Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry

The change in amino acid enrichment, an indicator of a change in protein sy nthesis and/or degradation, is usually measured using gas chromatography-ma ss spectrometry and/or (GC-combustion) isotope ratio mass spectrometry. Unf ortunately, often a complex and sensitive derivatization procedure and/or a large amount of sample is required. Also, these techniques are less suited to study intermediary metabolism, in which the simultaneous application (a nd thus measurement) of multiple amino acid tracers is preferred. Alternati vely, in this study the possibilities of the coupling of liquid chromatogra phy and mass spectrometry were explored, resulting in the measurement of bo th the concentration and isotope enrichment of o-phthaldialdehyde (OPA)-der ivatizated plasma amino acids in one run. This was achieved by the injectio n of OPA-derivatizated amino acids into an automated HPLC system. After the elution of buffer salts and reagent excess to drain using column switching , the column effluent was directed via a fluorescence detector into a Therm oquest Model LCQ benchtop LC-RIS, Mass spectrometric measurements were perf ormed in "zoom-scan" mode, employing multiple scan events if the target com ponents were not baseline separated. Best signal-to-noise ratio's were obta ined using the LCQ's electrospray probe in the negative mode, Still, when w orking under standard conditions the total ion current of OPA-amino acid de rivatives eluting at the beginning of the chromatogram (e.g., citrulline, a rginine and glycine) was by a factor of 5 lower, compared to components elu ting in the last part of the chromatogram (leucine, valine, and ornithine). These differences could be minimized by increasing the temperature of the healed capillary to 260 degrees C and by applying 5% collision energy (betw een the skimmer and the first octapole) to the first eluting components. A further improvement could not be obtained by the addition of makeup liquids like ammonia, acetic acid, methanol, or acetonitrile (up to 25% of column effluent flow). Considering these results and the fact that the first eluti ng amino acid derivatives are the most polar ones, we hypothesized that hyd ration of these components interferes with the ionization process. A linear calibration curve was obtained for both fluorescent response mid total ion current (TIC) for all amino acids in the range from 5 to 1000 pmol per inj ection. The coefficient of variation of the fluorescent response was typica lly on the order of 1-4%, for the TIC this was between 4 and 9%. However, m easurement of isotope ratios requires not only the determination of the are a of the base peak, but also of the area of the (enriched) isotopomeric pea k(s), having a much lower abundance. Therefore, isotope ratio measurements require the injection of at least 25 pmol of the amino acid derivative of i nterest (except for ARG 50 pmol) to obtain true ratio's. The accuracy of th e isotope enrichment measurement was determined by the injection of a stand ard containing all major physiological amino acids (400 pmol each) and a st andard at physiological concentrations (ranging from 50 pmol (CIT) to 350 p mol (VAL), Standard deviation of the isotopic ratios ranged from 0.1 to 0.5 % for the high (400 pmol) standards and from 0.2 to 0.8% for the low (physi ological) standard, which is comparable with GC-MS. A plot of the results a gainst the theoretical values gave a linear curve for all isotopes studied (R-2 ranged from 0.9984 to 0.9997). However, the [1-C-13]-enriched amino acids measured (LEU, CLY, and VAL)) ga ve a closer agreement to the expected values as was found for [ureido-C-13- 5,5-H-2(2)]-enriched citrulline and [guanidino-N-15(2)]-enriched arginine. We could not determine whether this was due to the measurement procedure it self or resulting from an instability of the tracers in solution. Neverthel ess, the results were reproducible and the theoretical value could be calcu lated using the tangent of the enrichment curves. Considering the easier la nd cheaper) derivatization procedure and instrumentation, the simultaneous collection of isotopomeric distribution spectra (enabling the application o f multiple labeled components) and concentration data, the method presents an attractive alternative to traditional GC-MS applications. (C) 1999 Acade mic Press.