Throughout the drug development process, potential new drug candidates (or new chemical entities; NCEs) and their metabolites
must be subjected to rigorous and extensive pharmacokinetic evaluations to determine their rates accumulation, metabolism,
and excretion from the body. With regards specifically to the excretion, urinary excretion is typically the predominant route
for the elimination of drugs and their metabolites, and urinary excretion profiling is an integral portion of the pharmacokinetic
characterization of NCEs.
Liquid chromatography tandem mass spectroscopy (LC–MS-MS) allows scientists to rapidly and accurately quantify specific drugs
and their metabolites at extremely low levels from various biological matrices, such as urine. On the liquid chromatography
side, the ultra-high efficiency delivered by sub-2 μm UHPLC core–shell media provides analysts with the ability to run their
samples in extremely short periods of time while maintaining excellent resolution from sample interferences. In this application
note, we present an example of the ability of the Kinetex® 1.7 μm core–shell particle to deliver significantly improved performance
over larger core–shell particles and conventional fully-porous media.
Column: Kinetex 1.7 μm C18 30 × 2.1 mm Fused-core 2.7 μm C18 50 × 2.1 mm Fully-porous 3.5 μm C18 50 × 2.1 mm
Figure 1: XIC for oxazepam glucuronide (MRM 463.1 -> 287.0) in urine.
Mobile phase: A: 10 mM Ammonium formate B: Acetonitrile
Gradient: (95:5) A/B to (0:100) A/B in 2 min, then re-equilibrate at (95:5) A/B for 1 min
Flow rate: 700 μL/min
Detection: MS using API 4000 detector
HPLC system: Agilent 1200 SL
Concentration: 100 ng/mL for active drug and 50 ng/mL for metabolite.
Analytes: Lorazepam-glucuronide spiked into urine at a concentration of 50 ng/mL.
In many instances, suitable MRM transitions for an analyte of interest are hidden because of significant matrix interference.
Oxazepam-glucuronide did not suffer from urinary interference (Figure 1), however the 497.2 -> 320.9 MRM transition for the
glucuronide metabolite of lorazepam does show significant isobaric interference (Figure 2). In cases such as this, the ultra-high
efficiency of the core–shell Kinetex 1.7 μm particle can provide significantly greater peak capacity than standard fully-porous
media (3.5 μm in this case) and a larger fused-core particle (2.7 μm fused-core).
Figure 2: Comparison of the performance of the Kinetex 1.7 μm C18 (30 × 2.1 mm) column vs. a fused-core 2.7 μm column (50
× 2.1 mm) and a fully-porous 3.5 μm C18 column (50 × 2.1 mm) for the glucuronide metabolite of lorazepam (MRM 497.2 -> 320.9).
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