Quantitative Chromatographic Methods

In vitro Agitation in simulated saliva and solvent extraction Idem Idem Impaction in simulated saliva and solvent extraction Ultrasonication in simulated saliva and solvent extraction 0.54-233 1.0 6.7-7.S (2.9-3.6, DEHP) 0.1. 4 7.9 31.4 

In vivo Saliva extracts from adult human volunteers Idem 10.9 (1.8-53.4) 26.0 (6.1-57.9) 

After Wilkinson and Lamb [58]. Reproduced fioraRegulatory Toxicology and Pharmacology 30, C.F. Wilkin.son etal., 140-155. Copyright (1999), with permission from Elsevier. 

A number of recommendations has been made in the development of quantitative chromatographic methods. ASTM disclosed substantial laboratory-to-laboratory differences in quantitative HPLC analyses [62]. A text describes the chromatographic integration methods for peak identification, validation and quantitation [63]. 

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Reaction monitoring is usually beneficial. Even for well-established syntheses, the progress of organic reactions is often monitored qualitatively by chromatographic techniques, most simply by TLC, to determine the reaction time , i.e. when the starting materials have been consumed. Application of standard quantitative chromatographic methods, e.g. GC or... 

This approach is only applicable for methods where SDbl can be estimated from replicate analysis of blank samples. It is therefore not applicable for most quantitative chromatographic methods, as here the response is usually measured in terms of peak area units, which can of course not be measured in a blank sample analysed with a selective method. 

In current industrial practice gas chromatographic analysis (glc) is used for quahty control. The impurities, mainly a small amount of water (by Kad-Fischer) and some organic trace constituents (by glc), are deterrnined quantitatively, and the balance to 100% is taken as the acetone content. Compliance to specified ranges of individual impurities can also be assured by this analysis. The gas chromatographic method is accurately correlated to any other tests specified for the assay of acetone in the product. Contract specification tests are performed on product to be shipped. Typical wet methods for the deterrnination of acetone are acidimetry (49), titration of the Hberated hydrochloric acid after treating the acetone with hydroxylamine hydrochloride and iodimetry (50), titrating the excess of iodine after treating the acetone with iodine and base (iodoform reaction). 

In addition to modem spectroscopic methods ( H nmr spectroscopy, ftir spectroscopy) and chromatographic methods (gc, hplc), HBr titration (29) is suitable for the quantitative analysis of ethyleneimine samples which contain relatively large amounts of ethyleneimine. In this titration, the ethyleneimine ring is opened with excess HBr in glacial acetic acid, and unconsumed HBr is back-titrated against silver nitrate. 

To determine the phosphoHpid and fatty acid compositions chromatographic methods (28) like gas chromatography (gc), thin-layer chromatography (tic), and high performance Hquid chromatography (hlpc) are used. Newer methods for quantitative deterrnination of different phosphoHpid classes include P-nmr (29). 

An analytical method vahdation study should include demonstration of the accuracy, precision, specificity, limits of detection and quantitation, linearity, range, and interferences. Additionally, peak resolution, peak tailing, and analyte recovery are important, especially in the case of chromatographic methods (37,38). 

Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments ia the types of columns and eluents for hplc appHcations, a significant amount of work has been done ia the kiads of detectioa methods for the vitamin. These detectioa methods iaclude direct detectioa by uv, fluoresceace after post-column reduction of the quiaone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

About 100 000 new chemical compounds are synthesized every year [6] these have to be recognized, identified and determined quantitatively. Ever more frequently this is only possible because of the employment of multiple chromatographic methods coupled with derivatization during or before the separation process. 

A gas chromatographic method is described in this work for the analysis of tetradecane-l,4-sultone (C14 5-sultone) and the combination of 2-chloro-tetradecane-l,3-sultone (C14 2-chloro-y-sultone) and l-tetradecene-l,3-sultone (C14 unsaturated y-sultone) in neutral oils isolated from alkenesulfonate. Samples of the neutral oil are diluted in hexane and injected directly into the gas chromatograph. Quantitative data are obtained by comparison to known amounts of the respective sultones. Through the use of silica gel column chromatography followed by GC of collected fractions, separation and individual quantitation of the 2-chlorotetradecane-l,3-sultone and l-tetradecene-l,3-sultone can be obtained. 

It follows that measurements must be made with a precision of about 0.2 second if quantitative results are to be of any value. It is seen from figure 4 that the experimental points lie very close to the line and a fairly accurate measurement of the distribution of the two isotopes can be obtained from retention time measurements. This method has very limited areas of application and is given here, more to demonstrate the effect of unresolved impurities on retention time, than to suggest it as an alternative to adequate chromatographic resolution. In some cases, however, particularly in the analysis of isotopes, it may be the only practical way to obtain a quantitative evaluation of the mixture by a liquid chromatographic method. 

Products 21 and 22 obtained in this reaction differ in their ESI-MS spectra, and the difference in the abundance of respective signals can be expressed quantitatively. Studies have shown that the pseudo-enantiomeric-excess values obtained in this way are in agreement 5% with the data obtained by chromatographic methods, which is sufficient for studying relative values and choosing most selective mutants. 

However, compared with the traditional analytical methods, the adoption of chromatographic methods represented a signihcant improvement in pharmaceutical analysis. This was because chromatographic methods had the advantages of method specihcity, the ability to separate and detect low-level impurities. Specihcity is especially important for methods intended for early-phase drug development when the chemical and physical properties of the active pharmaceutical ingredient (API) are not fully understood and the synthetic processes are not fully developed. Therefore the assurance of safety in clinical trials of an API relies heavily on the ability of analytical methods to detect and quantitate unknown impurities that may pose safety concerns. This task was not easily performed or simply could not be carried out by classic wet chemistry methods. Therefore, slowly, HPLC and GC established their places as the mainstream analytical methods in pharmaceutical analysis. 

Mlcrochromatographlc Methods During the past two years rapid. Inexpensive, miniaturized column chromatographic methods for the separation of hemoglobins have been developed These methods are designed for the qualitative detection and quantitative determination of hemoglobins In normal and abnormal conditions and cover the quantitation of Hb-A2 the detection of Hb-S, Hb-C other abnormal Hbs differentiation of various conditions In adults and the detection of hemoglobinopathies especially sickle cell anemia at birth (27, 28, 29, 30) ... 

The method has also been used In the Augusta area to quantitate Hb-S In nearly 300 AS cases and Hb-C + A2 In 150 AC cases. Percentages were comparable to those by more conventional chromatographic methods. 

A reliable chromatographic method has been developed for the quantitative aneilysis of hydrophobic impurities in water-soluble polymeric dyes. The method utilizes both the molecular sieve effect of normal gel permeation chromatography and solute-column packing interaction, modified by solvent composition. This method eliminates the need to extract the impurities from the polymeric dye with 100 extraction efficiency, as would be required for an ordinary liquid chromatographic analysis. 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

Farine, S., Villard, C., Moulin, A., Mouren, G. M., and Puigserver, A., Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods, Int. ]. Biol. Macromol., 21, 109, 1997. 

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