Quantitation of drug metabolites in the absence of pure metabolite standards by high-performance liquid chromatography coupled with a chemiluminescence nitrogen detector and mass spectrometer

Quantitative information on drug metabolites with pharmacological or toxicological activities is of great interest during the drug discovery and development process. Because the analyte response with mass spectrometry can change significantly due to small variations in chemical structure, pure standards are required to construct standard curves for quantitation. However, for most programs at the discovery stage, pure metabolite standards are not available. In this work, an evaluation was conducted using a chemiluminescent nitrogen detector (CLND) as a calibrator to obtain the response factor ratio on a mass spectrometer generated from a metabolite and its parent compound in biological fluids. Using the response factor ratio obtained from the CLND, the metabolite could be quantified with the liquid chromatography/tandem mass spectrometry (LC/MS/MS) response obtained from the parent drug's standard curve. For this evaluation, oxazepam and temazepam were chosen as a 'drug/metabolite' pair. Temazepam was treated as the methylated metabolite of oxazepam. A spiked dog urine sample with a known concentration of oxazepam and unknown concentration of temazepam was injected onto the HPLC system and detected by both the CLND and MS/MS. Taking advantage of the equimolar response feature of the CLND, a response factor ratio between temazepam and oxazepam on the mass spectrometer was obtained by comparing the peak areas generated on the CLND and the mass spectrometer. From this ratio, temazepam was quantified using the oxazepam standard curve. The difference between the concentration of temazepam obtained from the reconstructed standard curve and the concentration obtained directly from a real temazepam standard curve was within 13% except the least concentrated standard (31%). This methodology has been successfully applied to measure quantities of the metabolite of a proprietary compound in a dog pharmacokinetic (PK) study.