Detection limit, peptides

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

The detection limits for amino acids and peptides are between 50 and 200 pmol per chromatogram zone [9], 400 pg for 5-hydroxyindolylacetic acid [11] and 300 pg for dihydroxyergotoxin [19]. 

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

Miniaturizing the column i.d. is of great benefit to the sensitivity of ESl-MS, which behaves as a concentration-sensitive detection principle, because the concentration of equally abundant components in the LC mobile phase is proportional to the square of the column internal diameter. Column diameters from 150 to 15 jm with flow rates 20-200nL improve detection limits of peptides 1-2 orders magnitude over microliter flow rates. Several references referred to in other sections of this chapter discuss the use of LC-ESI MS to characterize separation products. and a sample chromatogram from Ito and coworkers. is seen in Figure 3.8. Table 3.4 provides additional and references that have used this technique. 

The classical methods for detection and quantitation of racemization require analysis of the chiral purity of the product of a peptide-bond-forming reaction. For example, the Anderson test is used to explore a variety of reaction conditions for the coupling of Z-Gly-Phe-OH to H-Gly-OEt (Scheme 6). 9 The two possible enantiomeric tripeptides are separable by fractional crystallization, so that gravimetric analysis furnishes the racemization data. This procedure has a detection limit of 1-2% of the epimerized tripeptide. A modification by Kemp,1"1 utilizing 14C-labeled carboxy components, extends the detection limit by two to three orders of magnitude by an isotopic dilution procedure. The Young test 11 addresses the coupling of Bz-Leu-OH to H-Gly-OEt, and the extent of epimerization is determined by measurement of the specific rotation of the dipeptide product. 

Kaiser and Krause (57) used HPLC to separate the tryptic peptides in cow s-milk and goat s-milk cheeses and cheeses made from mixtures of these milks. These authors reported that the quantitative detection limit could be as low as 1 % cow s milk in goat s-milk cheese. Mayer et al. (125a) have developed a procedure for the separation of bovine, ovine, and caprine para ic-casein using cation-exchange HPLC. 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

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