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Analysis of inorganic polyphosphates by capillary gel electrophoresis.

ABSTRACT: This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((P(i))(n)). Resolution of (P(i))(n) on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (P(i))(n) with n up to approximately 70; preparation and use of authentic standards enables the identification of peaks for (P(i))(n) with n = 1-10. The main advantages of this method over previously reported methods for analyzing mixtures of (P(i))(n) (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (P(i))(n) by suppressed conductivity. The method is useful for analyzing (P(i))(n) generated by the dehydration of P(i) at low temperature (125-140 degrees C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric, or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters).

SUBMITTER: Lee A

PROVIDER: S-EPMC2926810 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Analysis of inorganic polyphosphates by capillary gel electrophoresis.

Lee Andrew A Whitesides George M GM

Analytical chemistry 20100801 16

This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((P(i))(n)). Resolution of (P(i))(n) on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks repre ...[more]

PMID: 20704373

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Project description:BackgroundMolecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes.MethodsSamples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes.ResultsCapillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used.ConclusionsGenotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative risks of treatment failure.

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Analysis of inorganic polyphosphates by capillary gel electrophoresis.

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Analysis of inorganic polyphosphates by capillary gel electrophoresis.

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