Separation, simultaneous analysis

As discussed earlier, the simultaneous analysis of samples containing two analytes requires the isolation of two precipitates. As shown in Example 8.2, conservation of mass can be used to write separate stoichiometric equations for each precipitate. These equations can then be solved simultaneously for both analytes. 

Figure 15.7 Cliromatographic separation of cliiral hydroxy acids from Pseudomonas aeruginosa without (a) and with (h) co-injection of racemic standards. Peak identification is as follows 1, 3-hydroxy decanoic acid, methyl ester 2, 3-hydroxy dodecanoic acid, methyl ester 3, 2-hydroxy dodecanoic acid, methyl ester. Adapted from Journal of High Resolution Chromatography, 18, A. Kaunzinger et al., Stereo differentiation and simultaneous analysis of 2- and 3-hydroxyalkanoic acids from hiomemhranes hy multidimensional gas cliromatog-raphy , pp. 191 -193, 1995, with permission from Wiley-VCH. (continuedp. 419)...

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

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. 

Taylor and colleagues [98] at the Mayo Clinic published a method for the simultaneous analysis of urinary cortisol and cortisone. They used 2H4 cortisol as an internal standard and took a 0.5-ml urine sample. An API 2000 with Turboion-spray source was used in the positive-ion mode. Chromatography was conducted on a standard-bore C18 column with Q8 precolumn filter. MRM was conducted in the positive-ion mode monitoring m/z 363—>121 for cortisol, 367—>121 for 2TL, cortisol, and 361— -121 for cortisone. Cortisol and cortisone were separated and both were eluted within 2 min. Inter- and intra-assay variation for both compounds was < 9% for amounts above 2 pig/dl. The values obtained agree well with those of other studies, such as ours (Table 5.3.2) [62]. They found a range for cortisol for adult males of 4.2-60 pg/24 h and for adult females 3.0-43 pg/24 h. In summary, the 3-min run time of their method has allowed the Mayo group to completely transfer their cortisol and cortisone workload from RIA and HPLC to MS/MS. 

This method requires the least sophisticated equipment and relies heavily on the unique characteristics of the column to separate the carotenoids (Craft et al., 1992 Epler et al., 1992). It incorporates the use of a polymeric Cl 8 column, which has been shown to offer unique selectivity for structurally similar compounds such as geometric isomers. The addition of a second detector or use of a diode-array detector permits the simultaneous analysis of tocopherols, but not retinol. If the method is modified to incorporate a solvent gradient, retinol can be measured also (MacCrehan and Schonberger, 1987). 

Various methods have been developed for the simultaneous determination of several sweeteners in a single run. Most of the methods described in the literature and summarized in Table 3 have been developed for the separation of three sweeteners, especially for saccharin, acesulfame-K, and aspartame. Herrmann et al. (24), Veerabhadrarao et al. (27), and Hausch (66) developed methods for the simultaneous determination of four sweeteners. Prodolliet and Bruelhart (33) and Wu et al. (47) separated five sweeteners. The most comprehensive method is the one developed by Lawrence and Charbonneau (16), which allows the simultaneous analysis of seven sweeteners. With the increased number of sweeteners available and their use being approved for use in specified food products and beverages by different countries, methods capable of separating several sweeteners simultaneously are still needed. 

External Calibration When mass calibration is conducted in an entirely separate exercise from analysis of an unknown. External calibration can be performed infrequently, avoiding the potential problem of simultaneous analysis of calibrant and unknown (direct interferences, suppression, etc.). 

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%. 

Ion chromatography is not restricted to the separate analysis of only anions or cations, and, with the proper selection of the eluent and separator columns, the technique can be used for the simultaneous analysis of both anions and cations. 

Couchman et al. developed a method for simultaneous analysis of amisulpride, metamphetamine, and amphetamine in plasma by HPLC-MS/MS. To the sample (200 pi), ISTDs solution and NaOH solution were added to the extraction solvent (butyl acetate-butanol, 9 1). HPLC separation was performed by strong cation-exchange. The use of methanol as eluent improved sensitivity for analytes due to more efficient in-source desolvation when compared with aqueous eluents [99],... 

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