ARTICLE IN PRESS JID: CHROMA [m5G;January 3, 2020;15:54] Journal of Chromatography A xxx (xxxx) xxx Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Two dimensional chromatography mass spectrometry: Quantitation of chiral shifts in metabolism of propranolol in bioanalysis Lukas C. Harps a , Sonja Schipperges b , Felix Bredendiek a , Bernhard Wuest b , Andreas Borowiak b , Maria Kristina Parr a,∗ a Freie Universitaet Berlin, Institute of Pharmacy – Pharmaceutical Analysis, Königin-Luise-Str. 2+4, 14195 Berlin, Germany b Agilent Technologies R&D and Marketing GmbH and Co. KG, Waldbronn, Germany a r t i c l e i n f o Article history: Received 23 September 2019 Revised 20 December 2019 Accepted 24 December 2019 Available online xxx Keywords: Propranolol Bioanalysis Urinary excretion Enantiomers Two dimensional chromatography Beta-blocker 2D-LC-QQQ-MS/MS a b s t r a c t 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. Further- more, 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 suc- cessfully evaluated as robust. Calibration was performed achieving accuracy between 80% and 120%. Max- imal excretion rates of (S)-propranolol, (R)-propranolol, (S)-4 ′ –hydroxy propranolol, (R)-4 ′ –hydroxy pro- pranolol, (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. © 2019 Elsevier B.V. All rights reserved. 1. Introduction Racemic propranolol (PL) is successfully used in treatment of hypertension, cardiac disease but also migraine prophylaxis et cetera. Its spectrum of therapeutic indications has recently been expanded by treatment of children suffering from hemangioma. Even if already approved for human use, a better understanding of its metabolism concomitant with its pharmacokinetic behaviour is desirable. Chiral recognition of drugs and their metabolites plays an im- portant role for comprehension of pharmacodynamic effects and pharmacokinetic behaviour of chiral biologically active compounds. Published by the FDA and EMA in 1992 and 1993, respectively, guidelines shall ensure to consider each enantiomer of pharma- ceuticals as single active compound [1,2]. 2D-LC techniques with ∗ Corresponding author. E-mail address: maria.parr@fu-berlin.de (M.K. Parr). a chiral dimension offer an opportunity for enantiomeric discrimi- nation of analytes in complex mixtures in biological analysis [3,4]. Highly sensitive mass detectors hyphenated to 2D-LC enable the detection of trace amounts of drugs and metabolites in biological specimen. Enantiomeric discrimination of analytes in complex mix- tures is relevant in doping control, forensic investigations and ther- apeutic drug monitoring. Propranolol undergoes extensive enantioselective phase-1 and phase-2 metabolism. According to literature, cytochrome P450 en- zymes isoforms 2D6 (CYP2D6), CYP1A2, and CYP2C19 are in- volved in catalysing ring oxidation, side chain dealkylation and fur- ther side chain oxidation of propranolol [5]. Reportedly, phase-1 metabolites in humans are 4 ′ –hydroxy propranolol (4-HOPL), 5 ′ – hydroxy propranolol (5-HOPL), 7 ′ –hydroxy propranolol (7-HOPL), N-desisopropyl propranolol (NDP) and 3-(1-naphtoxy)lactic acid (NLA) [6,7]. According to literature (R)-propranolol is preferred in human phase-1 metabolism [8,9] and consequently exhibits a shorter half-life [6]. (S)-Propranolol is about 100-times more https://doi.org/10.1016/j.chroma.2019.460828 0021-9673/© 2019 Elsevier B.V. All rights reserved. Please cite this article as: L.C. Harps, S. Schipperges and F. Bredendiek et al., Two dimensional chromatography mass spectrometry: Quantitation of chiral shifts in metabolism of propranolol in bioanalysis, Journal of Chromatography A, https://doi.org/10.1016/j.chroma. 2019.460828