|= Emerging. More than 5 years before clinical availability.|
|= Expected to be clinically available in 1 to 4 years.|
|= Clinically available now.|
Authors: Jonathan P. Danaceau (1), Gareth Hammond (2) and Lisa J. Calton (2)
Using HILIC chromatography for very polar analytes is prone to peak tailing, due to low ionic strength mobile phases and immiscibility of organic mobile phases with high salt concentration aqueous mobile phases. Here we describe the use of mobile phase preparation protocols, which ensure full mobile phase miscibility and high salt concentration solubility, when using HILIC chromatography for the separation of catecholamines.
Excessive tailing of catecholamines in HILIC chromatography:
Catecholamines are polar molecules that can be retained by HILIC chromatography, but are prone to peak tailing in low ionic strength mobile phases. However, high salt concentrations in low aqueous mobile phases can cause phase separation between the aqueous and acetonitrile phases. In addition, consistently preparing buffered mobile phases has proved challenging for many clinical labs. We were able to minimize peak tailing for catecholamines and maintain the miscibility of the organic mobile phase by balancing the solute concentration with adequate aqueous content to ensure full solubility. Consistent chromatography between batches was achieved by preparing concentrated buffer solutions and following strict/consistent protocols when preparing working mobile phases.
400 µL acidified urine samples were buffered with ammonium acetate buffer followed by mixed-mode weak cation exchange (WCX) solid phase extraction
Waters ACQUITY I-Class UPLC system
Waters Xevo-TQD MS
MPA: 50 mM ammonium formate, pH 3.0 in 95:5 Water:ACN
MPB: 30 mM ammonium formate, pH 3.0, in 15:85 water:ACN
4 minute gradient, 0.6 mL/min flow rate. Initial conditions were 100% MPB, which was held for 1 minute and then ramped to 90% B over 1 minute.
Column: BEH Amide 1.7 µm; 2.1 x 100 mm
Column Temp: 30 °C
Injection volume: 10 µL
Quantitative MRM acquisition
Initial chromatographic results showed excessive tailing for catecholamines. Increasing the solute concentration in MPB improved this, but resulted in phase separation. We were able to achieve good peak shape with 30 mM ammonium formate. This required the concentration of water to be increased to 15% to prevent phase separation. The final chromatographic parameters reduced both peak width and peak tailing for catecholamines, enabling accurate integration and increasing sensitivity. Preparing a 400 mM concentrated buffer solution enabled us to prepare many batches of mobile phase that resulted in consistent chromatography. This was combined with water and acetonitrile and sonicated to aid in dissolution.
This method of preparing mobile phase resulted in a rugged and reliable solvent system for the HILIC separation of catecholamines. Increasing the aqueous content of the mobile phase to 15% maintained adequate retention while ensuring good peak shape with minimal tailing. Preparing mobile phases from concentrated buffer stock resulted in consistent mobile phases with minimal batch to batch variability. Additional tips and tricks to obtain good chromatography for these analytes will be presented.
For Research Use Only. Not for use in diagnostic procedures.
References & Acknowledgements:
|Salary||yes||Micromass UK Ltd|
IP Royalty: no
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||