Determination of Synacthen in human plasma using immunoaffinity purification and liquid chromatography/tandem mass spectrometry

Abstract

Synacthen is a synthetic analogue to human adrenocorticotropin, which plays an important physiological role by stimulating production of cortisol. In sports, corticosteroids as well as releasing factors (corticotropins) are prohibited according to the regulations of the World Anti-Doping Agency, and the misuse of Synacthen has been reported several times. Hence, an assay enabling the detection of Synacthen in doping control samples has been developed using immunoaffinity chromatographic isolation of Synacthen from human plasma combined with a concentration of collected fractions using solid-phase extraction. Unambiguous determination of the target analyte was accomplished using microbore liquid chromatography/electrospray ionization tandem mass spectrometry. Diagnostic product ions such as m/z 223 were characterized using high-resolution/high-accuracy Orbitrap mass spectrometry and employed for triple quadrupole MS/MS analysis. The established assay requiring 2 mL of plasma allowed a lower limit of detection (LLOD) at 100 fmol/mL, a recovery of 97% and a precision at the LLOD < 20%. Authentic plasma samples obtained from a patient undergoing a standard short Synacthen test were used to prove the applicability of the developed procedure.

Quantification of human insulin‐like growth factor‐1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionisation tandem mass spectrometry

“…The described procedure allows the valid quantification of human IGF‐1 as well as the unequivocal identification of its analogues LONG™R 3 IGF‐1 and des(1‐3)IGF‐1. The combination of immunoaffinity isolation and purification with LC/ESI‐MS/MS provides utmost specificity for the analysis of banned peptides as recently demonstrated 50. It enables absolute quantification without distortion caused by, e.g., cross‐reactions.…”

No variation of physical performance and perceived exertion after adrenal gland stimulation by synthetic ACTH (Synacthen®) in cyclists

“…Very recently, a direct method has been published allowing the mass spectrometric Synacthen ® detection in human plasma (Thevis et al 2006). However, urine is still the preferred biological Xuid used for antidoping test and the Wnding of any urinary secondary markers indicative of Synacthen ® abuse by athletes would be very useful.…”

Orbitrap mass spectrometry: Instrumentation, ion motion and applications

“…The high resolving power (>150,000) and excellent mass accuracy (specified as $2-5 ppm, but demonstrated to be as low as 0.2 ppm under favorable conditions) (Makarov et al, 2006a,b), significantly reduce false positive peptide identifications in bottom-up protein analyses (Adachi et al, 2006;Charneau et al, 2007;Dave et al, 2007;de Souza, Godoy, & Mann, 2006;Forner, Arriaga, & Mann, 2006;Graumann et al, 2008;Hanke et al, 2008;Hoopmann, Finney, & MacCoss, 2007;Kamphorst et al, 2007;Li et al, 2007b;Lu et al, 2007Lu et al, , 2008Manes et al, 2008;Mayampurath et al, 2008;Park et al, 2008;Scherl et al, 2008;Scigelova & Makarov, 2006;Shi et al, 2007;Swatkoski et al, 2007Swatkoski et al, , 2008Thevis et al, 2007b;Usaite et al, 2008;Venable et al, 2007;Wilson-Grady, Villén, & Gygi, 2008;Wisniewski et al, 2007;Zougman et al, 2008), and improve the accuracy of de novo interpretations of MS/MS spectra (DiMaggio et al, 2008;Ford et al, 2008;Frank et al, 2007;Scigelova & Makarov, 2006;Yates et al, 2006). The LTQOrbitrap has also been used to analyze intact small (3-10 kDa) (Bredehöft, Schänzer, & Thevis, 2008;Thevis et al, 2006a;Thevis, Thomas, & Schänzer, 2008b), medium (10-25 kDa) (Macek et al, 2006;…”