Carbohydrate quantitative analysis

The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes. 

Tourn, M. L., Lombard, A., Belliardo, F., and Buffa, M. (1980). Quantitative analysis of carbohydrates and organic acids in honeydew, honey and royal jelly by enzymic methods. J. Apicult. Res. 19,144 146. 

Difficulties are encountered in the qualitative and quantitative analysis of carbohydrate mixtures because of the structural and chemical similarity of many of these compounds, particularly with respect to the stereoisomers of a particular carbohydrate. As a consequence, many chemical methods of analysis are unable to differentiate between different carbohydrates. Analytical specificity may be improved by the preliminary separation of the components of the mixture using a chromatographic technique prior to quantitation and techniques such as gas-liquid and liquid chromatography are particularly useful. However, the availability of purified preparations of many enzymes primarily involved in carbohydrate metabolism has resulted in the development of many relatively simple methods of analysis which have the required specificity and high sensitivity and use less toxic reagents. 

Fermentation tests are based on the ability of yeast to oxidize the sugar to yield ethanol and carbon dioxide, although only the D-isomers are fermentable and only relatively few of these. Modem chromatographic techniques are, however, much more acceptable and paper and thin-layer techniques are useful for routine separation and semi-quantitation of carbohydrate mixtures, although GLC or HPLC techniques may be necessary for the more complex samples or for quantitative analysis. 

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. 

The original publication by Sweeley and coworkers5 was concerned with the separation of a wide range of carbohydrates, from mono- to tetra-saccharides. Most of the subsequent publications have considered the quantitative analysis of mixtures of varied complexity, although two studies have demonstrated the separation of the protium from the deuterium fonns of monosaccharides.200,201 The study of mutarotational equilibria by gas-liquid chromatography has been discussed in Section IV (see p. 38). 

The application of gas-liquid chromatography to the qualitative and quantitative analysis of carbohydrate derivatives has been increasing... 

Mazzoni, V., Bradesi, P., Tomi, F., and Casanova, J. (1995). Direct qualitative and quantitative analysis of carbohydrate mixtures using 13C NMR spectroscopy Application to honeys. Magn. Res on. Chem. 35, S81-S90. 

The shift in the two tensors is expected to be effective for carbohydrate molecules bearing a number of polar groups and hydrogen-bonding centers. Hence, serious difficulty for quantitative analysis may arise if the molecule does not contain three or more nonequivalent C—H vectors that relax predominantly via the overall motion. If this fact is ignored, qualitative treatment may lead to an erroneous motional description. Thus, one should be very cautious in interpreting the relaxation data for overall motion, especially when discrepancies well outside the experimental error are observed for the T, values. When the relaxation times are nearly similar and within the experimental error, isotropic motion may be considered as a first approximation to the problem. 

R. J. Smith, Methods Carbohydr. Chem., 4, 36-41 (1964) K. Fischer, Angew. Chem., 48,394-396 (1935) J. Mitchell and D. M. Smith, Aquametry. Application of the Karl Fischer Reagents to Quantitative Analysis Involving Water, Interscience, New York, 1948. 

The main area of application of gas-liquid chromatography is for quantitative analysis of compounds in mixtures of volatile substances in organic media. If the compounds are not sufficiently volatile, as is the case with carbohydrates, amino acids, steroids etc., they can usually be converted into suitable volatile compounds by derivativisation by methylation, acetylation or trimefhylsilylation and other similar treatments. 

Carbohydrate-specific hepatic receptor-mediated clearance mechanisms include the asialoglycoprotein, S04-GalNAc and Man receptors [235,236]. The asialoglycoprotein receptor on liver hepatocytes is most significant and accounts for the short circulatory half-life of proteins lacking terminal SA [237]. Reliable quantitative analysis of SA will be an important aspect of quality control for glycoprotein pharmaceuticals. For glycoproteins produced in recombinant S. cerevisiae and insect cells which possess terminal Man or Gn moieties, the Man receptor presumably represents a major clearance mechanism [229]. 

This Section Ls restricted to a description of some of the work of Ander-gon, 8-a> who has ably applied the quantitative analysis of vapors by infrared spectroscopy to analytical problems in carbohydrate chemistry, principally to the Zeisel alkoxyl determination. In this particular application, the usual Zeisel apparatus was used, and the volatile iodide liberated was carried by a flow of nitrogen into a cold trap where it was collected quantitatively Anhydrone (magnesium perchlorate) was used for removing water vapor which would otherwise interfere in the spectrum. The contents of the trap were allowed to vaporize into an evacuated gas-cell, and air was then admitted through the trap to sweep all the vapor into the gas-cell. Double-beam compensation of atmospheric water vapor and carbon dioxide was not upset by this procedure, which also served the purpose of increasing the sensitivity of the infrared method by the well known pressure-broadening effect. The complete spectrum of the vapor... 

Sinner, M., Simatupang, M.H. and Dietrichs, H.H., 1975. Automated quantitative analysis of wood carbohydrates by borate complex ion exchange chromatography. Wood Sci. Technol., 9 307—322. 

The quantitative analysis of oxidation of fuels shows that when most carbohydrates are oxidized, the ratio of carbon dioxide produced to oxygen consumed is 1. On the other hand, this ratio, which is called the respiratory quotient (RQ) or the respiratory exchange ratio, is -0.7 when fatty acids are oxidized. The measurements of O2 consumption and CO2 emission only indicate which fuels are being oxidized on average by the whole body and do not necessarily reflect which fuel is being used selectively by particular cell types or tissues. As noted in Chap. 11, the synthesis of triglycerides raises the RQ above 1. 

---