Detection of Synthetic Insulin Analogues in Human Serum

Doping laboratories detect abuse of synthetic analogs of insulin. Mass spectral analysis can differentiate between endogenous insulin and synthetic analogs of insulin.

A peptide hormone, insulin contains a couple of peptide chains cross-linked together by a pair of disulfide bridges, Figure 1.

Insulin amino acid sequence with disulfide bonds. Yellow highlighted amino acids indicate a change from human insulin

Figure 1. Insulin amino acid sequence with disulfide bonds. Yellow highlighted amino acids indicate a change from human insulin.

Humalog, Apidra, Lantus, Novorapid, and Levemir were analyzed utilizing LC/MS/MS. Synthetic insulin analogs are easily distinguished from endogenous insulin due to a modified sequence of amino acids. Apidra, Novorapid, Levemir, and Lantus are distinguished easily because each exhibits well-defined precursor ions due to the difference in the product ions and molecular weight. Humalog cannot be easily distinguished by LC/MS/MS because of the same precursor molecular ions ([M + nH] n). However, the product ions can be easily distinguished.

To use techniques such as liquid chromatography-mass spectrometry (LC-MS) to detect synthetic insulins requires that they are extracted and concentrated prior to LC-MS analysis. This requires at least one evaporation of a solution with high water content. It has been found for insulin-containing solutions that a vacuum concentrator is superior to nitrogen evaporation as it is much faster and gives better recoveries.

Results and Discussion

The Turbovap had a 6 hour drying time, increasing preparation time to days. While, the miVac DUO had a 2 hour drying time via the alcohol technique with 40°C heating, preparing samples ready for LC/MS/MS analysis in one day. Additionally, the recovery reproducibility of all insulin analogs was greatly enhanced (Figure 2).

Recovery for six replicate extractions by immunoaffinity chromatography.

Figure 2. Recovery for six replicate extractions by immunoaffinity chromatography.

Conclusion

Synthetic analogs of insulin in human serums are quickly detected using the miVac DUO with Quattro DUO pump. In addition to being faster, the miVac DUO system provides more reliable recoveries than the nitrogen evaporation technique.

Acknowledgments

Produced from materials authored by Dr. Catrin Goebel from the Australian Sports Drug Testing Laboratory, National Measurement Institute, Pymble NSW, Australia.

References and Further Reading

  1. Thevis M, Thomas A, Delahuat P, Bosseloir A, Schanzer W. Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes. Anal Chem. 2005 Jun 1;77(11):3579-85.

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Last updated: Sep 4, 2020 at 8:43 AM

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