Peptides bioanalysis

Enfuvirtide (Fuzeon , T-20, Ro 29-9800, Hoffmann-La Roche) is a 36-amino acid synthetic peptide with a molecular weight of 4492 Da. It selectively inhibits human immunodeficiency virus (HIV) fusion to the host cell membranes [73]. The N-terminus of the molecule is acetylated and the C-terminus is amidated. A metabolite, M-20, is deamidated at the C-terminus. An ELISA method was initially used during drug development of this compound, but the decision was made to develop and validate an LC-ESI-MS/MS method for the simultaneous determination of enfuvirtide and M-20 for PK studies to support the NDA submission of this product [53]. Some of the issues of LC-ESI-MS/MS application for peptide bioanalysis are highlighted in the following. 

Silica-based restricted access materials (RAM) have been developed for cleanup in bioanalysis, first for low molecular weight compounds in biofluids (Rbeida et al., 2005) and subsequently for biopolymers such as peptides (Wagner et al., 2002). A classification of different types of RAM has been given by Boos and Rudolphi (1997). Novel RAMs with strong cation-exchange functionality have been synthesized and implemented in the sample cleanup of biofluids. Racaityte et al. (2000) have shown that this type of RAM is highly suitable for the online extraction and analysis of... 

It is well known that a great variety of biomolecules exist where metals and metalloids are bound to proteins and peptides, coordinated by nucleic acids or complexed by polysaccharides and small organic ligands such as organic acids.55 Most proteins contain amino acids with covalently bonded heteroelements such as sulphur, selenium, phosphorus or iodine.51 Several reviews have been published on the development of mass spectrometric techniques for bioanalysis in metal-lomics , which integrate work on metalloproteins, metalloenzymes and other metal containing biomolecules.1 51 53 54 56-59 The authors consider trace metals, metalloids, P and S (so-called... 

As each resin bead contains only a single molecule the beads can be screened individually for bioactivity by either screening for activity of bound peptide in the biological assay or by cleaving the resultant peptide from the bead before undertaking the bioanalysis. The identity of any active compounds can then be determined by using mass spectrometry to sequence the active peptide. 

While straight MS analysis can yield important information in terms of identification, characterization and quantification of biomolecules, it becomes a much more powerful tool with further MS or when combined with other separation technologies. As noted earlier, these approaches include MS/MS, GC-MS, LC-MS and CE-MS. These methods have been extensively exploited in virtually all aspects of bioanalysis, and while fundamentally useful for peptide and protein analysis, these methods have also been used in the analysis of lipids, nucleic acids and a wide range of small molecules and drugs. The range of applications is obviously outside the scope of a book like this, but some indications of the uses of each of these techniques are given below. 

The typical column-switching setup for on-Une SPE-LC-MS is shown in Figure 1.3. In a typical application, the sample is loaded by the autosampler onto a precolunm. The sample volume can be larger than the typical injection volume of an analytical column. Analytes are adsorbed onto the chosen stationary phase under weak solvent conditions, while more hydrophilic sample constituents are flushed through. A washing step of the SPE column may be included in the procedure. Next, the valves are switched from the load to the inject position. The SPE coluum is eluted, in most cases in backflush mode, and the analytes are transferred to the LC coluum for separation and subsequent LC-MS detection. Examples of on-line SPE-LC-MS are discussed in Ch. 7.3.2 in enviroiunental analysis, in Ch. 11.6.4 for quantitative bioanalysis, and in Ch. 17.5.2 for peptide analysis. 

Various methods were evaluated for the targeted proteomics of human growth hormone (hGH) in human plasma [111]. hGH was spiked in plasma 10-fold above natural level ( 16 pg/pl). Iiutially, the full plasma proteome was reduced, alkylated, and digested prior to LC-MS via DDA on an ion-trap instrument. hGH could be identified from its T, peptide. Next, the plasma proteome was fractionated by RPLC and GE prior to digestion and LC-MS analysis. hGH could be identified with higher confidence. Finally, an LCxLC-MS approach was apphed, which enabled hGH identification from five tryptic peptides. An important conclusion was that hGH could be detected in a complex sample at the low femtomole level among proteins that were 40,000 x more abundant. The results show that a multidimensional approach may be taken for targeted proteomics and quantitative protein bioanalysis. 

The application area of LC-MS is rapidly growing. LC-MS is now regularly used for the analysis of many different types of compound drugs and metabolites, herbicides-pesticides and metabolites, surfactants, dyes, saccharides, lipids-phospholipids, steroids, and many others. In our opinion, the area that profits more from the development of LC-MS is bioanalysis natural products, proteins, peptides, nucleosides, and metabolic studies. Despite the current trends toward immunoassays-biospecific assays and capillary electrophoresis, LC-MS is an extremely powerful analytical technique that is considered complementary to the above mentioned, rather than competitive. 

Van den Broek I, Sparidans RW, Schellens JH, Beijnen JH. Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2008 872(1—2) 1—22. 

With the increasing number, diversity, and complexity of compounds being analyzed, UPLC presents the possibility to extend and expand the utility of separation science. Today, UPLC is widely used for metabolite identification analysis of natural products and herbal medicines pharmacokinetic, toxicity, degradation, bioanalysis, and bioequivalence studies quality control and in drug discovery determination of pesticides and separation of various pharmaceutical-related small organic molecules, proteins, and peptides. UPLC is also used for impurity profiling, method development, and validation performed in quality assurance and quality control laboratories [46,47,56-69]. 

---