Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes.
Synthetic insulins such as Humalog Lispro, Novolog Aspart, or Lantus Glargine, are commonly employed for the treatment of insulin-dependent diabetes mellitus owing to convenient handling and fast or prolonged bioavailability. However, the misuse of insulin in sports has been reported often, and the international doping control system requires a reliable and robust assay to determine the presence or absence of related drugs prohibited by the World Anti-Doping Agency. Qualitative evidence of administered substances, which is preferably obtained by mass spectrometry, is of utmost importance. Plasma specimens of 2 mL were fortified with three synthetic insulin analogues and purified by immunoaffinity chromatography, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This top-down sequencing-based assay allows the assignment of individual fragment ions, in particular, of those comprising modifications that are originating from C-termini of B-chains. Recoveries of synthetic insulins from plasma aliquots ranged from 91 to 98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes.
Thevis
Mario
M
Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, 50933 Cologne, Germany. m.thevis@biochem.dshs-koeln.de
Thomas
Andreas
A
Delahaut
Philippe
P
Bosseloir
Alain
A
Schänzer
Wilhelm
W
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007328:Insulin / Q000031:analogs & derivatives* / Q000097:blood*; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D007328:Insulin