Chromatographic methods HPLC

Table 6.1 Ge neral differences between chromatographic methods (HPLC and LC-MS/MS) and ligand-binding assays (immunoassays).

The key to the early assessment of instability in formulations is the availability of analytical methods to detect low levels of degradation products, generally less than 2%. With the aid of thermal analysis and chromatographic methods [HPLC and... 

The other steroids, such as bile acids (cholane), vitamin D, saponin steroids, steroid alkaloids, cardiac glycosides, and brassinosteroids, also have biologically important activities. Due to the metabolic versatility of steroid molecules, extremely complex mixtures are often encountered, necessitating the use of chromatographic methods (HPLC, TLC, GC) for their analyses. 

Chromatography. - Reviews of chromatographic methods (HPLC, GLC, capillary electrophoresis) for the quantitative determination of aminoglycoside antibiotics and of sugars, amino acids and carboxylic acids in foods (175 refs.) have been published. 

High performance liquid chromatographic methods (HPLC) were applied to analyse carotenoid [3], organic acid [4] and tocopherols [5]. The activity of lipoxygenase was spectrophotometrically determined by measuring A absorbance at 234 nm using linoleic acid as the substrate [6]. 

Most of the previous studies have used small electrodes (tenths of cm ) in large volumes of solution containing relatively high amounts of reactants the modification of the initial concentrations is thus insignificant and the formation of the intermediate species cannot be followed. In contrast, large area electrodes (tens of cm ) allow the detection of organic species in the bulk of solution by chromatographic methods (HPLC, GC) and also that of the quantity of OH" (or H+) ions involved in the whole oxidation process. 

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. 

Integration of the peaks for the two diastereomers accurately quantifies the relative amounts of each enantiomer within the mixture. Such diastereometic derivatives may also be analy2ed by more accurate methods such as gc or hplc. One drawback to diastereometic detivatization is that it requites at least 15 mg of material, which is likely to be material painstakingly synthesized, isolated, and purified. The use of analytical chiral chromatographic methods allows for the direct quantification of enantiomeric purity, is highly accurate to above 99.8% ee, and requites less than one milligram of material. 

Chromatographic Method. Progress in the development of chromatographic techniques (55), especially, in high performance Hquid chromatography, or hplc, is remarkable (56). Today, chiral separations are mainly carried out by three hplc methods chiral hplc columns, achiral hplc columns together with chiral mobile phases, and derivatization with optical reagents and separation on achiral columns. All three methods are usehil but none provides universal appHcation. 

Chromatographic methods, notably hplc, are available for the simultaneous deterrnination of ascorbic acid as weU as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as diketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of their accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid deterrnination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. ExceUent reviews of these methods have been pubHshed (73,88,89). 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

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). 

For selective estimation of phenols pollution of environment such chromatographic methods as gas chromatography with flame-ionization detector (ISO method 8165) and high performance liquid chromatography with UV-detector (EPA method 625) is recommended. For determination of phenol, cresols, chlorophenols in environmental samples application of HPLC with amperometric detector is perspective. Phenols and chlorophenols can be easy oxidized and determined with high sensitivity on carbon-glass electrode. 

Current interest is, however, mainly in the coupling of HPLC and TLC, to which considerable attention has been devoted for the solution of difficult separation problems. Since Boshoff et al. (39) first described the direct coupling of HPLC and TLC, several papers (40-43) have been published describing the on-line coupling of liquid chromatographic methods and PC, usually with different interfaces, depending on the first technique applied. If PC is used as the second method, all the MD methods discussed above can be applied to increase the separating power. 

Despite the difficulties caused by the rapidly expanding literature, the use of chiral stationary phases (CSPs) as the method of choice for analysis or preparation of enantiomers is today well established and has become almost routine. It results from the development of chiral chromatographic methods that more than 1000 chiral stationary phases exemplified by several thousands of enantiomer separations have been described for high-performance liquid chromatography (HPLC). 

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. 

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