Determination of hydroxybupropion in human serum for routine therapeutic drug monitoring in psychiatry: A tool for dose-individualization in treatment with bupropion.

Therapeutic drug monitoring (TDM) has become a clinical routine in psychiatry. Nevertheless, for bupropion there is only one method available that is suitable for routine use. However, it involves a complex sample clean-up. Owing to the instability of bupropion in serum, the main and active metabolite hydroxybupropion was chosen as the target substance. Therefore, a simple and robust high-performance liquid chromatography method for the quantification of hydroxybupropion in serum was developed and validated. A volume of 30 µL serum was used for easy sample clean-up, based on protein precipitation with acetonitrile followed by online solid-phase extraction. As hydroxybupropion was present in high serum concentrations, UV detection was possible. Owing to the commonly available instrumentation, the method could easily be integrated in routine TDM. The newly developed method was validated following the guidelines for bioanalytical method validation of the European Medicines Agency and US Food and Drug Administration. The lower limit of quantification was 100 ng/mL (0.391 µm) and linearity was shown between 100 and 2500 ng/mL. Intraday and interday precision ranged from 1.17 to 6.79% and from 6.07 to 9.41%, respectively. Intraday and interday accuracy ranged from 89.97 to 110.86% and from 95.05 to 101.2%. The method was shown to be selective, accurate and precise. Additionally, the method was successfully implemented in the therapeutic drug monitoring laboratory of the Department of Psychiatry, Psychosomatics and Psychotherapy at the University Hospital of Würzburg, Germany. Six months of routine analysis showed a rather low correlation between applied dose and serum concentration and therefore the necessity of TDM for dose-individualization in the treatment with bupropion.

Impurity profiling of N,N'-ethylenebis-l-cysteine diethyl ester (Bicisate).

A HPLC-UV-CAD method with a HILIC column for impurity profiling of the 99mTc chelating agent bicisate has been developed and evaluated. Bicisate and its impurities were separated by means of isocratic elution on a zwitterionic stationary phase using a mixture of 7.5mmol/L trifluoroacetic acid and acetonitrile (47.5:52.5 V/V) as the mobile phase. Five different bicisate batches of a manufacturer were tested using the method. In addition LC-MS experiments were conducted in order to identify the impurities. The predominant impurities found were the oxidation product (disulfide), the monoester of ethylene dicysteine and an unknown compound with an m/z of 293 in ESI positive mode. A new degradation product of bicisate, bicisate lactam, was identified during sample solution stability assessment.

Evaluation of enantiomeric purity of magnesium-L-aspartate dihydrate.

Magnesium supplementation in form of organic magnesium salts is a very popular practice. We examined the enantiomeric purity of "Magnesium aspartate dihydrate" monographed in the European Pharmacopeia. A chiral capillary zone electrophoresis using (2-hydroxypropyl)-ß-cyclodextrin coupled to laser induced fluorescence detection and a HPLC-fluorescence method with chiral derivatization using o-phthaldialdehyde and N-acetyl-L-cysteine as an orthogonal method were developed and validated. Two batch samples of this substance and three drug products containing the salt were analyzed by means of both methods. The concentration of the D-enantiomer of aspartic acid ranged from 0.03 to 0.12%. Simulations of the synthesis revealed that the d-aspartic acid content is elevated if the dissolution of L-aspartic acid was performed at acidic pH values.

Impurity profiling of carbocisteine by HPLC-CAD, qNMR and UV/vis spectroscopy.

For the impurity profiling of the mucolytic and anti-inflammatory drug carbocisteine a high performance liquid chromatographic (HPLC) method using corona charged aerosol detection (CAD) was developed and fully validated following the ICH guideline Q2(R1). The response was linear (R²>0.995) over a small concentration range (0.05-0.25 or 0.10-0.60% respectively) and a detection limit of at least 0.03% was registered. The separation was achieved on a mixed mode column combining hydrophobic C18 and strong cation exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid 10 mM and acetonitrile 12% (V/V). Impurities, not assessable by HPLC-CAD such as the volatile chloroacetic acid and the unstable cysteine, were determined by quantitative NMR (qNMR) with maleic acid as internal standard and UV/vis spectroscopy after reaction with Ellman's reagent, respectively. Six batches of three different manufacturers were tested by means of those methods. The purity varied from below 99.0 to higher than 99.8 per cent. The major impurities of all batches were the starting material cystine and N,S-dicarboxymethylcysteine being a synthesis by-product.

Anti-inflammatory effects of clopidogrel intake in renal transplant patients: effects on platelet-leukocyte interactions, platelet CD40 ligand expression, and proinflammatory biomarkers.

BACKGROUND: Increased platelet activation has been described during treatment with various immunosuppressive agents and may contribute to the high cardiovascular mortality rate in renal transplant patients. Platelets are thought to propagate the inflammatory process of atherosclerosis by interaction with leukocytes. METHODS: We tested an experimental therapy with clopidogrel in renal transplant patients treated with either tacrolimus (n = 20) or cyclosporine (INN, ciclosporin) (n = 19). All patients took low-dose steroids and had stable transplant function. Untreated healthy volunteers (n = 11) were included as the reference group. Degranulation (CD62P), glycoprotein IIb/IIIa receptor activation (PAC1), formation of platelet-leukocyte aggregates (monocyte-platelet-leukocyte aggregate, CD11b, mean fluorescence intensity), and platelet CD40 ligand (CD40L) expression (percent positive) were assessed by flow cytometry before therapy (visit 1) and after 4 weeks of clopidogrel (75 mg/d) intake (visit 2). To assess systemic anti-inflammatory effects, we measured levels of high-sensitivity C-reactive protein, soluble CD40L (sCD40L), monocyte chemoattractant protein 1, and matrix metalloproteinase 9 (MMP-9) by enzyme-linked immunosorbent assay. RESULTS: At visit 1, cyclosporine-treated patients had significantly enhanced CD62P and PAC1 expression and platelet-leukocyte aggregate formation, as well as elevated sCD40L concentrations, compared with tacrolimus-treated patients (all P < .03). Clopidogrel intake led to a significant decrease in platelet-leukocyte aggregate formation in tacrolimus-treated patients (median, 237 [interquartile range, 177-510] to 194 [interquartile range, 159-275] mean fluorescence intensity; P < .035) and cyclosporine-treated patients (median, 450 [interquartile range, 362-782] to 254 [interquartile range, 211-458] mean fluorescence intensity; P < .035). CD40L expression was reduced in tacrolimus-treated patients (median, 34 [interquartile range, 28-41] to 21 [interquartile range, 12-26] mean fluorescence intensity; P < .002) and cyclosporine-treated patients (median, 33 [interquartile range, 30-37] to 26 [interquartile range, 19-26] mean fluorescence intensity; P < .02). In addition, CD62P, PAC1, and CD11b were significantly reduced in both groups at visit 2 (P < .02). MMP-9 decreased from 88 ng/mL (range, 49-135 ng/mL) to 57 ng/mL (range, 38-73 ng/mL) (P < .05) in tacrolimus-treated patients and from 79 ng/mL (range, 54-148 ng/mL) to 66 ng/mL (range, 41-97 ng/mL) (P < .01) in cyclosporine-treated patients. The sCD40L concentration decreased significantly only in cyclosporine-treated patients (P < .004). In contrast, high-sensitivity C-reactive protein and monocyte chemoattractant protein 1 were not affected. CONCLUSION: The P2Y(12) receptor antagonist clopidogrel inhibits the expression of platelet activation markers and the interaction of platelets and leukocytes. Because the synthesis of vascular disease markers and inflammatory products such as sCD40L and MMP-9 has been inhibited, anti-inflammatory properties of clopidogrel are likely to be a result of decreasing platelet activation.