Carbohydrates detection limits

In the case of carbohydrates blue chromatogram zones are produced on a yellow background that slowly fades [2]. Steroids, vitamins, antioxidants, phenols and aromatic amines yield, sometimes even at room temperature, variously colored chromatogram zones [5]. -Blockers and laxatives also acquire various colors [7, 10]. The detection limits are in the nanogram to microgram range [S]. 

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. 

Also known as the mass detector, this is an evaporative analyzer in which the mobile phase is removed by nebulization and evaporation prior to the determination of nonvolatile carbohydrates by light scattering (44). Unlike the refractive index detector, it allows gradient elution (eluent is removed before detection) and is more sensitive. The detection limit can go up to a few tens of nanograms injected. 

This detection system is coupled with anion-exchange chromatography (48,49), which has the same alkaline pH requirements. The sensitivity of PAD is considerably higher than that yielded using the refractometric detector, with detection limit being of the order of 10 pmol (29). On the other hand, its response also depends heavily on the nature of the carbohydrate. Finally, other alternatives described in the literature include the use of nickel-based or copper-based electrodes and catalytic oxidation (50). 

Indirect conductimetry with a mobile phase containing polyol-borate complexes allows a detection limit of 10-5 mol of carbohydrate (53). A lower sensitivity is reached with the moving-... 

An example of a CILA using optical fibers has been described by Wang et al. [114] for the analysis of 6-mercaptopurine (6-MP). The template, 6-MP, was oxidized to a strong fluorescent compound by H202 in alkaline solution. Upon optimization of the H202 and NaOH concentrations and of the assay temperature, the sensor showed a linear response in the 1.0 x 10 s to 6.0 x 10 6 g mL-1 range with a detection limit of 3.0 x 10-9 g mL-1. Cross reactivity to metal ions, amino acids, and carbohydrates was tested and the sensor was applied to the analysis of 6-MP in spiked serum. 

Acrylate, propionate and acetate were separated by HPLC on a Benson carbohydrate column (30 cm by 0.6 cm i.d.) (Cnromtec, Fort Worth, FL). The HPLC system used a Waters UK6 injector and a Waters Model 6000A pump (Waters Associates, Milford, MA) with a Conductomonitor III Detector (Laboratory Data Control, Riviera Beach, FL) and a Shimadzu Data Processor. The solvent was 0.15 mM H2S04 at a flow rate of 0.5 ml/min. Typical retention times (min) were acrylate, 13.3 acetate, 12.3 propionate, 14.5. The detection limits for acrylate, acetate and propionate were 10, 30 and 40 pM respectively. 

There are many substances which would appear to be good candidates for LC-EC from a thermodynamic point of view but which do not behave well due to kinetic limitations. Johnson and co-workers at Iowa State University used some fundamental ideas about electrocatalysis to revolutionize the determination of carbohydrates, nearly intractable substances which do not readily lend themselves to ultraviolet absorption (LC-UV), fluorescence (LC-F), or traditional DC amperometry (LC-EC) [2], At the time that this work began, the EC of carbohydrates was more or less relegated to refractive index detection (LC-RI) of microgram amounts. The importance of polysaccharides and glycoproteins, as well as traditional sugars, has focused a lot of attention on pulsed electrochemical detection (FED) methodology. The detection limits are not competitive with DC amperometry of more easily oxidized substances such as phenols and aromatic amines however, they are far superior to optical detection approaches. 

Electrochemical detection has matured considerably in recent years and is routinely used by many laboratories, often for a very specific biomedical application. The most popular applications include acetylcholine, serotonin, catecholamines, thiols and disulfides, phenols, aromatic amines, macrocycUc antibiotics, ascorbic acid, nitro compounds, hydroxylamines, and carbohydrates. As the last century concluded, it is fair to say that many applications for which LC-EC would be an obvious choice are now pursued with LC-MS-MS. This only became practical in the 1990s and is clearly a more general method applicable to a wider variety of substances. In a similar fashion, LC-MS-MS has also largely supplanted LC-F for new bioanalytical methods. Nevertheless, there remain a number of key applications for these more traditional detectors known for their selectivity (and therefore excellent detection limits). 

All samples were at the detection limit of the method, approximately 0.1% by weight. In comparison, humic substances in soil commonly vary from 5 to 10% carbohydrates (Stevenson, 1982). The isolation method with XAD resin does not isolate polysaccharides unless they are part of the structure of the humic material (see Chapter 14). In a previous study (see Chapter 7) carbo-... 

From studies on numerous compounds in aqueous alkaline solutions, Johnson concluded that all aldehydes, alcohols, polyalcohols, and carbohydrates are electrochemi-cally detected by the pulsed amperometric scheme described above [61-63] these compounds include the oligosaccharides [64, 65]. Detection limits are typically less than 0.1 ppm for early eluting peaks when the sample volume is approximately 50 pL. 

There is also in the literature a reference to the solubility of carbohydrates in CO2. Specifically, Giddings, Myers, and King (1969) measured the migration of various compounds in ultrahigh-pressure CO2. At a pressure of 2,100 bar ( 30,000 psia), for example, they detected sucrose through flame ionization detection. Table 13.1 indicates that the detection limit of flame ionization is 10 g. For practical purposes, sugar is insoluble in CO2. Therefore, when... 

Thiamine, a soluble vitamin, is an essential nutrient for humans and is important in carbohydrate metabolism, maintaining normal neural activity and preventing beriberi. Various analytical techniques have been reported for the determination of thiamine in pure form, in pharmaceutical preparations, or in biological fluids. Spectrophotometric methods suffer from poor sensitivity (mg/L detection limit). Spectrofluorometric methods usually involve the conversion of thiamine to thiochrome.2 High performance liquid chromatography requires a post-column derivatization step3 and instrumentation for electrophoresis-based methods is expensive.4... 

MS, especially in combination with advanced separation techniques, is one of the most powerful and versatile techniques for the structural analysis of bacterial glycomes. Modern mass spectral ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) provide detection limits in the high atto- to low femto-mole range for the identification of peptides and complex carbohydrates. Structural characterization of these trace level components can be achieved using tandem MS. This provides a number of specific scanning functions such as product, precursor ion, and constant neutral loss scanning to... 

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