Reversed-phase liquid chromatography quantification

Vandecasteele, K., Gaus, I., Debreuck, W., and Walraevens, K., Identification and Quantification of 77 Pesticides in Groundwater Using Solid Phase Coupled to Liquid-Liquid Microextraction and Reversed-Phase Liquid Chromatography, Anal. Chem. 72, 3093, 2000. 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

P. V. Bondarenko, D. Chelius, and T. A. Shaler, Identification and relative quantification of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography tandem mass spectrometry, Anal. Chem. 74, 4741-4749 (2002). 

Veloso, A.C.A. Teixeira, N. Ferreira, I.M.P.L.V.O. Separation and quantification of the major casein fractions by reverse-phase liquid chromatography and urea-polyacrylamide gel electrophoresis Detection of milk adulterations. J. Chromatogr. A, 2002, 96, 209-218. 

Grebenstein, N. and J. Frank. 2012. Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver. J. Chromatogr. A 1243 39-46. 

Seifar, R. M., C. Ras, J. C. van Dam, W. M. van Gulik, J. J. Heijnen, and W. A. van Winden. 2009. Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry. Anal. Biochem. 388 213-219. 

Identification and quantification of natural dyes need high performance analytical techniques, appropriate for the analysis of materials of complicated matrices containing a small amount of coloured substances. This requirement perfectly fits coupling of modern separation modules (usually high performance liquid chromatography in reversed phase mode, RPLC, but also capillary electrophoresis, CE) with selective detection units (mainly mass spectrometer). 

Endo, Y., Tagiri-Endo, M., Seo, H. S., and Fujimoto, K. (2001). Identification and quantification of molecular species of diacylglyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry. J. Chro-matogr. A 911, 39-45. 

Lim CK, Peters TJ (1984) Urine and faecal porphyrin profiles by reversed-phase high-performance liquid chromatography in the porphyrias. Clin Chim Acta 139 55-63 Minder El, Vuilleumier JP, Vonderschmitt DJ (1992) Prototype application of robot in the clinical laboratory enabling fully automated quantification of fecal porphyrins. Clin Chem 38 516-521... 

According to the modified procedure (602), milk is thoroughly mixed in its storage container immediately before transfer of the 1 ml aliquot in the extraction tube. This is necessary because approximately 50% of phenylbutazone in milk is associated with the cream. The sample is extracted with 2.4 ml diethyl ether and 2.4 ml petroleum ether in presence of 1 ml ethanol and 100 1 25% ammonia solution. The organic layer that contains the milk lipids is discarded. Five ml hexane-tetrahydro furan (4 1) is added to the aqueous layer, which is tiien acidified with hydrochloric acid and the layers are mixed. Under the acidic conditions, phenylbutazone partitions quantitatively into tlie organic layer, which is collected, evaporated, and dissolved in the mobile phase to be analyzed by liquid chromatography. Separation is performed on a reversed-phase column using an isocratic 0.02 M phosphate buffer/methanol mobile phase, and determination is by ultraviolet detection at 264 nm (Fig. 29.18.2). The limit of detection and limit of quantification were 3.0 and 5.4 ppb, respectively (Table 29.17). 

In the Basic Protocol, gas-liquid chromatography (GLC) using an open tubular wall, coated, fused silica column with nonpolar liquid phase is described. In Alternate Protocol 1, reversed-phase HPLC (RP-HPLC) is applied for separation and quantification of cholesterol. In Alternate Protocol 2, enzymatic measurement is applied for determination of cholesterol. 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

In this unit, methods for reversed-phase high-performance liquid chromatography (HPLC) are described for the analysis of polyphenolics. HPLC analysis can be employed in an easy and fast manner to obtain an accurate elucidation and quantification of individual polyphenolic compounds found in plant-based materials. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and uses various combinations of mobile and stationary phases. 

Gibson, C.R. et al. Electrospray ionization mass spectrometry coupled to reversed-phase ion-pair high-performance liquid chromatography for quantification of sodium boroc-aptate and application to pharmacokinetic analysis. Anal. Chem. 2002, 74, 2394—2399. 

Liquid chromatography is a separation technique which is used widely in many different areas of analytical chemistry and provides a powerful tool for the separation and quantification of substances in various matrices. Nowadays, the majority of the high-performance liquid chromatographic methods are carried out in reversed-phase mode using a nonpolar stationary phase and a polar mobile phase. 

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