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

The Stable Isotope Facility (SIF) will be closing, effective July 26, 2026.

June 3, 2026
An Update on the Plant Sciences Stable Isotope Facility

Dear Faculty, Staff, Students, and Supporters,
After a lengthy review process and careful consideration, we have made the difficult decision to sunset the Stable Isotope Facility (SIF) in its current form, effective July 26, 2026.

The Department of Plant Sciences continues to face hard decisions surrounding funding allocation given the campus-wide request to reduce budgets. Over the past several years SIF has been operating with a significant and growing deficit and, despite extensive efforts over the last several months to find a solution that would allow the facility to continue to provide services to the research community, we have not found a model that is financially sustainable.

I want to thank SIF’s staff for their excellent work and dedication these past 25 years, and everyone who has played a role in supporting this facility.

We are committed to doing our best to support the researchers who rely on the facility during this transition, and will be in touch with individual clients about details of specific plans for handling existing orders over the next several weeks.

Sincerely,
Daniel Potter
Professor and Chair, Department of Plant Sciences
University of California, Davis

Original Letter

Returning SIF Samples (Facility closes July 26, 2026)

Dear Valued Client, 

After a lengthy review process and careful consideration, we have made the difficult decision to sunset the Stable Isotope Facility (SIF) in its current form, effective July 26, 2026.

The Department of Plant Sciences continues to face hard decisions surrounding funding allocation given the campus-wide request to reduce budgets. Over the past several years, SIF has been operating with a significant and growing deficit and, despite extensive efforts over the last several months to find a solution that would allow the facility to continue to provide services to the research community, we have not found a model that is financially sustainable.

We regret to inform you that SIF staff will be unable to process your submitted samples before the facility's closure date. As a result, we would like to offer the option of returning your samples. If you would like your samples returned, please submit the request via return form on the SIF website.   If you do not wish to have the samples returned, please notify us, and we will arrange for their appropriate disposal in accordance with established protocols.

We sincerely appreciate your support of the Stable Isotope Facility and the opportunity to have served your research needs.

Sincerely,
Daniel Potter
Professor and Chair, Department of Plant Sciences
University of California, Davis

Amino Acid Sample Preparation



Protein Hydrolysis

Acid hydrolysis is performed to liberate individual amino acids from proteinaceous samples. Dry, homogenized sample materials are placed in new borosilicate vials with heat- and acid-resistant caps, 0.5 mL (animal tissues) or 2 mL (plant tissues) of 6 M hydrochloric acid added, and vial threads wrapped with PTFE-tape. Vials are then flushed with N2, sealed, and placed in an oven at 150 °C for 70 minutes. After cooling, 200 μL of heptane:chloroform (6:5, v:v) is added to the acid hydrolysates of sample materials, the vials briefly vortexed, and the organic layer discarded in order to remove any remaining lipophilic compounds prior to drying. Samples are then dried in a heating block at 60 °C under a gentle stream of N2. For samples of purified proteins, such as collagen, the lipid removal step is not performed. For samples with a significant inorganic matrix, additional clean-up steps may be required, including cation-exchange chromatography.

 

Ethanolic (80%) extraction of free amino acids is also available, please contact us for details.

 

Amino Acid Derivatization
First, the sample acid hydrolysates (<10 μmoles total) and 10 μL of the internal reference solution are combined and then dried under a stream of nitrogen. 1 mL of 1.85 M acidified methanol is then added to each reaction vial and the solution heated at 100 °C for 1 hour. The remaining methanol is evaporated under nitrogen at room temperature. Dichloromethane (DCM) is added (250 μL) and evaporated under nitrogen at room temperature to remove remaining excess reagents. The partial derivatives are then acetylated with a mixture of acetic anhydride, trimethylamine, and acetone (1 mL; 1:2:5, v/v/v; 10 min., 60 °C) and the reagents evaporated under nitrogen gas at room temperature . Once dry, ethyl acetate is added (2 mL), along with a saturated NaCl solution (1 mL), and the solution vortexed. Following phase separation, the aqueous phase is discarded and the ethyl acetate removed under nitrogen gas . Trace water is removed with two additions of DCM (1 mL). Finally, ethyl acetate is added (100 μL) and the N-acetyl methyl esters transferred to a GC vial with insert. Prepared samples may be stored up to 4 weeks at -20 °C prior to analysis, but are generally analyzed within 24 hours of preparation.

 

The resulting N-acetyl amino acid methyl esters are then suitable for GC-C-IRMS analysis. The 16 amino acids suitable for derivatization by this method include: Ala, Asp, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, and Val. Measurement error is variable between amino acids, but is generally < ±1‰ [1,2].

 

General considerations
Following acid hydrolysis, some amino acids cannot be detected by GC-C-IRMS. They include glutamine and asparagine, which are converted to their respective acid forms (i.e. Glu, Asp), resulting in Glx and Asx. Tryptophan is destroyed during acid hydrolysis, while cysteine is only partially released as cystine. Arginine cannot be derivatized by nearly all techniques, regardless of experimental context. Please be aware that sample materials are inherently variable in amino acid composition and not all amino acids may be measureable from all samples. Further, variation in the amino acid composition of samples will impact relative measurement error among the individual amino acids.

 

References

1C.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]

 

2L.T. Corr, R. Berstan, P.O. Evershed. Optimisation of derivatisation procedures for the determination of δ13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry. Rapid Commun. Mass Spectrom. 2007, 21, 3759. [doi: 10.1002/rcm.3252]