Analysis of Free Drug Fractions Using Near-Infrared Fluorescent Labels and an Ultrafast Immunoextraction/Displacement Assay

Abstract

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the free fraction of phenytoin in serum or samples containing the binding protein human serum albumin (HSA). Items considered in the design of this method included the dissociation rate of HSA-bound phenytoin, the rate of capture of free phenytoin by immunoextraction microcolumns, the behavior of NIR fluorescent labels in a displacement format, and the overall response and stability of the resulting assay. In the final UFIDA method, the free fraction of phenytoin was extracted in approximately 100 ms by a microcolumn containing a small layer of anti-phenytoin antibodies. This gave a displacement peak for a NIRfluorescent labeled analog of phenytoin that appeared within 2−3 min of sample injection, creating a signal proportional to the amount of free phenytoin in the sample. The UFIDA method provided results within 1−5% of those determined by ultrafiltration for reference samples. The lower limit of detection was 570 pM and the linear range extended up to 10 μM. This approach is not limited to phenytoin but can be adapted for other analytes through the use of appropriate antibodies and labeled analogs.

Chromatographic Immunoassays: Strategies and Recent Developments in The Analysis of Drugs and Biological Agents

“…Even lower detection limits are again possible when fluorescence detection is combined with an enzyme label [8]. Nearinfrared fluorescent tags have also been employed in chromatographic immunoassays [26][27][28], resulting in detection limits as low as 10 -10 to 10 -13 M [26,27]. Two other detection methods that usually require the use of a label are those based on chemiluminescence or radioisotopes.…”

“…First, the response due to the displaced label increases as the amount of the target is increased, and often does so in a linear manner at low-to-moderate target concentrations [7,8]. Second, the displacement peak often appears with or just after the nonretained sample components, which can lead to a method with short analysis times [8,27,30]. Third, this method can be utilized for relatively high throughput measurements, especially if the amount of label that is present in the column is sufficient for use with multiple sample injections before the column must be regenerated and reloaded with the label [7,8,27,30,73].…”

“…Second, the displacement peak often appears with or just after the nonretained sample components, which can lead to a method with short analysis times [8,27,30]. Third, this method can be utilized for relatively high throughput measurements, especially if the amount of label that is present in the column is sufficient for use with multiple sample injections before the column must be regenerated and reloaded with the label [7,8,27,30,73]. Like the simultaneous and sequential injection methods, a displacement immunoassay can often be used to obtain lower limits of detection than can be acquired with chromatographic immunoassays that employ direct detection.…”

“…A variety of applications have been reported for displacement immunoassays [7,27,30,74,75]. One recent application has been the use of this format in combination with ultrafast immunoaffinity extraction for the analysis of free drug or free hormone fractions in serum or in mixtures of these agents with transport proteins [27,30].…”

“…One recent application has been the use of this format in combination with ultrafast immunoaffinity extraction for the analysis of free drug or free hormone fractions in serum or in mixtures of these agents with transport proteins [27,30]. An example of such an application, which was combined with chemiluminescent detection to measure the free fraction of thyroxine in serum, is illustrated in Figure 5B.…”