|Analysis of amine-containing compounds has applications in many areas of biochemical research. Samples of interest include plasma, urine, cerebrospinal fluid, and tissue extracts. In addition to amino acids, there are other amine-containing compounds of interest in many of these samples, such as thyroxines, glutathione, and polyamines. The level of these amines can provide important diagnostic information.|
We previously developed a quantitative amine assay based on the iTRAQ® reagent technology which uses two iTRAQ reagents, one to tag the amines in the sample and one to tag the internal standards. Quantitation of amines is done using HPLC separation followed by ESI MRM analysis on either triple quadrupole or QTRAP® MS/MS system operated in MRM mode. For MRM analysis, MRM transitions are made up of the labeled mass of the amine and the reporter ion fragment generated for the particular reagent used in the MS/MS. The reporter ions from these reagents differ by one mass unit. There is a small overlap of signal between these reporter groups which is not apparent unless the concentration of the analyte is either much smaller or much larger than the internal standard concentration. However, in some disease states, the concentration of the analytes of interest can be small or very large and accurate quantitation of these values is difficult.
We have modified this approach by using aTRAQ™ reagents which have the same basic chemical structure and reactivity as the iTRAQ reagents but have reporter ions that are separated by 8 mass units. Since there is no overlap of reporter ion signals, the lower and upper limits of quantitation have been improved allowing for accurate quantitation in samples with amine concentrations of below 1 to above 10,000 ìM (using the 3200 QTRAP® LC/MS/MS System).
Other improvements to this quantitative assay of amines and amino acids include increasing the throughput by decreasing the analysis time to 18 min using standard HPLC systems or as low as 10 min using a UPLC system and by combining separate assays into a single run.