Retention of carbon and alteration of expected 13C-tracer enrichments by silylated derivatives using continuous-flow combustion-isotope ratio mass spectrometry

Continuous-flow inlets from oxidation reactors are commonly used systems for biological sample introduction into isotope ratio mass spectrometers (IRMS) to measure C-13 enrichment above natural abundance. Because the samples must be volatile enough to pass through a gas chromatograph, silylated derivatization reactions are commonly used to modify biological molecules to add the necessary volatility. Addition of a tert-butyldimethylsilyl (TBDMS) group is a common derivatization approach. However, we have found that samples do not produce the expected increment in measured C-13 abundance as the TBDMS derivatives. We have made measurements of C-13 enrichment of leucine and glutamate standards of known C-13 enrichment using derivatives without silicon (N-acetyl n-propyl ester), with silicon (TBDMS), and an intermediate case. The measurements of MC in amino acids derivatized without silicon were as expected. The C-13 enrichment measurements using the TBDMS derivative were higher than expected but could be corrected to produce the expected C-13 enrichment measurement by IRMS if one carbon was removed per silicon. We postulate that the silicon in the derivative forms silicon carbide compounds in the heated cupric oxide reactor, rather than forming silicon dioxide. Doing so reduces the amount of CO2 formed from the carbon in the sample. Silylated derivatives retain carbon with the silicon and must be used carefully and with correction factors to measure C-13 enrichments by continuous-flow IRMS.