Ultrafast Amino Acid Analysis – Maximizing Speed, Sample Throughput and Cost Effectiveness of Established Assays
Microflow chromatography with column diameters ≤ 1 mm is an exciting approach for sensitive, high-throughput LC/MS/MS within the clinical research arena. High-throughput workflows, such as the analysis of amino acids from physiological fluids, require fast, high-performance separations, as well as low carryover and fast injection-to-injection cycle times. Such performance is particularly critical within the field of amino acid analysis, due to the large number of structurally similar, isobaric compounds present that demands extended chromatography in order to achieve the required separation for quantitation.
The Eksigent ekspert™ microLC 200 System is a dedicated microflow UHPLC system that has been designed with these considerations in mind, for optimal performance in the microflow regime, including a new autosampler injection system with modifications for very small volume sample handling with minimal sample waste as well as very low carryover.
Robust and simple sample preparation using SCIEX aTRAQ™ reagent technology
Sample preparation for amino acid analysis using the Eksigent ekspert™ microLC 200 System is identical to the approach used for the application running with conventional chromatography. No modifications or additional steps are needed.
Current dedicated amino acid analyzer systems have long run times, are prone to interferences from buffers, matrices, and other co-eluting amino acids, and can be difficult to maintain. An LC/MS/MS method for the analysis of amino acids in physiological fluids using amine-reactive isotope-coded tags (SCIEX aTRAQ™ Reagents) has been established. Because only compounds with the same mass require chromatographic separation, the analysis time can be significantly reduced versus traditional methods. The combination of LC/MS/MS with SCIEX aTRAQ™ reagents also provides better sensitivity (therefore lower detection limits), a wider dynamic range, and the ability to use labeled internal standards for more accurate and robust quantitation.
Separation of the isobaric amino acids is critical and one of the reasons that a long conventional column is used in the traditional SCIEX aTRAQ™ Reagents kit. In developing the microflow LC application, care was taken to ensure that separation was maintained for these compounds.
The use of the reduced diameter columns allows significantly reduced flow rates and injection volumes, reducing sample consumption, instrument downtime and cost of ownership. The Eksigent ekspert™ microLC 200 System shows potential for the analysis of amino acids in physiological fluids using the SCIEX aTRAQ™ Reagent kits. The improvements to the method given by the use of microflow LC allow the quantitation of 45 amino acids in samples in approximately 8 min, including column re-equilibration time, compared to 18 min using conventional HPLC.
Microflow LC allows dramatic savings in solvent consumption and hence cost of ownership. Sample preparation remains unchanged and assay performance, in terms of sensitivity based on injection volume comparisons, is greatly improved.