Researchers at Fukuoka University have been employing fluorous derivatization extensively for the detection and analysis of specific classes of compounds from biological samples. To date they’ve used several techniques including tagging and scavenging and enriched samples in targeted analytes from plasma, serum and urine. We highlighted their work using fluorous separations in biomolecule detection in a recent F-Blog post.
The same researchers, this time with Nohta as the corresponding author, have published a new paper on the detection of eight biogenic amines from urine (five catecholamines and three indolamines, pictured below).
The approach is relatively straightforward: derivatize the amines in a pre-treated urine sample with fluorous isocyanate; separate with F-HPLC; and analyze with UV detection. All the non-tagged species will elute with the solvent front while the fluorous tagged species will be longer retained. By using a non-fluorescent isocyanate only those fluorous tagged compounds that have native fluorescence will be observed. By employing this strategy all non-tagged compounds (those without an amine) and non-fluorescent compounds will not be detected thereby simplifying analysis.
The researchers first pretreated an urine sample by adding THF (9 volumes), vortexing, centrifuging, and filtering the supernatent to remove proteins and other water soluble compounds. This sample was then treated with 2-perfluorooctyl-ethyl isocyanate. The sample was then analyzed by F-HPLC using a fluorous silica gel column and analytes detected at 280 nm and 320 nm excitation and emission wavelengths. They validated the method by using various concentrations of spiked standards for each of the amines to build calibration curves and determine limits of detection, linearity ranges, etc. The demonstrated good repeatability both intra- and interday between samples. Below is a chromatogram obtained from a urine sample. Note that all eight compounds are fairly well retained and separated. All non-tagged compounds are less well retained.
The researchers also ran an underivatized sample and found that no fluorescent compounds were detected in the region which contained the eight biogenic amines. They also compared the fluorous derivatization with the non-fluorous version by using dodecyl isocyanate and RP-HPLC. The results were not as good as there were other endogenous compounds which eluted in the same region as the analytes of interest.
The authors conclude by claiming that the method described is the most selective method presently available for the detection of native fluorescent amines demonstrating once again the power of fluorous separations in sample enrichment and bioanalytical applications.