Spectrometry Coupled with Chemical Methods

Structural studies of the saponins can be broadly divided into three stages, viz. conventional methods, spectrometry coupled with chemical methods and modem spectrometric methods. With the advent of modem spectroscopic methods, examination of the intact glycoside itself may lead to determination of the complete structure. 

We will first describe briefly the main experimental techniques coupled with electrochemical methods Infrared Reflectance Spectroscopy (IRS), Electrochemical Quartz Crystal Microbalance (EQCM), Differential Electrochemical Mass Spectrometry (DEMS), Chemical Radiotracers and High Performance Liquid Chromatography (HPLC). 

The exact chemical composition of a plant extract is not always completely known. Many articles published in recent years attempt to identify the compounds structure by coupling chromatography with spectro-metric methods. Modern densitometers are able to record the in situ ultraviolet-visible (UV-vis) spectra of a separated substance on a TLC plate [6]. Thin-layer chromatography can be also coupled with other methods in order to enhance the identification of compounds, such as mass spectrometry (MS) or nuclear magnetic resonance (NMR). There are devices able to record the in situ spectra on the TLC plate, or the separated substance is removed from the plate together with the layer, then extracted in a small volume of an adequate solvent, and the sample can be used for obtaining the spectra [6,7]. 

Other spectroscopic methods such as infrared (ir), and nuclear magnetic resonance (nmr), circular dichroism (cd), and mass spectrometry (ms) are invaluable tools for identification and stmcture elucidation. Nmr spectroscopy allows for geometric assignment of the carbon—carbon double bonds, as well as relative stereochemistry of ring substituents. These spectroscopic methods coupled with traditional chemical derivatization techniques provide the framework by which new carotenoids are identified and characterized (16,17). 

Laser desorption methods (such as LD-ITMS) are indicated as cost-saving real-time techniques for the near future. In a single laser shot, the LDI technique coupled with Fourier-transform mass spectrometry (FTMS) can provide detailed chemical information on the polymeric molecular structure, and is a tool for direct determination of additives and contaminants in polymers. This offers new analytical capabilities to solve problems in research, development, engineering, production, technical support, competitor product analysis, and defect analysis. Laser desorption techniques are limited to surface analysis and do not allow quantitation, but exhibit superior analyte selectivity. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

NMR) [24], and Fourier transform-infrared (FT-IR) spectroscopy [25] are commonly applied methods. Analysis using mass spectrometric (MS) techniques has been achieved with gas chromatography-mass spectrometry (GC-MS), with chemical ionisation (Cl) often more informative than conventional electron impact (El) ionisation [26]. For the qualitative and quantitative characterisation of silicone polyether copolymers in particular, SEC, NMR, and FT-IR have also been demonstrated as useful and informative methods [22] and the application of high-temperature GC and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) is also described [5]. 

Pluym et al. compared the use of CE to that of HPLC in chemical and pharmaceutical quality control. They stated that CE could be considered as a complementary technique to HPLC because of its large separation capacity, its simplicity, and its economical benefits. Jimidar et al. decided that CE offers high separation efficiency and can be applied as an adjunct in HPLC method validation. Mol et al. evaluated the use of micellar electrokinetic chromatography (MEKC) coupled with electrospray ionization mass spectrometry (ESI—MS) in impurity profiling of drugs, which resulted in efficient separations. 

A striking feature of the ILs is their low vapor pressure. This, on the other hand, is a factor hampering their investigation by MS. For example, a technique like electron impact (El) MS, based on thermal evaporation of the sample prior to ionization of the vaporized analyte by collision with an electron beam, has only rarely been applied for the analysis of this class of compounds. In contrast, nonthermal ionization methods, like fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), atmospheric pressure chemical ionization (APCI), ESI, and MALDI suit better for this purpose. Measurement on the atomic level after burning the sample in a hot plasma (up to 8000°C), as realized in inductively coupled plasma (ICP) MS, has up to now only rarely been applied in the field of IE (characterization of gold particles dissolved in IE [1]). This method will potentially attract more interest in the future, especially, when the coupling of this method with chromatographic separations becomes a routine method. 

Electrochemical detection is better suited to the analysis of erythromycin and lincomycin. This method of detection has been applied for the determination of erythromycin A (139) and lincomycin (154) residues in salmon tissues. Liquid chromatography coupled with mass spectrometry is particularly suitable for confirmatory analysis of the nonvolatile macrolides and lincosamides. Typical applications of this technique are through thermospray mass spectrometry, which has been used to monitor pirlimycin in bovine milk and liver (141,142), and chemical ionization, which has been applied for identification of tilmicosin (151) in bovine muscle, and for identification of spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in the same tissue (150). 

For the confirmation of PMFs in Valencia orange peel oil and juice, an HPLC method coupled with a thermospray mass spectrometry (HPLC-TSP-MS) detection system was utilized (112). A C 8 column (/zBondapak, 300 X 6-mm ID) was used with a mobile phase of H20-ACN (60 40, v/v) at a flow rate of 1.0 ml/min. Extract (20 fi1) was injected into the HPLC-TSP-MS system, and positive-ion spectra from m/z 100 to 700 were recorded at 1360 ms. Mass spectro-metric identification was done using positive chemical ionization (ICP). This technique allowed confirmation of the presence of eight flavones in the peel oils and seven flavones in the juice. 

Inductively coupled plasma-mass spectrometry (ICP-MS) has been utilized as a bulk technique for the analysis of obsidian, chert and ceramic compositional analyses 12-14). However, due to the high level of spatial variation of ceramic materials, increased sample preparation is necessary with volatile acids coupled with microwave digestion (MD-ICP-MS) to properly represent the variability of ceramic assemblages IS, 16). Due to the increased sample preparation and exposure to volatile chemicals, researchers have continued to utilize neutron activation analysis (INAA) as the preferred method of chemical characterization of archaeological ceramics (77). 

Wet chemical methods involve sophisticated sample preparation and standardization with National Bureau of Standards reference materials but are not difficult for the analytical chemist nor necessarily time consuming (Figure 1). The time from sample preparation to final results for various analytical methods, such as GFAA (graphite furnace atomic absorption), ICP (inductively coupled plasma spectroscopy), ICP-MS (ICP-mass spectrometry), and colorimetry, ranges from 0.5 to 5.0 h, depending on the technique used. Colorimetry is the method of choice because of its extreme accuracy. Typical results of the colorimetric analysis of doped oxides are shown in Tables I and II, which show the accuracy and precision of the measurements. 

Sekine et al. [27] used a-spectrometry to determine plutonium (and americium) in soil. The chemical recovery of plutonium was 51-99% and averaged 81%, while for americium the recovery was 60-70%. The method is coupled with the liquid-liquid extraction stage, taking about two days less than the ion exchange method a complete analysis takes about one week. 

Currently, the most commonly used method for determining drug impurities is HPLC-MS. Such analysis requires proper preparation of the sample, adequate adjustment of separation parameters, and use of an ionization method. The use of soft ionization produces the molecular ion of the impurity and enables its molecular weight to be established. Use of tandem MS/MS spectrometry allows the chemical structure of the impurity to be established by marking its fragmentation schemes. Liquid chromatography coupled with MS detection was used in purity studies of zaleplon [63], etoricoxib [64], ethanediol, diacetate [65], dicloxacillin [66], quinapril [67], and others described in several review articles [68-70]. 

Although platinum was introduced to Europe in the mid-18th century, it was first made commercially available in large quantities and in malleable form in 1805 by the English chemist William Hyde Wollaston. Previous attempts at consistently producing malleable metal were hindered by chemical purification techniques that gave platinum contaminated with deleterious metallic impurities. Richard KnighPs improved process of 1800 was carried out on a suitable sample of crude ore, and analysis of the purified platinum by spark source mass spectrometry (SSMS) indicates an impurity level of about 6%. Reconstruction of Wollaston s purification procedures, coupled with SSMS analysis, indicates that his product was over 98% pure. His superior chemical purification techniques, coupled with improvements in the powder consolidation method, explain Wollastons success. 

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