Sample Preparation for the Detection of Synthetic Analogs of Insulin in Human Serum

The detection of the abuse of synthetic insulins by doping laboratories is likely to become a routine requirement. The World Anti-Doping Authority (WADA) code normally requires the use of mass spectrometry to identify prohibited drugs, but peptide hormones are currently excluded because of the difficulty of obtaining mass spectra from such large molecules at very low physiological concentrations. Recent developments in applying mass spectrometry to proteomics means that it is becoming feasible for doping laboratories to routinely apply such methodology to detect and confirm the abuse of peptide hormones. The methodology to detect and confirm the abuse of peptide hormones by mass spectrometry is preferred to the current use of immunoassays or other immunoreactive techniques. Insulin is a clear example of how both endogenous insulin and its synthetic analogs can give a positive result with some immunological assays; however, mass spectral analysis can easily distinguish between them. The use of all types of insulin is prohibited by nondiabetic athletes, but it is desirable if possible to identify which form of insulin has been used.

The use of 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, since it is much faster and gives better recoveries.

Figure 1 - Insulin amino acid sequence with disulfide bonds. The yellow-highlighted amino acids indicate the change from human insulin.

Insulin is a peptide hormone consisting of two peptide chains that are cross-linked by two disulfide bridges (Figure 1). The two peptide chains are denoted as chain A and chain B. Five synthetic insulins were studied—Apidra (Aventis, Bridgewater, NJ), Humalog (Eli Lilly & Co., Indianapolis, IN), Lantus (Aventis), Levemir (Novo Nordisk Pharmaceuticals Pty. Ltd., Baulkham Hills, NSW, Australia), and Novorapid (Novo Nordisk Pharmaceuticals Pty. Ltd.)—for analysis in serum samples by liquid chromatography tandem mass spectrometry (LC-MS-MS) with electrospray ionization. The synthetic insulins can be differentiated from endogenous human insulin because their amino acid sequence has been modified. The simplest modification is the swap of positions for the proline and lysine from human insulin to Humalog (B-chain residue 28 and 29), as highlighted in Figure 1. Humalog is therefore the hardest to differentiate by LC-MS-MS, since the observed precursor molecular ions ([M + nH]+n) are the same, but for the product ions there is a clear distinction. The m/z 217 is observed for Humalog fragment y2 (ProThr, B29B30), while for human insulin the m/z 226 is observed representing y3–y1 (ProLys, B28B29).

Apidra, a rapid-acting human insulin analog, has asparagine at position B3 replaced by lysine, and lysine at position B29 replaced by glutamic acid (Figure 1). Three modifications have been made for the insulin analog Lantus: Glycine replaces asparagine at A21 and two arginine amino acids are added to the COOH-terminal of the B chain. Threonine has been omitted from Levemir and a C14 fatty acid chain has been attached to the B29 amino acid, resulting in a long-acting analog. Novorapid has the amino acid proline at B28 replaced with aspartic acid (Figure 1), which introduces an additional negative charge within the insulin molecule, causing the rapid action of the product.

Apidra, Lantus, Levemir, and Novorapid are easily distinguished by LC-MS-MS analysis because each has distinct precursor ions due to the variation in molecular weight as well as distinct product ions.

Extraction, concentration, and analysis

Insulin was extracted from 2-mL serum samples using immunoaffinity chromatography columns followed by solid-phase extraction. The final volume of the purified samples is 1.2 mL in aqueous 2% acetic acid with 50% acetonitrile. The extraction technique implemented within the Australian Sports Drug Testing Laboratory (ASDTL) had been developed by Thevis et al.1

Figure 2 - The miVac DUO concentrator system.

The extracts were dried using either Instrument A (from a prominent manufacturer) with nitrogen gas supply and water bath set to 35 °C or a miVac DUO with Quattro DUO pump (Genevac, Ipswich, U.K.) (Figure 2). The samples were analyzed on a 4000 Q TRAP (Applied Biosystems, Foster City, CA ) with a gradient separation (Agilent 1100, CapPump Binary, Agilent Technologies, Santa Clara, CA) using an XBridge Shield RP18 3.5-μm 1.0 × 150 mm column (Waters Corp., Milford, MA). Solvent A consisted of 95% H2O, 5% acetonitrile and solvent B consisted of 90% acetonitrile, 10% H2O; both A and B had 0.2% formic acid. The gradient was constant from 0 to 2 min at 85% A, then reduced to 32.5% A at 14 min, solvent A increased back to 85% at 15 min, and the column was equilibrated at 85% A for 20 min.

Results

Table 1 - Recovery and standard deviation for six replicates by immunoaffinity chromatography

The samples required up to 6 hr drying time using Instrument A, which resulted in the preparation taking two days before samples would be ready for analysis by LC-MS-MS. In comparison, the miVac DUO dried samples in 2 hr using the alcohol method, with heating set to 40 °C. The samples could then be extracted and ready for analysis within a single day. Furthermore, the recoveries and recovery reproducibility of all five insulin analogs, Apidra, Humalog, Lantus, Levemir, and Novorapid, were significantly improved, as shown in Table 1.

Summary

The use of the miVac DUO with Quattro DUO pump significantly improved the method for the detection of synthetic insulins in serum by being both much faster and by giving better and more consistent recoveries than obtained with nitrogen evaporation.

Reference

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

Dr. Goebel is an Analyst, Australian Sports Drug Testing Laboratory, National Measurement Institute, 1 Suakin St., Pymble, New South Wales, 2073, Australia; tel.: +61 2 9449 0111; fax: +61 2 9449 1653; e-mail: catrin.goebel@nmi.gov.au.

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