As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.

Bundesen's theory of visual attention (TVA) task allows the adaptive and independent measurement of storage capacity and processing speed in visual short term memory. This study investigates how cognitive enhancing effects of psychostimulants depend on baseline performance and individual plasma levels. 18 healthy volunteers (aged 20–35 years) received single oral doses of either methylphenidate 40mg, modafinil 400mg or placebo in a double-blind crossover design. A semi-computerised version of the TVA task requiring whole report of visually presented letter arrays with ultra-short presentation times between 22 and 86ms was performed 2.5–3.5h after drug administration. Single doses of both psychostimulants enhanced visual perceptual processing speed. Differenzial plasma-dependent effects of methylphenidate were observed in low- and high-performing participants: Higher plasma levels resulted in greater improvement of processing speed in low-performing participants and decreasing improvement in high-performing participants. Modafinil enhanced visual short term memory storage capacity in low performing subjects. Processing speed improvements after both psychostimulants correlated with increases of subjective alertness (all effects significant at p<0.05). Our results confirm previous findings of cognitive enhancing effects of modafinil in lower performing (or sleep-deprived) participants and extent the model of baseline dependency to cognitive effects of methylphenidate.

Dopamine plays an important role in working memory. It has been hypothesized that D1 receptor signalling in the prefrontal cortex supports the stable maintenance of information in working memory, while D2 receptor signalling in the striatum facilitates the flexible updating and manipulation of new information in working memory. In the present study we tested the hypothesis that striatal dopamine is important for the manipulation of information in working memory in a pharmacological, event-related fMRI study. Twenty healthy human subjects were scanned twice, once after placebo and once after sulpiride 400mg, a selective D2 receptor antagonist, while performing a working memory task requiring different levels of manipulation. We observed a significant correlation between sulpiride plasma levels and BOLD signal related to manipulation bilaterally in the putamen, suggesting a dose-dependent effect of D2 antagonism on a striatally-based manipulation process. These effects on BOLD signal occurred in the context of a drug-induced improvement in performance on trials requiring the manipulation of information in working memory but not on simple retrieval trials. No significant drug effects were observed in the PFC. These results support models of dopamine function that posit a 'gating' function for dopamine D2 receptors in the striatum, which enables the flexible updating and manipulation of information in working memory.

A sensitive and simple liquid chromatography/mass spectrometry (LC/MS) method was developed for the determination of terephthalic acid isopropylamide, the final metabolite of procarbazine in human urine. A solid-phase extraction with C(18) cartridges was used followed by LC/MS with a single mass spectrometer (SSQ 7000 from Finnigan). Terephthalic acid isobutylamide was the internal standard. The quantification limit was 30 ng/mL in urine (6 x noise). This assay was applied for drug monitoring of terephthalic acid isopropylamide in urine after oral administration of procarbazine in children and adolescents with Hodgkin lymphomas.

For a correct determination of acrolein amounts generated in in vitro turnover experiments with oxazaphosphorines, it is necessary to characterize the interaction of acrolein with liver microsomal proteins. Acrolein, a highly reactive metabolite of oxazaphosphorines, readily forms covalent adducts with proteins by electrophilic attack on nucleophiles, such as the sulfhydryl group of cysteine, imidazole group of histidine, and amino group of lysine. The current investigations were mainly directed toward determination of the degree of acrolein-protein binding under conditions of in vitro experiments with liver microsome preparations. The acrolein concentration in protein dilution was determined by a fluorescence method. Moreover, the influence of sucrose and glycerine on the extent of acrolein-protein binding commonly used for the stabilization of microsomal preparations during storage was investigated. The current investigations show evidence that the chemical reaction of acrolein with liver microsomal proteins strictly follows first order kinetics. The main part of the formed acrolein in the in vitro attempts is available as bound part. Results of these investigations indicate that the calibration should be carried out with mixtures from liver microsome preparations and known amounts of acrolein under the same conditions as the in vitro experiments to record the entirely formed acrolein part (free and bound) in oxazaphosphorine turnover experiments. Glycerine is recommended as a preservative to store liver microsomes instead of sucrose because the latter reacts with acrolein.

The plasma kinetics of procarbazine (PCB) and its major metabolite azo-procarbazine (azo-PCB) were systematically investigated in humans for the first time. Eight therapy-refractory tumor patients with normal liver and renal function were given a single oral dose of 300 mg PCB hydrochloride as a drinking solution under fasting conditions. With the exception of the single i.v. administration of 10 mg ondansetron hydrochloride immediately before the administration of PCB, the patients were free of any co-medication 4 weeks before and during the study. PCB and azo-PCB were determined by a specially developed HPLC-UV method. PCB was absorbed very rapidly. Mean maximum plasma concentration was 12.5 min. A high elimination rate of PCB from plasma was found. The mean apparent oral systemic clearance and the plasma elimination half-life were estimated at 35.8 l/min and 9.2 min, respectively. Considerable amounts of azo-PCB are found in the plasma of the eight tumor patients. The mean C-max and AUC ratios of azo-PCB/PCB were estimated at 5.5 and 45.2. Azo-PCB is formed very rapidly from PCB, but eliminated much more slowly from plasma than PCB. Considerable interindividual differences in the conversion rate of azo-PCB to its further metabolites were observed which should have consequences for the individual tumor therapeutic efficiency of PCB. No toxic side-effects or symptoms such as nausea or vomiting were observed during the entire study.

Ifosfamide is a prodrug that requires bioactivation by cytochrome P450 for antitumour activity. Up to now, little is known, to what extent in addition to the liver the ifosfamide metabolism may occur intratumorally. For this purpose, we investigated the expression of CYP3A4, CYP2C9 and CYP2B6 in breast cancer tissue using Western Blotting. Ifosfamide turnover was determined by detection of metabolites of the ifosfamide 4-hydroxylation and N -dechloroethylation in tumour microsomal incubations using HPLC/UV and LC/MS. The results demonstrate that all mammary tumours ( n =11) reveal CYP3A4 expression; contents varied from 0.5 to 63 pmol mg protein −1 . CYP2C9 ( n =9) was present in all tested breast tumour samples, too, while CYP2B6 ( n =10) protein could not be detected. All measured breast cancer microsomes ( n =4) showed an ifosfamide N -dechloroethylation capacity in the range from 0.04 to 0.21 pmol min −1 mg protein −1 , while metabolites of the 4-hydroxylation could not be determined. In conclusion, the detected presence of CYP3A4 and CYP2C9 in breast tumours offers the possibility of intratumoral turnover of ifosfamide. For the first time in the literature, we could demonstrate a turnover of ifosfamide by microsomal preparations from human breast cancer tissue. A calculated modulation of intratumoral ifosfamide turnover could considerably influence its therapeutic efficiency.

This article presents an overview of the methods of bioanalysis of oxazaphosphorines and their metabolites, in particular liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS–MS) assays. Especially for the molecules that do not contain chromophores as oxazaphosphorines the mass spectrometry took an important place in chromatographic analysis. For trace analysis the LC–MS–MS methods are particularly well suited. Above all gas chromatography–mass spectrometry (GC–MS) assays were applied for the determination of these substances, in addition, LC–MS methods and other high-performance liquid chromatography (HPLC) or GC methods were included in the consideration.

This article presents an overview of the methods of bioanalysis of oxazaphosphorines, in particular, cyclophosphamide, ifosfamide, and trofosfamide as well as their metabolites. The metabolism of oxazaphosphorines is complex and leads to a large variety of metabolites and therefore the spectrum of methods used is relatively broad. The various methods used are shown in a table and the particularly important assays are described.

Pharmacokinetic gender-dependent differences in cytochrome P450-mediated drug metabolism, especially CYP3A4, and their clinical implications are increasingly apparent. CYP3A4 seems to be the most important CYP isoform in both bioactivation and N-dechloroethylation of the alkylating prodrug ifosfamide, but informations about possible gender-related differences are lacking. Therefore we compared in 10 male and 10 female liver microsomal preparations the contents and activities of specific isoenzymes, involved in both metabolic pathways, especially CYP3A4, further CYP2A6, CYP2C9 and CYP2B6 and measured the in vitro activities of these microsomes in the ifosfamide 4-hydroxylation and N-dechloroethylation using high-sensitive HPLC/MS and--UV detection methods.

A sensitive HPLC-MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C18 cartridges followed by HPLC-MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.