Detecting Impurities

The fermentation-derived food-grade product is sold in 50, 80, and 88% concentrations the other grades are available in 50 and 88% concentrations. The food-grade product meets the Vood Chemicals Codex III and the pharmaceutical grade meets the FCC and the United States Pharmacopoeia XK specifications (7). Other lactic acid derivatives such as salts and esters are also available in weU-estabhshed product specifications. Standard analytical methods such as titration and Hquid chromatography can be used to determine lactic acid, and other gravimetric and specific tests are used to detect impurities for the product specifications. A standard titration method neutralizes the acid with sodium hydroxide and then back-titrates the acid. An older standard quantitative method for determination of lactic acid was based on oxidation by potassium permanganate to acetaldehyde, which is absorbed in sodium bisulfite and titrated iodometricaHy. 

For each specific appHcation of a mbber compound as an iasulating material, there is a minimum value of resistivity below which it does not function satisfactorily. In addition, iasulating compounds are required to withstand the effect of water, moist atmosphere, or heat without their resistivity values falling below a satisfactory level. Insulation resistance measurements frequently serve as useful control tests to detect impurities and manufactuting defects ia mbber products. 

Tetrachlorodiben2o- >-dioxin. Purified 2,4,5-trichlorophenol (50 grams, 0.26 mole) was converted to its potassium salt and dissolved in 100 ml of bEEE. After addition of the copper catalyst and ethylene diacetate, the mixture was transferred to the bottom of a 300-ml sub-limer with chloroform. Sublimation (200°C/2 mm) yielded 14 grams (39% yield) of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mass spectral analysis revealed trace quantities of pentachlorodibenzo-p-dioxin, tetrachloro-dibenzofuran, and several unidentified substances of similar molecular weight. The combined impurity peaks were estimated to be <1% of the total integrated GLC area. The product was further purified by recrystallizations from o-dichlorobenzene and anisole. The final product had an estimated 260 ppm of trichlorodibenzo-p-dioxin as the only detected impurity. 

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. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Impure substances have melting points that are very dependent upon the amount of impurity present. For a few substances this is quantified as the molal freezing point depression constant. The result is that melting points can be a very useful indicator of purification efforts. As long as each purification step in a process results in a higher melting point, the substance has been made more pure. This same concept allows the quality control chemist to have a very sensitive method for detecting impurities that is lower than anticipated. 

Ethoxylated sorbitan ester surfactant mixtures like Tween 20 (cf. Fig. 2.9.38) were often used in biochemical applications. Detergents of this type were analysed by MALDI MS. The aim was to compare the separation results of TLC and RP-LC and to detect impurities within these ethoxylated sorbitan esters [30], Tween 20, the ethoxylated sorbitan carboxylate was ionised resulting in [M + Na]+ and [M + K]+ ions. The Tween 20 isomeric and homologue molecules contained a varying number of ethoxylate units. The number of EO units (-CH2CH2O-) was determined from 18 to 34 resulting in Am/z 44 equally spaced signals [30]. 

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

The host cell impurity assay can be used as a tool for detecting impurities in the various in-process purification steps as well as the final product. When using the assay for the in-process steps, it is important to test the samples in the appropriate buffers or matrix. Any sample recovery problems from buffers need to be determined and accounted for in the analysis. 

For DS Methods should separate the API, synthetic process impurities, and DS degradation products. Methods should be able to detect impurities and degradation products present at levels greater than 0.05% relative to the API. Impurities and degradation products present at levels greater than 0.1% should be identified and specifications should be placed on limits. 

For organic impurities summaries of all experiments used to detect impurities should be available and should include comparisons between batches produced during development, those from the proposed commercial process, and results from any stress testing. Any differences between these should be discussed. Studies used to identify and qualify impurities should also be discussed. The ICH has published guidelines depending on... 

The monograph of levocarbastine has already been revised. The determination of the related substances is performed by means of MEKC using an electrolyte solution composed of sodium dodecyl sulfate as a micelle-forming agent in addition to hydroxypropyl-/ -cyclodextrin in a boric acid buffer of pH 9.0. Due to the very good specificity and robustness the method is able to baseline separate the nine specified and detectable impurities and the drug substance. It is easy to meet the system suitability (Rs>4) the resolution between levocarbastine and impurity D was found to be 6.4 and the content of related substances less than 0.5% (see Figure lA and B). 

Vapor-phase chromatography shows no detectable impurities under conditions where <1% of isomeric material would be easily visible. A melting point of 108-109° has been reported. ... 

The product was shown to contain <1% of the starting material and no other detectable impurity by vapor-phase chromatographic analysis. 

Quinones and Hydroqulnones. In the analysis of quin-ones and hydroqulnones, the use of two different dual detector systems was required. The retention data for hydroqulnones shows the normal behavior of hydroxyl groups associating with the solvent, THF. Thus octyl quinone and hydroquinone elute almost together. Similarly dioctylquinone and octyl hydroquinone elute together (Figure 7). The UV/RI response ratio for benzoquinone is 3.75. Hydroquinone and dioctylquinone show similar disparities in the UV/RI responses. This information provides a very good method for detecting impurities in dioctyl hydroquinone. 

In the monitoring of impurities by UV detection, impurities that elute at the solvent front and late- or noneluters may not be observed. Impurities that have a chromophore that is significantly different from that of the drug substance may not be accurately quantified unless a correction factor is used. Generally, attempts are made to isolate, characterize, and synthesize the impurity to create analytical impurity standards that can be used to accurately quantify the impurity. In the... 

Finally, the deposits were heat treated at U00°C for 5 minutes under a N2 atmosphere. X-ray fluorescence data confirmed the presence of Cd and Se in the deposits with no other detectable impurities. CdSe photoelectrodes, 10 cm in area (2x5 cm) were prepared in this manner with a success rate of about 50. ... 

The submitters found that analysis of the undistilled aldehyde by gas chromatography indicated a purity of 98.6%. The analysis was conducted on a column packed with 16% LS-40 on Chromosorb P/AW at 100°. N.m.r. analysis indicated an isotopic purity of 97.9% (trace of impurity at 9.60 p.p.m. due to CHO, carbon tetrachloride solution, internal tetra-methylsilane as reference). If the crude aldimine is hydrolyzed, the aldehyde is obtained in 96% overall yield however, the purity is only 94% by gas chromatography analysis. The checkers found no detectable impurity by gas chromatography in the distilled aldehyde, and the n.m.r. spectrum indicated very high isotopic purity. 

Blank samples to detect impurities in the reagents or interferences ... 

Comparative values for several physicochemical analytical methods for establishing stability of the drug solutions and detecting impurities (including colorimetry) was reported [45]. 

The natural Mn minerals in our study consisted of four samples. The laser-induced time-resolved technique enables us to detect impurity and intrinsic luminescence of Mn " and Nd " " (Fig. 4.67). 

The breakdown spectra of graphite may be very different for pieces from the same deposit. Spectra with higher resolution enable us to detect impurities of Ca, Al, Mg and Si (Fig. 8.22). 

This method is used to detect impurities that show UV absorption. The UV cutoff points for various solvents are listed in Table 10.1. Usually it is not easy to identify the impurities that cause UV absorption. However, if the impurities are removed by purification, better results can be obtained in electrochemical mea-... 

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