Detection of Synthetic Insulin Analogues in Human Serum

Introduction

Doping laboratories detect abuse of synthetic analogues of insulin. Mass spectral analysis can differentiate between endogenous insulin and synthetic analogues 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. Image credit: SP Scientific

Humalog, Apidra, Lantus, Novorapid, and Levemir were analyzed utilizing LC/MS/MS. Synthetic insulin analogues 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 be 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, recovery reproducibility of all insulin analogues was greatly enhanced (Figure 2).

Recovery for six replicate extractions by immunoaffinity chromatography.

Figure 2. Recovery for six replicate extractions by immunoaffinity chromatography. Image credit: SP Scientific

Conclusion

Synthetic analogues 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.

Acknowledgements

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

References

  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: May 16, 2020 at 6:59 PM

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