High-precision measurement of phenylalanine δ15N values for environmental samples: A new approach coupling high-pressure liquid chromatography purification and elemental analyzer isotope ratio mass spectrometry

RATIONALECompound-specific isotope analysis of individual amino acids (CSI-AA) is a powerful new tool for tracing nitrogen (N) source and transformation in biogeochemical cycles. Specifically, the N-15 value of phenylalanine (N-15(Phe)) represents an increasingly used proxy for source N-15 signatures, with particular promise for paleoceanographic applications. However, current derivatization/gas chromatography methods require expensive and relatively uncommon instrumentation, and have relatively low precision, making many potential applications impractical. METHODSA new offline approach has been developed for high-precision N-15 measurements of amino acids (N-15(AA)), optimized for N-15(Phe) values. Amino acids (AAs) are first purified via high-pressure liquid chromatography (HPLC), using a mixed-phase column and automated fraction collection. The N-15 values are determined via offline elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). RESULTSThe combined HPLC/EA-IRMS method separated most protein AAs with sufficient resolution to obtain accurate N-15 values, despite significant intra-peak isotopic fractionation. For N-15(Phe) values, the precision was 0.16 parts per thousand for standards, 4x better than gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS; +/- 0.64 parts per thousand). We also compared a N-15(Phe) paleo-record from a deep-sea bamboo coral from Monterey Bay, CA, USA, using our method versus GC/C/IRMS. The two methods produced equivalent N-15(Phe) values within error; however, the N-15(Phe) values from HPLC/EA-IRMS had approximately twice the precision of GC/C/IRMS (average stdev of 0.27 parts per thousand +/- 0.14 parts per thousand vs 0.60 parts per thousand +/- 0.20 parts per thousand, respectively). CONCLUSIONSThese results demonstrate that offline HPLC represents a viable alternative to traditional GC/C/IMRS for N-15(AA) measurement. HPLC/EA-IRMS is more precise and widely available, and therefore useful in applications requiring increased precision for data interpretation (e.g. N-15 paleoproxies). Copyright (c) 2013 John Wiley & Sons, Ltd.