Baseline separation, determination

In a flame-dried Schlenk tube 0.37 g(1.88 mmol) of (-)-3-exo-(dimethylamino)isoborneol (C) and 200 mL of dry toluene are placed under an atmosphere of argon. 27 mL of 4.2 M diethylzinc (113 mmol) in toluene are added and the resulting solution is stirred at 15°C for 15 min. After cooling to — 78°C, lOg (94.2 mmol) of benzaldehyde are added and the mixture is wanned to O C. After stirring for 6 h, the reaction is quenched by the addition of sat. NH4C1 soln. Extractive workup is followed by distillation yield 12.4 g (97%) 98% ee [determined by HPLC analysis. Baseline separation of rac-1 -phenyl-1 -propanol was achieved on a Bakerbond dinitrobenzoyl phenylglycine column (eluent 2-propanol/hexanc 1 3 flow rate l.OmL/ min detection UV 254 nm)] [a] 0 —47 (c = 6.11, CHC13). 

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

In a further application of MI-SPE, theophylline could be separated from the structurally related caffeine by combining the specific extraction with pulsed elution, resulting in sharp baseline-separated peaks, which on the other hand was not possible when a theophylline imprinted polymer was used as stationary phase for HPLC. A detection limit of 120 ng mb1 was obtained, corresponding to a mass detection limit of only 2.4 ng [45]. This combination of techniques was also used for the determination of nicotine in tobacco. Nicotine is the main alkaloid in tobacco and is the focus of intensive HPLC or GC analyses due to its health risk to active and passive consumers. However, HPLC- and GC-techniques are time-consuming as well as expensive, due to the necessary pre-purification steps required because the sample matrices typically contain many other organic compounds besides nicotine. However, a simple pre-concentration step based on MI-SPE did allow faster determination of nicotine in tobacco samples. Mullett et al. obtained a detection limit of 1.8 jig ml 1 and a mass detection limit of 8.45 ng [95]. All these examples demonstrate the high potential of MI-SPE to become a broadly applicable sample pre-purification tool. 

The methods described above were completed by tandem mass spectrometry and applied for the determination of MG in various edible aquatic animals. The baseline separation of MG and LMG extracted from different tissues is illustrated in Fig. 3.27. 

Ponceau 4R, E-124 and Erythrosine, E-127) using a buffered mobile phase. Separation of dyes was performed in an ODS column (150 X 3.9 mm i.d. particle size 3 pm). Components of the mobile phase were methanol (eluent A) and 0.1 M NaH2P04/Na2HP04 buffer (pH = 7). The gradient elution started with 20 per cent A and reached 100 per cent in 2 min, final hold 4 min. The flow rate was 2 ml/min and dyes were detected at 520 nm. The baseline separation of dyes in 6 min is illustrated in Fig. 3.34. Commercial samples were diluted and injected into the analytical column without any pretreatment. The amounts of dyes found in the samples are compiled in Table 3.20. It was concluded from the good validation parameters that the technique is specific, sensitive, accurate and rapid. Consequently, its application for the determination of these synthetic dyes in drinks was proposed [112],... 

The electropherogams demonstrate the baseline separation of dyes even in the presence of complicated accompanying matrices such as mint syrup. The LOD and LOQ values were between 1.0 - 1.7 and 3.2 - 5.5 jUg/ml, respectively, and the RSD of migration time and peak areas ranged 1.3 - 1.8 per cent and 2.0 - 3.1 per cent. The migration times of the dyes were Tartrazine, 9.97 min Sunset yellow FCF 6.73 min Amaranth, 8.41 min New coccine, 7.49 min Allura red AC, 5.43 min Patent blue V calcium salt, 4.23 min. The amounts of synthetic dyes found in commercial samples are compiled in Table 3.38. It has been stated that the analysis time is rapid and the method makes possible the separation and determination of these dyes at ppm levels in various foodstuffs [186],... 

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). 

Coumarins are pharmacologically active and have been used in the treatment of a diverse range of diseases. The great diversity of coumarin structures and their wide range of polarities present special problems for their simultaneous analysis. The separation of seven closely related coumarins by CZE was studied. Optimized conditions tallied with a 200 mM boric acid—50 mM tetraborate buffer pH 8.5 and were applied to the determination of coumarins in extracts from roots and aerial parts from the plant Chrysanthemum segetum. Baseline separation of six coumarins was achieved in 10 min. 

An MEKC method for the determination of ibuprofen, codeine phosphate hemihydrate, their nine potential degradation products, and impurities in a commercial tablet formulation was developed, optimized, and fully validated according to ICH guidelines and submitted to the regulatory authorities. The optimized system containing ACN as organic modifier allowed baseline separation of ibuprofen, codeine, and nine related substances within 12 min. 

Baseline separation of the cephalosporin antibiotic cephradine, its main impurity cephalexin, and other related impurities was achieved by MEKC. The method was validated in compliance with the USP XXII analytical performance parameters and the results were comparable with a validated LC method, depicting CE to be a valuable alternative technique to LC in pharmaceutical quality control. In most cases, the amount of impurities relative to the main compound measured by MEKC is similar to that obtained by LC. However, some reports reveal that there are differences in number and amount of impurities between MEKC and LC analysis. MEKC permitted the determination of seven known and three unknown impurities in cefotaxime and the results were in good agreement with those of LC. ° MEKC yielded a higher amount of the cefotaxime dimer but a lower amount of an unidentified impurity with respect to LC. The differences may be due to the easier formation of the dimer in the aqueous sample solvent used in MEKC compared to the hydroorganic... 

Determination of the odor character and intensity of enantiomers relies heavily on complete separation of the components of the sample where there is no coelution and baseline separation of enantiomers is seen (see Fig. Gl.4.4). If these ideal conditions are not met, considerable errors will be incurred in making odor measurements, particularly in cases where both enantiomers have similar odors, or where one is odorless. Traces of odorants coeluting with analytes under investigation, tailing of peaks, and low resolution all seriously affect chromatographic odor data. If the retention times of two enantiomers differ by <1 min, quantitative odor data may be inaccurate. 

Lin and Wu [137] established a simple capillary zone electrophoresis method for the simultaneous analysis of omeprazole and lansoprazole. Untreated fused-silica capillary was operated using a phosphate buffer (50 mM, pH 9) under 20 kV and detection at 200 nm. Baseline separation was attained within 6 min. In the method validation, calibration curves were linear over a concentration range of 5-100 /iM, with correlation coefficients 0.9990. RSD and relative error were all less than 5% for the intra- and interday analysis, and all recoveries were greater than 95%. The limits of detection for omeprazole and lansoprazole were 2 fiM (S/N = 3, hydroxynamic injection 5 s). The method was applied to determine the quality of commercial capsules. Assay result fell within 94—106%. 

Capillary electrophoresis Capillary electrophoresis (CE) is used to analyze sodium, potassium, calcium, and magnesium in water samples. The detection is conducted by reverse absorbance measurements. Sufficient separation of the four cations is established with an electrolyte solution of 5 mM imidazole/6.5 mM a-hydroxyisobutyric acid/2 mM 18-crown-6 ether of pEI 4.1 [42]. CE with a contactless conductometric detector is used to determine small anions and cations in water samples from different sources. 2-(N-Morpholino)ethanesulfonic acid/histidine-based (Mes/Elis) electrolytes are used for direct conductivity detection of anions and cations, while ammonium acetate is used for indirect conductivity determination of alkylammonium salts. Eor the simultaneous separation procedure, involving dual-opposite end injection, an electrolyte consisting of 20 mM Mes/EIis, 1.5 mM 18-crown-6 and 20 mM cetyltri-methylammonium bromide provides baseline separation of 13 anions and cations in less than 6 min [43]. Also CE with a capacitively coupled... 

Chromatographic Separation. With respect to chromatographic techniques, specificity can be demonstrated by a sufficient separation of the substances present. For the assay, appropriate separation means an adequate resolution between the peak of interest and other peaks (e.g., impurities, placebo or matrix components), which need not to be separated from each other. In contrast, universal procedures for the determination of impurities require a sufficient separation of all relevant impurity peaks. The required resolution is strongly dependent on the difference in the size of the corresponding peaks as well as on their elution order. Therefore, if separation factors are determined, the typical concentration levels or the specification limits (as worst case) of the impurities should be maintained. Resolution factors can be calculated according to EP [Eq. (3)] and USP [Eq. (4)] at half height and at the baseline, respectively. However, this is only sensible for baseline-separated peaks. The USP approach is less sensitive toward tailing, but more complex to determine. 

If all the components of a sample loaded on an HPLC column are baseline-separated, any conventional detector will work, unless the object of the separation is to determine or confirm the stereochemical conformation of an enantiomer. In achiral systems, (solvent and/or stationary phase) enantiomers have identical retention times and are not separable. The problem has a solution if two detectors are used in series, e.g., CD and absorbance. Because the enantiomers elute together, the absorbance detector measures the sum of their concentrations, and the CD detector measures the difference AA. Solving the simultaneous equations gives the concentrations for both enantiomers. 

Peak purity software incorporating routines for data acquisition and reduction, extraction and comparison of spectra, and display of analytical results can be utilized in either an interactive or automated fashion. A desired sequence of operations can be recorded in a peak purity analysis method file and can run unattended after input parameters are selected. As the quality of the determination is highly dependent on the preanalysis data treatment, results should be inspected visually to verify that peaks are properly baseline-separated and correctly integrated. 

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