Quantitative analysis with capillary electrophoresis

With capillary electrophoresis (CE), it is possible to carry out simultaneous quantitative analysis with small sample volumes (1-10 nl) the amount of solvent waste generated is in the order of 1-2 ml/day and requires much less analysis time, with outstanding resolution. CE may be able to resolve a component of interest more quickly and with less... 

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. 

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. 

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. 

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. 

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. 

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. 

The same authors also applied capillary electrophoresis to the study of benazepril hydrochloride and several angiotensin-converting enzyme inhibitors [43]. Separation of the compounds was performed by means of two phosphate buffers (each 0.1 M) at pH 7 and 6.25, respectively [42], Due to the highest selectivity of the first mentioned running buffer, the same system has been applied for the quantification of benazepril and other compounds in their corresponding pharmaceutical formulations. It was found that the possibility of simultaneous identification and quantification of the active ingredient in the finished products was especially attractive, and that excipients do not adversely affect the results. This article deals with the validation of some parameters of the quantitative analysis, namely linearity, precision, accuracy, and robustness [43],... 

Furthermore, online chromatographic coupling techniques such as HPLC and CE coupled to ICP-MS with the isotope dilution technique have been used for element quantification in specia-tion analysis. Species analysis by ICP-SFMS in medical research has been performed by Prange et a/. on separated isoforms of metallothionein (e.g., of rabbit liver) by capillary electrophoresis (CE). For quantitative determination of S, Cd, Cu and Zn in isoforms, highly enriched " S, Cu, Zn and Cd spikes are added to the analyte solution in the interface region of CE. 

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