Two dimensional chromatography mass spectrometry: Quantitation of chiral shifts in metabolism of propranolol in bioanalysis

In this study a heart-cutting 2D-LC method was successfully developed and optimized in order to discriminate and quantitate (S)-propranolol, (R)-propranolol, and its hydroxy metabolites, namely the isomeric (S)-4'‑hydroxy propranolol, (R)-4'‑hydroxy propranolol, (S)-5'‑hydroxy propranolol, (R)-5'‑hydroxy propranolol, (S)-7'-hydroxy propranolol, and (R)-7'‑hydroxy propranolol in one chromatographic run. Thereby, experiments investigating chiral discrimination in ring hydroxylation of propranolol were made feasible. Analysis of human urine samples after administration of a single oral dose of 40 mg of propranolol clearly revealed considerable chiral shifts in propranolol and its 4'-, 5'-, and 7'-hydroxy metabolites. Furthermore, the excretion rates of the individual (S)- and (R)-enantiomers were continuously monitored over 24 h post administration. Studies were performed utilizing a 2D-LC system hyphenated to a triple quadrupole mass spectrometer. The chromatographic system was endued with a reversed phase column (phenyl-hexyl) in first dimension and a teicoplanin based chiral column in second dimension. The method was basically validated and successfully evaluated as robust. Calibration was performed achieving accuracy between 80% and 120%. Maximal excretion rates of (S)-propranolol, (R)-propranolol, (S)-4'‑hydroxy propranolol, (R)-4'‑hydroxy propranolol, (S)-5'‑hydroxy propranolol, (R)-5'‑hydroxy propranolol, and (R)-7'‑hydroxy propranolol were 237 ng/min, 281 ng/min, 4 ng/min, 4 ng/min, 1 ng/min, 9 ng/min, and 3 ng/min, respectively.

2D-LC-QQQ-MS/MS; Beta-blocker; Bioanalysis; Enantiomers; Propranolol; Two dimensional chromatography; Urinary excretion.