor its biochemical behavior during the firefly luciferin–luciferase reaction was affected by laser irradiation with NROF. Consequently, no evidence was found supporting the postulation that NROF-induced alternations on the charge distribution of the phosphate chain affect the reactivity of ATP.
The effect of disturbed root nodulation on the quantitative and qualitative composition of the ma... more The effect of disturbed root nodulation on the quantitative and qualitative composition of the main isoflavonoid glucosideYmalonates, glucosides, and aglycones in the leaves of Trifolium pratense L. grown under waterlogging conditions was investigated. Isoflavonoids are involved in the regulation of root nodule activity and the establishment of the mycorrhizal association. Isoflavonoid determination was performed using reversed-phase liquid chromatography coupled to mass spectrometric and UV absorbance detection. In response to waterlogging, the concentrations of biochanin A and biochanin AY7-O-glucosideYmalonate, biochanin AY7-O-glucoside, and gen-isteinY7-O-glucoside in the leaves increased two-to threefold after a lag period of 3 wk because of disturbed root nodulation. The other isoflavones detectedVformononetin, formononetinY7-O-glucosideYmalonate, and for-mononetinY7-O-glucosideVdid not show any significant changes related to waterlogging. After restoring normal soil water conditions, the concentrations of biochanin A and its glucoside and glucosideYmalonate rapidly returned to the initial values, whereas the concentration of genisteinY7-O-glucoside remained high.
... Correspondence: Gerard J. Stroomberg,, Vrije ... Compared to PAH metabolism studies on benzo[... more ... Correspondence: Gerard J. Stroomberg,, Vrije ... Compared to PAH metabolism studies on benzo[a]pyrene in other invertebrates, such as sea star (Asterias rubens) [17] or shore crab ... 5Van der Oost R, Van Schooten FJ, Ariese F, Heida H, Satumalay K, Vermeulen NPE: 1994. ...
Samples of sediment and eel taken from six sites in Amsterdam with different levels of water poll... more Samples of sediment and eel taken from six sites in Amsterdam with different levels of water pollution were ana lyzed for 16 parental PAHs In addition, biliary PAH metabolites and hepatic PAH-DNA adducts were determined in the eel to evaluate biomonitoring techniques for PAH exposure There was a clear difference between PAH profiles in sediments and eel Mainly two-and three-ring PAHs were detected in eel, whereas four-ring PAHs predominated in the sediments Because PAH bioaccumulation was highest in eel from the reference sites, tissue levels of the parental PAH are probably not the most accurate monitor of PAH exposure in fish An elevated excretion of 1 OH pyrene (determined by synchronous scan fluores cence) was observed in the bile of fish from three of the four polluted sites, indicating that this parameter may be used as a biomarker for PAH exposure A significant increase in PAH-DNA adduct levels (determined by 3zP postlabeling) was observed in the liver of eel from all polluted sites Therefore, this parameter seems to be a sensitive biomarker for exposure to mutagenic and carcinogenic PAHs
A new CE detection method was developed for the chiral drug bupropion (a second-generation antide... more A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.
The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum al... more The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom-induced room-temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern-Volmer plots based on the phosphorescence lifetimes indicate that (R)-FBP causes a stronger Trp quenching than (S)-FBP. For the methyl esters of FBP, the opposite is observed: (S)-(FBPMe) quenches more than (R)-FBPMe. The Stern-Volmer plots of (R)-FBP and (R)-FBPMe are similar although their high-affinity binding sites are different. The methylation of (S)-FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10(7) M(-1) s(-1) of the R-enantiomers and 2.5 × 10(7) M(-1) s(-1) for the S-enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs.
A Raman instrument was assembled and tested that rejects typically 98-99% of background fluoresce... more A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm À1 in the blue and 15 cm À1 in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (k exc ¼ 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (k exc ¼ 405 nm). Advantages and disadvantages of this approach for RRS are discussed.
In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid ch... more In earlier studies, it was demonstrated that the sensitivity of absorbance detection in liquid chromatography (LC) can be improved significantly by using cavity ring-down spectroscopy (CRDS). Thus far, CRDS experiments have been performed using visible laser light at fixed standard wavelengths, such as 532 nm. However, since by far most compounds of analytical interest absorb in the ultraviolet (UV), it is of utmost importance to develop UV-CRDS. In this study, as a first step towards the deep-UV region, LC separations with CRDS detection (using a previously described liquid-only cavity flow cell) at 457 and 355 nm are reported for standard mixtures of dyes and nitro-polyaromatic hydrocarbons (nitro-PAHs), respectively. For the measurements in the blue range a home-built optical parametric oscillator (OPO) system, tunable between 425 and 478 nm, was used, achieving a baseline noise of 2.7 3 10 À6 A.U. at 457 nm, improving upon the sensitivity of conventional absorbance detection (typically around 10 À4 A.U.). An enhancement of the sensitivity can be seen at 355 nm as well, but the improvement of the baseline noise (1.3 3 10 À5 A.U.) is much less pronounced. The sensitivity at 355 nm is limited by the quality of the UV-CRDS mirrors that are currently available: whereas the ring-down times as obtained at 457 nm are around 70-80 ns for the eluent, they are only 20-25 ns at 355 nm. Critical laser characteristics for LC-CRDS measurements, such as pulse length and mode structure, are given and prospects for going to shorter wavelengths are discussed.
Uploads
Papers by Freek Ariese