Suitability of collection tubes with separator gels for collecting and storing blood samples for therapeutic drug monitoring (TDM).

In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0-5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5-30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0-13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5-20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins.

Metabolism of [123I]epidepride may affect brain dopamine D2 receptor imaging with single-photon emission tomography.

Iodine-123 labelled epidepride is a novel radiopharmaceutical for the study of cerebral dopamine D2 receptors using single-photon emission tomography (SPET). A lipophilic labelled metabolite of [123I]epidepride which may enter the brain and hamper the quantitation of receptors has been observed in human plasma. In the present study, gradient high-performance liquid chromatography (HPLC) was used to investigate the plasma concentration of the lipophilic labelled metabolite and its correlation to SPET imaging of striatal dopamine D2 receptors. A linear regression fit showed a negative correlation between the amount of the lipophilic labelled metabolite and the striatum to cerebellum ratio (n=16, R=-0.58, P<0.02), suggesting that plasma metabolite analysis is essential when imaging dopamine D2 receptors with SPET using [123I]epidepride.

Fentanyl decreases beta-CIT binding to the dopamine transporter.

Evidence from animal studies suggest that centrally acting opiates increase synaptic dopamine (DA) concentration. However, the interaction between mu-opioid receptors and the DA system is unclear. We report here an effect of fentanyl on striatal [123I]beta-CIT binding to the DA transporter in a patient and in rats. A female patient underwent [123I]beta-CIT single-photon emission tomography (SPET) study after intrathecal injection of fentanyl for her back pain. After a 2-week drug-free period, the SPET study was repeated. In the experimental study, male Wistar rats were treated with fentanyl either acutely (50 micrograms/kg, i.p.) before imaging study or subacutely for 4 days (10 micrograms/kg, twice a day, i.p.). Brain planar imaging was performed at 3.5 hours after an intravenous injection of [123I]beta-CIT with gamma camera with a pinhole collimator. In a female patient, [123I]beta-CIT binding in the basal ganglia was decreased by 37% during fentanyl as compared to the binding after 2-week drug-free period. Similarly in rats, acute fentanyl treatment decreased [123I]beta-CIT binding to the striatum by 30% as compared to that of with the control rats. After subacute administration of fentanyl, no significant difference was observed compared to the control group. According to the present data, fentanyl decreases [123I]beta-CIT binding in the basal ganglia both in human and rats, suggesting that opiates possibly directly affect DA reuptake.

High-performance liquid chromatography method for analyzing citalopram and desmethylcitalopram from human serum.

This report describes a sensitive and specific method for analyzing a serotonin reuptake blocker, citalopram, and its active metabolite, desmethylcitalopram, in human serum. For high-performance liquid chromatography (HPLC) analysis, samples and standards are prepared with ASPEC automatic sample preparator using 100 mg Bond-Elut C-18 solid-phase extraction columns. The method is an isocratic HPLC method with a mobile phase of acetonitrile:methanol:50 mM dipotassium hydrogenphosphate, pH 4.7 (40:100). Detection is performed with diode array detector at 220 nm and the peak purity analyses at 210 to 365 nm. The intraassay coefficient of variation ranges from 3.7% to 7.3%, and the interassay coefficient of variation ranges from 6.9% to 9.9% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method is suitable for therapeutic drug monitoring in a clinical laboratory. A clear correlation, r = 0.72 (y = 0.36x + 17.94), between citalopram and its metabolite levels is observed in routine therapeutic drug monitoring service. A linear correlation between serum concentration and daily dose of citalopram in patient groups is also observed.

Iodine-123 labeled nor-beta-CIT as a potential tracer for serotonin transporter imaging in the human brain with single-photon emission tomography.

Iodine-123 labelled 2beta-carbomethoxy-3beta-(4-iodophenyl) (nor-beta-CIT) is an analogue of beta-CIT, which has high affinity to the serotonin transporter. Initial single-photon emission tomography (SPET) studies with [123I]nor-beta-CIT were performed in five healthy volunteers. In addition, its metabolism in plasma was investigated with gradient high performance liquid chromatography. [123I]nor-beta-CIT was prepared by a method which gave a specific radioactivity of more than 180 GBq/micromol. Unchanged [123I]nor-beta-CIT in plasma accounted for 43% and 19% of total radioactivity after 30 and 180 min, respectively. The dynamic SPET studies demonstrated a high and rapid uptake of radioactivity in the brain (6%/ID at 30 min). Highest accumulation was observed in the striatum, the mid-brain and the thalamus. The specific binding in the mid-brain was 33% higher compared with that of [123I]beta-CIT. The high radioactivity in the mid-brain is assumed to represent the accumulation of [123I]nor-beta-CIT in the serotonin transporter-rich regions, which indicates that [123I]nor-beta-CIT might be a potential tracer for visualization of serotonin transporter sites in the human brain with SPET.

Analysis of clozapine and norclozapine by high-performance liquid chromatography.

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C18 SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C8 reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210-365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 to 8.0% and the inter-assay C.V.s were from 1.1 to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.

Striatal and extrastriatal imaging of dopamine D2 receptors in the living human brain with [123I]epidepride single-photon emission tomography.

The iodine-123 labelled ligand benzamide epidepride was evaluated as a probe for in vivo imaging of striatal and extrastriatal dopamine D2 receptor sites in the human brain. Four healthy males were imaged with a high-resolution single-photon emission tomography scanner. Striatal radioactivity peaked at 3 h after injection. The specific binding in the striatum was 0.91+/-0.03 at 3 h and this ratio steadily increased with time. Extrastriatal radioactivity was highest in the thalamus, in the midbrain and in the temporal cortex, and peaked at 45-60 min after injection of tracer. A smaller amount of radioactivity was found in the parietal, frontal and occipital cortices. Two radioactive metabolites were observed, of which one was more lipophilic than the parent compound. The radiation burden to the patient was 0.035 mSv/MBq (effective dose equivalent). The preliminary results showed that [123I]epidepride can be used for imaging striatal and extrastriatal dopamine D2 receptor sites in the living human brain.

Analysis of clobazam and its active metabolite norclobazam in plasma and serum using HPLC/DAD.

This report describes a simple reversed-phase high-performance liquid chromatographic (HPLC) method with automated solid-phase extraction (SPE) for analysing clobazam and norclobazam concentrations in human serum or plasma. For the HPLC analysis the samples and standards are prepared with an ASPEC automatic sample preparer using 100-mg Bond-Elut C-18 solid-phase extraction columns. The HPLC method is an isocratic method with a mobile phase of acetonitrile:methanol:10 mmol l-1 dipotassium hydrogen phosphate, pH 3.7 (30:2:100), at a flow rate of 1.5 ml min-1. The benzodiazepines are detected with a diode array detector (DAD) at 240 nm and the peak purity analyses are performed at 210-365 nm. The recovery is over 97% for both analytes, and it is independent of the drug concentration. The intra-assay CVs vary between 0.7 and 2.2% and inter-assay CVs between 3.8 and 4.6% at therapeutic drug concentrations. The detection limit is 15 nmol l-1. The assay is linear from 30 to 20,000 nmol l-1 (clobazam) and from 170 to 105,000 nmol l-1 (norclobazam). This method leads to a very good separation of norclobazam from carbamazepine and phenytoin. None of the anti-epileptic or antidepressant drugs tested interfere with the assay.

Analysis of low-dose benzodiazepines by HPLC with automated solid-phase extraction.

We describe a simple, specific, and sensitive reversed-phase HPLC method with automated solid-phase extraction (SPE) for analyzing alprazolam, clonazepam, and nitrazepam concentrations in human serum or plasma. We prepare samples and calibrators with an automated sample preparer, using 100-mg Bond-Elut C18 SPE columns. The HPLC method uses isocratic elution with acetonitrile:methanol:dipotassium hydrogen phosphate, 10 nmol/L, pH 3.7 (30:2:100 by vol), at a flow rate of 1.5 mL/min to separate the drugs. We detect the benzodiazepines with diode-array detector at 240 nm; analyses of peak purity are performed at 210-365 nm. The recoveries were between 94% and 100%. The intraassay and interassay CVs were between 1.0% and 4.1%. The detection limit is 5 nmol/L. The antiepileptic and antidepressant drugs tested did not interfere with the assay. We developed the method for use in a clinical laboratory for therapeutic drug monitoring.

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.