CE-MS with electrokinetic supercharging and application to determination of neurotransmitters

Abstract

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI-MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI-MS. Using EKS, the sensitivity of the method was improved 5000-fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE-ESI-MS/MS to analyze biological samples.

Keywords: Biological samples; CE; CE-MS; Electrokinetic supercharging; Neurotransmitters.

Figure 1.

(A) Effect of buffer on CE separation of 1 μM DA, 5HT, EPI, and NE at 20 kV in 60 cm long capillary. Increasing ionic strength and methanol allow for increased selectivity to resolve overlapping peaks in the original separation. (B) Comparison of 200 mM ammonium formate and 100 mM sodium chloride as LE during a tITP injection. Analytes 1 μM DA, E, and NE dissolved in water. Separation was performed at 20 kV in a 60 cm capillary using the previously chosen background electrolyte. Ammonium formate LE offers increased peak height and decreased broadness.

Figure 2:

(A) Shows the comparison of a 15 s injection with and without a loaded terminating electrolyte (5 mM taurine). Substantial peak broadening and loss of resolution can be seen without a TE loaded after the injection, indicating a loss of tITP. (B) This figure shows the EKS method with the only adjustment of different injection durations. As injection duration increases, more resolution is gained and the peaks become narrower, indication the formation of a system-induced TE and subsequent tITP at longer injection durations.

Figure 3.

Comparison of EKS method with conventional HDI injection and other common forms of preconcentration in CE-MS. Concentrations of standards chosen to fit well within dynamic range for each method. For all, EKS background electrolyte was used with 30 kV separation voltage in an 80 cm capillary. Leading electrolyte only used in the EKS method. FASI performed at 30 kV for 30 s and EKS injection performed at 30 kV for 150 s. HDI and FASS injections performed using 50 mbar to fill 5% and 15% of the capillary volume, respectively.

Figure 4:

(A) Quantification of compounds in the rat brain stem. Concentrations determined by LC-MS/MS assay used for verification (grey bars). Each EKS injection used 100 μL of sample and LC injections used 5 μL of sample per injection. Measurements were made from three different aliquots of a single brain stem homogenate supernatant for each method. The error bars represent the standard deviation in each method determined for each compound from triplicate measurements. (B) Extracted electropherograms overlaid to show separation (Ch trace reduce by a factor of 250 for scaling).