On-column derivatization of single mammalian cells with capillary electrophoretic separation and laser-induced fluorescence detection is described. Individual cells are electrophorectically injected into the front end of the separation capillary, which is used as a chamber to lyse the cell and derivatize its contents for subsequent separation and detection. Reagents to lyse the cell and derivatize its contents are electrophoretically injected into the front end of the capillary (2.7 mm, 600-pL volume for a 17-microns-i.d. capillary) after the cell has been injected. Dopamine and five amino acids have been quantitatively determined in individual rat pheochromocytoma cells after on-column derivatization with naphthalene-2,3-dicarboxaldehyde and CN-. Average values of compounds determined in these cells range from 180 +/- 110 amol/cell for aspartic acid to 5.1 +/- 1.5 fmol/cell for taurine.

Electroosmotic flow has been monitored in a capillary using a method based on periodic photobleaching of a neutral, fluorescent buffer additive. Rhodamine B was determined to be neutral between pH 6.0 and 10.8 and was added to the running buffer at a concentration of 400 nM. Rhodamine B was photobleached by opening a shutter under computer control for 250 ms every 5.00 s, to expose the dye to a laser beam and create a photobleached zone. The time was measured for the photobleached zone to migrate 6.13 mm to a downstream laser-induced fluorescence detector, to determine the rate of electroosmotic flow in the entire capillary. The flow rate was sampled every 5.00 s, and the precision of the flow measurements was 0.7% or better. Three fluorescent compounds were separated and detected by capillary electrophoresis with laser-induced fluorescence detection, while simultaneously monitoring the electroosmotic flow rate.

Electroosmotic flow (EOF) was monitored in glass microfluidic devices at rates up to 2 Hz with a precision of 0.2-1.0% using a technique based on the periodic photobleaching of a dilute, neutral fluorophore added to the running buffer. This EOF monitoring method was used to examine the performance of the current monitoring technique for measuring an average electroosmotic flow in a microfluidic device with a cross-T design. Flow measurements made with the current monitoring method gave a precision of 0.4-2.2%, but the periodic photobleaching method shows that the current monitoring technique causes changes in EOF as high as 41% during a single experiment. The periodic photobleaching method for EOF monitoring was also used to study EOF in channels on opposite sides of a cross-channel intersection. The opposite channels were shown to exhibit substantially different EOF dynamics during a current monitoring experiment as well as different steady-state EOF rates during normal operating conditions.

Metabolites of atrazine were measured in human urine after dermal exposure using HPLC to separate and identify metabolites and accelerator mass spectrometry (AMS) to quantify them. Ring-labeled [14C]atrazine was applied for 24 h with a dermal patch to human volunteers at low (0.167 mg, 6.45 muCi) and high (1.98 mg, 24.7 muCi) doses. Urine was collected for 7 days. The urine was centrifuged to remove solids, and the supernatant was measured by liquid scintillation counting prior to injection on the HPLC to ensure that < 0.17 Bq (4.5 pCi) was injected on the column. A reversed-phase gradient of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile became less polar with increasing time and separated the parent compound and major atrazine metabolites over 31 min on an octadecylsilane column. Peaks were identified by coelution with known standards. Elution fractions were collected in 1-min increments; half of each fraction was analyzed by AMS to obtain limits of quantitation of 14 amol. Mercapturate metabolites of atrazine and dealkylated atrazine dominated the early metabolic time points, accounting for approximately 90% of the 14C in the urine. No parent compound was detected. The excreted atrazine metabolites became more polar with increasing time, and an unidentified polar metabolite that was present in all samples became as prevalent as any of the known ring metabolites several days after the dose was delivered. Knowledge of metabolite dynamics is crucial to developing useful assays for monitoring atrazine exposure in agricultural workers.