The detection and measurement of multi-analytes in clinical and basic biological research samples has been a subject of investigation for several years. This has lead to a growing need to measure multiple analytes in the same sample in addition to reducing analytical time. Comparison of the sample's unknowns to standards is time-consuming and often leads to inconsistencies, especially in specialized techniques such as capillary electrophoresis (CE). In the clinical arena, the need for timely analyses is of great necessity and the ability to simultaneously detect several internal standards along with the analytes of interest holds great potential.? ? The introduction of laser-induced fluorescence (LIF) detection to separation science has greatly improved detection sensitivity for a variety of analytes. The range and application of this type of detector has been further enhanced by the development of fluorophore labels with absorption spectra matched to known laser lines. Although a number of commercial LIF detectors are available, laboratory-designed, purpose-built systems can offer improved sensitivity as well as added function. In recent years, the DBEPS Instrumentation Research and Development Resource (IRDR) developed an ultrasensitive LIF capillary detector for applications in the separation and biological sciences that achieved a sensitivity of 450 fg/ml of Substance P.? ? To address the need for internal standards, IRDR introduced a second wavelength capability into the design for simultaneous measurement of internal standards and unknown analytes within the same sample. The internal standards were labeled with Bimane while human plasma samples were labeled with AlexaFluor633. Each sample was spiked with a mixture containing 100 pg of each standard and injected into a capillary electrophoresis system. The samples were run at 75 mA constant current and the resolved peaks detected on-line with a flow-cell set 60 cm from the inlet. The fluorescent signals were measured by the two-color detector, consisting of a 408-nm diode pumped solid state and a 633-nm helium-neon laser co-linearly combined and brought to common focus at the flow-cell. Emitted light was collected with an optical fiber, positioned at a 90-degree angle and in close proximity to the flow-cell. A collimated beam was passed through a 417-nm long pass Raman edge filter combined with a 633-nm laser notch filter. The resulting signal was transmitted via a second optical fiber to the entrance slit of a CCD spectrometer equipped with a data acquisition board and a Labview interface. The two resulting chromatograms were plotted as fluorescence units versus time with stacked traces. ? ? This new LIF detector improves on our previous work by allowing the simultaneous detection of internal standards that are labeled with one fluorophore (Bimane) and serum samples labeled with a different fluorophore (AlexaFluor633). This enables investigators to perform multi-analyte analyses on a variety of biological specimens. Quantification of the natural materials is determined by calculating peak areas and directly comparing them with those obtained with the standards. The advantage of the two-color detector is that direct calculation of unknowns can shorten analytical time and negate the need for additional standard or calibration runs. An added advantage of two-color LIF detection is that a high degree of sensitivity can be achieved during detection and that several standards can be introduced thus allowing for multi-analyte identification and quantification. Additionally, the simultaneous detection of standards and unknowns, within the same sample, greatly reduces analytical time. Finally, the reduced analytical time plus the in-built quality control makes this approach ideal for clinical studies and patient monitoring.? ? This year, we have begun systematic studies of different configurations for the collecting optics and photodetectors for an improved version of the two-color detector. The hope is to improve upon sensitivity by using high numerical aperture collimating lenses, together with a pinhole to exclude unwanted background, and directing the collected light onto photomultiplier tubes, in contrast to the CCD detector of the earlier design. In addition, this configuration should permit the use of smaller-diameter capillary, allowing detection on systems with lower flow rates. These changes, together with the incorporation of newly available solid-state lasers, should also allow a substantial decrease in the overall size of the detector, and hence the distance from the separation to the detection in the on-line system. In the next year, we plan to incorporate this detector into a microflow HPLC system in DBEPS's Ultramicro Analytical Immunochemistry Resource.
| Morgan, Nicole Y; Wellner, Edward; Talbot, Thomas et al. (2006) Development of a two-color laser fluorescence detector. On-line detection of internal standards and unknowns by capillary electrophoresis within the same sample. J Chromatogr A 1105:213-9 |
