A method that enables metabolomic profiling of single cells and subcellular structures is described using capillary electrophoresis coupled to electrospray ionization time-of-flight mass spectrometry. A nebulizer-free coaxial sheath-flow interface completes the circuit and provides a stable electrospray, yielding a signal with a relative standard deviation of under 5% for the total ion electropherogram. Detection limits are in the low nanomolar range (i.e., < 50 nM (< 300 amol)) for a number of cell-to-cell signaling molecules, including acetylcholine (ACh), histamine, dopamine, and serotonin. The instrument also yields high efficiency separations, e.g., ~600,000 for eluting ACh bands. The utility of this setup for single cell metabolomic profiling is demonstrated with identified neurons from Aplysia californica-the R2 neuron and metacerebral cell (MCC). Single cell electropherograms are reproducible, with a large number of metabolites detected; more than 100 compounds yield signals of over 10 4 counts from the injection of only 0.1% of the total content from a single MCC. Expected neurotransmitters are detected within the cells (ACh in R2 and serotonin in MCC), as are compounds that have molecular masses consistent with all of the naturally-occurring amino acids (except cysteine). Tandem MS using a quadrupole time-of-flight tandem mass spectrometer distinguishes ACh from isobaric compounds in the R2 neuron and demonstrates the ability of this method to characterize and identify metabolites present within single cells.

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A laser-induced native fluorescence detection system optimized for analysis of indolamines and catecholamines by capillary electrophoresis is described. A hollow-cathode metal vapor laser emitting at 224 nm is used for fluorescence excitation, and the emitted fluorescence is spectrally distributed by a series of dichroic beam-splitters into three wavelength channels: 250-310 nm, 310-400 nm, and >400 nm. A separate photomultiplier tube is used for detection of the fluorescence in each of the three wavelength ranges. The instrument provides more information than a single-channel system, without the complexity associated with a spectrograph/charge-coupled device-based detector. With this instrument, analytes can be separated and identified not only on the basis of their electrophoretic migration time but also on the basis of their multichannel signature, which consists of the ratios of relative fluorescence intensities detected in each wavelength channel. The 224-nm excitation channel resulted in a detection limit of 40 nmol L-1 for dopamine. The utility of this instrument for single-cell analysis was demonstrated by the detection and identification of the neurotransmitters in serotonergic LPeD1 and dopaminergic RPeD1 neurons, isolated from the central nervous system of the well-established neurobiological model Lymnaea stagnalis. Not only can this system detect neurotransmitters in these individual neurons with S/N>50, but analyte identity is confirmed on the basis of spectral characteristics.

A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B, B, G, and G; deoxynivalenol; fumonisins B, B, and B; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 C uniformly labeled mycotoxins (C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. Using their available LC-MS/MS platform, each laboratory developed in-house instrumental conditions for analysis. The majority of recoveries ranged from 80 to 120% with relative standard derivations (RSDs) <20%. Greater than 90% of the average recoveries of the participating laboratories were in the range of 90-110%, with repeatability RSD (within laboratory) < 10% and reproducibility RSD (among laboratory) < 15%. All Z scores of the results of certified reference materials were between -2 and 2. Using C-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of multiple mycotoxins in a simple LC-MS/MS procedure.