Capillary electrophoresis quantitative analysis

We have developed the method for quantitative analysis of urinary albumin with CE. A capillary electrophoresis systems Nanophor 01 (Institute of Analytical Instmmentation, Russian Academy of Sciences, Saint-Petersburg) equipped with a UV-detector was used to determine analyte. Separation was achieved using 45 cmx30 p.m I.D. fused silica capillary column with UV-detection at 214 nm. 

Schmidt, T., Friehs, K., Schleef, M., Voss, C., and Hasche, E., Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis, Anal. Biochem., 274, 235, 1999. 

Hurst, W.J. and Martin, R.A. Jr, The quantitative determination of caffeine in beverages using capillary electrophoresis analysis, 21,389-91,1993. 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

As the name implies, capillary electrophoresis is electrophoresis that is made to occur inside a piece (50 to 100 cm) of small-diameter capillary tubing, similar to the tubing used for capillary GC columns. The tubing contains the electrolyte medium, and the ends of the tube are dipped into solvent reservoirs, as is the paper in paper electrophoresis. Electrodes in these reservoirs create the potential difference across the capillary tube. An electronic detector, such as those described for HPLC (Chapter 13), is on-line and allows detection and quantitative analysis of mixture components. 

Capillary electrophoresis is an electrophoresis technique in which the mixture components are separated in a capillary tube and detected with an on-line detector after the separation occurs. The advantages include smaller quantity of sample and qualitative and quantitative analysis in a much shorter time. 

Because of their high separation capacity, short analysis time, low reagent consumption and simplicity, various electrophoretic methods have found application in the separation and quantitative determination of anthocyanins in various complex matrices [267].The different techniques used for the measurement of anthocyanins in beverages [268], the application of capillary electrophoresis (CE) for the analysis of natural food pigments [269], the use of CE for the determination of anthocyanins in foods [270] and in medicinal plants [271] have been previously reviewed. 

This chapter deals with the validation of capillary electrophoresis (CE) methods. It describes the various validation characteristics, namely accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range in accordance with the official guidelines. Practical aspects related to the calculation of these parameters and factors affecting them in CE analysis have also been described. Validation requirements have been described according to the goal of the method. The chapter contains numerous tables and diagrams to illustrate these ideas. It also covers other related aspects such as instrument qualification, revalidation, and method transfer. 

Lozano, R., Warren, F. V., Perlman, S., and Joseph, J. M. (1995). Quantitative analysis of fosinopril sodium by capillary zone electrophoresis and liquid chromatography. /. Pharm. Biomed. Anal. 13, 139-148. 

Altria, K. D. (1993). Quantitative aspects of the application of capillary electrophoresis to the analysis of pharmaceuticals and drug-related impurities. /. Chromatogr. 646, 245—257. 

Dedicated applications of capillary zone electrophoresis (CZE) coupled to MS are discussed, particularly in the field of drug analysis. Development of other capillary-based electrodriven separation techniques such as non-aqueous capillary electrophoresis (NACE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) hyphenated with MS are also treated. The successful coupling of these electromigration schemes with MS detection provides an efficient and sensitive analytical tool for the separation, quantitation, and identification of numerous pharmaceutical, biological, therapeutic, and environmental compounds. 

Juan-Garcia, A., Font, G., and Pico, Y. (2005). Quantitative analysis of six pesticides in fruits by capillary electrophoresis-electrospray-mass spectrometry. Electrophoresis 26, 1550—1561. 

Soga, T., Kakazu, Y., Robert, M., Tomita, M., andNishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry. Electrophoresis 25, 1964—1972. 

To establish chiral separation method for donepezil hydrochloride enantiomers by capillary electrophoresis (CE) and to determine the two enantiomers in plasma [39], alkalized plasma was extracted by isopropa-nol-n-hexane (3 97) and L-butefeina was used as the IS. Enantioresolution was achieved using 2.5% sulfated-beta-cyclodextrin as chiral selector in 25 mmol/1 triethylammonium phosphate solution (pH 2.5) on the uncoated fused-silica capillary column (70 cm x 50 fim i.d.). The feasibility of the method to be used as quantitation of donepezil HC1 enantiomers in rabbit plasma was also investigated. Donepezil HC1 enantiomers were separated at a baseline level under the above condition. The linearity of the response was evaluated in the concentration range from 0.1 to 5 mg/1. The linear regression analysis obtained by plotting the peak area ratio (A(s)/A(i)) of the analyte to the IS versus the concentration (C) showed excellent correlation coefficient The low limit of detection was 0.05 mg/1. The inter- and intra-day precisions (RSD) were all less than 20%. Compared with chiral stationary phase by HPLC, the CE method is simple, reliable, inexpensive, and suitable for studying the stereoseletive pharmacokinetics in rabbit. 

Schmitt-Kopplin, P., Garmash, A. V., Kudryavtsev, A. V., Menzinger, F., Perminova, I. V., Hertkorn, N., Freitag, D., Petrosyan, V. S., and Kettrup, A. (2001a). Quantitative and qualitative precision improvements by effective mobility-.scale data transformation in capillary electrophoresis analysis. Electrophoresis 22,77-87. 

Traditional biochemical techniques such as liquid chromatography (LC), gel electrophoresis, capillary electrophoresis (CE), and mass spectrometry (MS) have been widely used for the complete analysis of salivary proteins and peptides. The recent advances in these technical approaches applied to peptidomics have allowed a better comprehensive analysis of peptides in human whole saliva, envisioning the identification of potential salivary biomarkers of oral and systemic diseases. Sample preparation is a critical experimental step for the successful identification of peptides using MS-based approaches, for their quantitation and identification of PTMs. 

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