Determination of very low stable isotope enrichments of [2H5]-phenylalanine in chicken liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the determination of very low enrichment of protein-bound l-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC–MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and l-phenylalanine (phe) (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanolas eluent B. The method was calibrated with calibration standard solutions in the range of 0.01–0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography–mass spectrometry (GC–MS) method and reanalyzed with the developed LC–MS/MS method one year later. Compared to GC–MS, the main advantages of the LC–MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.