Analyzer chromatographic profiles

Figure 2 Chromatographic profile of separation of proteins in rat serum on a DEAE anion-exchange column at pH 4.5. The conditions of the separation are detailed in Sec. III. Individual fractions were pooled as indicated into eight major pools and analyzed by 2-D PAGE (Fig. 3).

The activity was obtained from a lysate of red blood cells. The reactions were terminated with a boiling water bath (45 s), and the samples clarified by centrifugation. Samples of 5 fiL were analyzed. Figure 9.94B, C, D shows the chromatographic profiles obtained after incubation times of 3,30, and 50 minutes, respectively, with the enzyme. The loss of adenosine is noted, but the effect of the nucleoside phosphorylase is seen, since hypoxanthine, not inosine, is the final product. 

Experiments have been carried out to demonstrate the method described above by means of a multienzyme complex. This complex was assayed first for the AMP kinase activity. A reaction mixture was prepared containing unlabeled ATP (1 mM) and radioactive [3H]AMP only. The reaction was started by the addition of the complex, and samples were removed and analyzed by HPLC. Chromatographic profiles, each representing the analysis of a sample removed from an incubation mixture at increasing times after the start of the incubation, are shown in Figure 10.4. Both optical density and radioactivity were determined. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

Numerous studies done on conjugated proteins were oriented toward the differentiation of the material without performing special separations. For example, direct pyrolysis of several enzymes showed a significant difference in the chromatographic profile of the pyrolysate. The enzymes analyzed by this procedure included a-chymotrypsin, creatine kinase, lactate dehydrogenase, catalase, acetylcholinesterase, and urease [19,20]. 

Extraction of Residues in Bile for HPLC Profiling. Bile recovered from poultry dosed with l4C-semduramicin sodium was diluted with distilled water, extracted with chloroform and the extract was concentrated to dryness. The residue was reconstituted in ethyl acetate/ isooctane (8 2), and aliquots of this solution were analyzed by HPLC with liquid scintillation counting of fractions to derive the chromatographic profile of radioactivity. 

FIGURE 11.5 Chromatographic profiles of omeprazole samples analyzed by a Q-TOF LC/MS system and after examination by the MDF approach, (a) and (b) are TIC profiles of plasma spiked with omeprazole metabolites obtained without and with MDF processing, respectively. Reprinted from Zhu et al. (2006) with permission of the American Society for Pharmacology and Experimental Therapeutics. 

The increasing application of LC to generate chromatographic profiles enables more-polar compounds such as acids to be analyzed without derivatization. Sensitive LC-MS systems have been developed but reported clinical applications are not yet as numerous as GC-MS. Two LC-MS techniques have been widely accepted for MS applications, namely ESI and APCI interfaces. These have almost completely replaced earlier thermospray and flow-EAB interfaces (Table 6). 

Hydrolysis of Jerusalem artichoke syrup by enzyme preparation PC-EXEN was carried out, and sugar composition was also analyzed by HPLC. Chromatographic profile of FOS contained in the syrup from the tubers of Jerusalem artichoke is... 

Figure 8.7 Chromatographic profile of released glycans from a monoclonal antibody following fluorescent labeling. Sample was analyzed with a Waters BEH Glycan colunm (1.7 jim, 1.5 x 150 mm) and fluorescent detection. Reproduced with permission from Cook, K.S., et al. Biologicals. 2012.

By contrast, choosing between a quadrupole and an ion trap is far harder in most analytical contexts. The comparison of quadrupolar analyzers in this chapter is based on the criteria of spectral repeatability, response functions, detection thresholds, chromatographic profiles, and maintenance needs. 

De Bruijn et al.26 30 used chromatographic and spectroscopic techniques to analyze the effect of reaction variables (such as pH and monosaccharide concentration) on the product profile and developed a reaction model (see Fig. 9) that emphasized the role of a-dicarbonyl compounds. Some of the features of the model shown in Fig. 9 are ... 

The metabolic and pharmacokinetic profile of sucralose (this is a novel intense sweetener with a potency about 600 times that of sucrose) in human volunteers was studied by Roberts and coworkers [82]. Part of this study was realized using PLC in the following chromatographic system in which the stationary phase was silica gel and the mobile phase was ethyl acetate-methanol-water-concentrated ammonia (60 20 10 2, v/v). Separated substances were scraped off separately, suspended in methanol, and analyzed by filtration, scintillation counting, or enzymatic assay. It was shown that the characteristics of sucralose include poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bio-accumulative potential. 

The transformation of cyclopentanol-cyclohexanone mixture was carried out in a fixed-bed reactor at 200°C and 250°C under atmospheric pressure and in the presence of nitrogen (nitrogen/reactant molar ratio = 4). The reactant was an equimolar mixture of cyclopentanol and cyclohexanone. The reaction products were analyzed on line by GC (VARIAN 3400 chromatograph, equipped with a SGE CIDEX B 25 m x 0.22 mm column and a flame ionization detector). The deactivation profile was obtained by analyzing reaction effluent for various times-on-stream (TOS). 

---