Determination of N-desethylamodiaquine by hydrophilic interaction liquid chromatography with tandem mass spectrometry: application to in vitro drug metabolism studies.

The antimalarial drug amodiaquine is extensively metabolized to N-desethylamodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). DEAQ formation is an enzyme specific reaction that is used to quantify in vitro CYP2C8 activity. A rapid and sensitive method for the determination of DEAQ in human liver microsomes was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC-MS/MS). Microsomal incubation samples were processed by protein precipitation with acetonitrile. The analytes were separated on a BETASIL Silica-100 (50mmx2.1mm, 5microm) column by isocratic elution at a flow rate of 220microl/min with a mobile phase consisting of 85% acetonitrile containing 5mM ammonium acetate and 0.1% formic acid. Detection was by positive electrospray ionization on a TSQ Quantum Discovery triple quadrupole mass spectrometer operated in the selective reaction monitoring mode. The precursor-product ion pair was m/z 328-->283 for DEAQ and m/z 331-->283 for DEAQ-d(3). The lower limit of quantification was 10nM for DEAQ and linearity was observed over the concentration range of 10-1500nM. Intra- and inter-day accuracy and precision were within 3.4 and 7.0%, respectively. The method was successfully applied to CYP2C8 drug metabolism studies in pooled human liver microsomes.

Assessment of oral bioavailability and preclinical pharmacokinetics of DRF-6196, a novel oxazolidinone analogue, in comparison to linezolid.

The aim of this study was to determine the bioavailability of a novel oxazolidinone, DRF-6196, in mice and rats following intravenous (i.v) and oral dosing and to compare the pharmacokinetics with those obtained following linezolid dosing. Blood samples were drawn at predetermined intervals up to 24 h post-dose after either DRF-6196 or linezolid administration. The concentrations of DRF-6196 and linezolid in various plasma samples were determined by a HPLC method. Following oral administration maximum concentrations of DRF-6196 were achieved within 0.5 h irrespective of the species. While the doses increased in the ratio of 1 : 3 : 10, mean Cmax and AUC(0-infinity) values in mice for DRF-6196 increased in the ratio of 1 : 3.87 : 8.53 and 1 : 2.51 : 9.24, respectively. Both the Cmax and AUC(0-infinity) values increased almost proportional to the dose administered in mice. Following i.v administration, the concentration of DRF-6196 declined in a bi-exponential fashion with terminal elimination half-life of 1.5 h irrespective of the species. The systemic clearance and volume of distribution of DRF-6196 in mice were 1.14 L/h/kg and 0.66 L/kg, respectively after i.v administration, while the respective values in rats were 0.61 L/h/kg and 0.41L/kg, respectively. Elimination half-life ranged between 0.8-1.5 h. Absolute oral bioavailability of DRF-6196 was found to be 80-96% across the test dose range. Although plasma levels of DRF-6196 were lesser compared to linezolid in the initial hours, it may not have any consequences on the clinical effectiveness of the molecule.

Validation of a simple bioanalytical method for the determination of DRF-6196, a novel oxazolidinone, in mouse plasma: application to a single-dose pharmacokinetic study.

An isocratic simple, specific, sensitive and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the estimation of DRF-6196, a novel oxazolidinone in mouse plasma. This method involves a simple liquid/liquid extraction of DRF-6196 and the internal standard (IS; chlorzoxazone, CAS 95-25-0) from plasma into dichloromethane/ethyl acetate mixture that was evaporated under nitrogen. The HPLC analysis was carried out on an Inertsil ODS 2 column using 0.01 mol/L potassium dihydorgen ortho phosphate (pH 3.2) and acetonitrile (65:35, v/v) as mobile phase. The eluate was monitored using an UV detector set at 266 nm. Ratio of peak area of analyte to IS was used for quantification of plasma samples. The retention time of DRF-6196 and IS were 8.2 and 11.1 min, respectively. The assay was linear (r2 > 0.999) in the concentration range 0.1-50 microg/ml. Absolute recovery for analyte and IS was > 94 % from mouse plasma. The lower limit of quantification (LLOQ) of DRF-6196 was 0.1 microg/ml. The inter- and intra-day precision in the measurement of quality control (QC) samples, 0.1, 0.3, 15.0 and 40.0 microg/ml, were in the range 3.64 to 9.51 % relative standard deviation (RSD) and 0.92 to 6.23 % RSD, respectively. Accuracy in the measurement of QC samples was in the range 88.15 to 106.05 % of the nominal values. The analyte and IS were stable in the stability studies viz., benchtop, autosampler and freeze/thaw cycles. The stability of DRF-6196 was established for 1 month at -80 degrees C. The assay method was successfully applied to a pharmacokinetic study of DRF-6196 in mice.