A new [2H]-labelled α-trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates.

A new reaction pathway for the synthesis of a [(2)H]-labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope-labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis.

LC-MS/MS method for the simultaneous quantification of artesunate and its metabolites dihydroartemisinin and dihydroartemisinin glucuronide in human plasma.

Artesunate (AS), a hemisuccinate derivative of artemisinin, is readily soluble in water and can easily be used in formulations for parenteral treatment of severe malaria. AS is rapidly hydrolyzed to the active metabolite dihydroartemisinin (DHA) and primarily eliminated by biliary excretion after glucuronidation. To investigate systematically the AS metabolism and pharmacokinetics, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of AS and its metabolites DHA and DHA glucuronide (DHAG) in human plasma samples was developed. Compared to previous methods, our method includes for the first time the quantification of the glucuronide metabolite using a newly synthesized stable isotope-labeled analogue as internal standard. Sample preparation was performed with only 50 µL plasma by high-throughput solid-phase extraction in the 96-well plate format. Separation of the analytes was achieved on a Poroshell 120 EC-C18 column (50*2.1 mm, 2.7 µm, Agilent Technologies, Waldbronn, Germany). The method was validated according to FDA guidelines. Calibration curves were linear over the entire range from 1 to 2,500 nM (0.4-961.1 ng/mL), 165 to 16,500 nM (46.9-4,691.8 ng/mL), and 4 to 10,000 nM (1.8-4,604.7 ng/mL) for AS, DHA, and DHAG, respectively. Intra- and interbatch accuracy, determined as a deviation between nominal and measured values, ranged from -5.7 to 3.5% and from 2.7 to 5.8%, respectively. The assay variability ranged from 1.5 to 10.9% for intra- and interbatch approaches. All analytes showed extraction recoveries above 85%. The method was successfully applied to plasma samples from patients under AS treatment.

Simultaneous quantification of mefloquine (+)- and (-)-enantiomers and the carboxy metabolite in dried blood spots by liquid chromatography/tandem mass spectrometry.

Mefloquine (MQ), a racemic mixture of (+)-(11S,12R)- and (-)-(11R,12S)-MQ, has been used for treatment and prophylaxis of malaria for almost 30 years. MQ is metabolized by the cytochrome P450 3A subfamily to 4-carboxymefloquine (CMQ), which shows no antimalarial activity in vitro. Highly stereospecific pharmacokinetics of MQ have been reported, although with contradictory results. This might be due to incorrect assignment of the absolute configuration as shown only recently. Gastrointestinal as well as neuropsychiatric adverse events were described after prophylaxis and treatment with MQ. Data are indicating that the tolerability of the enantiomers may vary considerably. An involvement of the main metabolite CMQ in the development of neuropsychiatric adverse events has also been supposed. Due to these inconsistent results we established a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of MQ enantiomers and the metabolite CMQ to investigate the attribution of efficacy and adverse effects to the single enantiomers as well as the main metabolite. Separation of the MQ enantiomers was achieved on a quinidine-based zwitterionic chiral stationary phase column, CHIRALPAK(®) ZWIX(-) (3.0×150mm, 3µm) in an isocratic run using a pre-mixed eluent consisting of methanol/acetonitrile/water (49:49:2 v/v) with 25mM formic acid and 12.5mM ammonium formate. We used stable isotope-labelled analogues as internal standards. The method was validated according to the FDA guidelines. With a linear calibration range from 5 to 2000nM for the MQ enantiomers and from 13 to 2600nM for CMQ respectively, the method was successfully applied to dried blood spot (DBS) samples from patients under prophylactic MQ treatment. The method was also applicable for plasma samples.