Compound Specific 13C & 15N analysis of Amino Acids by GC-C-IRMS


GC-C-IRMS analysis of 13C and 15N in amino acids

We provide CSIA for both δ13C and δ15N of amino acids from organic samples (e.g. animal and plant tissues) by GC-C-IRMS. Sample preparation involves acid hydrolysis for the liberation of amino acids from proteins or ethanolic extraction of free amino acids, followed by derivatization as either methoxycarbonyl methyl esters (MOC; 13C) or N-acetyl isopropyl esters (NAIP; 15N) to produce compounds amenable to GC analysis. Additional information is provided on our Sample Preparation page.


Gas Chromatography
MOC amino acid derivatives (13C) are injected at 240°C (splitless, 1min.) and separated on a DB-23 capillary column (30m, 0.25mm O.D., 0.25µm film) at constant flow rate of 1.2 mL/min under the following temperature program: 50°C (hold 2min.); 120°C (15°C/min.); 175°C (2°C/min., hold 5min.); 195°C (2°C/min.); 250°C (8°C/min., hold 9min.).


NAIP amino acid derivatives (15N) are injected at 255°C (splitless, 1 min.) and separated on an Agilent DB-1301 column (60m X 0.25mm ID, 1 µm film thickness) at constant flow rate of 1.2 mL/min under the following temperature program: 70°C (hold 2min.); 140°C (15°C/min., hold 4min.); 240°C (12°C/min., hold 5min.); 255°C (8°C/min., hold 35min.).


Isotope-ratio Mass Spectrometry
GC-C-IRMS is performed on a Thermo Trace GC 1310 gas chromatograph coupled to a Thermo Scientific Delta V Advantage isotope-ratio mass spectrometer via a GC IsoLink II combustion interface. The combustion reactor is composed of a NiO tube containing CuO/NiO wires maintained at 1000°C. Water is subsequently removed through a nafion dryer and the analyte gases transferred to the IRMS. During 15N analysis, CO2 is removed from the post-combustion carrier stream through the use of a liquid nitrogen trap to prevent isobaric interferences within the ion source.


Calibration procedures are described in detail elsewhere1 2, but may be briefly described as follows. First, a pure reference gas (CO2 or N2) is used to calculate provisional isotopic values of each sample peak during analysis. Next, isotopic values for all amino acids are adjusted to an internal reference compound (e.g. norleucine) such that its known isotopic composition is obtained. Isotopic values of the individual amino acids are then adjusted based on the normalization mixtures such that that their known isotopic composition is obtained; in the case of 13C analysis, this includes accounting for the influence of carbon from the derivatization reagents. Through each calibration step, quality assurance materials, both amino acid mixtures and natural materials, are monitored for accuracy and precision. The normalization and quality assurance mixtures are composed of pure amino acids that have been calibrated separately by EA-IRMS. EA-IRMS results are produced using secondary reference materials calibrated against NIST Standard Reference Materials (Gaithersburg, MD, U.S.A.; IAEA-N1, IAEA-N2, IAEA-N3, USGS-40, and USGS-41).


All samples are analyzed in duplicate by GC-C-IRMS; triplicate measurements are made if the average standard deviation of the duplicates falls outside expected measurement error (≈±1‰). Precision estimates from the co-measured calibrated amino acid mixtures and quality assurance materials are provided with data reports.


References

1G. Docherty, V. Jones, R.P. Evershed. 2001. Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry δ13C analysis of small polyfunctional compounds. Rapid Communications in Mass Spectrometry 15: 730-738. [doi: 10.1002/rcm.270]


2C.T. Yarnes and J. Herszage. 2017. The relative influence of derivatization and normalization procedures on the compound-specific stable isotope analysis of nitrogen in amino acids. Rapid Communications in Mass Spectrometry 31: 693-704. [doi: 10.1002/rcm.7832]






 
e-mail: sif@ucdavis.edu | phone: 530-752-8100 | fax: 530-752-4361
UC Davis Stable Isotope Facility | Department of Plant Sciences
One Shields Avenue | Davis, California, 95616 | USA