Quantitation of the Chiral Shift in Metabolism of Propranolol Using 2D-LC/MS/MS

Importance of the Topic

The chirality of pharmaceutical compounds profoundly influences their pharmacokinetics and pharmacodynamics. Enantiomeric discrimination of drugs and metabolites is crucial for safety and efficacy assessment. Conventional one-dimensional separations often lack the resolution and selectivity needed for complex biological matrices. Two-dimensional liquid chromatography (2D-LC) with a dedicated chiral dimension overcomes these limitations, streamlining analysis and reducing sample handling.

Objectives and Study Overview

This application focused on developing an online 2D-LC/MS/MS workflow combining achiral separation in the first dimension with chiral discrimination in the second dimension. The method was applied to quantify enantiomeric shifts in propranolol and its hydroxy metabolites in human urine following a single oral 40 mg dose of racemic propranolol.

Methodology and Instrumentation

• Sample Preparation: Post-dose urine samples collected over seven days, diluted (1:4) with methanol, centrifuged at 6 484 g for 10 minutes.
• Chromatography – 1st Dimension: Agilent Poroshell 120 Phenyl-Hexyl column (2.1×100 mm, 2.7 µm), gradient elution with 10 mM ammonium formate (pH 3) and methanol.
• Chromatography – 2nd Dimension: Agilent Poroshell 120 Chiral-T column (4.6×100 mm, 2.7 µm), isocratic ammonium formate/methanol mobile phase.
• 2D Mode: Heart-cutting transfers of propranolol and hydroxy metabolite fractions using 40 µL loops.
• Detection: Agilent 6495 triple quadrupole MS with Jet Stream ESI, MRM transitions for propranolol (m/z 260→183, 116, 56) and hydroxy metabolites (m/z 276→116, 72, 58).

Main Results and Discussion

• Baseline enantiomeric resolution (Rs 2.4–3.1) achieved for propranolol and its three hydroxy metabolites.
• First-dimension retention time precision (RSD <0.11%) ensured reproducible heart-cut transfer.
• Calibration linearity (R² >0.97) over 0.1–1000 ng/mL; LODs 8.3–46.7 pg/mL; LOQs 48.7–265 pg/mL.
• Urinary excretion profiling revealed enantioselective metabolism: (R)-propranolol and ring-hydroxylated (R)-metabolites showed higher excretion rates and retention in the chiral dimension.
• Maximum excretion rates for parent drug reached ~281 ng/min at 7.3 h; metabolite rates were 10–100× lower.

Benefits and Practical Applications

• Online 2D-LC eliminates manual fraction collection and second-run reanalysis, increasing throughput.
• High selectivity and resolution support detailed pharmacokinetic and metabolite profiling.
• Applicable to complex biological matrices in pharmaceutical development, clinical studies, and QA/QC.

Future Trends and Applications

• Coupling 2D-LC with high-resolution MS for broader metabolite identification and structural elucidation.
• Automation and miniaturization of chiral 2D workflows for routine bioanalysis.
• Extension to other chiral drug classes and diverse matrices (plasma, tissues).

Conclusion

The described online 2D-LC/MS/MS method provides a robust, time-efficient platform for quantifying enantiomeric shifts in propranolol metabolism with minimal sample handling. High resolution in both dimensions enables precise enantioselective pharmacokinetic studies, and the approach is adaptable to other chiral pharmaceuticals.

Used Instrumentation

• Agilent 1290 Infinity II 2D-LC System (high-speed pumps, multisampler, diode array detectors, valve modules).
• Agilent 6495 Triple Quadrupole LC/MS with Jet Stream Electrospray ionization.

References

1. EMA. Investigation of Chiral Active Substances. 1993.
2. FDA. Development of New Stereoisomeric Drug. 1992.
3. Woiwode U. et al. Enantioselective Multiple Heartcut 2D UHPLC, J. Chromatogr. A 2018, 1562, 69–77.
4. Joseph S.; Subramanian M.; Khera S. Simultaneous and Stereospecific Analysis of Warfarin Oxidative Metabolism Using 2D LC/Q-TOF, Bioanalysis 2015, 7, 2297–2309.
5. Harps L.C. et al. Quantitation of Chiral Shifts in Propranolol Metabolism by 2D-LC/MS/MS, J. Chromatogr. A 2019, in press.
6. Bichara N. et al. Propranolol Hydroxylation by CYP2D6, Drug Metab. Dispos. 1996, 24, 112–118.
7. Yoshimoto K. et al. Identification of Human CYP Isoforms in Propranolol Metabolism, Br. J. Clin. Pharmacol. 1995, 39, 421–431.
8. Masubuchi Y. et al. P450 Isozymes Involved in Propranolol Metabolism, Drug Metab. Dispos. 1994, 22, 909–915.
9. Narimatsu S. et al. Species Difference in Enantioselectivity for Propranolol Oxidation by CYP2D Enzymes, Chem. Biol. Interact. 2000, 127, 73–90.
10. Gu H. Selecting Correct Weighting Factors for Calibration in Bioanalytical LC-MS/MS Assays, Anal. Chem. 2014, 86, 8959–8966.