Mass spectrometric detection of siRNA in plasma samples for doping control purposes.
Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies.
Kohler
Maxie
M
Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
Thomas
Andreas
A
Walpurgis
Katja
K
Schänzer
Wilhelm
W
Thevis
Mario
M
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D004300:Doping in Sports / Q000517:prevention & control*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D055435:Myostatin / Q000235:genetics; D034741:RNA, Small Interfering / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D055815:Young Adult
D055435:Myostatin; D034741:RNA, Small Interfering