Written by: Lisa M. Roth, Ph.D.

I recently read an interesting article about how critical it is to utilize stable isotope labeled bioactive compounds as internal standards for quantitative GC-MS assays. The article demonstrates that it is important to consider the type and location of the stable isotope in the internal standard.  The researchers were conducting equine urinalysis for local and overseas racing authorities using testosterone-16,16,-17-d₃ as an internal standard.  They observed a consistent false positive in a biological sample that had specific enzymatic and microbial content.  Through further research, they demonstrated that the internal standard was undergoing oxidation followed by H/D exchange to form the analytes resulting in the false positive analysis.  The authors emphasize the need for caution when using internal standards and recommend not including internal standards when performing a qualitative confirmatory test.  However, an alternative to consider in this situation, would be to use to an internal standard where the isotope cannot be exchanged, for example testosterone-2,3,4-¹³C₃.  Using this material would preserve the M+3 mass shift and hopefully make it easier to prevent false positives from occurring due to this reaction pathway. 

Reference:

Rapid Communications in Mass Spectrometry.22:682-686,2008.Wok WH, et al. Unusual observations during steroid analysis.