Ion exchange in supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS): Application for polar and ionic drugs and metabolites in forensic and anti-doping analysis

Abstract

Supercritical fluid chromatography (SFC) is an orthogonal technique to UHPLC. In recent years, SFC has demonstrated potential for use in the analysis of a broad variety of analytes of different polarities, if modifiers and additives are utilized as additional mobile phase constituents. However, to date, little research has been carried out on ion-exchange separation of highly polar and ionic analytes using SFC.The objective of this work was to investigate the elution characteristics of polar compounds using SFC combined with tandem mass spectrometry. Highly polar and even ionic drugs and metabolites, with a diversity of functional groups such as gamma-hydroxybutyrate (GHB), gamma-butyrolactone, GHB-glucuronide, ethyl sulfate, ethyl glucuronide as well as meldonium and gamma-butyrobetaine, were selected for the study. To investigate the chromatographic behavior of the solutes using SFC, a systematic chromatographic method development workflow including a basic validation in human urine was implemented. To ensure the best selectivity, columns with different stationary phase chemistries (silica, NH2, CN, SCX, Diol, EP and amide) were screened. Furthermore, different modifier compositions were evaluated, including pure methanol and methanol with various additives (ammonium acetate or ammonium formate, water, and/or ammonia or formic acid in varying amounts). Trends in retention time shifts were investigated by the systematic variation of gradients, backpressure and column compartment temperature. The quality and reliability of the method has been tested in the course of a basic validation by evaluation of selectivity, linearity, limit of identification, limit of quantification, carry-over, precision, and matrix effect.The highest chromatographic selectivity (especially with respect to the separation of the critical pair of zwitterionic meldonium and γ-butyrobetaine) and suitable peak shapes for all target analytes were obtained using the strong cation exchange column. We observed that increasing buffer concentrations improved the peak shape. Ion-exchange stationary phase and polar additives (buffers, bases, acids and water) mixed with organic modifiers enabled separation of small ionic molecules using SFC. Hence, SFC further demonstrated its wide applicability in small molecule analysis, and may be considered an alternative separation technique to improve the separation of polar analytes.