SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

Abstract

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

A rapid method for quantification of persistent and mobile organic substances in water using supercritical fluid chromatography coupled to high-resolution mass spectrometry

“…However, due to the possibilities of using reversed phase as well as normal-phase stationary phases and to mix a polar co-solvent into supercritical (non-polar) CO 2 in the mobile phase, SFC is a very versatile separation technique, even encompassing applications for highly polar and mobile analytes [17]. Recent examples include environmental water pollutants [18,19], polar urinary metabolites [20], and polar compounds in anti-doping control [21]. Desfontaine and coworkers [22] compared matrix effects in SFC and RPLC coupled to tandem mass spectrometry for analysis of doping agents and pharmaceuticals in urine.…”

Simultaneous enantioseparation and simulation studies of atenolol, metoprolol and propranolol on Chiralpak® IG column using supercritical fluid chromatography

“… Technique Column Mobile phase composition Run time (min) Thermodynamic study & molecular modeling Application Refs. 1 Capillary electrophoresis Fused silica capillary (40 cm length × 50 μm) – 32 Yes; yes – [ 15 ] 2 a HPLC-UV Vancomycin-bonded column (150 mm × 2.1 mm, 5 μm) Methanol:triethylamine:glacial acetic acid in the volume ratio of 100:0.01:0.02 ( V/V/V ) ∼20 Yes; yes – [ 30 ] 3 b HPLC-UV Chiralpak IB (250 mm × 4.6 mm, 5 μm) n -hexane-ethanol/isopropanol-0.1% diethylamine ∼35 --; yes – [ 31 ] 4 HPLC-MS/MS Chirobiotic V vancomycin-based chiral column (250 mm × 4 . 6 mm, 5 μm) Methanol:water with 0.1% triethylammonium acetate adjusted to pH 4.0 with acetic acid (90:10, V/V ) 20 --; -- Quantification in wastewater treatment plant influents and effluents [ 32 ] 5 c SFC-MS/MS Chiralpak®IB-3 (100 mm × 4.6 mm, 3 μm) 82% carbon dioxide and 18% of a modifier, consisting of methanol & 0.5% ( V/V ) of the additives trifluoroacetic acid (TFA) & ammonia (NH 3 ) in a 2:1 M ratio 10 --; -- Monitored the enantiomeric fraction change over time in a laboratory scale wetland degradation study [ 40 ] …”

“…Several analytical techniques have been used for enantiomeric separation of these drugs, namely, thin layer chromatography [ 10 ], surface enhanced Raman scattering [ 11 ], counter current chromatography [ 12 ], electrochromatography [ 13 , 14 ], capillary electrophoresis [ [15] , [16] , [17] , [18] ], high performance liquid chromatography (HPLC) [ 9 , [19] , [20] , [21] , [22] , [23] ], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [ [24] , [25] , [26] , [27] , [28] , [29] ], and supercritical fluid chromatography (SFC) [ [30] , [31] , [32] , [33] ]. Among different chiral stationary phases (CSPs), polysaccharide (cellulose or amylose) based phases are the most widely used with these techniques [ 20 , 21 , 23 , 32 , 33 ].…”

Supercritical fluid chromatography – Mass spectrometry: Recent evolution and current trends

“…A second application area that is being under constant development is the implementation of SFC-MS in the forensic and anti-doping control analysis. Indeed, there has been an important number of papers recently released and focusing on several classes of compounds: amphetamines [45,69,70], stimulants and sympathomimetic drugs [51,71,72] or anabolic agents and steroids [44,54,73,74] (Fig. 4).…”