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Interlaboratory comparisons for the analysis of mineral oil in polluted soil using the GC-FID method indicate that extraction and cleanup conditions have significant effects on the analytical results. In this investigation a ruggedness test was performed on the extraction and cleanup method for the determination of total petroleum hydrocarbons in soil. A two-level Plackett-Burman design was utilized to study the effect of 11 different method parameters on the extraction recovery of total petroleum hydrocarbons (TPH) in soil. Both qualitative and quantitative factors were investigated. The results indicate that total petroleum hydrocarbons can be relatively reliably monitored through strict implementation of the ISO and CEN draft standards. However, variation in certain method parameters readily affects the validity of the results. The most critical factors affecting TPH recovery were the solvent and co-solvent used for extraction, the extraction time, adsorbent and its weight and sample TPH concentration. Because adaptation of the draft standards especially with respect to these factors easily leads to TPH recoveries higher than 200% or lower than 70%, the validity of the adapted method should always be verified. A proper estimate of the expanded uncertainty should also be appended to TPH results, because only then can the reliability of the results be guaranteed and further justification is gained to support the end-use of the data. This also supports the credibility of the analytical services and prevents the data end-users from drawing misleading conclusions concerning the environmental risks and potential remediation requirements.

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Diuron, a widely used herbicide and antifouling biocide, has been shown to persist in the environment and contaminate drinking water. It has been characterized as a "known/likely" human carcinogen. Whereas its environmental transformation and toxicity have been extensively examined, its metabolic characteristics in mammalian livers have not been reported. This study was designed to investigate diuron biotransformation and disposition because metabolic routes, metabolizing enzymes, interactions, interspecies differences, and interindividual variability are important for risk assessment purposes. The only metabolic pathway detected by liquid chromatography/mass spectometry in human liver homogenates and seven types of mammalian liver microsomes including human was demethylation at the terminal nitrogen atom. No other phase I or phase II metabolites were observed. The rank order of N-demethyldiuron formation in liver microsomes based on intrinsic clearance (V(max)/K(m)) was dog > monkey > rabbit > mouse > human > minipig > rat. All tested recombinant human cytochrome P450s (P450s) catalyzed diuron N-demethylation and the highest activities were possessed by CYP1A1, CYP1A2, CYP2C19, and CYP2D6. Relative contributions of human CYP1A2, CYP2C19, and CYP3A4 to hepatic diuron N-demethylation, based on average abundances of P450 enzymes in human liver microsomes, were approximately 60, 14, and 13%, respectively. Diuron inhibited relatively potently only CYP1A1/2 (IC(50) 4 microM). With human-derived and quantitative chemical-specific data, the uncertainty factors for animal to human differences and for human variability in toxicokinetics were within the range of the toxicokinetics default uncertainty/safety factors for chemical risk assessment.

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ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and computational methods (DFT, MP2, and COSMO) were used to investigate the hydrolysis products of aluminium chloride as a function of sulfate concentration at pH 3.7. With the aid of computational chemistry, structural information was deduced from the chemical compositions observed with ESI-MS. Many novel types of hydrolysis products were noted, revealing that our present understanding of aluminium speciation is too simple. The role of counterions was found to be critical: the speciation of aluminium changed markedly as a function of sulfate concentration. Ab initio calculations were used to reveal the energetically most favoured structures of aluminium sulfate anions and cations selected from the ESI-MS results. Several interesting observations were made. Most importantly, the bonding behaviour of the sulfate group changed as the number of aqua ligands increased. The accompanying structural rearrangement of the clusters revealed the highly active role of sulfate as a ligand. The gas phase calculations were expanded to the aquatic environment using a conductor-like screening model. As expected, the bonding behaviour of the sulfate group in the minimum energy structures was distinctly different in the aquatic environment compared to the gas phase. Together, these methods open a new window for research in the solution chemistry of aluminium species.

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In order to provide additional information for risk assessment of the fungicide metalaxyl, the main objectives were (1) to elucidate the interactions of metalaxyl with different human liver cytochrome P450 enzymes, (2) to tentitatively identify and (semi)quantify metabolites in vitro and (3) to identify human CYP enzymes responsible for metabolism. The mean inhibitory concentrations (IC(50)) for 7-pentoxyresorufin-O-dealkylation (CYP2B) and bupropion hydroxylation (2B6) were 48.9 and 41.7µM, respectively. The biotransformation reactions were hydroxylation, (di)demethylation and lactone formation. In human liver microsomes predominant metabolites were two hydroxymetalaxyl derivatives or atropisomers of one of the derivatives. On the basis of previous rat studies these could be N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester and/or N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester. The amounts of didemethylmetalaxyl N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine and lactone 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione were higher in homogenates than microsomes. The carcinogenic 2,6-dimethylaniline was not detected. Among the nine major human CYPs, CYP3A4 was the only one responsible for metalaxyl hydroxylation, while CYP2B6 was the major isoform responsible for (di)demethylation and lactone formation.

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The hydrolysis reactions of FeCl3 in 0.1 mM aqueous solutions were monitored by electrospray ionisation time-of-flight mass spectrometry (ESI-ToF-MS). In contrast to the other ionisation techniques, electrospray ionisation provides information on the composition of the elemental cores even in water and salt cluster ions. Therefore, the technique facilitates detection of the bonding of the chloride ions in the oxo-hydroxo-iron cores. A variety of mononuclear and polynuclear iron-oxohydroxo-chloride complexes were characterised from the cationic and anionic mass spectra of iron(III) solutions. Oxidation and reduction reactions of iron, as well as competition between OH- and Cl- ions within one iron core, were also observed.

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Phenolic compounds from the aerial parts of medicinal plant Rhodiola rosea were identified using LC/MS experiments with time-of-flight and triple quadrupole instruments, providing accurate mass and CID fragmentation data about the compounds. Supercritical fluid extraction (SFE) was used to remove non-polar compounds from the samples, followed by liquid extraction of the flavonoids. Flavonoids were the main constituents in aerial parts of the plant, and no phenylpropanoids were detected. In addition to usual fragment ions providing the size of the attached glycosides in flavonoids, ions due to radical cleavage of glycosides were observed in the negative ion mode with relatively high collision energies. Use of these ions for elucidating the glycosylation site in the aglycone part was evaluated and was found to give some tentative information, but their use in unambiguous identification of unknown flavonoids is not recommended. Fifteen flavonoids, of which 10 were previously unreported from the plant, were identified.

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A new infrared spectroscopic method suitable for determining total fatty alcohol and fatty acid ester concentrations in industrial oils has been developed. Oil samples were diluted with toluene (1:3 w/w), the toxicity and volatility of which are relatively low compared with more commonly used IR solvents, like carbon tetrachloride or carbon disulfide. Mixture standards were prepared from dodecanol, tetradecanol, octadecanol, methyl stearate and methyl palmitate. Some analytical and statistical tests were performed on the developed method. The recoveries and the repeatability of the method proved to be sufficient for the quantitative determination of fatty alcohol and fatty acid ester additives in industrial oils. Reproducibility testing in another laboratory also produced satisfactory results. The developed method also proved to be relatively quick and simple. This method was developed to satisfy industry's need to determine the concentrations of these oil additives, and it has already been applied successfully in machinery oil analysis.

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AIMS: Our objective was to study in vivo the role of CYP2C and CYP3A4 in the disposition of 3-keto-desogestrel after administration of desogestrel, by using the selective inhibitors fluconazole (CYP2C) and itraconazole (CYP3A4). METHODS: This study had a three-way crossover design and included 12 healthy females, the data from 11 of whom were analyzed. In the first (control) phase all subjects received a single 150 microg oral dose of desogestrel alone. In the second and third phases subjects received a 4 day pretreatment with either 200 mg fluconazole or 200 mg itraconazole once daily in a randomized balanced order. Desogestrel was given 1 h after the last dose of the CYP inhibitor. Plasma 3-keto-desogestrel concentrations were determined for up to 72 h post dose. RESULTS: Pretreatment with itraconazole for 4 days significantly increased the area under the plasma concentration-time curve (AUC) of 3-keto-desogestrel by 72.4% (95% confidence interval on the difference 12%, 133%; P = 0.024) compared with the control phase, whereas fluconazole pretreatment had no significant effect (95% CI on the difference -42%, 34%). Neither enzyme inhibitor affected significantly the maximum concentration (95% CI on the difference 14%, 124% for itraconazole and -23%, 40% for fluconazole) or elimination half-life (95% CI on the difference -42%, 120% for itraconazole and -24%, 61% for fluconazole) of 3-keto-desogestrel. CONCLUSIONS: According to the present study, the biotransformation of desogestrel to 3-keto-desogestrel did not appear to be mediated by CYP2C9 and CYP2C19 as suggested earlier. However, the further metabolism of 3-keto-desogestrel seems to be catalyzed by CYP3A4.

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OBJECTIVE: Our objective was to study the effect of the antiplatelet agents clopidogrel and ticlopidine on bupropion (INN, amfebutamone) hydroxylation, a probe reaction for cytochrome P450 (CYP) 2B6 activity. METHODS: Twelve healthy male volunteers took a single 150-mg oral dose of bupropion either alone or after pretreatment with 75 mg clopidogrel once daily or 250 mg ticlopidine twice daily for 4 days. On day 4, a single 150-mg oral dose of bupropion was administered. Plasma concentrations of bupropion and its CYP2B6-catalyzed metabolite, hydroxybupropion, were measured for up to 72 hours. RESULTS: The mean area under the plasma concentration-time curve (AUC) of hydroxybupropion calculated from time 0 to infinity was reduced by 52% ( P = .001; 95% confidence interval [CI], 39% to 66%) by clopidogrel and by 84% ( P < .0001; 95% CI, 73% to 94%) by ticlopidine. Clopidogrel reduced the AUC ratio of hydroxybupropion over bupropion by 68% ( P = .002; 95% CI, 58% to 77%) and ticlopidine by 90% ( P = .001; 95% CI, 85% to 96%). The AUC of bupropion was increased by 60% ( P = .02; 95% CI, 21% to 98%) and by 85% ( P < .0001; 95% CI, 48% to 85%) with clopidogrel and ticlopidine, respectively. CONCLUSIONS: Both clopidogrel and ticlopidine significantly inhibited the CYP2B6-catalyzed bupropion hydroxylation. Patients receiving either clopidogrel or ticlopidine are likely to require dose adjustments when treated with drugs primarily metabolized by CYP2B6.

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The dramatically increased number of new chemical entities (NCE) used in drug discovery has raised a demand for efficient and rapid drug metabolism screening techniques. The aim of this study was to develop a global in vitro metabolic interaction screening test utilising the N-in-1 approach. A cocktail consisting of 10 CYP-selective probes with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were analysed simultaneously using LC/TOF-MS. Performance of the method was assessed with cDNA expressed P450s and diagnostic CYP-specific inhibitors. The results were in good accordance with literature and our previous studies. The cocktail developed is suitable for fast and reliable in vitro screening of the interaction potential and characteristics of NCEs.

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Cinnamyl alcohol was added to the media of compact callus aggregates (CCA) of Rhodiola rosea for stimulating the production of cinnamyl glycosides. The biotransformation reaction produced high amounts of rosin, while only a very low amount of rosavin was produced. As the consumption rate of cinnamyl alcohol was much higher than production of rosin, the aqueous methanol extracts of compact callus aggregates were studied by liquid chromatography-mass spectrometric methods and four new unexpected biotransformation products of cinnamyl alcohol were identified.

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Endophytes are found in meristematic bud tissues of Scots pine (Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial (Methylobacterium extorquens, Pseudomonas synxantha) and one fungal endophyte (Rhodotorula minuta) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.

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The hydrolysis reactions of AlCl3 in 0.1 M aqueous solutions at pH 3.27-4.20 were monitored by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS) as a function of time. The cationic and anionic ESI mass spectra of aluminum(III) solutions gave strong evidence of the presence of a variety of monomeric and polymeric complexes. Competition between the OH- and Cl- ligands within the same aluminum core was observed. The influence of the sample cone voltage on the product distribution was also explored. The optimum sample cone voltage for the cationic spectra was 70 V. For the anionic spectra no optimum sample cone voltage was found and the appearance of the anionic spectra was strongly dependent on the sample cone voltage within the whole range explored.

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Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.

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Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. A liquid chromatographic (LC) method with mass spectrometric (MS) detection based on selected ion monitoring (SIM) was developed for determining salidroside, sachaliside 1, rosin, 4-methoxycinnamyl-O-beta-glucopyranoside, rosarin, rosavin, cinnamyl-(6'-O-beta-xylopyranosyl)-O-beta-glucopyranoside, 4-methoxy-cinnamyl-(6'-O-alpha-arabinopyranosyl)-O-beta-glucopyranoside, rosiridin and benzyl-O-beta-glucopyranoside from the callus and plant extracts in one chromatographic run. Good linearity over the range 0.5-500 ng ml(-1) for salidroside, 2-2000 ng ml(-1) for rosavin and 2-500 ng ml(-1) for benzyl-O-beta-glucopyranoside was observed. The intra-assay accuracy and precision within quantitation ranges varied between -10.0 and +13.2% and between 0.7 and 9.0%, respectively. Optimization of the ionization process was performed with electrospray and atmospheric pressure chemical ionization techniques using four different additive compositions for eluents in the LC/MS scan mode, using both positive and negative ion modes. The best ionization sensitivity for the compounds studied was obtained with electrospray ionization when using pure water without any additives as the aqueous phase.

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The concentration of 7-methyljuglone was studied in the round-leaved sundew Drosera rotundifolia L. collected from different regions in Northern Finland. Samples for analysis were collected from peat bogs and sandpit habitats. The mean concentration of 7-methyljuglone varied from 1.0 to 2.3% of dry weight. Variation between years in the amount of 7-methyljuglone was significant in plants growing on sand, and in the northernmost region studied. Overall, the variation in the production of 7-methyljuglone among different populations of round-leaved sundew in Northern Finland was rather low. The variation between years in the production of 7-methyljuglone was more significant.

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Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. Along its known secondary metabolites tyrosol (1), salidroside (rhodioloside) (2), rosin (3), rosarin (4), rosavin (5), sachaliside 1 (6) and 4-methoxy-cinnamyl-O-beta-D-glucopyranoside (7), four compounds were isolated from aqueous methanol extract of the plant and identified as cinnamyl-(6'-O-beta-xylopyranosyl)-O-beta-glucopyranoside (8), 4-methoxy-cinnamyl-(6'-O-alpha-arabinopyranosyl)-O-beta-glucopyranoside (9), picein (10) and benzyl-O-beta-glucopyranoside (11) by UV, MS and NMR methods. Compounds 8 and 9 are new natural compounds whereas compounds 10 and 11 were isolated first time from R. rosea. Also the compounds 6 and 7 are isolated earlier only from the callus cultures of the plant but not from the differentiated plant.

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Liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI/TOF/MS) and a novel NMR technique, developed to maximise the sensitivity obtained from the standard NMR spectrometer, have been applied to the identification of the phenolic constituents of Eleutherococcus senticosus. In addition, molecular modelling and dihedral bond angle calculations based on the vicinal 3JHH-coupling constants have been used in the unambiguous assignment of signals in the 1H-NMR spectra. 5'-O-Caffeoylquinic acid and three isomeric compounds, 1',5'-O-dicaffeoylquinic acid, 3',5'-O-dicaffeoylquinic acid and 4',5'-O-dicaffeoylquinic acid, have been isolated and identified from a sample. The isolation and structure determination of the latter two compounds are reported for the first time from this plant.

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Hypericum perforatum L. (St. John's Wort) has long been known as a medicinal plant, and has been used for the treatment of depression and neuralgic disorders. Its main active constituents are believed to be a naphthodianthrone, hypericin, and a phloroglucinol, hyperforin. A sensitive high performance liquid chromatography (HPLC)/electrospray tandem mass spectrometric method for fast simultaneous determination of six major naphthodianthrones and phloroglucinols of Hypericum perforatum extract has been developed. The method, based on multiple dissociation reaction monitoring (MRM), allows the analysis of hypericin, protohypericin, pseudohypericin, protopseudo-hypericin, hyperforin and adhyperforin from the extract in less than 5 min. Good linearity over the range 0.1-1000 ng/mL for hyperforin and 2-500 ng/mL for hypericin was observed. Intra-assay accuracy and precision varied from 2 to 19% within these ranges. Lower levels of quantitation for hyperforin were 0.5 ng/mL and 2 ng/mL for hypericin.

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