The present work presents a fast and sensitive capillary liquid chromatography (µLC) column-switching method with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection for the simultaneous determination of dopamine (D), epinephrine (E), norepinephrine (NE) and serotonin (SE). A sample volume of 100 µl was loaded on a 25 x 0.32 (i.d.) mm 5 µm Hypercarb column at a flow rate of 25 µl/min. The loading mobile phase contained 99.9% H2O and 0.1% pentafluoropropionic acid (PFPA) as ion-pairing agent. A water–acetonitrile gradient containing 0.1% acetic acid (AcOH) in both reservoirs backflushed the compounds onto a 34 x 0.32 (i.d.) mm 5 µm Hypercarb analytical column with a flow rate of 5 µl/min. The analytes were separated in less than 5 min and the total time for one injection, including reconditioning was only 22 min. The analytes were detected in positive ion modus with the molecular ion masses [M+H]+ of 154.1, 184.1, 170.1 and 177.1 for D, E, NE and SE, respectively. Additionally fragments of masses [M+H-17]+ for D and SE and [M+H-18]+ for E and NE were used for detection. The limit of detection (LOD) was 1.5 ng/ml, 0.75 ng/ml, 3.0 ng/ml and 1.5 ng/ml for D, E, NE and SE, respectively. The method was validated using a concentration range of 7.5–70 ng/ml for D, 3.0–70 ng/ml for E and 10–125 ng/ml for NE and SE. The calibration curves had R2-values of 0.992, 0.997, 0.985 and 0.998 for D, E, NE and SE, respectively. The within-day (n = 3) and the between-day (n = 3) precision of the retention times were in the range of 0.7–5.9% and 1.2–3.7%, respectively. The within-day (n = 3) and the between-day (n = 3) precision of the peak areas was in the range of 4.8–59% and 8.0–72%, respectively.
Limitations of the stationary phase material and efforts to eliminate oxidation of both the stationary phase and the analytes are discussed.