Impact of Reversed-Phase Chiral Chromatography on the LC-MS Analysis of Drugs in Biological Fluids

Importance of the Topic

Chiral separation techniques are essential in analytical chemistry because many drugs are chiral substances whose enantiomers can differ significantly in biological activity, metabolism, and toxicity. Accurate differentiation and quantification of each enantiomer in complex biological matrices such as plasma or tissue are therefore crucial for pharmacokinetic studies, clinical monitoring, and regulatory compliance.

Objectives and Study Overview

This investigation aimed to compare reversed-phase and normal-phase chiral chromatographic methods for the liquid chromatography mass spectrometry analysis of selected drugs and metabolites in biological fluids. Key goals included developing enantioselective methods, assessing their compatibility with LC-MS detection, and evaluating performance in real sample analyses including ketorolac and a series of beta blocker compounds.

Methodology and Instrumentation

The study began with a chiral screening program using twelve different columns and six mobile phase systems to identify conditions that achieved baseline resolution of racemic ketorolac. Normal-phase separations employed an amylosic stationary phase with heptane and 2-propanol plus triethylamine and trifluoroacetic acid as modifiers detected by UV at 254 nm. Reversed-phase trials utilized macrocyclic glycopeptide based phases with aqueous organic mobile phases compatible with MS detection.

Sample preparation for ketorolac analysis involved direct injection after chromatographic optimization. For beta blockers in rat plasma, a HybridSPE protein precipitation and phospholipid removal protocol was applied. Plasma samples spiked with enantiomeric standards were treated with acidified acetonitrile, centrifuged, filtered through HybridSPE plates, and analyzed directly by LC-MS.

Used Instrumentation

• Waters Alliance 2690 pump with single quadrupole Waters/Micromass ZQ MS using ESI positive mode
• Agilent 1200 Rapid Resolution HPLC coupled to Agilent 6210 TOF MS with ESI positive ion source
• Various chiral columns including Kromasil AmyCoat, Astec CHIROBIOTIC TAG, and CHIROBIOTIC T with particle sizes of 5 microns

Main Results and Discussion

Normal-phase chiral separations successfully resolved ketorolac enantiomers on the amylosic column but reliance on triethylamine and trifluoroacetic acid additives rendered the method unsuitable for LC-MS due to severe ion suppression. Removal of these modifiers prevented analyte elution, confirming that traditional normal-phase conditions cannot be directly transferred to MS detection.

In contrast, reversed-phase methods on macrocyclic glycopeptide phases achieved enantioselectivity with mobile phases inherently compatible with MS. A ketorolac method using 0.1 percent formic acid in a water methanol mixture markedly improved MS signal in positive ESI mode. Retention times of approximately 18 and 21 minutes for the two enantiomers yielded sensitive and reproducible quantification.

For beta blocker analysis, the CHIROBIOTIC T column with ammonium formate in methanol provided clear enantiomeric separation and robust MS response. Extracted ion chromatograms demonstrated baseline resolution of alprenolol, metoprolol, clenbuterol, pindolol, and salbutamol in rat plasma, supporting applicability to pharmacokinetic and toxicology studies.

Benefits and Practical Applications

• Reversed-phase chiral methods allow direct coupling to LC-MS, minimizing method transfer challenges
• Macrocyclic glycopeptide and cyclodextrin based phases offer broad enantiomeric selectivity in polar mobile phases
• Adaptable buffer and additive selection can enhance MS sensitivity with minimal impact on chromatographic selectivity
• Suitable for clinical pharmacokinetics, ADME-Tox profiling, quality control, and regulatory bioanalysis

Future Trends and Opportunities

Ongoing developments in stationary phase chemistry are expected to expand the range of enantiomeric separations under MS friendly conditions. Integration of automated sample preparation like HybridSPE or microfluidic extraction will improve throughput. Emerging mass spectrometers with higher sensitivity and resolution will further lower detection limits. Green solvent strategies and ultrafast chiral LC-MS platforms may address sustainability and time efficiency in high volume laboratories.

Conclusion

The conversion of chiral separations to LC-MS-friendly reversed-phase formats using macrocyclic glycopeptide chiral stationary phases provides a practical and sensitive approach for enantiomeric drug analysis in biological fluids. By selecting appropriate mobile phases and additives, these methods overcome the ion suppression and transfer limitations of normal-phase systems, offering robust solutions for pharmaceutical research and bioanalysis.

References

• Ing Lorenzini KR et al Journal of Chromatography A 1216 2009 3851
• Bell DS Aurand CR Claus J Schollenberger D Jones J Pittcon 2009 Chiral LC MS Analysis of Drug Substances from Plasma Using Macrocyclic Glycopeptide CSPs