Introduction: A promising candidate in the field of pharmacological treatment options regarding major depressive disorder (MDD) is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO is crucial for neurosteroid synthesis, which is in turn important for the regulation of emotions. It has already been shown that TSPO expression in platelets of depressed patients is reduced compared to healthy subjects. Methods: We measured TSPO levels in platelets of 37 depressed patients before and after 6 weeks of pharmacological treatment to test the hypotheses that i) such treatment would increase TSPO expression and ii) that this increase would be correlated with therapeutic response. Results: Surprisingly, TSPO levels in platelets of all patients were significantly reduced after 6 weeks of treatment (p=0.044). Within the responder group, a non-significant trend towards greater TSPO level reduction could be observed. Discussion: These results challenge our hypotheses that TSPO levels might increase during antidepressant therapy along with a decrease in depressive symptoms. Thus, we assume that TSPO expression in platelets is not a suitable state marker for MDD.

Introduction: The treatment of anxiety disorders is still a challenge; novel pharmacological approaches that combine rapid anxiolytic efficacy with fewer side effects are needed. A promising target for such compounds is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO plays an important role for the synthesis of neurosteroids, known to modulate GABA(A) receptors, thereby exerting anxiolytic effects.Methods: We investigated the pharmacological profile of 2 well established TSPO ligands (XBD173 and etifoxine) compared to the benzodiazepine diazepam with regard to TSPO binding affinity, TSPO expression and neurosteroidogenesis.Results: In BV-2 microglia and C6 glioma cells all compounds significantly enhanced TSPO protein expression. Radioligand binding assays revealed the highest binding affinity to TSPO for XBD173, followed by diazepam and etifoxine. Pregnenolone synthesis was most potently enhanced by etifoxine.Discussion: Etifoxine turned out to be the most potent enhancer of neurosteroidogenesis, although its binding affinity to TSPO was lowest. These results indicate that the efficacy of TSPO ligands to stimulate neurosteroid synthesis, thereby leading to anxiolytic effects cannot be concluded from their binding affinity to TSPO.

Aims: The use of low cost equipment that nevertheless yields drug concentrations within reasonable running times is essential for routine TDM. We focus our work on the development of analytical methods that allow the quantification of as many drugs as possible by HPLC under the same conditions. So far we measured antidepressants under three different conditions. Our aim was to combine these methods. Methods: The chromatographic separation was achieved on a PFP column with acetonitrile/potassium phosphate buffer as a mobile phase in automated column-switching HPLC using an UV detector. Results: Specimens were stable for > six months. The analytical method yielded good separation of O-desmethylvenlafaxine, amisulpride, lamotrigine, mirtazapine, venlafaxine, oxcarbazepine, quetiapine, donepezil, desmethylcitalopram, citalopram, paroxetine, fluvoxamine, dihydroaripiprazole, desmethylsertraline, aripiprazole, sertraline, norfluoxetine, and fluoxetine within 40 minutes in specimens obtained from patients that were treated either with sertraline or with fluoxetine. The method was validated according to the recommendations of GTFCH (Gesellschaft für toxikologische und forensische Chemie), for all substances tested recovery was >80%, the limit of detection 10ng, intraday precision <5%, and selectivity proofed not to interfere with 100 drugs usually prescribed for depression. TDM evaluation by KONBEST classified just 20% of the specimens as both in line with the prescribed dose and within the therapeutic reference ranges of the drugs. Conclusion: This method allows efficient quantification of a variety of often prescribed psychotherapeutic agents.

Introduction: We have been able to routinely measure concentrations of psychotropic drugs in human blood by using HPLC/UV in our laboratory since 2006. Currently, we have a diversity of 52 substances that can be separated and detected by several validated methods. An optimization of this sector is the use of UHPLC (Ultra High Pressure Liquid Chromatography) which allows faster separation of more substances. Methods: We use a reversed phase column and phosphate buffer with methanol and as variable part acetonitrile for the measurement. Substances were measured with a UHPLC/UV method. A protein precipitation step was required for pre-purification of human blood. Results: To measure 21 pharmaceutical drugs by HPLC, the laboratory uses actually four different methods for clinical routine. The UHPLC/UV method could measure 20 of these drugs plus 2 additional compounds. The detection limit was lower than the therapeutic range of all the substances. Therapeutic ranges of pharmaceutical drugs such as haloperidol that have very small concentrations in human blood could be measured by this method. The analytical measurement of 22 substances required 23 minutes. Time for measurement and costs for chemicals could be reduced significantly. Conclusion: UHPLC gives us the opportunity to separate und detect many substances under the same chromatographically conditions. A transfer of the routine analytics of HPLC to an UHPLC reduces costs and needs less time. Besides, this method can be used for routine analysis even though substances have only a very low concentration of active agents.

Measuring serum concentrations in patients' blood does not only give information about therapeutic effects but also – and even more so – about individual abnormalities in metabolism or compliance problems. This is possible if the measured concentration is not only referred to the well-known therapeutic reference range but also to a dose-related reference range. The last is calculated with data of clearance, bioavailability, daily dose and interval of taking. We're reporting about a female patient born in 1970, where we measured the serum concentrations of quetiapine several times. Following table shows the course of our results. In all measurings, except for the last one, the quetiapine concentration was 4–8/9fold too high, referred to the respective dose-related reference ranges. In the comedication amitriptyline and in the samples 2, 3 and 4 simvastatine are listed in literature as enzyme inhibitors. However, in our experience these interactions cannot explain the dose-related markedly increased concentrations. That is why we stated a poor metabolizer status for this patient and recommended continuous measuring of serum concentrations. Six weeks after the last very high concentration, another sample was measured. We found a surprisingly weakly increased concentration (just 1.5 fold above the dose-related reference range). Since we expect an increased concentration because of the poor metabolizer status of the patient, we now assume lack of compliance.

Introduction: Therapeutic drug monitoring is an essential tool to optimize pharmacotherapy through relating drug concentration to two reference ranges: the therapeutic reference range and the dose related reference range. To achieve this purpose a specific method to determine the concentration of fluoxetine, desmethylfluoxetine, sertraline and desmethylsertraline in serum was developed. It was applied in routine TDM with adding a clinical pharmacological report using Konbest (computer program). Methods: The chromatographic separation was achieved on a core shell column with acetonitrile/methanol/potassium phosphate buffer as mobile phase in automated column-switching HPLC using an UV detector. All chemicals used were of HPLC grade. The drugs used were obtained from different pharmaceutical companies in Germany. Drug free serum was spiked with the standard stock solutions of drugs to get the final concentrations needed. The later concentrations were then injected in the HPLC system. Results: The method was satisfactory, the compounds were well separated with a very good recovery. It was validated according to the recommendations of GTFCH (Gesellschaft für toxikologische und forensische Chemie). Conclusion: This method allows efficient and rapid determination of fluoxetine, sertraline and their metabolites in serum. It is practicable for the routine TDM and for the emergency cases. The Konbest information could guide the treatment of the patients.

In former years it was generally accepted to use therapeutic drug monitoring (TDM) to prevent toxic side effects of drugs with a narrow therapeutic window. Very often it was just used to confirm that a side effect that had already occurred was due to an elevated drug plasma concentration. For this purpose the quantified drug concentration was related to the therapeutic reference range of the drug. We relate the drug concentration to both the therapeutic reference range and the dose-related reference range. Dose-related reference ranges are calculated according to the mathematical equation De = Clt * c, where De is the maintenance dose and Clt the total clearance taken as x ± SD from phase II trials of the drug. A drug concentration outside this range is taken as a signal that the patient does not belong to the study population because of a comedication or a pharmacologically active food or drug component (drug-drug-interaction), an age below 18 years (children), an age above 65 years (old people), a genetically determined alteration in drug metabolism (fast/slow metabolizers), diseases in the elimination organs, or non-compliance. KONBEST, an internet platform that is programmed to allow clinical pharmacological commenting of drug concentrations in TDM, contains the relevant pharmacological data to calculate among others the dose-related reference ranges. By this approach we are able to prevent adverse drug reactions before the drug concentration is high enough to cause them.

Background: The therapeutic usage of Flupentixol is due to its efficacy as an antipsychotic drug. The approximate concentration for a satisfactory antipsychotic effect is 1–10 ng/ml. This low therapeutic range made the serum therapeutic drug monitoring of this drug very important. It will contribute in areas of clinical and forensic applications in optimising the patients' therapy. Material and Method: Choosing a suitable HPLC-Column for a given method is one of the factors that determines the reliability and accuracy of the measurements during a method development. A high-performance liquid chromatographic method with an UV-Detector was used. The pool human serum was donated by the university clinic in Regensburg (Germany). The columns used were Luna-Phenyl-Hexyl, Thermo-Betasil, SphereClone ODS und Nucleodur. The analytic solvent was a mixture of Acetonitrile-Methanol-Water. Result: For the determination of Flupentixol in the Serum sample, the use of Nucleodur column and 43% of organic solvent was found to be optimal. The serum levels were linear and the amount yielded corresponds to the given and expected amount. Conclusion: The accuracy and precision of this method is satisfactory. Pre-treatment procedures of the samples were not necessary. For the further improvement of the individual patients' therapy, it is to be recommended as a faster method in the therapeutic drug monitoring of Flupentixol.

Introduction: Butyrophenones are widely used for the treatment of psychoses and are frequently encountered in clinical toxicology, clinical pharmacology and forensic chemistry. This chemical class of antipsychotics tend to have a narrow therapeutic range and the quantity required to be effective is near to the quantity that causes significant side effects. Maintaining their steady state poses therefore a problem for the therapy. The rates at which individuals absorb, metabolize and eliminate drugs are based upon their age, general state of health, genetic makeup, and the interference of other medications. Therefore, the therapeutic drug monitoring of the selected butyrophenone serum concentrations plays an important role in the selection of the optimum dose for the therapy. This research method is aimed at the simultaneous quantification of benperidol, bromperidol and haloperidol without any pre-treatment step, which will help for a faster routine control. It is also aimed to be as specific as possible in separating other butyrophenones and similar substances in human serum for a better control of their different therapeutic concentrations. Materials and Methods: The chromatographic separation of the antipsychotics used in the experiment so far was performed with a C18 reversed-phased column with acetonitril and water (milli-Q) as eluent and with the help of an UV detector. The pool serum was obtained from the university clinic Regensburg. All chemicals used were of the highest purity commercially available. The raw materials used so far for the experiment was obtained from different pharmaceutical companies in Germany. The standard stock solutions of these raw substances were prepared by dissolving a properly weighed amount of the named substances in methanol to produce a standard solution. The diluted solutions were spiked in serum to yield the final concentrations for the measurement. The prepared butyrophenone serum concentrations were then injected in the HPLC-System for their chromatographic measurements. Result: The obtained results were very encouraging, the compounds were well separated reproducing a very good percentage amount in comparison with the expected added amount. So far, the three compounds showed good linearity with a detection limit of 2 ng/ml. Imprecision and inaccuracy that may occur during the experimental work has not yet been fully validated, because the research work is still successfully going on and very promising. The aim of getting a lower limit of detection is being targeted at and it is progressing. Conclusion: The advantage of this research work lies not only on its effectivity, but it is also a quicker method in the determination of serum antipsychotic concentrations. It is very practicable in case of any emergency and it also opens the possibility for quicker results of TDM routine measurements. It will also minimize the expenditure for the routine determination of a large set of individual and varying drug combinations.

Introduction: Interpreting an individual patient's drug concentration in relation to a therapeutic and a dose-related reference range is suitable for controlling compliance, lack of clinical response, adverse effects at recommended doses, drug interactions and genetic variations of metabolism. Therefore it is an important contribution to complement pharmacovigilance programs [1–2]. Bupropion is an antidepressant and smoking cessation aid. It acts as a dopamine and norepinephrine reuptake inhibitor, as well as α3β4-nicotinic receptor antagonist. Bupropion is extensively metabolized. Three pharmacologically active metabolites were identified in human plasma; hydroxybupropion, threo-hydrobupropion and erythro-hydrobupropion. We aim to establish a simple method for automated quantitative analysis of bupropion and its major metabolites in serum using column-switching high performance liquid chromatography (HPLC). Several routine methods were evaluated in reference to their prospective capacity to determine bupropion and its major metabolites, and UV-spectra of bupropion, hydroxybupropion, threo-hydrobupropion and erythro-hydrobupropion were recorded to determine the ideal detection wavelengths. Methods: The chromatographic analyses were performed on a Dionex system with Betasil C6 (250 × 4,6 mm, 5 µm, Method 1 and 2) or PerfectSil 120 ODS-L (250 × 4,6 mm, 5 µm, Method 3) analytical columns. The mobile phase consisted of 0,04 M KH2PO4/acetonitrile (pH 3.0) for Method 1, 0,02 M HCOOCH3/acetonitrile (pH 3.0) for Method 2, and acetonitrile/MeOH/TEMED/H2O mixture at pH 6.5 for Method 3. For sample clean-up we used online-extraction where serum was injected onto a LiChristopher ADS RP-4 precolumn and retained analytes were eluted by back-flush flow onto the analytical column. Results and Conclusion: Bupropion and hydroxybupropion are easily separated by all tested methods. The separation of threo-hydrobupropion and erythro-hydrobupropion was better for the low pH measurements (Method 1 and 2) but insufficient for those under neutral pH conditions (Methods 3). However, Method 1 and Method 2 yielded almost identical retention times for erythro-hydrobupropion and bupropion. As for the detection wavelengths, bupropion has absorption maxima at 210 nm and 254 nm. The UV-absorption spectra of hydroxybupropion, threo-hydrobupropion, and erythro-hydrobupropion all show absorption maxima at 211 nm and only very weak absorption at higher wavelengths. Thus, a bupropion peak measured at 254 nm would be hardly affected by underlying metabolite peaks. The method development is still in progress. Mobile phase components that are nontransparent above 210 nm – e.g. carbon acids – need to be replaced. The separation problem for threo- and erythro-hydrobupropion is yet to be solved. References: [1] Baumann P, Hiemke C, Ulrich S, et al. The AGNP-TDM expert group consensus guidelines: Therapeutic drug monitoring in psychiatry. Pharmacopsychiatry. 2004 Nov; 37(6): 243–65. [2] Haen E, Greiner C, Bader W, Wittmann M. Wirkstoffkonzentrationsbestimmungen zur Therapieleitung: Ergänzung therapeutischer Referenzbereiche durch dosisbezogene Referenzbereiche. Nervenarzt 2008 May; 79(5): 558–66.

Interpreting an individual patient's drug concentration in relation to a therapeutic and a dose-related reference range is suitable for controlling compliance, lack of clinical response, adverse effects at recommended doses, drug interactions and genetic variations of metabolism. Therefore it is an important contribution to complement pharmacovigilance programs. [1–2]

Aim: A novel high performance liquid chromatography(hplc)assay for the purification of rivastigmine from serum, its detection and estimation has been developed and validated. Methods: A solid phase extraction using Oasis HLB-columns(Waters Ltd,Ger)was optimized to purify rivastigmine from serum followed by HPLC separation with UV detection. Chromatographic analyses were performed on a Dionex system with a Phenomenex Luna Phenyl-Hexyl analytical column. The mobile phase constituted of 0,02mol/l K2HPO4/acetonitrile(80/20)and the flow rate was kept at 0,4ml/min. The detection wavelength was kept at 210nm. Results: The retention time was 10,5min for rivastigmine. Our method recovered >92% of rivastigmine from the serum samples. The calibration curve was linear (r=0,9999, n=9) over rivastigmine concentrations ranging from 5 to 640ng/ml. No endogenous compounds were found to interfere with the analyte. The method had an accuracy of >90%. Intra- and interday precision were <5% and <3%, resp., at three different concentrations of 5, 80 and 640ng/ml. The limit of quantification(LOQ) was found to be 2,5ng/ml. The accuracy and precision at the LOQ level are in agreement with the guidelines of GTFCh(Society of Tox. and Forensic Chemistry) in consideration of ISO 5725(International Organization for Standardization). The method reported here is simple, reliable, precise, and accurate and has the capacity to be used for detection of rivastigmine in serum samples in routine analysis.