Characterization of Performance-Enhancing Peptides via Inlet Ionization on DART-TOF/MS Award No. 2013-DN-BX-K020

Consumers can purchase performance-enhancing peptides and cosmetic peptides online via portals similar to those for emerging or “designer” drugs – anonymously and at a low price. Such products are frequently marketed as being “for research purposes only,” presumably in an effort to avoid legal ramifications; the sole non-medically-supervised use is to promote muscle growth or cosmetic enhancement. Though the sale of these peptides may not be legally prohibited, their identification remains of analytical concern. The detection of counterfeit peptide products, including those reportedly containing recombinant human growth hormone (rHGH), is of interest. These products may contain a different peptide or no active substance whatsoever. The authenticity of the peptides being sold to the public lead to health and safety, as well as legal, concerns. In general, crime laboratories do not have protocols for the identification of peptides and large biomolecules. The gold standard of forensic drug analysis, gas chromatography-mass spectrometry (GC-MS), is typically limited to the analysis of small molecules that are readily vaporized at the inlet. High-resolution mass spectrometers capable of ambient ionization, on the other hand, are well-suited for the analysis of peptides. These instruments, including the JEOL AccuTOF, are becoming more prevalent in forensic laboratories as ambient ionization techniques, such as Direct Analysis in Real Time (DART), continue to gain popularity. Through this project, we developed a protocol to analyze peptide samples utilizing a timeof-flight mass spectrometer through a variety of ionization methods. In many cases, this technique required no external ion source or additional equipment, theoretically enabling utilization with any mass spectrometer of appropriate mass analyzer range incorporating an atmospheric pressure inlet. Various peptide standards and former case samples were successfully analyzed using matrix-assisted inlet ionization mass spectrometry. Additionally, we purchased and tested an array of peptides from an online vendor to determine their authenticity. To characterize peptides via molecular mass, samples were dissolved in 1:1 acetonitrile:water with 0.1% formic acid. The matrix compound 3-nitrobenzonitrile (3-NBN) was added to samples just before analysis. Utilizing matrix-assisted inlet ionization, electrospray ionization (ESI)-like spectra were obtained in a matter of seconds. Molecular masses were calculated using a mass spectral interpretation software package. To further characterize the peptides, a simple enzymatic protein digestion procedure utilizing trypsin was performed. Matrix-assisted inlet ionization was utilized to analyze the resulting peptide fragments. Possible identifications for each peptide were assigned by comparing each digested spectrum against an online peptide database. Combined with the molecular mass attained from analysis of the intact peptides, identification of each peptide was made at a reasonable level of certainty. Finally, peptide analysis was explored utilizing paper spray ionization. This ionization method was favored for extremely large peptides in which inlet ionization was not feasible. By utilizing both inlet ionization and paper spray ionization as complementary techniques, the vast majority of peptide samples that could be encountered in a crime laboratory could be readily characterized.