Even-electron ions: a systematic study of the neutral species lost in the dissociation of quasi-molecular ions.

The collision-induced dissociations of the even-electron [M + H](+) and/or [M - H](-) ions of 121 model compounds (mainly small aromatic compounds with one to three functional groups) ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) have been studied using an ion trap instrument, and the results are compared with the literature data. While some functional groups (such as COOH, COOCH(3), SO(3)H in the negative ion mode, or NO(2) in both the positive and negative ion modes) generally promote the loss of neutrals that are characteristic as well as specific, other functional groups (such as COOH in the positive ion mode) give rise to the loss of neutrals that are characteristic, but not specific. Finally, functional groups such as OH and NH(2) in aromatic compounds do not lead to the loss of a neutral that reflects the presence of these substituents. In general, the dissociation of [M + H](+) and [M - H](-) ions generated from aliphatic compounds or compounds containing an aliphatic moiety obeys the even-electron rule (loss of a molecule), but deviations from this rule (loss of a radical) are sometimes observed for aromatic compounds, in particular for nitroaromatic compounds. Thermochemical data and ab initio calculations at the CBS-QB3 level of theory provide an explanation for these exceptions. When comparing the dissociation behaviour of the even-electron [M + H](+) and/or [M - H](-) ions (generated by ESI or APCI) with that of the corresponding odd-electron [M](+) ions (generated by electron ionization, EI), three cases may be distinguished: (1) the dissociation of the two ionic species differs completely; (2) the dissociation involves the loss of a common neutral, yielding product ions differing in mass by one Da, or (3) the dissociations lead to a common product ion.

Structure elucidation of phase II metabolites by tandem mass spectrometry: an overview.

The present paper provides a summary of the collision-induced dissociation of protonated and deprotonated phase II metabolites of drugs and pesticides. This overview is based on published literature and unpublished data from the authors. In particular, glutathione conjugates and their biotransformation products are discussed in detail. In addition, the fragmentation of the major classes of conjugates, i.e. glucuronides, glucosides, malonylglucosides, sulfates, acetates, methyl and glycine conjugates, is reported. Collision-induced dissociation, as studied by tandem mass spectrometry, allows the rapid identification of the type of conjugate, whereas the exact conjugation site can in general be determined only by additional NMR experiments.

Pyrethroids used indoor-ambient monitoring of pyrethroids following a pest control operation.

House dust and airborne particles (PM) were sampled before (T1) and 1 day (T2), 4-6 months (T3) as well as 10-12 months (T4) after a pest control operation (PCO). Cyfluthrin was applied in 11, cypermethrin in 1, deltamethrin in three and permethrin in four interiors. The pyrethroid concentrations in house dust and PM were measured by GC/MS with a detection limit for all pyrethroids of 0.5 mg/kg house dust and of 1 ng/m3 PM for deltamethrin and permethrin and 3 ng/m3 PM for cyfluthrin and cypermethrin. A general background concentration of permethrin (95th percentile: 5.9 mg/kg) and cyfluthrin (95th percentile: 34.9 mg/kg) in house dust was found. In general, an appropriately performed PCO lead to an increase of pyrethroids in house dust as well as in PM, in some cases up to 1 year after application. One day after the application the cyfluthrin concentration increased significantly from 0.25 (T1) to 33.8 mg/kg house dust (T2) and up to 4.9 ng/m3 in PM. The permethrin concentration increased significantly from 4.3 to 70 mg/kg in house dust and up to 18.1 ng/m3 in PM, deltamethrin increased to 54.5 mg/kg and 20.8 ng/m3 and cypermethrin to 14 mg/kg and 45.7 ng/m3. Thereafter a continuous decrease could be observed during the time course of 1 year. After 1 year the permethrin concentration in house dust was still 1/5 of the T2 concentration, whereas for cypermethrin and cyfluthrin only 1/14 and 1/23 of the T2 concentration were found. Deltamethrin was not detected at all after T2. Moreover, the data of this study showed significant, positive correlations between pyrethroids in house dust and in airborne particles especially one day after PCO.

Verapamil: new insight into the molecular mechanism of drug oxidation in the human heart.

Verapamil is a commonly prescribed cardiovascular drug, but surprisingly its metabolism in the target tissue of pharmacotherapy is basically unknown. We therefore investigated its biotransformation in human heart tissue and correlate the production of metabolites with the gene expression of major drug metabolising enzymes. Using electrospray LC-MS-MS and LC-MS3 experiments, a total of nine metabolites were observed in incubation experiments with verapamil and microsomes isolated from the human heart tissue, and this included a carbinolamine-, N-formyl-, ahemiacetale-, and formate-intermediate of N-demethyl- and O-demethylverapamil. We also observed a hydroxylation product at the benzylic position of atom C-7 (M9). Metabolites M5-M9 are novel and were not observed in previous studies with liver or other human tissues. A fine example of the considerable metabolic competence of human heart is the formation of M1-M4, e.g. dealkylverapamil, norverapamil and isomers of O-demethylverapamil, which were believed to be exclusively produced by the liver.

Bio-compatible in-tube solid-phase microextraction capillary for the direct extraction and high-performance liquid chromatographic determination of drugs in human serum.

A restricted access material (RAM), alkyl-diol-silica (ADS), was used to prepare a highly bio-compatible solid-phase microextraction (SPME) capillary for the automated and direct in-tube extraction of several benzodiazepines from human serum. The bifunctionality of the ADS extraction phase prevented fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. This the first report of a restricted access material utilized as an extraction phase for in-tube SPME. The approach simplified the required apparatus in comparison to existing RAM column switching procedures, and more importantly eliminated the excessive use of extraction solvents. The biocompatibility of the ADS material also overcame the existing problems with in-tube SPME that requires an ultrafiltration or other deproteinization step prior to handling biological samples, therefore further minimizing the sample preparation requirements. The calculated oxazepam, temazepam, nordazepam and diazepam detection limits were 26, 29, 22 and 24 ng/ml in serum, respectively. The method was linear over the range of 50-50 000 ng/ml with an average linear coefficient (R2) value of 0.9998. The injection repeatability and intra-assay precision of the method were evaluated with five injections of a 10-microg/ml serum sample (spiked with all compounds), resulting in an average RSD<7%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry.

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.

Indoor pyrethroid exposure in homes with woollen textile floor coverings.

In order to investigate human's exposure to permethrin from treated woollen textile floor coverings and possible adverse health effects, a study was carried out in 80 private homes in Hannover (Germany) equipped with woollen textile floor coverings (wool wall-to-wall carpets or woven or knotted rugs). For indoor monitoring, permethrin was determined both in house dust and on suspended particles. While permethrin concentrations in house dust (< 2 mm) were high (arithmetic mean: 53.7 mg/kg, 90th percentile 129.1 mg/kg), the permethrin concentrations in the air (suspended particles) were very low (arithmetic mean 2.8 ng/m3, 90th percentile 5.8 ng/m3, first sampling). Additional experiments demonstrate that permethrin on suspended particles result from carpet fiber abrasion (and not from an evaporation/re-condensation process). The internal exposure of the 145 inhabitants participating in the study was determined by biological monitoring (permethrin metabolites in urine). In a first sampling period almost 14% of the samples showed concentrations of the metabolite DCCA and almost 23% of the metabolite 3-PBA above the limit of detection (0.2 microgram/l). A model was developed which allows the calculation of the metabolite concentration in urine due to inhalative uptake of permethrin. Even for the worst case situation the calculated metabolite concentrations were ca. 30 times lower than the experimental results. The observed concentrations of metabolites are comparable to those of the background concentrations of the general population in Germany, suggesting that they must origin from other sources than woollen textile floor coverings. The indoor and biological monitoring data as well as the evaluation of the reported symptoms give no indication of an adverse health effect due to carpet treatment by permethrin.

Metabolism of verapamil in cultures of rat alveolar epithelial cells and pharmacokinetics after administration by intravenous and inhalation routes.

Administration of therapeutic entities by inhalation opens new possibilities for drug entry into systemic circulation, but this requires passage through the alveolar epithelium. Little is known about the pulmonary metabolism of verapamil. Specifically, this cardiovascular drug suffers from extensive first pass metabolism. We therefore evaluated the metabolism of verapamil in cultured alveolar epithelium and compared findings with results after administration by inhalation and intravenous routes. Specifically, cell culture of alveolar epithelium was characterized by gene expression of surfactant proteins A, B, C, and D, by immunohistochemistry of surfactant protein C, by staining for laminar bodies, and by gene expression of cytochrome P450 monooxygenases. During 6 days of culture expression, all cellular differentiation markers were obvious, albeit at different levels. With testosterone as substrate, we found alveolar epithelial cells to produce several stereo- and site-specific hydroxylation products. This provided evidence for metabolic competence of cultured alveolar epithelial cells. With verapamil as substrate, only limited production of metabolites was observed in cell culture assays, and similar results were recorded after administration by inhalation and intravenous routes. Likewise, elimination of verapamil from lung tissue and plasma was similar by both routes of administration. In conclusion, administration of verapamil by inhalation-abrogated extensive first pass metabolism frequently seen after oral application, and this may well be extended to the development of drugs with similar pharmacokinetic defects.

Application of restricted access material (RAM) with precolumn-switching and matrix solid-phase dispersion (MSPD) to the study of the metabolism and pharmacokinetics of Verapamil.

In the pharmaceutical industry, studies of the metabolism and pharmacokinetics of drugs are important routine applications which require the analysis of the precursor drug and its metabolites in various biological matrices, such as plasma, serum, urine, cell culture media and tissue samples. In this study, two new and simple methods of sample preparation were optimized and validated: on the one hand, a column-switching technique with a restricted access material (RAM) was used to analyze biological fluids, and on the other hand, matrix solid-phase dispersion (MSPD) was applied to the extraction of analytes from tissue samples. Identification of the metabolites was done with a LC-MS system (ion trap in the MS(n)mode) coupled both on-line (RAM) and off-line (MSPD). Using the common calcium antagonist Verapamil, it is shown that these two methods allow rapid identification of phase I and phase II metabolites from biological samples and are suitable for pharmacokinetic and pharmacodynamic studies of pharmaceuticals in biological matrices.

Verapamil: metabolism in cultures of primary human coronary arterial endothelial cells.

Endothelium is a metabolically active secretory tissue and an important barrier for metabolic products. Little is known about its contribution to drug oxidation. We investigated the gene and protein expression and enzyme activity of major cytochrome P450 monooxygenases in cultures of primary human coronary endothelial cells and studied its ability to metabolize verapamil, a commonly and widely prescribed calcium antagonist. Of the total 18 P450 monooxygenases investigated, transcripts for CYP1A1, CYP2A6/7, CYP2A13, CYP2B6/7, CYP2C8, CYP2E1, and CYP2J2 were expressed, albeit at different levels. Furthermore, metabolism of verapamil proceeded predominantly via N-desmethylation and/or N-desalkylation, i.e., production of D-617 [2-(3,4-dimethoxyphenyl)-5-amino-2-isopropylvaleronitrile], D-620 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], and norverapamil; but additional metabolites are the O-demethylated products, D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-703 [O-demethylverapamil; 5-N-(3,4-dimethoxyphenethyl)methylamino-2-(3'-methoxy-4'-hydroxyphenyl)-2-isopropylvaleronitrile]. We show endothelium to express an array of monooxygenases, and in view of its large body distribution, endothelium should be considered in the biotransformation of drugs, particularly when tissue-specific metabolism and/or metabolic inactivation are being investigated.

Automated in-tube solid-phase microextraction coupled with HPLC for the determination of N-nitrosamines in cell cultures.

An automated in-tube solid-phase microextraction (SPME) HPLC analysis method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several metabolites has been developed. NNK is one of the tobacco-specific N-nitrosamines (TSNA), which has been linked to cancers associated with the use of or exposure to tobacco products. In-tube SPME is an on-line extraction technique in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw/eject steps. In this study, a tailor-made polypyrrole (PPY)-coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures. Compared with commercial capillaries that were currently used for in-tube SPME, the PPY-coated capillary showed better extraction efficiency for all of the compounds studied. After optimization of the extraction conditions, NNK and five metabolite compounds were analyzed in spiked cell cultures, confirming the applicability of the developed method. Excellent linearity was observed for all compounds (av R2 = 0.9942) and detection limits that ranged from 20 to 250 ng/mL. The average within-day and between day variations (% RSD) were 2.9 and 3.6%, respectively. This automated extraction and analysis method simplified the determination of the TSNA, requiring a total sample analysis time of only approximately 30 min.