PAH biotransformation in terrestrial invertebrates--a new phase II metabolite in isopods and springtails.

Soil-living invertebrates are exposed to high concentrations of contaminants accumulating in dead organic matter, such as polycyclic aromatic hydrocarbons (PAHs). The capacity for PAH biotransformation is not equally developed in all invertebrates. In this paper, we compare three species of invertebrates, Porcellio scaber (Isopoda), Eisenia andrei (Lumbricidae) and Folsomia candida (Collembola), for the metabolites formed upon exposure to pyrene. Metabolic products of pyrene biotransformation in extracts from whole animals or isopod hepatopancreas were compared to those found in fish bile (flounder and plaice). An optimized HPLC method was used with fluorescence detection; excitation/emission spectra were compared to reference samples of 1-hydroxypyrene and enzymatically synthesized conjugates. Enzymatic hydrolysis after fractionation was used to demonstrate that the conjugates originated from 1-hydroxypyrene. All three invertebrates were able to oxidize pyrene to 1-hydroxypyrene, however, isopods and collembolans stood out as more efficient metabolizers compared to earthworms. In contrast to fish, none of the invertebrates produced pyrene-1-glucuronide as a phase II conjugate. Both Collembola and Isopoda produced significant amounts of pyrene-1-glucoside, whereas isopods also produced pyrene-1-sulfate. A third, previously unknown, conjugate was found in both isopods and springtails, and was analysed further using electrospray and atmospheric pressure chemical ionisation mass spectrometry. Based on the obtained mass spectra, a new conjugate is proposed: pyrene-1-O-(6"-O-malonyl)glucoside. The use of glucose-malonate as a conjugant in animal phase II biotransformation has not been described before, but is understandable in the microenvironment of soil-living invertebrates. In the earthworm, three other pyrene metabolites were observed, none of which was shared with the arthropods, although two were conjugates of 1-hydroxypyrene. Our study illustrates the great variety of the still unexplored metabolic diversity of invertebrate xenobiotic metabolism.

Flavonoids in Leguminosae: analysis of extracts of T. pratense L., T. dubium L., T. repens L., and L. corniculatus L. leaves using liquid chromatography with UV, mass spectrometric and fluorescence detection.

Reversed-phase LC on C-18 bonded silica with a methanol-ammonium formate gradient was used to determine the main flavonoids in leaves of four species of the Leguminosae family. The detection modes were diode-array UV absorbance, fluorescence, and (tandem) mass spectrometry. LC-UV was used for a general screening, sub-classification, and the calculation of total flavonoid contents. LC-FLU was included to identify isoflavones on the basis of their native fluorescence. Most structural information regarding aglycons, sugar moieties, and acidic groups was derived from LC-MS in both the full-scan and extracted-ion mode, using negative-ion atmospheric pressure chemical ionization. MS/MS did not provide much additional information, because the same fragments were observed as in full-scan MS. In T. pratense and T. repens, the main constituents were flavonoid glucoside-(di)malonates, while T. dubium and L. corniculatus mainly contained flavonoid (di)glycosides. Satellite sets comprising an aglycon, the glucoside and glucoside-malonates or -acetates, were abundantly present only in T. pratense. Generally speaking, the main aglycons and sugars in the four plant species are surprisingly different. In addition, while the results for T. pratense are similar to those reported in the literature, there is little agreement in the case of the other species. Finally, total flavonoid contents ranged from 50-65 mg/g for L. corniculatus and T. dubium, to 15 mg/g for T. pratense and only 1 mg/g for T. repens.

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage.

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%). The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.

Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triple-quadrupole and ion-trap instruments.

With 15 flavonoids as test compounds, the analytical performance of four modes of LC-MS, multiple MS (MSn) and tandem MS operation (atmospheric pressure chemical ionization (APCI), electrospray ionization, positive and negative ionization) was compared for two mass spectrometers, a triple-quadrupole and an ion-trap instrument. Two organic modifiers, methanol and acetonitrile, and two buffers, ammonium acetate and ammonium formate, were used. In general, the use of APCI in the negative ion mode gave the best response, with the signal intensities and the mass-spectral characteristics not differing significantly between the two instruments. The best results were obtained when methanol-ammonium formate (pH 4.0) was used as LC eluent. Under optimum conditions full-scan limits of detection of 0.1-30 mg/l were achieved in the negative APCI mode. Here it needs to be emphasized that up to 2-order response differences were found both between analytes and between modes of ionization. This implies that one should be very cautious when interpreting data on the screening of real-life samples. The main fragmentations observed in the MSn spectra on the ion-trap, or the tandem MS spectra on the triple-quadrupole were generally the same. The advantage of the former approach is the added possibility to ascertain precursor-->product ion relationships.