The ability to simultaneously collect quantitative and qualitative information from a DMPK analysis has the potential to significantly
increase productivity in pharmaceutical drug discovery and development. We present a single workflow allowing P450 drug clearance
values to be determined as well as metabolites identified, profiled, and their structures elucidated. To be able to do all
of this on a high throughput UHPLC chromatographic timescale is essential for the high levels of productivity required for
today's DMPK screening laboratories. Haloperidol provides a good example of what can be achieved.
C21H23NO2FCl M+H+ = 376.1474
Figure 1: In a single workflow, data dependent MS–MS spectra identify and elucidate metabolite structures and drug clearance
Workflow and Protocol
Microsomal incubations were carried out by Unilabs Bioanalytical Solutions at 1 µM drug concentration and a protein concentration
of 0.5 mg/mL. Aliquots were taken and quenched with acetonitrile containing propranolol as an internal standard at eight time
points over a period of 60 min.
Figure 2: Metabolite detection software compares the data file for the drug (in this case t60) with the corresponding control sample. A base peak chromatogram of the difference is created allowing the metabolites to
be easily observed and their mass determined to four decimal places.
Column: Fortis, 1.7 µm, H2O, 2.10 mm × 30 mm
Column temperature: 30 °C
MPA: 0.1% formic acid in 95% H2O/CH3CN
MPB: 100% CH3CN
Gradient: 0.0 0.3 2.0 2.5 2.6 3.0 min
MP %: 95 95 5 5 95 95 %
Flow rate: 300 µL/min
Injection volume: 5 µL
The high surface area and lipophilic ligand combined with a hydrophilic end cap give this stationary phase a broad selectivity
and resolving power for the target drug and the metabolites. The use of small particles allows UHPLC to compress the peak
into a tighter and taller peak, therefore enhancing detection of very low level analytes.
Figure 3: Time profiles for the disappearance of haloperidol and the appearance of three metabolites.
Metabolite detect software compares the data file for the drug (in this case t60) with the corresponding control sample. A base peak chromatogram of the difference is created allowing metabolites m/z 354, 212, and even 392 to be easily observed.
Metabolite detection software is able to detect the m/z = 392 metabolite even though it co-elutes with the internal standard.
Figure 4: Linear calibration of 50 pg/mL to 50 ng/mL (3 decades) was achieved using the XIC for the measured m/z of each metabolite ± 0.005 Da. R2= 0.9974.